Comp. Biochem. Physiol., 1976, Vol. 53B, pp. 499 to 503. Pergamon Press.
Printed in Great Britain
BIOCHEMICAL IDENTIFICATION OF THE MALLARD, A N A S P L A T Y R H Y N C H O S , AND BLACK DUCK, A. RUBRIPES RAYMOND P. MORGAN II', LINDA A. NOE' AND CHARLES J. HENNY 2 'Chesapeake Biological Laboratory, Center for Environmental and Estuarine Studies, University of Maryland, Solomons, MD 20688; and 2Migratory Bird and Habitat Research Laboratory, U.S. Fish and Wildlife Service, Laurel, MD 2081 l, U;S.A.
(Received 25 February 1975) A b s t r a c t - - l . Eleven tissue systems from mallards and black ducks were examined for soluble
proteins, lactate dehydrogenases and non-specific esterases through discontinuous polyacrylamide techniques. 2. Biochemical relationships between the black duck and mallard are extremely similar. 3. Hemoglobins and lactate dehydrogenase appear to be common in electrophoretic mobility between the two species. 4. Approximately 89% of the soluble proteins and 58% of the non-specific esterases are common among the two species, indicating both biochemical similarity at the genus level and speciesspecificity.
INTRODUCTION THE MAJOR o b j e c t i v e of this s t u d y is to c o m p a r e a n d c o n t r a s t , t h r o u g h the use of e l e c t r o p h o r e t i c t e c h n i q u e s , s o m e of t h e b i o c h e m i c a l s y s t e m s of the mallard, Anas platyrhynchos, a n d the b l a c k duck, A. rubripes, for t h e p u r p o s e of defining b i o c h e m i c a l c h a r a c t e r s useful in identification of the t w o species. MATERIALS AND METHODS Twenty-five black ducks and 25 mallards were obtained by the U.S. Fish and Wildlife Service and held at the Patuxent Wildlife Research Center, Laurel, Maryland until needed. Mallards were collected from Tulelake, California and black ducks from Sackville, New Brunswick. Both collection sites were assumed to be free of introductions and representative of the "pure" species. The following tissues were collected from the black ducks and mallards: blood (hemoglobin and plasma or serum proteins), muscle, liver, heart, spleen, brain, eye lens, gizzard, kidney, lung, and intestine. Black ducks and mallards were killed by concussion. Blood was collected, in 12 × 75 mm culture tubes containing a few crystals of heparin, by excising and severing of the carotid arteries. Heparin was used as an anticoagulant for half the blood samples in order to obtain hemoglobin and plasma. No anti-coagulant was used for the remaining blood samples which were allowed to clot. Serum was obtained from these samples. Hemoglobin was prepared by first centrifuging the blood at 1470g and then removing the plasma. The remaining RBC were exhaustively washed three times with 20 times their volume of physiological saline. A volume of distilled water equivalent to 10 times the RBC packed volume was added and the sample was frozen at -20°C prior to electrophoresis. The serum or plasma was also stored at -20°C. Other tissues used for enzyme and soluble protein electrophoresis were washed with physiological saline and frozen whole at -20°C. Prior to electrophoresis, the tissues were thawed and placed in Bellco tissue grinders. An equal volume of distilled water was added and the tissue was then triturated. The extract was then used immediately for enzyme and soluble protein electrophoresis. 499
Enzymes and proteins of ducks were separated electrophoretically on 7% acrylamide gels (Davis, 1964) with a Beckman Duostat and Canalco Model 66 bath. The acrylamide gels were formed in glass tubes (5 mm i.d., 7mrn o.d., 67ram long). The sample (Table l) was separated at room temperature (20-23°C) with a freshly prepared Tris (0.005 M)-glycine (0.039 M) buffer, pH 8.3. A few drops of bromphenol blue (0.005% w/v) were added prior to electrophoresis. The electrophoretic run was terminated when the dye band had migrated to within 5 mm of the gel end. A constant d.c. of 4.2 mA/column was used for all column electrophoresis. Voltage for the system was initially 290-325 V and finally 210-250 V. After the gel was removed from the glass tube, the front end of the dye band was cut. The cut end of the gel allowed measurements of relative mobility. Following electrophoresis, gels were stained for a minimum of l hr in 0.1% Buffalo Black NBR (in 7% acetic acid) for soluble proteins. Gels were doubly destained with a Canalco horizontal destainer using 7% acetic acid. Following destaining, gels were stored in 7% acetic acid. In addition to characterization of soluble proteins, two Table 1. Sample volumes (in /z l) used in column and slab electrophoresis for separating soluble proteins and enzymes Tissue/Proteln
Soluble proteins
Esterase
Hemoglobin
5
.
Serum/Plasma
5
i0
i0
Muscle
i0
20
20
Liver
i0
20
20
Heart
5
5
5
Spleen
5
5
5
Brain
5
I0
5
Eye
5
I0
5
Gizzard
I0
I0
I0
Kidney
i0
i0
i0
Lung
i0
i0
I0
Intestine
i0
i0
i0
.
LDH
.
.
500
R. P. MORGAN II, L. A. NOE AND C. J. HENNY
enzyme systems, non-specific esterases and lactate dehydrogenases (LDH), were assayed. Following electrophoresis the columns or slabs used for enzyme localization were washed exhaustively with the buffer used in the assay and incubated at room temperature in the dark for approximately 30 min. Following the staining process, the gels were washed with tap water and then stored in 7% acetic acid. Non-specific esterases were identified using the method of Markert & Hunter (1959). Gels were incubated in a solution consisting of 4 ml of 0.20 M Tris-HC1 buffer (pH7.4), 94ml of distilled water, 2ml of anaphthylacetate (1% in acetone), and 50 mg of Fast Blue RR. Inhibitor reactions were not employed. The technique developed by Goldberg (1963) for LDH localization consisted of gel incubation in a solution comprised of 14 ml of 0.014 M Tris-HCl buffer (pH 8-3), 6ml of 0.1M lactic acid sodium salt (pH 8.3), 16rag of nitro blue tetrazolium salt, 6mg of diphosphopyridine nucleotide, and 3 mg of phenazine methosulfate, RESULTS
The biochemical characters (soluble proteins, esterases and LDH) of the black duck and the mallard are extremely similar. Fortunately, discrete differences between the two species do occur and are useful in species identification. Results obtained for two systems are not diagrammed. Hemoglobin patterns from the black duck and mallard are identical in electrophoretic migration. The basic pattern for each species is a single hemoglobin migrating at a slow electrophoretic rate, Biochemical polymorphism (more than one molecular form of a functional protein type) is absent in the hemoglobin systems. A similar situation to hemoglobin is the LDH patterns in the black duck and mallard. Only one slowly migrating LDH is present in the majority of tissues tested for the two species. LDH polymorphism is absent, Serum proteins of the black duck and mallard are extremely similar (Fig. 1). Fifteen proteins are present in the serum of the black duck and fourteen in the mallard after electrophoresis on the 7% acrylamide gels. Minor differences occur in the post-albumin region (the area between the second anodal protein in both the mallard and black duck and fifth in the black duck and the sixth in the mallard) where two post-albumins occur in the black and three in the mallard. Variation also is present in the region past the second cathodal protein (Fig. 1) in both species. A comparison of this gel area for the species shows that black ducks have four proteins, presumably as two alleles each controlling a set of two proteins, and mallards contain two proteins. Basically, the electropherograms of the two species are very similar for serum protein patterns, yet differences are present. Polymorphism of serum proteins is not observed in either set of ten "pure" ducks. A larger sample of perhaps 25-50 ducks is needed to determine the extent of polymorphism in both species. Serum esterases of the black duck and mallard are quite distinct (Fig. 1). Three esterase isoenzymes are present in the black duck and two in the mallard. Electrophoretic mobilities of the isoenzymes are quite distinct. Occasional minor indistinct esterases may be present. Nine soluble muscle proteins are present in the
black duck and mallard (Fig. I). The pattern of protein migration is identical for both species. Typically, muscle proteins are more conservative evolutionally (Manwell & Baker, 1970) so the results are not unexpected. Muscle esterase patterns appear to be similar for one set of three isoenzymes (Fig. 1) in both the black duck and mallard. However, black duck muscle contains an extra pair of two esterases. Gizzard soluble proteins and esterases are identical for both species (Fig. 1 and 1). Again, polymorphism is not present. A similar situation exists in the soluble proteins of the liver (Fig. 1) where the patterns are almos! identical even though many more proteins are found in the liver than gizzard tissue. Only one extra protein was observed in the mallard. Fourteen soluble liver proteins are present in the black duck and 15 occur in the mallard (Fig. 1). l~iver esterase patterns of black and mallard ducks are quite distinct (Fig. I). A major esterase system, migrating equally from cathode to anode, is present in the mallard. One set of three cathodal esterases appears to be identical among the two species. In the anodal region of the gel one minor and a set of three esterases are common to the two species. The black duck contains four extra esterase isoenzymes and the mallard contains one additional esterase. It is easy to immediately distinguish the two species just on the presence of the major esterase system in the mallard (Fig. 1). Patterns of the soluble heart proteins from the black duck are identical to those of the mallard (Fig. 1). Two heart esterases are found in the black duck (Fig. 1). No heart esterases are present in the mallard for the amount of sample used in electrophoresis (Table 1). Eye proteins of the black duck and mallard are quite distinct (Fig. l). Five eye proteins are found in the black duck and four in the mallard. However, the overall pattern or spacing of soluble eye proteins is quite different (Fig. I). We did not detect esterase activity at the sample volumes (Table I) used for electrophoresis. Soluble brain proteins and brain esterases appear to be identical in the two species (Fig. I). Identical patterns for both systems are commonly observed (Fig. 1). Soluble lung proteins of the black duck and the mallard differ (Fig. 1). Six proteins in the black duck are identical to proteins observed in the mallard. Differences between the two species consist of one unique lung protein in the black duck and two unique proteins in the mallard. No lung esterases are present in the black duck for the amount of sample used (Fig. 1). Two lung esterases are present in the mallard (Fig. 1). Soluble intestinal proteins of the black duck and mallard are very similar. Eighteen intestinal proteins are present in black ducks and mallards, however, the pattern is slightly different in the cathodal end of the diagram (Fig. 1). Esterase patterns of the intestine in black ducks and mallards is quite distinct (Fig. l). Only one esterase out of the combined total of 16 observed (four in black duck, two in mallards) is common to both species (Fig. 1), Sixteen identical soluble kidney proteins are
Biochemical identification of the mallard, Anas platyrhynchos, and black duck, A. rubripes
501
SOLUBLE PROTEINS
se,.. I I+ I+G I; I)~ l lll+~:i: ~iIli m°,+le I II II II Ii I II II II II ++,z+r+ II II II II liver I llllllIllN +I I) l l l l l l l l l l II heart I II Nil II II I In I l l II II eyeiens II I !1 I I bran I I I I ill I I I I III ,.ng 11111 II I I I IH II +.,.~i.. I )1~ Ilti~ I II i ~! II I: I ) ii I ill II I I II H ii m.. +ll+il[]i::~[g+ ii I i+I+~!:++ ] spleen I) III II] II II II II ESTERASES
ill
serum
.-.~ o,~z-d liver
h,r, m,n
ill Ii III .11 II
!1 il
I1:11111
spleen
: -
II
II
lung
,.t.tm ki+.ev
III II I!1 ][i;I;!
I I!
III I I
II II
Ill BLACK
MALLARD
Fig. 1. Electropherograms of protein and enzyme systems of the mallard and black duck. present in the black duck and mallard (Fig. 1). Esterase patterns of the two species are also similar except that the black duck kidney contains an extra set of three esterases (Fig. 1). The soluble spleen proteins of the black duck are quite distinct from the mallard (Fig. 1). Four proteins are present in the mallard and these proteins are comparable to four of the spleen proteins in the black duck. In addition to the four similar proteins, the black duck contains an additional nine proteins (Fig. 1). This system appears to be one of the most distinct observed during the study. Three spleen esterases (Fig. 1) are observed in the black duck. No esterases are present in the mallard for the amount of sample used.
In review, a total of 119 soluble proteins are found in 11 tissue systems of the black duck (Table 2). In the mallard, there are 110 proteins from the same number of systems. One hundred and two soluble proteins are common to both species, or approx, 89% of the soluble protein gene loci investigated are identical. This is an estimate since several of the proteins may be controlled by one locus--usually proteins all with one function, or in some cases one protein may be regulated by more than one locus. Thirty-nine esterases (Table 2) are present in 10 tissue systems of the black duck, 27 esterases occur in 10 tissue systems of the mallard. Nineteen esterases are common to both species for a coefficient of approx 58% similarity in gene loci for
502
R. P. MORGAN 11, L. A. NOE AND C. J. HENNY Table 2. S u m m a r y of the n u m b e r of either soluble proteins or esterases o b s e r v e d in black d u c k s and mallards. Included in the table is the n u m b e r of proteins identical and distinct a m o n g the two species Soluble proteins Tissue
Black d u c k
Identical
Esteraues
Distinct
Mallard
Black duck
Identical
Distinct
Mal]ard
Serum
15
12
5
14
~
O
5
Muscle
9
9
0
9
5
3
2
2 3
Gizzard
4
4
0
4
2
2
0
2
Liver
14
14
]
15
Ii
7
7
]0
Heart
ii
ll
0
ii
2
0
2
0
5
2
5
4
Brain
7
7
0
7
2
2
D
2
Lung
7
6
3
8
O
O
2
2
Intestine
18
17
2
18
4
i
4
2
Kidney
16
16
0
16
7
4
3
4
Eye
Spleen TOTAL
13
4
9
4
3
0
3
0
119
102
25
ii0
39
19
28
27
esterases. Obviously, there are more differences in the esterase systems between the black duck and mallard than in the soluble proteins investigated, however, the total number of enzymes observed versus the total number of soluble proteins is low (66 vs 229). DISCUSSION
Johnsgard (1961) presents a very thorough treatment of the systematic relationships concerning North American mallards--a complex of six forms (four species and two subspecies) including the mallard and black duck. In his work, Johnsgard (1961) carefully discusses the arguments for and against defining the black duck as a "good" species in relation to the mallard. After carefully reviewing the arguments--both pro and con, Johnsgard states "I, therefore, believe that the most satisfactory status for rubripes is to consider it a subspecies of Arias platyrhynchos, but one that exhibits a greater degree of differences in eclogical preference and social segregation than normally occurs in subspecies. Neither a subspecific nor a specific relegation can be entirely satisfactory at present, and only time and continued investigations are likely to prove or disprove the conclusions reached here." (Johnsgard, 1961, p. 38). In support of Johnsgard's hypothesis, Sibley (1960) had concluded that the mallard and black duck are very similar based on electrophoretic comparison of egg-white proteins, a system which Sibley (1960, p. 227) describes, "the egg-white proteins, although adaptive, are phylogenetically conservative and change very slowly compared with other parts of the organism." Johnsgard (1961), in cooperation with Sibley, observed that egg-white profiles (from densitometry) of the mallard and black duck are very similar. Results of the present study suggest that the two ducks are distinct in bi,,chemical systems that are typically liberal in evolulion such as serum proteins and esterases. However, mallards and black ducks are similar in those systems that are more conservative such as muscle and other structural
proteins (the majority of soluble proteins analyzed). This closeness in the "soluble" systems may also reflect the fact that the two species are indeed very closely related--a point to consider when hybridization between the two species is discussed. The differences (and similarities) observed in both soluble proteins and enzymes of the two ducks is similar to other studies. Baker & Hanson (1966) observed little variation in serum proteins from 11 species of geese (Anser and Branta), Shaughnessy (1970) found no differences in serum proteins from two sibling species of the giant petrel, Brown et al. (1970) noted a close relationship between two sparrows based on hemoglobins, and Ford et al. (1974) discussed the extremely close relationship among five species of Darwin's finches (primarily work done on serum proteins and esterases). We conclude that, although the two ducks are closely related, there is sufficient biochemical evidence present to warrant their description at the species level and not as subspecies rank.
Acknowledgements--Mrs. Fran Younger and Mr. Michael J. Reber assisted in the preparation of figures. This is contribution No. 634 from the Center for Environmental and Estuarine Studies, University of Maryland. Support was through Contract No. 14-16-008805 from Migratory Bird and Habitat Research Laboratory, Fish and Wildlife Service, Department of the Interior. William R. Whitman, Canadian Wildlife Service, Sackville N.B. and Edward O'Niell, U.S. Fish and Wildlife Service, Tulelake, CA, kindly assisted by providing the black ducks and mallards. REFERENCES
BAKER C. M. A. & HANSON H. C. (1966) Molecular genetics of avian p r o t e i n s - - V l . Evolutionary implications of blood proteins of l! species of geese. Comp. Biochem. Physiol. 17, 997-1006. BROWN I. R. F., BANNISTER W. H. & DE LUCCA C. (1970) A comparison of Maltese and Sicilian sparrow h a e m o g lobins. Comp. Biochem. Physiol. 34, 557-562. DAVlS B. J. (1964) Disc electrophoresis--II. Method and application to h u m a n s e r u m proteins. Ann. N. Y. Acad. Sci. 121,404-427. FORD H. A., EWING A. W. & PIn, KIN D. T. (1974) Blood
Biochemical identification of the mallard, Anas platyrhynchos, and black duck, A. rubripes proteins in Darwin's finches. Comp. Biochem. Physiol. 47B, 369-375. GOLDBERG E. (1963) Lactic and malic dehydrogenase in human spermatozoa. Science, N.Y. 139, 602-603. JOHNSGARDP. A. (1961) Evolutionary relationships among the North American mallards. Auk 78, 3-43. MANWELL C. & BAKER C. M. A. (1970) Molecular Biology
and the Origin of Species: Heterosis, Protein Polymorphism and Animal Breeding. Sidgwick & Jackson, London.
C.B.P.(B)53/4~--v
503
MARKERT C. L. & HUNTER R. L. (1959)The distribution of esterases in mouse tissues. J. Histochem. Cytochem. 7, 42-49. SHAUGHNESSY P. P. (1970) Serum proteins of two sibling species of the giant petrel (Macronectes spp.) Comp. Biochem. Physiol. 33, 721-723. SIaLEY C. G. (1960) The electrophoretic patterns of avian egg-white proteins as taxonomic characters. Ibis 101, 215-284.
Printed in Great Britain
BIOCHEMICAL IDENTIFICATION OF THE MALLARD, A N A S P L A T Y R H Y N C H O S , AND BLACK DUCK, A. RUBRIPES RAYMOND P. MORGAN II', LINDA A. NOE' AND CHARLES J. HENNY 2 'Chesapeake Biological Laboratory, Center for Environmental and Estuarine Studies, University of Maryland, Solomons, MD 20688; and 2Migratory Bird and Habitat Research Laboratory, U.S. Fish and Wildlife Service, Laurel, MD 2081 l, U;S.A.
(Received 25 February 1975) A b s t r a c t - - l . Eleven tissue systems from mallards and black ducks were examined for soluble
proteins, lactate dehydrogenases and non-specific esterases through discontinuous polyacrylamide techniques. 2. Biochemical relationships between the black duck and mallard are extremely similar. 3. Hemoglobins and lactate dehydrogenase appear to be common in electrophoretic mobility between the two species. 4. Approximately 89% of the soluble proteins and 58% of the non-specific esterases are common among the two species, indicating both biochemical similarity at the genus level and speciesspecificity.
INTRODUCTION THE MAJOR o b j e c t i v e of this s t u d y is to c o m p a r e a n d c o n t r a s t , t h r o u g h the use of e l e c t r o p h o r e t i c t e c h n i q u e s , s o m e of t h e b i o c h e m i c a l s y s t e m s of the mallard, Anas platyrhynchos, a n d the b l a c k duck, A. rubripes, for t h e p u r p o s e of defining b i o c h e m i c a l c h a r a c t e r s useful in identification of the t w o species. MATERIALS AND METHODS Twenty-five black ducks and 25 mallards were obtained by the U.S. Fish and Wildlife Service and held at the Patuxent Wildlife Research Center, Laurel, Maryland until needed. Mallards were collected from Tulelake, California and black ducks from Sackville, New Brunswick. Both collection sites were assumed to be free of introductions and representative of the "pure" species. The following tissues were collected from the black ducks and mallards: blood (hemoglobin and plasma or serum proteins), muscle, liver, heart, spleen, brain, eye lens, gizzard, kidney, lung, and intestine. Black ducks and mallards were killed by concussion. Blood was collected, in 12 × 75 mm culture tubes containing a few crystals of heparin, by excising and severing of the carotid arteries. Heparin was used as an anticoagulant for half the blood samples in order to obtain hemoglobin and plasma. No anti-coagulant was used for the remaining blood samples which were allowed to clot. Serum was obtained from these samples. Hemoglobin was prepared by first centrifuging the blood at 1470g and then removing the plasma. The remaining RBC were exhaustively washed three times with 20 times their volume of physiological saline. A volume of distilled water equivalent to 10 times the RBC packed volume was added and the sample was frozen at -20°C prior to electrophoresis. The serum or plasma was also stored at -20°C. Other tissues used for enzyme and soluble protein electrophoresis were washed with physiological saline and frozen whole at -20°C. Prior to electrophoresis, the tissues were thawed and placed in Bellco tissue grinders. An equal volume of distilled water was added and the tissue was then triturated. The extract was then used immediately for enzyme and soluble protein electrophoresis. 499
Enzymes and proteins of ducks were separated electrophoretically on 7% acrylamide gels (Davis, 1964) with a Beckman Duostat and Canalco Model 66 bath. The acrylamide gels were formed in glass tubes (5 mm i.d., 7mrn o.d., 67ram long). The sample (Table l) was separated at room temperature (20-23°C) with a freshly prepared Tris (0.005 M)-glycine (0.039 M) buffer, pH 8.3. A few drops of bromphenol blue (0.005% w/v) were added prior to electrophoresis. The electrophoretic run was terminated when the dye band had migrated to within 5 mm of the gel end. A constant d.c. of 4.2 mA/column was used for all column electrophoresis. Voltage for the system was initially 290-325 V and finally 210-250 V. After the gel was removed from the glass tube, the front end of the dye band was cut. The cut end of the gel allowed measurements of relative mobility. Following electrophoresis, gels were stained for a minimum of l hr in 0.1% Buffalo Black NBR (in 7% acetic acid) for soluble proteins. Gels were doubly destained with a Canalco horizontal destainer using 7% acetic acid. Following destaining, gels were stored in 7% acetic acid. In addition to characterization of soluble proteins, two Table 1. Sample volumes (in /z l) used in column and slab electrophoresis for separating soluble proteins and enzymes Tissue/Proteln
Soluble proteins
Esterase
Hemoglobin
5
.
Serum/Plasma
5
i0
i0
Muscle
i0
20
20
Liver
i0
20
20
Heart
5
5
5
Spleen
5
5
5
Brain
5
I0
5
Eye
5
I0
5
Gizzard
I0
I0
I0
Kidney
i0
i0
i0
Lung
i0
i0
I0
Intestine
i0
i0
i0
.
LDH
.
.
500
R. P. MORGAN II, L. A. NOE AND C. J. HENNY
enzyme systems, non-specific esterases and lactate dehydrogenases (LDH), were assayed. Following electrophoresis the columns or slabs used for enzyme localization were washed exhaustively with the buffer used in the assay and incubated at room temperature in the dark for approximately 30 min. Following the staining process, the gels were washed with tap water and then stored in 7% acetic acid. Non-specific esterases were identified using the method of Markert & Hunter (1959). Gels were incubated in a solution consisting of 4 ml of 0.20 M Tris-HC1 buffer (pH7.4), 94ml of distilled water, 2ml of anaphthylacetate (1% in acetone), and 50 mg of Fast Blue RR. Inhibitor reactions were not employed. The technique developed by Goldberg (1963) for LDH localization consisted of gel incubation in a solution comprised of 14 ml of 0.014 M Tris-HCl buffer (pH 8-3), 6ml of 0.1M lactic acid sodium salt (pH 8.3), 16rag of nitro blue tetrazolium salt, 6mg of diphosphopyridine nucleotide, and 3 mg of phenazine methosulfate, RESULTS
The biochemical characters (soluble proteins, esterases and LDH) of the black duck and the mallard are extremely similar. Fortunately, discrete differences between the two species do occur and are useful in species identification. Results obtained for two systems are not diagrammed. Hemoglobin patterns from the black duck and mallard are identical in electrophoretic migration. The basic pattern for each species is a single hemoglobin migrating at a slow electrophoretic rate, Biochemical polymorphism (more than one molecular form of a functional protein type) is absent in the hemoglobin systems. A similar situation to hemoglobin is the LDH patterns in the black duck and mallard. Only one slowly migrating LDH is present in the majority of tissues tested for the two species. LDH polymorphism is absent, Serum proteins of the black duck and mallard are extremely similar (Fig. 1). Fifteen proteins are present in the serum of the black duck and fourteen in the mallard after electrophoresis on the 7% acrylamide gels. Minor differences occur in the post-albumin region (the area between the second anodal protein in both the mallard and black duck and fifth in the black duck and the sixth in the mallard) where two post-albumins occur in the black and three in the mallard. Variation also is present in the region past the second cathodal protein (Fig. 1) in both species. A comparison of this gel area for the species shows that black ducks have four proteins, presumably as two alleles each controlling a set of two proteins, and mallards contain two proteins. Basically, the electropherograms of the two species are very similar for serum protein patterns, yet differences are present. Polymorphism of serum proteins is not observed in either set of ten "pure" ducks. A larger sample of perhaps 25-50 ducks is needed to determine the extent of polymorphism in both species. Serum esterases of the black duck and mallard are quite distinct (Fig. 1). Three esterase isoenzymes are present in the black duck and two in the mallard. Electrophoretic mobilities of the isoenzymes are quite distinct. Occasional minor indistinct esterases may be present. Nine soluble muscle proteins are present in the
black duck and mallard (Fig. I). The pattern of protein migration is identical for both species. Typically, muscle proteins are more conservative evolutionally (Manwell & Baker, 1970) so the results are not unexpected. Muscle esterase patterns appear to be similar for one set of three isoenzymes (Fig. 1) in both the black duck and mallard. However, black duck muscle contains an extra pair of two esterases. Gizzard soluble proteins and esterases are identical for both species (Fig. 1 and 1). Again, polymorphism is not present. A similar situation exists in the soluble proteins of the liver (Fig. 1) where the patterns are almos! identical even though many more proteins are found in the liver than gizzard tissue. Only one extra protein was observed in the mallard. Fourteen soluble liver proteins are present in the black duck and 15 occur in the mallard (Fig. 1). l~iver esterase patterns of black and mallard ducks are quite distinct (Fig. I). A major esterase system, migrating equally from cathode to anode, is present in the mallard. One set of three cathodal esterases appears to be identical among the two species. In the anodal region of the gel one minor and a set of three esterases are common to the two species. The black duck contains four extra esterase isoenzymes and the mallard contains one additional esterase. It is easy to immediately distinguish the two species just on the presence of the major esterase system in the mallard (Fig. 1). Patterns of the soluble heart proteins from the black duck are identical to those of the mallard (Fig. 1). Two heart esterases are found in the black duck (Fig. 1). No heart esterases are present in the mallard for the amount of sample used in electrophoresis (Table 1). Eye proteins of the black duck and mallard are quite distinct (Fig. l). Five eye proteins are found in the black duck and four in the mallard. However, the overall pattern or spacing of soluble eye proteins is quite different (Fig. I). We did not detect esterase activity at the sample volumes (Table I) used for electrophoresis. Soluble brain proteins and brain esterases appear to be identical in the two species (Fig. I). Identical patterns for both systems are commonly observed (Fig. 1). Soluble lung proteins of the black duck and the mallard differ (Fig. 1). Six proteins in the black duck are identical to proteins observed in the mallard. Differences between the two species consist of one unique lung protein in the black duck and two unique proteins in the mallard. No lung esterases are present in the black duck for the amount of sample used (Fig. 1). Two lung esterases are present in the mallard (Fig. 1). Soluble intestinal proteins of the black duck and mallard are very similar. Eighteen intestinal proteins are present in black ducks and mallards, however, the pattern is slightly different in the cathodal end of the diagram (Fig. 1). Esterase patterns of the intestine in black ducks and mallards is quite distinct (Fig. l). Only one esterase out of the combined total of 16 observed (four in black duck, two in mallards) is common to both species (Fig. 1), Sixteen identical soluble kidney proteins are
Biochemical identification of the mallard, Anas platyrhynchos, and black duck, A. rubripes
501
SOLUBLE PROTEINS
se,.. I I+ I+G I; I)~ l lll+~:i: ~iIli m°,+le I II II II Ii I II II II II ++,z+r+ II II II II liver I llllllIllN +I I) l l l l l l l l l l II heart I II Nil II II I In I l l II II eyeiens II I !1 I I bran I I I I ill I I I I III ,.ng 11111 II I I I IH II +.,.~i.. I )1~ Ilti~ I II i ~! II I: I ) ii I ill II I I II H ii m.. +ll+il[]i::~[g+ ii I i+I+~!:++ ] spleen I) III II] II II II II ESTERASES
ill
serum
.-.~ o,~z-d liver
h,r, m,n
ill Ii III .11 II
!1 il
I1:11111
spleen
: -
II
II
lung
,.t.tm ki+.ev
III II I!1 ][i;I;!
I I!
III I I
II II
Ill BLACK
MALLARD
Fig. 1. Electropherograms of protein and enzyme systems of the mallard and black duck. present in the black duck and mallard (Fig. 1). Esterase patterns of the two species are also similar except that the black duck kidney contains an extra set of three esterases (Fig. 1). The soluble spleen proteins of the black duck are quite distinct from the mallard (Fig. 1). Four proteins are present in the mallard and these proteins are comparable to four of the spleen proteins in the black duck. In addition to the four similar proteins, the black duck contains an additional nine proteins (Fig. 1). This system appears to be one of the most distinct observed during the study. Three spleen esterases (Fig. 1) are observed in the black duck. No esterases are present in the mallard for the amount of sample used.
In review, a total of 119 soluble proteins are found in 11 tissue systems of the black duck (Table 2). In the mallard, there are 110 proteins from the same number of systems. One hundred and two soluble proteins are common to both species, or approx, 89% of the soluble protein gene loci investigated are identical. This is an estimate since several of the proteins may be controlled by one locus--usually proteins all with one function, or in some cases one protein may be regulated by more than one locus. Thirty-nine esterases (Table 2) are present in 10 tissue systems of the black duck, 27 esterases occur in 10 tissue systems of the mallard. Nineteen esterases are common to both species for a coefficient of approx 58% similarity in gene loci for
502
R. P. MORGAN 11, L. A. NOE AND C. J. HENNY Table 2. S u m m a r y of the n u m b e r of either soluble proteins or esterases o b s e r v e d in black d u c k s and mallards. Included in the table is the n u m b e r of proteins identical and distinct a m o n g the two species Soluble proteins Tissue
Black d u c k
Identical
Esteraues
Distinct
Mallard
Black duck
Identical
Distinct
Mal]ard
Serum
15
12
5
14
~
O
5
Muscle
9
9
0
9
5
3
2
2 3
Gizzard
4
4
0
4
2
2
0
2
Liver
14
14
]
15
Ii
7
7
]0
Heart
ii
ll
0
ii
2
0
2
0
5
2
5
4
Brain
7
7
0
7
2
2
D
2
Lung
7
6
3
8
O
O
2
2
Intestine
18
17
2
18
4
i
4
2
Kidney
16
16
0
16
7
4
3
4
Eye
Spleen TOTAL
13
4
9
4
3
0
3
0
119
102
25
ii0
39
19
28
27
esterases. Obviously, there are more differences in the esterase systems between the black duck and mallard than in the soluble proteins investigated, however, the total number of enzymes observed versus the total number of soluble proteins is low (66 vs 229). DISCUSSION
Johnsgard (1961) presents a very thorough treatment of the systematic relationships concerning North American mallards--a complex of six forms (four species and two subspecies) including the mallard and black duck. In his work, Johnsgard (1961) carefully discusses the arguments for and against defining the black duck as a "good" species in relation to the mallard. After carefully reviewing the arguments--both pro and con, Johnsgard states "I, therefore, believe that the most satisfactory status for rubripes is to consider it a subspecies of Arias platyrhynchos, but one that exhibits a greater degree of differences in eclogical preference and social segregation than normally occurs in subspecies. Neither a subspecific nor a specific relegation can be entirely satisfactory at present, and only time and continued investigations are likely to prove or disprove the conclusions reached here." (Johnsgard, 1961, p. 38). In support of Johnsgard's hypothesis, Sibley (1960) had concluded that the mallard and black duck are very similar based on electrophoretic comparison of egg-white proteins, a system which Sibley (1960, p. 227) describes, "the egg-white proteins, although adaptive, are phylogenetically conservative and change very slowly compared with other parts of the organism." Johnsgard (1961), in cooperation with Sibley, observed that egg-white profiles (from densitometry) of the mallard and black duck are very similar. Results of the present study suggest that the two ducks are distinct in bi,,chemical systems that are typically liberal in evolulion such as serum proteins and esterases. However, mallards and black ducks are similar in those systems that are more conservative such as muscle and other structural
proteins (the majority of soluble proteins analyzed). This closeness in the "soluble" systems may also reflect the fact that the two species are indeed very closely related--a point to consider when hybridization between the two species is discussed. The differences (and similarities) observed in both soluble proteins and enzymes of the two ducks is similar to other studies. Baker & Hanson (1966) observed little variation in serum proteins from 11 species of geese (Anser and Branta), Shaughnessy (1970) found no differences in serum proteins from two sibling species of the giant petrel, Brown et al. (1970) noted a close relationship between two sparrows based on hemoglobins, and Ford et al. (1974) discussed the extremely close relationship among five species of Darwin's finches (primarily work done on serum proteins and esterases). We conclude that, although the two ducks are closely related, there is sufficient biochemical evidence present to warrant their description at the species level and not as subspecies rank.
Acknowledgements--Mrs. Fran Younger and Mr. Michael J. Reber assisted in the preparation of figures. This is contribution No. 634 from the Center for Environmental and Estuarine Studies, University of Maryland. Support was through Contract No. 14-16-008805 from Migratory Bird and Habitat Research Laboratory, Fish and Wildlife Service, Department of the Interior. William R. Whitman, Canadian Wildlife Service, Sackville N.B. and Edward O'Niell, U.S. Fish and Wildlife Service, Tulelake, CA, kindly assisted by providing the black ducks and mallards. REFERENCES
BAKER C. M. A. & HANSON H. C. (1966) Molecular genetics of avian p r o t e i n s - - V l . Evolutionary implications of blood proteins of l! species of geese. Comp. Biochem. Physiol. 17, 997-1006. BROWN I. R. F., BANNISTER W. H. & DE LUCCA C. (1970) A comparison of Maltese and Sicilian sparrow h a e m o g lobins. Comp. Biochem. Physiol. 34, 557-562. DAVlS B. J. (1964) Disc electrophoresis--II. Method and application to h u m a n s e r u m proteins. Ann. N. Y. Acad. Sci. 121,404-427. FORD H. A., EWING A. W. & PIn, KIN D. T. (1974) Blood
Biochemical identification of the mallard, Anas platyrhynchos, and black duck, A. rubripes proteins in Darwin's finches. Comp. Biochem. Physiol. 47B, 369-375. GOLDBERG E. (1963) Lactic and malic dehydrogenase in human spermatozoa. Science, N.Y. 139, 602-603. JOHNSGARDP. A. (1961) Evolutionary relationships among the North American mallards. Auk 78, 3-43. MANWELL C. & BAKER C. M. A. (1970) Molecular Biology
and the Origin of Species: Heterosis, Protein Polymorphism and Animal Breeding. Sidgwick & Jackson, London.
C.B.P.(B)53/4~--v
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MARKERT C. L. & HUNTER R. L. (1959)The distribution of esterases in mouse tissues. J. Histochem. Cytochem. 7, 42-49. SHAUGHNESSY P. P. (1970) Serum proteins of two sibling species of the giant petrel (Macronectes spp.) Comp. Biochem. Physiol. 33, 721-723. SIaLEY C. G. (1960) The electrophoretic patterns of avian egg-white proteins as taxonomic characters. Ibis 101, 215-284.
