Conservation of Arabidopsis MIRNA Families in Plants.
The cladogram (not drawn to scale) represents the plant tree of life with major phylogenetic groups noted. Each box is labeled with the inferred number of Arabidopsis MIRNA families conserved between all available taxa within the box, based on the representative species listed. The numbers of MIRNA families in A. thaliana and A. lyrata are listed at the bottom left.

Locus Conservation around Several Classes of MIRNA Genes.
All panels show 40-kb regions flanking MIRNA genes. Each illustration shows the A. thaliana genome in the top half and the A. lyrata genome in the bottom half. Small RNA abundance at each nucleotide position for each strand (Watson strand reads are plotted up and Crick strand reads are plotted down) is shown in the top and bottom histograms. Small RNA reads are color coded by length (turquoise = 20 nucleotides, blue = 21 nucleotides, green = 22 nucleotides, fuchsia = 23 nucleotides, and red = 24 nucleotides). Genes and exons are drawn as yellow chevrons and blue boxes, respectively. Transposable elements are drawn as green boxes. Nucleotide conservation is illustrated for both genomes with orthologous positions linked. Conservation (percentage) was calculated using the scrolling window method (100-nucleotide windows, 1-nucleotide scroll) and plotted as a histogram with black bars representing positions that overlapped exons in A. thaliana and gray bars representing all other sites. Arrows in each panel mark the positions of the MIRNA gene indicated in the heading.
(A) Conserved, orthologous MIRNA. The deeply conserved MIR171a (left) and Arabidopsis-specific MIR822 (right) are shown.
(B) Orthologous MIRNA genes with diverged mature miRNA sequences.
(C) Unique MIRNA. MIR843 and MIR3439 are specific to A. thaliana and A. lyrata, respectively.
(D) Conserved families with species-specific, duplicated (paralogous) MIRNA genes. MIR395e and MIR395f are duplicated in A. lyrata (left). MIR399a has three additional copies in A. lyrata (right).

Sequence Divergence within Foldbacks of Orthologous A. thaliana and A. lyrata MIRNA.
(A) Arabidopsis MIRNA foldbacks were divided into five regions. Note that the miRNA sequence can occur on either the 5′ or 3′ arm.
(B) Sequence divergence of Arabidopsis MIRNA. Divergence was measured by adding nucleotide substitutions to insertion/deletion events for each A. lyrata/A. thaliana foldback alignment and dividing the sum by the regional sequence length. Arabidopsis MIRNA families were grouped into conservation groups based on whether or not the MIRNA family was conserved in P. trichocarpa. Listed P values are from pairwise permutation tests. Standard error bars are shown.

The Genomic Context of A. thaliana and A. lyrata MIRNA.
(A) The number of unique positions within 20,000 nucleotides upstream and downstream of MIRNA genes in A. thaliana and A. lyrata for which orthologous loci were identified. Scatterplots compare unique nucleotides in A. thaliana to A. lyrata for MIRNA families conserved to P. trichocarpa (black dots, Group 1), conserved only to A. thaliana and A. lyrata (red dots, Group 2), or MIRNA families that are unique to A. thaliana or A. lyrata (green dots, Group 3). The top left plot shows all three conservation groups.
(B) Notched boxplots of unique positions shown in (A). Notches mark the 95% confidence interval of the median for each group.

Orthology of MIRNA Genes in A. thaliana, A. lyrata, and C. rubella.
(A) Ortholog conservation matrix between A. thaliana (A.t.), A. lyrata (A.l.), and C. rubella (C.r.). MIRNA genes with orthologs are shown by a dark-blue box or as a light-blue box in the cases where the mature miRNA sequence has diverged in sequence. Lack of an ortholog is indicated as a white box. MIRNA genes found in only one species are shown with a gray box. C. rubella MIRNA that could not be confidently identified as present or absent are shown with a red slash. Genes with underlined names are from families conserved in P. trichocarpa. Note that the A. lyrata ortholog of ath-MIR775 is named aly-MIR3433.
(B) Conservation scenarios. Species vertices are labeled in the first diagram as in (A). Orthologous MIRNA genes are depicted as dots connected by solid lines. Dots connected by undulating lines indicate a mature miRNA that has diverged in sequence. Absent or missing orthologs are depicted as “X.” Scenarios under a question mark indicate that insufficient C. rubella data are available. The number of MIRNA observed for each scenario is listed below each diagram.
(C) Summary of MIRNA families and genes in A. lyrata and A. thaliana. The number of MIRNA families or genes conserved between (overlap region), or unique to, A. thaliana (gray region) and A. lyrata (blue region) are shown (Venn diagrams, top). The black region is the number of diverged orthologs (Venn diagrams, bottom). The numbers of multi- and single-gene MIRNA families are shown for both species (bar chart).

Identification of Intragenomic Loci with Extended Similarity to MIRNA Genes.
(A) Detection of MIRNA-related loci in the A. thaliana and A. lyrata genomes. The expected values for the top four FASTA alignments between each MIRNA foldback and the respective genome sequence are plotted. A. thaliana MIRNAs aligned to the A. thaliana genome are plotted as red dots. A. lyrata MIRNAs aligned to the A. lyrata genome are plotted as black dots. MIRNA are grouped on the x axis based on conservation. The horizontal gray line marks the false discovery rate (FDR) = 0.05 boundary, where points above the line have a FDR < 0.05.
(B) Proportion of A. thaliana and A. lyrata MIRNA families conserved with both Arabidopsis species and P. trichocarpa, conserved between A. thaliana and A. lyrata but not with P. trichocarpa, or MIRNAs unique to either A. thaliana or A. lyrata (inner pie chart). The percentage of MIRNA families with evidence of foldback origination from another locus (see [A]) in each conservation group is shown in the outer ring.
(C) MIRNA formation by inverted duplication. A generic model (top) and three example MIRNA loci that were formed by inverted duplication events.
(D) MIRNA formation by direct duplication of a locus containing a previous intralocus inverted duplication. A generic model (top) and three examples from A. lyrata are shown. In (C) and (D), the MIRNA-related genomic locus is on top and the MIRNA genomic locus is on the bottom of each diagram. Direct and inverted duplications are shown by gray and blue shadings, respectively, connecting the MIRNA-related locus to the MIRNA locus. Annotated features are shown as highlighted boxes in each region (genes = yellow, exons = blue, transposable elements = green, and MIRNA = red boxes). Arrows indicate the duplicated segments in each locus. In (D), sequences produced by inverted intralocus duplication in the MIRNA-related loci are connected by black boxes.
(E) Proportion of A. thaliana and A. lyrata MIRNA families that have detectable MIRNA-related loci in their respective genomes (inner pie chart). The proportion of MIRNA-related loci in each class (inverted or direct duplication) forming transcripts that are predicted or validated targets of the miRNA formed from the duplication event (outer ring).
(F) Proportion of MIRNA-related loci that are exons, introns, intergenic sequences, or MITEs (inner pie chart). Proportion of MIRNA-related loci forming transcripts that are predicted or validated targets of the miRNA formed from the duplication event (outer ring).

Gene and Repeat Density in the A. thaliana and A. lyrata Genomes.
In (A) to (C), annotated genes in A. thaliana and A. lyrata were placed in two-dimensional bins based on the length of their 5′ and 3′ intergenic lengths.
(A) Gene density. The number of genes per bin is color coded.
(B) Repeat density. The percentage of total nucleotides in the 5′ and 3′ intergenic regions of the genes in (A) occupied by transposable elements and repeat sequences is color coded.
(C) MIRNA loci. Bins that contain MIRNA that have orthologs in A. thaliana and A. lyrata are colored black. Bins that contain unique A. thaliana or A. lyrata MIRNA are colored red.
(D) Percentage of positions occupied by transposable elements in 20,000-nucleotide segments upstream and downstream of A. thaliana and A. lyrata MIRNA. Percentages were calculated in scrolling windows (100-nucleotide windows, 20-nucleotide scroll). MIRNA foldbacks are plotted on a relative scale.

Conservation of A. thaliana miRNA-Target Pairs in A. lyrata.
(A) Conserved and nonconserved A. thaliana miRNA-target pairs in A. lyrata (inner pie chart). Numbers in parentheses indicate pair counts for each category. The outer ring shows criteria for inclusion into a conservation category.
(B) Conservation or degradation of A. thaliana miRNA-target pairs. Pairs from (A) were binned into those present in P. trichocarpa, A. lyrata, and A. thaliana (Group 1), those present in A. lyrata and A. thaliana but not P. trichocarpa (Group 2), or those unique to A. thaliana (Group 3). The portion of conserved targeting, degraded targeting, and nonconserved targeting within each group is plotted. Note that degraded targeting can occur by substitutions in either the miRNA or target sequence.