SUPPLEMENTARY MATERIAL
APPENDIX I – Taxon sampling
Frozen tissues of six snake species (Ramphotyphlops braminus YPM 13663,
Agkistrodon contortrix YPM 13252, Micrurus fulvius YPM 14096, Dendroaspis jamesoni
YPM 13380, Lampropeltis getula YPM 13574, and Elaphe obsoleta YPM 13086) and two
limbless species (Ophisaurus apodus YPM 12870, and Isopachys gyldenstolpei YPM
11726) were obtained from the Peabody Museum at Yale University. Eggs of the snake
Coluber constrictor were purchased from herp trades (Glades Herps Inc., USA), and eggs
of Alligator mississipiensis were purchased from Clarkbrook Farm/Jungle Adventures.
Tissues were removed from the embryos of these two species for DNA extraction.
Published DNA sequences of Hoxa-13, c-mos, RAG-1 and BDNF available online were
used for the remaining species included in the present study: Hoxa-13 – Agama agama
(AF083099), Cyclura nubila (DQ119608), Leiocephalus barahonensis (DQ119607),
Varanus dumerilii (AF083102), Heloderma suspectum (DQ119604), Typhlops richardii
(AF083103), Liasis sauvensis (DQ119006), Charina trivirgata (AF083104), Rhineura
floridana (DQ119600), Trogonophis wiegmanii (DQ119603), Amphisbaena cubana
(DQ119600), and Anniella pulchra (DQ119605); RAG-1 – Alligator missisipiensis
(AY239171), Agama agama (AY487356), Cyclura nubila (DQ119625), Leiocephalus
barahonensis (DQ119624), Varanus dumerilii (AY487354), Heloderma suspectum
(AY662606), Typhlops lumbricalis (AY487387), Ramphotyphlops braminus (AY662612),
Liasis sauvensis (AY487397), Charina trivirgata (AY487394), Micrurus surinamensis
(AY487411), Dendroaspis angusticeps (AY487395); C-mos – Alligator missisipiensis
(AY910585), Agama agama (AF137530), Cyclura nubila (DQ119595), Leiocephalus
barahonensis (DQ119594), Varanus salvator (AF435017), Heloderma suspectum
(AY662566), Typhlops brachycephalus (AY099981), Rhamphotyphlops braminus
(AY099980), Liasis sauvensis (AF544726), Charina trivirgata (AY099974), Agkistrodon
piscivorus (AF471096), Micrurus fulvius (AY058935), Dendroaspis anguisticeps
(AF544735), Coluber constrictor priapus (AY486937), Elaphe quatuorlineata
(AY486955); BDNF – Alligator missisipiensis (AF416631), Japalura splendida
(AF497713), Leiocephalus carinatus (AY987970), Polychrus marmoratus (AY987966),
Brachylophus fasciatus (AY987980), Rhamphotyphlops sp. (AY988043), Charina bottae
(AY988042), Morelia spilota (AY988035), Acrochordus javanicus (AY988036),
Cylindrophis ruffus (AY988037), Exiliboa placata (AY988034).
For the analysis of Hoxa-13 along Gnathostomata, data was obtained also from
tissues obtained for Gymnopis multiplicata (Museum of Vertebrate Zoology, UC Berkeley,
MVZ228795), Protopterus annectens (tissue donated from a pet owner), Geochelone nigra
(tissue donated by Dr. G. Caccone, EEB-Yale University), and published sequences
available online for the following species: Heterodontus francisci (AF479755), Polyodon
spathula (AY661749), Polypterus senegalus (‘Bichir’, obtained from BAC genomic
library, Chiu et al. 2004), Latimeria chalumnae (AC147788), Ambystoma mexicanum
(U20942), Gallus gallus (AY030050).
APPENDIX II - Phylogenetic Analysis for Squamata
Public sequences of mitochondrial genomes available online were downloaded from
GenBank for the following taxa: Amphisbaena schmidti (AY605475.1), Geocalamus acutus
(AY605476.1), Bipes biporus (AY605480.1), Bipes canaliculatus (AY605482.1), Bipes
tridactylus (AY605477.1), Diplometopon zarudnyi (AY605474.1), Gecko gecko
(AY282753.1), Teratoscincus keyserlingii (AY753545.1), Iguana iguana (AJ278511.2),
Sceloporus occidentalis (AB079242.1), Cordylus warreni (AB079613.1), Eumeces
egregius (AB016606.1), Varanus komodoensis (AB080275.1 and AB080276.1),
Shinisaurus crocodilurus (AB080274.1), Abronia graminea (AB080273.1), Boa constrictor
(AB177354.1), Python regius (AB177878.1), Acrochordus granulatus (AB177879.1),
Cylindrophis ruffus (AB179619.1), Leptotyphlops dulcis (AB079597.1), Ovophis
okinavensis (AB175670.1), Xenopeltis unicolor (AB179620.1), Sphenodon punctatus
(AF534390.1), Alligator mississippiensis (Y13113.1), Gallus gallus (X52392.1), and
Casuarius casuarius (AF338713.2). All sequences were aligned using the Clustal-V
algorithm (Higgins et al. 1992), implemented by the software MegAlign 3.15 (DNASTAR).
Indels were manually removed and the alignment was manually inspected to identify
regions that were ambiguously aligned by MegAlign; these regions were also removed. All
characters were equally weighted.
Phylogenetic analyses were performed using maximum parsimony (MP) and
maximum likelihood (ML) in PAUP* version 4.0b10 (Swofford 2003), and Bayesian
inference (BPP) in MrBayes version 3.0b4 (Ronquist and Huelsenbeck 2003). Phylogenetic
inference was performed with the concatenated data set of 10,683bp. Models of evolution
were estimated by MrModeltest 2.1 (Nylander 2004). A single model was estimated for the
concatenated dataset for ML (HKY85 model). In the Bayesian analyses, the model used
was GTR+I+G, but the data was partitioned into 1st, 2nd and 3rd codon positions with each
partition having its own GTR+I+G parameters, estimated in MrBayes from the data using
default priors.
Statistical confidence in nodes was evaluated using 10,000 bootstrap replicates for
MP (Felsenstein 1985; Hillis and Bull 1993) and 100 bootstrap replicates for ML (due to
computational constraints). For ML and MP analyses we set the maxtrees command in
PAUP* to 5,000 with automatic increase every time the maximum of number of trees was
reached, followed by a heuristic search with TBR branch swapping. Heuristic searches
were unrooted and consensus trees were visualized with TreeView (Page 1996). In the
Bayesian phylogenetic inference, five chains of 1,000,000 generations were conducted,
sampling every 1,000th tree. The parameters of each run were visually inspected with
Tracer version 1.0.1 (Rambaut and Drummond 2003) to determine the number of
generations until stationary trees; the first 100 trees were discarded. All parameters were
linked between gene datasets in Bayesian analyses, but gamma and number of invariable
sites were estimated for each gene individually.
The phylogenetic tree generated by the Bayesian inferences (Fig. 1 of Appendix II;
bootstrap values from ML and MP heuristic searches are mapped on to nodes with support
values greater than 50%) was strongly supported by values of posterior probabilities higher
than 0.97, and the topology obtained was congruent among the three methods applied
(Maximum Parsimony, Maximum Likelihood and Bayesian inferences). In the
phylogenetic hypothesis generated by data from mitochondrial genomes, Serpentes are
sister clade of amphisbaenids, and this whole clade (snakes plus amphisbaenids) occupies a
sister position of a clade including iguanids and anguimorphs (Fig. 1 of Appendix II).
Gekkonids are placed in a basal position within squamates, with Sphenodon and the clade
grouping Alligator and birds placed in the most ancestral positions in the phylogenetic trees
(Fig. 1 of Appendix II), as they were determined a priori as outgroups in the analyses.
The phylogenetic hypothesis obtained in this study using molecular data from
mitochondrial genomes agrees with some aspects of topologies proposed from molecular
data (Vidal and Hedges 2005; Fry et al. 2006; Wiens et al. 2006) by placing a clade
composed by iguanids and anguimorphs as a sister group of the clade that contains snakes,
but disagrees with Townsend et al. (2004), which assumed Serpentes as a sister clade of
Anguimorpha, Iguanidae as a sister clade of this group (Serpentes + Anguimorpha), and
amphisbaenids placed in a separate group of Serpentes. It also disagrees with some
phylogenetic studies based on morphological data that placed iguanids in basal positions in
the topologies (e.g. Caldwell 1999; Lee 2000), although some few studies using
morphological characters also place iguanids and anguimorphs closer to each other, as a
separate group from the clade including snakes (e.g. Caprette et al. 2004 using eye
anatomy).
Literature Cited (Appendix II)
Caldwell MW (1999) Squamate phylogeny and the relationships of snakes and
mosasauroids. Zool. J. Linn. Soc. 125:115-147.
Carprette CL, Lee MSY, Shine R, Mokany A, Downhower JR (2004) The origin of snakes
(Serpentes) as seen through eye anatomy. Biol. J. Linn. Soc. 81:469-482.
Felsenstein J (1985) Phylogenies and the comparative method. Am. Nat. 125:1-15.
Fry BG, Vidal N, Norman JA, Vonk FJ, Scheib H, Ramjan FR, Kuruppu S, Fung K,
Hedges SB, Richardson MK, Hodgson WC, Ignjatovic V, Summerhayes R, Kochva E
(2006) Early evolution of the venom system in lizards and snakes. Nature 439(7076):
584-588.
Higgins DG, Bleasby AJ, Fuchs R (1992) CLUSTAL-V – Improved software for multiple
sequence alignment. Comp. Appl. Biosci. 8:189-191.
Hillis DM, Bull JJ (1993) An empirical test of bootstrapping as a method for assessing
confidence in phylogenetic analysis. Syst. Biol. 42:182-192.
Lee MS (2000) Soft anatomy, diffuse homoplasy, and the relationships of lizards and
snakes. Zool. Scripta 29(2):101-130.
Nylander JAA (2004) MrModeltest 2.1. Program distributed by the author. Evolutionary
Biology Centre, Uppsala University.
Page RD (1996) TREEVIEW: An application to display phylogenetic trees on personal
computers. Comput. Appl. Biosci. 12:357-358.
Rambaut A, Drummond A (2003) Tracer v1.0, available from evolve.zoo.ox.ac.uk.
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mixed models. Bioinformatics 19:1572-1574.
Swofford D (2003) PAUP*: Phylogenetic Analysis Using Parsimony (*and other methods),
Version 4. Sinauer Associates.
Townsend TM, Larson A, Louis E, Macey JR (2004) Molecular phylogenetics of
Squamata: the position of Snakes, Amphisbaenians, and Dibamids, and the root of the
Squamate tree. Syst. Biol. 53(5):735-757.
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amphisbaenians) inferred from nine nuclear protein-coding genes. C.R. Biologies
328:1000-1008.
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Appendix II
Alligator mississippiensis
1.00
Casuarius casuarius
-/96
1.00
100/100
Gallus gallus
Varanus komodoensis
Abronia graminea
1.00
64/94
1.00
78/57
Shinisaurus crocodilurus
Anguimorphs
Sphenodon punctatus
Cordylus cordylus
-/76
0.97
90/94
0.97
Eumeces egregius
Iguana iguana
58/82
1.00
100/100
Sceloporus occidentalis
Iguanids
1.00
Reptilia
Leptotyphlops dulcis
88/83
Boa constrictor
1.00
0.99
Ovophis okinavensis
100/100
-/-
Cylindrophis ruffus
1.00
96/72
Python regius
1.00
96/81
1.00
99/99
1.00
Xenopeltis unicolor
87/58
Amphisbaena schmidti
1.00
100/100
1.00
-/72
1.00
85/100
1.00
Geocalamus acutus
Bipes tridactylus
100/100
1.00
Bipes biporus
100/100
1.00
100/100
Bipes canaliculatus
Gekko gecko
1.00
100/100
PP
MP/ML
Teratoscincus keyserlingii
Amphisbaenias
Diplometopon zarudnyi
Squamata
1.00
100/100
Serpentes
Acrochordus granulatus
1.00