Supplementary materials and methods
Study species
Linaria glacialis Boiss. (Fam. Plantaginaceae, tribe Antirrhineae) is an annual or
perennial narrow endemic toadflax inhabiting schistose screes of the alpine vegetation
belt of Sierra Nevada (2700-3400 m). It has an occluded corolla similar to that of
snapdragons (Antirrhinum), but with a long spur. The corolla is similar in size and
shape to that of closely related congeneric species (subsect. Supinae, sect. Supinae;
Blanco-Pastor et al. 2012). L. glacialis is mainly differentiated from other Linaria species
by its large leaf-like calyx lobes and bracts (see Fig. 1B). The conservation status of this
species is Vulnerable –VU D2; UICN category– (Blanca et al. 1998) and the number of
individuals has been estimated to be less than 10,000 (Blanca et al. 2001). The scarcity of
adequate habitat is considered the main threat for this species (Blanca et al. 1998; Blanca
et al. 2001).
Study area
Sierra Nevada is located in the southern part of Spain nearby the Mediterranean Sea
(see Fig. 1A) and has the highest altitude of the Iberian Peninsula (Mulhacen, 3481 m
a.s.l.). This mountain range was covered with glaciers only in areas above ~2,500 m
a.s.l. during Quaternary glaciations (Gómez-Ortiz et al. 1996), while large areas
remained free of permanent ice. Sierra Nevada was a refuge area for many European
species during glacial ages (Blanca et al. 1998; González-Sampériz et al. 2010). Indeed,
location and altitude of Sierra Nevada has interested scientist to test speciation patterns
in plants (Gutiérrez Larena et al. 2002; Kropf et al. 2006).
The upper vegetation belt (alpine or cryoromediterranean) of Sierra Nevada has an
extension of 3875,7 ha and its lower boundaries span between 2750 m in northern and
western zones and 3290 m in southern and eastern zones (Fernández Calzado &
Molero Mesa 2011). It shelters an abrupt landscape with steep slopes, screes and rocky
areas and has a metamorphic substrate composed by feldspar, micaschist, slates and
quartzites. The effect of glacier phenomena is still present in the northern part of the
range, although at the beginning of 20th century the last ice mass (Veleta glacier) was
strongly reduced and since 1995 there are no traces left (Gómez-Ortiz et al. 2009). In
this area the vegetation is exposed to stressful climate conditions with large daily
temperature oscillations and a pronounced summer drought.
Sampling and DNA sequencing strategy
Apart from the L. glacialis dataset, an additional dataset (species-level dataset) was
generated for the present study. The species-level dataset included DNA sequences of
two L. glacialis individuals and 15 individuals of other closely-related species, which
were chosen based on previous phylogenetic studies (Blanco-Pastor et al. 2012;
Fernández-Mazuecos et al. 2013): L. aeruginea (two individuals), L. almijarensis, L.
anticaria, L. lilacina, L. platycalyx, L. tristis, L. amoi, L. verticillata, L. depauperata, L.
polygalifolia (3 individuals), L. supina, L. arvensis (two individuals) and L. saxatilis (see
above). The species-level dataset comprised both previously published and newly
generated sequences of the ITS region (Blanco-Pastor et al. 2012; Fernández-Mazuecos
et al. 2013) and the low copy nuclear gene AGT1 (partial intron) (Blanco-Pastor et al.
2012) (see Supplementary table 1 and 2).
Deciphering of haplotypes from unphased genotypes and test for recombination
In addition to AGT1 sequences of the L. glacialis dataset, AGT1 and ITS sequences of
the species-level dataset were also analyzed with PHASE. In the species-level dataset,
when haplotypes inferred were not statistically supported, we obtained haplotype data
by cloning the PCR products as done in Blanco-Pastor et al. (2012). In the cloning
process several paralogous copies were obtained for the ITS region from one species (L.
aeruginea) and for the AGT1 region from another species (L. verticillata). We then
assessed the orthology of amplification products following Whittall et al. (2006).
Recombination was also tested within the AGT1 and ITS alignments of the specieslevel dataset using the software RDP 3.44 (Martin et al. 2010). Methods and parameter
settings were as described in Blanco-Pastor et al. (2012).
Species tree estimation
In order to investigate the evolutionary framework for L. glacialis we performed a
*BEAST species tree analysis (Heled & Drummond 2010) as implemented in the BEAST
software v.1.7.2 (Drummond & Rambaut 2007; Drummond et al. 2012) using ITS and
AGT1 intron haplotype sequences of the species-level dataset after excluding two
AGT1 haplotypes with hard incongruence (L. amoi, L. lilacina) subject to be caused by
hybridization/introgression (as the *BEAST model does not account for such
processes). The crown age of section Supinae was calibrated with a normal distribution
prior with mean 1.95 Ma and standard deviation 0.66. This date was previously
obtained in a three-locus species tree phylogeny of Linaria sect. Supinae with detection
and exclusion of plausible hybrids (Blanco-Pastor et al. 2012). The remaining prior
settings of the analysis were equal to those used in that study.
Population size history
Extended Bayesian skyline plot
Extended Bayesian skyline plot (EBSP) analysis allows the joint analysis of multiple
loci and uses Bayesian stochastic variable selection to select the appropriate
smoothness of the demographic function (number of groups of coalescent intervals).
The analysis was carried out combining information of the four regions of the L.
glacialis dataset two linked plastid loci (ptDNA) and two linked regions of the AGT1
gene. We included the four regions as independent partitions for the analysis with
distinct substitution models as obtained with jModeltest 0.1.1 (Posada 2008) (see
Supplementary table 1). The two plastid regions were linked for the partition tree and
the clock model priors. AGT1 exon and intron regions were also linked for the partition
tree but unlinked for the clock model priors. Upper limits for the mean.rate parameters
were set to 5 s/s/Ma for the AGT1 regions and 1 s/s/Ma for the ptDNA loci. Operators
of the analysis were also modified following the recommendations of the EBSP tutorial
available in http://beast.bio.ed.ac.uk/Tutorials.
Supplementary results
Haplotype data gathering and recombination test
The species-level dataset included 36 ITS and 33 AGT1 sequences. Recombination was
neither detected in the ITS or AGT1 alignments of the species-level dataset by any of
the five methods used. This allowed us to perform the species tree analysis without
discarding any recombinant region.
Species tree
Despite the low resolution and the requirement of additional loci to unravel the species
relationships within this group (sect. Supinae subsect. Supinae), the higher number of
species and sequences used here compared to the analysis of Blanco-Pastor et al. (2012)
extends the previous phylogenetic information obtained of this recently diversified
group of Linaria. The species tree obtained from dataset A (Supplementary Fig. 1)
showed L. glacialis within subsect. Supinae sensu Blanco-Pastor et al. (2012) and forming
a monophyletic group together with eight southern Iberian endemics (0.99 PP).
Additionally it also showed L. glacialis as sister to the sub-endemic low-land species of
Sierra Nevada L. verticillata (with moderate support; 0.84 PP) (see Supplementary Fig.
1), with a divergence time 0 – 230 ka 95% HPD (60 ka median).
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