Fig 1.
Cross section of Erycina pusilla fruit 8 weeks after pollination reflecting the split carpel model according to Rasmussen and Johansen (2006).
Abbreviations: F1 = fertile valve originating from labellum; F2 = fertile valve originating from lateral petal 1; F2 = fertile valve originating from lateral petal 2; S1 = sterile valve originating from lateral sepal 1; S2 = sterile valve originating from dorsal sepal; S3 = sterile valve originating from lateral sepal 2; Ca = carpel; DVB = dorsal vascular bundle; LVD = lateral vascular bundle.
Fig 2.
Ancestral state reconstruction of fruit ripening period (A), fruit orientation (B), fruit dehiscence type (C), number of slits in mature fruit (D), fusion of valves (E), and lignification of dehiscence zone (F) characters from stochastic mapping analysis based on joint sampling of nrITS, matK and rbcL (10,000 mapped trees). Posterior probabilities (pie charts) are mapped in a random stochastic character map. Note: * nodes have bootstrap values < 70%. Illustrations of fruits: (A-B) Character 1: Erycina pusilla (ripe in ≤4 months), Phalaenopsis equestris (ripe in >4 months); character 2: Habenaria eustachys (erect), Papilionanthe teres (pendant). (C-D) Character 3: Vanilla pompona (indehiscent), Arundina graminifolia (dehiscent); character 4: Lockhartia acuta (<3 slits), Guarianthe skinneri (≥3 slits). (E-F) Character 5: A. graminifolia (valves fused at the base and apex), Ornithocephalus valerioi (valves fused at the base only); character 6: E. pusilla (non-lignified dehiscence zones), Cynorkis fastigiata (lignified dehiscence zones).
Fig 3.
Heatmap of correlations between each character analyzed for the ancestral state analyses.
Criteria: Bayesfactors > 10 very strong correlation, 5–10 strong correlation, >2 positive correlation, and <2 weak correlation.
Fig 4.
Ancestral state reconstructions of (A) character 2 (fruit orientation) vs 1 (ripening period), (B) character 4 (number of slits in mature fruit) vs 3 (dehiscence type, (C) character 5 (fusion of valves) vs character 2 (fruit orientation), (D) character 6 (lignified dehiscence zone) vs character 2 (fruit orientation), (E) character 4 (number of slits in mature fruit) vs character 3 (dehiscence type), and (F) character 5 (fusion of valves) vs character 6 (lignified dehiscence zone) from stochastic mapping analyses based on a joint sampling of nrITS, matK and rbcL (10000 map trees). The numbers represent the estimated number of evolutionary changes and the time spent in each state, arrows represent the direction of the transition between the states, and character combinations indicated with a square are interpreted as having co-evolved.
Fig 5.
Venn diagrams of identified genes (cutoff TPM ≥ 5) in 1 WAP and 3 WAP fruits of Erycina pusilla.
Fig 6.
Changes in gene expression profile of 1 WAP and 3 WAP fruits of Erycina pusilla.
Upregulated genes are shown in red, while the number of downregulated genes is shown in blue.
Fig 7.
Relative expression of MADS-box (A-D), fruit development and ovule, and carpel development (E-I), and lipid biosynthesis (J-M) genes from qPCR analysis in one week after pollination (1 WAP) and three weeks after pollination (3 WAP) fruit valves of E. pusilla. The x-axis shows the two samples of 1 WAP and 3 WAP. The y-axis represents the relative expression (in log2) of genes as the mean of three biological replicates’ expression and normalized relative to floral bud tissues. The asterisks denote statistically significant differences in the relative expression of the various developmental stages (*p≤ 0.05).
Fig 8.
Summary of expression patterns assessed by RNA-seq and qPCR of genes involved in Erycina pusilla fruit development.
Expression of EpRPL gene is higher (indicated in the purple triangle) in 1 WAP fruits but lower in 3 WAP fruits. Expression of EpBs, EpKCS6, EpGL1, EpPDH-E1, and EpMAH1 genes is higher (indicated in the yellow triangle) in 3 WAP fruits (right side) but lower in the 1 WAP fruits. Expression of EpAG2, EpAGL6-2, EpSEP3, EpKOR1, EpSLR1, EpCRC/DL1, and EpTPL genes is high (indicated by the white box) in the 1 WAP and 3 WAP fruits.