ARTICLE IN PRESS

Organisms, Diversity & Evolution 5 (2005) 25–45

www.elsevier.de/ode

Squamate phylogeny, taxon sampling, and data congruence

Michael S.Y. Leea,b,
a
Earth Sciences Section, South Australian Museum, North Terrace, Adelaide SA 5000, Australia
b
School of Earth and Environmental Sciences, University of Adelaide, Adelaide SA 5005, Australia

Received 16 December 2002; accepted 25 May 2004

Abstract
To investigate the affinities of snakes, amphisbaenians and dibamids, the phylogenetic relationships among the
major lineages (families) of extinct and extant squamates are assessed through a combined analysis of 248 osteological,
133 soft anatomical, and 18 ecological traits. The osteological data set represents a revision of previous data, taking
into account recent criticism; the ecological data set is new. In addition, potentially critical fossil taxa
(polyglyphanodontids and macrocephalosaurs) are included for the first time. The osteological and soft anatomical
data sets each place snakes within anguimorphs, with dibamids and amphisbaenians near gekkotans. The putative
primitive fossil amphisbaenian Sineoamphisbaena groups with macrocephalosaurs and polyglyphanodontids, together
the sister group to scleroglossans. All three data sets are congruent, and these results are reinforced by combined
analyses. In these, as in the osteological analyses, snakes are nested within marine lizards. However, exclusion of fossil
taxa from the osteological data set results in a ‘limbless clade’ consisting of snakes, amphisbaenians and dibamids, and
introduces significant conflict between osteology and soft anatomy. Also, deletion tests and character weighting reveal
that the signal in the reduced osteological data set is internally contradictory. These results increase confidence in the
arrangement supported by the all-taxon osteological, the soft anatomical, and the combined data, and suggest that
exclusion of fossils confounds the signal in the osteological data set. Finally, the morphological data support the
nesting of snakes within marine lizards, and thus a marine origin of snakes. This result still holds when relationships
between living forms are constrained to the topology suggested by molecular sequences: if marine lizards are allowed to
‘float’ within this molecular framework, they form the stem group to snakes, and do not group with varanids as
previously suggested.
r 2005 Elsevier GmbH. All rights reserved.

Keywords: Squamata; Snakes; Amphisbaenians; Dibamids; Partitioned branch support; Character congruence

See also Electronic Supplement at: http://www.senckenberg.de/odes/05-04.htm.

Introduction

It has been eight decades since Charles Camp (1923)

Corresponding author. Earth Sciences Section, South Australian
Museum, North Terrace, Adelaide SA 5000, Australia.
E-mail addresses: michael.s.lee@adelaide.edu.au,
lee.mike@saugov.sa.gov.au (M.S.Y. Lee).
produced his landmark monograph, ‘Classification of
the lizards’. Yet, many long-standing uncertainties
remain regarding higher-level squamate relationships,
despite detailed studies of a plethora of informative

1439-6092/$ - see front matter r 2005 Elsevier GmbH. All rights reserved.
doi:10.1016/j.ode.2004.05.003
 ARTICLE IN PRESS
26 M.S.Y. Lee / Organisms, Diversity & Evolution 5 (2005) 25–45

traits from a variety of systems: osteology (Estes et al. major squamate groups, even when only recent cladistic
1988; Wu et al. 1996; Lee 1998), musculature (Russell morphological studies are considered. Major areas of
1988), visceral anatomy (Gabe and Saint-Girons 1972), uncertainty include the position of gekkotans, relation-
oral glands (Kochva 1978), the brain (Northcutt 1978), ships (and monophyly) of the scincomorph taxa, and
olfactory organs (Parsons 1970), the ear (Miller 1966; basal relationships within Scleroglossa and Anguimor-
Wever 1978; Manley 2003), reproductive structures pha. Gekkotans have been associated with scinco-
(Gabe and Saint-Girons 1965; Arnold 1984), tongue morphs (Presch 1988; Schwenk 1988), or positioned
and hyoid apparatus (Schwenk 1988, 1993), spermato- more basally, as the sister group of scincomorphs plus
zoa (Jamieson 1995), and behaviour (Stamps 1977; anguimorphs (Camp 1923; Estes et al. 1988). Of the
Cooper 1996). Recent molecular studies of squamates scincomorph groups, xantusiids are particularly proble-
are promising (see Forstner et al. 1995; Saint et al. 1998; matic: though traditionally allied with lacertoids (e.g.
Harris et al. 2001; Vicario et al. 2002; Whiting et al. Estes et al. 1988; Wu et al. 1996; Evans and Barbadillo
2003; Townsend et al. 2004; Vidal and Hedges 2004), 1997), they also have been aligned with gekkotans (e.g.
but relationships between many major lineages remain McDowell and Bogert 1954; Northcutt 1978; Greer
poorly supported. While additional sequence data will 1985a); molecular data suggest scincoid affinities
undoubtedly clarify some remaining problems, it cannot (Vicario et al. 2002; Whiting et al. 2003; Townsend et
resolve all outstanding uncertainties (Lee 2005). Many al. 2004). Inferred relationships within the remaining
radiations, such as basal metazoan divergences, have scincomorph groups also differ between morphological
proven notoriously intractable to molecular (as well as studies (e.g. Estes et al. 1988; Presch 1988; Evans and
morphological) resolution despite initial optimism. Barbadillo 1997), and this diverse assemblage might not
The major areas of consensus and contention in even be monophyletic (Lee 1998; Townsend et al. 2004).
higher-level squamate phylogeny are illustrated in Within anguimorphs, the relationships between anguids,
Fig. 1. The following major clades of limbed squamates xenosaurids and varanoids are uncertain, with all
are widely accepted, based primarily on morphological three possibilities having some support in certain
data (e.g. Estes et al. 1988; Schwenk 1988; Wu et al. morphological analyses (Rieppel 1980; Presch 1988;
1996; Evans and Barbadillo 1997; Lee 1998; Lee and Wu et al. 1996).
Caldwell 2000): Iguania, Acrodonta, Scleroglossa, Gek- In addition to the above problems involving limbed
kota, Lacertoidea, Anguimorpha, and Varanoidea. All squamates (lizards), there remains uncertainty regarding
except Scleroglossa are also corroborated (or at least the phylogenetic position of three highly modified limb-
currently uncontradicted) by molecular data (e.g. reduced taxa (Fig. 1). Estes et al. (1988) conceded that
Donnellan et al. 1999; Vicario et al. 2002; Whiting et relationships of snakes, dibamids and amphisbaenians
al. 2003; Vidal and Hedges 2004). However, much could not be resolved beyond the likelihood that each
uncertainty remains regarding the affinities of many belonged ‘somewhere within Scleroglossa’. Snakes have
Gymnophthalmidae
Chamaeleonidae

Diplodactylinae
Gekkonines s.l.
Eublepharinae

Pygopodidae
Leiolepidinae

Lanthanotus
Xenosaurus
Xantusiidae

Shinisaurus

Heloderma
Cordylidae
Lacertidae
Agaminae

Scincidae
Iguanidae

Anguidae

Varanus
Teiidae

A S
A,D
A Varanoidea
Acrodonta Lacertoidea
Gekkota

Iguania A,D Scincoidea

Anguimorpha

Scleroglossa
A,D,S
A,S

Fig. 1. Major areas of agreement or disagreement between morphological analyses of squamate phylogeny. Arrows denote
proposed positions of the three highly modified limb-reduced groups: amphisbaenians (A), dibamids (D), and snakes (S).
 ARTICLE IN PRESS
M.S.Y. Lee / Organisms, Diversity & Evolution 5 (2005) 25–45
been allied with either anguimorphs, burrowing scinco-
morphs, amphisbaenians and dibamids, or placed out-
side lizards altogether (see Rieppel 1988 for review).
Dibamids have been placed with scincomorphs (Rieppel
1984), gekkotans (Wu et al. 1996), or amphisbaenians
(Greer 1985b; Hallermann 1998), or outside other
squamates altogether (Greer 1985b; see also Vidal and
Hedges 2004). Amphisbaenians have been associated
with lacertoids (Wu et al. 1996), teioids (Schwenk 1988),
gekkotans (Presch 1988) or dibamids (Greer 1985b;
Hallermann 1998).
The above disagreements between morphological
studies appear substantial, and might suggest that few
higher-level groupings of squamates are strongly sup-
ported by this data set. However, much of this apparent
incongruence might be a methodological artefact. Most
studies of squamate phylogeny have focused on
particular sources of data to the exclusion of other
sources: for instance, Schwenk (1988, 1993) focused on
tongue characters, Jamieson (1995) on sperm ultra-
structure, and even Estes et al.’s (1988) comprehensive
analysis relied mainly on osteological data, which
comprised 130 out of their 148 characters. It is not
surprising, therefore, that studies that have considered
different and often small subsets of characters have
often yielded different phylogenies. However, it is quite
possible that a very similar common signal underlies all
these data sets, but in some (or even all) of them might
be swamped by homoplasy and thus ‘hidden’ (Gatesy et
al. 1999). Thus, the ‘taxonomic congruence’ approach
(Miyamoto and Fitch 1995) of analysing data sets
separately and looking for groupings in common might
not merely underestimate the amount of agreement
between data sets, but completely overlook such
‘hidden’ groupings (Kluge 1989; Barrett et al. 1991;
Nixon and Carpenter 1996; Gatesy et al. 1999). A
simultaneous analysis of all data needs to be performed
to see if any common signal underlies these apparently
conflicting data sets, and if the overall data strongly
support a particular phylogenetic arrangement. These
insights cannot be gained from comparing the results of
separate analyses of small, isolated data sets. However,
analyses of subsets of characters or taxa can also be
valuable to probe the influence of particular portions of
the data. Thus, a combination of the simultaneous (e.g.
Kluge 1989; Nixon and Carpenter 1996) and partitioned
(Miyamoto and Fitch 1995) approaches can yield more
insights than adherence to a single method.
An analysis of all the (non-molecular) data bearing on
squamate phylogeny is attempted here. Informative
osteological, soft anatomical, and ecological characters
have been compiled, scored across all the major
squamate lineages, and analysed in a simultaneous
parsimony analysis. The amount of congruence between
these three data sets is also investigated, using standard
tests that assess overall levels of agreement between data
27
sets, and partitioned branch support (Baker and deSalle
1997) which assesses congruence with respect to
particular groupings. All methods indicate that, con-
trary to expectation, all three data sets are consistent
with the same phylogenetic arrangement. The effect of
fossils, both on tree topology and congruence, is also
investigated. If fossils are ignored, the osteological data
imply a tree which is intrinsically problematic, as well as
highly incongruent with the tree from the soft anatomy
and ecology. When fossils are added, the osteological
data yield a more biologically reasonable tree, and one
which is almost identical to the tree implied by soft
anatomy. Other tests involving successive deletion of
long convergent branches (see Scanlon 1996; Siddall and
Whiting 1999) also suggest that the fossils are not just
changing the osteological tree, but are changing it in
arguably the ‘right’ direction, by amplifying hidden
signals that are congruent with the soft anatomy.
If the resultant tree from the simultaneous analysis is
taken as the most highly corroborated phylogeny, the
relative ‘quality’ of each of the data sets can also be
evaluated. Ecological (including behavioural) traits are
no different from morphological traits in terms
of phylogenetic informativeness (e.g. Proctor 1996;
Wimberger and de Queiroz 1996). In both cases, initial
hypotheses of homology are formulated based on
several criteria (internal similarity, location or context,
existence of intermediate forms), and tested via con-
gruence with other characters in a phylogenetic analysis.
In both behaviour and morphology, there can be
problems with delimiting character states where a
continuum exists. Both behavioural and morphological
characters show ontogenetic variability, can be geneti-
cally fixed at birth or phenotypically plastic, can evolve
very rapidly or slowly, and exhibit similar ranges of
heritability. Empirical studies show that behaviour and
morphology are equally informative or, conversely,
equally likely to be homoplastic (e.g. Wenzel 1992; de
Queiroz and Wimberger 1993), and that neither class of
characters is consistently more useful in phylogenetic
reconstruction (e.g. Sanderson and Donoghue 1989).
Here, the reliability of osteology, soft anatomy and
behaviour is assessed by comparing their consistency
and retention indices in the context of a combined
analysis.
Character analysis
Character selection
A previous study of squamate phylogeny (Lee and
Caldwell 2000) compiled a data set of 258 cladistically
informative osteological characters. Most of these
characters had been employed in previous studies (e.g.
 ARTICLE IN PRESS
28 M.S.Y. Lee / Organisms, Diversity & Evolution 5 (2005) 25–45
Pregill et al. 1986; Estes et al. 1988; Presch 1988; Wu
et al. 1996; Evans and Barbadillo 1997; Lee 1998;
Reynoso 1998). Criticisms of certain character codings
in this data set (Rieppel and Zaher 2000) are here
evaluated: suggested recodings of taxa and redefinitions
of character states are assessed and, if valid, incorpo-
rated into the current matrix. Ten characters (corre-
sponding to characters 12, 41, 50, 58, 71, 89, 124, 134,
136, and 256 in Lee and Caldwell 2000) have been
deleted, for reasons discussed in a section concluding the
character list (see the accompanying Organisms Diver-
sity and Evolution Electronic Supplement: http://
www.senckenberg.de/odes/05-04.htm, Part 1). This re-
vised osteological data set has been supplemented with
soft anatomical and ecological characters. Many of
these characters were included in a recent phylogenetic
analysis of squamate soft anatomy (Lee 2000). However,
some terminal taxa of dubious monophyly in that
analysis (agamids, gekkonids, xenosaurids; see below)
have been subdivided in the present analysis, and as a
result additional characters have been added that help
resolve the relationships among these smaller terminal
taxa. Ecological (including behavioural) characters have
also been added, although this data set is much smaller
than the previous two. These include chemosensory
characters (Cooper 1995, 1996, 1997), territoriality
(Stamps 1977; Carpenter and Ferguson 1977), and
reproductive mode (Lee and Shine 1998).
The character list and data matrix are presented in
Electronic Supplement 05-04, Parts 1 and 2, respectively.
Nearly all the osteological characters have been
discussed more fully, and the relevant sources cited, in
previous papers (Lee 1998; Lee and Caldwell 2000) and
thus are listed only briefly here. However, characters
that have been debated recently (Rieppel and Zaher
2000) are discussed more fully to address these
criticisms. Most of the soft anatomical characters have
been discussed elsewhere (Lee 2000), and this informa-
tion is not repeated here. New soft anatomical
characters included in this study (see above) are
identified with an asterisk, and discussed and referenced
more fully. Ecological characters were not included in
the above analyses of squamate phylogeny, and thus are
also discussed and referenced more fully.
The phenotypic character set compiled in this
analysis, though one of the largest of this kind, has by
no means exhausted the possibilities. Many other
informative characters no doubt exist and should be
added to the data set in the future. For instance,
relatively few visceral characters have been included,
largely because of the previous lack of interest in
phylogenetic information contained in this system with
respect to higher-level squamate phylogeny. However,
studies on relationships within snakes have shown that
many visceral characters are overly variable (e.g.
Wallach 1985, 1998; Cundall et al. 1993; Keogh 1996).
Similarly, only a few cranial, trunk, and limb muscles
were included. Without doubt, a comprehensive survey
will reveal many more informative myological charac-
ters in this region; similar surveys of the tongue and
genital musculature have revealed many such characters.
However, some studies suggest that both visceral and
general myological features might not be very informa-
tive. Lee (1997) reported that in elapid snakes there was
extensive character conflict between visceral characters,
and no clear phylogenetic signal. In Russell’s (1988)
extensive survey of limb muscles across squamates, few
appeared as phylogenetically informative, there being
extensive variability for these muscles in many taxa.
Terminal taxa, polarity, and character-state coding
The terminal taxa used in this analysis (see Electr.
Suppl. 05-04, Part 2) are very similar to those used in
previous analyses of higher-level squamate phylogeny
(Estes et al. 1988; Presch 1988; Wu et al. 1996; Evans
and Barbadillo 1997). They include all extant lineages
traditionally termed families. However, the terminal
taxa have been refined and expanded in two important
ways.
Firstly, many extant squamate ‘families’ used in
previous analyses were of uncertain monophyly. Here,
the evidence for monophyly of each of these groups is
reassessed based on the most recent information
available. If the evidence is found to be still weak, the
group is subdivided into smaller (and demonstrably
monophyletic) lineages. Use of only such clades as
terminals addresses the criticism (Zaher and Rieppel
1999) that some terminal taxa employed in previous
analyses (e.g. Estes et al. 1988; Lee 1998) were of
dubious monophyly.
Iguanidae: Until recently, the monophyly of iguanids
with respect to acrodontans was uncertain (Estes et al.
1988; Frost and Etheridge 1989). However, molecular
analyses now have strongly supported the monophyly of
iguanids (Harris et al. 2001; Vidal and Hedges 2004):
Zaher and Rieppel’s (1999) statement that the mono-
phyly of iguanids remains poorly supported appears to
be based on the older studies. Iguanids are treated here
as a single terminal taxon.
Agamidae: The monophyly of ‘agamids’ remains
uncertain, as chameleons might be nested within them.
The most comprehensive analysis of the problem in
terms of taxon sampling (Frost and Etheridge 1989)
identified a trichotomy between chamaeleons and two
subgroups of agamids: leiolepidines (Leiolepis and
Uromastyx) and agamines sensu lato (all other aga-
mids). There is both morphological (Moody 1980; Frost
and Etheridge 1989) and molecular (Macey et al. 2000)
evidence for monophyly of agamines. Leiolepidine
monophyly is strongly corroborated by morphological
 ARTICLE IN PRESS
M.S.Y. Lee / Organisms, Diversity & Evolution 5 (2005) 25–45
data (Moody 1980; Frost and Etheridge 1989) and has
variable support from molecular data (Macey et al.
2000; Townsend et al. 2004). Thus, the metataxon
Agamidae is divided here into these two putatively
monophyletic subgroups in order to attempt to resolve
this trichotomy.
Gekkonidae: ‘Gekkonids’ (limbed gekkotans) might
be monophyletic (Estes et al. 1988) or, more likely,
paraphyletic (Kluge 1987; Donnellan et al. 1999; Harris
et al. 2001; Townsend et al. 2004) with respect to
pygopodids. For this reason, limbed gekkotans have
been divided into three sub-groups whose monophyly is
widely accepted and well-corroborated (Kluge 1987;
Donnellan et al. 1999): eublepharines, diplodactylines,
and gekkonines (sensu lato, including sphaerodactyines
and Teratoscincus).
Scincidae: It has been suggested that dibamids might
be nested within scincids, thus making the latter
paraphyletic (Rieppel 1984; Greer 1985b). However, a
recent cladistic analysis (Hallermann 1998), which coded
dibamids and their possible long-bodied, limb-reduced
skink relatives as separate terminal taxa, retrieved a
monophyletic Scincidae. Molecular data also support
monophyly, although taxon sampling remains sparse
(Harris et al. 2001; Townsend et al. 2004). Accordingly,
Scincidae is treated here as a single monophyletic
terminal taxon.
Xenosauridae: Though widely assumed in the past
(e.g. McDowell and Bogert 1954; Estes et al. 1988),
monophyly of the Xenosauridae has been questioned,
with molecular studies suggesting that xenosaurids
might not be monophyletic (Townsend et al. 2004).
Thus, Xenosauridae is divided here into two terminal
taxa of uncontested monophyly, Xenosaurus and Shini-
saurus.
Serpentes: The monophyly of modern snakes (here
called Serpentes), to the exclusion of the Cretaceous
marine snake-like squamates, has been suggested in the
past (e.g. Haas 1980; Lee and Caldwell 1998) but
questioned recently (Tchernov et al. 2000), with the
suggestion that Pachyrhachis and Haasiophis are deeply
nested within modern snakes as the sister group of
advanced snakes (macrostomatans). However, as dis-
cussed more fully elsewhere (Lee and Scanlon 2002), the
latter study is problematic. The characters proposed as
linking Pachyrhachis and Haasiophis with macrostoma-
tans are based on unlikely interpretations of poorly
preserved skull elements. Furthermore, even if these
interpretations are accepted, the proposed characters are
not unique to Pachyrhachis, Haasiophis, and macro-
stomatans, but are present in other snakes and in
‘lizards’, and thus might be primitive for snakes. A
recent re-analysis of snake phylogeny (Lee and Scanlon
2002), using a larger suite of characters, confirms that
pachyophiids are basal to all other snakes (Serpentes).
This result holds even if the data of Tchernov et al.
29
(2000) are added. Serpentes, excluding the pachyophiids,
thus is treated here as a single, monophyletic terminal
taxon (fide Haas 1980; Rage and Escuillié 2000; Lee and
Scanlon 2002). The primitive condition in modern
snakes is inferred based on taxa identified as basal in
recent analyses (e.g. Lee and Scanlon 2002; Slowinski
and Lawson 2002; Vidal and Hedges 2002, 2004;
Scanlon 2005): scolecophidians, ‘anilioids’, trophido-
phiines, Dinilysia, and madtsoiids.
The second change in the present study involves the
inclusion of additional fossil taxa of potential relevance
to the affinities of snakes, amphisbaenians and diba-
mids. These additional taxa obviously could be coded
for very few of the soft anatomical and behavioural
characters. Sineoamphisbaena has been interpreted pre-
viously as a basal amphisbaenian (Wu et al. 1996), the
sister group to the amphisbaenian–dibamid clade (Lee
1998), or as only distantly related to amphisbaenians
and dibamids (Kearney 2003). Affinities between snakes
and Cretaceous marine lizards (aigialosaurids, mosa-
saurids, dolichosaurs, and Adriosaurus) have also been
proposed (Cope 1869; Nopcsa 1923; McDowell and
Bogert 1954; Lee and Caldwell 2000) and disputed
(Zaher and Rieppel 1999; Rieppel and Zaher 2000).
These taxa, along with well-known Cretaceous marine
snakes, Pachyrhachis and Haasiophis, are all included.
Character data from Dinilysia and madtsoiids are
incorporated as well (see Serpentes above). Most
previous analyses of squamate phylogeny have not
included all these taxa (e.g. Estes et al., 1998; Wu et al.
1996; Evans and Barbadillo 1997), and the only study
which did (Lee and Caldwell 2000) only considered
osteological characters. In addition, affinities between
polyglyphanodontids, macrocephalosaurs, Sineoamphis-
baena and amphisbaenians have been proposed (Wu
et al. 1996). Polyglyphanodontids are represented here
by Polyglyphanodon, and macrocephalosaurs by Macro-
cephalosaurus, the eponymous and most completely
known forms. These taxa were scored based on
reexamination of very complete and undistorted materi-
al in Washington and Warsaw, respectively (see Estes
et al. 1988). There are, of course, additional fossil taxa
which could have been included (e.g. Evans and
Barbadillo 1997; Evans and Chure 1998; Reynoso
1998). However, none of these fossil taxa have ever
been proposed to be closely related to snakes, amphis-
baenians or dibamids. Thus they are unlikely to have an
impact on which taxa emerge as the immediate relatives
of the limb-reduced taxa. A preliminary analysis of some
of the better-known excluded fossil lineages confirmed
this: Estesia and paramacellodids were added in the
analysis based on descriptions in the literature, but had
very little effect on tree topology and support, and did
not influence the positions of the limb-reduced taxa. For
this reason, their exclusion in this study is not likely to
have an important effect on the main question
 ARTICLE IN PRESS
30 M.S.Y. Lee / Organisms, Diversity & Evolution 5 (2005) 25–45
addressed, i.e. the affinities of snakes, amphisbaenians
and dibamids.
The monophyly of the ingroup (Squamata) is
corroborated by a large suite of derived characters from
a variety of trait classes (Estes et al. 1988; Gauthier et al.
1988a; Pianka and Vitt 2003). The successively more
distant outgroups used to polarise characters were
rhynchocephalians, Marmoretta, and kuehneosaurs
(see Gauthier et al. 1988a; Evans 1991). The tree was
rooted with an ancestral taxon possessing the inferred
primitive states for all characters; where more than one
state could be primitive (e.g. because it was highly
variable, or not applicable, in the outgroups); the
ancestral taxon was coded with all possible primitive
states.
In some previous analyses (e.g. Wu et al. 1996; Zaher
and Rieppel 1999), where multiple character states
occurred within a terminal taxon, that taxon was often
coded as polymorphic (‘all polymorphisms’ coding).
Here, an attempt was made to determine, on the basis of
well-corroborated phylogenetic relationships within the
terminal taxon, which state was primitive (‘inferred-
ancestor’ coding). For diverse terminal taxa (e.g.
iguanids, agamines, scincids, gekkonines, snakes), the
primitive condition was that found in basal lineages as
identified in recent cladistic analyses. Only if the
primitive state could not be determined readily (due to
variability of the character in basal representatives, or
poor knowledge of relationships and/or character
distribution within the terminal taxon) was the terminal
taxon coded as polymorphic. The inferred-ancestor
method is widely used (e.g. Estes et al. 1988; Frost and
Etheridge 1989; Lee 1998) and in simulations has been
shown to give better results than the all-polymorphisms
method, which allows characters occurring only in a
single highly nested species to be potentially primitive
for a large terminal higher taxon (Bininda-Emonds et al.
1998; Wiens 1998a). These studies contradict claims
(Zaher and Rieppel 1999, p. 833) that the all-poly-
morphisms coding method is superior, and that the
inferred-ancestor method, as employed in Lee (1998),
‘fails to recognise variability (polymorphism) of char-
acters within families’, giving results that ‘continue to
obscure the debate’. The latter method, in fact, better
accounts for variability by recognising highly derived
states within terminal taxa as unrepresentative of the
basal condition.
Many of the characters in the present analysis are
multistate. Two approaches to analysing these were
employed. In one analysis, all multistate characters were
treated as unordered (non-additive). In another, multi-
state characters were ordered, where possible, into
morphoclines. The extremes in morphoclines were coded
as being derivable from each other only via intermediate
stages (the transformation from 0 to 2 entails two steps:
0 to 1, and 1 to 2), i.e. they were coded as ordered
(additive). Only characters that could not be ordered
into clear morphoclines were left unordered. Because the
latter approach discriminates against large changes
within a character (e.g. those between extremes in a
morphocline), it has been argued to result in cladograms
that entail less overall evolutionary change than
cladograms constructed by treating all multistate
characters as unordered (Lipscomb 1992; Wilkinson
1992; Slowinski 1993). The phylogenetic results from the
unordered and unordered analyses were compared to
ascertain any possible effects of the alternative treat-
ments of multistate characters.
The following multistate characters formed clear
morphoclines and thus were ordered 0–1–2 (or
0–1–2–3–4–5, etc., where applicable): 23, 31, 37, 41,
42, 57, 73, 85, 109, 118, 119, 122, 123, 125, 126, 141, 144,
147, 164, 165, 171, 172, 181, 185, 186, 203, 216, 218, 222,
225, 228, 240, 246, 267, 269, 272, 277, 280, 290, 297, 303,
308–311, 317, 321, 327, 329, 334, 336, 337, 351, 353, 355,
363, 369, 371, 376–378, 390.
The following multistate characters formed clear
morphoclines and thus were ordered 1–0–2 (or
1–0–2–3, 1–0–2–3–4, 1–0–2–3–4–5, as applicable): 56,
110, 145, 180, 223.
The following multistate characters did not form clear
morphoclines and were left unordered, even in the
‘ordered’ analyses: 55, 63, 67–70, 83, 94, 124, 134, 152,
153, 157, 257, 285, 299, 340, 351, 354, 356, 361, 380, 394,
395.
The complete character list and data matrix are
available from the two-part Electronic Supplement, the
matrix (Part 2) in Nexus (PAUP and MacClade) file
format. The character list and a similar matrix (with
additional molecular data) are also available from
TreeBASE (http://www.treebase.org/treebase/; acces-
sion number SN1715).
Phylogenetic analyses and results
In each analysis below, two runs were performed, one
with multistate characters ordered, the other, unor-
dered. Data were entered using MacClade (Maddison
and Maddison 2000), and tree searches were performed
using PAUP* (Swofford 2000); the heuristic algorithm
employing 100 random addition sequences was used.
Tree(s) were rooted with a taxon coded with the inferred
primitive state for each character, as discussed above.
For tree length calculations, variability within terminal
taxa was interpreted as uncertainty over the ancestral
condition, rather than as polymorphism in the ancestral
lineage. The robustness of each grouping was ascer-
tained using branch support (Bremer 1988) and, in
combined analyses, partitioned branch support (Baker
and deSalle 1997), calculated in PAUP using commands
 ARTICLE IN PRESS
M.S.Y. Lee / Organisms, Diversity & Evolution 5 (2005) 25–45
generated by TreeRot Version 2 (Sorenson 1999). These
commands were modified so that each heuristic reverse
search employed 100 rather than 20 random addition
sequences. Bootstrapping (Felsenstein 1985) was used as
well, employing 1000 replicates each with 100 random
addition sequences.
Partitioned analyses
The trees obtained from the separate analyses of
osteology, soft anatomy, and behaviour are shown in
Figs. 2–5. Two analyses of the osteological partition were
performed: with all taxa (including fossils), and with
extant taxa only. Only extant taxa were included in
analyses of the soft anatomical and behavioural partitions,
since fossils could not usually be scored for these traits.
The trees obtained from osteology (all taxa included)
and soft anatomy agree remarkably. The strict con-
sensus trees are shown in Figs. 2 and 3. The monophyly
of many traditionally accepted squamate groupings is
upheld: Iguania, Acrodonta, Scleroglossa, Gekkota,
Anguimorpha, Xenosauridae, and Varanoidea. In both
analyses, amphisbaenians and dibamids are sister
groups, together related to gekkotans, xantusiids are
more closely related to gekkotans than to lacertiforms,
scincids and cordylids occupy a heterodox position
nearer to anguimorphs than to lacertiforms, and snakes
are related to varanids. The marine varanoids form a
paraphyletic assemblage leading from varanids to snakes
in the osteological analysis (they could not be included in
the soft anatomical analysis). Sineoamphisbaena groups
not with amphisbaenians but with Polyglyphanodon and
Macrocephalosaurus (fide Kearney 2003), as a basal clade
of scleroglossans. These taxa are thus not closely related
to teioids, as often assumed (e.g. Wu et al. 1996), and
their dental similarities are convergent.
The two trees only differ in three areas. In the
osteological tree, xantusiids are the sister group to
gekkotans plus the amphisbaenian–dibamid clade,
whereas in the soft anatomical tree, xantusiids are the
sister group to gekkotans alone. Also, amphisbaenians
and dibamids are the sister group of pygopodids alone
in the osteological tree, but of all gekkotans in the soft
anatomical tree. Finally, in the osteological tree, scincids
and cordylids are successive outgroups to anguimorphs,
whereas in the soft anatomical tree a scincid–cordylid
clade forms the sister group to anguimorphs. However,
in no case are incompatible clades strongly supported.
The exact positions of xantusiids and the amphisbae-
nian–dibamid clade are poorly supported in both data
sets, suggesting that the differences might be the result
of sampling error and a weak overall signal, rather than
of strongly incongruent data sets. Similarly, while the
scincid–cordylid clade is strongly supported in the soft
anatomical data set, the conflicting scincid–anguimorph
31
clade in the osteological data set is very weak. Again, it
would be reasonable to view the result in the latter
analysis as an unresolved trichotomy involving scincids,
cordylids, and anguimorphs, an arrangement consistent
with the strong scincid–cordylid clade obtained from
soft anatomy. Thus, where there is disagreement, either
or both of the conflicting clades are poorly resolved. If
poorly resolved nodes are collapsed into polytomies,
there is no conflict between data sets.
Obviously, there are too few behavioural and
ecological characters in the present analysis (18) to
obtain robust phylogenetic results. An heuristic search
yielded over 20,000 equally parsimonious trees before
the computer ran out of memory. The consensus tree
obtained (Fig. 4) is largely unresolved, although all the
resolved clades except one are congruent with those
obtained by both osteology and soft anatomy. The
remaining clade (the gekkotan-xantusiid clade) was
obtained by soft anatomy but not osteology. As noted
before, however, this incongruence is not significant,
since the conflicting clades in the behavioural (and soft
anatomical) and the osteological trees are not well-
supported. The limited behavioural data, therefore,
appear to be consistent with the other two (much larger)
data sets. Future studies might identify new behavioural
characters allowing more detailed comparisons between
behaviour and the other data sets. One might predict
that, as more and more behavioural characters are
added, the unresolved polytomies in the behavioural tree
will gradually resolve to a topology similar to those in
the other analyses.
When fossil taxa were deleted, the results were as
follows (Fig. 5). In the ‘ordered’ analysis, six MPTs
resulted. In all of these, amphisbaenians and dibamids
grouped robustly with snakes, and this ‘limbless clade’
grouped with varanids. Pygopodids, in contrast,
grouped with other gekkotans. However, trees a single
step longer unite the amphisbaenian–dibamid-snake
clade with pygopodids, forming a single limbless
radiation. The movement of a limbless clade across
from varanids to pygopodids with only a single extra
step means that all intervening branches are very weakly
supported (Bremer 1, bootstrap o50%). In the
‘unordered’ analysis, the results were very similar. Two
MPTs resulted, and the relationships were largely as
before. Again, a limbless clade (amphisbaenians, diba-
mids and snakes) is retrieved and clusters either with
varanoids or, with a single extra step, pygopodids,
leading to weak branch supports within Scleroglossa.
Osteological convergence in limbless forms – further
tests
It was suspected that, when fossils are ignored, the
limbless taxa, namely amphisbaenians, dibamids, snakes
 ARTICLE IN PRESS
32 M.S.Y. Lee / Organisms, Diversity & Evolution 5 (2005) 25–45

Macrocephalosaurus
A

Sineoamphisbaena

Gymnophthalmidae
Polyglyphanodon
Chamaeleonidae

Dolichosauridae
Aigialosauridae
Gekkonines s.l.

Diplodactylinae
Eublepharinae

Amphisbaenia

Mosasauridae

Pachyrhachis
Pygopodidae
Leiolepidinae

Lanthanotus

Adriosaurus
Xantusiidae

Shinisaurus
Xenosaurus

Haasiophi s
Dibamidae

Heloderma
Cordylidae

Serpentes
Lacertidae
Agaminae
Iguanidae

Scincidae

Anguidae

Varanus
Teiidae
85 35,1 100
2 19 59,1
97,3 56,2 95 100
51,2 10 100,10
93,5 96 6
68,4 81, 2
4
85,4
93,14
52,2
49,1 70,2 91,10
71,1 91,12
69,1
79,4

82,5
64,4
98,10
Macrocephalosaurus

B
Sineoamphisbaena

Gymnophthalmidae
Polyglyphanodon
Chamaeleonidae

Dolichosauridae
Aigialosauridae
Gekkonines s.l.

Diplodactylinae
Eublepharinae

Amphisbaenia

Mosasauridae

Pachyrhachis
Pygopodidae
Leiolepidinae

Lanthanotus

Adriosaurus
Xantusiidae

Shinisaurus
Xenosaurus

Haasiophi s
Dibamidae

Heloderma
Cordylidae

Serpentes
Lacertidae
Agaminae
Iguanidae

Scincidae

Anguidae

Varanus
Teiidae

85,2 18,1
27,1 100,13 100
96,3 33,1 98 10
86,3 30,1 99,8
46,1 5 85
35 ,1 4 74,1
84,5
66 92,9
67,2 1
69,1 82,6
29,1
88,9
52,2
57,2
63,2
43,1
43,1
98,8

Fig. 2. Cladograms resulting from analysis of the osteology data set with both living and fossil taxa included. At each node, first
number refers to bootstrap frequency, second number to branch support. (A) Multistate characters ordered; tree is strict consensus
of nine most-parsimonious trees, each with length 669, consistency index 0.47, retention index 0.71. (B) Multistate characters
unordered; tree is strict consensus of two most-parsimonious trees, each with length 634, consistency index 0.49, retention index
0.71.

and (sometimes) pygopodids, might cluster together neously present in an analysis. Therefore, an analysis
spuriously based on characters correlated with body was performed on the extant osteological data, but
elongation and limb reduction. To test this possibility, including only snakes and excluding the other limbless
two further analyses were performed on the ‘extant- taxa (pygopodids, dibamids, amphisbaenians). Similar
only’ osteological data set. analyses were performed sequentially, including only
If taxa are ‘attracting’ one another due to convergence pygopodids, only dibamids, and only amphisbaenians
or long-branch attraction, and thus their relationships (Fig. 6). Snakes emerged within anguimorphs, as the
are obscured, this confounding signal can be eliminated sister taxon to varanids. Pygopodids emerged within
by including only one problematic taxon at a time in an gekkotans, in a polytomy with diplodactylines and
analysis (Scanlon 1996; Siddall and Whiting 1999). Taxa eublepharines. Dibamids emerged as the sister taxon
cannot attract one another unless they are simulta- to Gekkota. There was strong support for the positions
 ARTICLE IN PRESS
M.S.Y. Lee / Organisms, Diversity & Evolution 5 (2005) 25–45 33

Gymnophthalmidae
A

Chamaeleonidae

Gekkonines s.l.
Diplodactylinae

Eublepharinae
Amphisbaenia

Pygopodidae

Xenosaurus

Lanthanotus
Leiolepidinae

Shinisaurus
Xantusiidae

Heloderma
Cordylidae
Dibamidae

Serpentes
Lacertidae

Scincidae

Anguidae
Agaminae
Iguanidae

Varanus
Teiidae
100,10
95 ,4
72,4
67,1
98,7

94,9 10 0, 11
93 ,4

80,3
37,1 32 ,1
71,3

89,5

Gymnophthalmidae
B
Chamaeleonidae

Gekkonines s.l.
Diplodactylinae
Eublepharinae
Amphisbaenia

Pygopodidae

Xenosaurus

Lanthanotus
Leiolepidinae

Shinisaurus
Xantusiidae

Heloderma
Cordylidae
Dibamidae

Serpentes
Lacertidae

Scincidae

Anguidae
Agaminae
Iguanidae

Varanus
Teiidae

51,1 65,1
95,8 100, 7 94,5
49 ,1 96 ,7.2

100,11 92,4
29,1 51,1.2
31,1 29 ,1.2
86 ,4.1
41,2 68 ,3.2
32,2

90,5

Fig. 3. Cladograms resulting from analysis of the soft anatomy data set. At each node, first number refers to bootstrap frequency,
second number to branch support. (A) Multistate characters ordered; tree is strict consensus of 84 most-parsimonious trees, each
with length 350, consistency index 0.50, retention index 0.67. (B) Multistate characters unordered; tree is strict consensus of eight
most-parsimonious trees, each with length 329, consistency index 0.53, retention index 0.66.
Gymnophthalmidae
Chamaeleonidae

Gekkonines s.l.
Diplodactylinae
Eublepharinae

Amphisbaenia
Pygopodidae

Xenosaurus

Lanthanotus
Leiolepidinae

Shinisaurus
Xantusiidae

Heloderma
Cordylidae

Serpentes
Dibamidae

Lacertidae

Scincidae

Anguidae
Agaminae
Iguanidae

Varanus
Teiidae

50 ,1
48,1 58,1
47,1
63,1
66 ,1

Fig. 4. Cladogram resulting from analysis of the ecology data set. Both ordered and unordered analyses produced 420,000 most-
parsimonious trees, all with length 26, consistency index 0.77, retention index 0.90. upper pair of numbers refers to ordered analysis,
lower pair to unordered analysis; within each pair, first number refers to bootstrap frequency, second number to branch support.
 ARTICLE IN PRESS
34 M.S.Y. Lee / Organisms, Diversity & Evolution 5 (2005) 25–45

Gymnophthalmidae
A

Chamaeleonidae

Diplodactylinae
Gekkonines s.l.

Amphisbaenia
Eublepharinae

Pygopodidae
Leiolepidinae

Shinisaurus
Xenosaurus
Xantusiidae

Lanthanotus
Lacertidae

Heloderma

Dibamidae
Cordylidae

Serpentes
Anguidae
Agaminae

Scincidae
Iguanidae

Varanus
Teiidae
85 ,2 96 ,4 100,8
96 ,5 100,9
84 ,6 45 ,1 100,18
69 ,1 34,1
72, 2
71,2
50,1
45 ,1 33 ,1

40 ,1

42 ,1

92 ,8
Gymnophthalmidae

B
Chamaeleonidae

Diplodactylinae
Gekkonines s.l.

Amphisbaenia
Eublepharinae

Pygopodidae
Leiolepidinae

Shinisaurus
Xenosaurus
Xantusiidae

Lanthanotus
Lacertidae

Heloderma

Dibamidae
Cordylidae

Serpentes
Anguidae
Agaminae

Scincidae
Iguanidae

Varanus
Teiidae

84 ,2 98 ,5
93, 3 99,10
50 ,1 99,15
76, 4 98 ,6 91,2

62 ,1 76 ,1 70 ,1 8,1
69 ,2 27,1
42, 1
34 ,1

33 ,1
33,1

83 ,6

Fig. 5. Cladograms resulting from analysis of the osteology data set, with fossils deleted. (A) Multistate characters ordered; six
most-parsimonious trees, each with length 513, consistency index 0.53, retention index 0.69. (B) Multistate characters unordered;
two most-parsimonious trees, each with length 482, consistency index 0.55, retention index 0.69.

of each of these taxa. When only amphisbaenians limbless taxa attract each other. It is of interest that the
among the limbless taxa were included in an analysis, hidden phylogenetic signal for the limbless taxa in the
they clustered weakly with varanoids. Thus, even when ‘extant-only’ osteological data set revealed using this
only extant taxa are considered, there is an osteological deletion procedure matches closely the phylogenetic
signal placing snakes with varanids, dibamids with signal that emerges when fossils are added, i.e. the signal
gekkotans, and pygopodids with diplodactylines. The present in the full osteological data set. It also matches
relationships of amphisbaenians are more obscure, as the signal present in the soft anatomical data set. The
the smallest clade that they fall robustly within is ecological data set has very little signal due to the low
Scleroglossa (Bremer 15/12, bootstrap 98/97 in ordered/ number of characters, thus useful comparisons are not
unordered analyses). However, when all the limbless possible. The same analyses were performed on the full
taxa are simultaneously present in the analysis, these osteological, and soft anatomical data sets, where only a
secondary signals (which place them in different regions single limbless taxon was included at a time: the
of the squamate tree) are overwhelmed and hidden: the interrelationships between the remaining taxa were
 ARTICLE IN PRESS
M.S.Y. Lee / Organisms, Diversity & Evolution 5 (2005) 25–45 35

Gekkonines s.l.
Correlated with elongation of respective body region:

Diplodactylinae
Eublepharinae

Pygopodidae
Lanthanotus
Heloderma

increased number of presacral vertebrae (character 180),

Serpentes
Varanus
increased number of cervical vertebrae (181).
Correlated with the more uniform trunk region
and more complex axial musculature in long-
86/3,75/2
bodied animals: presence of cervical rib on third
presacral vertebra (197), anteroventral pseudotubercu-
95/8,91/4 72/1,69/1 lum of rib (198), posterodorsal pseudotuberculum of rib
97/13 96 /11 100/12 100/12 (199).
Correlated with loss of sacral contact in taxa with

Amphisbaenia
reduced pelvis: free lymphapophyses (200).
Lanthanotus
Gekkonines s.l.

Diplodactylinae
Eublepharinae

Heloderma
Pygopodidae

Correlated with reduction of appendicular skeleton:

Varanus
scapulocoracoid reduction or loss (203), clavicle loss
(207), interclavicle loss (210), sternum reduction or loss
(213), reduction of number of rib attachment points to
100/15 sternum (216), reduction or loss of forelimbs (218),
73/1,73 /1 100/12
84/3,97/5
reduction or loss of pelvis (222), reduction or loss of
99/6,98/4 hindlimbs (228).
80/5,87/4 76/5,38/1 Limb reduction and body elongation is functionally
relevant in small, burrowing squamates. Thus, the
Fig. 6. Subtrees showing positions of limb-reduced taxa in the following characters linked to miniaturisation and
‘extant-only’ data set, when only one taxon is included in each
fossoriality can be considered to be correlated with limb
analysis. Respective first pair of numbers refer to ordered
reduction and body elongation as well. Not all limb-
analysis, second pair to unordered analysis; within each pair,
first number refers to bootstrap frequency, second number to reduced squamates possess these traits, since limb
branch support. Well-corroborated nodes containing the reduction and body elongation also occur in large
limbless taxon are indicated. aquatic squamates (for anguilliform swimming) and in
large surface-active terrestrial squamates (for sliding
through dense vegetation). Furthermore, these traits can
occur in tetrapodal squamates. Thus, the correlation
largely unchanged in each of these analyses (results not between these traits and limb reduction/body elongation
shown). is less precise than for the previous set of traits.
The other way to account for functional convergence Nevertheless, they are at least partly correlated with
is to identify, and downweight, the characters suspected limb reduction and body elongation, and all these traits
to be functionally correlated. This approach, however, is occur in two or more of the limb-reduced groups
invariably subjective and imprecise. Characters will be considered here, but not in the majority of squamate
correlated with a particular function to varying degrees, groups.
and dividing the data into characters which are totally Correlated with miniaturisation of the skull, resulting
‘independent’ or totally ‘correlated’ with that function in fewer centres of ossification and thus reduced number
results in an artificial dichotomy. Furthermore, of of discrete bones: lacrimal loss (character 11), jugal loss
course, there will be other functions with associated (15), postfrontal loss (31), postorbital loss (35), supra-
suites of correlated characters, and these suites can temporal loss (51), splenial loss (125), angular loss (137),
overlap to a greater or lesser extent. To arbitrarily single articular loss (144).
out a single function, and then arbitrarily divide Correlated with reduction in skull size: maxillary teeth
characters ‘correlated’ with that function and ‘indepen- reduced in number (164). dentary teeth reduced in
dent’ ones, will often represent an oversimplification. number (165).
However, some characters will tend to be more strongly Correlated with the relatively larger size of the brain
correlated than others. Downweighting them can have in small animals, pushing the jaw musculature onto the
great heuristic value (e.g. McCracken et al. 1999) and skull roof and the region of the temporal arch: parietal
thus was attempted here. The method is not circular, jaw adductors cover skull table (42), loss of temporal
since the weighting scheme is not derived from existing arch (45).
phylogenetic assumptions, but from an independent Correlated with cranial consolidation needed for
source of evidence (e.g. functional morphology, devel- head-first burrowing: frontoparietal suture complex
opmental genetics). (30).
The following osteological characters appear (almost Correlated with the cylindrically symmetrical trunk
by definition) to be tightly correlated functionally with region in burrowing animals: neural spines reduced
body elongation and limb reduction, respectively. (176).
 ARTICLE IN PRESS
36 M.S.Y. Lee / Organisms, Diversity & Evolution 5 (2005) 25–45

Correlated with the proportionally large braincase in Table 1. Results (P values; highly significant results in italics)
small forms pushing the opisthotic towards the quad- for the ILD tests comparing various data partitions; note that
rate, and the supraoccipital upwards and backwards the osteological comparison is congruent with the soft
against the skull roof: suspensorial process of parietal anatomy when all taxa are considered, but highly incongruent
when only extant taxa are considered—this trend influences
short (44), quadrate articulates directly with opisthotic
the three-way tests, which are insignificant when all taxa are
(55), supraoccipital situated behind parietal (86), post-
included, but highly significant when only extant taxa are
temporal fenestra closed (89). considered
Correlated with adaptation in fossorial animals to
detect ground-borne rather than airborne sounds: ILD comparison Ordered Unordered
tympanic crest lost (57), stapes robust (78).
Three-way (all taxa) 0.91 0.89
Correlated with eye reduction in subterranean ani-
Three-way (no fossils) 0.023 0.030
mals: scleral ossicles lost (241). Three-way (no fossils, 0.79 0.57
When the ‘extant’ osteological data are re-analysed weighted)
with the above characters deleted, the topology changes. Osteology (all taxa) vs. soft 0.62 0.78
Again, snakes become separated from the amphisbae- anatomy
nian–dibamid clade. In both the ordered and unordered Osteology (no fossils) vs. soft 0.0020 0.063
analyses, snakes cluster with varanids, and pygopodids anatomy
with the three groups of limbed gekkotans, but amphis- Osteology (no fossils, 0.92 0.47
baenians and dibamids remain sister groups. Thus, the weighted) vs. soft anatomy
signal in the weighted ‘extant’ osteological data set seems Osteology (all taxa) vs. ecology 0.83 0.75
to produce a tree congruent with the tree produced by the Osteology (no fossils) vs. 0.88 0.59
ecology
unweighted (and weighted) complete osteological data set,
Osteology (no fossils, 0.86 0.43
and with the tree produced by the soft anatomy.
weighted) vs. ecology
Soft anatomy vs. ecology 0.94 0.99
Congruence between data sets

The incongruence length difference (ILD) test (Farris

et al. 1995; for caveats see Lee 2001; Barker and Lutzoni
2002; Darlu and Lecointre 2002), implemented in
PAUP* as the partition homogeneity test, was used to
assess congruence between the three data sets, in a three-
way test and in pairwise tests. As before, two separate
treatments of the osteological data (with and without
fossils) were used. Uninformative characters were
deleted before each ILD test (Lee 2001).
As might be expected from examination of the tree
topologies obtained in the partitioned analyses, the
osteological data are congruent with the soft anatomical
data when fossils are included, but incongruent when
fossils are excluded (Table 1). Again it appears that the
fossils are not only changing the nature of the signal in
the osteological data, but changing it so that it matches
the signal in the soft anatomy. The ‘extant-only’
osteological data set, however, becomes congruent with
the soft anatomy if characters associated with burrow-
ing are deleted; thus it appears that the weighting
scheme has increased congruence. The tests comparing
ecological traits with the other two data partitions did
not detect significant incongruence, which might have
been expected due to the small number of characters and
weak phylogenetic signal in the ecological data set.
Fossils and phylogeny
The signal in the osteological data, whether measured
by tree topology or by congruence with other data sets,
therefore is affected drastically by the inclusion of fossil
taxa. While all the limbed taxa stay in approximately the
same position, the affinities of the limb-reduced taxa
change significantly. If both living and fossil taxa are
considered, amphisbaenians and dibamids cluster with
Sineoamphisbaena and gekkotans, and snakes are
embedded within varanoids. If only extant taxa are
considered, amphisbaenians, dibamids, snakes (and
sometimes pygopodids) cluster together.
It is reasonable to interpret the results in the following
fashion (although other interpretations are possible).
Living amphisbaenians, dibamids and snakes share a
host of similarities correlated with limb reduction and
fossoriality. Fossils exhibit combinations of character
states that reveal the convergent acquisition of these
traits in amphisbaenians and dibamids, and in snakes.
In particular, the aquatic varanoids and Cretaceous
marine snakes share many synapomorphies with snakes,
but also retain well-developed limbs and lack almost all
fossorial specialisations. When these fossils are included,
therefore, the amphisbaenian–dibamid clade, and
snakes, do not cluster together. In the absence of these
critical fossils, however, the evidence for convergence
disappears and all the limbless taxa form a spurious
grouping based on traits correlated with body elonga-
tion and limb reduction. This reasonable hypothesis,
that fossils are not just affecting the phylogenetic signal
in the osteological data, but improving it, is supported
by several observations.
 ARTICLE IN PRESS
M.S.Y. Lee / Organisms, Diversity & Evolution 5 (2005) 25–45
Firstly, the phylogenetic signal in the osteology with
only extant taxa considered is highly incongruent with
the signal in the soft anatomy (whether measured by
comparing tree topologies in partitioned analyses, or via
the ILD test). However, when fossils are considered, the
osteological signal becomes almost identical to the
signal in the soft anatomy. This strong coincidence of
signals suggests they are reflecting common evolutionary
history, although some other common (confounding)
factor cannot be ruled out. Secondly, when only extant
taxa are considered, analyses including problematic taxa
one at a time reveal that there is a strong but hidden
signal grouping snakes with varanoids, and amphisbae-
nians and dibamids with gekkotans. This signal is
overwhelmed by an even stronger signal uniting all
limb-reduced groups. The hidden signal, however,
coincides exactly with the signal found in the full (fossil
plus extant) osteological data set, and in the soft
anatomical data set. Again, it increases the confidence
in the tree from the full osteological data set, since the
same signal is not only present in other data sets, but
also present (albeit latent) in the extant-only osteological
data. Thirdly, if characters functionally correlated with
limb reduction and fossoriality are deleted, the topology
of the extant-only osteological analysis again changes to
more closely match the topology found in the full (fossil
plus extant) osteological data set, and in the soft
anatomical data set—at least in the ordered analysis.
In other words, the hidden signal is again revealed, and
supports a topology found in the full osteological and
the soft anatomical data set.
These results together strongly suggest that when only
extant taxa are considered, the osteological data set is
misleading (at least regarding the affinities of snakes,
amphisbaenians and dibamids) due to extensive con-
vergence, and fails to produce relationships consistent
with the most highly corroborated tree for all taxa.
When fossils are included, a very different tree, with a
different arrangement of extant taxa, is retrieved. The
soft anatomy, however, produces the latter tree based on
extant taxa alone. There is thus the question of why,
when only extant taxa are considered, convergence
apparently confounds the osteological data, but not the
soft anatomical data. In this example, there is a
plausible reason. Limb reduction and fossoriality is
one of the most pervasive adaptive trends in squamates,
and involves an extremely large suite of characters. A
large proportion of the osteological characters used in
this analysis are reductions or losses of bones correlated
with limb reduction and fossoriality: for instance, many
deal with the reduction or loss of cranial, girdle, and
limb elements. In contrast, a smaller proportion of soft
anatomical traits appears to be correlated with limb
reduction and fossoriality: this problem only appears to
seriously influence a few of the cranial cartilage and
visceral characters. The tongue, hemipenial, histological
37
and spermatozoal characters which dominate the soft
anatomical data set are not as obviously linked to limb
reduction and fossoriality (though they are undoubtedly
correlated with other functional complexes). Thus, for
this particular data set there is some reason to expect
that convergence would affect the osteological data set
more severely.
Combined analyses
In cases with significant conflict between data subsets,
the validity of the combined-analysis approach has been
debated (e.g. Kluge 1989; Bull et al. 1993; Miyamoto
and Fitch 1995; Nixon and Carpenter 1996; Wiens
1998b). In the present study, the (complete) osteological,
soft anatomical and behavioural data partitions are not
in significant conflict, and indeed the two informative
data sets give almost identical phylogenetic signals.
Therefore, combined analyses were performed, with all
characters included, and both with and without the
fossil taxa. It has been demonstrated that fossil taxa can
have major effects even in analyses where nearly all soft
anatomical, ecological, and molecular traits must be
scored as unknown (e.g. Gauthier et al. 1988b; O’Leary
1999).
The support for a particular clade from each data
partition in the context of a combined analysis is the
partitioned branch support, or PBS (Baker and deSalle
1997). A positive PBS means that the data partition
supports that node in the context of a combined
analysis, a negative PBS means that the data partition
contradicts that node in the context of a combined
analysis. Note that partitioned branch supports are not
identical to branch supports in partitioned analyses (for
a fuller discussion, see Gatesy et al. 1999). For each
clade, PBS was calculated for the osteology, the soft
anatomy, and the ecological partitions. TreeRot version
2 (Sorenson 1999) was used in all these calculations.
In the combined analyses that included all taxa, three
most-parsimonious trees were obtained when multistate
characters were ordered, and 12 when multistate
characters were unordered. The consensus trees for
both analyses (Fig. 7) are almost identical, the only
difference concerning a lack of resolution of relation-
ships within gekkotans in the unordered analysis. As
expected, the topology of the combined tree is almost
identical to those found in the (all-taxon) osteological
and soft anatomical analyses. It is also consistent with
the (few) resolved nodes in the ecological tree. Regard-
ing the two areas where there were disagreements
between the three data sets, xantusiids emerge as the
sister group of the gekkotan–dibamid–amphisbaenian
clade (fide osteology, contra soft anatomy and beha-
viour), whereas scincids and cordylids emerge as sister
taxa (fide soft anatomy, contra osteology).
 ARTICLE IN PRESS
38 M.S.Y. Lee / Organisms, Diversity & Evolution 5 (2005) 25–45

Macrocephalosaurus
A

Gymnophthalmidae
Sineoamphisbaena
Polyglyphanodon
Chamaeleonidae

Dolichosauridae
Aigialosauridae
Gekkonines s.l.
Diplodactylinae
Eublepharinae

Mosasauridae
Amphisbaenia

Pachyrhachis
Pygopodidae

Lanthanotus
Leiolepidinae

Shinisaurus
Xenosaurus

Adriosaurus
Xantusiidae

Heloderma

Haasiophis
Cordylidae
Dibamidae

Lacertidae

Serpentes
Scincidae

Anguidae
Agaminae
Iguanidae

Varanus
Teiidae
84 52 100 58,1/0/0
2/0/0 100 100
37 17/0/0 0.5/0/0.5 3.5/11/0.5 93 100,10/0/0
97 1/0/0 61,0/0/1 6/0/0 10/0/0
97 97 84,2/0/0
6/2/1 95,-2/11/1
5/0/1 5/0/1 88,4/0/0
57,2/0/0
41 98,17/0/1
95
1/0/0 58
97 3.5/5/0.5 97,11/10/0
3.3/4.3/1.3 2/0/0
97,14/0/0
78,2/1/0
93,7.5/2.5/0
74,5/-2/0
79,6/0/0
55,2/0/0
99,11/0/0
Macrocephalosaurus

B
Gymnophthalmidae
Sineoamphisbaena
Polyglyphanodon
Chamaeleonidae

Dolichosauridae
Aigialosauridae
Gekkonines s.l.
Diplodactylinae
Eublepharinae

Mosasauridae
Amphisbaenia

Pachyrhachis
Pygopodidae

Lanthanotus
Leiolepidinae

Shinisaurus
Xenosaurus

Adriosaurus
Xantusiidae

Heloderma

Haasiophis
Cordylidae
Dibamidae

Lacertidae

Serpentes
Scincidae

Anguidae
Agaminae
Iguanidae

Varanus
Teiidae

85 100
2/0/0 2/7.5/0.5
95 91 100 99,8/0/0
5/1/1 92 4/1/0 10/0/0 78, 1/0/0
3/3.5/0.5 98
6/0/1 92,5/0/0
97,15/0.5/0.5
99 97
100 97,16/0/0
10.9/1/0.1 0/6/0
96 11/0/0 97,9/0/0
1.8/5.2/1
64,2/0/0
86,4.7/1.8/-0.5
66, 5/-2/0
65,4.2/0.3/0.5

99, 9/0/0

Fig. 7. Cladograms resulting from analysis of the combined data set, with both extant and fossil taxa included. Respective first
number refers to bootstrap frequency, next three numbers to partitioned branch support for osteology, soft anatomy, and ecology,
respectively; overall branch support is the sum of these three numbers. (A) Multistate characters ordered; strict consensus of three
most-parsimonious trees, each with length 1057, consistency index 0.48, retention index 0.70. (B) Multistate characters unordered;
strict consensus of 12 most-parsimonious trees, each with length 1002, consistency index 0.50, retention index 0.69.

Combining data sets greatly increased the support for as robust in the combined analysis. In particular, the
almost all groupings, whether measured by branch position of dibamids, amphisbaenians, and snakes is
support or bootstrap frequency. This was expected, reinforced. It is notable that, for nearly every clade in
given that usually the same groupings were found in the both the ordered and unordered analyses, partitioned
two separate analyses. Combining data sets thus Bremer support is positive or zero for all data sets. This
reinforces these common signals, whereas conflicting indicates that nearly all clades are supported by (or at
signals (presumably random noise) are unlikely to least compatible with) all three data sets. There are only
reinforce each other. Many groupings which were two clades in the ordered analysis, and three clades in
present in both analyses, but only weakly supported the unordered analysis, with negative partitioned branch
(and thus might be interpreted as insignificant), emerge supports. Furthermore, these negative values have very
 ARTICLE IN PRESS
M.S.Y. Lee / Organisms, Diversity & Evolution 5 (2005) 25–45 39

Gymnophthalmidae
A

Chamaeleonidae

Gekkonines s.l.
Diplodactylinae

Amphisbaenia
Eublepharinae

Pygopodidae
Leiolepidinae

Xenosaurus

Lanthanotus
Shinisaurus
Cordylidae

Heloderma
Xantusiidae

Lacertidae

Dibamidae
Serpentes
Scincidae

Anguidae
Agaminae
Iguanidae

Varanus
Teiidae
82 , 62 100 96 100,14/4/0
2/0/0 1/0/0 4/12/0 98
98 9.5/2.5/0
98, 78,1/1/1 0/8/0 79,22/-12/1
6/3/1 5/0/1
98,18/-1/2 97 57,2/1/0
3/6/0
78,22/-12/1

98 71,4.5/0/2.5 63,2/2/0
2.5/6.5/1
76,13.5/-3/0.5

61,5/-2/0
63,0/1/5

100,16.5/6.5/1
Gymnophthalmidae

B
Chamaeleonidae

Gekkonines s.l.
Diplodactylinae

Amphisbaenia
Eublepharinae

Pygopodidae
Leiolepidinae

Xenosaurus

Lanthanotus
Shinisaurus
Cordylidae

Heloderma
Xantusiidae

Lacertidae

Dibamidae
Serpentes
Scincidae

Anguidae
Agaminae
Iguanidae

Varanus
Teiidae

87 53 100 95 99,10/5/0
2/0/0 1/0/0 3/9/0 99 18/-8.5/1.5
66,1/0/1 5/641/1 79,16/-9.5/1.5
95, 6 /2/1 89 2/1/0
100,15/2/2 3.5/3.5/0 64,14/-9.5/1.5
98 ,
0/7/0 52, 2 /0/0
96,2/5/1
76, 2 /1/2 73,16/-9.5/1.5

53,5/-2/0

48, - 2/0/5

100,13.5/2.5/1

Fig. 8. Cladograms resulting from analysis of the combined data set, with only extant taxa included. Respective first number refers
to bootstrap frequency, next three numbers to partitioned branch support for osteology, soft anatomy, and ecology, respectively;
overall branch support is the sum of these three numbers. (A) Multistate characters ordered; single most-parsimonious tree with
length 908, consistency index 0.52, retention index 0.68. (B) Multistate characters unordered; single most-parsimonious tree with
length 857, consistency index 0.54, retention index 0.67.

small (absolute) values, the largest being only 2. obtained (Fig. 8). Not only is the tree very poorly
Agreement between the data sets therefore is very good, supported, but there is great disagreement between the
with nearly all the phylogenetic signal in the three data data sets. The number of nodes with at least one
sets being retained as branch support in the combined negative PBS is five in the ordered analysis, four in the
tree. unordered analysis. Despite there being fewer nodes in
When the combined analyses are performed with the extant-only data sets (due to fewer taxa), there
fossils deleted, however, a very different result is are more nodes with negative PBS. Moreover, these
 ARTICLE IN PRESS
40 M.S.Y. Lee / Organisms, Diversity & Evolution 5 (2005) 25–45
negative values are often rather large: in the ordered
analysis, two nodes have a PBS of 12; in the unordered
analysis, three nodes have a PBS of 9.5, and another
has a PBS of 8.5. Thus, a smaller proportion of the
phylogenetic signal in the three data sets is retained as
support for the combined tree. The amount of disagree-
ment between the data sets appears to be substantial; it
is also rather localised. In particular, all the high
negative values (implying disagreement between data
sets over particular clades) occur within the clade
consisting of anguimorphs and three limbless taxa
(snakes, amphisbaenians and dibamids), indicating that
there is severe disagreement between data sets concern-
ing this clade. This is explicable because the (extant-
only) osteology strongly supports this arrangement,
while the soft anatomy strongly suggests that snakes
nest within anguimorphs, but that amphisbaenians and
dibamids are more basal; the combined data set
preserves the osteological signal over the conflicting soft
anatomical signal.
These two combined analyses, with and without
fossils, reinforce the results obtained in the separate
analyses and the ILD tests. When fossils are included in
the analyses, the data sets are congruent with each other,
the phylogenetic signal in the separate data sets is
retained in the combined analysis, and the resultant tree
is well-supported. When fossils are ignored, there is a
great deal of incongruence between data sets, much of
the phylogenetic signal in the separate data sets is lost in
the combined analysis, and the resultant tree is weakly
supported. Again, the strong and arguably positive
effect that fossils have on data congruence and tree
topology is upheld.
Relative informativeness of osteology, soft anatomy
and behaviour
The relative phylogenetic informativeness of osteol-
ogy, soft anatomy and behaviour was investigated in the
context of the combined analyses. As there is no
significant incongruence between the three data sets,
the tree from the combined analysis can be accepted as
the most highly corroborated estimate of squamate
phylogeny. The congruence of osteology, soft anatomy,
and behaviour on this ‘best-estimate’ tree can be
evaluated by comparing the average consistency and
retention indices for each set of characters when
optimised on this combined tree. All three classes of
characters have very similar values that do not differ
significantly (Table 2). While osteology has slightly
lower CIs and RIs than soft anatomy and ecology, this
might be due to osteological characters being observable
on more taxa (varying numbers of fossils), and thus
having more opportunity for homoplasy.
Table 2. The relative informativeness of each data partition,
as measured by their consistency and retention indices (CU,
RI); Farris, 1989 in the context of the overall combined tree;
note that all means fall within one standard deviation of each
other, and thus are not significantly different
Data Ordered analysis Unordered analysis
partition
CI RI CI RI
Osteology 0.6170.30 0.7570.48 0.6270.30 0.7170.30
Soft anatomy 0.6470.30 0.7270.49 0.6670.30 0.6870.32
Ecology 0.7970.25 0.8070.31 0.7670.25 0.7770.32
Conclusions, prospects, and snake origins
When different data sets strongly support radically
different phylogenies, at least one of them is wrong (i.e.
does not reflect evolutionary history). There are
instances where both data sets can be right, involving
lineage sorting, hybridisation, and other processes (e.g.
Wiens 1998b). However, when dealing with higher taxa,
these processes generally can be assumed to be rather
minor. It has been shown that increased taxon sampling
is one of the factors most important to retrieving the
correct tree topology (e.g. Hillis 1996), and that the
critical additional sampled taxa are often fossils (e.g.
Gauthier et al. 1988b). In an analysis of higher taxa, if
two data sets disagree, at least one of them is wrong.
Increasing the taxon sampling in both data sets should
cause both to converge on the true tree, and thus on
each other (cf. Cunningham 1997). This prediction is
upheld by the above analyses. If only extant taxa are
considered, the osteological data set contains a phylo-
genetic signal that is driven by a suite of correlated
characters, and is incongruent with the soft anatomical
data set. When additional (fossil) taxa are sampled,
however, the osteological data set becomes congruent
with the soft anatomical data. This suggests that the
fossil taxa are not merely changing the signal in the
osteological data, but changing by retreiving secondary
signals that match those in other data sets. More
empirical studies are needed, however, because another
recent study (Mitchell et al. 2000) found that adding
taxa decreased congruence.
Assessing phylogenetic accuracy is usually proble-
matic because (with a few special exceptions) one can
never know the true phylogeny. Increased congruence
between data sets, however, is a valuable indicator that
the data sets are each converging on the real phylogeny.
Previous studies have used this assumption to test the
validity of tree construction methods (Cunningham
1997) and coding and weighting criteria (Allard and
Carpenter 1996; Wiens 1998c). If a method produces the
same tree when applied to different sources of data, this
 ARTICLE IN PRESS
M.S.Y. Lee / Organisms, Diversity & Evolution 5 (2005) 25–45 41

would suggest that the method is converging on the
real phylogeny. Conversely, if a method produces
radically different trees when applied to different data
sets, this would suggest that the method fails (at least in
some applications). There is, however, the added
complication (not considered by Cunningham 1997)
that different methods might be applicable to different
data sets (e.g. likelihood to molecules, parsimony to
morphology), and this might be suggested if applying
different methods to different data sets increases tree
congruence, compared to using a single method on all
data sets.
Tree congruence can be used to evaluate other
problems. There currently is a debate over whether
adding characters or adding taxa is more important to
recovering the correct tree topology. Most arguments
for either position have been based on rather simplified
simulations that might be of limited relevance to the
biological world. However, if congruence between data
sets is an indicator of phylogenetic accuracy, there might
be a way to evaluate the problem using real data after
all. If adding taxa is the better way to improve chances
at retrieving the real phylogeny, then one would expect
that doubling the number of sampled taxa (e.g. species
sequenced) will be better for reducing incongruence than
doubling the number of characters (e.g. getting twice as
much sequence data for each taxon). Conversely, if
adding characters is the better way, then one would
expect that doubling the number of characters will be
better for reducing incongruence than doubling the
number of sampled taxa. The current study shows that
adding taxa can have a great effect on reducing
incongruence and presumably improving phylogenetic
accuracy. However, it remains to be seen if adding
characters can have just as profound an effect. A more
comprehensive study using multiple data sets and
varying both taxon sampling and character sampling is
required.
In particular, recent molecular work on the higher-
level phylogeny of squamates (e.g. Saint et al. 1998;
Harris et al. 2001; Vicario et al. 2002; Vidal and Hedges
2004; Townsend et al. 2004) should soon offer a large
independent data set to test these ideas. These molecular
studies tend to place dibamids, then gekkotans, as the
basal squamates (an arrangement with some morpholo-
gical support: Greer 1985b; Underwood and Lee 2001),
challenging the idea of a basal iguanian–scleroglossan
dichotomy. The poor resolution at the base of squa-
mates found in the present analysis is mirrored in recent
molecular studies, which have also found short branches
and/or poor support in this region. The failure of both
morphology and molecules to resolve many of these
branches is consistent with the hypothesis that basal
divergences within Scleroglossa occurred rapidly, gen-
erating short internal branches that are difficult to
reconstruct.
The new molecular data also tend to group snakes
with iguanians (Vidal and Hedges 2004; Townsend et al.
2004). This is highly heterodox, given that the two taxa
represent opposite morphological and behavioural
extremes within squamates (e.g. Schwenk 2000; Pianka
and Vitt 2003). However, the snake–iguanian clade is
weakly supported: snakes and iguanians essentially form
a polytomy with anguimorphs, and a snake–angui-
morph clade (strongly supported by morphology) thus
remains consistent with the molecular data (Townsend

Fig. 9. Strict consensus of the two trees (753 steps) that result if osteology data are analysed with relationships between extant taxa
(for which gene sequences are known) constrained to the molecular topology suggested by Vidal and Hedges (2004); other taxa
(indicated by asterisks) are allowed to ‘float’ within this molecular backbone. Note that marine lizards and pachyophiids both group
with snakes, implying a marine phase in snake origins. See Lee (2005) for further discussion.
 ARTICLE IN PRESS
42 M.S.Y. Lee / Organisms, Diversity & Evolution 5 (2005) 25–45
et al. 2004). The nesting of snakes within marine lizards
found here strongly supports an aquatic origin of snakes
(Nopcsa 1923; Lee and Caldwell 2000). The recent
molecular evidence has been interpreted as refuting this
scenario (Vidal and Hedges 2004), suggesting that
snakes are only distantly related to living varanid
lizards. As marine lizards such as mosasaurs and
dolichosaurs usually are considered to be varanoids
(e.g. Lee and Caldwell 2000), this would seem to
preclude them being close relatives of snakes. However,
these marine lizards share similarities with both snakes
and living varanids. In fact, the phylogeny found here –
(varanids (marine lizards (snakes))) – indicates that
derived traits shared between marine lizards and
varanids will also tend to be found in snakes, and that
marine lizards must share additional synapomorphies
with snakes alone. Thus, if the molecular phylogeny is
accepted and varanids and snakes are assumed to be
widely separated, marine lizards must ‘choose’ what
other group they align with; they could be expected to
cluster more strongly with snakes than with varanids.
Accordingly, an analysis of the osteological data
suggests that, if relationships between living taxa are
constrained to the proposed molecular topology, and
marine lizards are allowed to ‘float’, they group most
parsimoniously with snakes, rather than with varanids
(Lee 2005; see present Fig. 9). This tree is significantly
better than the one resulting when relationships between
living taxa are constrained to the molecular topology,
and when marine lizards are forced to cluster with
varanids (the arrangement suggested by Vidal and
Hedges 2004). Very similar results occur if the full
morphological data set (not just the osteology) is used;
this is to be expected given that the majority of ‘floating’
taxa (including all fossils) are scored for osteology only.
Thus, contrary to recent suggestions, the molecular data
do not refute the idea that marine lizards are related to
snakes. Instead, it contradicts the idea that marine
lizards are related to varanids.
Acknowledgements
The author thanks the late Garth Underwood, and
Michael Caldwell, John Scanlon, Jean-Claude Rage,
Tod Reeder, Ken Kardong, Susan Evans and Nick
Arnold for discussion, as well as anonymous reviewers
for comments on two versions of this manuscript. The
author is especially grateful to Kurt Schwenk for his
help in clarifying some tongue characters, and to
Magdalena Borsuk-Bialynicka (Polish Academy of
Sciences, Warsaw) and Bill Purdy (US National
Museum of Natural History, Washington) for access
to macrocephalosaur and polyglyphanodontid material.
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