SUPPLEMENTARY MATERIALS
Here you will find supporting methods, data, and figures for Roda et al.
“Genomic evidence for the parallel evolution of coastal forms in the Senecio
lautus complex”, published in Molecular Ecology in 2013. In separate excel
sheets you will find:
Table S1. Description of populations used in this study.
Table S2. Voucher specimens previously sampled in or close to our sampling localities.
Table S3. Senecio sequences retrieved from public databases.
Table S4. Herbarium specimens sequenced in this study.
Table S5. Marker, primer and sample information for plastid and nuclear genomic regions
sequenced in this study.
Table S6. Available phylogenetic information from relevant Senecio samples. We provide
information from species sampled in this study as well as for other Australian Senecio
species.
Table S7. Estimates of population parameters as evaluated in pairwise genomic
comparisons.
Table S8. Mantel Test results.
SUPPLEMENTARY METHODS
Sampling considerations
A combination of biological characteristics, such as its wide and diverse
bio-geographic distribution, the correlation between the morphology of its
populations and the habitat they occupy, but the continuous distribution of some
traits within and among its populations, makes the Senecio lautus species
complex (Senecio lautus G.Forst) taxonomically problematic (Ali 1964a; b,
1969; Thompson 2005; Radford & Cousens 2006). The complex has been
repeatedly split into multiple species based on flower morphology (Ali 1964a; b,
1969; Thompson 2005; Radford & Cousens 2006), geographic distribution
(Ornduff 1964; Belcher 1993, 1994), reproductive system (Ornduff 1964; Ali
1966) or the existence of post-zygotic barriers to gene flow (Ornduff 1964; Ali
1966). However, subspecies and varieties have been defined according to
habitat and plant morphology (mostly leaf morphology but also growth habit and
flower morphology) (Ali 1964a; Ober & Hartmann 1999; Thompson 2005;
Radford & Cousens 2006).
This taxonomic flux brings enormous challenges to the study of
evolutionary processes where the distinction between taxa relies on gene
exchange and its consequences. The scope of the present study is to contribute
to our understanding of how adaptation to similar environmental conditions
drove the divergence of members of the S. pinnatifolius/lautus taxa rather than
clarifying the taxonomy of the complex, which will be the focus of a different
study.
In 2008 and 2009 we collected leaf samples from plants belonging to 49
populations of the Senecio lautus complex distributed across Australia (Table
S1). We identified collection sites using two complementary strategies. First, we
retrieved all collection sites from the Australia’s Virtual Herbarium server
(http://avh.ala.org.au/) for all members of the Senecio lautus complex,
specifically from S. spanomerus I.Thomps., S. hamersleyensis I.Thomps., S.
warrenensis I.Thomps., S. depressicola I.Thomps., S. condylus I.Thomps., S.
eremicola I.Thomps., S. lacustrinus I.Thomps., S. lautus G.Forst. ex Willd., S.
pinnatifolius A.Rich., S. brigalowensis I.Thomps., S. condylus I.Thomps., S.
halophilus I.Thomps., S. biserratus Belcher, and S. spathulatus A.Rich.
specimens. Second, we sampled in reported locations for headland, dune,
alpinus, lanceolatus, dessert and Inland ecotypes as described in Radford et al
(2004).
We obtained leaf material of 13 individuals from the Queensland
Herbarium and the Western Australian Herbarium to complement sampling of
known species and variants from the S. lautus complex, including samples from
S. brigalowensis, S. lacustrinus and S. pinnatifolius var. dissectifolius (see
Table S4 for details). We distinguished S. lautus plants from other Senecio
species, including S. madagascariensis, by counting the number of phyllaries
(Thompson, 2005; members of the S. lautus species complex in Australia have
13 phyllaries whereas S. madagascariensis plants have 20 phyllaries; S.
brigalowensis has 18-20 phyllaries, but it does not co-occur with S.
madagascariensis). We currently have a germplasm collection in the laboratory
for most of these populations and they are available upon request.
The RADs-based phylogeny (Figure 2) was rooted using Senecio
madagascariensis Poir., an invasive plant of African origin (Scott, Congdon, &
Playford, 1998) that is present in Australia and was classified previously as a
member of the S. lautus complex (Radford et al. 2000; Scott et al., 1998;
Thompson, 2005). According to our results (Figures 2, S1 and S2) and the
literature (Pelser, 2002; Pelser et al., 2010; Pelser et al. 2007) the closest
relatives to the members of the S. lautus complex are not in Australia but
contained in clades from America (including S. eremophilus and S.
polygaloides) and Africa (including S. madagascariensis, S. nevadensis, S.
cadiscus and S inaequidens). This trend holds despite the numerous
incongruences between nuclear and plastid phylogenies. Therefore, we
considered that any species from these clades, including S. madagascariensis
would make a good outgroup for phylogenetic analysis of S. lautus complex
populations using RADs markers. Genus level ITS and ETS phylogenies were
rooted with Senecio flavus (Coleman & Liston 2003; Kadereit et al. 2006; Pelser
et al. 2012). We obtained very similar results when we used Crassocephalum
(Pelser et al. 2010) as an alternative outgroup (data not shown). Chloroplast
phylogenies were rooted using Senecio nemorensis L. (Pelser 2002; Pelser et
al. 2012). Results were very similar when we used other suitable outgroups
such as Senecio gregorii F.Muell. and Senecio oerstedianus Benth.
Chloroplast DNA and nuclear sequences
We sequenced three chloroplast intergenic spacers (ndhF-rpl32, psbJpetA and rps16-trnK; Shaw et al. 2007), and two nuclear regions (the internal
transcribed spacer, ITS, and the external transcribed spacer, ETS, of ribosomal
DNA (Bayer, et al. 2002), see table S5) in individuals belonging to 45
populations of the S. lautus complex. Due to technical limitations associated to
uneven PCR efficiency across samples and markers some samples were not
sequenced with all markers.
PCR reactions from individual samples were sequenced by the Sanger
method at Macrogen inc., Korea. Chromatograms were edited and aligned
using the Codon-Code Aligner software (CodonCode Corporation, Dedham,
MA, USA), as well as manually. We considered an individual heterozygous at a
specific site if the lower peak of the chromatogram represented more than 30 %
of the highest peak. Ambiguity codes were used for these heterozygous sites.
Since sequences from individuals belonging to the same populations were
almost identical and appear in the same phylogenetic clusters (Figure S3) we
created consensus sequences for each population using the Codon-Code
Aligner by calling major alleles for each polymorphic site. We defined a major
allele as the variant present as homozygous in more than 70% of the individuals
sampled. For those sites where we could not define a major allele ambiguity
codes were used.
We performed phylogenetic analyses using the Phylogeny.fr server
(Dereeper et al. 2008) following these steps: Sequences were first aligned with
MUSCLE v3.7 (Edgar 2004) in the default run mode with a maximum of 16
iterations. After alignment, ambiguous regions (i.e. containing gaps and/or
poorly aligned) were removed with Gblocks (v0.91b) using a minimum block
length after gap cleaning of 10, rejecting all segments with more than 8
contiguous nonconserved positions and selecting positions with a gap in less
than 50% of the sequences and being located within an appropriate block. The
phylogenetic tree was reconstructed using the maximum likelihood method
implemented in the PhyML v3.0 aLRT program (Guindon et al. 2010; Guindon &
Gascuel 2003). The best substitution model (HKY+G) was selected outside the
Phylogeny.fr server using FINDMODEL
(http://www.hiv.lanl.gov/content/sequence/findmodel/findmodel.html), that uses
the procedure first described in Model Test (Posada & Crandall 1998). In
FINDMODEL we reconstructed gene phylogenies using weighted neighbor
joning, and determined the best substitution model using the Akaike Information
Criterion measurement. Back in the Phylogeny.fr server, the gamma shape
parameter, proportion of invariable sites estimated and transition/transversion
ratio were estimated directly from each dataset. Reliability for internal branch
was assessed using the aLRT test (Chi2-based parametric). Gaps were
removed from the analysis. Graphical representation and edition of the
phylogenetic tree were performed with TreeDyn (v198.3). FigTree (v1.3.1) was
used to create figures.
We used BLASTn (Altschul et al.1990) at the NCBI server to find
homologous sequences of close Senecio relatives (Table S3) in public
databases. The sequences were edited and trimmed in Codon-Code Aligner to
produce consensus sequences for those species with several entries in
databases. Phylogenetic analyses of these sequences were performed as
described above.
We defined the geographic distribution and taxonomic status (i.e.,
synonyms and accepted names) of the species from which we had sequence
data using the Plant List server (http://www.theplantlist.org/) (Table S6).
Additionally we retrieved from the Global Compositae Checklist the names of all
Senecio species inhabiting the Australian subcontinent (Table S6) and
conducted a literature review to define which of these species were reported as
being closely related to members of the S. lautus complex (Ornduff 1960, 1964;
Ali 1964b, 1969; Belcher 1993, 1994; Thompson 2005; Radford & Cousens
2006). According to this analysis we had sequence data from a third of
Australian Senecio species (41 out of 110) but have sampled most of S.
pinnatifolius and S. lautus close relatives (13 out of 17 species were sampled).
Senecio repangae de Lange & B.G.Murray, S. condylus I.Thomps., S.
eremicola I.Thomps., S. hamersleyensis I.Thomps. and S. warrenensis
I.Thomps. were the only putative members of the complex that were missing
from our analyses.
References
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SUPPLEMENTARY DATA
Monophyly of Senecio lautus
Adaptation to contrasting environments can occur rapidly and produce
striking morphological and species diversity. Our phylogenetic analyses
indicate that the S. lautus complex, a set of groundsels endemic to Australia
and New Zealand (Ali 1966; Ornduff 1964; Radford et al. 2004; Thompson
2005), is an example of such a fast diversification. In agreement with earlier
studies (Pelser et al. 2007, 2010), our results indicate that the closest
relatives of S. lautus are in Africa and America (Figures 1B, S1 and S2),
which suggests that the diversification of these plants could have followed a
long-distance dispersal event. Despite numerous incongruences between
nuclear and plastid phylogenies (Figures S2 and S4) our results consistently
show that all samples of the complex included in this study belong to the
same clade.
Populations of the S.lautus complex found in different habitats (Figures
1 and S2) diverged recently. According to a calibrated phylogeny the most
recent common ancestor between S. pinnatifolius and the mediterranean
species S. nevadensis lived about 1.25 million years ago (Pelser et al. 2010),
placing the divergence of the complex in the Pleistocene or later. In fact
populations adapted to different environments have very similar genomes
(one SNP per 152, bases which is in the range of intra-specific variation in
humans and Arabidopsis) and are often undistinguishable using widely used
markers (Figures S1 and S2). Different ecotypes have striking geneticallybased morphological differences (Ali 1964a; Ornduff 1964; Radford &
Cousens 2000; Radford et al. 2004; Thompson 2005) and show evidence of
local adaptation (Radford & Cousens 2000; Melo et al. Unpublished results).
Nevertheless, populations inhabiting the same continental mass (either
Australia or New Zealand) are largely inter-fertile (Ornduff 1964), so relatively
little post-zygotic isolation has accompanied local adaptation.
Genetic diversity in the Senecio lautus complex is highly
structured over large but not small geographic scales
Changing climates can lead to barriers to gene flow after colonization,
leading to genetic differentiation. For instance, climate instability during the
Pleistocene and the subsequent expansion of the central desert deeply
affected the Australian flora. Mesic species that survived were confined to
refugia in mountains and along the coast, and typically suffered reductions in
range and population size (Burbidge 1960; Crisp et al. 2001; Hopper 1979).
This pattern was more pronounced in the central and southern part of the
continental mass, an observation that led pioneers of work in S. lautus and S.
pinnatifolius to suggest that these conditions might have fueled the rapid
diversification of the species complex (Ornduff 1964).
Consistent with this biogeographic scenario, RAD genetic diversity in
S. lautus populations differed noticeably between northeastern (NE in Figure
1), southern (SE, T and S) and western (W) Australia. Specifically, the
genomes of NE populations carry more polymorphic sites (F[1, 20] = 38.01, p <
0.0001) and show an excess of derived allelic variants (F[1, 20] = 4.68, p =
0.04; sequence coverage was similar between populations in both regions).
Similarly, genetic variability at eight microsatellites shows that populations
from the south are largely homogenous, whereas those from the north are
genetically similar but heterogeneous (Figure 2D). These results highlight the
significant differences in genetic variability between regions and suggest that
northeastern and southern populations have, despite their sometimesremarkable phenotypic similarity (Figure 3A), experienced quite different
evolutionary trajectories, possibly reflecting differences in demography and
ecology (habitats are more homogenous in the south) between the two
regions (Ornduff 1964).
SUPPLEMENTARY FIGURES
Figure S1: Phylogenetic analysis of Senecio populations: Maximum
Likelihood (ML) phylogenetic analysis of Senecio species (based on
consensus concatenated sequences from ITS and ETS nuclear markers)
showing the fast diversification, common origin and regional stratification
(regions with same codes as A) within Australia and New Zealand of the S.
lautus clade (shown in red). This clade includes several species that were
originally described as part of S. lautus sensu lato G.Forst. ex Willd (S.
pinnatifolius, S. spathulatus, S. halophilus, S. radiolatus and S. spanomerus).
Colored circles indicate the environment inhabited by members of the
complex and abbreviations indicate the geographic region of S. lautus
samples (as in Figure 1A). Senecio flavus was used as outgroup (not shown).
Node support is presented only when lower than 0.95. Letters and symbols at
the end of species names indicate the geographic distribution of the species:
(SA) South America; (NA) North-America; (M) Mediterranean area; (NZ) New
Zealand; (@) Australia, member of the S. lautus complex; (*) Australia, not
member of the S. lautus complex;
Figure S2: Similitudes and discrepancies between nuclear and plastid
phylogenies. Maximum likelihood cladograms of Senecio species based on
consensus sequences from the ITS nuclear intergenic spacer (A) and the
psbJ-petA chloroplast marker (B). Senecio nemorensis was used as outgroup.
The color of branches indicates the environment inhabited by S. lautus
populations and the letters at the end of names indicate he geographic
distribution of taxa. Although the two phylogenies present several
discrepancies both reveal that S. lautus complex is an independent
Australasian lineage that includes the lautusoid species S. lautus var.
lanceolatus, S. brigalowensis and S. lacustrinus. Despite the great diversity
shown by members of the complex populations cluster according to their
geographic distribution (regions shown in the right; E east; NE north-east; S
south; SW south-west; T tasmania; W west) rather than their morphology or
habitat. Coastal and alpine forms appear in several clusters, which suggests
that these environments were colonized multiple times. Posterior probabilities
are presented for all nodes.
Figure S3: Individual based nuclear and plastid phylogenies. Maximum
likelihood cladograms of Senecio species based on individual sequences from
the ITS nuclear intergenic spacer (A) and the psbJ-petA chloroplast marker
(B). Senecio nemorensis was used as outgroup. The color of branches
indicates the environment inhabited by individuals (as in Figure 1). Branch
names are composed of the population followed by a serial number. Members
of the S. lautus complex cluster according to their geographic distribution
(regions shown in the right; E east; NE north-east; S south; SE south-east;
SW south-west; T Tasmania; W west) rather than the environment they
inhabit (shown by colors in branches).
Figure S4: Genetic structure of S. lautus populations. (A) Principal
components analysis of allelic frequencies at 2270 neutral SNPs. Colors
indicate geographic regions: Blue: Northeast; Dark green South-east; Light
green: South; Red West; Black Inland. Shapes indicate the environment
inhabited by the populations: Filled circles: sand dunes; Empty circles: Sea
bird rockeries; Filled squares: rocky headlands; Empty squares; dessert.
Filled Triangles: alpine meadows; Empty triangles: mountain forests. (B)
Microsatellite based STRUCTURE analysis of six parapatric pairs confirming
the similarity of parapatric populations and the higher diversity of northeastern
taxa. Populations are organized according to their distribution along the coast
(see figure 1A).
Figure 1
Figure 2
Figure 3
Figure 4