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The taxonomic status of Nicator: Evidence from feather protein electrophoresis
BiochemicalSystematicsand Ecology,Vol. 15, No. 5, pp. 629-634, 1987. Printed in Great Britain.
0305-1978/87 $3.00+0.00 PergamonJournalsLtd.
The Taxonomic Status of Nicator: Evidence from Feather Protein Electrophoresis O. HANOTTE*, A. G. KNOXt and A. PRIGOGINE1: DOpartement de Chimie G6n~rale Iet Laboratoire de Biologie Animale et Cellulaire, Universit~ Libre de Bruxelles, 50 avenue F. D. Roosevelt, B 1050 Brussels, Belgium; tSub-department of Ornithology, British Museum (Natural History), Tring, Herts. HP23 6AP, England; ~lnstitut Royal des Sciences Naturelles de Belgique, 31,'rue Vautier, B 1040 Brussels, Belgium
Key Word Index--Pycnonotidae; Laniidae; Nicator; keratin; electrophoresis; phylogenetic relationship. Abstract--Electrophoresis of soluble feather proteins was used to investigate the controversial taxonomic status of the genus Nicator. Nicatorwas compared to species in four genera of Pycnonotidae and two sub-families of Laniidae. Electrophoretic similarity values were calculated and then transformed to Nei D distances. Dendrograms were constructed using the Fitch-Margoliash, unweighted pair-group, Farris and modified Farris algorithms. The results clearly show the affinities of Nicatorare with Pycnonotidae and not with the Laniidae. The modified Farris tree gave the best fit. Within the Pycnonotidae, our data do not support a close relationship between Pycnonotus and Andropadus, as had been previously suggested.
Introduction The genus Nicator contains two [1] or three [2] species, all endemic to Africa. Based largely on morphological criteria, Nicator has been placed in the shrike family Laniidae (sub-family Malacotinae [3-6]) or with the bulbuls, Pycnonotidae [1, 2, 7]. "Like many of the greenbuls, the Nicators are olive with greyish underparts and they have yellow under-tail coverts like most forms of Pycnonotus barbatus. They are distinct in having yellow spots on the wings, yellow thighs and yellow tips to the tails. The bill is stronger and more hooked at the tip than in other bulbuls. The nestling of N. chloris is distinctive in having the head and neck bare except for a strip of feathers on the crown: that of N. vireo is not known." [1]. According to Chappuis [8], the vocalization of Nicator also indicate an affinity with the Pycnonotidae. DNA-DNA hybridization techniques also place Nicatorwith this family [9]. Feathers are composed mainly of keratin, a family of similar proteins of small M r (about *Present address: Laboratoire de Biochimie Mol~culaire, Facult~ de M~decine, Universit6 de I'Etat ~ Mons, 24, avenue du Champ de Mars 7000 Mons, Belgium. (Received 19 December 1986) 629
10 500). Their organization and evolution is becoming better understood [10-13]. The advantages of keratins over other proteins for biochemical study are clear: they are readily obtainable, and samples can be taken from museum skins or collected from live birds without killing the donor. Storage and transportation could not be easier [14]. The first attempts to compare solubilized feather keratins (see Experimental) from different species were made by Schroeder et al. [15] and Harrap and Woods [16-18], but it as only with the work of Brush [19] that the technique was seriously applied to taxonomic problems. Knox [14, 20, 21] and Brush and Witt [22] extended and confirmed the taxonomic value of the methodology. It has even been used in the discovery of a new species of birds (Knox, [23]). Results Several electrophoretic gels were prepared and critical comparisons made on the one giving the maximum resolution and clarity. Similarity values (I) were obtained between pairs of species by calculating the number of bands with the same mobility in both divided by the total number of band positions for the pair.
630
O. HANOTTE, A. G. KNOX AND A. PRIGOGINE
These values were then transformed to Nei genetic distances (D) using D =--log EI From the genetic distances, dendrograms were constructed according to the Fitch-Margoliash (F-M, [24]), UPGMA (unweighted pairgroup, [25]) and Wagner, [26, 27] procedures. UPGMA assumes a uniform evolutionary rate in all lineages, whereas this is not necessary for the F-M or Farris methods. The Tateno et al. [27] modification of the Farris approach [26] is suitable for use in cases where the distance measure is proportional to evolutionary time. The percent standard deviation (SD) of the trees were compared [24] to assess their goodness of fit to the original data. Visual inspection of the electrophoresis gels indictes a closer similarity of Nicator to the Pycnonotidae than to Laniidae (Fig. 1). This is confirmed by the calculated electrophoretic similarities and Nei D values (Table 1). The genetic distances between Nicator and the shrikes are several times greater than between Nicator and the bulbuls, and are comparable with the distances between the four control Pycnonotidae and the Laniidae. Dendrograms were constructed from these data (Fig. 2, a-d). Nicator clearly clusters with the Pycnonotidae rather than with the Laniidae. Of the four procedures used, the modified Farris gives the closest fit (SD=11.0%; F-M SD=17.4%; UPGMA SD=20.5% and Farris SD=23.6%).
Discussion During an investigation of the feather proteins TABLE 1. SIMILARITY AND NEI D VALUES Genus* Genus
And
Andropadus Ixonotus 0.24 Phyllastrephus 0.53 Nicator 0.43 Pycnonotus 0.58 Dryoscopus 1.24 Lanius 1.10
Ixo
Phi
Nic
Pyc
Dry
Lan
0.79
0.59 0.64
0.65 0.67 0.50
0.56 0.69 0.85 0.86
0,29 0.26 0.24 0.17 0.14
0.33 0.31 0.28 0.20 0.17 0.64
0.45 0.40 0.37 1.35 1.17
0.69 0.16 1.43 1.27
0.58 1.77 1.60
1.96 1.77
0.44
*As in first column. Electrophoretic similarity values (above the diagonal) and genetic distances (below) for Nicator, four bulbuls (Andropadus, Ixonotus, Phyllastrephus and Pycnonotus) and two shrikes (Dryoscopus and Lanius). See Experimental.
of the Pelicaniformes and Cuculiformes, Brush and Witt [22] found that the tree topology differed considerably depending on the clustering algorithm employed. In the present study, the UPGMA and F-M trees (Fig. 2a, b) have the same structure, which disagrees only in one linkage from the two Wagner trees. This difference does not affect our primary conclusion--Nicator is unambiguously closer to the Pycnonotidae than the Laniidae, on the basis of their feather proteins. We suspect the poor concordance between Brush and Witt's trees [22] resulted from their smaller sample of electrophoretic bands, particularly on their low pH gels. We further suggest that the feather protein techniques, as presently applied, are only suitable for examining the relationships of fairly closely related taxa. In the past it has been suggested that Andropadus and Pycnonotus were closely related (they were treated as congeneric in ref. [21]). This is not supported by our feather protein evidence, which indicates a closer affinity between other Pycnonotid genera than between these two. Because only one species in each genus has been examined, this result should be considered with caution. At present, the technique of gradient gel electrophoresis represents a most efficient way to compare avian feather proteins in a relatively quick and inexpensive manner. However, it must be remembered that the patterns are of whole protein, and it is not possible to precisely determine the relationships between the keratin monomers of different mobility in different species. Nor is there sufficient discrimination to permit population genetic techniques to be utilized, although this may be possible with projected improvements in the resolution. Thorpe and Giddings [28] and Thorpe [29] obtained good results using isoelectric focusing of reptilian keratins, and preliminary studies (Knox, A. G. unpublished) indicate that the technique may offer enhanced definition with birds.
Experimental Feather s a m p l e s w e r e o b t a i n e d f r o m the Institut Royal des Sciences Naturelles de Belgique (IRSNB). The species e x a m i n e d w e r e Andropadus virens, Ixonotus guttatus, Pycnonotus barbatus and Phyllastrephus xavieri f r o m the
631
!
5
FIG. 1 SCMK ELECTROPHORETIC PATTERNS OF: 1. ANDROPADUS VIRENS:2. IXONOTUSGUTTATUS; 3. PHYLLASTREPHU$XAVIER/;4. NICATOR CHLORIS; 5. PYC/VONOTUSBARBATUS; 6. DRYOSCOPUSCUB/A; 7. LANIUS COLLURIO.
THE TAXONOMIC STATUS OF NICATOR:EVIDENCE FROM FEATHER PROTEIN ELECTROPHORESIS
633
0.12
Andropadus
0.12
0.07
I
Ixonotus
o.21
Nicator
0.49 0.08 0.20 t0.08
O.
Pycnonotus
0.22
55
(a)
Phy~(asfrephus
Dryoscopus
0.22
Lonius ~ndropodus
,<[.._ C) II - O.0 7 / ~''''~;'LL~
Ixonotus
IV/ha(or
/
(
b
~o.3o
~
L.on/us
Shrikes
Butbuts
Dryoscopus
0.:52
Phy( ~ostrephus Andropodus
Ixonotus
o.o.z_~=.. 0.98
~)
0.23
T
~0"09
0.12
0.32
1"0'04 P yc nonotus
I0.30
Lan/us
(c)
I Nicator
FIG. 2, DENDROGRAMS CONSTRUCTED FROM THE SCMK NEI D VALUES FOR N/CATOR, FOUR BULBULS (ANDROPADUS, IXONOTUS, PHYLLASTREPHUSAND PYCNONOTUS)AND TWO SHRIKES (DRYOSCOPUSAND LANIUS). (a), UPGMA; (b), Fitch-Margoliuh; (c) Farris; (d),
modified Farris,
634
O. HANOTTE,A. G. KNOX AND A. PRIGOGINE
Shrikes
BuLbuLs
Oryoscopus
Phy/(os~rephus
0.30 Andropodus [xonotus
0%
0.92
0.28
~0.12 / O . 04
0.14
Pycnonotus
Lonms
(d) N/cotor
FIG. 2. Continued Pycnonotidae; Dryoscopus cubla (Malacotinae) and Lanius collurio (Laniidae) from the Laniidae; and Nicator chloris. Although the protein from the different morphological components of a feather give different electrophoretic patterns [16, 24, 25], there is no significant intraspecific variation when corresponding parts are used from different individuals [14, 19]. For this reason, only one individual was sampled from each species. The 6th primary feather from each bird was washed and degreased and the pennaceous barbs removed for solubilization. Barbs were used as they have previously been demonstrated to give the most complex electrophoretic patterns [16, 30, 31]. They were rendered soluble and converted to their S-carboxymethyl form (SCMK). Electrophoresis was then carried out in polyacrylamide gradient gels with a Tris-glycine buffer pH 8.3. After pptn with 7.5% trichloracetic acid, the gels were stained with 0.25% Coomassie Blue in 10% acetic acid, followed by destaining in 10% acetic acid. Full experimental details are given in ref. [14]. Acknowledgements--We wish to thank P. Devillers and J. Pasteels for helpful discussion and criticism of this research, and A. G. Schneck for his constant encouragement. D. W. Snow commented on the manuscript.
References 1. Hall, B. P. and Moreau, R. E. (1970) An Atlas of Speciation in African Passerine Birds. Brit. Mus Nat. Hist., London. 2. Rand, A. L. and Deignan, H. G. (1980) in Check-List of Birds of the WorldVol. 9, p. 221. Museum of Comparative Zoology, Cambridge, Mass. 3. Reichenow, A. (1902-1903) Die Vogel Afrikas Vol. 2. Neumann, Neudamm. 4. Sclater, W. L. (1930) Systema Avium Aethiopicarum Part 2. British Ornithologists' Union, London. 5. Wolters, H. E. (1977) Die Vogelarten der Erde Vot. 3. Paul Parey, Hamburg.
6. Wolters, H. E. (1979) Die Voge/arten der Erde VoI. 4. Paul Parey, Hamburg. 7. Chapin, J. P. (1953) Bull. Am. Mus. Nat. Hist. 75A, 1. 8. Chappuis, C. (1975) Alauda 43, 427. 9. Sibley, C. G. and Ahlquist, J. E. (1985) in Proc. Intern. Syrup. African. vertebr. (Schuchmann, K. L., ed.) p, 115. Bonn. 10. Kemp, D. J. (1975) Nature, Lond. 254, 573. 11. Lockett, T. J., Kemp, J. and Rogers, G. E. (1979) Biochem. 18, 5654. 12. Molloy, P. L., Powel, B. C., Gregg, K., Barone, E. D. and Rogers, G. E. (1982) Nucl. Acids Res. 10, 6007. 13. Gregg, K., Wilton, S. D., Parry, D. A. D. and Rogers, G. E. (1984) EMBO J. 3, 175. 14. Knox, A. G. (1980) Comp. Biochem. Physiol. 65B, 45. 15. Schroeder, W. A., Kay, L. M., Lewis, B. & Munger, N. (1955) J. Am. Chem. Soc. 77, 3901. 16. Harrap, B. S. and Woods, E. F. (1964) Biochem. J. 92, 8. 17. Harrap, B. S. and Woods, E. F. (1964) Biochem, J. 92, 19. 18. Harrap, B. S. and Woods, E. F. (1967) Cornp. Biochem. Physiol. 20, 449. 19. Brush, A. H. (1976) J. Zoo/. Lond. 179, 467. 20. Knox, A. G. (1977) Ph.D. Thesis, University of Aberdeen. 21. Knox, A. G. (1980) Ibis 122, 72. 22. Brush, A. H. and Witt, H. H. (1983) Ibis 125, 181. 23. Snow, D. W. (1980) Bull. Brit. Ore. CI. 100, 213. 24. Fitch, W. M. and Margoliash, E. (1967) Science 155, 279. 25. Sneath, P. H. A. and Sokal, R. R. (1973) Numerical taxonomy. Freeman, San Francisco. 26. Farris, J. S. (1972) Am. Nat. 106, 645. 27. Tateno, Y., Nei, M. and Tajima, F. (1982) J. Mol. Evol. 18, 387. 28. Thorpe, R. S. and Giddings, M. R. (1981) Experientia 37, 700. 29. Thorpe, R. S. (1985) Biochem. Syst. Ecol. 13, 63. 30. Brush, A. H. (1972) Biochemo Genet. 7, 87. 31. Kemp, D. J. and Rogers, G. E. (1972) Biochemistry 11, 969.
0305-1978/87 $3.00+0.00 PergamonJournalsLtd.
The Taxonomic Status of Nicator: Evidence from Feather Protein Electrophoresis O. HANOTTE*, A. G. KNOXt and A. PRIGOGINE1: DOpartement de Chimie G6n~rale Iet Laboratoire de Biologie Animale et Cellulaire, Universit~ Libre de Bruxelles, 50 avenue F. D. Roosevelt, B 1050 Brussels, Belgium; tSub-department of Ornithology, British Museum (Natural History), Tring, Herts. HP23 6AP, England; ~lnstitut Royal des Sciences Naturelles de Belgique, 31,'rue Vautier, B 1040 Brussels, Belgium
Key Word Index--Pycnonotidae; Laniidae; Nicator; keratin; electrophoresis; phylogenetic relationship. Abstract--Electrophoresis of soluble feather proteins was used to investigate the controversial taxonomic status of the genus Nicator. Nicatorwas compared to species in four genera of Pycnonotidae and two sub-families of Laniidae. Electrophoretic similarity values were calculated and then transformed to Nei D distances. Dendrograms were constructed using the Fitch-Margoliash, unweighted pair-group, Farris and modified Farris algorithms. The results clearly show the affinities of Nicatorare with Pycnonotidae and not with the Laniidae. The modified Farris tree gave the best fit. Within the Pycnonotidae, our data do not support a close relationship between Pycnonotus and Andropadus, as had been previously suggested.
Introduction The genus Nicator contains two [1] or three [2] species, all endemic to Africa. Based largely on morphological criteria, Nicator has been placed in the shrike family Laniidae (sub-family Malacotinae [3-6]) or with the bulbuls, Pycnonotidae [1, 2, 7]. "Like many of the greenbuls, the Nicators are olive with greyish underparts and they have yellow under-tail coverts like most forms of Pycnonotus barbatus. They are distinct in having yellow spots on the wings, yellow thighs and yellow tips to the tails. The bill is stronger and more hooked at the tip than in other bulbuls. The nestling of N. chloris is distinctive in having the head and neck bare except for a strip of feathers on the crown: that of N. vireo is not known." [1]. According to Chappuis [8], the vocalization of Nicator also indicate an affinity with the Pycnonotidae. DNA-DNA hybridization techniques also place Nicatorwith this family [9]. Feathers are composed mainly of keratin, a family of similar proteins of small M r (about *Present address: Laboratoire de Biochimie Mol~culaire, Facult~ de M~decine, Universit6 de I'Etat ~ Mons, 24, avenue du Champ de Mars 7000 Mons, Belgium. (Received 19 December 1986) 629
10 500). Their organization and evolution is becoming better understood [10-13]. The advantages of keratins over other proteins for biochemical study are clear: they are readily obtainable, and samples can be taken from museum skins or collected from live birds without killing the donor. Storage and transportation could not be easier [14]. The first attempts to compare solubilized feather keratins (see Experimental) from different species were made by Schroeder et al. [15] and Harrap and Woods [16-18], but it as only with the work of Brush [19] that the technique was seriously applied to taxonomic problems. Knox [14, 20, 21] and Brush and Witt [22] extended and confirmed the taxonomic value of the methodology. It has even been used in the discovery of a new species of birds (Knox, [23]). Results Several electrophoretic gels were prepared and critical comparisons made on the one giving the maximum resolution and clarity. Similarity values (I) were obtained between pairs of species by calculating the number of bands with the same mobility in both divided by the total number of band positions for the pair.
630
O. HANOTTE, A. G. KNOX AND A. PRIGOGINE
These values were then transformed to Nei genetic distances (D) using D =--log EI From the genetic distances, dendrograms were constructed according to the Fitch-Margoliash (F-M, [24]), UPGMA (unweighted pairgroup, [25]) and Wagner, [26, 27] procedures. UPGMA assumes a uniform evolutionary rate in all lineages, whereas this is not necessary for the F-M or Farris methods. The Tateno et al. [27] modification of the Farris approach [26] is suitable for use in cases where the distance measure is proportional to evolutionary time. The percent standard deviation (SD) of the trees were compared [24] to assess their goodness of fit to the original data. Visual inspection of the electrophoresis gels indictes a closer similarity of Nicator to the Pycnonotidae than to Laniidae (Fig. 1). This is confirmed by the calculated electrophoretic similarities and Nei D values (Table 1). The genetic distances between Nicator and the shrikes are several times greater than between Nicator and the bulbuls, and are comparable with the distances between the four control Pycnonotidae and the Laniidae. Dendrograms were constructed from these data (Fig. 2, a-d). Nicator clearly clusters with the Pycnonotidae rather than with the Laniidae. Of the four procedures used, the modified Farris gives the closest fit (SD=11.0%; F-M SD=17.4%; UPGMA SD=20.5% and Farris SD=23.6%).
Discussion During an investigation of the feather proteins TABLE 1. SIMILARITY AND NEI D VALUES Genus* Genus
And
Andropadus Ixonotus 0.24 Phyllastrephus 0.53 Nicator 0.43 Pycnonotus 0.58 Dryoscopus 1.24 Lanius 1.10
Ixo
Phi
Nic
Pyc
Dry
Lan
0.79
0.59 0.64
0.65 0.67 0.50
0.56 0.69 0.85 0.86
0,29 0.26 0.24 0.17 0.14
0.33 0.31 0.28 0.20 0.17 0.64
0.45 0.40 0.37 1.35 1.17
0.69 0.16 1.43 1.27
0.58 1.77 1.60
1.96 1.77
0.44
*As in first column. Electrophoretic similarity values (above the diagonal) and genetic distances (below) for Nicator, four bulbuls (Andropadus, Ixonotus, Phyllastrephus and Pycnonotus) and two shrikes (Dryoscopus and Lanius). See Experimental.
of the Pelicaniformes and Cuculiformes, Brush and Witt [22] found that the tree topology differed considerably depending on the clustering algorithm employed. In the present study, the UPGMA and F-M trees (Fig. 2a, b) have the same structure, which disagrees only in one linkage from the two Wagner trees. This difference does not affect our primary conclusion--Nicator is unambiguously closer to the Pycnonotidae than the Laniidae, on the basis of their feather proteins. We suspect the poor concordance between Brush and Witt's trees [22] resulted from their smaller sample of electrophoretic bands, particularly on their low pH gels. We further suggest that the feather protein techniques, as presently applied, are only suitable for examining the relationships of fairly closely related taxa. In the past it has been suggested that Andropadus and Pycnonotus were closely related (they were treated as congeneric in ref. [21]). This is not supported by our feather protein evidence, which indicates a closer affinity between other Pycnonotid genera than between these two. Because only one species in each genus has been examined, this result should be considered with caution. At present, the technique of gradient gel electrophoresis represents a most efficient way to compare avian feather proteins in a relatively quick and inexpensive manner. However, it must be remembered that the patterns are of whole protein, and it is not possible to precisely determine the relationships between the keratin monomers of different mobility in different species. Nor is there sufficient discrimination to permit population genetic techniques to be utilized, although this may be possible with projected improvements in the resolution. Thorpe and Giddings [28] and Thorpe [29] obtained good results using isoelectric focusing of reptilian keratins, and preliminary studies (Knox, A. G. unpublished) indicate that the technique may offer enhanced definition with birds.
Experimental Feather s a m p l e s w e r e o b t a i n e d f r o m the Institut Royal des Sciences Naturelles de Belgique (IRSNB). The species e x a m i n e d w e r e Andropadus virens, Ixonotus guttatus, Pycnonotus barbatus and Phyllastrephus xavieri f r o m the
631
!
5
FIG. 1 SCMK ELECTROPHORETIC PATTERNS OF: 1. ANDROPADUS VIRENS:2. IXONOTUSGUTTATUS; 3. PHYLLASTREPHU$XAVIER/;4. NICATOR CHLORIS; 5. PYC/VONOTUSBARBATUS; 6. DRYOSCOPUSCUB/A; 7. LANIUS COLLURIO.
THE TAXONOMIC STATUS OF NICATOR:EVIDENCE FROM FEATHER PROTEIN ELECTROPHORESIS
633
0.12
Andropadus
0.12
0.07
I
Ixonotus
o.21
Nicator
0.49 0.08 0.20 t0.08
O.
Pycnonotus
0.22
55
(a)
Phy~(asfrephus
Dryoscopus
0.22
Lonius ~ndropodus
,<[.._ C) II - O.0 7 / ~''''~;'LL~
Ixonotus
IV/ha(or
/
(
b
~o.3o
~
L.on/us
Shrikes
Butbuts
Dryoscopus
0.:52
Phy( ~ostrephus Andropodus
Ixonotus
o.o.z_~=.. 0.98
~)
0.23
T
~0"09
0.12
0.32
1"0'04 P yc nonotus
I0.30
Lan/us
(c)
I Nicator
FIG. 2, DENDROGRAMS CONSTRUCTED FROM THE SCMK NEI D VALUES FOR N/CATOR, FOUR BULBULS (ANDROPADUS, IXONOTUS, PHYLLASTREPHUSAND PYCNONOTUS)AND TWO SHRIKES (DRYOSCOPUSAND LANIUS). (a), UPGMA; (b), Fitch-Margoliuh; (c) Farris; (d),
modified Farris,
634
O. HANOTTE,A. G. KNOX AND A. PRIGOGINE
Shrikes
BuLbuLs
Oryoscopus
Phy/(os~rephus
0.30 Andropodus [xonotus
0%
0.92
0.28
~0.12 / O . 04
0.14
Pycnonotus
Lonms
(d) N/cotor
FIG. 2. Continued Pycnonotidae; Dryoscopus cubla (Malacotinae) and Lanius collurio (Laniidae) from the Laniidae; and Nicator chloris. Although the protein from the different morphological components of a feather give different electrophoretic patterns [16, 24, 25], there is no significant intraspecific variation when corresponding parts are used from different individuals [14, 19]. For this reason, only one individual was sampled from each species. The 6th primary feather from each bird was washed and degreased and the pennaceous barbs removed for solubilization. Barbs were used as they have previously been demonstrated to give the most complex electrophoretic patterns [16, 30, 31]. They were rendered soluble and converted to their S-carboxymethyl form (SCMK). Electrophoresis was then carried out in polyacrylamide gradient gels with a Tris-glycine buffer pH 8.3. After pptn with 7.5% trichloracetic acid, the gels were stained with 0.25% Coomassie Blue in 10% acetic acid, followed by destaining in 10% acetic acid. Full experimental details are given in ref. [14]. Acknowledgements--We wish to thank P. Devillers and J. Pasteels for helpful discussion and criticism of this research, and A. G. Schneck for his constant encouragement. D. W. Snow commented on the manuscript.
References 1. Hall, B. P. and Moreau, R. E. (1970) An Atlas of Speciation in African Passerine Birds. Brit. Mus Nat. Hist., London. 2. Rand, A. L. and Deignan, H. G. (1980) in Check-List of Birds of the WorldVol. 9, p. 221. Museum of Comparative Zoology, Cambridge, Mass. 3. Reichenow, A. (1902-1903) Die Vogel Afrikas Vol. 2. Neumann, Neudamm. 4. Sclater, W. L. (1930) Systema Avium Aethiopicarum Part 2. British Ornithologists' Union, London. 5. Wolters, H. E. (1977) Die Vogelarten der Erde Vot. 3. Paul Parey, Hamburg.
6. Wolters, H. E. (1979) Die Voge/arten der Erde VoI. 4. Paul Parey, Hamburg. 7. Chapin, J. P. (1953) Bull. Am. Mus. Nat. Hist. 75A, 1. 8. Chappuis, C. (1975) Alauda 43, 427. 9. Sibley, C. G. and Ahlquist, J. E. (1985) in Proc. Intern. Syrup. African. vertebr. (Schuchmann, K. L., ed.) p, 115. Bonn. 10. Kemp, D. J. (1975) Nature, Lond. 254, 573. 11. Lockett, T. J., Kemp, J. and Rogers, G. E. (1979) Biochem. 18, 5654. 12. Molloy, P. L., Powel, B. C., Gregg, K., Barone, E. D. and Rogers, G. E. (1982) Nucl. Acids Res. 10, 6007. 13. Gregg, K., Wilton, S. D., Parry, D. A. D. and Rogers, G. E. (1984) EMBO J. 3, 175. 14. Knox, A. G. (1980) Comp. Biochem. Physiol. 65B, 45. 15. Schroeder, W. A., Kay, L. M., Lewis, B. & Munger, N. (1955) J. Am. Chem. Soc. 77, 3901. 16. Harrap, B. S. and Woods, E. F. (1964) Biochem. J. 92, 8. 17. Harrap, B. S. and Woods, E. F. (1964) Biochem, J. 92, 19. 18. Harrap, B. S. and Woods, E. F. (1967) Cornp. Biochem. Physiol. 20, 449. 19. Brush, A. H. (1976) J. Zoo/. Lond. 179, 467. 20. Knox, A. G. (1977) Ph.D. Thesis, University of Aberdeen. 21. Knox, A. G. (1980) Ibis 122, 72. 22. Brush, A. H. and Witt, H. H. (1983) Ibis 125, 181. 23. Snow, D. W. (1980) Bull. Brit. Ore. CI. 100, 213. 24. Fitch, W. M. and Margoliash, E. (1967) Science 155, 279. 25. Sneath, P. H. A. and Sokal, R. R. (1973) Numerical taxonomy. Freeman, San Francisco. 26. Farris, J. S. (1972) Am. Nat. 106, 645. 27. Tateno, Y., Nei, M. and Tajima, F. (1982) J. Mol. Evol. 18, 387. 28. Thorpe, R. S. and Giddings, M. R. (1981) Experientia 37, 700. 29. Thorpe, R. S. (1985) Biochem. Syst. Ecol. 13, 63. 30. Brush, A. H. (1972) Biochemo Genet. 7, 87. 31. Kemp, D. J. and Rogers, G. E. (1972) Biochemistry 11, 969.