Sampling geographical distribution.

<p>Location of <i>I. triloba</i>, <i>I. trifida</i>, <i>I. batatas</i> and polyploid <i>Ipomoea</i> sp. accessions used in the present study and current taxon distribution ranges, as determined from GBIF records (<a href="http://data.gbif.org/species/" target="_blank">http://data.gbif.org/species/</a>) are provided. Accessions with no geographical information are not shown; details on sampling are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062707#pone.0062707.s005" target="_blank">Tables S1</a>.</p

Two possible scenarios about the origins of <i>Ipomoea batatas.</i>

<p>a) Scenario A which represents according to us, the most parsimonious scenario explaining the clear-cut phylogeographical pattern inferred from both nuclear and chloroplast data: 1) Multiple independent events of autopolypoidy within several polymorphic and pre-differentiated wild populations (phylogeographical differentiation), and then 2) multi-local domestication within each polyploid population, followed by 3) gene flow between the two cultivated genepools and between cultivated and wild forms. b) Scenario B: 1) Hybridization between differentiated conspecific wild populations (in contact because of potential climate-induced or human-induced range shift) and polyploidization, followed by 2) the domestication of these polyploids forms and then 3) patterns of post-domestication human expansion may have been responsible for the clear-cut phylogeographical pattern found within cultivated <i>I. batatas</i> in tropical America. Finally, 4) Gene flow between the two cultivated genepools and between cultivated and wild forms may also have occurred.</p

Contingency table comparing cpDNA haplotype “lineages” with DAPC clusters among the different taxa <i>I. batatas</i>, <i>I. trifida</i>, <i>I. triloba</i>, <i>Ipomoea</i> sp. (including <i>I. tabascana</i>).

<p><i>I. trifida</i> haplotypes correspond to hap2, 5, 9, 10, 14, 15, 16, 17 and <i>I. triloba</i> haplotypes to hap3, 8 and 18.</p

Taxa boundaries as accessed with DAPC analysis for nuclear SSR data.

<p>Diagram representing the proportion of membership probabilities in nuclear five clusters (K1, K2, K3, K4 and K5) as determined by the DAPC analysis. Each individual is represented as a vertical bar, with colours corresponding to membership probabilities to the five clusters.</p

Genetic diversity of the four geographically well-sampled taxa as revealed by nuclear SSRs.

<p>Values for the number of alleles (<i>NA</i>), its rarefied value over 25 individuals (1000 resamplings; <i>Ar</i>) and the observed and expected heterozygosities <i>Ho</i> and <i>He</i>, are provided both per locus and as mean values averaged over all loci. <i>D</i> corresponds to the intra-taxon mean Lynch distance between genotypes.</p

Genetic relationships of <i>I. batatas</i>, five wild relatives and <i>Ipomoea</i> sp. accessions based on chloroplast DNA analyses.

<p>Statistical Parsimony Network of rpl32-trnL(UAG) haplotypes. Circle size is proportional to the number of individuals per haplotype. Substitutions and inversions are represented using full lines and indels are displayed using broken lines. Intermediate, unsampled haplotypes appear as dots. The posterior probability of two nodes, as obtained through a Bayesian tree reconstruction (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062707#pone.0062707.s001" target="_blank">Figure S1</a>), is reported in italics. The ploidy level of <i>Ipomoea</i> sp. accessions is indicated.</p

<i>Igf2</i> Pm mRNA levels in mouse tissues and <i>Igf2</i> promoter usage in transfected <i>H19</i>KO myoblasts.

<p>(A) Pm <i>Igf2</i> mRNA levels relative to total <i>Igf2</i> mRNA level (100%) in different mouse tissues. Total <i>Igf2</i> mRNA levels were determined by RT-qPCR using a PCR primer pair (Igf2exon6 PCR amplicon) located in the exon 6 common to all <i>Igf2</i> transcripts. (B) <i>Igf2</i> promoter usage in untransfected/transfected <i>H19</i> KO myoblasts. Relative mRNA levels (%) are calculated relative to the P3 <i>Igf2</i> transcript level in the control cells (+/−) (100%).</p

Comparison between the endogenous <i>Igf2</i> transcription levels and the steady state levels of the ectopic RNAs.

<p>In these graphs, we compared, for each transfected <i>H19</i> KO clones, <i>Igf2</i> transcription data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037923#pone-0037923-g005" target="_blank">Figure 5B</a> with the steady state <i>91H</i> and <i>H19</i> ectopic RNA levels shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037923#pone-0037923-g004" target="_blank">Figure 4B</a>. (A) <i>Igf2</i> transcription <i>versus 91H</i> RNA levels. (B) <i>Igf2</i> transcription <i>versus H19</i> RNA levels. In untransfected H19 KO myoblasts (−/−), both <i>91H</i> and <i>H19</i> are not expressed (RNA levels  = 0) and <i>Igf2</i> transcription level is below the “empty plasmid” background (see Fig. 5A) which is inferior to 0.11. (C) <i>Igf2</i> transcription level <i>versus</i> the ratio of <i>91H/H19</i> RNA levels. Clones expressing large amount of ectopic <i>H19</i> RNA (clones 4, 11 and 12ND, black diamonds) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037923#pone-0037923-g004" target="_blank">Figure 4B</a>) were analysed separately from the others (ratio of <i>91H</i>/<i>H19</i> RNA levels >0.2; open diamonds). In clones expressing high <i>H19</i> RNA levels (black diamonds), the ectopic <i>H19</i> RNA level relative to the <i>91H</i> RNA level (which leads to a decrease of the <i>91H/H19</i> RNA ratio) is inversely proportional to <i>Igf2</i> transcription levels (R² = 0.8173).</p

Map of the mouse <i>H19/91H</i> region showing the PCR amplicons used in RT-qPCR experiments.

<p>The region corresponding to the sequence removed by the H19^Δ3 deletion in the <i>H19</i> KO myoblasts (see below) is indicated in blue. The <i>H19/91H</i> insert transfected into the <i>H19</i> KO myoblast cells is also shown in the figure (green lane). The insert is a 16 kb BamHI-BamHI fragment encompassing the <i>H19</i> endodermic enhancers and the whole <i>H19</i> gene. PCR amplicons used to quantify the ectopic RNAs are indicated (<i>H19</i> RNA, mI1-mI3, mJ and mC’). DNA methylation is indicated by black lollipops and RNAs are depicted in red. Positions of restriction sites and PCR amplicons used for real-time PCRs are indicated relative to the <i>H19</i> transcription start site. The mA and mB PCR amplicons have been used in a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037923#pone.0037923-Berteaux1" target="_blank">[34]</a> and are indicated here solely for clarity of our PCR nomenclature. For primer sequences see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037923#pone.0037923.s008" target="_blank">Table S1</a>.</p

Nuclear Run-on experiments.

<p>(A) Autoradiographies of nuclear Run-on experiments on transfected and untransfected <i>H19</i> KO myoblasts. Nuclear Run-on experiments were performed as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037923#pone.0037923-Milligan1" target="_blank">[38]</a> and the α³²P UTP labelled transcripts were hybridized on filters to denatured plasmids containing the insert DNA of genes indicated on the figure. <i>91H</i>/<i>H19</i> transcription was assayed using an insert corresponding to the <i>H19</i> sequence and <i>Igf2</i> with a genomic 2.4 kb BamHI-BamHI DNA fragment encompassing the exon 4-exon 6 region. Such nuclear run-on experiments were performed on undifferentiated (ND) and differentiated (D) cells either on the whole hygromycin-resistant transfected <i>H19</i> KO myoblast cell population (“Whole”) and transfected clones. (B) The same filters as those used for the autoradiographies shown in A were used for PhosphorImager quantifications. The ectopic <i>91H/H19</i> transcription levels (open bars) were compared to the endogenous <i>Igf2</i> transcription levels (black bars). For each hybridized filter, the relative transcription levels were determined for each gene by normalizing to the <i>Gapdh</i> transcription level.</p