a molecular phylogeny of anseriformes based on mitochondrial dna analysis

A molecular phylogeny of anseriformes based onmitochondrial DNA analysis

Carole Donne-Gouss�ee,a Vincent Laudet,b and Catherine H€aannia,*

a CNRS UMR 5534, Centre de G�een�eetique Mol�eeculaire et Cellulaire, Universit�ee Claude Bernard Lyon 1, 16 rue Raphael Dubois,

Baat. Mendel, 69622 Villeurbanne Cedex, Franceb CNRS UMR 5665, Laboratoire de Biologie Mol�eeculaire et Cellulaire, Ecole Normale Sup�eerieure de Lyon,

45 All�eee d’Italie, 69364 Lyon Cedex 07, France

Received 5 June 2001; received in revised form 4 December 2001

Abstract

To study the phylogenetic relationships among Anseriformes, sequences for the complete mitochondrial control region (CR)

were determined from 45 waterfowl representing 24 genera, i.e., half of the existing genera. To confirm the results based on CR

analysis we also analyzed representative species based on two mitochondrial protein-coding genes, cytochrome b (cytb) and NADH

dehydrogenase subunit 2 (ND2). These data allowed us to construct a robust phylogeny of the Anseriformes and to compare it with

existing phylogenies based on morphological or molecular data. Chauna and Dendrocygna were identified as early offshoots of the

Anseriformes. All the remaining taxa fell into two clades that correspond to the two subfamilies Anatinae and Anserinae. Within

Anserinae Branta and Anser cluster together, whereas Coscoroba, Cygnus, and Cereopsis form a relatively weak clade with Cygnus

diverging first. Five clades are clearly recognizable among Anatinae: (i) the Anatini with Anas and Lophonetta; (ii) the Aythyini with

Aythya and Netta; (iii) the Cairinini with Cairina and Aix; (iv) the Mergini with Mergus, Bucephala, Melanitta, Callonetta, So-

materia, and Clangula, and (v) the Tadornini with Tadorna, Chloephaga, and Alopochen. The Tadornini diverged early on from the

Anatinae; then the Mergini and a large group that comprises the Anatini, Aythyini, Cairinini, and two isolated genera, Chenonetta

and Marmaronetta, diverged. The phylogeny obtained with the control region appears more robust than the one obtained with

mitochondrial protein-coding genes such as ND2 and cytb. This suggests that the CR is a powerful tool for bird phylogeny, not only

at a small scale (i.e., relationships between species) but also at the family level. Whereas morphological analysis effectively resolved

the split between Anatinae and Anserinae and the existence of some of the clades, the precise composition of the clades are different

when morphological and molecular data are compared. � 2002 Elsevier Science (USA). All rights reserved.

Keywords: Anseriformes; mtDNA; Control region; Waterfowl

1. Introduction

Among avian orders, the Anseriformes (screamersand waterfowls) are a morphologically and biologicallydiverse group containing ca. 150 species distributedworldwide. This order contains the screamers of SouthAmerica, the magpie goose of Australia and Asia, andthe ducks, geese, and swans known worldwide. Fossilrecords indicate that the first Anseriformes (genusPresbyornis) was present during Upper Paleocene (61–62 million years ago), whereas the first Anatidae wasfound in the Upper Eocene (40–50 million years ago) in

North America (Olson and Feduccia, 1980). Accordingto these paleontological data, the main radiation ofmodern ducks has taken place during Miocene, 5–23million years ago (Olson, 1985).

The Anseriformes are traditionally divided into twofamilies, Anhimidae (2 genera and 3 species) andAnatidae (approximately 41 genera and 147 species).The taxonomic division is rather complex and has beenmuch disputed and revised. Most available data con-cerning Anseriformes phylogeny came from morpho-logical, anatomical, and behavioral analyses (Delacourand Mayr, 1945; Del Hoyo et al., 1992; Livezey, 1986,1997b). Molecular data such as DNA–DNA hybridiza-tion studies were also used to decipher the relationshipsbetween these birds (Sibley and Ahlquist, 1990). More

Molecular Phylogenetics and Evolution 23 (2002) 339–356

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E-mail address: [email protected] (C. Hanni).

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recently, phylogenies based on DNA sequence analysisof mitochondrial genes were proposed for some taxa ofthe Anseriformes such as the Australasian waterfowl(Sraml et al., 1996), the genus Anas (Johnson andSorenson, 1998, 1999), some extinct species such asmoa-nalos (Sorenson et al., 1999a), or the endangeredLaysan ducks (Cooper et al., 1996). The phylogeniesobtained by these different approaches differ mostly on(i) the relative positions of Anhimidae (with Ansera-natidae) and Anatidae, (ii) the grouping of Anatidae intwo main subfamilies, Anatinae and Anserinae, and (iii)the phylogeny inside each of these subfamilies, in par-ticular the composition and relationships of so-called‘‘tribes’’ clustering several genera within Anatidae.

The traditional classification of the Anseriformes ofDelacour and Mayr (1945), based on morphological andbehavioral characters, later modified by Johnsgard(1978), has been followedbymanyothers (DelHoyo et al.,1992). According to these authors, the genus Chauna,representative of the Anhimidae, diverged first, and wasthen followed by Anseranas and the crown group con-taining ducks, geese, and swans (Fig. 1A and AppendixA). According to these authors the whistling-ducks(Dendrocygna) are placed inside the subfamily Anserinae.This scheme has been mainly confirmed by Livezey(1997b), who nevertheless proposed that Dendrocygnarepresents an independent lineage, unrelated to Anseri-

nae (Fig. 1B). A radically different view has been pro-posed by Sibley andAhlquist (1990), who cluster togetherChauna and Anseranas to form the suborder Anhimides.As Livezey (1997b), these authors consider Dendrocygnaan independent lineage. Because of these discrepanciesthe composition of the family Anatidae is still a matter ofdiscussion, as for example the inclusion of Anseranas.

The Anatidae have been traditionally divided into twosubfamilies, Anatinae and Anserinae, the latter includinggeese, swans, and Dendrocygna (Delacour and Mayr,1945; Del Hoyo et al., 1992; Fig. 1A and Appendix A).This view as been challenged by Livezey (1997b), whorecognized four main clades (Fig. 1B): (i) the Anatinae;(ii) a clade called Tadornini including the genera Tad-orna,Chloephaga, andAlopochen; (iii) the Anserinae; and(iv) Dendrocygna. In addition other minor clades (Tha-lassorninae, Stictonettinae, and Plectropterinae) werealso noticed inside Anatidae. The relationships betweenall these clades remain poorly resolved. According to thisauthor, the Tadornini, which were previously includedinside the Anatinae by Del Hoyo et al. (1992), representan independent lineage. The definition of Anserinae byDel Hoyo et al. (1992) and Livezey (1997b) differs onlyby the inclusion, or not, ofDendrocygna inside this clade.The DNA–DNA hybridization results of Sibley andAhlquist (1990) give rise to an even different scheme withthree, main lineages (Fig. 1C and Appendix A): (i) the

Fig. 1. Schematic phylogenies of the Anseriformes according to (A) Del Hoyo et al. (1992); (B) Livezey (1997b); and (C) Sibley and Ahlquist (1990).

Only the 24 genera analyzed in our study plus Anseranas are depicted in these trees. The subfamilies Anatinae and Anserinae (or the tribes Anatini

and Anserini) defined by the various authors are indicated.

340 C. Donne-Gouss�ee et al. / Molecular Phylogenetics and Evolution 23 (2002) 339–356

Anatini, which has a composition different from that ofthe Anatidae of Del Hoyo et al. (1992) and Livezey(1997b); (ii) the Anserini, including Tadornini and Cai-rina; and (iii) the genera Cygnus and Coscoroba whichtogether form an independent grouping called Cygninae.

Given the strong level of discrepancy existing betweenthe three main published phylogenies at the family andsubfamily levels, it is not surprising that the relation-ships inside each subfamily are also a matter of con-tention (Fig. 1). As shown in Fig. 1A, Del Hoyo et al.(1992) found five main tribes in the Anatinae. Some ofthese tribes such as Mergini (Mergus, Bucephala, Mel-anitta, Somateria, and Clangula) were also found byLivezey (1997b). The tribe called Aythyini has differentcompositions for the two authors since Livezey (1997b)includes Marmaronetta in this clade, whereas Del Hoyoet al. (1992) consider it as more closely related to Anas.Other tribes such as those containing the genera Anas,Aix, or Cairina were completely different for the twoauthors. Concerning the Anserinae, Del Hoyo et al.(1992) cluster together Anser, Branta, Cygnus, and Co-scoroba with the exclusion of Cereopsis and Dendrocy-gna which form two independent lineages (Fig. 1A).Livezey (1997b) clusters Anser and Branta on one handand Cygnus and Coscoroba on the other hand. He alsoconsiders Cereopsis as an independent lineage (Fig. 1B).Using DNA–DNA hybridization, Sibley and Ahlquist(1990) cannot resolve the phylogeny at the generic level.

This complex situation calls for the completion of amolecular phylogeny of Anseriformes using DNA se-quence analysis. Several recent reports have proposedpartial schemes that were mainly focused on Anatinae oreven on the genus Anas. Using complete cytochrome b(cytb) and NADH dehydrogenase subunit 2 (ND2)genes, Johnson and Sorenson (1998, 1999) found thatAnas is not a monophyletic genus since it also containsspecies of the genera Lophonetta, Amazonetta, Specul-anas, and Tachyeres. Despite the use of extensive out-groups containing sequences of 11 genera of Anatidae,no information with regard to the grouping of these taxawas given. The analysis of a short segment of the cytbgene of Australasian waterfowl suggests some groupinginside the Anatidae despite the fact that, because of thesmall size of the sequenced fragment, the overall ro-bustness level of this analysis is weak (Sraml et al., 1996).These authors found that Anseranas and Dendrocygnawere two independent lineages diverging early from otherAnatidae. The Anatidae are then split into two mainlineages that correspond to Anatinae and Anserinae.Given the weak resolution of this analysis, within thesetwo subfamilies only two groups, namely Cairina andAix in Anatinae andCygnus andBranta in Anserinae, arerobustly found (Sraml et al., 1996). A more recent anal-ysis of a longer set of mitochondrial sequences containingclustered fragments of 12S, cytb, and tRNAs gave rise tothe same scheme with Anseranas and Dendrocygna di-

verging first and then a split between Anserinae (Anser,Branta, and Cygnus) and Anatinae (Sorenson et al.,1999a). Within Anatinae four main clades were found: (i)Anas and related genera; (ii) a clade containing Tador-nini (Tadorna and Chloephaga) allied with Cairina andAix (this group is then clustered with Mergini (Bucep-hala)); (iii) Aythyini (Aythya, Netta and Marmaronetta)with other genera such asCyanochen and Pteronetta; and(iv) Chenonetta and Sarkidiornis. Despite their interest inclearly indicating that a robust phylogenetic signal existsin mtDNA to resolve waterfowl phylogeny, most of thesemolecular analyses were based on a limited sampling ofspecies or only marginally discuss the relationships be-tween the various groups of Anseriformes. For thesereasons we sequenced and analyzed the mtDNA controlregion from 45 Anseriformes representing 24 genera. Oursampling includes representatives of all of the main tribesof Anseriformes and half of the existing genera, allowingus to propose a phylogeny of this order. In addition, wesequence the cytb and ND2 genes in a more limited set ofspecies to constitute, in addition to the sequencing doneby Johnson and Sorenson (1998), a data set containingthe mitochondrial control region (CR), cytb, and ND2for 18 species of the crown group Anatinae. This allowsus to compare the resolution and robustness of phylog-enies based on CR or protein-coding genes. This clearlyshows that the CR appears to be an efficient tool withwhich to decipher the phylogeny of Anseriformes, notonly at the species and genus levels but also at the familylevel. This analysis allowed us to test the various sce-narios proposed by other authors based on morpholog-ical or DNA–DNA hybridization analysis.

2. Materials and methods

2.1. Taxa examined

Investigation of 45 Anseriformes species from 24 gen-erawasdone.Cytochrome bandND2of 14 sequences andthe complete mitochondrial DNAof the redhead (Aythyaamericana) were obtained from GenBank whereas 4 cytband 4 ND2 sequences were determined (see Table 1;Johnson and Sorenson, 1998; Sorenson et al., 1998).The complete control region of the snow goose (Ansercaerulescens) was published by Quinn andWilson (1993).

2.2. DNA extraction

Feather samples were collected from live birds in thewild and in captivity in France at the ‘‘Parc de Cleres’’ ofMNHN, Zoological Museum of Lille, and the ‘‘ParcOrnithologiqueKerAnas’’ (Table 1).DNAwas extractedfrom feathers using themethod described by Taberlet andBouvet (1991). Feathers were digested in a total volume of400 ll of buffer (10mMTris–HCL, pH 8.0, 2mMEDTA,

C. Donne-Gouss�ee et al. / Molecular Phylogenetics and Evolution 23 (2002) 339–356 341

pH 8.0, 10mM NaCl, 1% sodium dodecyl sulfate, and0.4mg/ml proteinase K) by incubation with constantagitation at 42 �C for 1–3 h. Samples were extracted twiceto standard phenol/chloroform extraction and isopro-panol precipitation and dissolved in 100ml of distilledwater (H€aanni et al., 1995). For some samples, genomicDNA was also isolated with a QIAamp DNeasy Kit(QIAGEN) according to the manufacturer’s protocol.

2.3. Gene amplification and sequencing

The complete mitochondrial control region of eachspecies was amplified with the primers listed in Table 2.To complete the range of species available, the mito-chondrial cytb (1047 bp) and ND2 (1041 bp) genes wereamplified for four species, goldeneye (Bucephala clan-gula), Magellan goose (Chloephaga picta), black scoter

Table 1

Scientific name, common name, region of mtDNA sequenced, origin and accession number of the relevant sequences for the various samples studied

Species Common name Region of mtDNA sequenced Origin of sample Accession number

Aix galericulata Mandarin duck D loop Lille Museum AY112953

Alopochen aegyptiacus Egyptian goose D loop Ker Anas Park AY112964

Anas acuta Pintail D loop Cytba ND2a Ker Anas Park AY112939, AF059055, AF059116

Anas bahamensis

bahamensis

Bahama pintail D loop Cytba ND2a Lille Museum AY112940, AF059058, AF059119

Anas clypeata Nothern shoveler D loop Cytba ND2a Ker Anas Park AY112941, AF059062, AF059174

Anas crecca Eurasian greenwinged teal D loop Cytba ND2a Ker Anas Park AY112942, AF059064, AF059124

Anas platyrhynchos Mallard D loop Cytba ND2a Lille Museum AY112938, AF059081, AF059141

Anas sibilatrix Chilo�ee wigeon D loop Cytba ND2a MNHN AY112943, AF059108, AF059168

Anas strepera Gadwall D loop Cytba ND2a Ker Anas Park AY112944, AF059109, AF059169

Anser albifrons White-fronted goose D loop MNHN AY112967

Anser anser Greylag goose D loop MNHN AY112966

Anser caerulescens Snow goose D loopc ACMTTPGF

Anser erythropus Lesser white-fronted goose D loop MNHN AY112970

Anser indicus Bar-headed goose D loop Lille Museum AY112971

Anser rosii Ross goose D loop Ker Anas Park AY112972

Anser canagicus Emperor goose D loop MNHN AY112969

Aythya americana Redhead D loopb Cytbb ND2b NC000877

Aythya marila Greater saup D loop Lille Museum AY112947

Aythya nycora Ferruginous duck D loop Ker Anas Park AY112948

Branta bernicla Brent goose D loop Lille Museum AY112973

Branta canadensis Canada goose D loop Lille Museum AY112974

Branta leucopsis Barnacle goose D loop Ker Anas Park AY112975

Branta ruficollis Red-breasted goose D loop MNHN AY112976

Bucephala clangula Goldeneye D loop Cytb ND2 MNHN AY112959, AF515261, AF515265

Cairina moschata Muscovy duck D loop Cytba ND2a Toulouse (INRA) AY112952, AF059098, AF059158

Callonetta leucophrys Ringed teal D loop Cytba ND2a Lille Museum AY112960, AF059157, AF059097

Cereopsis

novaehollandiae

Cape barren goose D loop MNHN AY112977

Chauna torquata Crested screamer D loop MNHN AY112982

Chenonetta jubata Maned goose D loop Cytba ND2a Lille Museum AY112951, AF059100, AF059160

Chloephaga picta Magellan goose D loop Cytb ND2 Lille Museum AY112965, AF515262, AF515266

Clangula hyemalis Long-tailed duck D loop MNHN AY112954

Coscoroba coscoroba Coscoroba swan D loop MNHN AY112979

Cygnus atratus Black swan D loop Lille Museum AY112978

Dendrocygna bicolor Fulvous whistling duck D loop MNHN AY112980

Dendrocygna eytoni Eyton whistling duck D loop Lille Museum AY112981

Lophonetta

specularoides

Crested duck D loop Cytba ND2a MNHN AY112945, AF059102, AF059162

Marmaronetta

angustirostris

Marbled teal D loop Cytba ND2a Ker Anas Park AY112950, AF059104, AF059164

Melanitta nigra Black scoter D loop Cytb ND2 MNHN AY112961, AF515263, AF515267

Mergus albellus Smew D loop Ker Anas Park AY112957

Mergus cucullatus Hooded merganser D loop MNHN AY112958

Mergus serrator Red-breasted merganser D loop Lille Museum AY112956

Netta rufina Red-crested pochard D loop Ker Anas Park AY112949

Somateria mollissima Eider D loop Cytb ND2 MNHN AY112955, AF515264, AF515268

Tadorna tadorna Common shelduck D loop Cytba ND2a Ker Anas Park AY112962, AF059113, AF059173

Tadorna tadornoides Australian shelduck D loop Lille Museum AY112963

a Johnson and Sorenson (1998).b Sorenson et al. (1998).cQuinn and Wilson (1993).


(Melanitta nigra), and eider (Somateria mollissima), us-ing the primers listed in Table 2. PCR amplification wasmade in 50 ll total volume with 1 unit AmpliTaq DNAPolymerase (Sigma), the manufacturer’s buffer, 2mMMgCl2, 0.25mM each dNTP, 200 lg=ml bovine serumabumin, and 1 lM each primer. The PCR cycle was asfollows: denaturation at 94 �C for 1min, annealing at55 �C for 1.5min, and extension at 72 �C for 2min for 40cycles. Five-microliter aliquots of the amplificationproducts were electrophoresed in 2% agarose gels andvisualized via ethidium bromide staining. PCR productswere purified with QIAquick PCR Purification Kit.PCR products were cloned with TOPO cloning (Invi-trogen). Double-stranded PCR product was used incycle sequencing reactions using fluorescent dye termi-nators and AmpliTaq (Applied Biosystems). Reactionproducts were run on an ABI 373 automated DNAsequencer.

2.4. Authenticity of the sequences

To avoid contamination between sample extrac-tions, PCR amplifications and sequence analysis wereperformed in different dedicated rooms. When possi-

ble, complete cytb, ND2, or control region sequenceswere systematically compared with partial sequencesdetermined by other authors, available in GenBank.The presence of nuclear insertions of mtDNAsequences (called Numts) in our amplified sequences,which can lead to the wrong phylogeny (Quinn,1997; Sorenson and Quinn, 1998; Zhang and He-witt, 1996), has been tested using the following cri-teria. (i) All the sequences were sequenced directlyfrom the PCR product, cloned, and sequenced.All the clones exhibit the same sequences which areidentical to the direct sequence of the PCR product.This suggest that only one fragment was amplifiedfrom each sample. (ii) DNA was extracted fromfeather and not blood, a tissue known to be prone toamplification of Numts since it is poor in mtDNA(Arctander, 1995; Quinn, 1992; Sorenson andFleischer, 1996). (iii) The cytb and ND2 fragmentsthat we amplified are coding proteins of regularsize, indicating that no mutations disrupting thereading frame took place. (iv) The control regionsequences are relatively rich in transitions, a situation that is reminiscent of mtDNA, but not ofNumts.

Table 2

Primers used for amplification and sequencing of D loop, Cytb, and ND2

Namea Sequenceb Refc

D loop

L16722 50-ACTACCCGAGACCTACGGCT-30

H1254 50-TCTTGGCAGCTTCAGTGCCA-30

L128 50-CATGCACGGACTAAACCCAT-30

L481 50-CCCCCTAAACCCCTCGCCCT-30

L718 50-TAAGCCTGGACACACCTGCG-30

H738 50-CGCAGGTGTGTCCAGGCTTA-30

H501 50-AGGGCGAGGGGTTTAGGGGG-30

H319 50-TGAATGCTCTAATACCCAAC-30

Cytb

L14990 50-AACATCTCCGCATGATGAAA-30 1

H16064 50-CTTCGATTTTTGGTTTACAAGACC-30 1

L15191 50-ATCTGCATCTACCTACACATCGG-30 1

L15517 50-CACGAATCAGGCTCAAACAACC-30 1

L15710 50-CCMMCMCAYATCAARCCMGAATG-30 2

H15742 50-TGCTAGTACGCCTCCTAGTTTGTTTGGGATTGA-30 1

H15545 50-GTATGGGTGAAATGGAATTT-30 1

H15298 50-CCCTCAGAATGATATTTGTCCTCA-30 1

ND2

L5219 50-CCCATACCCCGAAAATGATG-30 1

H6313 50-CTCTTATTTAAGGCTTTGAAGGC-30 1

L5524 50-AGGCCTGGTCCCATTTCACT-30

L5758 50-GGCTGAATRGGMCTNAAYCARAC-30 1

L6022 50-CCAAAGTGACTCATCATCCA-30

H6031 50-CACTTTGGTATAAACCCTGT- 30

H5766 50-GGATGAGAAGGCTAGGATTTTKCG-30 1

H5544 50-AGTGAAATGGGACCAGGCCT-30

a L and H numbers designate the location of the 30 base in the light or heavy strand, respectively, of the published chicken mtDNA sequence

(Desjardins and Morais, 1990).bDegenerate primer positions are as follows: M¼A or C; Y¼C or T; R¼A or G; N¼A, C, T, or G; K¼G or T.c 1, Johnson and Sorenson (1998); 2, Sorenson et al. (1999b).


2.5. Phylogenetic analysis

Sequences were aligned by eye using SEAVIEW(Galtier et al., 1996) and CLUSTAL_W (Thompsonet al., 1994). All positions containing gaps were excludedfrom the analysis using either a pairwise or a globalremoval scheme (Hillis et al., 1996).

To estimate saturation, scatter plots that comparedpairwise percentage sequence divergence to pairwisetransversion (TV) and pairwise transition (TS) diver-gences were drawn. Saturation plots using Kimura’s(1980) two-parameter genetic distances were drawn.According to Hackett (1996) saturation is determined tohave occurred if the scatter of points shows a clearleveling off of changes as sequence divergence increases.

The aligned sequences were treated by distance(neighbor-joining (NJ); Saitou and Nei, 1987) andmaximum-parsimony (MP) analyses as implemented inPHYLO_WIN (Galtier et al., 1996) and PAUP (version3.1) (Swofford, 1993). We employed the random taxonaddition option to prevent the tree from getting stuck ina local optimum during the heuristic search. For dis-tance analysis we employed log-determinant (LogDet)distances that allow correction for heterogeneous basecomposition (Steel, 1993). The parsimony analysis wasassessed using the heuristic search method (characteroptimization ACCTRAN, MULPARS, and TBRbranch-swapping options) with gaps treated as a fifthbase and all uninformative positions excluded. We em-ployed the random taxon addition option to prevent thetree from getting stuck in a local optimum during theheuristic search. Parsimony analyses weighted all char-acters equally. ‘‘Classical’’ maximum-likelihood (ML)analysis, as implemented in DNAML of PHYLIP forexample, excludes all positions with gaps and is thusvery lowly resolved, as are the distance and parsimonyanalyses excluding these positions. Maximum-likelihoodestimation was thus performed using quartet puzzling(Strimmer and Von Haeseler, 1996) as implemented inTREE-PUZZLE which allows pairwise gap removal.The robustness of inferences was assessed throughbootstrap resampling (BP) (Felsenstein, 1985) with thedistance (1000 replicates) and parsimony (100 replicates)with one random addition per replicate.

3. Results

3.1. Control region sequence analysis

The Anseriformes control region has many avianfeatures that have been reported in other birds (Des-jardins and Morais, 1990). Typically, the vertebrate CRis subdivided into three domains (domain I, centraldomain, and domain II), characterized primarily bydifferent structural features (e.g., conserved sequence

blocks; CSBs). Following this division, on our align-ment, domain I runs from the 50 end of the CR lightstrand to position 470, the central domain runs fromposition 471 to 1050, and domain IIs runs from position1051 to the 30 end of the CR (Fig. 2). The conservedstructural features are clearly evident on our alignment(1424 bp). Within the control region, four CSBs wereclearly identified (Fig. 2): F-, D-, and C-boxes are lo-cated in the central domain, and CSB-1 is located indomain II (Baker and Marshall, 1997).

The complete sequences of the control regions ofAnseriformes range in size from 970 bp in the manedgoose (Chenonetta jubata) to 1230 bp in the crestedscreamer (Chauna torquata), with an average size of1100 bp. Within Anatidae, there is a deletion of ca. 100–130 bp in Anatinae (Aix, Anas, Alopochen, Aythya, Bu-cephala, Cairina, Callonetta, Chenonetta, Chloephaga,Clangula, Lophonetta, Marmaronetta, Melanitta, Mer-gus, Netta, Somateria, Tadorna) compared to othergroups of Anserinae (Anser, Branta, Cereopsis, Cosco-roba, Cygnus, and the divergent genera Chauna andDendrocygna). Other small (1–20 bp) deletions in do-main I are also present in Anatinae versus other species(Fig. 2). Quinn and Wilson (1993) also reported rela-tively large deletions in both 50 (61 bp) and 30 (38 bp)regions of the lesser snow goose compared to the do-mestic chicken. This has been confirmed also by Ra-mirez et al. (1993), who reported large deletions in bothadjacent regions in the Peking duck versus the domesticchicken. The average sequence divergences between se-quences in different genera used in this study in domainI, central region, and domain II are 25%, 9%, and 22%,respectively.

The complete alignment of the control region se-quences of the 45 Anseriformes used in this study gaverise to an alignment containing 1424 sites, among whichare 1143 variable sites, 836 sites containing at least onegap, and 937 sites that are informative for parsimonywhen all events, transitions, transversions, and gaps areconsidered. When all positions with gaps are deletedfrom the analysis, 201 parsimony-informative charactersremain. The mean frequency of nucleotides in thecompared sequences show a paucity of G compared tothe other bases (28% A, 31.2% C, 15.4% G, and 25.5%T) this compostion does not vary among the 45 se-quenced species but it varies among regions of the Dloop. Domain I is rich in A and C, the central domain isrich in C and T, and domain II is AT rich and very lowin G as observed for other birds (Baker and Marshall,1997). The average TS/TV ratio is 1.1. Characters werethus equally weighted for the parsimony analysis.

Two data sets were used in the phylogenetic analysis:(i) a data set containing the complete control regionsequence of the 45 sequenced species and (ii) a reduceddata set containing only 1 sequence for each genus, i.e.,only 24 sequences. This last data set contains 1424 sites


of which 1114 are variable and 790 informative forparsimony when all events are considered. To testwhether a robust phylogenetic signal was present in thisdata set, we recorded g1 statistic values after con-structing 10 independent sets of 1000 random trees usingPAUP. We obtained g1 statistics �1:0502772�0:0463358 for the complete data set of 45 species and of�0:5362543 � 0:0982904 for the reduced data set. Bothvalues are robust according to Hillis and Huelsenbeck(1992), suggesting that the control region of Anserifor-mes contains some structured signal. To investigatesaturation we used the method developed by Hackett(1996) (see Section 2). We obtained a linear increase ofboth transitions and transversions as sequence diver-gence increases (Fig. 3). We thus conclude that there isno evidence of saturation in our data set, an observationthat is in accordance with the range of sequence diver-gence that we observed (ca. 30% at most). There are no

significant rate differences among our sequences(P > 0:05), which suggests that long-branch attraction isnot a problem in this data set. We notably comparedsuspicious groupings by relative-rate tests (Robinson-Rechavi and Huchon, 2000). Taken together, all thesedata indicate the existence of a phylogenetic signal evenfor the profound dichotomies in the tree and clearlysupport the use of the control region for Anseriformesphylogeny.

3.2. Phylogenetic reconstruction using the control regionsequences

The pairwise deletion scheme results in a much betterresolved tree of the reduced data set (compare Figs. 4 Aand B). For example, in the tree constructed after globalgap removal, the clustering of Melanitta, Callonetta,Bucephala, Mergus, Somateria, and Clangula that weobserved in the pairwise deletion scheme supported by66% bootstrap values is not found. We also found thatthe resolution of the tree containing the whole controlregion is much better than any isolated domain orcombination of domains (not shown). For all otheranalyses we thus used the complete control region with apairwise deletion scheme when applicable. For neigh-bor-joining, with corrections for multiple substitutions,we observed very little influence on topology or ro-bustness (not shown). We used the LogDet distance forall subsequent distance analyses.

The overall topology of the tree is identical for NJ(Fig. 4B), MP (Fig. 4C), and ML (Fig. 4D) analyses. Wefound Chauna torquata and Dendrocygna bicolor at ba-sal positions in both types of analysis. This basalplacement was confirmed by the rooting of the tree withoutgroup sequences of other bird orders (Galliformes,Gruiformes, and Passeriformes). In a tree based on thecentral region only, due to sequence divergence of the

Fig. 3. Analysis of the saturation present in the 45-species data set. For

each pair of species the number of observed differences in the distance

matrix was plotted against the number of inferred substitutions that is

given by the patristic distance after a parsimony analysis. The upper

points separated from the main plot correspond to the comparison of

Anserinae with Chauna torquata, whereas the lower points corresponds

to the comparison of Anatinae with Chauna torquata.

Fig. 2. Structure of the mitochondrial DNA replication control region in three representative species of Anseriformes used in our study: an anatine,

the mallard Anas platyrhynchos; an anserine, the common goose Anser anser; and an animid, the screamer Chauna torquata. The tRNA Glu and Phe

that surround the control region are indicated. The three domains discussed in the text are differentially shaded with the central conserved domain

depicted with a darker shading. The various conserved sequence blocks, F-, D-, and C-boxes, and the CSB-1 are indicated as small boxes. The

numbering system refers to the alignment of the 45 species. The gaps that are present in domain I and domain II are discussed in the text and shown

as small slashed boxes. Sizes of the CR sequence for the three regions are indicated below each species name.


Fig. 4. Phylogenetic reconstruction of the relationships among the various genera of waterfowl from the reduced data set containing CR sequences

from only 24 species. (A) Distance analysis calculated with the neighbor-joining method using a LogDet distance and a global gap removal option. A

total of 630 sites remain in this analysis; 1000 bootstrap replicates were calculated. (B) Neighbor-joining analysis using a LogDet distance and a

pairwise gap removal option (Hillis et al., 1996). A total of 988 sites remain in this analysis; 1000 bootstrap replicates were calculated. (C) Maximum-

parsimony analysis calculated using PAUP with a pairwise gap removal option. A total of 1100 sites remains; only 100 bootstrap replicates were

performed and the bootstrap tree is shown. Two equally parsimonious trees were obtained (tree length 4259). The tribes discussed in the text and the

two subfamilies Anatinae and Anserinae are indicated when they are supported by bootstrap values above 50%. Only bootstrap values above 50% are

indicated. (D) Maximum-likelihood estimation performed using quartet puzzling as implemented in TREE-PUZZLE. The numbers on each branch

indicate quartet puzzling support values. Unresolved branches according to likelihood criteria were polytomized.


domains I and II, we consistently found C. torquatabasal to all the Anseriformes. In all subsequent analysesChauna was thus used as the outgroup sequence. It isthus clear from these results that D. bicolor does notbelong to the Anserinae as proposed by Del Hoyo et al.(1992). Most species split into two groups, which cor-respond to the Anatinae and Anserinae subfamilies.

Within Anserinae all trees also give congruent androbust results, with two groups of species: the geese(Anser and Branta) in one group and the swans andCape barren goose (Cygnus, Coscoroba, and Cereopsis)in the other. Within this group Coscoroba coscoroba andCereopsis novae-hollandiae are sister species.

The situation is more complex for the larger Anatinaesubfamily. Groups found in all types of analysis includeAnas+Lophonetta, Aix+Cairina, Bucephala+Mergus,Somateria+Clangula, and Alopochen+Tadorna+Chlo-ephaga. Melanitta and Callonetta group with highbootstrap support in MP. The grouping of Cairina andAix with the Anas, Lophonetta, Netta, Aythya, Chenon-

etta, and Marmaronetta group is strongly supportedwith MP (94% bootstrap), although weakly with NJ(BP < 50%) and not at all in ML. Tadorna and relatedspecies appear as a basal offshoot of Anatinae, withstrong support in MP (99%) but low in NJ, and is notfound in ML.

The analysis of the complete data set by NJ (Fig. 5A)or MP (Fig. 5B) gives essentially the same results, sug-gesting a very weak influence of species sampling for thisphylogeny. The NJ and MP trees again found Chaunaand then the two Dendrocygna as basal species and thenthe Anatinae/Anserinae split. Within Anserinae, thevarious Anser are clearly monophyletic and closely re-lated to Branta. In both analyses Coscoroba and Cere-opsis cluster together, suggesting that, in this group,Cygnus diverged first. Within Anatinae both analysesfound the tribes Anatini (Anas and Lophonetta), Ay-thyini (Aythya and Netta), Cairinini (Cairina and Aix),and Mergini (Mergus, Bucephala, Melanitta, Callonetta,Somateria, and Clangula). The topology inside the

Fig. 5. Phylogenetic reconstruction of the relationships among the 45 studied species of waterfowl from the complete data set of CR sequences. (A)

Distance analysis calculated with the neighbor-joining method using a LogDet distance and a pairwise gap removal option. A total of 997 sites

remain in this analysis; 1000 bootstrap replicates were calculated. (B) Maximum-parsimony analysis calculated using PAUP with a pairwise gap

removal option. A total of 1218 sites remains; only 100 bootstrap replicates were performed. The tribes discussed in the text and the two subfamilies

Anatinae and Anserinae are indicated when they are supported by bootstrap values above 50%. Only bootstrap values above 50% are indicated.


Mergini is different in NJ and MP and this tribe was notfound in the MP analyses of the reduced data set (seeFig. 4C) but is observed in the ML analyses (Fig. 4D).Both MP and NJ analyses also found Lophonetta insidethe Anas genus, an observation that was already madeby Johnson and Sorenson (1998) using the ND2 andcytb genes. The tribe Tadornini is found in NJ with 82%support, but not in MP. Yet, this tribe was robustlyfound in the MP analysis of the reduced data set.

Taken together these results suggest the existence offive tribes in the Anserinae (Fig. 6): (i) Anatini and (ii)Aythyini which are linked; (iii) Cairinini which forms amonophyletic group with Anatini, Aythyini, Chenonetta,

and Marmaronetta; (iv) Mergini with six genera; and (v)Tadornini which is the first to diverge inside theAnatinae.

3.3. Comparison of control region-based phylogeny withND2 and cytb data

Since ND2 and cytb, two protein-coding mito-chondrial genes, were sequenced and analyzed in 18Anatinae species belonging to the main tribes (Johnsonand Sorenson, 1998) we compared the topologies foundusing these two genes with those found using the controlregion (Fig. 7). We also analyzed a data set containingcytb, ND2, and the control region together. The NJ treebased on control region sequences for the 18 speciesfound the same clustering as the 24- or 45-species datasets (compare Fig. 7A with Figs. 4B and 5A, respec-tively), with the tribes Anatini and Mergini well sup-ported and Tadornini recovered with less than 50% BP.

The alignment of the two protein-coding genes (Cytb/ND2) comprised a total of 2103 sites, of which 790 werevariable and 623 phylogenetically informative for par-simony. Since Johnson and Sorenson (1998) show thatthere are no differences between cytb and ND2 withrespect to their phylogenetical signal, we combinedthem. Comparing TS and TV, and first and second co-don position versus the three positions, we found thatthe most robust result was found using the three codonpositions and all differences (not shown), by NJ withLogDet distance (Fig. 7B), or by maximum-parsimony(not shown). The topologies of the trees that we ob-tained with cytb and ND2 are comparable with thosedescribed by Sorenson et al. (1999a) using a largenumber of sequences. In both NJ and MP analyses, theresolution power of these two genes appears very weakcompared to that of the control region. The onlygrouping found using NJ or MP was that of Anas withLophonetta (Anatini) and Bucephala with Melanitta,which is not observed using the control region. In theMP tree we also noticed the grouping of Marmaronettawith Aythya, which is not found in the control region.From these data it appears that the control region is amuch better marker with which to trace back phyloge-netic relationships among Anatinae than the protein-coding cytb and ND2 genes.

Of note, the combined analysis of cytb, ND2, and CRdoes not improve (and even appears to decrease) theresolutive power of CR alone (Fig. 7C). This analysisagain recovers the Anatini, but the Mergini are notsupported when the three genes are used together.Ironically, Aythya and Marmaronetta on the one handand Melanitta and Bucephala on the other hand arefound together as for cytb/ND2 alone. We thus concludefrom these data that the analysis of the control regionwhich contains only ca.1400 bp alone is a better strategywith which to resolve Anseriformes phylogeny than theanalysis of cytb and ND2 which contains ca. 2103 bp.

Fig. 6. Schematic phylogeny of the Anseriformes that summarizes the

main conclusions of our study. For each branch, bootstrap values

found in the complete data set are indicated. The values found by the

distance analysis (Fig. 5A) are indicated above the branch, whereas

those found by MP (Fig. 5B) are indicated below. The star for the

value (55) found by MP in the branch connecting the three genera of

the Tadornini indicates that this value was found only by the study of

the reduced data set (Fig. 4C). The value of the corresponding branch

for the complete data set is below 50%. Branches that are unstable and/

or for which all bootstrap values are below 50% are collapsed. The

various tribes and subfamilies are indicated by brackets.


Fig. 7. Comparison of the resolutive power of the mtDNA control region (CR) and two mitochondrial proteins-coding genes ND2 and cytb. A data

set of 18 species of Anatinae for which both CR and cytb/ND2 sequences were available has been studied. In all cases the analysis was performed

using the neighbor-joining method using a LogDet distance and a pairwise gap removal option. To assess the robustness of the branches 1000

bootstrap replicates were calculated. (A) Tree obtained with the CR sequences. A total of 990 sites remain in this analysis. (B) Tree obtained with the

ND2/Cytb sequences, A total of 2103 sites remain in this analysis. (C) Tree obtained with the ND2/Cytb associated with the CR sequences. A total of

3100 sites remain in this analysis. The tribes discussed in the text and the two subfamilies Anatinae and Anserinae are indicated when they are

supported by bootstrap values above 50%. Only bootstrap values above 50% are indicated.


4. Discussion

4.1. The control region as a phylogenetic marker for birdphylogeny

The control region has been classically divided intothree subregions: domain I in 50, domain II in 30, and acentral domain. These regions differ in their base com-position and in rate and mode of evolution (Baker andMarshall, 1997; Lee et al., 1995). Due to its relatively fastrate of evolution, the CR has been typically found to bemore appropriate for intraspecific studies, especially inmammals (see Quinn (1997) for a review). It is less wellappreciated that it can also resolve phylogenetic rela-tionships at much deeper levels. Nevertheless, severalrecent studies have highlighted its potential in recoveringphylogeny at the family level (see Douzery and Randi(1997) and Saunders and Edwards (2000) for specificexamples in Cervidae and Corvidae, respectively). Themain structural and evolutionary features of the controlregion of Anseriformes, such as the division into threeregions with different base composition, the variableamounts of gaps in these regions, the structure of theconserved blocks, the respective amounts of transitionsand transversions, or the average intrageneric diver-gence, are similar to those described for other birds suchas Corvidae (Saunders and Edwards, 2000) and othergroups (Baker and Marshall, 1997). Indeed, the CR hasbeen recently demonstrated to be very efficient in re-covering the phylogeny of New World jays (Saundersand Edwards, 2000). The comparison of the dynamics ofCR and cytb made by these authors has revealed thatsaturation of transitions is less of a problem in the CRdata than in the third codon positions of cytb.

In accordance with these recent studies, our resultsclearly show that the control region is a useful tool withwhich to construct a robust phylogeny even at a rela-tively deep level, such as families, in Anseriformes. Weobserved that the trees obtained using the control regioneither by the NJ or by the MP methods are consistentlymore stable (i.e., less variable when the sampling or thetree reconstruction methods are changed), more re-solved (i.e., fewer nodes with bootstrap values below50%, irrespective of the sampling or method used), andmore robust (i.e., the resolved nodes are supported byhigher bootstrap values) than the trees constructed usingprotein-coding genes such as ND2 or cytb. As discussedabove, the phylogeny that we obtained with the CR datais reasonable given the debated issues with regard toAnseriformes phylogeny. Even if a detailed comparisonis still impossible given the large difference of taxonomicsampling between the CR and the cytb/ND2 data sets, itseems that well-resolved nodes in both phylogenies arein agreement, suggesting that no obvious conflict existsbetween the two types of data. These points support theidea that fast-evolving DNA sequences such as those of

the CR may be valuable in relatively deep phylogenyreconstruction. This is true even with divergences ashigh as 20%, as long as alignment is satisfactory, whichis the case for Anseriformes. Thus, the CR, althoughshorter than the association of cytb and ND2, appearsas a promising tool for future phylogenetic studies.

Recent studies suggest that increased taxonomicsampling may improve recovery of higher-level trees,although the importance of increased taxon sampling isdebated (Graybeal, 1998; Lecointre et al., 1993; Poe andSwofford, 1999). Our results suggest that the resolutionof phylogeny is effectively better when more samples areincluded, since the bootstrap value are improved, par-ticularly at deeper nodes in the tree (compare Fig. 5 withFig. 4). Although this remains to be systematically tes-ted in the case of Anseriformes, it suggests that taxonsampling has a much more visible affect on phylogeneticresults than, for example, the type of distance correctionused in NJ analysis. Again this confirms the analysisdone on New World jays that suggests that the partic-ular weighting scheme used has a much more modestimpact on tree robustness than taxon sampling (Saun-ders and Edwards, 2000). Our data set also confirmsthat increasing sequence size increases resolution sincethe use of the three domains gives rise to better-resolvedtrees than the separate use of each domain.

4.2. Chauna and Dendrocygna as three early diverginggenera

All the tree topologies based on CR, irrespective ofthe sampling or the method used, place C. torquata at thebasal position of Anseriformes. The position of thescreamers as an early offshoot within Anseriformes hasbeen recognized widely by morphological studies (DelHoyo et al., 1992; Livezey, 1997b; Sibley and Ahlquist,1990) and has lead to comparison with other avian or-ders in attempts to discover the origin of Anseriformes(Olson and Feduccia, 1980). It is generally believed thatthere are enough synapomorphies for screamers to bedesignated a distinct family, comprising three exclusivelySouth American species. Another molecular analysis hasalso confirmed this basal placement (Sraml et al., 1996).

The whistling ducks (Dendrocygna) diverged more re-cently from the main lineage and represent one of themost distinctive genera of the Anatidae. Several mor-phological features such as erect posture, relativelyelongated necks and legs, and conspicuous perching treehabits distinguish them from most other waterfowl(Delacour, 1954). Our molecular results corroboratemorphological phylogenies, suggesting that this groupdiverged from other Anatidae earlier than the Anatinae/Anserinae split (Livezey, 1997b;Madsen et al., 1988). Theseparation of Dendrocygna from Anserinae is also con-sistent with an early divergence of the whistling ducksbased on allozyme data (Numachi et al., 1983), DNA–


DNA hybridization (Sibley et al., 1988; Sibley and Ahl-quist, 1990), and analysis of concatenated mtDNA frag-ments from three different genes (Sorenson et al., 1999a).

4.3. Two subfamilies: Anserinae and Anatinae

Within Anatidae, our analysis supports the conven-tional division between Anatinae (Anas, Lophonetta,Netta, Aythya, Chenonetta, Marmaronetta, Aix, Cairina,Melanitta, Callonetta, Bucephala, Mergus, Somateria,Ciangula, Alopochen, Tadorna, and Chloephaga) andAnserinae (Anser, Branta, Cereopsis, Coscoroba, andCygnus). This basal dichotomy, is on the one handstrongly supported in all of our analyses with highbootstrap values and on the other hand confirmed byseveral insertion/deletion events. For example, weobserved a large deletion on the CR sequence of ca. 100–130 bp in Anatinae compared to Anserinae. This di-chotomy between Anserinae and Anatinae was alsoobserved by other molecular studies (Sorenson et al.,1999a), but based on a relatively small set of Anserinae.

Our results strongly favor the definition of Anserinaegiven by Livezey (1986), with Anserinae paraphyletic tothe rest of the family, in contrast to the monophylysuggested by Delacour and Mayr (1945). The majorityof the convergences of this group are associated withadaptations for diving (see Fig. 1B). Indeed, in all othermorphological analyses Anserinae either containsDendrocygna (Delacour and Mayr, 1945; see Fig. 1A) oris totally different (Sibley and Ahlquist, 1990).

4.4. Relationships within Anserinae: The problem ofCygnus, Coscoroba, and Cereopsis

In all of our analyses Cygnus diverged first, and C.Coscoroba and C. novae-hollandiae are sister species,whereas traditionally Coscoroba and Cygnus are con-sidered sister species (Del Hoyo et al., 1992; Livezey,1997b; Sibley and Ahlquist, 1990). The unique species ofthe genus Cereopsis, the Cape barren goose (C. novae-hottandiae), is an Australian endemic goose of disputedaffinities. It was formerly considered an aberrant shel-duck and thus included in the tribe Tadornini (Delacourand Mayr, 1945). It is now more commonly regarded asdistantly related to the swans and true geese: it is oc-casionally included in the tribe Anserini, but more oftenseparated in its own tribe, Cereopsini (Del Hoyo et al.,1992; Livezey, 1997b). This species has never been in-cluded in molecular analyses. The position that we ob-serve, closely related to C. coscoroba, was neverobserved previously. However, Livezey (1997b) men-tions an unpublished phylogeny of Anseriformes byHarshman, which places Coscoroba and Cereopsis assister genera as in our CR-based trees. This cluster is inaccordance with the geographical origin of these speciessince C. Coscoroba and C. novae-hollandiae come from

the Southern Hemisphere, and Cygnus comes from theNorthern Hemisphere. It would be interesting to study alarger sample of species from Cygnus to confirm thisposition, notably to test the monophyly of Cygnus.

The position of Coscoroba has also been much dis-puted. Johnsgard (1978) used behavioral characteristicsto place this species in the Anserini tribe (geese andswans). In an extensive morphological study, Livezey(1986) found only 6 characters of 120 studied, supportinga sister group relationship between Coscoroba andswans, but his topology of Cygnus remains unresolved.More recently, the complete mitochondrial srRNA genewas shown to support the branching of Coscoroba priorto the divergence of geese and swans or, depending onthe method used, the association with Cygnus (Zimmeret al., 1994). These authors discuss the relatively closebranching times among Coscoroba, swans, and geese.Our data allow a firm resolution of the branching ordersamongCoscoroba andCygnus, since in all cases we foundCygnus splitting out first and then the clade Cereopsisand Coscoroba. Nevertheless, the short length of thebranch connecting Cygnus, Coscoroba, and Cereopsisand the low bootstrap value of this branch in parsimonysuggest that the Cygnus lineage diverged rapidly after thesplit between geese and Cygnus/Coscoroba/Cereopsis.

4.5. Five main clades inside Anatinae

Within the Anatinae, we found five consistent cladeswhen all types of analyses and/or data sets are consid-ered (Fig. 6): (i) Anatini (Anas and Lophonetta) and (ii)Aythyini (Aythya and Netta), which form a larger clade;(iii) Cairinini (Cairina and Aix) +Anatini +Aythy-ini +Chenonetta+Marmaronetta; (iv) Mergini, with sixgenera (Mergus, Bucephala, Melanitta, Callonetta, So-materia, and Clangula); and (v) Tadornini (Tadorna,Alopochen, and Chloephaga), which is the first to splitfrom the basal Anatinae lineage.

Phylogenetic relationships of the tribe Anatini (dab-bling ducks) remain controversial despite intensive study(Johnson and Sorenson, 1998, 1999; Livezey, 1991).Livezey recognized the tribe Anatini in which he in-cluded all of the dabbling ducks and many of theperching ducks (Anas, Lophonetta, Cairina, Aix, Cal-lonetta, and Chenonetta; see Fig. 1B). He classified thegenus Anas and a few other closely related genera(Amazonetta, Callonetta, Lophonetta, Speculanas, andMareca) in the subtribe Anateae. Other authors eitherdid not resolve the distribution of Anatinae into tribes(DNA–DNA hybridizations; Sibley and Ahlquist, 1990)or found Anas allied to Lophonetta and Marmaronettain the tribe Anatini (Del Hoyo et al., 1992). Our mo-lecular phylogenies do not support any of these views,but are in accordance with a recent detailed phylogenyof dabbling ducks based on ND2 and cytb (Johnson andSorenson, 1999; Sorenson et al., 1999a). We found that


Lophonetta is closely related to (parsimony; see Fig. 5B)or even located inside (NJ analysis, 79% bootstrap; seeFig. 5A) the Anas genus. In the study of Johnson andSorenson (1999) using ND2 and cytb the position ofLophonetta related to Anas is not robustly resolved. Inour trees using ND2 and cytb (see Fig. 7) based on amore limited number of species we found that Lophon-etta is included within Anas with a relatively low boot-strap support. The close relationship betweenLophonetta and Anas was also found in morphologicalanalysis since in some works the crested duck Lophon-etta specularoides is called Anas specularoides. Cairina,Aix, and Callonetta are clearly excluded from the Ana-tini in all our trees. The case of Marmaronetta andChenonetta is less clear since the position of these speciesremains unresolved. It is clear that both genera are re-lated to Anatini, Aythyini, and Cairinini but their pre-cise affiliation remain unknown. We thus cannotformally reject the definition of Anatini proposed by DelHoyo et al. (1992) (Anas, Lophonetta, and Marmaron-etta) although we find no statistical support for it.

Relationships within the genus Anas are rather intri-cate, as some species have very wide geographical rangesand occur in a number of strains such as the mallard(Anas platyrhynchos). Molecular phylogeny divides thedabbling ducks into several groups that are stronglysupported (Johnson and Sorenson, 1999). The pintails(Anas bahamensis/Anas acuta), the wigeons (Anas stre-pera/Anas sibilatrix), and the mallard (A. platyrhynchos)represent the major clade of Anatini. The remainingspecies, green-winged teals (Anas crecca) and blue-win-ged ducks (Anas clypeata), are unresolved in the tribeAnatini. Our analyses based on CR and on cytb/ND2also found that A. acuta grouped with A. bahamensisand that A. sibilatrix grouped with A. strepera. Thepositions of the other studied species are less clear,whereas we consistently found A. crecca and A. clypeataas sister species, an association which is not resolvedusing cytb/ND2 (Johnson and Sorenson, 1999). Thisagain highlights the strong resolutive power of the CRwhen compared with the protein-coding genes.

The second tribe that we recover is Aythyini, withAythya and Netta. Del Hoyo et al. (1992) divided themodern pochards (Aythyini) into these two genera,whereas Livezey (1996), by the analysis of skeleton,trachea, natal plumage, and definitive integument,placed Marmaronetta inside this tribe, a suggestion firstmade by Johnsgard (1961). Our molecular analysis is inaccordance with the association of Netta and Aythya butwe found no support for the inclusion of Marmaronettain this tribe since the position of this species remainunresolved in our analysis. It will be probably importantto sample other species closely related to Aythya, Netta,and Marmaronetta to correctly resolve this issue.

The third tribe, Cairinini, grouping Aix and Cairina,forms a large clade with Anatini, Aythyini, Marmaron-

etta, and Chenonetta. According to Del Hoyo et al.(1992), Aix and Cairina are clustered with Chenonettaand Callonetta since these birds have more characteris-tics in common with each other than they have with themembers of any other tribe, particularly in the aspects ofgeneral behavior and breeding biology. This group has acosmopolitan distribution and is most closely related tothe dabbling ducks (Delacour and Mayr, 1945; DelHoyo et al., 1992). Livezey (1997b) includes Aix andCairina in the Anatini but proposes a subtribe, Cairin-ina, clustering these two species together on the basis ofa single osteological synapomorphy. Our molecular re-sults supported this view since we found that the twogenera always grouped with high bootstrap support, andthis tribe grouped with Anatini and Aythyini. Othermolecular analyses based on three concatenated shortmtDNA fragments confirmed the close association be-tween Aix and Cairina (Sorenson et al., 1999a; Sramlet al., 1996) but, in contradiction with most morpho-logical studies, found this group related to Tadorniniand Mergini with low bootstrap support (52 and 54%;Sorenson et al., 1999a). Our data are in accordance withmorphological data, although the relatively low boot-strap support suggests that a more thorough analysis,including a more complete sampling, may be needed toconfirm or exclude this proposal.

The fourth clade comprises the Mergini (Mergus,Bucephala, Melanitta, Clangula, Somateria, and Cal-lonetta). Traditionally, Callonetta, which contains aunique species, Callonetta leucophrys, is associated withthe Anatini (Livezey, 1997b; Sibley and Ahlquist, 1990)or the Cairinini (Del Hoyo et al., 1992; Johnsgard, 1978)but this species has never been ascribed to the tribeMergini and is closely related to Melanitta, as suggestedin our strongly supported MP analysis. The unambigu-ous position of Callonetta within Mergini in our CRphylogeny was confirmed in the cytb/ND2 tree, since wefound Callonetta associated with either Melanitta orBucephala, and is found using distance, MP, and MLanalyses (Fig. 4). The position of this species in an in-dependent study (Johnson and Sorenson, 1999) clearlyexcludes the placement that we observed in the CR treeas the result of a misidentification or a contamination.The phylogenetic relationships of the remaining speciesof modern sea ducks (Mergini) based on control regionsequence confirmed the previously reported compositionof the group (Del Hoyo et al., 1992; Livezey, 1997b).The sawbills, Mergus, is monophyletic and despite theirmarkedly different external appearance, they seem to beclosest to the goldeneyes (Bucephala; Johnsgard, 1978;Livezey, 1995). The eiders (Somateria) are sometimesseparated from the rest of the sea ducks in their owntribe Somateirini, (Delacour, 1959; Cramp and Sim-mons, 1977). More recently, Livezey (1995) presented aphylogenetic analysis of modern Mergini using charac-ters of the skeleton, trachea, and natal and definitive


plumage. On that analysis, Somateria is monophyleticand constitutes the sister group of all other sea ducks ina subtribe Somaterina. Our analysis clearly suggests thatSomateria is close to Clangula and that both generaform an early offshoot inside the Mergini.

Tadornini contains Tadorna, Alopochen, and Chlo-ephaga and is the sister group of all other Anatinaetribes with moderate support in our study (51–72%bootstrap). The monophyly of the tribe itself is bettersupported (from 55 to 89% bootstrap). Our phyloge-netic relationships inferred in the molecular analysis ofthe CR agree with most recent classifications, separat-ing the sheldgeese (Chloephaga and Alopochen) andshelducks (Tadorna) (Livezey, 1997a). The southernhemisphere shelgeese are considered ‘‘intermediate’’between Anserinae and Anatinae in anatomy and be-havior by Delacour and Mayr (1945) and Livezey(1986), Alopochen had been clearly separated fromTadorna by the allozyme study of Numachi et al.(1983) and associated with the Anserini on behavioralgrounds by Johnsgard (1961). Nevertheless, the twomain morphological classifications depicted Fig. 1consider Tadorna, Chloephaga, and Alopochen to forma monophyletic group, in accordance with our molec-ular analysis. The case of the other genera of theTadornini tribe such as Cyanochen, which we have notstudied, is probably more problematic (see Sorensonet al., 1999a). In our phylogeny we cannot resolvecorrectly the trichotomy among Tadorna, Alopochen,and Chloephaga, which suggests that the three generaoriginated from a rapid cladogenesis event.

The detailed analysis of the relationships among the24 studied genera of Anseriformes inferred from ouranalysis of the mtDNA control region supports a phy-

logeny which is reasonably congruent with previousmorphological analysis. This suggests that analysis ofthe remaining species with the same method and usingother genes, including nuclear genes, will probablycontribute to further clarify the relationships inside thisgroup. It is interesting to note that specific problems ofrelationships between living species of Anseriformes canalso benefit from the study of extinct species using an-cient DNA analysis. This kind of analysis has alreadyproven to be useful in the study of the moa-nalos fromHawaii (Sorenson et al., 1999a) and will probably befruitful for other extinct taxa.

Acknowledgments

We are grateful to Michel Saint Jalme, PatrickRambaud, Yves Gaumetou, and G�eerard Guy for help incollecting the specimens used in this study and toAur�eelie Th�eenot for invaluable technical help. We thankC�eecile Mourer-Chauvire Marc Robinson-Rechavi andLudovic Orlando for critical reading of the manuscriptand two anonymous reviewers for helpful comments.We warmly appreciate the implication of the grand-mothers for babysitting during the redaction of themanuscript. We thank CNRS, MENRT, UCBL, IBL,and ENS-Lyon for financial support.

Appendix A

Different Taxonomic Arrangements According to (A)Del Hoyo et al. (1992), (B) Livezey (1997b), and (C)Sibley and Ahlquist (1990)

(A)Suborder Anhimae

Family Anhimidae Anhima, ChaunaSuborder Anseres

Family Anatidae

Subfamily Anseranatinae AnseranasSubfamily Anserinae

Tribe Dendrocygnini Dendrocygna, ThalassornisTribe Anserini Branta, Anser, Cygnus, CoscorobaTribe Cereopsini CereopsisTribe Stictonettini Stictonetta

Subfamily Anatinae

Tribe Tadornini Cyanochen, Chloephaga, Alopochen, Neochen, TadornaTribe Tachyerini TackyeresTribe Cairinini Sarkidiornis, Pteronetta, Cairina, Plectropterus, Nettapus, Callonetta,

Amazonetta, Chenonetta, AixTribe Merganettini MerganettaTribe Anatini Anas, Lophonetta, Hyemenolaimus, Malacorhynchus, MarmaronettaTribe Aythyini Netta, AythyaTribe Mergini Somateria, Polysticta, Melanitta, Histrionicus, Clangula, Bucephala, MergusTribe Oxyurini Oxyura, Biziura, Heteronetta


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(B)Suboder Anhimae

Family Anhimidae Anhima, ChaunaSuboder Anseres

Family Anseranatidae; AnseranasFamily Anatidae

Subfamily DendrocygninaeTribe Dendrocygnini DendrocygnaTribe Thalassornithini Thalassornis

Subfamily Anserinae

Tribe Cereopsini CereopsisTribe Anserini Branta, AnserTribe Cygnini Cygnus, Coscoroba

Subfamily Stictonettinae StictonettaSubfamily Tadorninae

Tribe Merganettini Hyemenolaimus, Merganetta, TachyeresTribe Plectropteini Plectropterus, SarkidiornisTribe Tadornini Subtribe Tadornina (Tadoma), Subtribe Chloephagina (Cyanochen, Alopochen,

Neochen, Chloephaga)Subfamily Anatinae

Tribe Malacorhynchini MalacorhynchusTribe Anatini Subtribe Cairinina (Cairina, Pteronetta, Aix), Subtribe Nettapodina (Chenonetta,

Nettapus), Subtribe Anatina (Amazonetta, Callonetta, Lophonetta, Anas)Tribe Aythyini Subtribe Marmaronettina (Marmaronetta), Subtribe Rhodonessina (Netta,

Rhodonessa), Subtribe Aythyina (Aythya)Tribe Mergini Subtribe Somaterina (Somateria, Polysticta), Subtribe (Histrionicus Melanitta,

Clangula, Bucephala, Mergellus, Mergus, Lophodytes, Camptorhychus)Tribe Oxyurini Subtribe Heteronettina (Heteronetta), Substribe Oxyurina (Nomonyx, Oxyura,

Biziura)

(C)Infraoder Anhimides

Superfamily Anhimoidae

Family Anhimidae Anhima, ChaunaSuperfamily Anserantoidea

Family Anseranatidae AnseranasInfraoder Anserides

Family Dendrocygnidae Dendrocygna, ThalassomisFamily Anatidae

Subfamily Oxyurinae Oxyura, BiziuraSubfamily Stictonettinae StictonettaSubfamily Cygninae Cygnus, CoscorobaSubfamily Anatinae,

Tribe Anserini Branta, Anser, Cereopsis, Cyanochen, Chloephaga, Alopochen, Neochen,Tadorna, Tachyeres, Plectropterus, Cairina, Pteronetta, Sarkidiornis, Nettapus

Tribe Anatini Callonetta, Aix, Chenonetta, Amazonetta, Merganetta, Hyemenolaimus,Salvadorina, Anas, Malacorhynchus, Marmaronetta, Rhodonessa, Netta,Aythya, Somateria, Polysticta, Histrionicus, Clangula, Melanitta, Bucephala,Mergellus, Lophodytes, Mergus, Heteronetta


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a molecular phylogeny of anseriformes based on mitochondrial dna analysis

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