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BMC Evolutionary Biology
Open Access
Research article
Multilocus perspectives on the monophyly and phylogeny of the
order Charadriiformes (Aves)
Matthew G Fain and Peter Houde*
Address: Department of Biology, New Mexico State University, Box 30001 MSC 3AF, Las Cruces New Mexico 88003 USA
Email: Matthew G Fain - mgfain@yahoo.com; Peter Houde* - phoude@nmsu.edu
* Corresponding author
Abstract
Background: The phylogeny of shorebirds (Aves: Charadriiformes) and their putative sister
groups was reconstructed using approximately 5 kilobases of data from three nuclear loci and two
mitochondrial genes, and compared to that based on two other nuclear loci.
Results: Charadriiformes represent a monophyletic group that consists of three monophyletic
suborders Lari (i.e., Laridae [including Sternidae and Rynchopidae], Stercorariidae, Alcidae,
Glareolidae, Dromadidae, and Turnicidae), Scolopaci (i.e., Scolopacidae [including Phalaropidae],
Jacanidae, Rostratulidae, Thinocoridae, Pedionomidae), and Charadrii (i.e., Burhinidae, Chionididae,
Charadriidae, Haematopodidae, Recurvirostridae, and presumably Ibidorhynchidae). The position
of purported "gruiform" buttonquails within Charadriiformes is confirmed. Skimmers are most
likely sister to terns alone, and plovers may be paraphyletic with respect to oystercatchers and
stilts. The Egyptian Plover is not a member of the Glareolidae, but is instead relatively basal among
Charadrii. None of the putative sisters of Charadriiformes were recovered as such.
Conclusion: Hypotheses of non-monophyly and sister relationships of shorebirds are tested by
multilocus analysis. The monophyly of and interfamilial relationships among shorebirds are
confirmed and refined. Lineage-specific differences in evolutionary rates are more consistent across
loci in shorebirds than other birds and may contribute to the congruence of locus-specific
phylogenetic estimates in shorebirds.
Background
The order Charadriiformes is one of relatively few exam-
ples in which the phylogenetic relationships of a major
higher-level clade of birds are becoming successfully
resolved [1,2]. The order includes what have traditionally
been known as the shorebirds, a diverse and apparently
ancient group of non-passerine birds whose three subor-
ders are estimated to have diverged from one another in
the Cretaceous [3]. Earlier morphological and biochemi-
cal analyses produced conflicting pictures of shorebird
phylogeny. Morphological and biochemical studies were
in general agreement as to recognition of the suborders
Charadrii, Scolopaci, and Lari as clades. However, mor-
phological studies also recognized the Alci as distinct
[4,5], whereas DNA-DNA hybridization placed them near
gull-like birds in the Lari. In contrast, recent molecular
studies sampling both nuclear and mitochondrial
sequences have generated a remarkably consistent and
highly-resolved interfamilial tree for Charadriiformes
[2,3,6,7].
Published: 8 March 2007
BMC Evolutionary Biology 2007, 7:35 doi:10.1186/1471-2148-7-35
Received: 25 September 2006
Accepted: 8 March 2007
This article is available from: http://www.biomedcentral.com/1471-2148/7/35
© 2007 Fain and Houde; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0
),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
BMC Evolutionary Biology 2007, 7:35 http://www.biomedcentral.com/1471-2148/7/35
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While the monophyly of Charadriiformes is popularly
accepted, it has been questioned both implicitly and
explicitly. Olson and Feduccia hypothesized that charadri-
iform stilts (i.e., Recurvirostridae) were ancestral to both
waterfowl (Anseriformes) and flamingos (Phoenicopteri-
dae) based on their interpretation of fossils and compara-
tive anatomy [8-11]. Like some early anatomists of the
19
th
century, Olson portrayed the "gruiform" bustards
(Otididae) as Charadriiformes, closely related to the
coursers (Glareolidae) and in particular to the Egyptian
Plover [10]. He further advocated an ill-defined relation-
ship of ibises (Ciconiiformes: Threskiornithidae) "unit-
ing" Gruiformes and Charadriiformes [10,12]. Sibley et al.
found rails (Rallidae) to be statistically inseparable from
both Gruiformes and Charadriiformes using DNA-DNA
hybridization [13].
These hypotheses have been discredited piecemeal in
recent years. Specifically, there exists strong evidence for
the sistership of waterfowl and fowl as Galloanserae, the
sister to Neoaves [14-16]. There is also strong evidence for
a clade of flamingos and grebes [17,18] within Metaves,
one of two hypothesized basal clades of Neoaves (the
other being Coronaves, to which shorebirds belong)
[7,19]. All subsequent studies have upheld the novel
transfer of both Australian Plains-wanderer (Pedionomi-
dae) and buttonquails (Turnicidae) from the order Grui-
formes to Charadriiformes [2,3,7,20,21].
However, many of the proposed interrelationships of
gruiform, charadriiform, and ciconiiform taxa have not
yet been explicitly tested in a comprehensive molecular
phylogenetic framework with both evidence from multi-
ple independent loci and comprehensive taxon sampling.
Gruiformes or sandgrouse (Pterocliformes) have been
cited most commonly as potential sister groups of
Charadriiformes and representative members of these
groups have generally been used to root a presumed
monophyletic Charadriiformes. The monophyly of
Charadriiformes has been tested only with limited taxon
sampling [6]; with incomplete DNA-DNA hybridization
matrices [21]; or with single-locus studies [7]. In the proc-
ess of studying the phylogenetic relationships of Grui-
formes [22]we had the opportunity to characterize and
analyze more than 5 kb of DNA sequences from four loci
(mitochondrial and three nuclear) from a variety of puta-
tive sister groups of Gruiformes, including multiple repre-
sentatives of most families of Charadriiformes. Our novel
data include intronic and exonic sequences from beta-
fibrinogen, alcohol dehydrogenase-1, and glyceraldehyde-3-
phosphate dehydrogenase. These map to chromosomes 4
(position 20,917K), 4 (position 60,497K), and 1 (posi-
tion 71,016.5K), respectively, in chicken. We present the
results of phylogenetic analyses of these loci for Charadri-
iformes and compare our results to those of others, who
independently studied the relationships of Charadrii-
formes using DNA sequences from two other nuclear loci
(myoglobin chromosome 1, position 48,720.4K, and RAG-
1 chromosome 5, position 16,597.6K in chicken) and
nearly complete mitochondrial genomes [2,3,6]. We also
test the monophyly and sister relationships of Charadrii-
formes by multi-locus sequence analysis including all the
aforementioned putative ingroups or sister groups.
Of note, no single molecular phylogenetic analysis of
Charadriiformes has yet included representatives of all its
member families. The monotypic Ibisbill (Ibidorhynchi-
dae) has yet to be studied by anyone, although it is gener-
ally presumed to fall within the Charadrii, near stilts.
Paton et al. [3] and Paton & Baker [2] lacked DNA
sequences of Ibisbill and the monotypic Crab Plover
(Dromadidae). Ericson et al. [6] lacked these as well as
buttonquails (Turnicidae) and the monotypic Australian
Plains-wanderer (Pedionomidae). Thomas et al.
[23]lacked eight of the traditionally recognized charadrii-
form families in their study of mitochondrial cytochrome-b
DNA sequences. Likewise, we were unable to include Ibis-
bill, Crab-Plover, sheathbills (Chionididae), and the
genus Pluvianellus, the last of which is not a member of the
family Charadriidae in which it is traditionally included
[3]. Our results corroborate numerous novel family-level
relationships reported in the aforementioned recent stud-
ies, including the positions of the traditional gruiform
Turnicidae basal to a clade of glareolids, larids, alcids and
relatives (suborder Lari). In the present study, we include
two putative representatives of Glareolidae, the Double-
banded Courser (Rhinoptilus africanus) and the Egyptian
Plover (Pluvianus aegyptius). Pluvianus has not been
included previously in molecular analyses, and in contrast
to traditional classifications, we conclude that this genus
is distinct from the Glareolidae, with closer relations
within the Charadrii than the Lari.
Last, some interfamilial relationships have not been well-
resolved in previous single-locus nuclear or mitochon-
drial molecular data sets. For example, myoglobin intron 2
and RAG-1 suggested that Recurvirostridae and Haemat-
opodidae may be nested within Charadriidae, rendering
the latter paraphyletic. Also unresolved is whether skim-
mers ("Rynchopidae") are sister to either gulls (Larinae)
or terns (Sterninae) or both [2,3,6]. Further, a potential
conflict in topology exists between myo-2 and RAG-1 [6]
as to whether Jacanidae is sister to Thinocoridae (seed-
snipes) or to Rostratulidae (painted-snipes). The data at
hand address these relationships.
Results
Molecular Characterization
We sampled four independent, presumably unlinked loci
to test recent novel hypotheses of relationships within
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Charadriiformes and to test monophyly of the group com-
prehensively, particularly given our interest in proposed
interrelationships between shorebirds, various families
traditionally recognized as Gruiformes, and other groups.
For taxa sampled see Table 1. Having a variety of loci with
differing substitution rates and evolutionary properties is
particularly desirable in a case such as this, wherein the
relationships which interest us potentially span 60 to 80
million years. These include potentially relatively recent
divergences between shorebird sister families to diver-
gences between orders in the late Cretaceous [3]. Hence
our sampling includes relatively rapidly-evolving mito-
chondrial loci; nuclear introns which have proven useful
within a number of vertebrate clades including mammals,
birds, and snakes; and relatively constrained nuclear
exons, which may change slowly enough to retain some
phylogenetic signal deep within the tree.
The alignment of FGB7 is 1457 sites in length for the 48
included taxa (Table 2); 531 sites (36%) were removed
before analysis. This large percentage of sites should not
be taken to indicate overall difficulty in alignment. Most
of the removed sites (316 aligned positions) were due to
large apomorphic insertions in seedsnipes (Thinocoridae)
and Plains-wanderer (Pedionomidae). In fact, despite
length differences between sequences of Attagis, Thinoc-
orus, and Pedionomus, the alignment of these large inser-
tions could be taken as further evidence of the close
relationship of these two families. This intron is relatively
long with a slight excess of A and T nucleotides and rela-
tively even rates among substitution types. Also, most
sites are free to vary, allowing accumulation and retention
of more phylogenetic signal per site in comparison to pro-
tein-coding sequences [24,25]. These general evolutionary
properties explain why FGB7 is becoming widely used in
avian systematics and has been successfully employed for
even the deepest levels of avian phylogeny [7,19,26]. The
nucleotide substitution models selected for FGB7 were
HKY+G (hLRT) or TVM+ G (AIC) (Table 3). The latter is a
more general case of the former, with each of the four
transversion substitution types having a different rate. We
analyzed the data with the less complex HKY model to
minimize variance associated with estimating the addi-
tional rate parameters. However, an analysis with the
more complex model did not substantively change the
result (not shown).
ADH5 shares many of the desirable properties of FGB7,
such as even base composition and relatively uniform
rates among sites (Table 2). The overall alignment is 851
sites, and 148 (17%) were removed as autapomorphic
insertions or ambiguously-aligned. Of the remaining
alignment, 207 sites were retained from the flanking
exons. No significant heterogeneity in base composition
was found among lineages for either the intron or any of
the codon positions. Model selection for the entire align-
ment chose either the Tamura-Nei model with equal
nucleotide frequencies and gamma-distributed rate varia-
tion (TNef+G; hLRT) or the GTR+G model (AIC) (Table
3). In this case, we chose a "compromise" model by relax-
ing the assumption of equal base frequencies, because the
combination of intron and exon partitions masks some
nucleotide variation between them, and within exons at
each of the codon positions. In practice, this makes very
little difference to the analysis because of the small
number of and relatively low divergence of exon sites.
The GPD3-5 alignment is the most heterogeneous of the
nuclear sequences, consisting of introns 3, 4, and 5, and
exons 4 and 5 (Table 2). The introns, in sum, consist of
713 aligned positions, and the exons are 198 bp in length.
Of the total 911 aligned sites, 227 (25%) were removed
prior to phylogenetic analysis. GPD3-5 introns were short
compared to those from ADH-I or fibrinogen, and the most
problematic with respect to reliable alignment. The high
proportion of sites removed reflects the fact that polypyri-
midine tracts near the end of the introns made up a greater
proportion of total intronic length in GPD3-5.
We plotted relative rates among partitions of characters
that should be nearest to the ideal of neutral substitution
(Figure 1). This revealed that GPD3-5 intron substitutions
were evolving slightly faster than the other two introns
sampled in this study, while the rates of ADH5 and FGB7
were virtually identical. For this analysis, we also included
the 930-bp fragment of RAG-1 and myo-2 analyzed by
Ericson et al. (2003) for genera which overlapped in our
study (a total of 17 taxa). Surprisingly, third positions in
RAG-1 evolved faster than any of the introns, and myo-2
was actually the slowest of the introns. Not surprisingly,
transition:transversion saturation plots revealed that rates
of mitochondrial transitions far exceeded those of all
other partitions (Figure 2).
The mitochondrial data set included genes encoding three
structural RNAs, 12S rRNA, Valine tRNA, and 16S rRNA
(Table 2). The total alignment of 48 taxa consisted of
2903 sites, of which 674 (23%) were excluded due to
ambiguous alignment. Model selection resulted in the
most complex available model in ModelTest, with six sub-
stitution types, and a proportion of invariant sites plus a
gamma distribution of rate variation among sites free to
vary (GTR+I+G) (Table 3). Despite the fact that rDNA
sequences are generally considered the slowest evolving
mitochondrial genes and therefore appropriate for deeper
divergences, their component sites actually vary consider-
ably in rate with 37% of sites estimated to be invariable
and Chi-Square = 0.39. Typical saturation plots showed
no decline in the slope of transition or transversion dis-
tances with total distance, but divergences among some
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ingroup taxa were greater than ingroup-outgroup compar-
isons. Base composition is relatively even within the
rDNAs. For example, there is a higher percentage of G
nucleotides than expected based on overall mitochondrial
base composition. However, this apparent evenness
masks heterogeneity among sites, particularly between
stem and loop sites. When base composition is calculated
only for variable sites, a more usual mitochondrial signa-
ture is seen, with an excess of A and C, and G underrepre-
sented. This uneven composition at variable positions,
Table 1: Taxon sampling and GenBank accession numbers for loci
GenBank accession numbers
Higher Taxon Species 12S+Val 16S FGB7 ADH5 GPD3-5
Galliformes Callipepla gambelii [DQ485791] [DQ485829][DQ494145] [DQ485865] [DQ485912]
Anseriformes Anseranas semipalmata [DQ674553
] [DQ674593] [AY695132] [DQ485866] [DQ485913]
Phoenicopteriformes Phoenicopterus ruber [DQ674554
] [DQ674594] [AY695139] [DQ674631] [DQ674666]
Podicipediformes Aechmophorus sp [DQ674555
]N/A [AY695146] N/A [DQ674667]
Podylimbus podiceps [DQ674556
] [DQ674595] [AY695145] [DQ674632] [DQ674668]
Pterocliformes Pterocles bicintus [DQ674558
] [DQ674597] [AY695147] [DQ674634] [DQ674670]
Syrrhaptes paradoxus [DQ674559
] [DQ674598] [AY695148] [DQ674635] [DQ674671]
Mesitornithiformes Mesitornis unicolor [DQ674557
] [DQ674596] [AY695144] [DQ674633] [DQ674669]
Otidiformes Afrotis afra [DQ674591
] [DQ674629] [AY695149] [DQ674664] [DQ674699]
Ardeotis kori [DQ674590
] [DQ674628] [AY695150] [DQ674663] [DQ674698]
Eupodotis senegalensis [DQ674592
] [DQ674630] [AY695152] [DQ674665] [DQ674700]
Tetrax tetrax [DQ674589
] [DQ674627] [AY695151] [DQ674662] [DQ674697]
Ciconiiformes Plegadis chihi [DQ674561
]N/A [AY695215] [DQ674637] [DQ674673]
Ajaia ajaja [DQ674560
] [DQ674599] [AY695214] [DQ674636] [DQ674672]
Gruiformes Grus canadensis [DQ485815
] [DQ485853] [AY082410] [DQ485879] [DQ485925]
Fulica americana [DQ485827
] [DQ485863] [AY695244] [DQ485887] [DQ485933]
Charadriiformes
Burhinidae Burhinus bistriatus [DQ674587
] [DQ674625] [AY695198] [DQ674660] [DQ674695]
Charadriidae Charadrius vociferus [DQ485792
] [DQ485830] [AY695205] [DQ48586] [DQ485914]
Pluvialis dominica [DQ674562
] [DQ674600] [AY695201] [DQ674638] [DQ674674]
Vanellus resplendens [DQ674565
] [DQ674603] [AY695206] [DQ674641] [DQ674676]
Haematopodidae Haematopus palliatus [DQ674563
] [DQ674601] [AY695204] [DQ674639] [DQ674675]
Recurvirostridae Himantopus mexicanus [DQ674564
] [DQ674602] [AY695203] [DQ674640] [DQ485915]
Recurvirostra americana [DQ485793
] [DQ485831] [AY695202] [DQ485868]N/A
Turnicidae Turnix varia [DQ674575
] [DQ674613] [AY695197] [DQ674649] [DQ674685]
Glareolidae Rhinoptilus africanus [DQ674574
] [DQ674612] [AY695196] [DQ674648] [DQ674684]
Pluvianus aegyptius [DQ674588
] [DQ674626] [AY695199] [DQ674661] [DQ674696]
Stercorariidae Stercorarius pomarinus [DQ674573
] [DQ674611] [AY695195] N/A N/A
Alcidae Cepphus columba [DQ674572
] [DQ674610] [AY695193] N/A [DQ674683]
Uria aalge [DQ485794
] [DQ485832] [AY695192] [DQ485869] [DQ485916]
Rynchopidae Rynchops niger [DQ674567
] [DQ674605] [AY695191] [DQ674643] [DQ674678]
Laridae Larus atricilla [DQ485795
] [DQ485833] [AY695186] [DQ485870] [DQ485917]
Larus occidentalis [DQ674566
] [DQ674604] [AY695185] [DQ674642] [DQ674677]
Sterna antillarum [DQ674568
] [DQ674606] [AY695190] [DQ674644] [DQ674679]
Sterna caspia [DQ674569
] [DQ674607] [AY695188] [DQ674645] [DQ674680]
Sterna forsteri [DQ674570
] [DQ674608] [AY695187] [DQ674646] [DQ674681]
Sterna maxima [DQ674571
] [DQ674609] [AY695189] [DQ674647] [DQ674682]
Scolopacidae Actitis macularius [DQ674579
] [DQ674617] [AY695182] [DQ674653]N/A
Gallinago gallinago [DQ674576
] [DQ674614] N/A [DQ674650] [DQ674686]
Limosa fedoa [DQ674577
] [DQ674615] [AY695180] [DQ674651] [DQ674687]
Limnodromus sp [DQ674578
] [DQ674616] [AY695183] [DQ674652] [DQ674688]
Phalaropus tricolor [DQ674581
] [DQ674619] [AY695184] [DQ674655] [DQ674690]
Tringa melanoleuca [DQ674580
] [DQ674618] [AY695181] [DQ674654] [DQ674689]
Jacanidae Actophilornis africanus [DQ674582
] [DQ674620] [AY695178] [DQ674656]N/A
Jacana spinosa [DQ485796
] [DQ485834] [AY695179] [DQ485871] [DQ485918]
Rostratulidae Rostratula benghalensis [DQ674583
] [DQ674621] [AY695177] [DQ674657] [DQ674691]
Thinocoridae Attagis gayi [DQ674584
] [DQ674622] [AY695175] [DQ674658] [DQ674692]
Thinocorus orbignyanus [DQ674585
] [DQ674623] [AY695176] [DQ674659] [DQ674693]
Pedionomidae Pedionomus torquatus [DQ674586
] [DQ674624] [AY695174] N/A [DQ674694]
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combined with significant rate heterogeneity among sites
and constraints on paired stem sites, suggests that at
deeper levels, multiple substitutions at those sites free to
vary may obscure phylogenetic signal. Nevertheless, the
inferred phylogeny was largely congruent with estimates
based on present and previous data sets (see below).
Phylogenetic Reconstruction
Maximum likelihood, mixed-model Bayesian, and maxi-
mum parsimony phylogenetic analyses of the combined
data sets (Figure 3) are highly supported by bootstrap and
posterior probabilities at most nodes. Relationships based
on the combined data sets strongly support monophyly of
Charadriiformes, and division of the order into three
major clades including 1) Scolopaci (Scolopacidae, Jaca-
nidae, Rostratulidae, Thinocoridae; and Pedionomidae)
and its sister, 2) Lari (Laridae, Alcidae, Stercorariidae,
Glareolidae [i.e., Rhinoptilus, not Pluvianus], and Turnici-
dae), and 3) their combined sister Charadrii (Burhinidae,
Pluvianus, Charadriidae, Haematopodidae, and Recurvi-
rostridae). Within Charadrii, Charadriidae (i.e., Pluvialis)
is paraphyletic to Haematopodidae plus Recurvirostridae.
Table 3: Models and parameters
R- Matrix
locus model Pinv alpha A-C C-T A-G A-T C-G G-T
ADH-I intron 5 (with partial flanking exons)
TN93+GNA1.411.03.341.0 1.05.09 1
GAPD-H exons 4–5 and introns 3–5
TN93+I+G 0.20 1.96 1.0 5.04 1.0 1.0 7.08 1
FGB intron 7
HKY85+G NA 5.04 1.0 4.03 1.0 1.0 4.03 1
combined nuclear DNA
TN93+GNA1.701.03.921.0 1.04.98 1
combined mtDNA
GTR+I+G 0.37 0.39 4.50 19.25 3.09 0.34 60.68 1
total combined data
GTR+I+G 0.10 0.55 1.21 4.89 0.80 1.01 9.55 1
Table 2: Characteristics of loci
mean base frequencies
1
heterogeneity of base frequencies
2
locus # aligned sites (# analyzed) # PI sites
1
A C G T %AT Chi square df P value
ADH-I intron 5 (including partial flanking exons)
851 (703) 306 0.27 0.24 0.24 0.25 52 81.80 129 1.00 (NS)
ADH-I partial exons 5,6 (69 codons:1
st
position 17 variable, 2
nd
position 4 variable, 3
rd
position 44 variable)
207 (207) 57 0.25 0.24 0.28 0.23 48 52.18 129 1.00 (NS)
ADH-I intron 5
644 (499) 249 0.31 0.22 0.24 0.23 54 66.21 129 1.00 (NS)
GAPD-H exons 4–5 and introns 3–5
911 (684) 316 0.23 0.20 0.33 0.24 47 104.64 129 0.94 (NS)
GAPD-H exons 4–5 (65 codons: 1
st
position 5 variable, 2
nd
position 5 variable
2
, 3
rd
position 35 variable)
196 (196) 23 0.25 0.22 0.27 0.26 51 35.46 129 1.00
3
(NS)
GAPD-H introns 3–5
713 (486) 292 0.20 0.22 0.33 0.32 44 96.41 129 0.99 (NS)
FGB intron 7
1475 (926) 487 0.30 0.18 0.19 0.33 63 36.35 138 1.00 (NS)
combined mtDNA (12S rDNA, Valine-tDNA, 16S rDNA)
2903 (2229) 659 0.33 0.25 0.21 0.21 54 135.95 135 0.46 (NS)
12S rDNA
1074 (851) 271 0.31 0.26 0.22 0.21 52 79.23 141 1.00 (NS)
16S rDNA
1753 (1318) 375 0.34 0.24 0.20 0.22 56 94.30 135 1.00 (NS)
valine tDNA
76 (60) 15 0.36 0.21 0.19 0.24 60 59.05 135 1.00 (NS)
1
number remaining after ambiguously aligned sites removed
2
base composition heterogeneity based on variable sites only
3
2
nd
position P = 0.94 (NS)
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Within Scolopaci, Rostratulidae and Jacanidae are sisters,
and Phalaropus is sister to Tringa among those genera stud-
ied within Scolopacidae. These data provide further evi-
dence that the "gruiform" buttonquails (Turnicidae) are
sister to the remaining Lari, following Glareolidae (i.e.,
Rhinoptilus), then Alcidae, and last terns, gulls, and skim-
mers.
The combined data are also notable for what they do not
support. None of the proposed relationships between
shorebirds and taxa not considered charadriiform (in tra-
ditional classifications) are corroborated, with the excep-
tions of the "gruiform" Turnicidae and Pedionomidae.
Bustards (Otididae) are not near Burhinidae, in particular,
nor plovers, in general. The sister of the rails is not to be
found among Jacanidae or indeed any other shorebird
taxon. Ibises (Threskiornithidae) do not "link" Charadrii-
formes and Gruiformes. A sister relationship between
sandgrouse (Pteroclidae) and Charadriiformes is also not
found.
All three nuclear loci, ADH5, GPD3-5, and FGB7, analyzed
independently or in combination yield virtually identical
phylogenetic reconstructions (not shown). Individually,
there is moderate conflict among the nuclear loci with
respect to the position of Burhinidae. FGB7 supports the
placement of Burhinus as sister to Lari plus Scolopaci with
75% bootstrap in ML analysis, but weak support (57%) in
MP analysis. ADH5 does not resolve the position of Burhi-
nus with respect to other charadriiform lineages, while
GPD3-5 strongly supports it as sister to Charadrii, which
is also the result of the combined analysis. All three data
Substitution rates per locusFigure 1
Substitution rates per locus. Pairwise distances of each of five non-coding partitions of nuclear loci plotted against com-
bined pairwise distances with linear model regressions added, showing differences in evolutionary rates among loci. Closed dia-
monds, RAG-1 3
rd
positions; open squares, GPD3-5; closed triangles, ADH5; open circles, FGB7; and open triangles, myo-2. Note
faster rate of RAG-1 3
rd
positions than introns.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14
RAG-1, 3
rd
pos
myo-2
GAPD3-5
FGB7
ADH5
total percent divergence
pairwise distance
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sets, individually or combined, support the Egyptian
Plover (Pluvianus aegyptius) as sister to plovers in ML and
MP analyses, rather than to the other glareolid, Rhinop-
tilus.
Our ~3.5 kb of mitochondrial data alone recovered pre-
cisely the same higher-level charadriiform relationships as
did nuclear data alone, but with no bootstrap support to
vouchsafe either the positions or monophyly of the three
major clades. However, > 12 kb of mtDNA does recover
bootstrap support all these clades [2]. Conclusions sup-
ported by our smaller mtDNA data set include the non-
monophyly of Glareolidae, with Pluvianus nearer to
Charadrii than to Lari. Alone, our mtDNA data supported
Burhinus and Pluvianus as sister taxa (albeit with no boot-
strap support), a relationship that has been suggested
based on morphological data. The observation that boot-
strap support for this pair is higher in MP than in ML anal-
yses might suggest that long-branch attraction may be
playing a role in uniting these two relatively basal line-
ages.
Discussion
High bootstrap values clearly point to the monophyly of
Charadriiformes, in spite of the inclusion here of taxa that
have been suggested to be ingroups or sisters of Charadri-
iformes. Not surprisingly, waterfowl are recovered as sister
to fowl, rather than among Neoaves. Flamingos are sister
to grebes and sandgrouse are sister to mesites (Mesitorni-
thidae) among the taxa we sampled, and all are recovered
as monophyletic, consistent with their interpretation as
members of Metaves [7,19]. Metaves and Coronaves are
hypothesized basalmost sister clades of Neoaves, whose
convergent members have in some cases been classified in
polyphyletic orders. Among Coronaves, the remaining
putative relatives of Charadriiformes are all found to be
closer to one another than to Charadriiformes. Ibises are
sister to spoonbills, and they in turn are sister to a clade of
Transition:transversion plotsFigure 2
Transition:transversion plots. Uncorrected transition and transversion pairwise distances plotted against total distance for
each of four loci obtained from combined MP analysis, drawn to same scale. (a), ADH5; (b), FGB7; (c), GPD3-5; and (d), 16S
rDNA, 12SrDNA, and tRNA Valine. Closed circles, transition substitutions; and open circles, transversion substitutions. Note
accelerated rate of transition substitutions of mtDNA.
0
50
100
150
0 0.05 0.1 0.15 0.2 0.25
ADH5 % divergence
0
50
100
150
200
250
0 0.05 0.1 0.15 0.2 0.25
FGB7 % divergence
0
50
100
150
200
250
0 0.05 0.1 0.15 0.2
mtDNA % divergence
0
50
100
150
0 0.05 0.1 0.15 0.2
GPD3-5 % divergence
ab
c
d
pairwise distance
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Phylogeny of CharadriiformesFigure 3
Phylogeny of Charadriiformes. Optimal maximum likelihood phylogenetic reconstruction of Charadriiformes and selected
outgroups based on combined data of ADH5, GPD3-5, FGB7, 12S rDNA, 16S rDNA, and tRNA Valine using GTR + G. Both mixed
model Bayesian analysis and maximum parsimony produce trees of identical topology. Bootstrap values obtained from 500 ML
pseudoreplicates are indicated above branches or positioned by arrows. Asterisks indicate bootstrap values of 100%. Charadri-
iformes are indicated by bold font and subordinal epithets.
*
75
80
*
*
92
91
*
86
*
95
*
99
99
94
88
*
*
72
*
*
99
99
99
*
*
*
*
*
*
99
*
61
99
94
*
*
*
87
98
*
94
72
*
0.1 expected substitutions per site
Callipepla
Anseranas
Mesitornis
Syrrhaptes
Pterocles
Phoenicopterus
Aechmophorus
Podilymbus
Eupodotis
Afrotis
Ardeotis
Tetrax
Fulica
Grus
Plegadis
Ajaia
Burhinus
Pluvianus
Vanellus
Charadrius
Pluvialis
Haematopus
Recurvirostra
Himantopus
Pedionomus
Thinocorus
Attagis
Rostratula
Jacana
Actophilornis
Limosa
Gallinago
Limnodromus
Actitis
Phalaropus
Tringa
Turnix
Rhinoptilus
Stercorarius
Uria
Cepphus
Larus occidentalis
Larus atricilla
Rynchops
Sterna antillarum
Sterna caspia
Sterna maxima
Sterna forsteri
Charadrii
Scolopaci
Lari
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Residual plotsFigure 4
Residual plots. Residual plots of internodal distances of each of four nuclear loci obtained from regression against RAG-1
internodal distances (independent variable) on MP tree reconstructed from combined data sets of 5 nuclear loci. (a, b), FGB7;
(c, d), ADH5; (e, f), GPD3-5; and (g, h), myo-2. Note the higher variance of residuals in non-Charadriiformes (left panel) than
Charadriiformes (right panel), indicating better correlation of estimates of internode lengths in the latter across all loci.
-80
-60
-40
-20
0
20
40
60
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100 120 140 160 180
-80
-60
-40
-20
0
20
40
60
80
40 60 80 100 120 140 160 180
-80
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80
20 40 60 80 100 120 140 160 180
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-20
0
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60 80 100 120 140 160 180
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0
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20 40 60 80 100 120 140 160 180
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60 80 100 120 140 160 180
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non-Charadriiformes Charadriiformes
a
fe
dc
b
gh
FGB7 ResidualsADH5 ResidualsGPD3-5 Residualsmyo-2 Residuals
RAG-1 internode and terminal branch distances
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rails plus cranes. Bustards, too, are among this group.
Somewhat ironically, this begs the question of what is the
true sister of Charadriiformes, but the answer is not forth-
coming from the present data set. Ongoing analysis of a
larger sample of loci suggests that Charadriiformes are sis-
ter to all other coronavian waterbirds (not shown).
Other recent DNA studies of intraordinal charadriiform
phylogeny are in agreement with the results presented
here, lending credibility to the phylogenetic signal present
in our data and vice versa. Two other nuclear loci, RAG-1
and myo-2 [3,6] and nearly complete mitochondrial
genomes [2] yield trees of nearly perfect congruence to
those of this analysis to the extent that taxa overlap. In
particular, 1) Charadriiformes are comprised of three sub-
orders, Lari, Scolopaci, and their sister Charadrii, 2) Alci-
dae is nested well within the Lari rather than basal among
Charadriiformes as was suggested on morphological crite-
ria [4,5,27], 3) Turnicidae are recovered as Charadrii-
formes, sister to Lari. When analyzed alone, myo-2
produced some conflicts with our data [6]. Specifically,
our data do not corroborate a MP recovery of 1) a sister-
ship between Alcidae and Glareolidae to the exclusion of
other Lari, 2) a sistership of Rynchops and Larinae to the
exclusion of Sterninae, and 3) a sistership of Jacanidae
and Thinocoridae to the exclusion of Rostratulidae. These
myo-2 results are also incongruent with those obtained
from RAG-1. These differences are minor, and may be
attributed to three causes. myo-2 is relatively short com-
pared to the other introns we studied and RAG-1 third
positions, thus there may be a higher stochastic affect on
its fewer sites. This effect may be compounded by lower
rates of nucleotide substitution in myo-2 than in the other
loci. Alternatively, these could represent validly recon-
structed gene trees that differ due to incomplete lineage
sorting.
One recent study based solely on mtDNA cytb produced
markedly contrasting results that we consider problem-
atic[28]. The authors analyzed two data sets: the "pri-
mary" data set, which included 41 complete or largely
complete gene sequences, and the "expanded" data set,
which included an additional 50 partial sequences. They
claim to have found four major clades of Charadriiformes
(Charadrii, Scolopacii, Lari, and Alci). In fact, all of their
reconstructions show Lari as paraphyletic to Alci (in agree-
ment with the present study), so the latter should not be
considered distinct. The finding in their primary data set
that Charadrii is paraphyletic to other Charadriiformes is
not well supported and contradicts all other DNA
sequence studies [2]. Even more problematic, the
expanded data set recovered polyphyletic relationships of
indisputably monophyletic lower-level clades, e.g., within
Lari (Sterna sister to Glareolidae in MP tree or in a clade
including Jacanidae plus Rostratulidae in the Bayesian
tree) and Charadriidae (the genus Vanellus is included in
the Scolopacidae in the MP tree). Furthermore, Stercorar-
iidae are recovered as members of the Alcidae in the MP
tree but as sister to Lari plus Alcidae in the Bayesian tree.
These incongruencies receive no statistical support from
bootstrap analysis, and a Lento plot showed that conflict
equaled or exceeded support for most clades. Indeed, the
splits having the highest support/conflict ratios were
either congruent with our results and previous studies
(e.g., monophyly of a clade containing Charadrius, Hae-
matopus, and Recurvirostra) or represented closely related
taxa (e.g., species within genera).
Spurious associations of taxa from analyses of the
expanded cytb data set may be explained as attraction
between non-overlapping 5-prime and 3-prime partial
sequences (e.g., 5-prime sequence for Sterna and 3-prime
sequence for jacanids). More insidious problems with the
cytb locus for phylogenetic reconstruction are issues of
possible substitutional saturation and base composition
bias. Thomas et al. [28]report that they detected no non-
stationarity of base composition, nor did they apparently
assess the potential for saturation. We conducted a Chi-
Square test of their primary data set that shows third posi-
tions of codons to be significantly biased (Chi-Square =
151.267077, df = 120, P = 0.028; outgroups excluded).
We suggest that the difference in our results accrues from
partitioning the data by codon position and potentially
our exclusion of constant sites (Thomas et al. did not pro-
vide details on how they conducted the test). The disparity
index test in MEGA further revealed that 10.8% of all pair-
wise comparisons were significantly heterogeneous across
all codon positions in sequences of the primary data set
(outgroups excluded). We also note that two of their jaca-
nid sequences include a deletional frameshift, suggesting
that the sequences may represent nuclear pseudogenes if
they are free of errors.
Inclusion of cytb sequences in our own concatenated data
set resulted in reduced support for most clades, even
though the same phylogenetic relationships were gener-
ally recovered (not shown). Moreover, Paton & Baker [2]
found that cytb performed more poorly than most other
mitochondrial genes in recovering charadriiform phylog-
eny. In contrast, they found that 12S rDNA alone recov-
ered 12 of 19 nodes and 16S rDNA recovered 8 of 19
nodes in the combined mitochondrial tree. We found that
the rDNAs together were able to recover 15 of 17 nodes
listed by Paton and Baker [2], while the difference results
only from incomplete taxonomic overlap between our
study and theirs.
DNA-DNA hybridization [21] yielded a similar topology
for Charadriiformes as a whole, with exceptions as already
noted by Paton et al. [3] on the monophyly of Thinocori-
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dae plus Rostratulidae plus Jacanidae, the inclusion or
direct sistership of Pteroclidae, and the exclusion of Turni-
cidae. Unfortunately, Sibley & Ahlquist did not publish a
record of what subsets of taxa were used in pairwise com-
parisons to produce their supertrees.
Supertree analysis [23] based on morphology, DNA-DNA
hybridization, RAG-1, myo-2, and cytb is in near perfect
agreement with the combined analysis of the 4 loci here,
with the one exception of our recovery of Charadriidae as
paraphyletic to Haematopodidae and Recurvirostridae
that was also recovered in the study of myo-2. The super-
tree study did not include Turnicidae, and its authors
asserted that further work was needed to establish their
affinities[23]. Additional evidence for the placement of
Turnicidae within Charadriiformes provided by this
study, Fain & Houde [7], and Paton & Baker [2] suggests
this question is now irrefragably resolved.
myo-2, FGB7, ADH5, and GPD3-5, whether analyzed sep-
arately or combined, all recovered Charadriidae as para-
phyletic to Haematopodidae plus Recurvirostridae [6].
RAG-1 produced results that are consistent with these, but
sequence was unavailable for the genus Pluvialis, which
was essential in demonstrating charadriid paraphyly in all
of the other data sets. Reciprocal monophyly of Charadri-
idae and Haematopodidae plus Recurvirostridae in the
Thomas et al. supertree is clearly biased by the relative
abundance of taxa for which only morphological data
were available in that study. With bootstrap support as
high as 100% in our complete molecular data set, the par-
aphyly of Charadriidae is a hypothesis that warrants seri-
ous attention.
The position of skimmers has not been consistently
resolved in previous studies. Skimmers are traditionally
placed in their own family Rynchopidae, sister to both
terns and gulls (Laridae: Sterninae and Larinae, resp.).
Ericson et al. [6] recovered this relationship with little or
no support, but Paton et al. [3] obtained some support
(Bayesian posterior probability 87%) for the sistership of
skimmers to gulls alone. In contrast, our nuclear data
strongly support a sister relationship of skimmers to terns
alone (MP bootstrap = 100%), and this result is robust to
combined analysis with RAG-1 and myo-2.
The Egyptian Plover (Pluvianus aegyptius) is traditionally
placed within the family Glareolidae, although it has
always been acknowledged as being atypical of the family.
Some authors have suggested that the Egyptian Plover is
most closely related to stone curlew (Burhinidae) based
on osteological characters [4,5]. Dove [29] further noted
it is atypical of the Glareolidae in microscopic feather
characters. It shares with Rhinoptilus cinctus the peculiar
habit of incubating its eggs by burying them in sandy soil
[30]. At times, it has been given its own family rank, Plu-
vianidae [21,30].
Despite its distinctiveness as an adult, Lowe concluded
that the Egyptian Plover was "obviously an advanced
courser" on the basis of its natal plumage [31]. Jehl con-
cluded instead that " [t]he color pattern of the Egyptian
Plover chick is plover-like [32]" as is the relative length of
the tarsus [to wing] and lack of pectination of middle toe
of the adult. He further asserted that its tarsal scutellation
and relative lack of flattening of anterior toes was interme-
diate between those of glareolids and "charadriines."
Others have questioned the placement of the Egyptian
Plover in the Glareolidae, and it has even been afforded its
own familial status by some [33]. Strauch [4], Mickevich
& Parenti [34], and Chu [5] each studied variants of the
same osteological data set using different methods of
analysis. All concluded that the Egyptian Plover is sister to
stone curlew, although they differed on whether the Egyp-
tian Plover-stone curlew clade is closest to gulls, coursers,
or to plovers. Thomas et al.'s supertree study positioned
Egyptian Plover outside of the Glareolidae, as sister to
Burhinus, on the basis of morphological characters alone.
The authors lamented that, "Morphological studies have
failed to resolve the position of Glareolidae, placing the
family in a large polytomy with all other major groups
except Alcinae and the sandpipers and allies[23]." We sur-
mise that these earlier difficulties may have arisen from
the polyphyletic nature of the Glareolidae with Egyptian
Plover included.
Our data provide no unequivocal evidence in support of a
special relationship between the Egyptian Plover and
stone curlews. Both are fairly basal among Charadrii; thus,
it is possible that the morphological characters they share
are merely symplesiomorphies. The nuclear data obtained
here, analyzed separately and in combination, strongly
suggest that the Egyptian Plover is sister to a clade of plov-
ers and allies. This phylogenetic position is consistent
with previous proposals that it merits family status as Plu-
vianidae [33]. We were unable to compare Egyptian
Plover to sheath bills, Crab Plover, and Ibisbill, and no
one else has made these direct comparisons either. It is
conceivable that these missing taxa could affect the group-
ing of Egyptian Plover with these or even other taxa
among Charadriiformes.
While none of the aforementioned DNA sequence or
hybridization studies included the Egyptian Plover, they
all agree that the Glareolidae is sister to jaegers, auks, and
gulls plus terns plus skimmers. The Glareolidae tradition-
ally includes two subfamilies, the coursers (Cursoriinae)
and pratincoles (Glareolinae), which are sufficiently dis-
tinct to cause some to question their monophyly [10].
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Coursers have slender bills, long legs, and stubby tails,
whereas pratincoles are swallow-like with short wide bills,
short legs, and gracefully long pointed wings and tail.
Despite these anatomical disparities, the monophyly of
coursers plus pratincoles was strongly supported by the
one DNA sequence study that included representatives of
both [3]. Our treatment of the Egyptian Plover in no way
challenges that result.
It is remarkable that the phylogeny of Charadriiformes is
so consistently and congruently recovered from a variety
of loci, when in general, higher level relationships among
Aves are notoriously difficult to resolve [35]. Even though
rates of evolution differ dramatically between different
lineages of Charadriiformes, it appears that these rates are
lineage specific and independent of locus. For example,
all loci are congruent in recovering buttonquails on a long
branch, yet short branches among sandpipers. Thus, we
reasoned that there might be lesser conflict between data
sets within Charadriiformes than among other birds. To
quantify this, we constructed a MP tree using a superma-
trix data set of FGB7, ADH5, GPD3-5, RAG-1, and myo-2.
Distances were then determined for all internodes and ter-
minals for each of the loci individually, and these were
regressed against one another using the RAG-1 distances
as the independent variable. The residuals obtained from
regressing each locus against those of RAG-1 were next
segregated and plotted on the basis of whether the
branches were within (fig. 4, right panel) or outside (fig.
4, left panel) the Charadriiformes clade.
The sample variance of the residuals for Charadriiformes
was in every case significantly smaller than for non-
Charadriiformes as determined by two-tailed sample var-
iance ratio test (Table 4). Similarly, the sample means of
the absolute values of the residuals (absolute because
residuals are both positive and negative) are significantly
smaller for Charadriiformes than non-Charadriiformes as
determined using the two-tailed normal approximation
to the Mann-Whitney Test [36]. The lesser mean and vari-
ance of residuals reflect the relatively higher correlation of
estimation of internodal branch lengths between data sets
within Charadriiformes, not shorter distances (although
note r
2
is smaller for Charadriiformes than non-Charadri-
iformes for myo-2). We interpret these statistics to indicate
that there is significantly less conflict in the nuclear locus
data sets among Charadriiformes than among non-
Charadriiformes.
It is important to note that the values used to produce Fig-
ure 4 represent globally optimized internodes rather than
measured pairwise distances between taxa. In this regard
we have avoided issues of autocorrelation of pairwise dis-
tances along shared branches. Furthermore, the lengths of
the internodes are not correlated with their depth in the
tree. This can be appreciated by the broad overlap of data
on the horizontal axes between shorebirds (fig. 4, right
panel) and non-shorebirds (fig. 4, left panel) in all but the
myo-2 locus.
If character conflict among data sets is reduced in
Charadriiformes, then it might make the reconstruction of
their phylogeny more tractable than those of many other
birds. After all, the extent to which various loci yield con-
gruent phylogenetic reconstructions of Charadriiformes is
presumably reflective of the degree to which each is
informative individually. The reason(s) that certain clades
should be more readily recovered than others in phyloge-
netic analysis is not always self-evident. The age of line-
ages does not appear to be a primary factor because the
most ancient avian divergences, such as between paleog-
naths and neognaths or between Galloanserae and
Neoaves, are among those recovered with the greatest
reproducibility, irrespective of data set. The shortness of
Table 4: Descriptive statistics of internodal regression analysis
Residuals
Regression r
2
sample variance F ratio and significance n range mean of absolute residuals Z value and significance
b-fib7 X RAG-1
non-Charadriiformes 0.56 308.7 3.811 82 111.9 12.7 2.853
Charadriiformes 0.77 81.0 P < 0.001 45 42.9 6.7 P < 0.005
ADH-5 X RAG-1
non-Charadriiformes 0.29 426.0 8.192 19 91.4 15.5 3.779
Charadriiformes 0.67 52.0 P < 0.001 43 33.3 5.5 P < 0.001
GPD3-5 X RAG-1
non-Charadriiformes 0.13 605.5 7.915 19 101.5 17.3 3.468
Charadriiformes 0.42 76.5 P < 0.001 32 39.0 6.4 P < 0.001
myo-2 X RAG-1
non-Charadriiformes 0.31 210.7 5.077 66 80.6 11.0 4.706
Charadriiformes 0.10 41.5 P < 0.001 27 26.2 4.2 P < 0.001
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internodes has been implicated as an impediment to phy-
logenetic resolution at least in the extreme case of "explo-
sive radiations" [35]. While it might seem intuitive that
internode length should be a factor, our residual plots
suggest otherwise, at least in the case of shorebirds. This is
because RAG-1 has been shown to be clock-like in shore-
birds [3] and there is broad overlap between internode
distances (i.e., not
pairwise distances) within Charadrii-
formes versus non-Charadriiformes.
Hypothetically, high support might be obtained even for
congruent gene-phylogenies that yield vastly different
length estimates of the same branches. For instance,
despite relatively strong support for the rostratulid-jaca-
nid clade, FGB7 shows a very short internode to these
taxa; but, this is neither the case for ADH5 nor GPD3-5.
On the contrary, it seems in this case that there is lesser
support for parts of the tree where different loci yield
more conflict in estimates of branch length. This is intui-
tively satisfying, though the cause for conflicts in individ-
ual gene trees remains obscure. Conflict may be a result of
sampling error or of truly different gene genealogies. An
argument for sampling error might be made from the
observation that the substitution rates of "neutral" parti-
tions (i.e., introns and third positions) appear to be corre-
lated with support. Specifically, myo-2 had the lowest
substitution rate relative to other loci and recovered the
most nodes inconsistent with the combined tree, as well
as having lower bootstrap support values. Future studies
might profitably consider whether clade-specific differ-
ences such as genomic composition might adversely affect
phylogenetic analysis at multiple loci in a lineage specific
fashion.
Conclusion
Charadriiformes represent a monophyletic group that nei-
ther includes nor is sister to waterfowl, flamingos, bus-
tards, sandgrouse, ibises, cranes or rails. Exactly what their
sister relationships are remain obscure. Charadriiformes
consist of three monophyletic suborders Lari (i.e., Laridae
[including Sternidae and Rynchopidae], Stercorariidae,
Alcidae, Glareolidae, Turnicidae, and presumably Droma-
didae), Scolopaci (i.e., Scolopacidae [including Pha-
laropidae], Jacanidae, Rostratulidae, Thinocoridae,
Pedionomidae), and Charadrii (i.e., Burhinidae, Chio-
nididae, Charadriidae, [including Haematopodidae, and
Recurvirostridae], and presumably Ibidorhynchidae).
Skimmers are most likely sister to terns alone, and plovers
may be paraphyletic with respect to oystercatchers and
stilts. The Egyptian Plover is not a member of the Glareol-
idae, but is instead relatively basal among Charadrii.
Methods
Taxa sampled and GenBank accession numbers are listed
in Table 1. A galliform (Callipepla gambelii) and an anseri-
form (Anseranas semipalmata) were designated as the root,
as they represent the Galloanserae, widely accepted as the
monophyletic sister clade to Neoaves in which all other
taxa in this study are included.
Sequences of mitochondrial-encoded 12S rDNA, tRNA-
Valine, and 16S rDNA, and three nuclear loci; alcohol dehy-
drogenase-I intron 5, glyceraldehyde-3-phosphate dehydroge-
nase exons 4–5 and introns 3–5, and
β
-fibrinogen intron 7
were amplified from genomic DNA using primers as
described [22,24,37,38]. PCR reaction conditions were:
35 cycles of 94°C denature, 55°-60°C annealing
(depending on primer pair), 72°C extension, for one
minute each step. Amplicons were purified by agarose gel
electrophoresis and QIAquick gel extraction kit (Qiagen,
Inc., Valencia, CA) according to manufacturer's instruc-
tions. Cycle sequencing was performed using the above
primers, according to manufacturer's instructions using
BigDye v3.1 and read on an ABI 3100 DNA sequencer.
Sequences were aligned using Se-Al v2.0a11[39]. Align-
ments for mitochondrial RNA genes followed secondary
structures as templates to attempt to maximize homolo-
gous positions [22,40,41]. Alignments for the nuclear
introns were generally straightforward, but were algorith-
mically aligned with ClustalX 1.8 [42] and MUSCLE [43]
and adjusted by hand. Differences corresponded to
regions where a multiple alignments could justifiably be
produced; these regions of ambiguous alignment were
removed prior to the phylogenetic analysis.
Phylogenetic analyses were conducted by equally-
weighted maximum parsimony, with gaps treated as miss-
ing data, using PAUP*4.0b10 [44]. Maximum likelihood
analyses were performed using PHYML v2.44 [45], and
mixed-model analysis was implemented in MrBayes 3.0.
Compositional stationarity was explored using the Chi-
Square test in PAUP, and in some cases further investi-
gated by the pairwise disparity index analysis in MEGA
3.1. Trees for selection of nucleotide substitution models
were obtained in PAUP* from neighbor-joining analyses
using an F84 model. The best-fitting substitution model
for the ML analyses was chosen by hierarchical likelihood
ratio tests and the Akaike information criterion imple-
mented in ModelTest v3.06 [46]. For these data, where the
two methods differed, we chose the less parameter-rich
model. In the case of ADH5, we selected a "compromise"
model by relaxing the assumption of equal base frequen-
cies for the Tamura-Nei model. This appeared justified
because the selection of equal base frequencies by hLRT is
likely a consequence of combining short conserved seg-
ments of flanking exon sequence with the intron. Table 3
shows models and associated parameter values used in
tree-searches with PHYML. The best tree obtained was
submitted to PAUP for a further tree-bisection-reconnec-
BMC Evolutionary Biology 2007, 7:35 http://www.biomedcentral.com/1471-2148/7/35
Page 14 of 15
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tion search. Statistical support for the resulting phyloge-
nies was assayed by conducting 500 bootstrap
pseudoreplicate searches, also completed in PHYML.
Mixed-model Bayesian analysis also employed locus-spe-
cific models comparable to those chosen for ML analyses
of individual genes.
List of Abbreviations
12S rRNA – small subunit ribosomal ribonucleic acid
16S rRNA – large subunit ribosomal ribonucleic acid
ABI – Applied Biosystems Incorporated
ADH-I – alcohol dehydrogenase-1
ADH5 – alcohol dehydrogenase-1 intron 5
AIC – Akaike Information Criterion
FGB7 –
β
-fibrinogen intron 7
bp – base pairs
cytb – cytochrome-b
+G – gamma distributed
GAPD-H – glyceraldehyde-3-phosphate dehydrogenase
GPD3-5 – glyceraldehyde-3-phosphate dehydrogenase exons
4–5 and introns 3–5
GTR – general time reversible substitution model
HKY – Hasegawa, Kishino, and Yano 85 substitution
model
hLRT – hierarchical likelihood ratio test
+I – invariable proportion of sites
kb – kilobases
ML – maximum likelihood
MP – maximum parsimony
mtDNA – mitochondrial deoxyribonucleic acid
myo-2 – myoglobin intron 2
PCR – polymerase chain reaction
PI – parsimony informative
Pinv – proportion of invariant sites
RAG-1 – recombination activating gene
rDNA – ribosomal deoxyribonucleic acid
TNef – Tamura Nei equal frequencies substitution model
tRNA – transfer ribonucleic acid
TVM – transversional model
Authors' contributions
MGF performed the sequencing. Both MGF and PH per-
formed analyses and cooperatively wrote the manuscript.
Both authors read and approved the final manuscript.
Acknowledgements
For specimens we are indebted to S. Goodman, Field Museum of Natural
History, D. Lucio, Gladys Porter Zoo, F. Sheldon, Louisiana State University
Museum of Natural Science, J. Turnage, R. Papendick, and A. Gorow, San
Diego Zoo, R. Faucett, University of Washington Burke Museum, K.
Winker, University of Alaska Museum. We thank W. Boecklen for statisti-
cal advice. This research was supported by National Science Foundation
DEB 0108568 to PH.
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