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Supporting Information
Unraveling the Peculiarities of Island Life: Vicariance, Dispersal and the Diversification
of the Extinct and Extant Giant Galápagos Tortoises.
Nikos Poulakakis, Michael Russello, Dennis Geist, and Adalgisa Caccone
Material and Methods
Higher-level analyses were based on consensus mtDNA sequences for extinct and
extant lineages of Galápagos tortoises from three mitochondrial DNA regions: control
region (CR), 16s rDNA (16S) and cytochrome b (cyt b). A dataset of 108 OTUs [105 of
all extant and extinct populations throughout Galápagos and three outgroup taxa (G.
chilensis, G. carbonaria and G. denticulata)] was used for subsequent phylogenetic
analyses (Table S1), based on the most divergent mtDNA region of the giant tortoises,
which is the CR (see Caccone et al. 2002). This comprehensive dataset consists of 21
haplotypes recovered from all extinct populations (Floreana, Rabida, Santa Fe,
Fernandina, and San Cristóbal) (Poulakakis et al. 2008; Russello et al. 2005; present
study) and 84 haplotypes identified from almost 900 individuals sampled from all extant
populations throughout Galápagos (Beheregaray et al. 2004; Caccone et al. 2002;
Caccone et al. 1999; Russello et al. 2005) 1, excluding the 12 haplotypes (Burns et al.
2003; Russello et al. 2007) that originated from animals in captivity [Charles Darwin
Research Station in the Galápagos Islands (CDRS); Caloosahatchee Aviary and Botanical
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In the article of Beheregaray et al. (2004), the authors by mistake mentioned 83 different haplotypes as the
haplotypes 80 (AF548283) and 83 (AF548286) are identical.
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Garden (CABG); mainland Ecuador hotels, universities, zoological and private
collections (ECU); Prague Zoo (PRZ); Zurich Zoo (ZUZ)].
DNA extraction
A) Modern specimens: The 16S and cyt b sequences were available only for 48
of the 84 specimens that produced the 84 CR haplotypes. For this reason, total genomic
DNA was extracted from 36 fresh specimens (Table S1) following a previously described
protocol (Caccone et al. 1999) in order to augment the available mtDNA database of
giant Galápagos tortoises. DNA was extracted from blood stored in 100 mM Tris/100
mM EDTA/2% SDS buffer by using the DNeasy Blood & Tissue Kit (QIAGEN).
B) Museum samples: Bone samples of the Giant Galápagos tortoises were
obtained from the following museums: Museum of Comparative Zoology (MCZ),
California Academy of Science (CAS), American Museum Natural History (AMNH),
and National Museum of Natural History, Smithsonian Institution (USNM) (Table S1),
representing the extinct taxa of Floreana, Rabida, Santa Fe, and Fernandina, and
extirpated populations from western San Cristóbal, Pinta and Pinzón. In total we
processed 69 specimens, 16 from MCZ, 24 from AMNH, 9 from USNM, and 20 from
CAS. Homogenized bone powder was obtained by grinding the samples using a Spex
6750 freezer mill (Spex SamplePrep, Metuchen, NJ, USA).
All DNA extractions were carried out in a physically separate laboratory
dedicated to the study of ancient DNA at Yale University (USA), following all necessary
precautions to prevent contamination by extant specimens, following the bone extraction
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method of the DNA IQ System (Promega, USA) (Rohland & Hofreiter 2007) as in
(Poulakakis et al. 2008).
Mitochondrial DNA amplification and sequencing
A) Modern specimens: A fragment of 568 bp of the 16S rRNA and 386 bp of the
cyt b gene were amplified using two pairs of primers (16Sar-16Sbr for 16S rRNA and
GLU - H15149 for cyt b). All CR sequences were already available.
B) Museum samples: Partial sequences of the mitochondrial CR, cyt b, and 16S
rRNA were amplified in small overlapping fragments of ~200 (including the primers) in
length (Table S2). For each DNA extract, PCR amplification was initially performed with
a corresponding pair of external primers. The product of the primary reaction served as
the template for another PCR amplification (nested reaction) using the corresponding pair
of internal primers. Mitochondrial fragments were amplified in 20μL PCR reactions
containing: 1X High Fidelity PCR Buffer, 3.00mM MgSO4, 100μM of each dNTP,
0.30μM of each PCR primer, 1mg/ml BSA and 0.4 units of Taq polymerase (Platinum
Taq High Fidelity; Invitrogen). Reactions were carried out using an initial step at 94oC/5
min, followed by 40 cycles of 94oC for 45s, 50oC (CR), 48oC (cyt b), and 50oC (16S) for
60s, and 68oC for 90s, and a final extension of 68oC for 25 min. At least two sterile
negative controls were used for each reaction to detect contamination throughout
amplification. Sequences were obtained from at least two amplifications of individual
samples. Double-stranded PCR products were sequenced using Big Dye 3.1 terminators
on an ABI3730 DNA sequencer (Applied Biosystems, USA).
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Mito-phylogenetic analyses (combined dataset)
Of the 69 museum specimens sampled, only 21 specimens yielded DNA of
sufficient quality and quantity to produce scorable and repeatable sequence data (Table
S1). Sequences from the extinct lineages (Floreana, Santa Fe, Rabida, and Fernandina)
were combined with present and previously published sequences of the extant and
recently extirpated Giant Galápagos tortoise lineages and three outgroup taxa (C.
chilensis, C. carbonaria and C. denticulata) for subsequent phylogenetic analyses,
producing a dataset of 1,682 bp of CR, 16s, cyt b mtDNA regions for 108 extinct and
extant tortoises.
DNA sequences were aligned using default parameters in ClustalX (Thompson et
al. 1997). The best-fit model of DNA substitution and parameter estimates used for tree
constructions were chosen according to the Akaike Information Criterion (Akaike 1974)
(see Posada & Buckley 2004) as implemented in Modeltest v. 3.7 (Posada & Crandall
1998). The best-fit model for the concatenated dataset was GTR+I+G (-lnL=6219.8735,
I=0.62, α=0.53). The most suitable model for each target gene (D-loop, cyt b, and 16S)
was: TrN+I+G (-lnL= 3398.97, I=0.42, a=0.50), GTR+I+G (-lnL= 1205.26, I=0.62,
a=0.88), and GTR+I (-lnL=1170.02, I=0.77), respectively.
Phylogenetic analyses was conducted using Bayesian Inference (BI) as
implemented in MrBayes v3.1.2 (Ronquist & Huelsenbeck 2003), taking advantage of the
ability of this software package to handle a wide variety of data types and models, as well
as any mix of these models, based on the procedure described in manual. The analysis
was run with four chains for 107 generations, sampling from the chain every 100
generations. This generated an output of 105 trees. In order to confirm that the chains had
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achieved stationarity, we evaluated “burn-in” by plotting log-likelihood scores and tree
lengths against generation number using the software Tracer (v. 1.5.0) (Rambaut &
Drummond 2008). The –lnL stabilized after approximately 106 generations and the first
104 trees were discarded as a conservative measure to avoid the possibility of including
random, sub-optimal trees. The percentage of samples recovering any particular clade in
a BI analysis represents that clade’s posterior probability (Huelsenbeck & Ronquist
2001). A majority rule consensus tree ('Bayesian' tree) was then calculated from the
posterior distribution of trees, and the posterior probabilities calculated as the percentage
of samples recovering any particular clade, where probabilities ≥ 95% indicate significant
support (Fig S1).
Of the 108 haplotypes analyzed, nine tortoise haplotypes (non-native individuals;
Caccone et al. 2002; Poulakakis et al. 2008) belonging to five populations (Puerto Bravo,
PBR; Piedras Blancas, PBL; Santiago, AGO; Roca Union, RU; and Cerro Montura in
Santa Cruz; CM) stand out as being aberrant in that they are clearly different from the
majority of haplotypes in the population from which they were sampled while they differ
by only a few substitutions from haplotypes found in geographically distinct populations,
often located on different islands. Additionally, seven museum specimens (R-4477, R4478, R-11064, R-11070, R-213706, R-222475, and R-222478) cluster with lineages
other than that of their geographical origin (e.g., the last three specimens described by
museum records as being sampled on Pinta island actually cluster with the Pinzón
lineage). These individuals and the nine modern specimens previously recognized as
"non-native" were eliminated from the final dataset, as they are likely not the outcome of
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natural colonization events, but the result of either human mediated translocations
(Caccone et al. 2002; Poulakakis et al. 2008) or possibly error in the museum records.
The exclusion of the non-native and outlier specimens produced a dataset of 92
"pure" haplotypes that was used to repeat the Bayesian analysis (detailed in the main text)
as before and for the remaining analyses, such as 1) the delineation of genetic clusters
using a generalized mixed Yule coalescent (GMYC) model, 2) the estimation of sequence
divergences [(MEGA v.5.04 (Tamura et al. 2011), using the Tamura & Nei (1993) model
of evolution] for the current taxonomic units of the giant Galápagos tortoises (Table S3),
and the independently evolving mtDNA entities as indicated by SPLITS (Table S4), and
3) the estimation of divergence times.
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