Phylogeny construction
Sequences of five nuclear genes and five mitochondrial genes were downloaded from GenBank
for 202 lacertid species and one outgroup (the amphisbaenian Rhineura floridana) (table S1).
For species with multiple entries for the same gene, the most complete sequence was retained.
Due to uneven taxon sampling many species suffer from missing data, but all are represented by
at least two genes. Each gene was aligned and edited separately in Bioedit v.7.2 [1] and
ambiguous positions were identified and removed in GBLOCKS v.0.91b [2] using the following
parameters: a minimum of half the number of sequences for a conserved and flanking position, a
maximum of eight contiguous non-conserved positions, a minimum of two sites for the block
length after gap cleaning and all gap positions selected. Resulting alignments were concatenated
in SeaView v.4 [3] to form a multigene supermatrix consisting of 10439 sites with 72% missing
data.
Molecular divergence dating analysis was performed in the Bayesian software package
BEAST v.1.7.4 [4] with the following fossil-based dates as calibrations:
1. Amphisbaenia-Lacertidae, "Lacertibaenia" (minimum: 64.2 Ma; maximum: 113.0 Ma): based
on the oldest known amphisbaenian fossil, Plesiorhineura [5]. The maximum bound was set to
113.0, which marks the oldest record of the clade’s sister-taxon, Teiioidea (see above). Because
there is currently no evidence of other truly amphisbaenian or lacertid fossils older than the K-T
boundary despite a decent Late Cretaceous fossil record for squamates, we applied an
exponential probability distribution decreasing towards older ages, i.e. a mean of 8.0 and an
offset of 0.
2. Gallotiinae-Lacertinae (minimum: 29.0 Ma; maximum: 64.2 Ma): based on the oldest known
occurrence of the fossil lacertid genus Dracaenosaurus (see [6] and references therein), which
clusters with gallotiines in a recent combined analysis by [7]. The maximum bound was set to
64.2, marking the age of the oldest known fossil for the split Amphisbaenia-Lacertidae (see
above). For this calibration, a uniform prior was used due to the current absence of other
phylogenetically informative lacertid fossils from the Paleogene.
3. Lacerta-Timon (minimum: 18.1 Ma; maximum: 64.2 Ma): based on the oldest known
occurrence of a fossil from the Lacerta viridis complex [8]. Again, the oldest date was set to
64.2, but this time with an exponential probability distribution decreasing towards older ages
(mean: 2.0; offset: 0.0), as a high number of modern-looking lacertid fossils from the early
Neogene (pers. obs.) makes it unlikely that the split between these two genera lies significantly
below the Paleogene/Neogene boundary.
4. Mesalina-Acanthodactylus (minimum: 13.1 Ma; maximum: 64.2 Ma): based on the record of
a middle Miocene lacertid from Beni Mellal, Morocco, originally assigned to Eremias by [9]. At
the time of description, however, Mesalina was considered part of Eremias and only later
became a separate genus. Given North Africa contains no other lacertids formerly assigned to
Eremias except for Mesalina, we consider it reasonable to assign this fossil to the Mesalina
lineage. As in the case of the Lacerta-Timon split, the maximum age was set to 64.2, with an
exponential probability distribution decreasing towards older ages (mean: 2.0; offset: 0.0).
5. Timon lepidus-Timon pater (minimum: 1.9 Ma; maximum: 64.2 Ma): based on the oldest
known occurrence of Timon lepidus from the Pleistocene of Spain [10]. The maximum bound
was set to 64.2, with an exponential probability distribution decreasing towards older ages
(mean: 2.0; offset: 0.0).
7. Acanthodactylus erythrurus-Acanthodactylus blanci (minimum 0.8 Ma; maximum: 64.2 Ma):
based on the oldest undoubted occurrence of Acanthodactylus erythrurus from the Pleistocene of
Spain [11]. The maximum bound was set to 64.2, with an exponential probability distribution
decreasing towards older ages (mean: 2.0; offset: 0.0).
Due to the still uncertain phylogenetic affinities within lacertine (palearctic) lacertids, a
preliminary run without calibrations was performed to obtain a reasonable hypothesis of
relationships. Based on this topology, which was also used as the template for the starting tree,
split 3 was identified and calibrated (see above).
Divergence dates were estimated using an uncorrelated lognormal relaxed clock with a
Yule process as tree prior. The following substitution models were applied for each individual
gene as determined by jmodeltest v.0.1.1 [12], always with empirical base frequencies: NDF,
RAG1, CO1, 12S, 16S, CYTB: GTR + I + Gamma; BDNF: HKY + I; CMOS: HKY + I +
Gamma; RAG2: HKY + Gamma; FGB: HKY. We conducted a run with 20000000 generations
and a sampling of every 1000th generation, as the large size of the supermatrix made it difficult
to reach stationarity at earlier stages. The first 25% of trees were discarded as burn in. Analysis
in Tracer 1.5 [13] revealed estimated sample sizes of more than 100 for all relevant parameters.
The final tree was generated in TreeAnnotator (implemented in the BEAST package), with a
posterior probability limit of 0.99 and mean node heights.
To perform phylogenetic comparative analyses on the morphological data set, some
species lacking sufficient sequence data were manually added to the phylogeny. Twenty-one
lacertid species were grafted to the final 99% maximum clade credibility tree based on
information from the literature (e.g., morphology-based hypotheses) or our own phylogenetic
analyses. In cases where only one species of a genus was included in the original tree thus
creating a long branch, we arbitrarily created a polytomy halfway along the branch and added
the additional taxa (Latastia boscai, L. siebenrocki, Philochortus hardeggeri, P. neumanni,
Pseuderemias brenneri, P. mucronata, and P. striatus). Phylogenetic placement of Darevskia
armeniaca, Eremias grammica, E. przewalskii and E. vermiculata (each represented by a single
gene) was based on preliminary PhyML trees generated in SeaView. Missing Ophisops taxa (O.
leschenaulti, O. microlepis) formerly assigned to the Indian genus Cabrita were placed with the
other former Cabrita in the tree, Ophisops jerdonii.
The above insertions resulted in the following tree used in all analyses, containing 223
species (added taxa are in shown red):
Morphological data
540 ethanol-preserved adults (1-9 individuals/species, median=4) were measured for
features of the head and body shown to be functionally related to habitat use (e.g., [14]):
snout-vent length, tail length, head depth, head width, head length, humerus length,
radius length, femur length, tibia length, foot length and toe length (table S2). Because
individuals varied considerably in snout-vent length (28-146 mm), we removed the
effects of body size using Mosimann’s [15] geometric mean method. An overall index of
size was calculated for each individual as the arithmetic mean of the log-transformed
variables (equal to the log of the geometric mean). The size index was then subtracted
from the log of each trait to generate a size-free shape variable. A reduced data set
containing 361 individuals (111 species) with whole, original tails was treated separately
in all analyses. The full data set containing species means and sample sizes is available at
http://morphobank.org/permalink/?P1174.
To reduce dimensionality of the data, we performed a principal component
analysis on the covariance matrix of the size-adjusted species means. The first five axes,
accounting for 92% of total shape variation, were retained for analyses (table S2).
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