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Resolving the phylogeny of lizards and snakes (Squamata)

with extensive sampling of genes and species
John J. Wiens, Carl R. Hutter, Daniel G. Mulcahy, Brice P. Noonan, Ted M. Townsend, Jack W.
Sites, Jr and Tod W. Reeder
Biol. Lett. published online 19 September 2012
doi: 10.1098/rsbl.2012.0703

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Biol. Lett.
doi:10.1098/rsbl.2012.0703
Published online

Phylogeny

Resolving the
phylogeny of lizards
and snakes (Squamata)
with extensive sampling
of genes and species
John J. Wiens1,*, Carl R. Hutter1,
Daniel G. Mulcahy2, Brice P. Noonan2,
Ted M. Townsend3, Jack W. Sites Jr2
and Tod W. Reeder3
1

Department of Ecology and Evolution, Stony Brook University,

Stony Brook, NY 11794-5245, USA
2
Department of Biology and Bean Life Science Museum,
Brigham Young University, Provo, UT 84602, USA
3
Department of Biology, San Diego State University, San Diego,
CA 92182-4614, USA
*Author for correspondence (wiensj@life.bio.sunysb.edu).

Squamate reptiles (lizards and snakes) are one of

the most diverse groups of terrestrial vertebrates.
Recent molecular analyses have suggested a very
different squamate phylogeny relative to morphological hypotheses, but many aspects remain
uncertain from molecular data. Here, we analyse
higher-level squamate phylogeny with a molecular dataset of unprecedented size, including 161
squamate species for up to 44 nuclear genes
each (33 717 base pairs), using both concatenated
and species-tree methods for the first time. Our
results strongly resolve most squamate relationships and reveal some surprising results. In
contrast to most other recent studies, we find
that dibamids and gekkotans are together the
sister group to all other squamates. Remarkably,
we find that the distinctive scolecophidians (blind
snakes) are paraphyletic with respect to other
snakes, suggesting that snakes were primitively
burrowers and subsequently re-invaded surface
habitats. Finally, we find that some clades
remain poorly supported, despite our extensive
data. Our analyses show that weakly supported
clades are associated with relatively short
branches for which individual genes often show
conflicting relationships. These latter results
have important implications for all studies that
attempt to resolve phylogenies with large-scale
phylogenomic datasets.
Keywords: phylogeny; phylogenomics; reptiles

1. INTRODUCTION
Squamate reptiles are one of the most diverse and wellknown vertebrate groups, with approximately 9000
species among 61 families [1]. Squamates offer outstanding model systems in ecology and evolution,
especially for studying origins of asexuality, viviparity,
body form and venom [1]. Some squamates are also
Electronic supplementary material is available at http://dx.doi.org/
10.1098/rsbl.2012.0703 or via http://rsbl.royalsocietypublishing.org.
Received 27 July 2012
Accepted 29 August 2012

an important cause of human mortality, with tens of

thousands of snakebite deaths every year [2].
Understanding these diverse aspects of squamate
biology requires a well-resolved phylogeny. Recent
molecular analyses have suggested a phylogeny that
differs dramatically from morphological hypotheses, especially in placing iguanians with snakes
and anguimorphans [3 5]. Although these molecular
studies are generally concordant, several issues
remain unclear [3 9], including: (i) the sister group
to other squamates, (ii) the sister group to snakes,
(iii) interrelationships of major snake clades and
(iv) relationships of iguanian families. Many of these
questions have resisted resolution even with datasets
of 20 genes or more [5,7,9].
Here, we analyse squamate phylogeny using extensive
sampling of taxa (161) and characters (44 loci), the
largest dataset yet assembled. We also present the first
analysis of higher squamate relationships using
species-tree methods [10,11]. We generate a strongly
supported hypothesis and reveal some surprising results.
However, some branches remain weakly supported, and
our analyses shed light on this unexpected pattern.
2. MATERIAL AND METHODS
We sampled 161 squamate species and 10 outgroup taxa, including mammals (Homo, Mus, Tachyglossus), crocodilians (Alligator,
Crocodylus), birds (Dromaius, Gallus), turtles (Chelydra, Podocnemis)
and a rhyncocephalian (Sphenodon). We included all extant squamate
families (excepting a few recently recognized groups, such as Cadeidae, Blanidae, Phyllodactylidae and Xenophiidae [1]), with two or
more representatives from most families. We sequenced portions of
44 nuclear genes (exons approx. 5001500 base pairs in length)
carefully selected based on comparisons of vertebrate genomes
[12], targeting single-copy genes evolving at appropriate rates. Standard methods of DNA extraction, amplification and sequencing were
used. Nucleotide sequences were translated to amino acids to aid
alignment, and alignment was straightforward. The total alignment
consisted of 33 717 base pairs (available through the Dryad data
depository (doi:10.5061/dryad.g1gd8)). Voucher and GenBank
numbers and the names, lengths and sampling of genes are provided
in the electronic supplementary material, appendices S1 S3).
On average, each gene had data for 143 species (84% complete).
Simulations and empirical analyses suggest that missing data
need not be problematic for concatenated phylogenetic analyses,
particularly when sampling many characters [13].
Two general approaches to data analysis were used (concatenated
and species tree). First, we performed a concatenated analysis of all
taxa using likelihood (RAxML, v. 7.2.0; [14]), using 1000 bootstrap
replicates integrated with 200 searches for the optimal tree. We used
the GTR G model and partitioned the data by genes and codon positions (see the electronic supplementary material, appendix S4). We
also performed a Bayesian concatenated analysis using MRBAYES v.
3.1.2 [15] (see the electronic supplementary material, appendix S4),
with branch support based on posterior probabilities (Pp).
Second, we performed Bayesian species-tree analyses (using
*BEAST v. 1.6.2 [11]). We used 31 species (29 squamates, two
outgroups, Sphenodon and Gallus) for computational feasibility.
The selected species represented all major squamate clades, and
had relatively complete sampling of genes (mean 41.5 genes),
given that the impact of missing data on species-tree methods
remains poorly known. Details of methods are provided in the
electronic supplementary material, appendix S4.
To address why some clades are strongly versus weakly supported, we used 49 interfamilial clades from the concatenated-data
likelihood tree and analysed relationships between bootstrap support
(bs), branch lengths and congruence among genes [7]. Branch
lengths from the concatenated-data tree were used, and these lengths
were strongly correlated with mean lengths (for comparable clades)
from separately analysed genes (Rho 0.840; p 0.0001; see the
electronic supplementary material, appendix S5). We evaluated
the proportion of separately analysed genes supporting each node
in the concatenated tree, and the bootstrap support for supporting
and conflicting clades. Relationships were tested using nonparametric Spearmans rank correlation (data in the electronic
supplementary material, appendix S5).

This journal is q 2012 The Royal Society

Downloaded from rsbl.royalsocietypublishing.org on October 22, 2012

J. J. Wiens et al.

Squamate reptile phylogeny

Anelytropsis papilosus

Dibamidae
Dibamus novaeguineae
Saltaurius cornutus
Carphodactylidae
Delma borea
Pygopodidae
Lialis burtonis
100
100
5
Gekkota
Rhacodactylus auriculatus
90
Diplodactylidae
Strophurus ciliaris
100
3
Aeluroscalabotes felinus
100
Eublepharidae
Coleonyx variegatus
100
Eublepharis macularius
88
6
Gekko gecko
Gekkonidae
65
Phelsuma lineata
100
7
Gonatodes albogularis
100
Sphaerodactylidae
Teratoscincus scincus
100
11
Cricosaura typica
Xantusiidae
Lepidophyma flavimaculatum
10
100
Xantusia
vigilis
100
Cordylosaurus subtesselatus
Gerrhosauridae
100
Zonosaurus ornatus
100
9
Cordylus mossambicus
100
Cordylidae
Platysaurus
pungweensis
100
100
Acontias meleagris
Amphiglossus splendidus
59
Feylinia polylepis
100
Eumeces schneideri
100
Scincus scincus
100
Plestiodon fasciatus
Scincidae
100
Plestiodon skiltonianus
100
Brachymeles gracilis
Eugongylus rufescens
70
Sphenomorphus solomonis
100
Tiliqua scincoides
100
Trachylepis quinquetaeniata
100
Alopoglossus angulatum
Gymnophthalmidae
Colobosaura modesta
14 100
Pholidobolus macbrydei
100
Aspidoscelis tigris
100
Teius teyou
100
13
Teiidae
Callopistes maculatus
99
Tupinambis teguixin
100
100
Rhineura floridana
Rhineuridae
8
15
Lacerta viridis
Lacertidae
Takydromus ocellatus
100
100
Bipes
biporus
16
Bipedidae
17
Bipes canaliculatus
100
Amphisbaena
fuliginosa
Amphisbaenidae
18
100
Geocalamus acutus
100
Diplometopon
zarudnyi
100
Trognophidae
Trogonophis wiegmanni
100
21
Leptotyphlopidae
Leptotyphlops humilis
Serpentes
Scolecophidia
Typhlops jamaicensis
Typhlopidae
20 99
Liotyphlops albirostris
Anomalepididae
Anilius scytale
24
100
Aniliidae
100
Trachyboa
boulengeri
22
Tropidophiidae
Tropidophis haetianus
100
100
26
23
Cylindrophis ruffus
Uropeltidae
Uropeltis melanogaster
100
100
Xenopeltis unicolor
Xenopeltidae
28
25
Loxocemus bicolor
Loxocemidae
29
Aspidites melanocephalus
12
Pythonidae
100 98 100 100
Python molurus
Bolyeriidae
Casarea dussumieri
100
31
Calabaria reinhardtii
Calabariidae
Eryx colubrinus
93 32
100
27
Boa constrictor
100
Epicrates striatus
100
Boidae
Lichanura trivirgata
100
30
Exiliboa placata
100
Ungaliophis continentalis
100
Acrochordidae
Acrochordus granulatus
Xenodermatidae
Xenodermus javanicus
33
Pareas hamptoni
Pareatidae
100
100
Causus defilippii
34
Daboia russelii
35
100
Azemiops feae
Viperidae
100
Bothrops asper
100
92
Agkistrodon contortrix
100
Lachesis muta
Homalopsis buccata
Homalopsidae
36
100
100
Aparallactus werneri
99
Atractaspis irregularis
Lamprophiidae
Lamprophis
fuliginosus
37
100
19
Lycophidion capense
100
Naja kaouthia
100 38
100
Micrurus fulvius
Elapidae
100
Laticauda colubrina
Notechis scutatus
100
39
Lampropeltis getula
100
Trimorphodon biscutatus
100
Coluber constrictor
100
Sonora semiannulata
40
100 76
Imantodes cenchoa
100
Diadophis punctatus
Colubridae
89
Heterodon platyrhinos
Natrix natrix
Thamnophis marcianus
100
Afronatrix anoscopus
100
Amphiesma stolata
100
Xenochrophis piscator
100
Shinisauridae
Shinisaurus crocodilurus
100
Lanthanotidae
43
Lanthanotus borneensis
44
Anguimorpha
Varanus exanthematicus
100
Varanus acanthurus
Varanidae
100
Varanus salvator
42
100
Heloderma horridum
100
Helodermatidae
Heloderma
suspectum
100
45
Xenosaurus grandis
Xenosauridae
100
Xenosaurus platyceps
100
46
Celestus enneagrammus
54
Anniella pulchra
Anguidae
100
Elgaria multicarinata
73
Ophisaurus apodus
100
Ophisaurus ventralis
100
Brookesia brygooi
Chamaeleonidae
48
41
100 Uromastyx aegyptia Chamaeleo calyptratus
67
Leiolepis belliana
100
Hydrosaurus sp.
Acrodonta
96
Phrynocephalus mystaceus
100
Agama agama
100
Trapelus agilis
100
100
Draco blanfordii
100
Calotes emma
100
Agamidae
Acanthosaura lepidogaster
100
76
Physignathus cocincinus
47
100
Chelosania brunnea
Moloch
horridus
100
100
Hypsilurus boydi
100
Istiurus lesueurii
87
Pogona vitticeps
Iguania
100
Chlamydosaurus kingii
98
Ctenophorus isolepis
100
Rankinia adelaidensis
100
Leiocephalus barahonensis
Leiocephalidae
Basiliscus basiliscus
Corytophanidae
Corytophanes cristatus
49
100
Crotaphytus collaris
Crotaphytidae
Gambelia wislizenii
100
100
Enyalioides laticeps
Hoplocercidae
Morunasaurus annularis
100
Pleurodonta
Diposaurus dorsalis
Iguanidae
Brachylophus fasciatus
100
Sauromalus obesus
77
Stenocercus guentheri
Tropiduridae
Tropidurus plica
100
Uranoscodon superciliosus
100
56
Anolis carolinensis
Dactyloidae
Polychrus marmoratus
Polychrotidae
Phrynosoma platyrhinos
63
Uma scoparia
100
Phrynosomatidae
Petrosaurus mearnsi
100
Sceloporus variabilis
100
Uta stansburiana
99
97
Phymaturus palluma
Liolaemus bellii
Liolaemidae
100
Liolaemus elongatus
100
Chalarodon madagascariensis
Opluridae
Oplurus cyclurus
100
Urostrophus vautieri
100
Leiosaurus catamarcensis
Leiosauridae
100
Pristidactylus
torquatus
100
100

76

1
100

100

0.01 substitutions per site

Figure 1. Phylogeny of squamate reptiles from concatenated likelihood analysis of 44 nuclear genes (see the electronic supplementary material, figure S1 for Bayesian tree). Uncircled numbers at nodes indicate bootstrap values .50% (branches
too short to depict here have clades indicated with an open circle); circled numbers correspond to clades in electronic supplementary material, appendix S5. Branch lengths are estimated by likelihood (length for root arbitrarily shortened to
facilitate showing ingroup branch lengths).

3. RESULTS
Analyses of the concatenated data using likelihood (figure 1) and Bayesian (see the electronic
Biol. Lett.

supplementary material, figure S1) methods yield similar phylogenies and support values and provide strong
support for most higher-level squamate relationships.

Downloaded from rsbl.royalsocietypublishing.org on October 22, 2012

Squamate reptile phylogeny J. J. Wiens et al.

Table 1. Relationships between likelihood branch lengths, support and congruence for 49 clades of squamate reptiles.
variables
branch
branch
branch
branch
branch
branch

length
length
length
length
length
length

versus
versus
versus
versus
versus
versus

bootstrap support
proportion genes supporting
proportion genes strongly supporting (bs 70%)
proportion genes strongly supporting (bs 95%)
proportion genes strongly rejecting (bs 70%)
proportion genes strongly rejecting (bs 95%)

Many aspects of the tree are consistent with other recent

molecular analyses [e.g. 35] such as the clade of snakes,
anguimorphs and iguanians (Toxicofera).
However, we also find some surprising relationships (figure 1; electronic supplementary material,
figure S1). First, we find that dibamids and gekkotans
are together the sister group to other squamates (bs
76%; Pp 1.00), whereas previous studies have generally placed either dibamids or gekkotans as sister to
other squamates [4,6,16], but not both (but see [5]).
Second, we find strong support for non-monophyly
of Scolecophidia, the blind snakes (Anomalepididae,
Leptotyphlopidae and Typhlopidae). Specifically,
leptotyphlopids typhlopids are strongly supported as
sister to all other snakes, whereas anomalepidids are
sister to all non-scolecophidians (bs 100%; Pp
1.00). We also find a strongly supported clade (bs
97%; Pp 1.00) within pleurodont iguanians that is
inconsistent with relationships from 29 nuclear loci
[9]. This clade includes oplurids, leiosaurids, polychrotids, dactyloids, liolaemids and phrynosomatids,
whereas the earlier study found phrynosomatids as
sister to other pleurodonts (bs 88%; Pp 1.00).
Unexpectedly, some aspects of squamate phylogeny
still remain weakly supported. These include placement of uropeltids among snakes and relationships
among many pleurodont iguanian families (figure 1;
see the electronic supplementary material, figure S1).
We find a strong relationship between bootstrap support and branch lengths, and between congruence
and branch lengths, such that shorter branches tend
to be weakly supported and have greater incongruence
among genes (table 1).
The phylogenetic estimate from the species-tree
analysis (see the electronic supplementary material,
figure S2) is generally similar to the concatenated-data
trees (but with some differences), and strongly supports
some of the more surprising relationships. Specifically,
there is very strong support for placing dibamids and
gekkotans as sister taxa, and for non-monophyly of scolecophidians (but with a different arrangement of taxa).
Interestingly, this analysis strongly supports iguanians
and anguimorphs as sister taxa, a particularly controversial aspect of squamate phylogeny. This clade is also
supported by the concatenated analyses (figure 1; see
the electronic supplementary material, figure S1), as
sister to snakes.

4. DISCUSSION
We present the most extensive analysis of higher squamate phylogeny to date (in terms of characters and
Biol. Lett.

Rho

0.667
0.921
0.931
0.897
20.491
20.266

0.0001
0.0001
0.0001
0.0001
0.0003
0.0646

taxa), and the first to apply species-tree methods to

these relationships. Our results support some aspects
of previous hypotheses, but also show some surprising
findings. First, both approaches support dibamids and
gekkotans as the sister group to all other squamates, in
contrast to most previous studies. Second, we find
strong support for paraphyly of scolecophidian snakes.
This result has appeared in some previous studies [e.g.
7], and we are unaware of molecular studies that have
both included all three families and strongly supported
scolecophidian monophyly.
Paraphyly of scolecophidians is surprising in that
these families share many morphological and ecological
traits, including highly reduced eyes [17]. All scolecophidians are specialized burrowers. Thus, paraphyly of
scolecophidians at the base of snake phylogeny suggests
that snakes may have been burrowers ancestrally, and
that most snake species evolved from an ancestor that
subsequently returned to surface dwelling. This hypothesis is also supported by the morphology of snakes
relative to other limb-reduced lizards: for example,
snakes have short tails and elongate trunks (as do burrowing snake-like lizards), whereas surface-dwelling
snake-like lizards have elongate tails relative to the
trunk [16]. However, some scolecophidian features
may have evolved convergently in the two clades,
rather than being ancestral for snakes.
Our results also show that some aspects of squamate
phylogeny remain weakly supported, especially relationships among some snake and iguanian families. Many of
these relationships were also weakly supported in analyses of 2029 loci [5,7,9]. Thus, roughly doubling
the number of loci fails to lead to strong support. In contrast, many relationships supported here were also found
in analyses of only one or two nuclear loci [3].
Our analyses of branch lengths, support and congruence suggest that these patterns are related to branch
lengths [7]. Specifically, we find weaker support and
greater incongruence among genes (and more strongly
supported conflicts) on shorter branches, suggesting
that these nodes may continue to be problematic as
more loci are added. This incongruence seems to arise
from incomplete lineage sorting on short branches [7]
and may plague many other phylogenomic studies
[18]. Importantly, many shorter branches are strongly
resolved by the coalescent-based species-tree approach,
which incorporates incomplete lineage sorting [10,11].
Our results highlight the potential value of this general
approach for resolving short branches with phylogenomic data, although further advances may be needed
to make these methods practical for large datasets with
incomplete sampling of genes.

Downloaded from rsbl.royalsocietypublishing.org on October 22, 2012

J. J. Wiens et al.

Squamate reptile phylogeny

This project was supported by a US National Science

Foundation-AToL grant, with awards to T.W.R. (EF
0334967), J.W.S. (EF 0334966) and J.J.W. (EF 0334923).
We thank the many institutions and individuals who
provided tissue samples (see the electronic supplementary
material, appendix S1), Jyoti Aggrawal and Arianna Klein for
assistance with congruence analyses, and two anonymous
reviewers for helpful comments on the manuscript.
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