Phylogeny of ‘‘core Gruiformes’’ (Aves: Grues) and ...biology-web.nmsu.edu/~houde/Fain_Krajewski_Houde.pdf · Phylogeny of ‘‘core Gruiformes’’ (Aves: Grues) and resolution

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Molecular Phylogenetics and Evolution 43 (2007) 515–529

Phylogeny of ‘‘core Gruiformes’’ (Aves: Grues) and resolutionof the Limpkin–Sungrebe problem

Matthew G. Fain a, Carey Krajewski b, Peter Houde a,*

a Department of Biology, New Mexico State University, Box 30001, MSC 3AF, Las Cruces, NM 88003-8001, USAb Department of Zoology, MC 6501, Southern Illinois University, Carbondale, IL 62901, USA

Received 18 April 2006; revised 29 December 2006; accepted 17 February 2007Available online 24 February 2007

Abstract

Opinions on the systematic relationships of birds in the avian order Gruiformes have been as diverse as the families included within it.Despite ongoing debate over monophyly of the order and relationships among its various members, recent opinion has converged on themonophyly of a ‘‘core’’ group of five families classified as the suborder Grues: the rails (Rallidae), the cranes (Gruidae), the Limpkin(Aramidae), the trumpeters (Psophiidae), and the finfoots (Heliornithidae). We present DNA sequence data from four mitochondrial(cytochrome b, 12S rRNA, Valine tRNA, and 16S rRNA) and three nuclear loci (intron 7 of b-fibrinogen, intron 5 of alcohol dehydro-genase-I, and introns 3 through 5 of glyceraldehyde-3-phosphate dehydrogenase) to test previous hypotheses of interfamilial relation-ships within Grues, with particular attention to the enigmatic family Heliornithidae. Separate and combined analyses of these genesequences confirm the monophyly of Grues as a whole, and of the five families individually, including all three species of Heliornithidae.The preferred topology unambiguously supports relationships among four of the five families, with only the position of Psophiidaeremaining equivocal. Bayesian ‘‘relaxed-clock’’ dating methods suggest that the divergences of the three heliornithid species occurredin the mid-Tertiary, suggesting that their present disjunct pantropical distribution is a result of early- to mid-Tertiary dispersal.� 2007 Elsevier Inc. All rights reserved.

Keywords: Gruiformes; Grues; Gruidae; Aramidae; Psophiidae; Heliornithidae; Rallidae; Cranes; Limpkin; Trumpeters; Sungrebe; Finfoots; Rails; Beta-fibrinogen; Alcohol dehydrogenase; Glyceraldehyde 3-phosphate dehydrogenase; Cytochrome b; 12S ribosomal RNA; 16S ribosomal RNA; Valine tRNA;Biogeography; Bayesian dating; Tertiary; DNA · DNA hybridization

1. Introduction

The classification of the avian order Gruiformes has beenone of the least stable in the taxonomic history of birds.Traditional classifications (e.g., Wetmore, 1960) include12 families distributed worldwide, ranging from the speci-ose Rallidae to morphologically distinct, monotypic fami-lies such as the neotropical Limpkin (Aramus guarauna).The combination of widespread geographical distributionand extensive morphological and ecological divergence sug-gests an ancient origin of gruiform families. This pattern,coupled with virtual monotypy within most families (gener-ally attributed to extinction), has presented difficulties in

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doi:10.1016/j.ympev.2007.02.015

* Corresponding author. Fax: +1 505 646 5665.E-mail address: [email protected] (P. Houde).

interpreting the evolutionary history of these birds. For wellover a century, avian systematists have debated their phylo-genetic relationships and even whether they constitute amonophyletic assemblage (Cracraft, 1981; Olson, 1985; Sib-ley and Ahlquist, 1990; Sibley et al., 1993; Houde et al.,1997; Livezey, 1998; Fain and Houde, 2004).

In an early cladistic study of morphology, Cracraft(1982) included Kagu, Sunbittern, trumpeters, seriemas,Limpkin, and cranes in his suborder Grues, with the lattertwo families as outgroups rooting the former as the‘‘Psophii’’ (Fig. 1a). Evidence from DNA hybridizationinverted Cracraft’s topology by rerooting it, suggestingthat cranes and Limpkin were the more recent phyletic lin-eages (Fig. 1b, Sibley and Ahlquist, 1990). Neither Cracraftnor Sibley and Ahlquist regarded rails as members of theirrespective suborders Grues. Remarkably, Sibley and

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Fig. 1. Previous estimates of phylogenetic relationships among gruiform families. (a) Morphological study of Cracraft (1982). (b) DNA hybridizationstudy of Sibley and Ahlquist. (c) Morphological study of Livezey (1998). (d) 12S rDNA sequence study of Houde et al. (1997).

516 M.G. Fain et al. / Molecular Phylogenetics and Evolution 43 (2007) 515–529

Ahlquist (1990) also reported evidence that the neotropicalSungrebe (Heliornis fulica), a heliornithid generallythought to be close to rails, was in fact sister to the sympat-ric Limpkin. This result was codified in the classification ofSibley and Monroe (1990) by including the Limpkin as atribe within the finfoot family Heliornithidae, and has sincebeen widely cited in popular literature (e.g., Bertram, 1996;Bryan, 1996). A sister relationship between these two verydifferent birds would imply incredible anatomical conver-gence (Houde, 1994), but the Sungrebe is divergent fromother finfoots in being smaller, having altricial hatchlings,and possessing the unique ability to carry its young inpaired axillary marsupia or ‘‘pouches’’ (Alvarez del Toro,1971). Unfortunately, Sibley and Ahlquist did not havesamples of the other finfoot species for comparison, leavingopen the question of heliornithid monophyly and theirrelationship to the rails and/or Limpkin. Further analysesby Sibley et al. (1993) excluded the Sungrebe, and attemptsto duplicate the original DNA hybridization result of Sib-ley and Ahlquist suggested instead that the Limpkin andSungrebe were not particularly close (Houde, 1994; Houdeet al., 1995).

In contrast to the overall taxonomic uncertainty sur-rounding many lineages considered gruiform, a consensushas begun to emerge in recent studies (Sibley et al., 1993;Houde et al., 1997; Fain and Houde, 2004; see Fig. 1cand d) that there is a monophyletic ‘‘core’’ consisting ofrails (Rallidae), Sungrebe and finfoots (Heliornithidae),trumpeters (Psophiidae), Limpkin (Aramidae), and cranes(Gruidae) i.e., the suborder Grues sensu Livezey (1998).Genetic evidence further suggests that Grues is inclusiveand not part of a larger monophyletic ‘‘Gruiformes,’’ astraditionally recognized (Houde et al., 1997; Fain and

Houde, 2004; Ericson et al., 2006). Houde et al. (1997)addressed the problem of gruiform relationships with mito-chondrial 12S rRNA sequence, and found the Sungrebeand African Finfoot (Podica senegalensis) to be sisters,while the Limpkin was supported as sister to cranes(Fig. 1d). However, the question remained whether the pre-ferred topology would be robust to further sampling ofcharacters and taxa. The first comprehensive morphologi-cal cladistic analysis of Gruiformes (Livezey, 1998) agreedwith the molecular sequence study in supporting the five‘‘core’’ gruiform families as monophyletic, suggesting thatthe morphologically disparate rails and cranes are moreclosely related than other gruiform families traditionallyconsidered ‘‘crane-like’’ (Fig. 1c). Livezey examined theanatomy of all three heliornithids, and concluded that theyare monophyletic, with the Masked Finfoot (Heliopais per-

sonata) sister to a clade consisting of the African Finfoot(Podica senegalensis) plus Sungrebe. This contrasts withan idea that the more similar African and Asian finfootsare sister species (Brooke, 1984). Livezey further foundstrong support for heliornithids and rails in superfamilyRalloidea, and for cranes, Limpkin, and trumpeters insuperfamily Gruoidea. The latter is interesting in thattrumpeters are noted for being ‘‘intermediate’’ betweenrails and cranes in many ways, and have been suggestedas sister to rails by other morphologists (Olson, 1973).Although the analyses of Houde et al. (1997) tended to sup-port trumpeters as sister to rails, they also noted intriguingconflict among sites in different 12S domains that indicatedeither a trumpeters–rails or a trumpeters–cranes relation-ship. Further, a study by one of us (Houde, P., unpublisheddata) indicates a greater degree of skeletal character con-flict for the position of trumpeters than found by Livezey

M.G. Fain et al. / Molecular Phylogenetics and Evolution 43 (2007) 515–529 517

(1998); this conflict may be partitioned between appendic-ular and axial characters.

Our goal in this study was to expand on the mitochon-drial DNA sequence data set of Houde et al. (1997) andadd sequence from independent nuclear loci in an effortto test current hypotheses of familial relationships in thesuborder Grues. In particular, we were interested in assess-ing the question of heliornithid monophyly and interfamil-ial relationships, and in resolving the phylogeneticrelationships of the Limpkin and trumpeters.

2. Methods

2.1. Taxon and locus sampling

We sampled all families ever included in the suborderGrues (Table 1), including all 15 species of Gruidae, theLimpkin (Aramidae), two of the three members of Psoph-iidae, all three species of Heliornithidae, and seven generaof Rallidae. The sampling of rails is not comprehensivebut represents a broad cross-section of rallid generic diver-sity. Outgroups (Table 1) include representatives of fivefamilies of Charadriiformes (Jacanidae, Laridae, Alcidae,Charadriidae, and Recurvirostridae), and two families ofCiconiiformes (Ciconiidae and Ardeidae). Charadriiformeshas generally been thought to be the closest order to Gru-iformes (Sibley et al., 1993; Livezey, 1998). However, initialanalyses of our mtDNA data were ambiguous with respectto the monophyly of the ingroup and the relative positionsof outgroup taxa, so we included representatives of Gallo-anserae as unambiguous outgroups to Neoaves. The rela-tionship of other gruiform families is beyond the scope ofthis study and will be addressed in further work. However,recent studies indicate that not all currently recognizedfamilies of Gruiformes form a natural group (Fain andHoude, 2004; Ericson et al., 2006).

We sequenced four complete mitochondrial genes: cyto-chrome b (cytb), small subunit ribosomal RNA (12S),tRNA-Valine (Val), and large subunit ribosomal RNA(16S). The heliornithids Heliopais and Podica were avail-able from museum specimens, and we obtained only cytb,12S, and Val for these species (Table 1). In addition, wesampled three nuclear loci: intron 7 from b-fibrinogen(bfib7), alcohol dehydrogenase-I (designated ADH5), andglyceraldehyde-3-phosphate dehydrogenase (designatedGPD3-5). The sequences analyzed for ADH5 includeintron 5 and small flanking sequences of exons 5 and 6;GPD3-5 includes complete introns 3-5 and exons 4-5.Mitochondrial sequences for Anseranas semipalmata weretaken from GenBank (Harrison et al., 2004). Sequencesfor crane cytochrome b (Krajewski and Fetzner, 1994; Kra-jewski and King, 1996), and fibrinogen intron and partial12S rRNA sequences for various gruiform taxa (Fain andHoude, 2004; Houde et al., 1997) were also available. Allother sequences were obtained in the course of this study,although not all loci were obtained for all taxa, as shownin Table 1.

2.2. Laboratory protocols

Whole genomic DNA was extracted either with a Qia-gen DNeasy DNA purification kit (Valencia, California)following the manufacturer’s protocol, or with a high-saltextraction protocol (Aljanabi and Martinez, 1997). Theloci were amplified by standard polymerase chain reaction(PCR) methods; Table 2 lists primer sequences for eachgene and references. To minimize the chance of amplifyingnuclear pseudocopies of the mitochondrial genes (numts;e.g., Sorenson and Quinn, 1998), we attempted to amplifyentire genes or large (1 kilobase or greater) fragments,and/or we used overlapping primer sets. Amplificationsthat produced single crisp bands were cleaned with Qia-gen’s QIAquick PCR product purification kit. Alterna-tively, the product was excised from a gel and cleanedwith Qiagen’s QIAquick Gel Extraction kit. Cleaned prod-ucts were sequenced directly with a BigDye Cycle Sequenc-ing kit (version 3.1; Applied Biosystems, Foster City, CA),and sequences were collected on an ABI 3100 capillaryautomated sequencer (Applied Biosystems).

2.3. Phylogenetic analysis

Manual alignment was straightforward for cytb and thenuclear sequences. Numerous studies have suggested thataligning RNA sequences according to conserved structureimproves the homology of aligned nucleotides andimproves resulting phylogenetic inference (Espinosa delos Monteros, 2003; Kjer, 1995, 2004). The alignment of12S rRNA proceeded according to the model of Houdeet al. (1997), alignment of tRNA-Valine followed the struc-ture of Desjardins and Morais (1990), and alignment of the16S rRNA sequences was inferred based on available ver-tebrate models (Cannone et al., 2002; Wuyts et al., 2001).Several highly-variable loop regions were subsequentlyconsidered unreliable and removed from further phyloge-netic analysis. These regions were delimited subjectivelyby inspection with boundaries of aligned sites that wereinvariant or nearly so; these sites corresponded well toand were at least as conservative as regions that wereremoved under various parameter conditions using theprogram Gblocks (Castresana, 2000). Alignments of com-plete sequences, those with ambiguous sites removed, andof stem and loop partitions of rRNAs in NEXUS formatare deposited at http://www.treeBASE.org.

Sequences were tested for homogeneity of nucleotidefrequencies using the v2-test implemented in PAUP* 4b8(Swofford, 1998). Tests were performed on all sites and var-iable sites for each gene individually, and for certain struc-tural/functional partitions within genes (e.g., codonpositions of cytb, ADH, and GPD; loops and helices inthe RNA genes). Further exploration of individual pair-wise comparisons of base composition was evaluated withthe disparity index test in MEGA 3.0 (Kumar and Gadag-kar, 2001; Kumar et al., 2004). An assessment of substitu-tional saturation for transitions and transversions was

http://www.treeBASE.org

Table 1Taxon sampling and GenBank accession numbers for loci

Taxon/species GenBank Accession numbers

Cytb 12S+Val 16S bfib7 ADH5 GPD3-5

GalliformesCallipepla gambelii DQ485889 DQ485791 DQ485829 DQ494145 DQ485865 DQ485912

AnseriformesAnseranas semipalmata NC005933 NC005933 NC005933 NC005933 DQ485866 DQ485913

CharadriiformesCharadrius vociferus DQ485890 DQ485792 DQ485830 AY695205 DQ485867 DQ485914Recurvirostra americana DQ485891 DQ485793 DQ485831 AY695202 DQ485868 NAHimantopus mexicanus NA NA NA AY695203 NA DQ485915Uria aalge DQ485892 DQ485794 DQ485832 AY695192 DQ485869 DQ485916Larus atricilla DQ485893 DQ485795 DQ485833 AY695186 DQ485870 DQ485917Jacana spinosa DQ485894 DQ485796 DQ485834 AY695179 DQ485871 DQ485918

CiconiiformesMycteria americana DQ485895 DQ485797 DQ485835 AY695227 DQ485872 DQ485919Ciconia maguari DQ485896 DQ485798 DQ485836 AY695228 DQ485873 DQ485920Botaurus lentiginosus DQ485897 DQ485799 DQ485837 AY695229 DQ485874 DQ485921Bubulcus ibis DQ485898 DQ485800 DQ485838 AY695233 DQ485875 NA

Gruiformes, GruidaeBalearica regulorum U27543 DQ485801 DQ485839 AY695251 NA NABalearica pavonina U27544 DQ485802 DQ485840 AY695251 DQ485876 DQ485922Bugeranus carunculatus U27556 DQ485803 DQ485841 DQ497625 DQ485877 DQ485923Anthropoides paradisea U27557 DQ485804 DQ485842 AY695252 DQ485878 DQ485924Anthropoides virgo U27545 DQ485805 DQ485843 AY695253 NA NAGrus leucogeranus U27549 DQ485806 DQ485844 DQ494146 NA NAGrus japonensis U27550 DQ485807 DQ485845 DQ494147 NA NAGrus americana U27555 DQ485808 DQ485846 AY695254 NA NAGrus grus U27546 DQ485809 DQ485847 AY695255 NA NAGrus monacha U27548 DQ485810 DQ485848 DQ494148 NA NAGrus nigricollis U27547 DQ485811 DQ485849 DQ494149 NA NAGrus vipio U11065 DQ485812 DQ485850 DQ494150 NA NAGrus antigone U11064 DQ485814 DQ485852 DQ494151 NA NAGrus rubicunda U11062 DQ485813 DQ485851 DQ494152 NA NAGrus canadensis U27553 DQ485815 DQ485853 AY082410 DQ485879 DQ485925

Gruiformes, AramidaeAramus guarauna DQ485899 DQ485816 DQ485854 AY695250 DQ485880 DQ485926

Gruiformes, PsophiidaePsophia crepitans DQ485900 DQ485817 DQ485855 AY695248 DQ485881 DQ485927Psophia viridis DQ485901 DQ485818 DQ485856 AY695249 NA NA

Gruiformes, HeliornithidaeHeliornis fulica DQ485902 DQ485819 DQ485857 AY695246 DQ485882 DQ485928Heliopais personata DQ485903 DQ485820 NA NA NA NAPodica senegalensis DQ485904 DQ485821 NA AY695247 NA NA

Gruiformes, RallidaePorphyrio porphyrio DQ485905 DQ485822 DQ485858 AY695240 DQ485883 DQ485929Laterallus melanophaius DQ485906 DQ485823 DQ485859 AY695238 DQ485884 DQ485930Gallirallus philippensis DQ485907 DQ485824 DQ485860 AY695241 NA NARallus longirostris DQ485908 DQ485825 DQ485861 AY695243 DQ485885 DQ485931Porzana carolina DQ485909 DQ485826 DQ485862 AY695239 DQ485886 DQ485932Fulica americana DQ485910 DQ485827 DQ485863 AY695244 DQ485887 DQ485933Gallinula chloropus DQ485911 DQ485828 DQ485864 AY695245 DQ485888 NA


carried out by plotting distances for each substitution typeagainst overall distance, with distances corrected under theF84 model (Felsenstein, 1993).

Weighted and unweighted maximum parsimony analy-ses were conducted with PAUP*4b8. Weighting involvedexcluding specific partitions (e.g., cytb 3rd positions) where

saturation could be seen to affect most pairwise compari-sons and/or where base composition among lineages wasfound to be nonstationary. In most cases, sites with gapswere included if located in otherwise unproblematic areasof the alignment and gap characters were treated as missingdata.

Table 2Primers used for amplification and/or DNA sequencing, and references

Gene Primer designation and sequence Reference

Cytb L14764: 50-TGRTACAAAAAAATAGGMCCMGAAGG-30 Sorenson et al. (1999)L14990: 50-AACATCTCCGCATGATGAAA-30 Kocher et al. (1989)L15087: 50-TACTTAAACAAAGAAACCTGAAA-30 Edwards et al. (1991)L15136: 50-ATAGCAACAGCATTTGTAGG-30 Krajewski and Fetzner (1994)L15517: 50-CACGAATCAGGCTCAAACAACC-30 Sorenson et al. (1999)L15560: 50-CTGACAAAATYCCATTYCACCC-30 Sorenson et al. (1999)H15541: 50-GGGTGGAAKGGRATTTTRTC-30 This studyH15767: 50-ATGAAGGGATGTTCTACTGGTTG-30 Edwards et al. (1991)H15915A: 50-AGTCTTCAGTCTCTGGTTTACAAGAC-30 This study

12S/Val/16S L1263: 50-AACCCACAAAGCATGGCACTGAA-30 Sorenson et al. (1999)L1267: 50-AAAGCATGGCACTGAAGHTG-30 Sorenson et al. (1999)L1753: 50-AAACTGGGATTAGATACCCCACTAT-30 Kocher et al. (1989)H1858: 50-TCGATTAYAGAACAGGCTCCTCTAG-30 Kocher et al. (1989)H2294: 50-CTTTCAGGTGTAAGCTGARTGCTT-30 Sorenson et al. (1999)L2258: 50-CGTAACAAGGTAAGTGTACCGGAAGG-30 Sorenson et al. (1999)L2724: 50-ACCGAGCTGGGTGATAGCTG-30 Sorenson et al. (1999)L3259: 50-GGTGATGCCTGCCCAGTG-30 Sorenson et al. (1999)L3827: 50-GCAATCCAGGTCGGTTTCTATC-30 Sorenson et al. (1999)H2826: 50-TTCTTTTTTAAWGGAGCTGTACC-30 Sorenson et al. (1999)H2902: 50-GACGCACTYTTTRTTGRTGGCTGCT-30 This studyH3292: 50-TGATTGCGCTACCTTYGCACGG-30 Sorenson et al. (1999)H3652: 50-GATTGCGCTGTTATCCCTGG-30 Sorenson et al. (1999)H4017: 50-GCTAGGGAGAGGATTTGAACCTC-30 Sorenson et al. (1999)

bfib7 Fib-BI7U: 50-GGAGAAAACAGGACAATGACAATTCAC-30 Prychitko and Moore (1997)Fib-BI7L: 50-TCCCCAGTAGTATCTGCCATTAGGGTTT-30 Prychitko and Moore (1997)FBE8L2: 50-TTCTTTGGAGCACTGTTTTCTTGGATC-30 Fain and Houde (2004)Fib-BI7U2: 50-ATATGTTTTATCCCTGCA-30 This studyFib-BI7L2: 50-TAAGCAAACAGATCAAC-30 This studyFBI7-470U: 50-CCTACTCAGAAGAYAGGAGCT-30 This studyFBI7-378L: 50-GGAGTGAGTACCAATATTCCT-30 This study

ADH5 ADH5F: 50-TCTGTTGTCATGGGCTGCAAG-30 This studyADH6R: 50-TCCAAAGACGGACCCTTTCCAG-30 This studyADH5F2: 50-TGCCAAGGCCAARGAGCTGGG-30 This studyADH6R2: 50-GGTCAAAAGTGATCTTYTGTGCTGC-30 This study

GPD3-5 GAPD.3/4.F: 50-TCTCTGGCAAAGTCCAAGTG-30 This studyGPD.e3F: 50-CTTCATTGATCTGAACTACATG-30 This studyGAPD.4/5.F: 50-ACACTTCAAGGGCACTGTC-30 This studyGAPD.4/5.R: 50-TGGACTCCACAACATACTCAG-30 This studyGPD.E5F: 50-CTGGTGCTGAATATGTTGTGG-30 This studyGAPD.5/6.R: 50-TCATGGTTGACACCCATC-30 This study


For loci where heterogeneous base composition amonglineages was judged to be problematic, LogDet (Lake,1994; Lockhart et al., 1994) distance analyses were per-formed with PHYLIP 3.6 (Felsenstein, 1993) for compari-son to other methods that assume homogenousfrequencies. Maximum likelihood analyses were conductedwith PHYML 2.3 (Guindon and Gascuel, 2003), with moreextensive tree searches in PAUP* using the PHYML topol-ogy as a starting tree. Bayesian analyses were performedwith MrBayes 3.0 (Huelsenbeck and Ronquist, 2001), run-ning 107 generations of the Markov chain, sampling every250 generations, and discarding the first 105 for burn-in.Four chains were run simultaneously, three ‘‘heated’’ andone ‘‘cold’’ to facilitate effective sampling of tree space.Confidence on nodes was evaluated with 1000 bootstrapreplicates for parsimony, distance and likelihood searches

(Felsenstein, 1985), and by summarizing the 30,000 treesretained in the Bayesian analyses as posterior probabilities.

Initially, individual genes were analyzed usingunweighted maximum parsimony and neighbor-joining(Saitou and Nei, 1987) with the F84 model of sequenceevolution (Felsenstein, 1993). The resulting topologies wereused as input for ModelTest 3.06 (Posada and Crandall,1998) to determine an appropriate model of sequenceevolution using hierarchical likelihood ratio test (hLRTs)and the Akaike information criterion (AIC). We emphasizethat the models tested will all misrepresent the actualevolutionary processes to some extent. We therefore chosethe least parameter-rich model justified by either of thesetests in order to capture the most relevant features ofsequence evolution. In most cases where there wasdisagreement, hLRTs chose a simpler model and this was


used for maximum likelihood phylogenetic analysis. Oneexception where we did not choose the simpler modelwas in the concatenation of nuclear and mitochondrialsequences: hLRTs chose a general-time reversible modelwith a gamma correction for variable rates among sites(GTR+C), whereas for the mtDNA sequences alone bothtests had chosen GTR+I+C, incorporating a proportionof invariant sites. This highlights that the differences inbase composition, rates, and nucleotide substitutionparameters among genes violate models that presumehomogeneity in evolutionary processes across the loci(Krajewski et al., 1999). To better account for differencesamong genes, we employed the weighted-average distancemethod implemented in the WAVEBOOT program (Kra-jewski et al., 1999) and mixed-model Bayesian analyseswith MrBayes 3.0. Models used for individual partitionsin MrBayes were chosen to be comparable to the modelused for the mixed-model distances. Currently, the mostcomplex model available in PHYLIP is F84+C. This modelwas used for all partitions, allowing partition-specific basecomposition, transition bias, and among-site rate distribu-tion. The transition/transversion ratio and shape parame-ter of the gamma distribution were estimated in PAUP.Partitions were chosen by structural/functional consider-ations for the mitochondrial data (codon positions in cytb,stems and loops in RNAs). Stems and loops in the rRNAsmay not be as well-defined as codon positions (see Houdeet al., 1997; Mindell et al., 1997), but partitioning in thismanner can be justified by differences in rates, base compo-sition, and substitutional biases (e.g., Krajewski et al.,1997). Each of the nuclear gene fragments were treated assingle partitions as there is no justification for dividing sitesin the introns more finely, and the exon segments includedfor ADH and GPD were small and highly conserved, pro-viding too few sites for reliable parameter estimation.

Divergence times were estimated using a Bayesianrelaxed-clock method as implemented in the program Mul-tidivtime (Thorne and Kishino, 2002; Wiegmann et al.,2003). Calibration constraints were set as follows. Thedivergence of the extant gruine radiation was constrainedto 3–5 million years ago (MYA), and a lower limit on thegruine/balearicine divergence was placed at 10 MYA(Feduccia and Voorhies, 1992; Krajewski and Fetzner,1994; Krajewski and King, 1996). A lower constraint of28 million years was placed on the divergence of Aramidaebased on an Oligocene fossil Limpkin (Olson, 1985); noupper bound was placed on this node. Analyses were runboth with and without this latter constraint to determinewhat effect it might have on the estimated dates. A con-straint external to Grues was placed on the alcid-larid splitbased on a 30-MY fossil stercorariid. Assuming that alcidsand stercorariids are sister taxa (Paton et al., 2003), thelarid-alcid divergence would necessarily predate 30 MY—this date therefore serves as a conservative lower boundthat should only result in larger confidence intervals onestimated dates, particularly deeper in the tree. An upperbound of 45 MY on larid-alcid divergence was assumed

(Paton et al., 2003). Effects of analysis with different nodeconstraints are discussed further below.

3. Results

3.1. Alignments and nucleotide substitution dynamics

Complete sequences of cytb were obtained for allincluded taxa, and the alignment was 1143 sites corre-sponding to 381 codons including the termination codon.Translated sequences of cytb showed no premature stopcodons, indels (in-frame or otherwise), nor frameshiftsresulting in stretches of radical amino acid replacements.Sequences of the rRNAs and the tRNA could be foldedinto presumably functional secondary sequences based onprevious or inferred models. These features, and the factthat different primer pairs amplified identical sequence inregions of overlap, minimizes concern that the resultingsequences originate from nuclear pseudogenes. Alignmentof the RNAs for these taxa is not trivial, and highly vari-able regions exist within specific loops. These ambigu-ously-aligned sites were excluded from phylogeneticanalyses. Sequences for 12S rRNA ranged from 964 to990 nucleotides, and the resulting alignment was 1079 sites.Of these, 145 sites (13%) were excluded as alignment-ambiguous. The tRNA-Valine alignment is 77 sites inlength, ranging from 68 to 74; 17 sites (22%) were excluded.The aligned 16S rRNA consisted of 1803 sites, rangingfrom 1575 to 1611; 477 sites (26%) were considered ambig-uously aligned. The sites excluded from 12S correspondedto regions (usually loops, or highly variable stem-loopcombinations) also considered problematic in previousstudies (Houde et al., 1997; Mindell et al., 1997). Presum-ably the same would be true for 16S, although there arefewer data available for comparison.

Alignments for the nuclear introns also included a sig-nificant insertion–deletion component, although lessambiguously placed than in the RNAs. The inferred gappositions in the nuclear loci are also remarkably consistentwith individual gene trees and the combined data topology.Lengths of bfib7 sequences range from 876 to 935 nucleo-tides, resulting in a total aligned length of 1043 sites. TheADH5 alignment consists of 752 sites, ranging from 656to 703 nucleotides. Of these, 144 sites were from exon 5and 60 sites from exon 6. The GPD3-5 alignment is 905sites, ranging from 676 bp to 781 bp. Of these, 41 sites ina polypyrimidine tract near the 30-end of intron 5 wereexcluded due to questionable alignment.

As expected, mtDNA genes were more variable thannuclear, with much higher percentages of informative sitesand greater homoplasy. This is best illustrated by cytb, inwhich 371 of 381 third positions are variable (Table 3);transitions for this partition are clearly saturated (notshown). Scatter made substitution plots difficult to inter-pret for first and second positions, but saturation appearsonly to marginally affect first position transition distancesin comparisons to outgroups. All plotted transversion

Table 3Parameters for mixed-model phylogenetic analyses shown in Fig. 4

Gene partition Total sites Variable sites Informative sites Ti/Tv a A:C:G:T

Cytochrome b 1143 557 480 NA NA 0.34:0.45:0.06:0.151st position 381 135 106 3.31 0.21 0.34:0.42:0.11:0.142nd position 381 51 29 3.65 0.07 0.12:0.37:0.12:0.383rd position 381 371 345 NA NA 0.38:0.47:0.03:0.12a

mt RNA 2312 830 554 NA NA 0.33:0.25:0.21:0.21Loops 1198 477 332 3.71 0.22 0.44:0.24:0.13:0.19Stems 1114 353 222 12.7 0.21 0.21:0.27:0.29:0.23

b-fibrinogen intron 7 1043 631 399 1.88 3.53 0.28:0.20:0.22:0.30ADH-I intron 5 752 418 220 2.05 1.53 0.27:0.25:0.26:0.22G3PD introns 3-5 864 397 229 2.82 0.79 0.19:0.25:0.30:0.26

a Significantly heterogeneous at the P < 0.0001 level, v2 test.


distances showed a linear increase with increasing totaldivergence. Saturation of transition substitutions wasapparent in rRNA loops, but again only for the greatestdivergences, while all substitution types appeared toincrease linearly in helices.

Beyond the potential for greater homoplasy obscuringphylogenetic signal deeper in the tree, a more insidiousproblem of the mtDNA sequences in this data set provedto be differences in base composition among lineages.Again, this was most evident for cytb third position sites,where differences in base composition were found to be sig-nificant (Table 3). In particular, storks and trumpeters haveelevated levels of cytosine and reduced adenine and thy-mine in cytb third positions. Despite the fact that no otherpartition showed significant differences in base composi-tion, similar trends were apparent in first positions and inthe RNAs. A test of pairwise nucleotide homogeneity usingthe disparity index in MEGA indicated that, even withthird positions removed, storks and trumpeters had thegreatest number of significant deviations from other taxa,in sharp contrast to the homogeneity inferred for trum-peter-stork comparisons. Trumpeters and storks also tendto ‘‘attract’’ in analyses of the 12S sequences for which basecomposition is more homogeneous overall. Sites in 12Sloops do not show any clear trends in base composition;although storks still have elevated C, trumpeters do not.In contrast, constraints that maintain pairing in helicesmay affect compositional convergence more severely. For12S stems, storks and trumpeters are nearly 30% C, com-pared to more even compositions for other taxa. Interest-ingly, 16S sequences do not show the same trends.Phylogenetic analyses of this gene alone and in combina-tion with the other mitochondrial genes are more consis-tent with the results of the nuclear loci. Because cytb 3rd

positions exhibit multiple attributes known to negativelyaffect phylogenetic analyses (e.g., high substitution ratesresulting in substitutional saturation and base composi-tional heterogeneity among taxa), this partition wasexcluded from all phylogenetic analyses presented.

In contrast, the nuclear loci were found to have proper-ties more amenable to standard phylogenetic analyses.

Rates of substitution were markedly lower in the nucleargenes compared to mtDNA and substitutional saturationwas not evident in any of the three genes (not shown). Basecomposition was slightly enriched in AT-content, consis-tent with expectations for nuclear noncoding sequences(Prychitko and Moore, 1997, 2003). However, these devia-tions were not nearly as dramatic as base compositionbiases seen in mtDNA. Further, there was no indicationthat base composition shifted significantly among taxafor the variable sites of bfib7 (P = 1.0), GPD3-5(P = 0.96), or ADH5 (P = 0.23). Neither was composi-tional heterogeneity observed to be problematic in parti-tions of the nuclear loci that included coding sequences,even for 3rd positions. Variation in rates among sites forboth sequences was also greatly reduced compared tomtDNA. While models including a gamma distributionof rates among sites were favored, the estimated values ofthe shape parameter were above 1.0, indicating a relativelyeven distribution, with a greater number of characters freeto change and hence more likely to retain informative var-iation. The lower values of the shape parameter estimatedfor ADH5 and GPD3-5 indicate greater rate variationamong sites than in bfib7, which can be attributed to theirsmaller size and to the inclusion of highly conserved exonsequences.

3.2. Phylogeny

Best trees from maximum-likelihood analyses and mixed-model distance and Bayesian analyses of mtDNA alone(treating cytb 1st and 2nd positions, and the the RNA stemsand loops as four separate process partitions) agreed inrecovering the monophyly of Grues (Fig. 2a). These analysesalso showed strong support for sister-group relationshipsbetween Limpkin and cranes, and heliornithids and rails,respectively. Psophiidae, the trumpeters, were included asmembers of this core gruiform clade, but their position withrespect to the ralloids or gruoids was not resolved and thebootstrap support on monophyly of the clade as a wholeremained low. The combination of cytb, 12S, and Valstrongly supported monophyly of Heliornithidae and a sister

0.1

Anseranas semipalmataCallipepla gambelii

Botaurus lentiginosusBubulcus ibis

Mycteria americanaCiconia maguari

Heliornis fulica

Rallus longirostris

Gallirallus philippensis

Porzana carolina

Fulica americana

Gallinula chloropus

Laterallus melanophaius

Porphyrio porphyrio

Psophia crepitans

Psophia viridis

Aramus guarauna

Balearica regulorum

Balearica pavonina

Grus leucogeranus

Grus antigone

Grus rubicunda

Grus vipio

Grus canadensis

Bugeranus carunculatus

Anthropoides virgo

Anthropoides paradisea

Grus japonensis

Grus americana

Grus grus

Grus monacha

Grus nigricollis

Larus atricillaUria aalge

Jacana spinosaCharadrius vociferus

Recurvirostra americana

100

69

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79100

100

99

100

80

98

100

100

83100

100

6789

100

100

100

57

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Podica senegalensis

Heliornis fulicaHeliopais personata

10099

a

b

Fig. 2. (a) Maximum-likelihood (GTR+I+C) phylogeny of Grues (taxa in boldface type) based on concatenated mtDNA sequence data (cytb excludingthird positions, 12S, Val, 16S). Bootstrap support is based on 1000 pseudoreplicates analyzed with PHYML. (b) Inset shows monophyly of Heliornithidaebased on cytb, 12S, Val.


relationship of Heliopais and Heliornis (Fig. 2b) as foundby Houde et al. (1995). Inclusion of 16S in the mtDNAdata set (unavailable for Podica and Heliopais) provedessential to achieve congruence with nuclear data sets inrecovering a monophyletic Grues, but bootstrap supportremained poor for deeper-level relationships. Even withreduced taxon sampling, Heliornis remained sister to rails,and Aramus sister to cranes. Analysis of bfib7 alone alsosupported the two heliornithids sampled, Podica andHeliornis, together as sister to rails (not shown), and Limp-kin sister to cranes.

The individual nuclear gene trees were strikingly similarto one another both topologically and in levels of bootstrapsupport for the monophyly of Grues, heliornithids plusrails, and Limpkin plus cranes, respectively, at nearly100% (Fig. 3a). They also routinely recovered the

monophyly of both Charadriiformes and Ciconiiformesat deeper levels of the tree, although support for the latterwas weak. The best gene-tree estimates from each of thenuclear loci and the entire mtDNA data set are congruentin that they do not conflict at well-supported nodes, andare topologically consistent across optimality criteria.One notable exception in these topologies is the positionof Psophiidae. The bfib7 topology moderately supportstrumpeters as sister to other gruoids (66% MP; 81% ML;Fig. 3b). ADH5 moderately supports trumpeters as sisterto ralloids (74% MP; 73% ML; Fig. 3b). GPD3-5 moder-ately to strongly supports trumpeters as the basal memberof the clade (89% MP; 82% ML; Fig. 3d). Hence, each pos-sible position for trumpeters relative to the other twoclades is represented in these gene trees. The conflict tendsnot to be strongly supported, and Shimodaira–Hasegawa

Fig. 3. (a) Combined maximum-likelihood (TN93+C) phylogeny of Grues (taxa in boldface type) based on the three nuclear loci. Insets show theindividual gene-tree conflict in the placement of Psophiidae (trumpeters). (b) bfib7 topology. (c) ADH5 topology. (d) GPD3-5 topology. Numbers at nodesindicate bootstrap support and asterisks indicate 100% support.


(1999) tests of the three possibilities show that the two sub-optimal trees are not significantly worse within each dataset (not shown). It is unclear whether the conflict resultsfrom lineage-sorting rather than some systematic bias inone or the other data set, but the possibility is an importantcaveat to bear in mind in the following combined analyses.

Because all genes were considered provisionally con-gruent (except the position of trumpeters), the loci werecombined in concatenated analyses. Two different setsof analyses were run. The first set examined the nuclearloci only, to examine the results of their combinationwithout the potentially confounding effects of the moreproblematic mtDNA data. The second combined allDNA sequences. We were particularly interested whethera stronger influence of one data set would resolve theposition of trumpeters as sister to either gruoids or ral-loids. Also, we were interested in testing the closer posi-tion of ciconiiforms to Grues than charadriiforms,

because the latter have long been thought to be the like-liest sister taxon to Gruiformes. Any change in the levelof bootstrap support might elucidate the nature of thephylogenetic signal between mitochondrial and nucleardata sets. An increase in bootstrap support for onetopology might indicate a congruent but weaker signalin the alternate data set, whereas a decrease could sug-gest real differences between the two that might be takenas an indication of gene-tree conflict.

Combination of the three nuclear loci resulted in con-flicting interpretations of the position of trumpetersdepending on the method of analysis. Maximum-parsi-mony analysis resulted in six most parsimonious trees thatdiffered in the position of the trumpeter; this in itself mightindicate that the genes contain conflicting signals for thisnode. ML and mixed-model analyses resulted in trumpetersbeing sister to other gruoids, but this was very weaklysupported.


Fig. 4 shows our best current estimate of phylogeneticrelationships for Grues, obtained from mixed-model dis-tance and Bayesian analyses of the combination of thethree nuclear loci with the mitochondrial data. The ‘‘core’’gruiforms are strongly supported as monophyletic, withbootstrap support of 92% for both ML and MP, and a pos-terior probability of 1.0. Aramus is sister to cranes, andHeliornis is sister to rails, both garnering 100% bootstrapsupport in MP and ML analyses, and both have a posteriorprobability of 1.0 in Bayesian mixed-model analysis.Clearly, the Limpkin is not sister to the Sungrebe, nor clo-sely related to heliornithids. In light of the morphologicalstudy of Livezey (1998) and the DNA hybridization resultsof Sibley et al. (1993), the majority of evidence appears tosupport Psophiidae as a basal gruoid, sister to the cranesplus Limpkin clade. However, our mixed-model Bayesiananalysis provides no support for this, and even the low

0.1

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Recurvir

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0.57/--

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1.00/--

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1

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3

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Fig. 4. Mixed-model Bayesian analysis of the combined DNA sequence datpositions, mt RNA loops, mt RNA stems, bfib7, ADH5, and GPD3-5. Ingrouposterior probability, and the second number is the bootstrap proportion obtainumbers on selected nodes refer to divergence times presented in Table 4.

posterior probability of 0.57 may be inflated relative tobootstrap support (e.g., Douady et al., 2003). Acceptingtrumpeters as sister to other gruoids is here considereda tentative conclusion that needs to be confirmed withadditional data.

3.3. Divergence time estimates

One has only to examine the trees presented to observestriking differences in branch lengths between clades. Railsand heliornithids have particularly long branches com-pared to the other gruiforms, and this is true for all genes,indicating a genome-wide effect rather than a gene-specificeffect, in these lineages. We used a Bayesian relaxed-clockmethod to estimate divergence times in the face ofthis among-lineage rate heterogeneity, and based it onlyon the nuclear genes to avoid problems arising from

Callipepla gambelii

rius vociferus

ostridae

Jacana spinosa

aalge

atricilla

otaurus lentiginosus

ia americana

maguari

Heliornis fulica

Porphyrio porphyrio

Laterallus melanophaius

/--

Rallus longirostris

Porzana carolina

Fulica americana.00/96

68

0

Psophia crepitans

us guarauna

arica pavonina

canadensis

ranus carunculatus

ropoides paradisea

a sets. The data are partitioned as follows: cytb 1st positions, cytb 2ndp taxa are in boldface type. The first number at each node is the Bayesianned in a weighted-average distance analysis of the same partitions. Circled


saturation in the mtDNA data. Interestingly, estimates oftimes including mtDNA in the analyses were only slightlyyounger than those from the nuclear genes alone; estimateswere far more affected by the inclusion or exclusion of spe-cific calibrations providing constraints on node times.Table 4 gives the estimated times and associated credibleintervals for the three nuclear loci. Including an upperbound of 20–25 MY on the balearicine-gruine divergence(Krajewski and Fetzner, 1994; Krajewski and King, 1996)tended to make estimates substantially younger overall,while exclusion of this upper bound and of the Limpkincalibration tended to result in somewhat older dates. Theresults are consistent with those of other recent studiesusing molecular data to estimate divergence times, manyof which imply that neoavian orders diverged before theend-Cretaceous extinction (Paton et al., 2003; Harrisonet al., 2004). The basal divergence among the core gruiformlineages appears to straddle this boundary, and the esti-mated split between gruoids and ralloids is estimated tobe as old as 73 MY (no upper bound enforced on the bal-earicine-gruine split) or as young as 51 MY (aforemen-tioned upper bound assumed at 20 MY; Krajewski andKing, 1996). The divergence of Heliornis from rails isplaced in the mid- to early-Eocene, 43 MY, roughly coevalwith the estimated Limpkin-crane divergence. The estimatefor the basal crane divergence is somewhat older thanrecently suggested on the basis of cytb divergence (Krajew-ski and King, 1996), but is broadly consistent with a newfossil ‘‘stem’’ gruoid found in the Oligocene of Europe(Mayr, 2005). The credible intervals are wide due to uncer-tainty in the fossil calibrations and to the disparate ratesbetween the ralloid and gruoid lineages, but they appearto rule out a Gondwanan divergence to explain the pan-tropical distribution of heliornithids.

4. Discussion

4.1. Molecular evolution and phylogenetic utility

Phylogenetic estimates from individual genes wereremarkably congruent, although the support for relation-ships varied among genes. Most dramatic was the differ-ence in support for basal nodes from the mtDNA genetree versus the individual nuclear gene trees. It is no longersurprising that some of the evolutionary properties of

Table 4Estimated divergence times for selected nodes in the phylogeny of Grues

Node Clade

1 Aramidae–Gruidae2 Gruinae–Balearicinae3 Psophiidae4 Heliornithidae–Rallidae5 Basal Rallidae6 Ralloidea–Gruoidea7 Ardeidae–Ciconiidae8 Basal Charadriiformes

mtDNA compromise its ability to resolve relationshipsfor deep divergences (Naylor and Brown, 1998; Springeret al., 2001). Mitochondrial sequences have been shownto have relatively high substitution rates and a high degreeof rate variation among sites, leading to substitutional sat-uration at those sites that are free to vary, and variation inbase composition among lineages has been shown at theordinal level in mammals (e.g., Gibson et al., 2005). Mostsampling for mtDNA phylogenies among birds has focusedon the generic level and below, or employed limited ordinalsampling. Hence, we are generally lacking informationfrom intermediate divergences that might link the twolevels and demonstrate the level at which the phylogeneticinformation in mtDNA begins to degrade. While this studyaddresses intermediate divergences, ranging from recent(5 MY and less for crane species) to ancient (approximatePaleocene–Eocene divergences among the included grui-form families), the peculiar substitutional properties themtDNA of some of the taxa involved may result in theconclusions not generalizing well to other groups.

Rate variation and compositional nonstationarityamong lineages are two properties of the mtDNAsequences in particular that are likely to have had a nega-tive impact on phylogenetic inference. While the latterappears responsible for a specific attraction between twounrelated taxa (trumpeters and storks), the formerundoubtedly contributed to the inability of mtDNA to sup-port Grues monophyly by tending to place the ralloids (fin-foots + rails) near the galloanseran outgroups.

Much attention has been paid to substitution rate vari-ation among sites leading to substitutional saturation andto rate variation among lineages leading to inconsistentphylogenetic estimates. Compositional nonstationarityhas received rather less attention except for those workingamongst the deepest branches of the tree of life. That situ-ation is changing and the necessity of looking for rate var-iation even among closely related taxa is becoming betterappreciated (e.g., Schmitz et al., 2002; Griffiths et al.,2004; Gibson et al., 2005). Recent studies of cytb alone(Johnson, 2001), 12S alone (Mindell et al., 1997), or com-plete mitochondrial genomes (Harrison et al., 2004) largelyfocused on rate variation rather than compositional biases.Harrison et al. (2004) attempted to ameliorate both prob-lems by coding saturated sites as purines (R) and pyrimi-dines (Y) so that only transversion information would be

Divergence time (MY) 95% credible interval

48.5 (31.3,73.8)31.4 (19.1,49.8)66.4 (44.8,97.8)42.6 (27.1,65.5)21.8 (12.9,35.6)73.2 (50.0,107)80.7 (55.9,117)74.3 (54.6,105)


retained. This strategy was not applicable to the currentstudy because A M C transversions contributed greatly tothe compositional heterogeneity among taxa, a phenome-non observed for other, similar data sets (e.g., Schmitzet al., 2002). Braun and Kimball (2002) analyzed then-available avian mitochondrial genomes and explicitlytested for base compositional variation. They found signif-icant differences among taxa, even when the data were lim-ited to transversions, but concluded that ‘‘convergence inoverall base composition is not driving the observed rela-tionships.’’ Our results suggest that compositional differ-ences may be exacerbating rate effects in misleadingphylogenetic analyses at deep divergences.

We suspected compositional convergence, particularlyin cytb and 12S, to be incorrectly uniting the trumpeterswith a taxon intended as an outgroup, the storks. However,the only data partition that failed the v2 test of composi-tional homogeneity implemented in PAUP* was cytb thirdpositions. In contrast, the disparity index test in MEGAshowed homogeneity between stork-trumpeter compari-sons for cytb and 12S, but nearly all comparisons betweenthese two taxa and others were significant. More interestingyet were differences between the 12S and 16S rRNAs. Thelatter showed little sign of the specific compositional con-vergence that appeared to affect cytb and 12S, and ratesbetween stem and loop partitions in 12S and 16S appearedsimilar. Phylogenetic analysis of 16S alone was able torecover Grues monophyly (not shown), though with lowbootstrap support and trumpeters appearing basal to theother families. LogDet analyses have been used for theirsupposed ability to correct for compositional convergence(Lake, 1994; Lockhart et al., 1994), but such methods con-tinued to link storks and trumpeters for cytb, for 12S, andfor the two combined. One can conclude from these analy-ses that (1) the LogDet method is not adequately overcom-ing base compositional convergence (Tarrıo et al., 2001), or(2) that other factors (e.g., rate variation among lineages oramong sites) are not taken into account, thus compromis-ing its ability to overcome systematic bias (Foster andHickey, 1999; Conant and Lewis, 2001). Further, basecomposition may also vary across the mitochondrial gen-ome (Faith and Pollock, 2003). This observation, and dif-ferences in the relative proportions of stems versus loopsin 12S and 16S, may explain the differences in composi-tional heterogeneity observed between these two genes.Regardless, some combination of base composition andrate variation likely contributed to the inability of mtDNAto robustly support monophyly of the ingroup or relation-ships among outgroup taxa.

In contrast to mtDNA, each of the individual nucleargenes sampled in this study performed well. In all threecases, monophyly of Grues received very high support, asdid sister relationships between Aramidae and Gruidae,and Heliornithidae and Rallidae. The exception was theposition of Psophiidae, which was moderately resolved invarious positions relative to the other two clades. The evo-lution of b-fibrinogen intron 7 in comparison to mitochon-

drial DNA has already been extensively discussed(Prychitko and Moore, 1997, 2000, 2003). In brief, itsexceptional performance for phylogenetic inference canbe attributed to more even substitution rates among sites,little taxon-specific compositional bias, and substitutionrates low enough that saturation does not affect even thehighest levels of avian phylogeny. ADH5 and GPD3-5appears to have similar properties, although introns inboth loci are apparently evolving somewhat more rapidlythan bfib7. Compositional nonstationarity did not signifi-cantly affect any nuclear gene, nor partitions of these, suchas 3rd codon positions. ADH5 had the lowest P-value, andthe disparity-index test revealed a high proportion of sig-nificant pairs. However, there was no obvious trend in con-vergence in particular taxa, and differences did not affectthe ability of ADH5 to infer a well-resolved topology con-gruent with that from other loci. We emphasize that ratedifferences among lineages were similar in all the genes,indicating some lineage-specific effect acting genome-wideto increase substitution rates in rails and heliornithids incomparison to cranes and the Limpkin. An increase ingenomic rate may explain why the original DNA hybrid-ization study of Sibley and Ahlquist (1990) placed railsbasal to other gruiform taxa based on UPGMA analysis.However, heliornithids have even longer branches, so thissimple explanation cannot account for the misplacementof Heliornis as sister to Aramus in the DNA hybridizationresult.

4.2. Phylogeny and biogeography

Analyses of all three nuclear genes alone, in combina-tion with one another, and in combination with mtDNAresult in a highly resolved phylogeny for the families ofGrues that is congruent with recent studies of the group.The single exception is the inferred position of the Psoph-iidae, which changed with the locus analyzed and for theestimation method employed (for example, parsimony ver-sus single-model likelihood analysis versus partitioned dis-tance and Bayesian analyses). Crossed Shimodaira–Hasegawa (1999) tests of tree topology were applied foreach nuclear gene (not shown), and in no case could anyof the three alternative placements of Psophia be rejectedas significantly worse for any gene. Other evidence suggeststhat this relationship could be difficult to resolve. Mito-chondrial sequences appear to have different sets of sitessupporting different alternatives. Houde et al. (1997) notedthat different protein-binding domains of 12S conflict inpreferring a ‘‘trumpeters-cranes’’ or a ‘‘trumpeters-rails’’clade. Furthermore, Houde (unpublished data) has col-lected a morphological data set which shows conflictbetween axial and appendicular characters, contrary tothe well-resolved morphological phylogeny obtained byLivezey (1998). Collection of further data to assess the nat-ure of these character conflicts should prove interesting.

The well-resolved mtDNA topology for the three helior-nithid species is congruent with the results of the DNA


hybridization study of Houde et al. (1995), indicating thatHeliornis is sister to the southeast-Asian Heliopais. Theaforementioned properties of mtDNA sequences precludeestimating heliornithid divergence times from this dataset. The estimated date from nuclear data indicates aheliornithid-rallid divergence of some 43 MYA, implyingmid-Tertiary divergences among heliornithids. No fossilheliornithids have been known until recently, leavingopen the possibility that extant species were old enoughfor the pantropical distribution to be a relict of Gondwa-nan continental drift. However, a fossil attributed to themodern genus Heliornis has recently been described fromthe eastern United States and dated to the middle Mio-cene, approximately 14 MYA (Olson, 2003). This demon-strates that heliornithids were present in North Americain the Tertiary, and may have shifted their range to Cen-tral and South America as the climate changed in the lateMiocene–Pliocene. This fossil and our molecular diver-gence analyses add heliornithids to a list of avian taxaincluding jacanas (Whittingham et al., 2000) and trogons(Espinosa de los Monteros, 1998; Moyle, 2005) thatapparently underwent dispersal from Asia to NorthAmerica during the early to mid-Tertiary. Derivation ofHeliornis from Old World ancestors is implied becauseOld World finfoots share characters to the exclusion ofHeliornis (the basis for traditional classifications thattreat them in separate subfamilies), but the African fin-foot is sister to both Heliornis and the Asian finfoot. Itis further corroborated by analyses of ADH5 and 12Sthat recover African flufftails (Rallidae: Sarothrura) assister to heliornithids (data not shown).

Our best estimate of Grues phylogeny comes from parti-tioned mixed-model combined analysis of all includedgenes. This includes trumpeters as basal gruoids tentativelysister to Limpkin plus cranes, which agrees with morpho-logical and DNA hybridization studies. The inferred posi-tions of Limpkin and finfoots do not agree with theoriginal DNA hybridization study of Sibley and Ahlquist(1990), but are perfectly congruent with both morphologi-cal and subsequent DNA hybridization results (Houdeet al., 1995). The Limpkin should not be classified as a tribein the family Heliornithidae (Sibley and Monroe, 1990;American Ornithologists’ Union, 1998) and we agree withLivezey (1998) in retaining it in its own family nearest thecranes as per most traditional classifications. More data willbe necessary in elucidating the nature of the character con-flict among genes and morphological partitions of trumpet-ers. Further examination of the phylogenetic affinities offossils attributed to Grues could also contribute to narrow-ing the credible intervals on divergence time estimates andimprove inferences regarding the late Cretaceous-earlyTertiary biogeography of lineages of core gruiforms.

Acknowledgments

Special thanks to Yolanda Chacon and Marilyn Gotb-eter for generating mitochondrial sequences for outgroups

and Justin T. Sipiorski for generating the gruioid 16Ssequences. Yolanda Chacon and Marilyn Gotbeter weresupported by MARC traineeships, NIGMS GM07667. Thisresearch was supported by National Science Foundationgrants DEB-0108656 (to CK) and DEB-0108568 (to PH).

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