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Supporting Information Methods S1, Notes S1 & S2, Tables S1-S8 and Supporting
references
Methods S1
Isolation of full-length elements
The BAC library of S. latifolia was characterized by Cegan et al., 2010. Screening was
performed by radioactive hybridization with labelled Ogre reverse transcriptase and integrase
genes as probes. BAC DNA was isolated and commercially sequenced using 454 sequencing
with Roche GS FLX (GATC Biotech, Konstanz). 454 reads were assembled using Mira3
(Chevreux et al., 2004), TGICL (Pertea et al., 2003) and Roche GS De novo Assembler
version 2.5.3. We also used 454 reads of S. latifolia genomic DNA (Macas et al., 2011).
Contigs within individual clusters were manually assembled to reconstruct consensus
sequences of Ogre elements. Basic sequence analyses were done in Geneious Pro
(Biomatters). Homology searches were performed with FASTA and BLAST online
applications. Full-length element prediction was carried out by an LTR finder (Xu & Wang,
2007), annotations and visualizations in Artemis (Rutherford et al., 2000). Sequence
similarities were identified by JDotter (Brodie et al., 2004).
Copy number estimation
The copy number in S. latifolia genome was estimated by hybridization of respective LTR
probes with the BAC library as follows: The number of elements per genome = Genome size
x Percentage of genome / Element size x 100. Percentage of genome = Number of
hybridizing clones x Element size / Total clone number x Average clone size. Average BAC
clone size is 125 kbp. Total genome size of Silene latifolia male is 2.879 x 109bp - 1C (Široký
et al., 2001; Lengerova et al., 2004). Additionally, copy numbers were estimated from
genomic DNA - Illumina libraries.
In silico copy numbers estimation
Copy numbers were estimated from the Illumina genomic reads (accessible under
ERX015036, ERX015035 in SRA) in the following manner: The reads were mapped uniquely
to the reference LTRs obtained from the BAC sequences of distinct Ogre, Retand and Athila
types with at least 90% overlap of the read to the reference sequence. The copy numbers were
subsequently estimated from the sequencing depth (library coverage, LC), depth of coverage
(DOC) and the genome size (GS) of Silene latifolia using the formula: DOC x GS/LC.
Sequencing of genomic and transcribed Ogre copies
Genomic DNA was extracted from young leaves using the DNeasy Plant Mini Kit (Quiagen).
RNA was extracted using the NucleoSpin RNA Plant kit (Macherey-Nagel) or RNA-Blue
(Top-Bio). DNA contaminations were removed using the Turbo DNA-free kit (Ambion).
Equal amounts of total RNA (1µg) were reverse transcribed using the High Capacity RNA-tocDNA kit (Applied Biosystems). Three degenerate primer pairs were designed to amplify the
integrase gene of the three Ogre families (Table S1a). To prevent potential chimeric PCR
products an emulsion PCR protocol (Williams et al., 2006) was followed with High fidelity
Herculase II Fusion DNA polymerase (Agilent Technologies – Stratagene). PCR products
were ligated into pDrive (Quiagen) or pCRII cloning vector (Invitrogen) and cloned into E.
coli DH5α strain. After PCR screening, selected PCR products were sequenced (Sanger
sequencing) from both sides. For analysis of Ogre CL5 splicing, we used primers described in
Table S1b and for amplification of LTR sequences, Table S1c primers.
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RACE mapping of transcription starts and ends
Total RNA was extracted by RNA-Blue (Top-Bio) from male flower buds less than 2mm in
diameter. 5’ and 3’ RACE ready first-strand cDNA was amplified by the SMARTer RACE
cDNA amplification kit (Clontech). 5’ and 3’transcription ends were amplified using
Advantage 2 Polymerase mix (Clontech). PCR products were cloned and sequenced as
described above. Gene specific primers used for amplification of transcription starts and ends
are described in Table S1d.
Computer processing of sequences
Raw sequence files were trimmed and assembled in Geneious Pro software (Biomatters Ltd,
Auckland, New Zealand). Sequence analyses and alignments were done in Geneious and
Bioedit (Hall, 1999) using MAFFT (Katoh et al., 2002), ClustalW (Thompson et al., 1994)
and manual refinement.
Fluorescene in situ hybridization (FISH)
To synchronize the germinating seeds of S. latifolia, the DNA polymerase inhibitor
aphidicolin was used, and mitoses were then accumulated with oryzalin. Slides were prepared
from root tips and treated as described in Lengerova et al. (2004) with slight modifications.
Slides were analyzed using the Olympus AX1 microscope, and image analysis using ISIS
software (Metasystems). To differentiate the arms of the Y chromosome, a cytogenetic FISH
marker X-43.1 accumulated at subtelomeric regions of the majority of chromosomes was used
(Buzek et al., 1997). Plasmid DNA clones containing integrase of respective Ogre families
were used as probes. LTR probes were prepared from PCR products with primers (Table S1c)
and genomic DNA.
In situ hybridization
Whole anthers and pistils of S. latifolia plants were fixed in 2% formaldehyde and 5% acetic
acid in 60% ethanol. After fixation, the reproductive organs were embedded by Cryomount
(HistoLab Products AB, Göteborg, Sweden) and frozen. Tissue blocks were cut longitudinally
into 7 μm sections using CM 1800 (Leica Microsystems, Germany), transferred to
microscopic slides and air dried. Probes cloned in pCR II-TOPO (Invitrogen - Life
Technologies, Grand Island, NY, USA) were subjected to in vitro transcription and labeling
with digoxigenin (DIG) using the DIG RNA Labeling Kit (Roche Applied Science,
Mannheim, Germany). mRNAs were detected in the sections according to Brewer et al.,
2006.
mRNA and small RNA isolation and sequencing by Illumina, transcript level and sRNA
abundance estimation
Pollen grains were isolated from male flowers using 0.3M mannitol according to
http://www.bio-protocol.org/wenzhang.aspx?id=67. High-molecular-weight and lowmolecular-weight RNA were isolated simultaneously according to Carra et al. (2007) from
young male and female leaves, unfertilized and fertilized pistils and pollen grains. RNA and
small RNA samples were sequenced at IGA Technology Services (Udine, Italy) on
HiSeq2000 by using standard Illumina sequencing workflow.
RNA reads treatment and mapping
The sequence reads from five different organs of Silene latifolia (accessible at BioProject
under PRJNA179506) from the Solexa RNA-Seq and miRNA-seq were clipped and filtered
according to their quality using the FASTX-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/).
In order to proof our hypotheses we downloaded external datasets of cDNAs from SRA (raw
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reads accessible under SRR316288, SRR316289, SRR404980, SRR404981, SRR404982,
SRR404983, SRR404984, SRR404985). Additionally we downloaded small RNA datasets of
flowers and leaves from NCBI GEO (http://www.ncbi.nlm.nih.gov/geo/, accessible under
GSM803576, GSM803577 and GSM803578). All the analysed cDNA reads were trimmed to
the same size of 30 nucleotides. The reads were then mapped to the BAC sequences of Ogre
and Retand elements using the LAST alignment tool (Kiełbasa et al., 2011). The parameters
were adjusted according to the LAST manual, allowing up to 2 mismatches, which means
setting the seed to 1110100 and initial match to 10 nucleotides for small RNAs and
1111101110010 and 15 nucleotides for cDNAs.
The small RNA alignment results were then sorted for each position in the reference sequence
based on the hit score using our own computational pipeline in bash. Low scored hits were
removed from the lists. Similar analysis was done for the cDNA alignment, but this time the
coverage of whole reads was recorded. The hit list values of cDNA reads were averaged
before plotting using sliding window of size 15.
As there was certain overlap in hit lists of two different types of Ogre subfamily (CL5_267
and CL5_277) and two types of Retand subfamily (Retand-1 and Retand-2) a method for
merging hit lists using Python programming language was developed. In order to merge two
elements, positions were normalized based on the element length using per mile scale and all
the redundant reads mapped to both elements were counted only once.
All the hit lists were normalized based on the library size and copy number estimated from the
in silico copy number estimation following the formula 1.5x1012/F, where F is the factor value
representing product of the library size, copy number value and reference sequence length.
The results were visualized using Gnuplot, Gimp and LibreOffice suite.
Genomic and transcriptomic proportions of three different types of Ogre (full length,
with deletion, and with insertion) - splicing
In order to count the proportions of different Ogre subfamilies, only regions unique for each
type of Ogre were selected. To see the differences, especially spliced sites and deleted sites, a
multiple alignment using the ClustalW algorithm was performed. For a spliced variant Ogre
and an Ogre with a deletion, a subsequence was made linking both flanking regions of the
splice site and the deleted site respectively. For the full length Ogre, the spliced site itself was
selected.
This small dataset was then analyzed in contrast to Solexa genomic reads (accessible under
ERX015036, ERX015035 in SRA) and transcriptomic reads (accessible at BioProject under
PRJNA179506) using the LAST alignment tool allowing up to two mismatches. The results
were then filtered, whereby only reads covered by at least 90% were considered correct. The
numbers of hits for different Ogre types were then summed using our own bash scripts.
Bisulphite sequencing
DNA from whole pollen grains, leaves and flower buds was modified by EpiTect Bisulfite Kit
(Quiagen) and BisulFlash DNA Modification Kit (Epigentek). As a control for successful
bisulfite conversion MROS1 gene was used (Janousek et al., 2002), all twenty sequenced
clones showed equal methylation. Primers were designed using Bisprimer software
(Kovacova & Janousek, 2012). At least 20 clones amplified on modified DNA samples and
20 clones amplified on non-modified DNA were sequenced.
Hierarchical cluster analysis
Hierarchical cluster analysis was used to separate DMR of sequenced pollen bisulfite treated
DNA samples (to distinguish vegetative cells from sperm cells, based on the hypothesis that
sperm cells have much lower methylation level than vegetative cells): we used R software -
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Euclidean method for counting to generate a distance matrix that was clustered by hclust basic
R tool with default settings (Akalin et al., 2012).
Statistical evaluation of methylation level in pollen
P-values from ANOVA test for linear modeling of an equation y = a + bx, where y is the
methylation level of the sample and x denotes the treatment indicator for sample (= 0 if
sample is in “control group” (sperm cells) and = 1 if sample is in “treatment group”
(vegetative cells)). If the null hypothesis (Ho: b=0) is rejected, the “control” and “treatment”
groups have different methylation levels (= differentially methylated region DMR) (Schultz et
al., 2012).
i = cytosine position in one sequence
Global methylation
n = number of seqs.
Sem = standard error of measurement
O5M1
Sd = standard deviation
Pollen
all
Cluster 1
Cluster 2
mean ∑(Ci/(Ci+Ti)) / n
0.2476
0.4529
0.1873
sd
0.1473
0.1098
0.0919
mean + - sd
0.1003 – 0.3949
sem
0.0222
number of clustres
∑Ci / (∑ Ci + ∑ Ti)
95% confidence interval: including continuity
correction
P-value
* statistically significant
0.3431 – 0.5628 0.0953 – 0.2792
0.2481
0.23 – 0.2672
0.0158
0.4494
0.1871
0.4051 – 0.4945 0.1686 – 0.2071
1.12E-010
yes
O5M3
Pollen
all
mean ∑(Ci/(Ci+Ti)) / n
sd
mean + - sd
0.0347
2
Cluster 1
0.5838
0.1257
0.4580 – 0.7095
Cluster 2
0.6182
0.0662
0.2743
0.1123
0.5519 – 0.6844 0.1619 – 0.3865
sem
0.0230
0.0127
0.0648
number of clustres
∑Ci / (∑ Ci + ∑ Ti)
95% confidence interval: including continuity
correction
P-value
* statistically significant
2
0.6943
0.7452
0.2439
0.678 – 0.7101
mean + - sd
sem
0.1992 – 0.2948
Cluster 1
Cluster 2
6.54E-003
yes
O5M5
Pollen
all
mean ∑(Ci/(Ci+Ti)) / n
sd
0.729 – 0.761
0.1629
0.1440
0.0189 – 0.3070
0.0244
0.4539
0.0576
0.1254
0.1022
0.3964 – 0.5115 0.0233 – 0.226
0.0288
0.0184
number of clustres
∑Ci / (∑ Ci + ∑ Ti)
95% confidence interval: including continuity
correction
P-value
* statistically significant
2
0.1729
0.1572 – 0.1898
0.1199
0.4933 – 0.6178 0.1057 – 0.1357
3.36E-007
yes
O6M1
Pollen
all
mean ∑(Ci/(Ci+Ti)) / n
sd
mean + - sd
0.5564
Cluster 1
0.7411
0.2079
0.5333 – 0.949
Cluster 2
0.4110
0.0671
0.8649
0.0183
0.3439 – 0.4781 0.8466 – 0.8832
sem
0.0313
0.0194
0.0032
number of clustres
∑Ci / (∑ Ci + ∑ Ti)
95% confidence interval: including continuity
correction
P-value
* statistically significant
2
0.7404
0.4118
0.8647
0.7242 – 0.756
2.20E-016
yes
O6M2
Pollen
all
mean ∑(Ci/(Ci+Ti)) / n
sd
mean + - sd
Seq 38
0.9048
0.1099
Cluster [-38]
0.2608
0.9222
0.0242
0.7948 – 1.0147
sem
0.0178
number of clustres
∑Ci / (∑ Ci + ∑ Ti)
95% confidence interval: including continuity
correction
P-value
* statistically significant
2
0.9091
0.899 – 0.9183
0.8979 – 0.9464
0.0040
0.2609
0.9221
0.1659 – 0.3828 0.9125 – 0.9307
2.20E-016
yes
O6M3
Pollen
all
mean ∑(Ci/(Ci+Ti)) / n
sd
mean + - sd
0.3779 – 0.4465 0.8494 – 0.8787
Cluster1
0.3721
0.2261
0.1460 – 0.5983
Cluster2
0.8103
0.0529
0.2774
0.1003
0.7573 – 0.8633 0.1771 – 0.3777
sem
0.0337
0.0187
0.0165
number of clustres
∑Ci / (∑ Ci + ∑ Ti)
95% confidence interval: including continuity
correction
P-value
* statistically significant
2
0.3739
0.8106
0.2779
0.3564 – 3917
2.20E-016
yes
0.774 – 0.8426
0.2602 – 0.2965
O6M4
Pollen
all
mean ∑(Ci/(Ci+Ti)) / n
sd
mean + - sd
Cluster1
0.3681
0.2541
0.1141 – 0.6222
0.8592
0.0206
0.2879
0.1688
0.8386 – 0.8799 0.1192 – 0.4567
sem
0.0337
0.0073
0.0241
number of clustres
∑Ci / (∑ Ci + ∑ Ti)
95% confidence interval: including continuity
correction
P-value
* statistically significant
2
0.3686
0.8593
0.2855
0.3537 – 0.3837
0.2705 – 0.301
Cluster1
Cluster2
yes
mean ∑(Ci/(Ci+Ti)) / n
sd
mean + - sd
0.8278 – 0.886
3.48E-013
O11M1
Pollen
all
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Cluster2
0.1531
0.2045
-0.0514 - 0.3562
0.6869
0.0349
0.1060
0.1317
0.6519 – 0.7219 -0.0257 - 2377
sem
0.0336
0.0202
0.0226
number of clustres
∑Ci / (∑ Ci + ∑ Ti)
95% confidence interval: including continuity
correction
P-value
* statistically significant
2
0.1608
0.6842
0.1096
0.1368 – 0.1869
0.5662 – 0.7834 0.089 – 0.1341
8.23E-009
yes
Nucleotide sequences for ancestral state reconstruction
Nucleotide sequences of fructose-2,4-bisphosphatase, spermidine synthase, CCLS1 and
eIF4A were obtained by PCR with the exception of spermidine synthase sequences of S.
latifolia and S. vulgaris and all sequences of fructose-2,4-bisphosphatase. Primers c2B12+1,
c2B12-2 (Filatov, 2005), CCLS1-F1, CCLS1-R1 (Zluvova et al., 2010), eIF4A-F and eIF4AR (Zluvova et al., 2005) were used to amplify the nucleotide sequence from genomic DNA.
PCR products were gel-purified using Gel Extraction Kit (Qiagen) and directly sequenced.
Other sequences were retrieved from database. Accession numbers are listed in the Table S3.
Nucleotide alignment and phylogenetic tree reconstruction
Rough alignment of Ogre sequences was performed using Clustal Omega (Sievers et al.,
2011). The nucleotide alignment was refined manually in Seaview (Gouy et al., 2010) using
the translated nucleotide sequences as a guide. The sequences serving for ancestral state
reconstruction phylogram and orthologues of sex-linked gene pairs were aligned using
ClustalX (Larkin et al., 2007) followed by a manual refinement in Seaview. Phylogenetic
trees were reconstructed using maximum likelihood and Bayesian methods.
Details on the Ogre alignment datasets
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Four different nucleotide alignment datasets were prepared. Alignment A contained GAG-Pol
coding sequences and comprised S. latifolia Ogre sequences obtained from sequencing of
BAC clones, S. viscosa, S. zawadzkii, S. vulgaris, S. pendula, S. otites and S. colpophylla
sequences obtained by PCR on genomic DNA, and outgroup sequences from Vitis vinifera,
Gossypium hirsutum, Pisum sativum, Medicago truncatula, Solanum lycopersicum, and
Populus trichocarpa obtained from database. To avoid problems caused by sequence
misalignment, the outgroup sequences covered only reverse transcriptase, RNase H and
integrase sequences, which are more conserved than Gag and proteinase. Alignment B was
prepared from the BAC-derived sequences of S. latifolia. Alignment C contained integrase
sequence and comprised sequences of S. latifolia obtained by PCR, and BAC-derived
sequences. Alignment D comprised one representative sequence of each species and each
group of Ogre retroelements and all outgroup sequences as in alignment A. It spanned the
region from the reverse transcriptase to the end of the integrase. Alignment A was also
translated using Seaview.
Phylogenetic tree reconstruction
For the maximum likelihood tree reconstruction, the appropriate model of nucleotide
substitution was used as proposed by MrAIC (Nylander, 2004) together with PhyML 3
(Guindon et al., 2010) using either Akaike information criterion (AIC; in the case of long
alignments) or second-order AIC (in the case of short alignments). The maximum likelihood
trees were reconstructed using PhyML 3. The tree topologies were estimated using the
approach BEST that estimates the phylogeny using both nearest neighbor interchange and
subtree pruning and regrafting. The tree search was started from BioNJ tree and ten random
starting trees. The branch support was estimated using a Shimodaira-Hasegawa-like
approximate likelihood ratio test (SH-aLRT) (Anisimova & Gascuel, 2006). Unlike
computationally expensive bootstrapping, the SH-aLRT is much faster and provides excellent
levels of accuracy and power, even under the violation of the model assumptions (Anisimova
et al., 2011).
The tree reconstruction by Bayesian inference was performed using PhyloBayes 3.3e
(Lartillot et al., 2009) for alignment D. The trees were reconstructed using the CAT-GTR + Γ
nucleotide substitution model. The search was started from random trees, and four
independent chains were run. The chains were stopped according to the PhyloBayes manual
with the exception that the minimal effective sample size reached 100.
To substantially decrease the computational time demand of the tree reconstruction based on
alignment A, a phylogenetic tree search was performed using MrBayes 3.1.2 (Ronquist &
Huelsenbeck, 2003). The appropriate substitution model was found using MrAIC similar to
the case of the maximum likelihood approach. Tree search was run for 10 million generations
with four MCMC chains and two independent runs with trees sampled every 100th generation.
The burn-in proportion was estimated using Tracer version 1.4 (Rambaut & Drummond,
2007). The convergence of the tree topologies was subsequently checked using AWTY
(Nylander et al., 2008).
Tree reconstruction of the translated alignments A and D were performed using maximum
likelihood. The appropriate model for the translated dataset D was found using ProtTest
(Darriba et al., 2011) and the phylogenetic tree was reconstructed using PhyML 3 with
gamma substitution parameter estimated and a JTT model of amino acid substitution. The tree
topologies were estimated using the approach BEST. The tree search was started from BioNJ
tree and ten random starting trees. The branch support was estimated similar to the case of the
nucleotide alignments. The appropriate model of amino acid substitution and tree
reconstruction of the translated alignment A was performed automatically using Phylogenetic
reconstruction by Automatic Likelihood Model selector (Chen et al., 2009). BEAST v1.6.1
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(Drummond & Rambaut, 2007) was used to construct chronograms. Input files for BEAST
were created with BEAUti (Drummond & Rambaut, 2007) using a relaxed clock model
(Drummond et al., 2006) with a Yule prior and the nucleotide substitution models proposed
by MrAIC, using the second-order AIC. A prior on the age of the split between the genera
Silene and Lychnis was set to 12.5 million years, and a prior of the age of the split between
the genera Silene and Petrocoptis was set to 20 million years, both with a normally distributed
standard deviation of one million years. Subgenus Silene, subgenus Behenantha and the
section Melandrium were forced to be monophyletic. Two MCMC chains were run for 10
million generations with trees and parameter values saved every 1000th generation. The
resulting log files were checked in Tracer version 1.4 (Rambaut & Drummond, 2007), and the
tree files were summarized using TreeAnnotator (Drummond et al., 2006) into one Maximum
credibility tree with median node heights. Trees were visualized using FigTree 1.3.1
(Rambaut, 2009).
Estimation of the time of mobilisation peaks
To estimate the approximate time of mobilisation peaks, we used methods based (i) on the
terminal branch lengths of Ogre elements in the chronogram counted using BEAST, and (ii)
on the branch lengths for synonymous substitutions counted using PAML. For the PAMLbased method, we used a set of chronograms of sex-linked genes. We took the advantage of
the fact that the recombination arrest between the sex chromosomes in S. latifolia is gradual
(Nicolas et al., 2004; Marais et al., 2011), and thus each X-Y gene pair diverged at a different
time. We used X-Y gene pairs that were sequenced from at least two dioecious species of the
section Melandrium and from several related non-dioecious Silene species (XY4 – Atanassov
et al., 2001; DD44 – Moore et al., 2003; Cyp – Bergero et al., 2007; XY1 – Delichère et al.,
1999; Rautenberg et al., 2008). From each dataset, we constructed a chronogram using
BEAST and computed a pairwise synonymous divergence (dS) of the S. latifolia X-Y gene
pair using the CODEML program of PAML. The chronograms served to assess the timing of
the split of the respective X-Y gene pair. Subsequently we constructed a linear regression of
X-Y split time and percentage of synonymous substitutions in Microsoft Excel that served to
assess the time of the maximal transposition activity of each Silene Ogre family.
PAML analyses
The CODEML program of PAML 4.5 (Yang, 2007) was used to estimate the ratio (ω) of the
non-synonymous substitution rate (dN) to the synonymous substitution rate (dS) after
removing frameshift insertions and recoding the stop codon as missing data following
Meredith et al., 2009. The maximum likelihood trees generated from the alignments B and/or
C were used as the reference trees. In the branch-site analyses, modified model A was
compared with both the corresponding null model with ω 2 = 1 fixed (test 2) and with the
model M1a (test 1). The chi2 program of PAML was used to estimate the P-values. The
results of branch models served to estimate the age distribution of the retrotransposon
insertions. Frequency histograms of terminal branch lengths for synonymous sites were
constructed in Microsoft Excel.
Estimation of the order of Silene Ogre mobilisation
To determine the order of Ogre mobilisation waves, we derived maximum-likelihood
estimates of the synonymous substitutions (dS) per branch using the CODEML program in
PAML 4.5 (Yang, 2007). The synonymous substitutions are defined as nucleotide
substitutions that do not lead to the change of the amino acid sequence. Because natural
selection acts mainly on protein sequences, synonymous codon positions are largely free from
selection and so accumulate changes in a neutral manner, at a rate similar to the mutation rate.
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Thus, it is generally assumed that the level of synonymous substitutions increases
approximately linearly with time, at least for relatively low levels of sequence divergence
before saturation with multiple substitutions becomes an issue. We estimated the number of
synonymous substitutions in terminal branches, because they reflect changes after the Ogre
insertion into the genome. For this reason, it was not possible to use exclusively sequences
with intact open reading frame because their use would bias the estimation of the substitution
number towards lower numbers. Thus, similarly to Meredith et al. (2009), we removed
frameshift insertions and recoded the stop codons as missing data.
The branch model of CODEML estimates the number of synonymous substitutions per each
branch in a phylogenetic tree. We used an unrooted tree based on the integrase sequence
(alignment C). We estimated codon-based branch lengths under a one-ratio model (model
M0) and used the tree with the estimated branch lengths for the modelling two-ratios branch
models. The two ratios branch model allows two different ratios (ω) of the non-synonymous
substitution rate (dN) to the synonymous substitution rate (dS) values to fit to the data – the
first value corresponds to the background branches, and the second value corresponds to the
foreground branches. Branches of interest are selected and called “foreground branches”. All
other branches in the tree are the “background branches”. We modelled three two-ratios
models, each with foreground branches corresponding to the terminal branches leading to
Ogre CL5, Ogre CL6 and Ogre CL11, respectively. We used the “CL5 two-ratios model” to
count the dS of terminal branches leading to Ogre CL5. We used an analogous procedure to
count the dS for Ogre CL6 and Ogre CL11. For each Ogre family, we constructed a frequency
histogram in Microsoft Excel.
Selection analyses
We estimated the ratio (ω) of the non-synonymous substitution rate (dN) to the synonymous
substitution rate (dS) under a one-ratio model in which the same ω ratio occurs across the tree,
and subsequently, we used the two-ratio branch model to compare the estimated ω ratio on
specific foreground branches in the phylogeny with the background ω ratio. Branch models
were applied to (i) branches leading from the most recent common ancestor (MRCA) of all
Silene Ogre retroelements to MRCA of Ogre CL5, Ogre CL6 and Ogre CL11 (hereafter
referred as to internal branches), and to (ii) terminal branches. Modelling the ω ratio along the
internal branches allowed us to assess the evolution of each of Ogre CL5, Ogre CL6 and Ogre
CL11 during their diversification from their MRCA. Because the modelling works on the
“reconstructed ancestral sequences”, it is not necessary to use exclusively Ogre elements with
intact ORFs – in the past the ORFs were intact to allow the mobilisation. Modelling the ω
ratio along the terminal branches allowed us to see what happened to each group of Ogre
elements after their insertion into DNA. Similar to the modelling internal branches, we used
all sequences of respective Ogre elements, including the sequences with disrupted ORFs.
Excluding sequences with disrupted ORFs would strongly shift the results towards low ω
values. Low ω values (ω is significantly lower than one) indicate purifying selection, ω
values that do not significantly differ from one indicate neutral evolution (i. e. degeneration),
and ω values significantly higher than one indicate positive selection. To see whether the
foreground ω significantly differs from the background ω, we compared the respective tworatios model to the one-ratio model by using likelihood-ratio tests (LRT) to obtain the
statistical significance of the difference.
Site models allow ω to vary along the sequence alignment. We implemented two pairs of site
models. The nearly neutral model (M1a) assumes two classes of sites: one is under purifying
selection with 0 <ωbackground <1, the other is under neutral evolution with ωforeground =1. We
compared this model to the positive selection model (M2a) in which an additional ω
parameter is included that allows positive selection where present (ω>1). We also used the
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M3 model (discrete model that uses an unconstrained discrete distribution to model
heterogeneous ω ratios among sites), and compared it to M0 model (one-ratio model), in
which positive selection is not allowed. The test M3 vs. M0 is generally used as a test of
variable ω among sites. Although the model of a single ω for all sites (M0) is unlikely in
functional proteins, it could fit data in degenerated sequences that are not functional for a long
time.
The limitation of branch- and site-models is that they are highly conservative. Testing the
lineages for positive selection, the  ratio is assumed to be identical across sites. In such a
case, positive selection is detected along a lineage only if the  ratio averaged over all sites is
significantly greater than one. The likelihood ratio test of positively selected sites is based on
the assumption that the  ratio is identical among all lineages on the tree. In this case, positive
selection is detected for a site only if the underlying  ratio averaged over all lineages is
significantly greater than one. If adaptive evolution occurs at a few time points and affects
only a few amino acids both classes of models might lack power to detect positive selection.
Branch-site models allows the  ratio to vary both among sites and among evolutionary
lineages. It is based on the basic model of codon substitution that. Further, we assume that the
phylogeny is known or independently estimated, and the branches expected to be under
positive selection are a priori specified. We assume a variable  ratio among sites and four
site classes in the sequence. The first class includes highly conserved sites in all lineages with
a small  ratio, 0. The second class includes neutral or weakly constrained sites at which 
= 1, 1 is near or smaller than one. In the third and fourth category, the background lineages
have 0 or 1, but the foreground lineages have 2, which may be greater than one. This
means that there are two site categories with ratios 0 or 1 along the background branches,
while along the lineages of interest, some sites are caused to come under positive selection
due to a certain event, having the ratio 2 >1. To model branch-site models, we ran PAML
with either internal or terminal branches of respective Ogre elements as the foreground
sequence and the other sequences as the background. Statistical significance was evaluated by
comparing a model with ω foregrounded for 2a and 2b categories as a free parameter with a
model with ω foregrounded for 2a and 2b categories set to 1 (neutral evolution).
Ancestral state reconstruction
The probability of the presence of Ogre CL5 in the ancestor of the subgenus Silene, in the
ancestor of the subgenus Behenantha, and in the ancestor of the genus Silene was estimated
using BayesTraits (Pagel et al., 2004). The phylogram constructed from the spermidine
synthase, CCLS1, eIF4A and fructose-2,6-bisphosphatase partitioned alignment was used as
the input tree. The taxon sampling used for the analysis and phylogram construction are
summarised below. The analyses were performed with BayesMultiState model and the model
allowed only the transition from Ogre CL5 absent to Ogre CL5 present. The maximum
likelihood analysis was performed with 100 optimization attempts. In the Bayesian analysis,
the rate deviation was increased to 15 to increase the acceptance rate to 34.7%. The chain was
run for 100 million generations and every 200,000th generation was sampled. The resulting
log file was checked in Tracer version 1.4 (Rambaut & Drummond, 2007). The probability
values were counted as median. Using a chronogram instead of a chronogram did not
significantly change the results (data not shown).
Taxon sampling for ancestral state reconstruction and for chronogram construction
The genus Silene L. (Caryophyllaceae) is divided into two subgenera Silene and Behenantha
(Otth) Endl of approximately equal size (Popp & Oxelman, 2004). Most of the sampled
species belong to the subgenus Behenantha. Three dioecious Silene species from the section
Melandrium (Röhl.) Rabeler. - S. latifolia Poir., S. dioica (L.) Clairv., and S. diclinis (Lag.)
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Lainz – were sampled. The dioecious species of the section Melandrium had been previously
classified together with the hermaphroditic species S. noctiflora L. in Silene sect. Elisanthe
(Fenzl ex Endl.) Ledeb. Thus, S. noctiflora was also used for our analyses. Previous studies
indicated a close relationship among the dioecious species in the section Conoimorpha Otth
(Oxelman & Lidén, 1995; Desfeux & Lejeune, 1996; Rautenberg et al., 2010). For this
reason, S. conica L. coming from this section was also included. Non-dioecious S. viscosa
(L.) Pers. reported as crossable to S. latifolia (Correns, 1928; Prentice, 1978) and S. zawadzkii
Herbich reported as crossable to S. diclinis and S. dioica (Prentice, 1978) were further
analysed. Both S. zawadzkii and S. viscosa belong to the section Physolychnis (Benth.)
Bocquet. Species S. vulgaris (Moench) Garcke and S. pendula L. reported as relatives
(Desfeux & Lejeune, 1996) and belonging to the section Behenantha Otth were further
analysed. The phylogenetic relationship of these sections is as yet unclear - three different
sister groups have been suggested - to the section Melandrium - the section Conoimorpha
(Desfeux & Lejeune 1996, Erixon & Oxelman 2008), S. viscosa and S. zawadzkii coming
from the section Physolychnis (Marais et al. 2011) and S. vulgaris coming from the section
Behenantha (Rautenberg et al. 2008). Three species from the subgenus Silene - S. colpophylla
Wrigley, S. otites (L.) Wibel, and S. saxifraga L. – were also added to the dataset. Petrocoptis
pyrenaica A.Braun ex Walpwas used as an outgroup.
Phylogenetic tree for ancestral state reconstruction
A phylogram for ancestral state reconstruction was generated using sequences of four nuclear
genes – CCLS1 (Barbacar et al., 1997), eIF4A (Zluvova et al., 2006), fructose-2,6bisphosphatase (Atanassov et al., 2001) and spermidine synthase (Filatov, 2005). The
sequence alignments of CCLS1 and eIF4A were processed using Gblocks (Castresana, 2000)
to remove divergent and ambiguously aligned blocks. After this procedure, the alignment
comprised of 5189 nucleotides partitioned into four partitions. The phylogenetic tree was
reconstructed by maximum likelihood approach using RAxML BlackBox (Stamatakis et al.,
2008) with nucleotide models as proposed by MrAIC (Erixon & Oxelman, 2008) together
with PhyML 3 (Desfeux & Lejeune, 1996), using second-order AIC. As the closest relatives
of the dioecious Silene from the section Melandrium are unknown, we also reconstructed the
tree by Bayesian inference to be sure that our phylogram is not an artifactual result caused by
long branch attraction. The Bayesian inference was performed using MrBayes 3.1.2 (Ronquist
& Huelsenbeck, 2003) with nucleotide models as proposed by MrAIC. Tree search was run
for 50 million generations with four MCMC chains and two independent runs with trees
sampled every 100th generation. The burn-in proportion was estimated using Tracer version
1.4 (Rambaut & Drummond, 2007). The convergence of the tree topologies was subsequently
checked using AWTY (Nylander et al., 2008). The resulting trees were visualised using
FigTree 1.3.1 (Rambaut, 2009).
Notes S1 & S2
Note S1
A. The maximum likelihood newick treefile based on the alignment A.
(((((((((((((((((((((((((Y185:0.0331290990,((a195:0.0199579189,Z095:0.0240624724)1.00000
00000:0.0153176844,(c215:0.0314028432,(d135:0.0357492936,((CL5ISVi5:0.0027260097,C
L5ISVi3:0.0005932827)1.0000000000:0.0723340909,((g295:0.0180373870,(h205:0.0205346
784,((CL5ISVi1:0.0466616356,(j145:0.0188600794,(((((o165:0.0136447230,o015:0.0107838
249)0.9950000000:0.0071867789,n315:0.0080168890)0.1690000000:0.0029290536,(m285:0
.0110250049,l175:0.0094352591)0.7410000000:0.0035900411)1.0000000000:0.0299464047,
(((n245:0.0000000578,m065:0.0000000001)1.0000000000:0.0066193773,p155:0.008106186
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6)1.0000000000:0.0068448446,q035:0.0169812623)1.0000000000:0.0096992161)0.9980000
000:0.0082854536,(k265:0.0014116427,p025:0.0006557607)1.0000000000:0.0146400953)0.
7090000000:0.0063089812)0.6250000000:0.0060521715)0.9900000000:0.0160076607,i045:
0.0215087719)0.9820000000:0.0053282440)1.0000000000:0.0090116067)1.0000000000:0.0
093300843,(f225:0.0027883942,e055:0.0000001343)0.9570000000:0.0252467643)0.929000
0000:0.0112850384)0.8590000000:0.0048690951)0.9720000000:0.0041395213)0.99500000
00:0.0067471834)0.7690000000:0.0023797948)0.8050000000:0.0017174742,(X275:0.00000
00001,W075:0.0000000496)1.0000000000:0.0232519125)0.2540000000:0.0042524726,b105
:0.0136428443)0.6220000000:0.0033456445,V305:0.0308694165)0.6510000000:0.00507056
23,U255:0.0267759588)0.0000000000:0.0016151273,CL5ISVi6:0.0659671025)0.112000000
0:0.0038488453,CL5ISVu6:0.0437647329)1.0000000000:0.0376054211,Q085:0.063224540
1)1.0000000000:0.0323580324,R125:0.0609658634)0.0000000000:0.0001606810,T235:0.06
04767518)0.8560000000:0.0169691809,(CL5ISZ2:0.0891684833,S115:0.0435834217)0.982
0000000:0.0318819817)1.0000000000:0.0646341726,((CL5ISZ3:0.0303510977,((CL5ISZ7:
0.0013799845,CL5ISZ4:0.0160067654)0.8920000000:0.0375976650,CL5ISZ5:0.072119244
0)0.9810000000:0.0174351986)0.9520000000:0.0219850452,(((CL5ISVi7:0.0000000818,CL
5ISVi2:0.0017352495)0.4930000000:0.0074791154,CL5ISVi4:0.0223279352)1.0000000000
:0.0443654615,CL5ISVi8:0.0487988501)0.9990000000:0.0609289725)0.9860000000:0.0363
580157)0.9240000000:0.0446565886,(((((CL5ISVu8:0.0000000573,CL5ISVu5:0.000000000
1)0.0000000000:0.0000000689,CL5ISVu4:0.0000000001)0.0000000000:0.0000000825,CL5I
SVu7:0.0000000001)0.0000000000:0.0000000706,(CL5ISVu2:0.0000000001,CL5ISVu1:0.0
008166690)0.8130000000:0.0008173570)1.0000000000:0.0810476144,((CL5ISP5:0.000000
0678,(CL5ISP2:0.0016916110,(CL5ISP7:0.0000007695,(CL5ISP6:0.0020212910,CL5ISP1:0
.0000000002)0.8900000000:0.0020218920)0.8050000000:0.0085937657)0.9990000000:0.01
29388604)0.8250000000:0.0018253648,CL5ISP8:0.0007215427)1.0000000000:0.153125731
7)0.9080000000:0.0401173533)1.0000000000:0.2963588374,(((SO2:0.1355970260,SO1:0.1
036587486)0.9980000000:0.0711560735,(CL6ISVi4:0.0000001182,CL6ISVi6:0.006940372
9)1.0000000000:0.1464723388)0.3350000000:0.0159602579,(((((CL6ISZ8:0.0161650688,(((
N376:0.1366563237,M346:0.0315585301)0.9890000000:0.0136227397,(CL6ISZ3:0.017858
0176,(CL6ISZ5:0.0126282573,CL6ISZ6:0.0290744753)0.8920000000:0.0026981597)0.3390
000000:0.0008660176)0.7390000000:0.0007144524,(L396:0.0454318528,(CL6ISZ7:0.01942
52828,CL6ISZ4:0.0280473292)0.0000000000:0.0027418133)0.0000000000:0.0000156855)0
.0000000000:0.0000000531)0.9240000000:0.0026194451,(CL6ISZ1:0.0522627795,(CL6ISV
i2:0.0749893286,(CL6ISVi1:0.0712905746,CL6ISVi3:0.0601736511)0.6890000000:0.00350
93580)0.6610000000:0.0009586269)0.7660000000:0.0028380477)0.9030000000:0.0043248
464,(((CL6ISP3:0.0646282278,(((((CL6ISVu4:0.0651242747,P356:0.0363144849)0.4070000
000:0.0026411247,((CL6ISVu2:0.0432918799,O326:0.0470177864)0.3160000000:0.003495
2758,(CL6ISVu3:0.0412854178,CL6ISVu8:0.0419824111)0.9120000000:0.0077140749)0.3
150000000:0.0033975142)0.0000000000:0.0000264996,CL6ISVu1:0.0538386384)0.919000
0000:0.0087822289,CL6ISVu5:0.0349374365)0.7650000000:0.0062608965,CL6ISVu7:0.04
63467833)0.7770000000:0.0032926349)0.9980000000:0.0182792919,((CL6ISVu6:0.069620
7023,CL6ISP7:0.0586132427)0.6490000000:0.0019531530,K366:0.0665709436)0.31600000
00:0.0034919157)0.9670000000:0.0059089007,((CL6ISVi7:0.0738409847,CL6ISVi5:0.1098
868724)0.3780000000:0.0097997090,CL6ISVi8:0.0705572929)0.9350000000:0.0121006383
)0.4870000000:0.0023438945)0.9480000000:0.0146083688,CL6ISZ2:0.0486313080)0.0000
000000:0.0003702035,J386:0.0879854851)0.7250000000:0.0291474235)1.0000000000:0.30
83301881)0.9910000000:0.1822432213,((((populus:0.4804481966,gossypium:0.4426181186
)0.6320000000:0.0908402160,(pisum:0.2669437076,medicago:0.2854273938)1.0000000000:
0.2268678731)0.9890000000:0.1241179225,solanum:0.6479915336)1.0000000000:0.233217
9000,Vitis:0.5926786205)1.0000000000:0.4989986362)0.9650000000:0.1208487671,(2SO8:
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0.0363587851,2SO2:0.0235792476)1.0000000000:0.1245109783)0.9900000000:0.10699180
95,((SO8:0.0382445945,((SO6:0.0109288499,SC2:0.0057441852)0.8060000000:0.00089786
54,SC5:0.0066718284)0.7930000000:0.0034735163)1.0000000000:0.0835922266,(SC6:0.00
20740998,SC1:0.0000001155)1.0000000000:0.0956739074)0.9910000000:0.0539151120)0.
4800000000:0.0255581438,(((((CL11ISZ7:0.0000000377,CL11ISZ1:0.0000000001)0.00000
00000:0.0000000520,CL11ISZ5:0.0000000001)0.0000000000:0.0000000092,CL11ISZ4:0.00
00000001)0.0000000000:0.0000000744,CL11ISZ8:0.0009505954)1.0000000000:0.09021672
12,(((CL11ISVu6:0.0009521462,CL11ISVu8:0.0000000001)1.0000000000:0.0096067862,C
L11ISVu4:0.0000000975)0.9340000000:0.0104076357,(CL11ISVu3:0.0067548792,(CL11IS
Vu5:0.0000001024,CL11ISVu2:0.0000000001)0.9320000000:0.0030727041)0.8720000000:
0.0066545028)1.0000000000:0.0612701814)0.9850000000:0.0466963106)0.9820000000:0.0
447015439,CL11ISZ6:0.1279998798)1.0000000000:0.1076750430,(CL11ISP4:0.016958149
7,(((((B411:0.0094046342,(((CL11ISP6:0.0009474005,SO7:0.0076747871)0.0000000000:0.0
000000932,CL11ISZ3:0.0047715611)0.9890000000:0.0089970170,(CL11ISVi8:0.00000010
12,CL11ISVi1:0.0000000001)0.9850000000:0.0094736322)0.5990000000:0.0036163485)0.7
170000000:0.0050320625,E441:0.0312669727)0.0000000000:0.0006454771,(CL11ISVi3:0.
0166323996,CL11ISZ2:0.0195189129)0.9990000000:0.0197613841)0.7680000000:0.003464
3030,((((((CL11ISP3:0.0000000940,CL11ISP2:0.0000000001)0.0000000000:0.0000001004,
CL11ISP1:0.0000000001)0.9990000000:0.0255340745,CL11ISVi4:0.0274152547)0.918000
0000:0.0063165928,(CL11ISP8:0.0256386286,CL11ISVu7:0.0311126689)0.9400000000:0.0
060477762)0.1280000000:0.0050991009,((CL11ISP7:0.0304019966,CL11ISVu1:0.0118517
198)0.8950000000:0.0081647666,A421:0.0178919062)0.9070000000:0.0083982816)0.9170
000000:0.0131308692,CL11ISVi2:0.0363481252)0.9790000000:0.0119763227)0.947000000
0:0.0054963510,(D431:0.0419351410,C401:0.0206974658)0.6420000000:0.0029794571)0.7
330000000:0.0040399612)0.7200000000:0.0067017945)0.0360000000:0.0054979692,CL11I
SVi6:0.0113242083)0.0000000000:0.0000212581,CL11ISVi7:0.0173353053)1.0000000000:
0.0846225544,F461:0.0207274994)0.9840000000:0.0178435032,H451:0.0315092513)0.0440
000000:0.0027926391,I471:0.0249223947,G481:0.0268999330);
B. The maximum likelihood nexus treefile presented in the figure 6.
#NEXUS
begin taxa;
dimensions ntax=104;
taxlabels
2SO8
2SO2
CL11ISZ6
CL11ISVu1
CL11ISVu7
CL11ISP8
CL11ISVi4[&!color=#-16777216]
CL11ISP2
CL11ISP3
CL11ISVi2[&!color=#-16777216]
CL11ISVi8[&!color=#-16777216]
CL11ISVi1[&!color=#-16777216]
CL11ISZ3
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541
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569
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573
574
575
576
577
578
579
580
581
582
CL11ISP6
SO7
A421
C401
B411
CL11ISVi3[&!color=#-16777216]
CL11ISZ2
CL11ISP4
CL11ISVi6[&!color=#-16777216]
CL11ISVi7[&!color=#-16777216]
CL11ISVu3
CL11ISVu4
CL11ISVu6
CL11ISZ1
CL11ISZ8
SC6
SC1
SO8
SC5
SO6
SC2
CL6ISVi6[&!color=#-16777216]
SO2
SO1
CL6ISZ2
M346
CL6ISZ3
CL6ISZ8
CL6ISZ6
CL6ISZ5
CL6ISZ1
CL6ISVi5[&!color=#-16777216]
CL6ISVi8[&!color=#-16777216]
CL6ISVi3[&!color=#-16777216]
CL6ISZ4
CL6ISZ7
CL6ISVi2[&!color=#-16777216]
CL6ISP7
CL6ISP3
CL6ISVu5
CL6ISVu4
P356
CL6ISVu3
CL6ISVu2
O326
CL6ISVu6
K366
CL5ISVu7
CL5ISVu5
CL5ISVu2
583
584
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586
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630
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632
CL5ISVu1
CL5ISP8
CL5ISP5
CL5ISP2
CL5ISP6
CL5ISP1
CL5ISVi4[&!color=#-16777216]
CL5ISVi7[&!color=#-16777216]
CL5ISZ3
CL5ISZ5
CL5ISZ7
CL5ISZ4
CL5ISZ2
R125
CL5ISVu6
CL5ISVi3[&!color=#-16777216]
CL5ISVi5[&!color=#-16777216]
l175
CL5ISVi1[&!color=#-16777216]
q035
p155
m065
n245
JatCur1
VitVin1
PopTri2
PopTri1
LotJap1
GlMax1
GlMax2
MedTru4
MedTru3
PisSat1
VicPan3
VicPan1
GosRai1
GosHir1
SolLyc1
CapFrut1
CapAnn2
CapAnn1
;
end;
begin trees;
tree tree_1 = [&R] (((((2SO8[&!color=#-6750055]:0.03421,2SO2[&!color=#6750055]:0.02191)[&aLRT=1.0,!rotate=false,!color=#6750055]:0.12008,(((CL11ISZ6[&!color=#-16737895]:0.12217,(CL11ISVu1[&!color=#65536]:0.02757,(((CL11ISVu7[&!color=#-65536]:0.02922,CL11ISP8[&!color=#52225]:0.02407)[&aLRT=0.913,!color=#-6710887]:0.0059,(CL11ISVi4[&!color=#-
633
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16777216]:0.02567,(CL11ISP2[&!color=#-52225]:0.0,CL11ISP3[&!color=#52225]:0.0)[&aLRT=1.0,!color=#-52225]:0.02375)[&aLRT=0.933,!color=#6710887]:0.00585)[&aLRT=0.392,!color=#-6710887]:0.00462,(CL11ISVi2[&!color=#16777216]:0.03427,((((CL11ISVi8[&!color=#-16777216]:0.0,CL11ISVi1[&!color=#16777216]:0.0)[&aLRT=0.983,!color=#-16777216]:0.00795,(CL11ISZ3[&!color=#16737895]:0.00449,(CL11ISP6[&!color=#-16711885]:8.9E-4,SO7[&!color=#6750055]:0.00722)[&aLRT=0.0,!color=#-6710887]:0.0)[&aLRT=0.993,!color=#6710887]:0.0094)[&aLRT=0.845,!color=#-6710887]:0.00445,(A421[&!color=#16711885]:0.05064,(C401[&!color=#-16711885]:0.03469,B411[&!color=#16711885]:0.01024)[&aLRT=0.603,!color=#-16711885]:0.00132)[&aLRT=1.0,!color=#16711885]:0.02094)[&aLRT=0.763,!rotate=true,!color=#6710887]:0.00576,((CL11ISVi3[&!color=#-16777216]:0.0133,CL11ISZ2[&!color=#16737895]:0.02054)[&aLRT=0.994,!rotate=true,!color=#6710887]:0.01755,(CL11ISP4[&!color=#-52225]:0.01651,(CL11ISVi6[&!color=#16777216]:0.00947,CL11ISVi7[&!color=#-16777216]:0.01745)[&aLRT=0.986,!color=#16777216]:0.01122)[&aLRT=0.875,!rotate=true,!color=#6710887]:0.00626)[&aLRT=0.415,!rotate=true,!color=#6710887]:0.00429)[&aLRT=0.985,!color=#6710887]:0.01652)[&aLRT=0.938,!rotate=true,!color=#6710887]:0.01539)[&aLRT=0.0,!color=#-16711885]:2.8E4)[&aLRT=1.0,!rotate=true,!color=#6710887]:0.08631)[&aLRT=0.964,!rotate=true,!color=#6710887]:0.03409,((CL11ISVu3[&!color=#-65536]:0.01106,(CL11ISVu4[&!color=#65536]:0.0,CL11ISVu6[&!color=#-65536]:0.00994)[&aLRT=0.957,!color=#65536]:0.01144)[&aLRT=0.999,!color=#-65536]:0.05217,(CL11ISZ1[&!color=#16737895]:0.0,CL11ISZ8[&!color=#-16737895]:8.9E-4)[&aLRT=1.0,!color=#16737895]:0.08584)[&aLRT=0.997,!color=#-6710887]:0.04605)[&aLRT=0.514,!color=#6710887]:0.02234,((SC6[&!color=#-6750055]:0.00196,SC1[&!color=#6750055]:0.0)[&aLRT=1.0,!color=#-6750055]:0.089,(SO8[&!color=#6750055]:0.0356,(SC5[&!color=#-6750055]:0.00633,(SO6[&!color=#6750055]:0.0104,SC2[&!color=#-6750055]:0.00546)[&aLRT=0.727,!color=#6750055]:7.7E-4)[&aLRT=0.743,!color=#-6750055]:0.00364)[&aLRT=1.0,!color=#6750055]:0.0777)[&aLRT=0.985,!rotate=true,!color=#6750055]:0.05088)[&aLRT=0.985,!color=#6710887]:0.08614)[&aLRT=0.992,!rotate=true,!color=#6710887]:0.11064,(((CL6ISVi6[&!color=#-16777216]:0.14668,(SO2[&!color=#6750055]:0.12415,SO1[&!color=#-6750055]:0.09458)[&aLRT=1.0,!color=#6750055]:0.06707)[&aLRT=0.554,!color=#-6710887]:0.01614,(CL6ISZ2[&!color=#16737895]:0.04674,(M346[&!color=#-16711885]:0.04158,((CL6ISZ3[&!color=#16737895]:0.01766,((CL6ISZ8[&!color=#-16737895]:0.01315,(CL6ISZ6[&!color=#16737895]:0.02719,CL6ISZ5[&!color=#-16737895]:0.01092)[&aLRT=0.763,!color=#16737895]:0.00178)[&aLRT=0.591,!color=#-6710887]:0.00244,((CL6ISZ1[&!color=#16737895]:0.04954,((CL6ISVi5[&!color=#-16777216]:0.10842,CL6ISVi8[&!color=#16777216]:0.06271)[&aLRT=0.932,!color=#-16777216]:0.0136,(CL6ISVi3[&!color=#16777216]:0.0591,CL6ISZ4[&!color=#-16737895]:0.02139)[&aLRT=0.439,!color=#6710887]:0.00337)[&aLRT=0.571,!color=#-6710887]:6.3E-4)[&aLRT=0.902,!color=#6710887]:0.00256,(CL6ISZ7[&!color=#-16737895]:0.01888,CL6ISVi2[&!color=#16777216]:0.0711)[&aLRT=0.698,!color=#-6710887]:0.00117)[&aLRT=0.871,!color=#6710887]:0.00151)[&aLRT=0.0,!color=#-16711885]:0.0)[&aLRT=0.867,!color=#6710887]:0.00527,(CL6ISP7[&!color=#-52225]:0.05191,((CL6ISP3[&!color=#-
683
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52225]:0.06172,(CL6ISVu5[&!color=#-65536]:0.03262,((CL6ISVu4[&!color=#65536]:0.06162,P356[&!color=#-16711885]:0.02682)[&aLRT=0.287,!color=#6710887]:0.00174,(CL6ISVu3[&!color=#-65536]:0.04483,(CL6ISVu2[&!color=#65536]:0.04105,O326[&!color=#-16711885]:0.02418)[&aLRT=0.214,!color=#6710887]:0.00177)[&aLRT=0.418,!color=#-6710887]:0.00201)[&aLRT=0.974,!color=#6710887]:0.01485)[&aLRT=0.583,!color=#-6710887]:0.00762)[&aLRT=0.994,!color=#6710887]:0.01837,(CL6ISVu6[&!color=#-65536]:0.06666,K366[&!color=#16711885]:0.05247)[&aLRT=0.0,!color=#-6710887]:0.00106)[&aLRT=0.884,!color=#6710887]:0.00693)[&aLRT=0.778,!color=#-6710887]:0.00767)[&aLRT=0.569,!color=#16711885]:0.00365)[&aLRT=0.901,!rotate=true,!color=#6710887]:0.01166)[&aLRT=0.876,!rotate=true,!color=#6710887]:0.02065)[&aLRT=1.0,!rotate=true,!color=#6710887]:0.2387,(((CL5ISVu7[&!color=#-65536]:0.0,(CL5ISVu5[&!color=#65536]:0.0,(CL5ISVu2[&!color=#-65536]:0.0,CL5ISVu1[&!color=#-65536]:7.7E4)[&aLRT=0.851,!color=#-65536]:7.7E-4)[&aLRT=0.0,!color=#65536]:0.0)[&aLRT=1.0,!color=#-65536]:0.07718,(CL5ISP8[&!color=#-52225]:5.5E4,(CL5ISP5[&!color=#-52225]:0.0,(CL5ISP2[&!color=#-52225]:0.0,(CL5ISP6[&!color=#52225]:0.00189,CL5ISP1[&!color=#-52225]:0.0)[&aLRT=0.915,!color=#52225]:0.00284)[&aLRT=0.909,!color=#-52225]:0.01383)[&aLRT=0.853,!color=#52225]:0.00185)[&aLRT=1.0,!color=#52225]:0.13911)[&aLRT=0.944,!rotate=true,!color=#6710887]:0.03954,(((CL5ISVi4[&!color=#-16777216]:0.02095,CL5ISVi7[&!color=#16777216]:0.00737)[&aLRT=1.0,!color=#-16777216]:0.10475,(CL5ISZ3[&!color=#16737895]:0.02707,(CL5ISZ5[&!color=#-16737895]:0.06708,(CL5ISZ7[&!color=#16737895]:0.00204,CL5ISZ4[&!color=#-16737895]:0.0143)[&aLRT=0.902,!color=#16737895]:0.03446)[&aLRT=0.99,!color=#-16737895]:0.01803)[&aLRT=0.915,!color=#16737895]:0.01709)[&aLRT=0.995,!color=#-6710887]:0.03842,(CL5ISZ2[&!color=#16737895]:0.11093,(R125[&!color=#-16711885]:0.07327,(CL5ISVu6[&!color=#65536]:0.05086,((CL5ISVi3[&!color=#-16777216]:0.00139,CL5ISVi5[&!color=#16777216]:0.00174)[&aLRT=1.0,!color=#-16777216]:0.05994,(l175[&!color=#16711885]:0.04805,(CL5ISVi1[&!color=#-16777216]:0.03788,(q035[&!color=#16711885]:0.01733,(p155[&!color=#-16711885]:0.01037,(m065[&!color=#16711885]:0.0,n245[&!color=#-16711885]:0.0)[&aLRT=1.0,!color=#16711885]:0.01428)[&aLRT=0.946,!rotate=true,!color=#16711885]:0.00743)[&aLRT=0.859,!rotate=true,!color=#16711885]:0.00551)[&aLRT=0.684,!rotate=true,!color=#6710887]:0.01099)[&aLRT=0.999,!rotate=true,!color=#6710887]:0.02712)[&aLRT=0.947,!rotate=true,!color=#6710887]:0.01328)[&aLRT=1.0,!rotate=true,!color=#6710887]:0.05498)[&aLRT=0.945,!rotate=true,!color=#6710887]:0.02712)[&aLRT=0.993,!rotate=true,!color=#6710887]:0.03806)[&aLRT=0.8,!rotate=true,!color=#6710887]:0.03369)[&aLRT=1.0,!rotate=true,!color=#6710887]:0.22392)[&aLRT=0.937,!color=#6710887]:0.10962)[&aLRT=1.0,!rotate=true,!color=#16777012]:0.31937,JatCur1[&!rotate=false,!color=#-16777012]:0.53081)[&!color=#16777012]:0.0622,(VitVin1[&!color=#-16777012]:0.53934,((((PopTri2[&!color=#16777012]:0.16588,PopTri1[&!color=#16777012]:0.25482)[&aLRT=1.0,!rotate=true,!color=#16777012]:0.28342,((LotJap1[&!color=#-16777012]:0.2266,(GlMax1[&!color=#-
733
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16777012]:0.01807,GlMax2[&!color=#16777012]:0.03916)[&aLRT=1.0,!rotate=true,!color=#16777012]:0.26733)[&aLRT=0.952,!rotate=true,!color=#16777012]:0.08313,(MedTru4[&!color=#-16777012]:0.20311,(MedTru3[&!color=#16777012]:0.15449,(PisSat1[&!color=#-16777012]:0.20657,(VicPan3[&!color=#16777012]:0.03473,VicPan1[&!color=#-16777012]:0.01707)[&aLRT=1.0,!color=#16777012]:0.13311)[&aLRT=0.763,!rotate=true,!color=#16777012]:0.04745)[&aLRT=0.931,!rotate=true,!color=#16777012]:0.06481)[&aLRT=0.999,!rotate=true,!color=#16777012]:0.14298)[&aLRT=0.93,!rotate=true,!color=#16777012]:0.07575)[&aLRT=0.092,!rotate=true,!color=#16777012]:0.06301,(GosRai1[&!color=#-16777012]:0.18093,GosHir1[&!color=#16777012]:0.08612)[&aLRT=1.0,!color=#-16777012]:0.23215)[&aLRT=0.981,!color=#16777012]:0.09899,(SolLyc1[&!color=#-16777012]:0.32702,(CapFrut1[&!color=#16777012]:0.02784,(CapAnn2[&!color=#-16777012]:0.05016,CapAnn1[&!color=#16777012]:0.02863)[&aLRT=0.597,!color=#-16777012]:0.00199)[&aLRT=1.0,!color=#16777012]:0.36216)[&aLRT=1.0,!rotate=true,!color=#16777012]:0.21)[&aLRT=1.0,!color=#-16777012]:0.23064)[&!rotate=true,!color=#16777012]:0.00898)[&aLRT=0.289,!color=#-1];
end;
begin figtree;
set appearance.backgroundColorAttribute="User Selection";
set appearance.backgroundColour=#-1;
set appearance.branchColorAttribute="User Selection";
set appearance.branchLineWidth=3.0;
set appearance.foregroundColour=#-16777216;
set appearance.selectionColour=#-2144520576;
set branchLabels.colorAttribute="User Selection";
set branchLabels.displayAttribute="Branch times";
set branchLabels.fontName="sansserif";
set branchLabels.fontSize=8;
set branchLabels.fontStyle=0;
set branchLabels.isShown=false;
set branchLabels.significantDigits=4;
set layout.expansion=0;
set layout.layoutType="RECTILINEAR";
set layout.zoom=0;
set nodeBars.barWidth=4.0;
set nodeLabels.colorAttribute="aLRT";
set nodeLabels.displayAttribute="aLRT";
set nodeLabels.fontName="Arial";
set nodeLabels.fontSize=12;
set nodeLabels.fontStyle=0;
set nodeLabels.isShown=true;
set nodeLabels.significantDigits=2;
set polarLayout.alignTipLabels=false;
set polarLayout.angularRange=0;
set polarLayout.rootAngle=0;
set polarLayout.rootLength=100;
783
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829
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831
832
set polarLayout.showRoot=true;
set radialLayout.spread=0.0;
set rectilinearLayout.alignTipLabels=false;
set rectilinearLayout.curvature=0;
set rectilinearLayout.rootLength=100;
set scale.offsetAge=0.0;
set scale.rootAge=1.0;
set scale.scaleFactor=1.0;
set scale.scaleRoot=false;
set scaleAxis.automaticScale=true;
set scaleAxis.fontSize=8.0;
set scaleAxis.isShown=false;
set scaleAxis.lineWidth=1.0;
set scaleAxis.majorTicks=0.1;
set scaleAxis.origin=0.0;
set scaleAxis.reverseAxis=false;
set scaleAxis.showGrid=true;
set scaleAxis.significantDigits=4;
set scaleBar.automaticScale=true;
set scaleBar.fontSize=10.0;
set scaleBar.isShown=true;
set scaleBar.lineWidth=1.0;
set scaleBar.scaleRange=0.0;
set scaleBar.significantDigits=4;
set tipLabels.colorAttribute="User Selection";
set tipLabels.displayAttribute="Names";
set tipLabels.fontName="sansserif";
set tipLabels.fontSize=8;
set tipLabels.fontStyle=0;
set tipLabels.isShown=false;
set tipLabels.significantDigits=4;
set trees.order=false;
set trees.orderType="increasing";
set trees.rooting=true;
set trees.rootingType="User Selection";
set trees.transform=false;
set trees.transformType="cladogram";
end;
C. The maximum likelihood newick treefile.
((CapFrut1:0.02752857,(SolLyc1:0.31773141,(((GosRai1:0.18470712,GosHir1:0.07900045)
100:0.22709542,(((PisSat1:0.14627829,((VicPan3:0.03487224,VicPan1:0.01624083)100:0.13
545534,(MedTru3:0.14661979,MedTru4:0.25011214)30:0.04085581)50:0.08177174)100:0.1
6143727,(LotJap1:0.22390005,(GlMax1:0.01786273,GlMax2:0.03846516)100:0.26219728)8
4:0.06101287)88:0.09926679,(PopTri2:0.16005690,PopTri1:0.25596940)100:0.26822448)49
:0.07260780)98:0.09920458,(VitVin1:0.53856494,(JatCur1:0.52288951,(((((CL5ISP8:0.0005
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859
860
861
862
863
864
865
866
867
868
869
870
871
872
873
874
875
876
877
878
879
880
881
882
3636,(CL5ISP5:0.00000006,(CL5ISP2:0.00000047,(CL5ISP1:0.00000009,CL5ISP6:0.00186
234)98:0.00279538)90:0.01365001)81:0.00183721)100:0.13751542,((CL5ISVu1:0.00076363
,CL5ISVu2:0.00000007)74:0.00076425,(CL5ISVu5:0.00000001,CL5ISVu7:0.00000009)40:
0.00000001)100:0.07622884)99:0.03869485,((CL5ISZ2:0.10954983,(R125:0.07234940,(CL
5ISVu6:0.05021236,((CL5ISVi3:0.00136961,CL5ISVi5:0.00171454)100:0.05918363,(l175:0
.04743181,(CL5ISVi1:0.03743915,(q035:0.01709469,(p155:0.01022837,(m065:0.00000001,
n245:0.00000001)100:0.01408645)100:0.00734724)68:0.00537135)69:0.01085388)94:0.026
80352)89:0.01314669)96:0.05427216)86:0.02671320)88:0.03759702,((CL5ISVi4:0.0206657
6,CL5ISVi7:0.00728836)100:0.10333995,(CL5ISZ3:0.02669302,(CL5ISZ5:0.06612340,(CL
5ISZ4:0.01410013,CL5ISZ7:0.00201794)92:0.03399969)96:0.01782066)95:0.01694809)95:0
.03799553)76:0.03363566)100:0.22292092,(((SO1:0.09411560,SO2:0.12217479)100:0.0666
4382,CL6ISVi6:0.14507424)73:0.01736060,(CL6ISZ2:0.04577839,(((((K366:0.05280943,C
L6ISVu6:0.06542704)57:0.00191614,(CL6ISP3:0.06084236,(CL6ISVu5:0.03252563,((CL6I
SVu4:0.06089993,P356:0.02628991)31:0.00166681,(CL6ISVu3:0.04430429,(O326:0.02371
032,CL6ISVu2:0.04064507)35:0.00182176)41:0.00200262)93:0.01424366)65:0.00745841)9
9:0.01734796)43:0.00846046,(CL6ISVi8:0.06242770,CL6ISVi5:0.10488907)38:0.01243498
)6:0.00608407,(((CL6ISZ8:0.01264597,(CL6ISZ6:0.02684400,CL6ISZ5:0.01077740)53:0.00
201548)46:0.00241688,((CL6ISZ7:0.01835557,(CL6ISZ4:0.02065176,CL6ISVi3:0.0580786
3)30:0.00634988)12:0.00104339,(CL6ISZ1:0.05130088,CL6ISVi2:0.07115376)10:0.000000
93)11:0.00168264)1:0.00000008,CL6ISZ3:0.01735618)15:0.00416124)7:0.00246363,(M346:
0.04020795,CL6ISP7:0.05351024)15:0.00329855)56:0.01260523)76:0.01934376)100:0.2368
0896)88:0.10855210,((2SO2:0.02169691,2SO8:0.03364531)100:0.11970760,(((SC1:0.00000
012,SC6:0.00192861)100:0.08790608,((SC5:0.00623571,(SO6:0.01025358,SC2:0.00538269)
72:0.00074951)85:0.00342392,SO8:0.03524288)100:0.07676824)98:0.05028615,(((CL11ISZ
8:0.00088171,CL11ISZ1:0.00000009)100:0.08472707,(CL11ISVu3:0.01093880,(CL11ISVu
4:0.00000006,CL11ISVu6:0.00979731)93:0.01122964)100:0.05137565)99:0.04572025,(CL1
1ISZ6:0.12081397,(CL11ISVu1:0.02724717,((CL11ISVi2:0.03379056,(((A421:0.04993698,(
B411:0.01010681,C401:0.03422397)48:0.00126419)91:0.02068592,((CL11ISZ3:0.00442453
,(SO7:0.00711739,CL11ISP6:0.00087902)45:0.00000007)100:0.00926795,(CL11ISVi1:0.00
000001,CL11ISVi8:0.00000001)100:0.00783382)87:0.00438684)57:0.00569334,((CL11ISP4
:0.01628102,(CL11ISVi7:0.01720890,CL11ISVi6:0.00934036)69:0.01105688)57:0.0061738
6,(CL11ISZ2:0.02026089,CL11ISVi3:0.01311638)95:0.01730529)22:0.00423153)53:0.0162
9134)66:0.01520525,((CL11ISVi4:0.02530609,(CL11ISP2:0.00000001,CL11ISP3:0.000000
01)100:0.02341600)71:0.00577227,(CL11ISVu7:0.02883624,CL11ISP8:0.02375794)90:0.00
577702)39:0.00456327)21:0.00018153)100:0.08533827)89:0.03364805)56:0.02174450)96:0.
08416878)100:0.10880324)100:0.31832700)47:0.06890368)100:0.22700889)100:0.2139156
4)100:0.36649038)67:0.00181258,CapAnn2:0.04947231,CapAnn1:0.02830782);
D. The maximum likelihood newick treefile.
(((((((((CL11ISVu1:0.0144673060,(CL11ISP8:0.0431760984,((((((((((((((((((((CL6ISVi3:0.1
239786416,(CL6ISZ1:0.0606550833,CL6ISVi7:0.1294288651)5:0.0131534217)0:0.0103528
086,CL6ISVi5:0.2279163187)0:0.0052483990,(((((CL6ISVu6:0.1004667723,K366:0.083810
7835)1:0.0047295347,((CL6ISZ7:0.0352504084,CL6ISZ2:0.0662523139)3:0.0031667041,C
L6ISVi2:0.1334326040)3:0.0060319704)0:0.0021922197,(CL6ISP7:0.0916894062,((CL6ISZ
5:0.0058269158,CL6ISZ3:0.0350972758)4:0.0000003484,CL6ISZ6:0.0386890916)9:0.0029
831594)0:0.0029410472)0:0.0000001395,CL6ISZ8:0.0217143070)0:0.0025711331,(CL6ISZ
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4:0.0376674051,CL6ISVi1:0.1144673904)3:0.0093225323)0:0.0000002251)0:0.0046831166
,CL6ISVu5:0.0377587110)0:0.0039128045,(((SO1:0.1878873258,SO2:0.2162293631)9:0.02
53294620,(CL6ISVi4:0.0000003478,CL6ISVi6:0.0141238654)10:0.1857619806)4:0.027265
8224,CL6ISVi8:0.1015624708)0:0.0034944998)0:0.0058333956,(((M346:0.0456880807,L39
6:0.0277433090)6:0.0045222575,N376:0.1013316733)6:0.0105881573,((P356:0.0488261989
,(CL6ISVu3:0.0655351947,O326:0.0500345788)3:0.0000005290)3:0.0038218333,CL6ISVu
2:0.0665789759)5:0.0045933508)4:0.0027126836)0:0.0000002680,(CL6ISP3:0.1042285649,
CL6ISVu4:0.1037556889)3:0.0096517904)0:0.0000014096,J386:0.0616664319)0:0.0035635
808,(CL6ISVu1:0.0595075302,CL6ISVu7:0.0697722990)0:0.0031206154)0:0.0085751850,
CL6ISVu8:0.0708667090)10:0.2199524383,((((((275:0.0000000001,W075:0.0000002036)10:0.0254857854,(b105:0.0064706775,((a195:0.026
9058834,Z095:0.0297612152)6:0.0045067223,((c215:0.0399646226,(((U255:0.0479375193,(
d135:0.0398662496,((f225:0.0086909495,e055:0.0000052937)5:0.0250918693,(g295:0.0233
378260,((i045:0.0181872942,((((p025:0.0012898397,k265:0.0028799102)9:0.0091535245,(((
n315:0.0064584839,(o165:0.0081625473,o015:0.0080075263)9:0.0037403346)5:0.00108730
63,l175:0.0062595900)9:0.0090036145,(((n245:0.0000000001,m065:0.0000000946)10:0.011
8675549,p155:0.0119558522)10:0.0023256821,q035:0.0167310795)9:0.0030844158)4:0.001
2838379)5:0.0021828137,CL5ISVi1:0.0255833501)2:0.0041784654,j145:0.0130502722)2:0.
0070651196)1:0.0036880310,h205:0.0164478550)0:0.0025082271)0:0.0031443637)0:0.0084
408359)0:0.0015333721)0:0.0010475549,T235:0.1125930042)0:0.0000006823,(CL5ISVi5:0.
0059563020,CL5ISVi3:0.0032204548)10:0.0796224666)0:0.0000002255)0:0.0015041407,Y
185:0.0434772553)0:0.0000001324)0:0.0000001555)0:0.0017042857)0:0.0000005961,CL5I
SVi6:0.1151228856)0:0.0079171108,CL5ISVu6:0.0655870932)0:0.0122882909,((Q085:0.08
53192741,(R125:0.0822762270,(CL5ISZ2:0.1101235427,S115:0.0679203999)3:0.01757348
56)1:0.0036466129)0:0.0096456471,V305:0.0288725457)0:0.0040470335)0:0.0147489944,(
((((CL5ISVi7:0.0000003490,CL5ISVi2:0.0062210203)5:0.0182429361,CL5ISVi4:0.0322262
397)10:0.1005366018,CL5ISVi8:0.1035305916)3:0.0115989410,(CL5ISZ3:0.0254587760,((
(CL5ISZ4:0.0305598736,CL5ISZ7:0.0034367580)10:0.0583848517,CL5ISZ5:0.0948160921
)0:0.0069994938,m285:0.0000000043)0:0.0064713001)0:0.0056120603)0:0.0079087335,((((
((CL5ISVu8:0.0000002289,CL5ISVu5:0.0000000001)2:0.0000002180,CL5ISVu2:0.0000000
001)5:0.0000001972,CL5ISVu7:0.0000000001)6:0.0000002605,CL5ISVu4:0.0000000001)7:
0.0000003060,CL5ISVu1:0.0029881963)10:0.0874529992,((CL5ISP5:0.0000003666,(CL5IS
P2:0.0079299858,(CL5ISP7:0.0144765580,(CL5ISP6:0.0036876205,CL5ISP1:0.0000000001
)7:0.0054961914)4:0.0102814645)6:0.0278179146)5:0.0065804123,CL5ISP8:0.0025116959)
10:0.1935714824)10:0.0393864177)0:0.0097959467)9:0.2375525811)5:0.1464653190,((((po
pulus:0.3801551695,gossypium:0.3571269997)5:0.0690863835,(pisum:0.1896681385,medic
ago:0.2164125732)10:0.1690750933)10:0.1394853770,solanum:0.5032430402)10:0.2476531
586,Vitis:0.4136047956)10:0.3811963007)10:0.1265306228,(2SO8:0.0844451817,2SO2:0.0
417873309)10:0.0479297144)10:0.0641054142,(((((CL11ISZ7:0.0000001796,CL11ISZ1:0.0
000000001)0:0.0000002735,CL11ISZ4:0.0000000001)0:0.0000001746,CL11ISZ5:0.000000
0001)5:0.0000003863,CL11ISZ8:0.0035006181)10:0.0743477914,((CL11ISVu6:0.00352156
92,CL11ISVu8:0.0000000001)5:0.0039666259,(CL11ISVu4:0.0000000001,((CL11ISVu3:0.
0036020115,CL11ISVu5:0.0000000001)8:0.0000003247,CL11ISVu2:0.0000000001)10:0.00
35978217)7:0.0031066167)8:0.0226978142)6:0.0150446403)2:0.0163176826,(SC6:0.003842
6544,SC1:0.0000000001)10:0.0772840662)2:0.0057026522,((SO6:0.0293986984,SC2:0.010
5200487)4:0.0000004161,(SO8:0.0612351364,SC5:0.0211889850)2:0.0033849180)10:0.033
2135256)8:0.0189626540,CL11ISZ6:0.1340450506)7:0.0445417623,CL11ISVu7:0.0501054
214)2:0.0000003111,((CL11ISP3:0.0000002742,CL11ISP2:0.0000000001)5:0.0000002847,
CL11ISP1:0.0000000001)10:0.0542096693)1:0.0000015275,(CL11ISP7:0.0503193715,CL1
1ISVi4:0.0383580075)4:0.0131819783)0:0.0044114087)0:0.0044819944)5:0.0135727648,C
933
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L11ISVi2:0.0319257125)0:0.0000007419,(((CL11ISVi6:0.0035176980,CL11ISP6:0.000000
0001)1:0.0000003800,SO7:0.0035633886)1:0.0000003417,CL11ISZ3:0.0106138996)3:0.003
5153171)0:0.0000003290,CL11ISVi7:0.0035144741)0:0.0000003702,(CL11ISVi3:0.007030
0520,CL11ISZ2:0.0216609028)4:0.0070953683)0:0.0000003182,(((CL11ISVi8:0.000000302
3,CL11ISVi1:0.0000000001)10:0.0105830195,B411:0.0148274918)0:0.0000006284,(CL11I
SP4:0.0106122411,A421:0.0315343092)0:0.0000012807)0:0.0017500551)1:0.0031214054,C
401:0.0112818370)0:0.0052699177,(F461:0.0208201037,(H451:0.0288903171,(G481:0.0317
150296,I471:0.0346151537)9:0.0085060692)7:0.0147042729)10:0.0909019480)2:0.0081389
757,E441:0.0062733244,D431:0.0149054046);
E. The MrBayes newick treefile.
(o015:0.010769,o165:0.014121,((((p025:0.000777,k265:0.001533)1.00:0.014569,((i045:0.02
2660,((((e055:0.009871,f225:0.005599)0.76:0.021189,(((((((W075:0.000432,X275:0.000779)
1.00:0.023866,(((Q085:0.064930,(((S115:0.043228,CL5ISZ2:0.096883)1.00:0.032447,((((((((
(((O326:0.037706,CL6ISVu1:0.048990)1.00:0.016170,(P356:0.035722,CL6ISVu2:0.047391)
0.50:0.003957,CL6ISVu4:0.068468)0.59:0.002749,CL6ISVu3:0.047247)0.62:0.004371,CL6I
SVu8:0.044782)1.00:0.008110,CL6ISVu5:0.038869)0.90:0.006933,CL6ISVu7:0.047571)0.6
7:0.003999,CL6ISP3:0.067513)0.76:0.011282,((M346:0.032709,(N376:0.042409,(SO1:0.108
199,SO2:0.134579)0.71:0.064827,(CL6ISVi6:0.008891,CL6ISVi4:0.001060)1.00:0.148792)
0.54:0.039362,L396:0.025678,(CL6ISVi1:0.074574,CL6ISZ2:0.059700,CL6ISVi3:0.061315)
0.64:0.004546,((CL6ISVi7:0.080629,CL6ISVi5:0.118571)0.71:0.012136,CL6ISVi8:0.07547
1)0.74:0.014235,CL6ISVi2:0.062766,CL6ISZ1:0.049887,CL6ISZ4:0.028465,(CL6ISZ6:0.02
9732,CL6ISZ5:0.013744)0.68:0.003819,CL6ISZ7:0.021633,CL6ISZ3:0.019624,CL6ISZ8:0.
015206,CL6ISP7:0.059308)0.56:0.006450,K366:0.061062,J386:0.085176,CL6ISVu6:0.0656
64)0.70:0.011156)1.00:0.342854,((((((((((C401:0.022494,D431:0.030584,CL11ISP4:0.01614
6,(CL11ISVi6:0.011480,CL11ISVi7:0.014668)0.70:0.012656)0.62:0.006217,(B411:0.009821
,((SO7:0.008723,CL11ISZ3:0.005957,CL11ISP6:0.002341)0.97:0.009470,(CL11ISVi1:0.001
218,CL11ISVi8:0.000890)1.00:0.010664)0.88:0.004744)0.96:0.006302,(CL11ISZ2:0.022956
,CL11ISVi3:0.017382)0.97:0.020466)0.76:0.009533,CL11ISVi2:0.040164)0.68:0.009123,(A
421:0.022144,(CL11ISVu1:0.011894,CL11ISP7:0.031768)0.88:0.008380)0.60:0.008662,E44
1:0.013296,((CL11ISVu7:0.032759,CL11ISP8:0.026576)0.83:0.006642,(CL11ISVi4:0.02878
9,(CL11ISP1:0.000748,CL11ISP2:0.001131,CL11ISP3:0.001263)0.97:0.025953)0.82:0.0067
96)0.51:0.006705)0.53:0.062668,((H451:0.026243,I471:0.023286,G481:0.024966)0.51:0.022
703,F461:0.017290)0.50:0.067366)0.50:0.103551,CL11ISZ6:0.137400)0.50:0.046653,((((CL
11ISVu2:0.000801,CL11ISVu5:0.001386)1.00:0.004286,CL11ISVu3:0.007916)0.98:0.00734
5,((CL11ISVu8:0.000803,CL11ISVu6:0.001909)1.00:0.011245,CL11ISVu4:0.001069)1.00:0
.012579)1.00:0.053644,(CL11ISZ8:0.001986,CL11ISZ5:0.000984,CL11ISZ4:0.001093,CL1
1ISZ1:0.001022,CL11ISZ7:0.001089)1.00:0.092593)0.51:0.047603)0.91:0.033295,((SC1:0.0
01320,SC6:0.003344)1.00:0.099741,(((SC2:0.006693,SO6:0.012440)0.77:0.002108,SC5:0.00
7998)0.81:0.005249,SO8:0.040124)1.00:0.087109)1.00:0.056733)1.00:0.126240,(2SO2:0.02
5083,2SO8:0.038228)1.00:0.121746)1.00:0.135419,(Vitis:0.594537,(((medicago:0.293921,pi
sum:0.269392)1.00:0.232706,(gossypium:0.444257,populus:0.488964)1.00:0.097048)1.00:0.
132598,solanum:0.659448)1.00:0.240094)1.00:0.502257)1.00:0.183440)1.00:0.311938,((((((
CL5ISP1:0.001302,CL5ISP6:0.003023)1.00:0.002928,CL5ISP7:0.003330)0.99:0.006964,CL
5ISP2:0.002895)1.00:0.014018,CL5ISP5:0.000934)0.96:0.002377,CL5ISP8:0.001726)1.00:0
.160057,(CL5ISVu4:0.000747,CL5ISVu7:0.000740,(CL5ISVu1:0.001720,CL5ISVu2:0.0008
70)0.98:0.001652,CL5ISVu5:0.000853,CL5ISVu8:0.000796)1.00:0.084402)1.00:0.050872)0.
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95:0.040532,(((CL5ISZ5:0.074816,(CL5ISZ4:0.017892,CL5ISZ7:0.002104)1.00:0.039030)1.
00:0.018696,CL5ISZ3:0.032561)1.00:0.023230,(CL5ISVi8:0.052679,(CL5ISVi4:0.023772,(
CL5ISVi2:0.002879,CL5ISVi7:0.001046)0.99:0.008620)1.00:0.046403)1.00:0.065003)1.00:
0.040601)0.96:0.067336)0.91:0.017251,(R125:0.033647,T235:0.035948)0.87:0.032661)0.96:
0.032341)1.00:0.042420,U255:0.017472,V305:0.019174,CL5ISVu6:0.035761,CL5ISVi6:0.0
64198)0.85:0.008216,b105:0.016796)0.61:0.005385)0.64:0.001883,Y185:0.032338)0.67:0.00
2768,(Z095:0.024668,a195:0.020442)1.00:0.015903)0.80:0.006553,c215:0.031777)0.79:0.00
4508,d135:0.037388)0.76:0.005611,(CL5ISVi3:0.001749,CL5ISVi5:0.003174)1.00:0.072798
)0.77:0.011425)1.00:0.009588,g295:0.018655)1.00:0.009341,h205:0.021569)1.00:0.005242)
1.00:0.020834,j145:0.019176)0.77:0.007010)0.76:0.009056,((q035:0.017321,((m065:0.00037
6,n245:0.000410)1.00:0.006911,p155:0.008339)1.00:0.007145)0.99:0.005681,CL5ISVi1:0.0
44608)0.76:0.006275)1.00:0.030393,(l175:0.009776,m285:0.011675)0.82:0.004608,n315:0.0
07536)1.00:0.008376);
F. The PhyloBayes newick treefile.
(2SO8:0.293923,((solanum:1.0295,((pisum:0.277071,medicago:0.382164)1:0.337606,(populu
s:0.659017,gossypium:0.606037)0.75:0.116687)0.9:0.216597)1:0.704556,Vitis:0.843983)1:1.
17319,(SO8:0.038795,SC2:0.008511)0.89:0.096541,(((P356:0.031717,CL6ISVu2:0.044871)
0.85:0.022577,(SO1:0.249435,(CL6ISZ1:0.054596,CL6ISVi1:0.077229)0.63:0.007465,CL6I
SP7:0.070728)0.82:0.029118)1:0.392319,((i045:0.110569,CL5ISZ2:0.152839)0.62:0.022381,
(CL5ISVi7:0.161878,(CL5ISVu4:0.077495,CL5ISP2:0.193428)1:0.079328)0.74:0.028987)1:
0.339361)1:0.306907,(CL11ISZ6:0.133941,((CL11ISVu1:0.025598,(CL11ISVi4:0.033257,C
L11ISP8:0.028412)0.95:0.009376)0.85:0.016796,C401:0.042634)0.83:0.063877)0.52:0.0465
84);
Note S2
Branching order of Silene Ogre retrotransposon groups
The phylogenetic tree of the translated alignment A was very similar to the tree constructed
from the nucleotide alignment (Note S1). The result of the Bayesian inference using MrBayes
(Ronquist & Huelsenbeck, 2003) performed on the dataset A was very similar to the results of
the maximum likelihood tree search (Note S1). To reduce the probability that the branching
order of Ogre CL5, Ogre CL6 and Ogre CL11 is affected by long branch attraction artifacts,
we used reduced alignment (alignment D) for phylogenetic tree reconstruction by PhyloBayes
(Lartillot et al. 2009) using the CAT-GTR + Γ model. The results are consistent with the
results of the analyses of the full alignment A (Note S1). Alignments can be found at
http://purl.org/phylo/treebase/phylows/study/TB2:S13636.
Tables S1-S8
Table S1. Primers used in this study. Silene latifolia specific primers. (*) PCR product length with/without intron.
Experiment
Name
Sequence
Ogre Cluster
a. Primers for phylogenetic
analyses
OgreINTdeg-F2
CGMACGAHGGTCAGAGCCAGC
Ogre CL5
OgreINTdeg-R2
OgreINTdeg-F1
OgreINTdeg-R1
OgreINTCL11-F
GCTKRGCCAGGGGCCGTTT
MGRAGCGAGCCAGCTCACATT
ARRTGCCGCAGCTTAGACTCG
CCGCTCAGTTCGTTAGAACC
Ogre CL5
Ogre CL6
Ogre CL6
Ogre CL11
1427
OgreINTCL11-R
AAACCGAGCTCGTAGTTCCA
Ogre CL11
1310
b. Primers for splicing
Ogre-F1
CTACACCCGAACCAGAAAAG
OgreCL5
TTTCCCGAACTACTGTGACTA
CATTTCTCCACACAGAAATC
ACAGCCAGAACTCACCCTTG
OgreCL5
OgreCL6
OgreCL5
2227/627*
c. Primers for LTR amplification
Ogre-R1
Ogre-F2
CL5-LTR-F
CL5-LTR-R
GGAGTCGCCACCAATTTTTA
OgreCL5
732
CL6-LTR-F
ACCGGGTTCAAATACCCATT
OgreCL6
CL6-LTR-R
CCCGTTCGAATTCCACTTTA
OgreCL6
CL11-LTR-F
TTCCCCAATGCTTGTAGGAG
OgreCL11
CL11-LTR-R
ATCGACTCGAGGTTCTTTCG
OgreCL11
CL2-RACE-F1
TCGACCACGTAAGCTCGGATCCTTC
Ogre CL5
CL2-RACE-R1n
GGCAGAAACGGAGTTCAGGGACAGA
Ogre CL5
CL2-RACE-R1
CCCGAAGTCTGTGGAGAGGCTCGTA
Ogre CL5
CL2-RACE-F2
TCGAGCCTTCAAAGCCCTGAAACTG
Ogre CL5
CL2-RACE-R2
TGCGGGAGGTGCAGAAATGAGGTAT
Ogre CL5
CL2-RACE-F3
GATCGGCCTGAGAACAGCAGCAAAT
Ogre CL5
CL2-RACE-R3
CCCATGGAGATAGACGCCGCTACTG
Ogre CL5
CL2-RACE-F4
ATGCGCACGGACCTAGTGGGATCTA
Ogre CL5
CL2-RACE-R4
GCAGGTCGGTTCTGACGGATTTTTG
Ogre CL5
CL5-RACE-F1
cgtgttcattggcatccacgagagt
Ogre CL5
d. Primers for RACE
PCR product
length
1383-1434
757
831
CL5-RACE-R1
cccgaagtctgtggagaggctcgta
Ogre CL5
CL2_RACE-F5
ACGGCTTGCAATTACGGCTTCACAG
Ogre CL5
CL2_RACE-F6
TTAGGGCCCCACACCCTAGCACAAT
Ogre CL5
CL2_RACE-F7
TCCCTCCAGACCTGAACCAGAGGAC
Ogre CL5
CL2_RACE-R5
AAAGGAAAGCATCGACGGGAAGGAG
Ogre CL5
CL2_RACE-R6
CCTTTTGTCGCTGAGGTCCTTCGAC
Ogre CL5
CL2_RACE-F8
ACTTTCGCCTTGTCCAAGCCTCAGTC
Ogre CL5
CL2_RACE-F9
CCTCTTCCCAGGTCCTTTTCTGCGTA
Ogre CL5
CL2_RACE-F10
CGAGGGCACTTTCGTTACATTCGAGTC
Ogre CL5
CL2_RACE-F11
GTGTTCATTGGCATCCACGAGAGTCA
Ogre CL5
CL2_RACE-R7
TGACTCTCGTGGATGCCAATGAACAC
Ogre CL5
CL2_RACE-R8
GCTCAGTTCTGGCTGTGAAGCCGTAAT
Ogre CL5
CL2_RACE-R9
TGCAAACGAGGCTGGTACTCAGAAGG
Ogre CL5
CL5-277C14R3
GCGGACACTCGCGTGAGAAATATGA
Ogre CL5
CL2_RACE-R8
GCTCAGTTCTGGCTGTGAAGCCGTAAT
Ogre CL5
CL5-267M19R2
GTGAAGCCGGAATTGCGTGTTGTT
Ogre CL5
CL5-277C14R1
TGGAACCGTTCGAATACCTCGTGTC
Ogre CL5
CL5-267M19R1
GGCGCTTGGGAGGAAAGAGAAACAA
Ogre CL5
CL6-24I12R3
AGGTCGTAAACACGCGTCGGATTGT
Ogre CL6
CL6-24I12-R-R2
GGGGCTTCTGCCTCAGACCAAAAAT
Ogre CL6
CL6-24I12R2
GTAAGGGGCCTCCCTCTGGTTTTGA
Ogre CL6
CL6-24I12-R-R1
GATCGGTCGGTTTTGTCTCGGTAAGG
Ogre CL6
CL6-24I12R1
GGTGCTTTCGACCGGATCGTTTTAG
Ogre CL6
CL6-24I12-R-R1
GATCGGTCGGTTTTGTCTCGGTAAGG
Ogre CL6
CL6-24I12-R-R2
GGGGCTTCTGCCTCAGACCAAAAAT
Ogre CL6
CL6-24I12-R-F1
GTCGGTCACTCGCCACAACCAAATA
Ogre CL6
CL6-24I12-R-F2
GCACCTCAAGCAGGCTCCTCATTTC
Ogre CL6
CL11-RACE-F1
ttacgacccaagacggtgtcaacga
Ogre CL11
CL11-RACE-R1
acacggccattcggcctagaaaaac
Ogre CL11
CL11-93L7c-R-R1
ACACCAGGCATAGTCGACTCGAGGTTC
Ogre CL11
e. Primers for qRT-PCR
CL11-93L7c-R-R2
GCTCCTACAAGCATTGGGGAACAACC
Ogre CL11
CL11-93L7c-R-R3
ATCGTTGACACCGTCTTGGGTCGTAA
Ogre CL11
CL11-93L7c-R-F1
TTACGACCCAAGACGGTGTCAACGAT
Ogre CL11
CL11-93L7c-R-F2
TGTTCCCCAATGCTTGTAGGAGCGTA
Ogre CL11
2F13Int-F
TCTTCCAATCGGCCTCCGGG
Ogre CL5
2F13Int-R
GCCCACCGGGGCTACTCCTT
Ogre CL5
1F3Int-F
TCGACGGGTCCATTCCGCCT
Ogre CL6
1F3Int-R
ACACCGACAGGAGCCACCCC
Ogre CL6
F1Int-F
GCAGCCAGTCAGCTTCAGGGA
Ogre CL11
F1Int-R
ACAGCCACTGGAGCAACTCCG
Ogre CL11
CL5-RT-F
CL5-RT-R
CL6-RT-F
CL6-RT-R
CL11-RT-F
CL11-RT-R
ActinS-F1
ACCGCCATGGGTGCTATGCT
GCCCACACAAGAGCGAGGCA
GCGCCTCCGATCACACCGA
TCCCCGGTTGAGGTGGCA
CCCCTCCCGTGCTCAGCC
GCGCCAGCATTGCCCCC
caggccgttctctccttgta
Ogre CL5
Ogre CL5
Ogre CL6
Ogre CL6
Ogre CL11
Ogre CL11
ActinS-R1
tccaccactgagcacacaat
Sl-EF1-F
GCGATCAGGTAAGGAGCTTG
Sl-EF1-R
TGCAGAGAAGGTCTCGACAA
Sl-TUB-F
CCTGAATGTGGATGTGAACG
Sl-TUB-R
GCTGCTCATGGTAAGCCTTC
121
128
123
134
105
90
348/203*
109
116
Table S2. Copy numbers of TEs in the Silene latifolia genome (1C) estimated by two methods – BAC library hybridization and in silico
read mapping. (*) Copy numbers based on reads mapped onto LTRs of Ogre CL5 within BAC clones 267M19 and 277C14 respectively.
Ogre CL5
Ogre CL6
Ogre CL11
Retand-1
Retand-2
Athila CL3
Athila CL10
BAC library hybridization
4990
4223
2879
2100
2700
Number of elements per genome
Illumina Female Illumina Male
3427/1722*
2898/1508*
3123
2925
2012
2017
1128
1019
764
687
7145
6765
1413
1425
454 Female
3053/4020*
3440
1311
454 Male
2829/3690*
3157
1435
Table S3. List of sequences used in the phylogenetic analyses of Ogre elements in Silene including their accession numbers.
species
gene
accession number
Silene latifolia
spermidine synthase X
AY705437
Silene vulgaris
spermidine synthase
AY705436
Silene latifolia
peptidyl-prolyl cis-trans isomerase (CypY)
EF408658
Silene latifolia
peptidyl-prolyl cis-trans isomerase (CypX)
EF408657
Silene vulgaris
peptidyl-prolyl cis-trans isomerase (Cyp)
JN394123
Silene dioica
peptidyl-prolyl cis-trans isomerase (CypX)
EU561052
Silene dioica
peptidyl-prolyl cis-trans isomerase (CypY)
EU561048
Silene noctiflora
peptidyl-prolyl cis-trans isomerase (Cyp)
EU561050
Silene diclinis
peptidyl-prolyl cis-trans isomerase (CypX)
EU561051
Silene diclinis
peptidyl-prolyl cis-trans isomerase (CypY)
EU561047
Silene conica
peptidyl-prolyl cis-trans isomerase (Cyp)
EU561054
predicted protein (peptidyl-prolyl cis-trans
Populus trichocarpa
isomerase)
XM_002312866
Silene latifolia
oligomycin sensitivity conferring protein (DD44Y)
AF543834
Silene latifolia
oligomycin sensitivity conferring protein (DD44X)
AF543833
Silene dioica
oligomycin sensitivity conferring protein (DD44X)
AY722065
Silene dioica
oligomycin sensitivity conferring protein (DD44Y)
AY720883
Silene diclinis
oligomycin sensitivity conferring protein (DD44X)
AY722091
Silene diclinis
oligomycin sensitivity conferring protein (DD44Y)
AY720879
Silene heuffelii
oligomycin sensitivity conferring protein (DD44X)
AY722078
Silene heuffelii
oligomycin sensitivity conferring protein (DD44Y)
AY720885
Silene vulgaris
oligomycin sensitivity conferring protein (DD44)
AY725028
Silene conica
oligomycin sensitivity conferring protein (DD44)
EU521734
Silene conica
fructose-2,6-bisphosphatase (XY4)
EU521734
Silene vulgaris
fructose-2,6-bisphosphatase (XY4)
AY084041
Silene diclinis
fructose-2,6-bisphosphatase (X4)
AJ632101
Silene diclinis
fructose-2,6-bisphosphatase (Y4)
AJ632102
Silene latifolia
fructose-2,6-bisphosphatase (X4)
AJ310660
Silene latifolia
fructose-2,6-bisphosphatase (Y4)
AY084039
Silene dioica
fructose-2,6-bisphosphatase (X4)
AY084046
Silene dioica
fructose-2,6-bisphosphatase (Y4)
AY084047
Silene viscosa
fructose-2,6-bisphosphatase (XY4)
AJ697610
Silene noctiflora
fructose-2,6-bisphosphatase (XY4)
EF674313
Vitis vinifera
V. vinifera orthologue of Silene Ogre
AM442918.2
Gossypium hirsutum
G. hirsutum orthologue of Silene Ogre
AC243134.1
Pisum sativum
P. sativum orthologue of Silene Ogre
AY299397.1
Medicago truncatula
M. truncatula orthologue of Silene Ogre
AC145061.27
Solanum lycopersicum
S. lycopersicum orthologue of Silene Ogre
AC240856.4
Populus trichocarpa
P. trichocarpa orthologue of Silene Ogre
AC210333.1
Table S4. P-values of Mann-Whitney test in Silene latifolia. The upper right part of the table shows results concerning synonymous
substitutions in terminal branches, the lower left part shows results concerning ages of terminal branches.
CL5
CL6
CL11
P-values of Mann-Whitney test
CL5
CL6
CL11
1
0.00808
0.96739
0.00003
0.00069 0
Table S5. Percentages of synonymous substitution per codon and time of X-Y chromosome split into four sex-linked Silene latifolia
genes. The time of split is in millions of years. Standard errors (SE) and 95% highest posterior density (95% HPD) are also given. *The time of
X-Y split and 95% HPD in the SlX1/SlY1 gene pair were taken from Rautenberg et al., 2008.
gene
X1/Y1*
CypX/CypY
DD44X/DD44Y
X4/Y4
ds S. latifolia X-Y ± SE X-Y split (95% HPD)
4.0% ± 1.1%
2.0 (0.9 - 3.3)
5.9% ± 1.3%
3.3 (1.2 - 5.3)
9.2% ± 2.1%
3.6 (2.1 - 5.2)
18.1% ± 2.3%
6.0 (2.6 - 10.5)
Table S6. Percentages of disturbed open reading frames (ORF) in each group of retrotransposons in Silene latifolia. The number of Ogre
BAC clones in each group is written in parentheses. 95% confidence intervals (CI) show that the percentages of disturbed Ogre CL11 clones is
significantly lower than the percentage of the disturbed Ogre CL5 or Ogre CL6 clones.
CL5
CL6
CL11
disturbed ORF
percentage
CI
84% (26)
67% - 93%
100 % (8)
68% - 100%
33% (3)
12% - 65%
stop codon(s)
61% (19)
100 % (8)
33% (3)
reasons
frameshift(s)
68% (21)
75% (6)
11% (1)
both
45% (14)
75% (6)
11% (1)
Table S7. Branch analysis of terminal and internal branches in Silene latifolia genome.
one ratio model
branch analysis of terminal branches
background ω foreground ω
lnL
0.11446
N/A
-52094.4791
result of the test
N/A
two ratios: background vs. CL5
0.08755
0.1974
-52007.9757
ωCL5 > ωbackground; P < 10⁻¹⁶
two ratios: background vs. CL6
0.10295
0.18526
-52060.2137
ωCL6 > ωbackground; P < 10⁻¹⁶
two ratios: background vs. CL11
0.11293
0.14011
-52092.8108
ωCL11 = ωbackground
branch analysis of internal branches
background ω foreground ω
lnL
result of the test
two ratios: background vs. CL5
0.12555
0.00197
-51985.2576
ωCL5 < ωbackground; P < 10⁻¹⁶
two ratios: background vs. CL6
0.12524
0.00634
-52004.4272
ωCL6 < ωbackground; P < 10⁻¹⁶
two ratios: background vs. CL11
0.12083
0.0045
-52040.0925
ωCL11 < ωbackground; P < 10⁻¹⁶
Table S8. Abundance of 19-24 nucleotide small RNAs (sRNAs) complementary to LTRs in sense (+) and antisense (-) orientation in
Silene latifolia. Counts of sRNA reads were normalized to size of the respective library, LTR length and copy number of respective element.
Male Leaves
Female Leaves
Unfertilized Pistils
Fertilized Pistils
Pollen
CL11 CL5 CL6 Retand CL11 CL5 CL6 Retand CL11 CL5 CL6 Retand CL11 CL5 CL6 Retand CL11 CL5 CL6 Retand
24+
2423+
2322+
2221+
2120+
2019+
19-
275 442
429 1558
16
66
34
84
26
87
84
93
24
59
41
43
2
32
6
15
4
11
0
32
570
354
32
37
78
71
46
29
15
15
4
14
1789
1894
96
220
240
223
87
209
22
69
13
80
102
187
4
8
2
22
2
6
0
4
0
4
270
917
14
52
46
35
29
26
4
10
3
17
255
206
8
14
16
12
11
7
7
7
0
4
1121
1273
64
120
177
127
61
114
23
50
3
41
662 3565
1032 5158
82 404
119 459
39 395
51 289
28 258
49 191
12 125
6
98
8
51
20
68
741
360
58
61
32
33
15
15
4
11
7
8
2939
3795
342
333
436
399
213
275
64
106
50
86
240 3481 1100
499 4392 412
59 514
86
102 449
79
28 393
40
43 315
54
18 313
12
24 214
30
4 144
8
14
68
7
4
73
9
4
58
4
4301
6360
579
527
674
735
350
680
115
145
51
130
337 948
621 1197
39 189
168 144
86 380
201 187
76 160
203 111
12
48
43
40
8
28
12
47
294
183
32
28
78
78
40
29
36
7
11
4
588
635
78
108
386
307
144
229
30
72
19
177
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