Evolutionary Genetics of Birds IV Rates of Protein Divergence in Waterfowl (Anatidae)

Evolutionary genetics of birds IV Rates of protein divergence in waterfowl (Anatidae)

J. C. Patton & J. C. Avise Department of Molecular and Population Genetics, University of Georgia, Athens, GA 30602, USA

Abstract

An electrophoretic comparison of proteins in 26 species of waterfowl (Anatidae), representing two major subfamilies and six subfamilial tribes, led to the following major conclusions: (1) the genetic data, analyzed phenetically and cladistically, generally support traditional concepts of evolutionary relationships, although some areas of disagreement are apparent; (2) species and genera within Anatidae exhibit smaller genetic distances at protein-coding loci than do most non-avian vertebrates of equivalent taxonomic rank; (3) the conservative pattern of protein differentiation in Anatidae parallels patterns previously reported in Passeriforme birds. If previous taxonomic assignments and ages of anatid fossils are reliable, it would appear that the conservative levels of protein divergence among living species may not be due to recent age of the family, but rather to a several-fold deceleration in rate of protein evolution relative to non-avian vertebrates. Since it now appears quite possible that homologous proteins can evolve at different rates in different phylads, molecular-based conclusions about absolute divergence times for species with a poor fossil record should remain appropriately reserved. However, the recognition and study of the phenomenon of apparent heterogeneity in rates of protein divergence across phylads may eventually enhance our understanding of molecular and organismal evolution.

Introduction cies elsewhere (Avise & Aquadro, 1982). Compara- ble data for non-passerine birds are limited (Bar- Species and genera within several families of pas- rowclough et al., 1981; Gutierrez et aL, 1983). seriform (perching) birds exhibit far smaller genetic Several hypotheses (not mutually-exclusive) can distances at protein-coding loci than do most non- be advanced to account for this conservative pat- avian vertebrates of equivalent taxonomic rank tern of protein divergence in Aves: birds could be (Avise et al., 1980a, b, c; Barrowclough & Corbin, taxonomically 'oversplit' relative to other groups; 1978; Corbin et al., 1974; Martin & Selander, 1975; hybridization and introgression between species Smith & Zimmerman, 1976). In each of several pas- could decelerate their differentiation; avian taxa seriform families that have been extensively studied could be younger than most other vertebrate taxa; with multi-locus electrophoretic techniques, levels avian proteins could be evolving more slowly, on of genetic distance between congeneric species ap- the average, than those of other vertebrates. These proximate those between conspecific populations hypotheses are difficult to test critically, in part be- of many fishes, mammals, and other non-avian cause the fossil record for most passeriform groups vertebrates, and genetic distances between con- is very poor. Avise et al. (1980c) tentatively con- familial avian genera are typically lower than or clude that protein evolution is decelerated in the equal to distances among very closely related spe- wood warblers (Parulidae). This conclusion de-

Genetica 68, 129-143 (1986). c~') Dr W. Junk Publishers. Dordrecht. Printed in The Netherlands. 130 pends heavily upon the validity of estimated diver- proteins encoded by 17-19 loci in a total of 206 gence times which were based primarily on zoogeo- specimens of 26 species of ducks, geese, and swans. graphic considerations (Mengel, 1964). These samples represent several major lines of In this report we describe and evaluate levels of divergence in waterfowl, including two subfamilies protein divergence in a large assemblage of non- and six distinct tribes. passeriform birds: the waterfowl (Anseriformes; Anatidae). We are primarily interested in two questions: (1) Is the conservative pattern of protein evo- The Anatidae lution in Passeriformes also exhibited by these non- Passeriforme birds?; (2) What are the rates of pro- The well-delimited, presumably monophyletic tein divergence in waterfowl relative to rates in oth- family Anatidae contains approximately 150 living er birds and in non-avian vertebrates? An ancillary species of ducks, geese, and swans distributed goal is to refine estimates of the genetic and sys- worldwide. Sibley and Ahlquist (1972) thoroughly tematic relationships~ of a large number of species review the history of waterfowl classification. The and genera of waterfowl. The basic rationale for family is generally divided into at least two major choice of waterfowl, described in detail below, is subfamilies, the Anserinae (tree ducks, geese, and that the family has been the subject of intense sys- swans) and Anatinae (most other ducks), and a tematic study, and by 'bird-standards' is compara- variable number of subfamilial tribes. Important tively well-represented in the fossil record. recent contributors to waterfowl taxonomy have We have examined electrophoretic variation in been Delacour and Johnsgard, whose classifica-

SPECIES TRIBE OLORCOLUMBIANUS BRANTACANAOENSIS ANSERALBIFRONS ANSERINJ ANSERCAERULESCENS ANSERROSSI / ~ OXYURAJAMAICENSIS] OXYURINI ~ MELANITIAOEGLANOl 1 ~ CLANGULAHYEMALIS MERGINI 8UCEPHALACLANGULA 8UCEPHALAALBEOLA AYTHYAAFFINIS /. AYTHYAMARILA AYTHYINI / AYTHYAVALISINERIA AYTRYAAMERICANA \ AYTHYACOLLARIS AIXSPONSA .] CAIRININI ANASPLATYRHYNCHOS ANASFULVIGULA ANASRUBRIPES ANASACUTA ANASSTREPERA ANATINI ANASAMERICANA ANASCAROLINENSIS ANASCLYPEATA ANASOISCORS ANASCYANOPTERA

Fig. I. 'Model" phylogeny of waterfovd based on behavioral and morphological considerations as discussed by Delacour and Mayr (1945) and Johnsgard (1968). 131 tions (reviewed in Delacour & Mayr, 1945; Detailed relationships among species and genera, Delacour, 1954; Johnsgard, 1968; Bellrose, 1976) based on considerations of morphology and be- will form the basis for our present discussion. havior, are discussed by Delacour and Mayr (1945). For those 26 species examined by us, Delacour A total of about 165 anseriform species are (1954) and Johnsgard (1968) are in fairly close known from fossils, and the majority of these are agreement on taxonomic (and evolutionary) rela- no longer extant (Howard, 1964). An early but dis- tionships. From their discussions and figures, we puted (Olson & Feduccia, 1980) anseriform fossil have distilled a summary phylogeny (Fig. I) which (Eonessa) dates to the middle Eocene (Wetmore, constitutes a 'model' against which to evaluate the 1938). By the Oligocene, representatives of both results of various methods of genetic data analysis. Anserinae and Anatinae (including the large extant The higher classification underlying the model genus Anas) were reportedly present. Additional phylogeny (see also Morony et al., 1975) is as fol- living genera (including Anser, Branta and Aythya) lows: date from the Miocene (Brodkorb, 1964; Howard, Subfamily Anserinae 1964; Romer, 1966). Thus several major evolution- Tribe Anserini - Olor, Branta, Anser ary lines of waterfowl are probably at least as old Subfamily Anatinae as the mid-Tertiary (Olson & Feduccia, 1980). Sever- Tribe Anatini - Anas al Pliocene remains appear indistinguishable from Tribe Aythyini - Aythya present-day species (Howard, 1964). Tribe Cairinini - Aix Using the fossil record as a rough time-scale, the Tribe Mergini - Bucephala, Melanitta, Clan- basal split between Anserinae and Anatinae (Fig. I) gula might be conservatively dated at about 25 million Tribe Oxyurini - Oxyura years ago, and many genera within the subfamilies

Table 1. Heterozygosity values determined by direct counts of proportions of individuals heterozygous per locus, averaged across the 17- 19 assayed loci for twenty-six species of waterfowl.

Species Common name Sample size IZl

(1) Olor columbianus Whistling Swan 2 0 (2) Branta canadensis Canada Goose 8 0.050 (3) Anser albifrons White-frothed Goose II 0.014 (4) Anser caerulenscens Snow andBlue Goose 14 0.025 (5) Anser rossi Ross' Goose 4 0.038 (6) A has platyrhynchos Mallard 10 0.037 (7) Anasfulvigula Mottled Duck 1 0 (8) Anas rubripes Black Duck 4 0.028 (9) Anas acuta Pintail 16 0.046 (10) Anas strepera Gadwall 6 0.008 (11) Anas discors Blue-winged Teal 1 0.056 (12) Anas c vanoptera Cinnamon Teal 6 0.025 (13) Anas carolinensis I Green-winged Teal 6 0.025 (14) A has americana Amerian Wigeon 16 0.016 (15) A nas clypeata Northern Shoveler 17 0.028 (16) Aix sponsa Wood Duck 7 0.045 (17) Aythya americana Redhead 1 0.050 (18) Ayth.va valisineria Canvasback 7 0.083 (19) Aythya collaris Ring-necked Duck 3 0.018 (20) Aythya marila Greater Scaup 15 0.028 (21) Aythya affinis Lesser Scaup 23 0.048 (22) Bucephala clangula Goldeneye 1 0.050 (23) Bucephala albeola Bufflehead 5 0.032 (24) Clangula hyemalis Oldsquaw 2 0.050 (25) Melanitta deglandi Surf Scorer 7 0.015 (26) Oxyura jamaicensis Ruddy Duck 14 0.030

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I i n e-. o ,,6 t~ r ,.6 ~a I i 134 are at least 15-20 million years old. As we will these species completely lack genetic variation. show, even with these conservative estimates ot Across all species, mean heterozygosity equals evolutionary age, protein differentiation in water. 0.033, both unweighted and weighted by sample fowl appears to have proceeded at a very slow pace. size. These heterozygosities are only slightly lower than mean values previously reported for birds and for other vertebrates (Powell, 1976). Materials and methods Electromorphs and their frequencies are presented in Table 2. Since particular electromorphs can Specimens were obtained through the coopera- be thought of as qualitative characters, either pres- tion of several state fish and game agencies and lo- ent or absent in samples of a given taxon, they can cal chapters of Ducks Unlimited, and by personal be directly employed to generate an evolutionary collecting. Freshly-killed specimens, or those which tree according to Hennigian principles. A cladistic had died on refuges from natural causes, were fro- zen and shipped to the laboratory for processing. tree for the waterfowl is shown in Figure 2. This tree was developed by procedures detailed by Pat- Sample sizes of the species examined are listed in ton and Avise (1983), who also discuss strengths Table 1. Extracts from heart, pectoral muscle, and liver and weaknesses of this approach. Perhaps the most were analyzed separately by horizontal starch-gel appealing aspect of this procedure is that individu- electrophoresis according to routine procedures al character states (electromorphs in this case) are described by Selander et al. (1971), Ayala et al. explicitly defined along all branches of the output (1972), and Arise et al. (1980a). The proteins as- tree. This in turn facilitates hypothesis testing be- sayed in this study, listed by the standard abbrevia- cause specific characters leading to points of am- tions employed in the above references, are given in biguity or discrepancy in the tree can be identified Table 2. All samples were compared on a side-by- and potentially subjected to further study. Charac- side basis, and scoring was accomplished by desig- ter states along branches of the cladistic tree in Fig- nating mobilities relative to the common electro- ure 2 are presented in Table 3. morph of the Mallard, which was arbitrarily la- In developing the cladistic network, plesio- beled '100' (anodal migration) or '-100' (cathodal morphs (electromorphs ancestral to the assayed migration). waterfowl) were disregarded in establishing geneal- ogies within the tree. A total of 19 such plesio- Mean genetic distances across loci were estimat- morphs were identified by the criterion that they ed by Nei's (1972) and Roger's (1972) formulas. were shared by any members of the two basally Nei's D values were used to generate a phenogram related subfamilies, Anserinae and Anatinae. Elec- by the Unweighted Pair-Group Method Analysis tromorphs unique to a taxon or taxa within Anati- (UPGMA; Sneath& Sokal, 1973). Roger's/) values nae, or within Anserinae, constitute derived charac- were employed to develop a phylogenetic tree by the ter states (apomorphs), and form the basis for distance Wagner procedure (Farris, 1972). The raw data (presence or absence of electromorphs) were clade identification. A total of 63 apomorphs con- used to develop yet another tree by the qualitative tributed to the structure of the tree. method of Hennig (1966; see Patton & Avise, 1983). The use of the qualitative phylogenetic approach All heterozygosities (H's) were determined by direct proposed by Hennig (1966) is a subject of con- counts of mean proportions of loci heterozygous troversy for many reasons, including the possibility per individual. of producing alternative phylogenetic trees (see Sokal, 1975). The figured relationship of Aythya-Bucephala (Fig. 2) can serve as a case in Results point. Since no synapomorphic (shared-derived) character was found to define Aythya, several Observed heterozygosities ranged from zero different branching sequences could be proposed to (Olor columbianus and Anas fulvigula) to 0.083 represent the relatedness of Aythya and Bucephala. (Aythya valisineria). Because the two species ex- Some of these representations would suggest a hibiting H=0 were represented by a total of only polyphyletic origin for Aythya, since minor syn- three specimens, our estimates do not imply that apomorphic alleles were shared by A. americana-B. 135

OLOR COLUMRIANUS BhANTA CANAOENSIS ANSER ALRIFRONS ~)_EI...,, ANSERCAERULESCENS ""~'L A~SERROSS1 OXYURAJAMAICENSIS. MELANITTA DEGLANOI

10 CLANGULA HYEMALIS AIX SPONSA /--.~ BUCEPHALACLANGULA 15 BUCEPHALA ALBEOLA 14 ~.~ 17 20 AYTHYAAFFINIS 16 ~ AYTHYAMARILA AYTHYAVALISINERIA AYTHYA AMERICANA AYTHYA COLLARIS 26 ANAS PLATYRHYNCHOS ANAS FULVIGULA 32 ANAS RURRIPES ANAS CAROLINENSiS ANAS ACUTA ANAS CLYPEATA 38 ANAS OISCORS ANAS CYANOPTERA ANAS STREPERA "~ ANASAMERICANA

Fig. 2. Phylogenetic tree for assayed waterfowl generated from qualitative analysis of electromorphs using the method o1 Hennig (1966) and Patton and Avise (1983). Character states along branches of the tree are listed in Table 3. Lines crossing branches indicate derived electromorphs whose ancestral states can be hypothesized, and circles crossing branches denote electromorphs whose presumed ancestral states remain undetermined.

Albeola (Pgdl2~ and by A. affinis-B, albeola with 'model' phylogenies based on independent (Gpd7~ In Fig. 2, we have opted to present a con- criteria. In the following sections, we discuss points servative interpretation which suggests that Pgd ~2~ of agreement and disagreement in the data ana- and Gpd 70 were ancestral alleles independently re- lyses, and evaluate them against the 'model' tained in these species. phylogeny for waterfowl. Two other 'trees', generated by Wagner analyses and UPGMA from genetic distance matrices, are Commonalities presented in Figures 3 and 4 respectively. The results summarized in Figures 2-4 represent only a Anatinae and Anserinae small sampling of numerous available methods of All methods of genetic data analysis recognize tree generation. Nonetheless, they do encompass a the basal split between the two subfamilies philosophically diverse array of approaches, rang- described by classical systematists (the exception is ing from cladistic to strictly phenetic, and utilizing the UPGMA placement of Oxyura, discussed both qualitative and quantitative data bases. There later). Anatinae and Anserinae cluster in UPGMA is no general concensus on the 'best' method of tree at a genetic distance of about 0.4, and they form generation, nor even on criteria by which to recog- major branches of the Wagner tree. Two synapo- nize a 'best' tree. We prefer to employ conceptually morphs (Idh-I I~176and Idh-I 2~176define the Anatinae diverse approaches, and to base our strongest con- clade in the Hennigian analysis; Pgd 2~176and Alb I~ clusions on commonalities in results. The commo- were unique to the Anserinae and shared by all its nalities are further strengthened when they agree members. 136

Table 3. Electromorphs defining branches of the phylogenetic only a small 2 distance units (Fig. 3) prior to the tree in Figure 2. Line 1 lists characters presumed plesiomorphic split of Olor. Three electromorphs (ldh-1125, (ancestral) to assayed waterfowl taxa, and successive lines list Gpd 6~ and Got-18~ were unique to Olor, and one apomorphic (derived) characters. Electromorphs in brackets are apomorphs whose presumed ancestral states remain undeter- synapomorph (Idh-U 2~ defines the Branta-Anser mined. clade.

(1) Mdh-I "~'. Mdh-2 ~. Ldh-U ~'. Ldh-2 tc~, Idh-l% A FIseF Idh-2 "~', Pgd "~J, Gpd::, Pgd ~', Pgi ~~ Pgm :~ Ck-2 t~176 Ck-3 ""~, Got-I Ic~J, Got-2 m~, lpo:% Pept-U ~, Pept-2 I~ All methods of analysis recognize the close Hb too genetic relationships among the three species of (2) Pgd e'~ [AIb j~ Anser reflected in the current classification. Baker (3) Idh-U::, Gpcl~', Got-I ~~ and Hanson (1966) have previously reported only (4) ldh-I t-" minor differences in hemoglobins and serum pro- (5) Mdh-I '~ Got-U:", [Alb 9~] (61 Ck-2 I~~ teins among eight Anser (and three Branta) species. (7) (Speciation) A synapomorph (Ck-214~ defines the Anser clade. (8) Gpd :~ (9) (Speciation) A nas (10) ldh-I ~', Idh-I ~~176 (11) Gpd Is~ Gpd s:, pgpo, Pgi4:, Pgrnl~o, Pgm~:O, Ck_2.,~. This genus is also consistently delineated in all Got-I -~oo, Got-l-'~, [AIb:O7] genetic summaries, as it is in morphological and (12) Gpd r2, Hb t4o [AIbl~] behavioral characteristics. Pgm/~176is synapo- (13) Hb I~o [AlbHO] morphic for the Anas assayed, and the group forms (14) Gpd I~176 a coherent assemblage in both the Wagner and (15) Gpd I'~ [Albl% AIM ~ (16) Pgd I'~ Gpd 7~ [Alb I~ UPGMA analyses. Genetic distances within the ge- (17) Idh-U ~~ nus are very small (ranging from Nei's D=0.001 be- (187 (Speciation) tween Anas platyrhynchos and A. rubripes to (19) (Speciation) D=0.186 between A. strepera and A. cyanoptera). (20) Gpd I:.~. pgylO, Pgil~o (21) Pept-2 ~: Even so, some genetic groupings within the genus (22) Pgrn I~ appear consistently and these are generally among (23) Ldh-I ~'~~ species thought by systematists to be particularly (24) (Speciation) closely related. (25) (Speciation) One such grouping involves A. clypeata (North- (26) Pgm j~176 (27) Pgd n~ ern Shoveler), A. discors (Blue-winged Teal) and A. (28) Pgi~' cyanoptera (Cinnamon teal). Delacour and Mayr (29) [AIb '~176 (1945) stress the 'extremely close relationship' (30) (Speciation) among these species, evidenced by the almost iden- (31) (Speciation) (32) (Speciation) tical and unique wing coloration, peculiar court- (33) (Speciation) ship methods, and shared feeding habits. Geneti- (34) ldh-U ~~ Idh-I :~ Got-I ~" [AIbg'S], Pgd ~" cally, the three species share a unique allele (35) Ldh-I ~: (Ldh-195) which defines the clade, and they form (36) Mdh-I ~~ [AIb/~ distinct branches in the Wagner and UPGMA ana- (37) [.4lb ~~ (38) (Speciation) lyses. (1Votwithstanding its common name, the (39) Pgct~~ HO ~o Green-winged Teal is not particularly closely relat- (40) Gpd ~: [~lib ~s] ed to the 'blue-winged ducks', and is placed in a (41) Mdh-2 ~', Gpd ~~ Gpd ~~ [AIb ~'] different group by Delacour and Mayr (1945).) Another consistent genetic grouping involves A. platyrhynchos (Mallard), A. fulvigula (Mottled Olor Duck), and A. rubripes (Black Duck). Some or- Except in the Wagner analysis, the Whistling nithologists consider this complex a 'superspecies" Swan appears basally related to the geese which with the Black and Mottled Ducks representing compose the remainder of Anserinae. Even in the sexually non-dimorphic forms of the Mallard; Gra- Wagner tree, the earlier branching to Branta occurs ham (1979) suspects the forms may someday be tax- 14 OtoeCOLOMRZANUS 137 ANSER ROSSI ANSER ALRIFRONS ANSER CAERULESCENS BRANTA CANADENSIS OXYURA JAMAICENSIS 26 11 MELANITTA DEGLANOI 6 - CLANGULA HYEMALIS AIX SPONSA AYTHYA COLLARIS BUCEPHALA CLANGULA BUCEPHALA ALBEOLA AYTRYA AFFINIS AYTRYA VALISINERIA AYTRYA AMERICANA AYTHYA MANILA ANAS CAROLINENSIS ANAS PLATYRHYNCHOS ANAS RUBRIPES ANAS FULVIGULA ANAS ACUTA ANAS STREPERA ANAS AMERICANA ANAS CLYPEATA ANAS OISCORS ANAS CYANOPTERA

Fig. 3. Wagner tree for assayed waterfowl, generated from a matrix of genetic distances. Branch lengths are in Roger's /3 (•

OLOR COLUMBIANUS BRANTA CANAOENSIS ANSER ALBIFRONS ANSER CAERULESCENS ANSER ROSSI q MELANITTA OEGLANDI J CLANGULA HYEMALIS AIX SPONSA BUCEPHALA ALREOLA AYTHYAAMERICANA AYTHYA VALISINENIA AYTHYA MARILA BUCEPHALA CLANGULA AYTHYAAFFINIS AYTHYA COLLARIS ANAS PLATYRHYNCHOS ANAS RURRIPES ANAS CAROLINENSIS ANAS FULVIGULA ANAS ACUTA ANAS STREPERA ANAS AMERICANA ANAS OISCORS ANAS CLYPEATA ANAS CYANOPTERA OXYURA JAMAICENSIS I I I I I I 0.5 0.4 0.3 0.2 0,4 0 Fig. 4. UPGMA dendrogram for assayed waterfowl, generated from a matrix of genetic distances. The scale is in Nei's/). 138 onomically merged. The three species, together with Melanitta, and Clangula is rather high (by 'water- the Green-winged Teal, form a minor branch in fowl and bird standards'): D=0.20. Much of the each of the genetic trees, and they all share an elec- genetic distinctness of Melanitta and Clangula tromorph (AIb 1~176not observed in other water- from one another and from other Anatinae stems fowl. The Green-winged Teal is not conventionally from their possession of several unique (autapo- thought to be particularly close to the Mallard morphic) alleles (Fig. 2). The remainder of Anati- group, but based on our sample of 19 loci it could nae (excluding Oxyura) are also united by posses- not be distinguished from the Mallard assemblage. sion of the synapomorph Gpd I~176in high frequency The other Anas species (acuta, strepera, in all species. americana) are placed in distinct subgeneric groups Other traditional classifications had considered by Delacour and Mayr (1945), and apart from their the bay ducks (Aythyini of Johnsgard and obvious close genetic ties to other Anas, they show Delacour) and sea ducks (Mergini) to be closely no consistent branching or clustering tendencies in related and in a subfamily Aythyinae (i.e., Robbins Figures 2-4. et al., 1966). Perhaps the close genetic ties of Bucephala and Aythya are compatible with this Aythya view. The genetic data also suggest that other sea Members of this compact genus of diving ducks duck genera, such as Melanitta and Clangula are or pochards are also very close in genetic composi- only basally related to one another. A more com- tion. They congregate (with Bucephala - see be- prehensive genetic investigation of the sea duck yond) in each method of genetic data summary. complex, including the Eiders (Somateria), Mer- Within the genus, Nei's/~'s are very small and fall gansers (Mergus) and Harlequin (Histrionicus), within a narrow range - 0.004 (A. affinis versus A. would now appear most desirable. collaris) to 0.055 (A. americana versus A. collaris). No electromorph was found to define Aythya. Aix The Wood Duck (Aix sponsa) is the sole Bucephala representative of the perching ducks, Cairinini, that The two assayed species of this genus, B. clangu- we have examined. Both Johnsgard (1968) and la (Goldeneye) and B. albeola (Bufflehead), are Delacour and Mayr (1945) suggest close evolution- very similar in genic composition: D=0.036 (al- ary ties between Cairinini and Anatini, based on though in the UPGMA clustering they are inter- coloration of the downy young and structure of the mingled with the Aythya). They do form a distinct syrinx. Woolfenden (1961) urged that Cairinini be minor branch in both the Wagner and Hennigian merged with Anatini. These relationships are not analyses, the latter determined by shared posses- evident in the genetic data. In the UPGMA pheno- sion of a derived electromorph, Idh-1 m~ gram, Aix clusters slightly closer to Aythya-Bucephala than to Anas; in the Wagner Discrepancies and ambiguities tree, Aix falls just outside the Aythyini-Anatini complex; and in the Hennigian tree, Aix stems from Mergini a common branch point with Aythyini and Anatini. This tribe, which includes Bucephala, Melanitta, ) and Clangula, is thought by Delacour and Mayr Oxyura (1945) to be well delineated, showing no close rela- The stiff-tailed ducks, Oxyurini, are thought to tionships to the pochards (Aythyim) or to other have branched very early from primitive Anatinae tribes. Therefore, it is surprising that Bucephala ap- stock, and to be only distantly related to other pears genetically close to Aythya, and furthermore tribes within that subfamily (Johnsgard, 1968; that the three assayed Mergini genera exhibit no Delacour & Mayr, 1945). Our genetic data confirm particular genetic ties to one another (Figs. 2-4). that Oxyura is a highly divergent anatid, although The Hennigian analysis suggests that Pgd 12~ different methods of genetic analysis do not entire- Gpd 7~ and Alb m are shared by Bucephala and ly agree on its exact placement within Anatidae. Aythya, yet were not observed in other waterfowl. Phenetically, Oxyura falls outside both the Anati- The mean genetic distance between Bucephala, nae and Anserinae clusters (Fig. 4), exhibiting a 139 mean genetic distance (Nei's D) of about 0.43 to the Table 4. Summary of genetic distances (Nei's/3) in waterfowl, other waterfowl species. In the Wagner and Hen- based on 17- 19 loci. nigian analyses, Oxyura stems from a common bas- Comparison Number /3 Range al branch point with Melanitta-Clangula, or with of species Melanitta-Clangula-Aythyini-Anatini, respective- comparisons ly. Oxyura does exhibit both ldh-11~176and Idh-12~176 electromorphs that were observed in most Anatinae Between species within genera species, but not in the Anserinae. These alleles ac- Anser 3 0.002 (0.001-0.003) Bucephala 1 0.036 count for its membership in the Anatinae clade in Aythya 10 0.023 (0.004- 0.055) the Hennigian analysis. Oxyura also exhibits a large Arias 45 0.092 (0.001 -0.186) number of autapomorphic alleles (Table 3). Between genera within tribes Overall, the agreement between the various Anserini 7 0.186 (0.116 - 0.262) Mergini 5 0.238 (0.188-0.288) genetic summaries of waterfowl relationships is Between tribes within subfamilies quite good. The agreement between classical Anatinae 149 0.207 (0.010- 0.565) phylogenies and the genetic phylogenies of water- Between subfamilies fowl is also very strong. The major area of poten- Anserinae-Anatinae 105 0.386 (0.152-0.611) tial disagreement concerns possible relationships of Totals 325 0.241 (0.001- 0.611) various sea ducks (Mergini) to one another and to the Aythyini. It must also be recognized, as discussed below, that all of the genetic distances in scales for widely different phylads could be provid- waterfowl are very small compared to genetic dis- ed by times of divergence (where these are known tances in most other families of non-avian ver- or suspected) or by estimates of divergence in tebrates. The members of Anatidae are clearly a ge- homologous molecules, for example. Concordance netically close-knit assemblage. or non-concordance between results of comparisons utilizing different scales can provide insights into general patterns of evolutionary change. Discussion Among the waterfowl, genetic distances between species at various levels of taxonomic recognition Conservative patterns of divergence are summarized in Table 4. Since this is among the few large-scale estimates of such genetic distances In discussions of comparative phylogeny, it is im- in a group of non-Passeriforme birds, it is of in- portant to ask whether the taxonomies of the terest to compare results with those previously pub- groups under consideration are equivalent. These lished for several Passeriforme families. This is questions can be best addressed when common done in Table 5. It is apparent that the An- scales for comparison are available. Such common seriformes exhibit the same conservative pattern of

Table 5. Summary of genetic distances (Nei's D) in Passeriformes and Anseriformes at comparable levels of the taxonomic hierarchy.

Comparison t Number of species D Range comparisons

Anseriformes Between species within genera 59 0.075 (0.001- 0.186) Between genera within subfamilies 161 0.207 (0.010- 0.565) Between subfamilies 105 0.386 (0.152 - 0.611) Passeriformes 2 Between species within genera 87 0.059 (0.000-0.279) Between genera within subfamilies 351 0.195 (0.011- 0.636) Between subfamilies ~ 27 0.483 (0.350- 0.693)

According to the classification of Morony et al. (1975). 2 Data from Avise et al., 1980a, b, c; for additional data (which reflect the same trend), see Barrowclough and Corbin (1978) and Smith and Zimmerman (1976). Assumes lcteria is subfamilially distinct from other assayed Parulidae. 140 protein differentiation as do other birds. In many mean genetic distances among rodents are 6-7 groups of Aves assayed to date, as a general rule times as large as mean genetic distances among mean genetic distances among congeners are typi- birds. cally about 0.10 or less; between subfamilial genera, /)s average about 0.20, and between subfamilies/)'s Rate of protein evolution are commonly in the range of about 0.35-0.50. These statements should not be interpreted as hard- One hypothesis to account for these trends is that ened guidelines for future taxonomic assignments. species of Anatidae are evolutionarily younger than They are simply empirical generalities from genetic most other vertebrates. This seems unlikely given data currently available. the fossil evidence presented earlier. An alternative A much larger literature exists on genetic dis- hypothesis is that protein evolution is decelerated in tances among non-avian vertebrates. Some phylads birds relative to non-avian vertebrates. This possi- do exhibit a conservative level of genetic differenti- bility has proved difficult to evaluate in the Pas- ation comparable to that summarized for Aves seriformes because of the poor fossil record, but above, but these are in the minority. Far more typi- with caution we can address the issue in the Anati- cally, even closely related congeners exhibit D's as dae. large or larger than those characteristic of avian Nei (1971, 1975) has shown that his measure of genera or subfamilies. The protein electrophoretic genetic distance can be related to time of diver- literature is summarized by Avise and Aquadro gence of species by the formula/)= t r, where r is (1982). a variable determined by several considerations In referring to comparisons of heterozygosity about protein structure and variation: c, the levels in different organisms, Selander (1976) argues proportion of amino acids which are electrophoret- that 'the major determinant of the span of varia- ically detectable; n, the average number of amino tion in estimates of polymorphism is the laboratory acids per protein; and X, the rate of amino acid sub- in which the survey was conducted!' This argument stitution per polypeptide per site per year cannot account for the emerging avian/non-avian (r=2cnk). Suppose we wish to compare two difference in mean level of genetic differentiation. phylads, A and B, which exhibit genetic distances First, the conservative Ayes pattern has been DA and D B. Then reported independently by several labs (Barrow- clough & Corbin, 1978, Smith & Zimmerman, DA /ACAnA)kA (1) 1976). Second, in our own laboratory we have as- Da tBCBnBXB sayed other vertebrates concurrently with members of Aves, employing similar buffers and elec- There is no evidence to suggest that c's or n's are trophoretic conditions to analyze comparable sets consistently different in birds than in other ver- of proteins. Results for 16 species and 9 genera tebrates (it is perhaps conceivable that for some within the single rodent subfamily Cricetinae (Pat- reason the electrophoretic buffers yield lower c's in ton et al., 1981) are contrasted with the avian results birds, and this possibility may be worth experimen- in Table 6. Both at the species and generic levels, tal test). Thus formula (1) simplifies to

Table 6. Summary of genetic distances (Nei's D) in birds versus rodents at comparable levels of the taxonomic hierarchy.

Comparison Number of species D Range comparisons

Birds' Between species within genera 146 0.065 (0.000- 0.274) Between genera within sub families 512 0.199 (0.010 - 0.636) Rodents-' Between species within genera 120 0.393 (0.002 -1.002) Between genera within subfamilies 83 1.256 (0.398 - 2.238)

Data from Avise et al., 1980a, b, c, and this paper. 2 Data from Avise et al., 1974 (and reference therein), and Patton et al., 1981. 141

certainties involve the reliability of estimates of/3, DA [A)kA (2) DB tB~kB and the representativeness of protein data to divergence in the total avian genome. From immunolog- As mentioned in the introduction, the fossil rec- ical comparisons of albumins and transferrins, ord suggests that many genera of waterfowl are Prager and Wilson (1975) conclude that these pro- about 20 million years old. Hibbard (1968) suggests teins 'have evolved about three times as slowly in that the earliest divergence of the genera of the birds as in other vertebrates'. Wilson et aL (1977) cricetine rodents examined by Patton et al. (1981 - later suggested that this conclusion may have been Table 6) also occurred about 20 million years ago. premature. Nonetheless, the evidence for an avian If we accept these estimates, that anseriform and protein deceleration remains sufficiently compel- rodent genera are of roughly equivalent age, the ra- ling to warrant further study. In the future it will be tio XA/XB equals about 6; rodents are diverging in especially valuable to assay other portions of the protein composition at rates several times faster avian genome as well as to develop and test hypoth- than waterfowl. If the rodent genera are in fact eses about the causal process responsible for any younger (or the waterfowl older) the ratio of rates possible deceleration of protein divergence in birds. of genetic divergence in the two groups would be One of the important observations of research in even greater than this. molecular evolution is that the pace of evolution The problem can be looked at in another way. among non-homologous proteins can vary tremen- Mean genetic distance between the waterfowl sub- dously (Sarich, 1977). Rates of amino acid substitu- families Anserinae and Anatinae is 0.386 (Table 5). tion per polypeptide site differ by more than In a large survey of protein comparisons in other 100-fold from rapidly evolving immunoglobulins organisms, Nei (1975) estimates r to be roughly and fibrinopeptides to slowly evolving histones 2• -7 per year for electrophoretically detectable (Dayhoff, 1972; Wilson et. al., 1977). Such observa- protein substitutions. Substituting these values in tions have stimulated a great deal of research lead- the formula t=D/r, and assuming that waterfowl ing to an increased recognition of the significance proteins evolve at the standard rate, the subfamilies of functional constraints (or lack thereof) on pro- should be about 2 million years old. Even if we tein divergence (Wilson et al., 1977). If indeed it plug these values into the slowest calibrated eventually proves true that homologous or analo- 'protein-clock' of which we are aware, that pro- gous proteins also evolve at distinct rates in differ- posed in 1977 by Sarich (and which assumes all as- ent phylads, our ability to use molecular informa- sayed proteins belong to a 'slowly-evolving' class), tion to reach definitive conclusions about absolute estimated divergence time would be about divergence times for species with a poor fossil rec- 12 million years. These estimates still contrast with ord will be comprised. In that case, an exploration the literal fossil interpretation which places the of possible reasons for heterogeneous rates across Anserinae-Anatinae divergence in waterfowl at per- phylads could ultimately lead to a fuller under- haps 25 million years. Again, at face value, it ap- standing of the factors governing protein differenti- pears that protein divergence is proceeding much ation. more slowly than in most other organisms. Gutier- rez et al. (1983) report very similar conclusions for another group of non-Passeriforme birds. Protein Acknowledgments clocks previously suggested for some non-avian vertebrates proved to be far to rapid (by a factor of We wish to express our deepest gratitude to the five) to yield divergence times compatible with ob- people of Ducks Unlimited, to the various Fish and served genetic distances and with the fossil record Game Agencies, and to others who so willingly for the Galliformes. provided specimens and suggestions which aided In view of the inevitable uncertainties about true the acquisition of specimens. Principal among divergence times in waterfowl, and in most other these were Carlton Woods, Sam Parish, Jack Kam- organisms, we are hesitant to draw firm conclu- man, Irv Kornfield, Howard Leach (California Fish sions about the exact magnitude of any possible and Game), and Larry Hindeman (Maryland Wild- slowdown in protein evolution in Ayes. Further un- life Administration). Wyatt Anderson, Joshua 142

Laerm, James Porter, and Whit Gibbons reviewed Gutierrez, R. J., Zink, R. M. & Yang, S. Y., 1983. Genic varia- and commented on the manuscript. Bob Chapman tion, systematic and biogeographic relationships of some gal- assisted with the data analysis and Joanne Zahner liform birds. The Auk 100:33 47. Hennig, W., 1966. Phylogenetic Systematics. Univ. Illinois Press, assisted with lab work. Work was supported by a Chicago. USPHS training grant from Univ. Ga., and by a Hibbard, C. W., 1968. Paleontology. Pp. 1-26 in: J. A. King University Research Committee Grant from Baylor (ed.), Biology of Peromyscus (Rodentia), Spec. Pub. Am. Soc. to JCP. Work was also supported by a grant from Marnm. 2. Howard, H., 1964. Fossil Anseriformes. Pp. 233-326 in: J. the Penrose Fund of the American Philosophical Delacour (ed.), The Waterfowl of the world, V. 4, Country Society and by NSF grant DEB 7814195. Life Ltd, London. Johnsgard, P. A., 1968. Waterfowl. Univ. Nebraska Press, Lin- coln, Nebraska. Martin, R. K & Selander, R. K., 1975. Morphological and bi- References ochemical evidence of hybridization between cave and barn swallows. Condor 77: 362-364. Avise, J. C. & Aquadro, C. E, 1982. A comparative summary of Mengel, R. N., 1964. The probable history of species formation genetic distances in the vertebrates. Evol. Biol. 15: 151-185. in some northern wood warblers (Parulidae). Living Bird 3: Avise, J. C., Patton, J. C. & Aquadro, C. E, 1980a. Evolutionary 9-43. genetics of birds I. Relationships among North American Morony, J. J., Bock, W. J. & Farrand J., Jr., 1975. Reference list thrushes and allies. The Auk 97: 135-147. of the birds of the world. Spec. Pub. Am. Mus. Nat. Hist., Avise, J. C., Patton, J. C, & Aquadro, C. E, 1980b. Evolutionary New York. genetics of birds I1. Conservative protein evolution in North Nei, M., 1971. lnterspecific gene differences an~ evolutionary American sparrows and relatives. Syst. Zool. 29: 323-334. time estimated from electrophoretic data on protein identity. Avise, J. C., Patton, J. C. & Aquadro, C. E, 1980c. Evolutionary Am. Nat. 105: 385-398. genetics of birds 111. Comparative molecular evolution in Nei, M., 1972. Genetic distance between populations. Am. Nat. New World warblers (Parulidae) and rodents (Cricetinae). J. 106: 283-292. Hered. 71: 303-310. Nei, M., 1975. Molecular population genetics and evolution. Avise, J. C., Smith, M. H. & Selander, R. K., 1974. Biochemical North Holland, Amsterdam. polymorphism and systematics in the genus Peromyscus. VI. Olson, S. L. & Feduccia, A., 1980. Presbyornis and the origin of The boylii species group. J. Mamm. 55: 751-763. the Anseriformes (Ayes: Charadriomorphae). Smithsonian Ayala, F. J., Powell, J. R., Tracey, M. L., Mourao, C. A. & Perez- Cont. Zool. 323: 1-24. Salas, S., 1972. Enzyme variability in the Drosophila willisto- Patton, J. C. & Avise, J. C., 1983. An empirical evaluation of ni group. IV. Genetic variation in natural populations of qualitative Hennigian analyses of protein electrophoretic Drosophila willistoni. Genetics 70: 113-139. data. J. mol. Evol. 19: 244-254. Baker, C. M. Ann & Hanson, H. C., 1966. Molecular genetics Patton, J. C., Baker, R. J. & Avise, J. C., 1981. Phenetic and of avian proteins. VI. Evolutionary implications of blood pro- cladistic analyses of biochemical evolution in peromyscine ro- teins of eleven species of geese. Comp. Biochem. Physiol. 17: dents. Pp. 288-308 in: Mammalian population genetics, M. 997-1006. H. Smith and J. Joule (eds.), Univ. Ga. Press, Athens. Barrowclough, G. E & Corbin, K. W., 1978. Genetic variation Powell, J. R., 1976. Protein variation in natural populations of and differentiation in the Parulidae. The Auk 95: 691-702. animals. Evol. Biol. 8: 79-119. Barrowclough, G. E, Corbin, K. W. & Zink, R. M., 1981. Genet- Prager, E. M. & Wilson, A. C., 1975. Slow evolutionary loss of ic differentiation in the Procellariiformes. Comp. Biochem. the potential for interspecific hybridization in birds: a Physiol. 69B: 629-632. manifestation of slow regulatory evolution. Proc. hath. Acad. Bellrose, F. C., 1976. Ducks, geese and swans of North America. Sci. U.S.A. 72: 200-204. Stackpole Books, Harrison, Penn. Robbins, C. S., Bruun, B. & Zim, H. S., 1966. Birds of North Brodkorb, P., 1964. Catalogue of fossil birds, part 2. Bull. Fla America. Golden Press, New York. St. Mus. 8: 195-335. Rogers, J. S., 1972. Measures of genetic similarity and genetic Corbin, K. W., Sibiey, C. G., Ferguson, A., Wilson, A. C., distance~)Studies in Genetics VII. Univ. Texas Publ. 7213: Brush, A. H. & Ahlquist, J. E., 1974. Genetic polymorphism 145-153. in New Guinea starlings of the genus Aplonis. Condor 76: Romer, A. S., 1966. Vertebrate Paleontology. Univ. of Chicago 307-318. Press, Ch!cago, 111. Dayhoff, M. O., 1972. Atlas of protein sequence and structure. Sarich, V. M., 1977. Rates, sample sizes, and the neutrality National Biomedical Research Foundation, Washington, D.C. hypothesis for electrophoresis in evolutionary studies. Nature Delacour, J., 1954. The Waterfowl of the world. Hamlyn Publ. 265: 24-28. Group Ltd., London. Selander, R. K., 1976. Genic variation in natural populations. Delacour, J. & Mayr, E., 1945. The family Anatidae. Wilson Pp. 2145 in: Molecular evolution, E J. Ayala (ed.), Sinauer, Bull. 57: 3-55. Sunderland, Massachusetts. Farris, J. S., 1972. Estimating phylogenetic trees from distance Selander, R. K., Smith, M. H., Yang, S. Y., Johnson, W. E. & matrices. Amer. Nat. 106: 645-668. Gentry, J. B., 1971. Biochemical polymorphism and systemat- Graham, F., Jr., 1979. Farewell, Mexican duck. Audubon 81: ics in the genus Peromyscus 1_ Variation in the old-field 24-26. 143

mouse (Peromyscus polionotus). Studies in Genetics Vl. Sokal, R. R., 1975. Mayr on cladism - and his critics. Syst. Zool. Univ. Texas Publ. 7103: 43-90. 24: 257-262. Sibley, C. G. & Ahlquist, J. E., 1972. A comparative study of the Wetmore, A., 1938. A fossil duck from the Eocene of Utah. J. egg-white proteins of non-passerine birds. Peabody Mus. Nat. Paleont. 12: 280-283. Hist. Bull. 39, Yale University, New Haven, Conn. Wilson, A. C., Carlson, S. S. & White, T. J., 1977. Biochemical Smith, J. K. & Zimmerman, E. G., 1976. Biochemical genetics evolution. Ann. Rev. Biochem. 46: 573-639. and evolution of North American blackbirds, family lcteri- Woolfenden, G. E., 1961. Postcranial osteology of the water- dae. Comp. Biochem. Physiol. 53B: 319-324. fowl. Bull. Florida State Mus. 6: 1-129. Sneath, P. H. A. & Sokal, R. R., 1973. Numerical Taxonomy. W. H. Freeman, San Francisco. Received 5.4.1984. Accepted 15.10.1984.