J.Mol.Evol.8,271-282

Journal of

(1976)

MolecularEvolution © by Springer-Vedag I976

Penguin Evolution: Protein Comparisons Demonstrate Phylogenetic Relationship to Flying Aquatic Birds* CHARLES Y.-K. HO"**, ELLEN M. PRAGER 2, ALLAN C. WILSON 2, DAVID T. OSUGA 3, and ROBERT E. FEENEY 3 1 Department of Food Science and Technology, University of California, Davis, Cal. 95616, USA 2

Department of Biochemistry, University of California, Berkeley, Cal. 94720, USA

Received May 29, 1976; July 20, 1976

Summary.

Quantitative immunological comparisons of three avian proteins,

transferrin, ovalbumin, and penalbumin, indicate that penguins are phylogenetically most closely related to loons, albatrosses, herons, and grebes. These data support the theory that the ancestors of penguins were flying oceanic birds and that flightlessness in penguins has evolved independently from flightlessness in ratites. Key words: Penguins/Birds/Bird Evolution/Protein Evolution/Transferrin/

Ovalbumin/Penalbumin/Sequence-Immunology Correlation~Micro-Complement Fixation/Phylogeny

Much

uncertainty

exists

regarding

the

evolutionary

relation-

ships among the major groups of birds (Sibley & Ahlquist, 1972). Of particular i n t e r e s t is t h e o r i g i n o f p e n g u i n s , f l i g h t l e s s s e a b i r d s of t h e S o u t h e r n H e m i s p h e r e comprising the order

*This work was supported in part by grants GB-42028X from NSF and GM-21509 from NIH to A.C.W. and by grants HD-OOI22 from NIH and GA-12607 from NSF to R.E.F. Part of the data is taken from the Ph.D.thesis of C.Y.-K. Ho submitted to the Graduate Division of the University of California at Davis. eePresent address: Department of Nutrition, University of California, Davis Cal. 95616. The following abbreviations are used in this work: MC'F = micro-complement fixation; OT = ovotransferrin.

271

Sphenisciformes. Penguins are so distinctive from other birds in anatomy and way of life that ornithologists often classify them in the superorder Impennes, separate from all other bird orders (Storer, 1960). The principal theory proposed concerning the ancestors of penguins is that they were flying oceanic birds which evolved into underwater fliers and became incapable o f aerial flight (Sibley & Ahlquist, 1972; Simpson, 1975, 1976). We present here biochemical evidence which shows that penguins are very closely related to certain orders of aquatic birds. Biochemical techniques, notably comparison of the amino acid sequences of proteins among living species, enable us to construct molecular phylogenies (Fitch & Margoliash, 1967; Sarich, 1969b). Such phylogenies, even those based on a single protein, show substantial agreement in branching order with those based on morphological evidence (Maxson & Wilson, 1975; Cronin & Sarich, 1975). This is most evident from studies with the Anthropoidea, for which there is good agreement among the cladograms produced from albumin (Sarich & Wilson, 1967; Sarich, 1970), hemoglobin (Wilson & Sarich, 1969; Goodman & Moore, 1973), fibrinopeptides (Doolittle et al., 1971), nonrepetitive DNA (Kohne et al., 1972), myoglobin (Romero Herrera et al., 1973), and transferrin (Cronin & Sarich, 1975). Thus we can derive evolutionary relationships among living species despite inadequate fossil data or uncertain interpretation of morphological evidence. The degree of amino acid sequence difference among proteins may be determined by direct sequencing or, much more readily when a large number of species and several proteins are to be compared, by quantitative immunological measurements. The immunological method of greatest utility for such comparisons is m i c r o - c o m p l e m e n t fixation (MC'F), a rapid, sensitive technique which requires little material (Champion et al., 1974, 1975). The degree of antigenic difference in the MC'F test is given in immunological distance units, which are roughly proportional to the degree of amino acid sequence difference between 2 genetically homologous proteins (Champion et al., 1974, 1975). Because of these technical advantages, a preponderance of molecular phylogenies has of late been done by MC'F, rather than by direct amino acid sequencing of proteins, in a wide variety of organisms ranging from bacteria (Cocks & Wilson, 1972) to vertebrates (Sarich, 1969a,b, 1970, 1973; Sarich & Wilson, 1967; Cronin & Sarich, 1975, 1976; Wallace et al., 1973; Maxson & Wilson, 1974, 1975; Maxson et al., 1975). Several biochemical techniques, principally electrophoresis and qualitative immunological measurements, have previously been used to compare penguin proteins with those of other birds (Feeney et al., 1966, 1968; Miller & Feeney, 1966; Allison & Feeney, 1968; Feeney & Allison, 1969; Osuga et al., 1974; Sibley & Ahlquist, 1972; Baker & Manwell, 1975). To determine 272

those orders of extant birds m o s t c l o s e l y a l l i e d w i t h penguins, we have now used M C ' F to c o m p a r e 3 p r o t e i n s found in penguins, t r a n s f e r r i n , ovalbumin, and p e n a l b u m i n , w i t h the h o m o l o g o u s p r o t e i n s of r e p r e s e n t a t i v e s of all the other bird orders.

MATERIALS

AND M E T H O D S

Avian Samples. Bird egg whites, sera, and tissue extracts were o b t a i n e d from the l o n g - e s t a b l i s h e d c o l l e c t i o n s of A.C. W i l s o n and R.E. F e e n e y and w e r e p r e p a r e d and stored as d e s c r i b e d (Prager et al., 1974). Protein Purification. S e r u m t r a n s f e r r i n was p u r i f i e d from 2 species (arctic l o o n and duck) and o v o t r a n s f e r r i n (OT) from the egg w h i t e s of 14 a d d i t i o n a l species (Table I). Each r e p r e s e n t e d a d i f f e r e n t order e x c e p t the 2 p e n g u i n s (Sphenisciformes) and the emu and c a s s o w a r y (both m e m b e r s of C a s u a r i i f o r m e s ) . B l a c k b i r d (Agelaius phoeniceus) , c h i c k e n ( Gallus gallus) , rhea ( Rhea americana) , kiwi (Apteryx australis), and o s t r i c h (Struthio camelus) OT were p u r i f i e d by CM- and D E A E - c e l l u l o s e c h r o m a t o g r a p h y (Osuga & Feeney, 1968; Prager et al., 1974); loon (Gavia ~ r c t i c a ) a n d duck (Anas platyrhynchos) t r a n s f e r r i n by R i v a n o l and a m m o n i u m s u l f a t e f r a c t i o n a t i o n and p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s (Prager & Wilson, 1975); and A d e l i e (Pygoscelis adeliae) and E m p e r o r (Aptenodytes forsteri) p e n g u i n OT by a c o m b i n a t i o n of CM- and D E A E - c e l l u l o s e c h r o m a t o g r a p h y , p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s at pH 8.9, and S e p h a d e x G - 1 O O gel f i l t r a t i o n (Osuga & Feeney, 1968; Ho, 1975). Great c r e s t e d grebe (Podiceps cristatus), owl (Asio otus) , W e s t e r n gull (Larus occidentalis) , p i g e o n (Columba livia) , t i n a m o u (Eudromia elegans) , emu ( Dromiceius novae-hollandiae) , and c a s s o w a r y (Casuarius aruensis) OT w e r e p u r i f i e d by 2 s u c c e s s ive p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s runs at pH 8.9 (Prager & Wilson, 1975) ; the leads w e r e reversed, so that m i g r a t i o n was t o w a r d the cathode, for the emu and c a s s o w a r y OT p u r i f i c a t i o n s in v i e w of their high i s o e l e c t r i c points (Osuga & Feeney, 1968). Prior to e l e c t r o p h o r e s i s the egg w h i t e s were d i a l y z e d a g a i n s t a s o l u t i o n c o n t a i n i n g 5xiO -4 M ferrous a m m o n i u m sulfate and citric acid (1:1), s u f f i c i e n t N a H C O 3 to reach a pH of 7.35, and e n o u g h N a O H to reach a final pH of 7.45 (Prager et al., 1974). This p r o c e d u r e was used rather than simply adding ferrous a m m o n i u m s u l f a t e d i r e c t l y to the egg w h i t e s to s a t u r a t e the OT w i t h iron (Prager & Wilson, 1975). O v a l b u m i n and p e n a l b u m i n from A d e l i e and E m p e r o r p e n g u i n egg w h i t e s w e r e i s o l a t e d by CM- and D E A E - c e l l u l o s e c h r o m a t o g r a p h y and S e p h a d e x G - I O O gel f i l t r a t i o n (Ho, 1975; Ho et al., in prep. ) . Antisera and Immunological Methods. Each of the 20 p u r i f i e d avian p r o t e i n s (immunogens) e x c e p t the 2 p e n a l b u m i n s was i n j e c t e d 273

into 3 or 4 N e w Z e a l a n d w h i t e or D u t c h B e l t e d r a b b i t s e s s e n t i a l l y as d e s c r i b e d (Prager & Wilson, 1975); each p e n a l b u m i n was i n j e c t e d into o n l y 2 rabbits. W h i l e as b e f o r e (Prager & Wilson, 1975) each r a b b i t g e n e r a l l y r e c e i v e d no m o r e than 250 ~g of t r a n s f e r r i n per injection, in the cases of o v a l b u m i n and p e n a l b u m i n 4 mg of i m m u n o g e n w e r e g i v e n w i t h each intrad e r m a l i n j e c t i o n and I mg w i t h each i n t r a v e n o u s i n j e c t i o n (Ho, 1975). The r e s u l t i n g a n t i s e r a to each i m m u n o g e n w e r e heated, pooled, e v a l u a t e d for purity, and u s e d in q u a n t i t a t i v e M C ' F (Champion et al., 1974; P r a g e r et al., 1974). The a n t i s e r a to b l a c k b i r d , chicken, and d u c k t r a n s f e r r i n s w e r e t h o s e d e s c r i b e d (Prager et al., 1974; P r a g e r & Wilson, 1975); m o s t of the d a t a in T a b l e I o b t a i n e d w i t h a n t i - b l a c k b i r d and a n t i - c h i c k e n OT have been r e p o r t e d p r e v i o u s l y (Prager et al., 1974). W h o l e egg w h i t e s w e r e m o s t o f t e n u s e d as a n t i g e n s o u r c e s in MC'F; sera, t i s s u e extracts, and p u r i f i e d p r o t e i n s w e r e also u s e d o c c a s i o n a l l y . Q u a l i t a t i v e i m m u n o l o g i c a l t e c h n i q u e s w e r e c a r r i e d out as in P r a g e r et al. (1974). A n t i b o d i e s e l i c i t e d by p r o t e i n s o t h e r than the i m m u n o g e n s p e c i f i e d w e r e so w e a k as not to i n t e r f e r e in M C ' F tests. The a n t i s e r a to A d e l i e and E m p e r o r p e n g u i n ovalb u m i n c o n t a i n e d a n t i b o d i e s to p e n a l b u m i n and v i c e versa. In each i n s t a n c e the titer of the a n t i b o d i e s e l i c i t e d by the c o n t a m i n a n t was 7-9% of the titer e l i c i t e d by the i m m u n o g e n specified. This d e g r e e of c o n t a m i n a t i o n will o f t e n not i n t e r f e r e in M C ' F tests w h e n u n p u r i f i e d a n t i g e n s (as in w h o l e egg w h i t e s or w h o l e sera) are used. However, in cases in w h i c h the cont a m i n a n t is a slowly e v o l v i n g p r o t e i n and the p r o t e i n of i n t e r e s t a r a p i d l y e v o l v i n g protein, one could, w i t h d i s t a n t l y r e l a t e d h e t e r o l o g o u s species, o b t a i n s p u r i o u s c r o s s - r e a c t i o n v a l u e s due to the c o n t a m i n a t i n g a n t i b o d i e s . Thus in our i n i t i a l s u r v e y for the p r e s e n c e of the n e w l y - d i s c o v e r e d p e n a l b u m i n in egg w h i t e s t h r o u g h o u t the class A y e s (Ho, 1975; Ho et al., in prep.) we u s e d a n t i s e r a to p e n a l b u m i n a b s o r b e d w i t h o v a l b u m i n in the O u c h t e r l o n y d o u b l e d i f f u s i o n test. O n l y t h o s e s p e c i e s g i v i n g a line of i m m u n o p r e c i p i t a t i o n w i t h the a b s o r b e d a n t i s e r a or 2 lines w i t h the u n a b s o r b e d a n t i s e r a w e r e d e s i g n a t e d as h a v i n g p e n a l b u m i n in their egg whites. T h e s e species w e r e t h e n t e s t e d by MC'F.

RESULTS

T a b l e I p r e s e n t s the r e s u l t s of r e c i p r o c a l M C ' F tests u s i n g the a n t i s e r a to t r a n s f e r r i n from 16 species in 14 of the 27 a v i a n o r d e r s I. We also i n c l u d e the u n i d i r e c t i o n a l m e a s u r e m e n t s to the

1

The present standard deviation from perfect reciprocity (Champion et al., 1975) is 13.8%.

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