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Supplementary data of article "Domestication has altered gene expression and secondary metabolites in pea seed coat".

Table S1. Excel- GO_terms_MF_selected_WGCNA_modules. Table S2. Excel- GO_terms_MF_DEGs_UP_and_DOWN. Table S3. Excel- GO_terms_MF_DEGs_summary. Table S4. Excel- List of DEGs involved in flavonoid pathway found in WILD gene set. Table S5. Protein recoveries calculated for individual pea protein samples. Numbers 1, 2, 3 denote treatment groups corresponding to seed developmental stages (D1, D2 and mature seeds, respectively). Letters a–d denote biological replicates within the treatment groups. Table S6. Excel- Annotation of proteins differentially expressed in wild and domesticated pea seed coat samples. Table S7. Primary metabolites identified by spectral similarity library search and/or co-elution with authentic standards in pea seed coats aqua methanolic extracts. Metabolite analysis relied on GC-EI-Q-MS analysis after derivatization of the lyophilized extracts with methoxamine hydrochloride (MOA) and N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). Table S8. Primary metabolites detected in the aq. methanolic extracts of mature Cameor seed coats demonstrating statistically significant up- and down-regulation in comparison to those of wild JI261. Table S9. Primary metabolites of mature JI92 seed coats demonstrating statistically significant up- and down-regulation in comparison with those of wild JI261. Table S10. Primary metabolites of mature JI1794 seed coats demonstrating statistically significant up- and down-regulation in comparison with those of JI261. Table S11. Primary metabolites of mature JI64 seed coats demonstrating statistically significant up- and down-regulation in comparison with those of JI261. Table S12. Mass analyzer settings applied for QqTOF-MS experiments in analysis of seed coat (cell wall) hydrolyzates and reference authentic standards. Table S13. Cell wall-bound metabolites extracted from the seed coats of wild (JI64, JI1794, JI261) and domesticated (Cameor, JI92) peas upon alkali hydrolysis of corresponding isolated and purified cell wall material. Table S14. Excel- Coordinates of markers in S-plot obtained from OPLS-DA analysis (FIA-ESI-HRTMS, negative ionization, lock mass uncorrected). Table S15. List of identified significantly differential metabolites rising during seed coat development. Table S16. List of identified significantly differential metabolites decreasing during seed coat development (positive ionization mode). Table S17. List of identified metabolites with significantly higher content in wild compared cultivated genotypes in older developmental stages (D5-6). Table S18. Excel- Expression of genes encoding enzymes of monolignol pathway in seed coats (SC) and embryos (E) of domesticated (Cameor, JI92 and Pisum abyssinicum PI358617) and wild (JI64, JI1794, JI261) peas over five seed developmental stages (13, 17, 20, 23, 28 DAP, labelled as 1-5). PAL: phenylalanine ammonia-lyase, C4H: cinnamate-4-hydroxylase, 4CL: 4-coumaroyl: CoA ligase, HCT: hydroxycinnamoyl CoA:shikimate hydroxycinnamoyltransferase, COMT: caffeic acid O-methyltransferase, CSE: caffeoyl shikimate esterase, CAD: cinnamyl alcohol dehydrogenase, CCR: cinnamoyl CoA reductase, CCoAMT: caffeoyl CoA-3-methyltransferase, F5H: ferulate-5-hydroxylase Table S19. Studied metabolites of phenylpropanoid pathway. Table S20. Instrument settings used in the proteomics LIT-Orbitrap-MS and -MS/MS experiments. Table S21. Procedures and specific settings for data processing and post-processing of the proteomics data. Table S22. Gas chromatographic (GC) separation conditions and electron ionization-quadrupole-mass spectrometry (EI-Q-MS) settings for GC-EI-Q-MS analysis of the primary metabolites in pea seed coats. Table S23. Chromatographic conditions used for UHPLC separation of seed coat (cell wall) hydrolyzates and reference authentic standards. Table S24. Variable parameters of MS/cIMS/MS measurements. Figure S1. The dynamics of gene expression between studied developmental stages within all genotypes (a) or among genotypes in particular developmental stages (b). Figure S2. Twelve representative groups of transcription factors described within 20 gene modules of pea SC. Visualized by Cytoscape 3.9.0. Figure S3. SDS-PAGE electropherograms of the total protein fractions isolated from the seed coats of JI92 (a, c, e) and JI64 (b, d, f) seeds before and after tryptic hydrolysis. Numbers 1, 2, 3 denote seed developmental stages: DS1, DS2 and mature seeds, respectively. Letters a-d denote biological replicates. The aliquots (10 μg) of samples before hydrolysis (a, b), the incompletely digested aliquots left on filter unit after peptide elution (c, d) and aliquots of tryptic hydrolysates (corresponding to 5 μg of protein), (e, f) were loaded on gels. Inter-gel normalization relied on the total density of the Protein Ladder (PageRuler™ Prestained Protein Ladder #26616, 10–180 kDa) lane (St); the ND (non-digested) sample represents a reference protein not subjected to hydrolysis. Figure S4. The numbers of tryptic peptides (a), possible proteins (b), and non-redundant proteins (protein groups) (c) identified in domesticated JI92 seed coats at developmental stages D1, D2 and D6. The tryptic digests (n =&thinsp;3) obtained from seed coats were analyzed by nano-high performance liquid chromatography-electrospray ionization linear ion trap-orbital trap mass spectrometry (nanoHPLC-ESI-LIT-Orbitrap-MS) operated in positive DDA mode. Figure S5. The numbers of tryptic peptides (a), possible proteins (b), and non-redundant proteins (protein groups, c) identified in wild pea JI64 seed coats at D1, D2 and D6 stages. The tryptic digests (n =&thinsp;3), obtained from pea seedlings, were analyzed by nano-high performance liquid chromatography-electrospray ionization linear ion trap-orbital trap mass spectrometry (nanoHPLC-ESI-LIT-Orbitrap-MS) operated in positive DDA mode. Figure S6. Principal component analysis (PCA) with score plot representation (a) accomplished for seed coat proteins differentially expressed at developmental stages D1 and D2 and in the mature state (D6) and hierarchical clustering with a heatmap representation (b). Figure S7. Functional annotation (accomplished with the Mercator MapMan v3.6 tool) of the pea seed coat proteins isolated in stage D1. White and black columns denote the functional groups of the proteins, which were more expressed in the developing seeds of domesticated JI92 and wild JI64, respectively. Figure S8. Functional annotation (accomplished with the Mercator MapMan v3.6 tool) of the pea seed coat proteins isolated in stage D2. White and black columns denote the functional groups of the proteins, which were more expressed in the developing seeds of the domesticated JI92 and wild JI64, respectively. Figure S9. Prediction of sub-cellular localization of the proteins more expressed in the developing seeds of JI92 and JI64 with the BUSCA prediction tool. Figure S10. Evaluation of the differences in the metabolic profiles of the mature seeds obtained from the wild JI261 and domesticated Cameor by principal component analysis (PCA). Figure S11. Representation of the differences in the metabolic profiles of the mature seed coats obtained from the wild JI261 and domesticated Cameor by the t-test with Volcano plot representation (a) and the top 30 differentially abundant metabolites demonstrating the most pronounced differences of corresponding GC-MS signals associated with seed dormancy (b). Figure S12. Evaluation of the differences in the metabolic profiles of the mature seeds obtained from the wild JI261 and domesticated JI92 by principal component analysis (PCA) with score plot representation (a) and hierarchical clustering with heatmap representation (b). Figure S13. Principal component analysis (PCA) illustrating distribution of metabolic profiles of mature seed coats of two wild pea genotypes, JI1794 and JI261. Figure S14. Principal component analysis (PCA) demonstrates the distribution of mature seed coat metabolic profiles of two wild pea genotypes, JI64 and JI261(control). Figure S15. Evaluation of the differences in the patterns of the cell wall-bound metabolites obtained from mature seed coats of wild JI261 and domesticated Cameor: principal component analysis (PCA) with score plot representation (a), hierarchical clustering with heatmap representation (b) and t-test analysis with the Volcano-plot representation (c). Figure S16. Statistical analysis (t-test with Volcano plot representation) characterizing the differences between the levels of mature seed coat cell wall-bound metabolites of Cameor compared with those of wild JI261. Figure S17. Principal component analysis (PCA) illustrates the distribution of mature seed coat metabolic profiles of domesticated JI92 and wild JI261. Figure S18. Principal component analysis (PCA) shows the distribution of metabolic profiles of mature seed coats of two wild pea genotypes, JI1794 and JI261, control. Figure S19. Principal component analysis (PCA) demonstrates the distribution of mature seed coat metabolic profiles of two wild genotypes, JI64 and control JI261. Figure S20. Annotated cell wall-bound metabolites extracted from the seed coats of the dormant wild pea genotype JI261 and the seed coats from several pea genotypes varying in their dormancy (Cameor, JI92, JI64, and JI1794) upon alkali hydrolysis of corresponding isolated and purified cell wall material.  Figure S21. Ion mobility separation of m/z 299.0841. Figure S22. Ion mobility separation of m/z 701.1907.  Figure S23. Ion mobility separation of m/z 619.1041. Figure S24. Ion mobility separation of m/z 631.1017. Figure S25. Ion mobility separation of m/z 641.1139. Figure S26. Ion mobility separation of m/z 771.1346.  Figure S27. Reconstructed chromatograms of p-hydroxybenzoic and salicylic acids in DS5 of dormant JI64 and domesticated landraces JI92 (LC/HRTMS, negative ionization mode). Figure S28. Module-trait relationship depiction showing the correlation between expression of the gene modules and the abundance of identified metabolites of the monolignol pathway.

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Text-fig. 6. Scanning electron micrographs of seeds of ericalean affinity (a–d) and seeds of uncertain affinity (e–i) from Zliv-Řídká Blana locality. a: Protovisnea sp. 1, rounded seed with the narrow elongate seed cavity flanked by two bulging regions of larger cells, no. NM-F 3177; b: Protovisnea sp. 2, angular seed with the narrow elongate seed cavity flanked by two bulging regions of larger cells, no. NM-F 3179; c, d: Eurya crassitesta, one seed split into two parts, no. NM-F 3211, c – surface cells of the seed coat are palisade, d – cross-section of the seed; e: Nympheaceae sp. 1, seed, no. NM-F 3636; f: Nympheaceae sp. 2, seed, no. NM-F 4634; g: Klikovispermum sp.1, seeds with irregular outline and smooth outer surface, no. NM-F 3203; h: Klikovispermum malechii, seed with an orange-segment shape, no. NM-F 3299; i: Taxon 35, seed, no. NM-F 3236. in Plant Mesofossils From The Late Cretaceous Klikov Formation, The Czech Republic

Text-fig. 6. Scanning electron micrographs of seeds of ericalean affinity (a–d) and seeds of uncertain affinity (e–i) from Zliv-Řídká Blana locality. a: Protovisnea sp. 1, rounded seed with the narrow elongate seed cavity flanked by two bulging regions of larger cells, no. NM-F 3177; b: Protovisnea sp. 2, angular seed with the narrow elongate seed cavity flanked by two bulging regions of larger cells, no. NM-F 3179; c, d: Eurya crassitesta, one seed split into two parts, no. NM-F 3211, c – surface cells of the seed coat are palisade, d – cross-section of the seed; e: Nympheaceae sp. 1, seed, no. NM-F 3636; f: Nympheaceae sp. 2, seed, no. NM-F 4634; g: Klikovispermum sp.1, seeds with irregular outline and smooth outer surface, no. NM-F 3203; h: Klikovispermum malechii, seed with an orange-segment shape, no. NM-F 3299; i: Taxon 35, seed, no. NM-F 3236.

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Text-fig. 4. Scanning electron micrographs (a, c, e–k) and X-ray microtomographic orthoslices (b, d) of capsular fruits compose of five carpels from Zliv-Řídká Blana locality. a–d: Taxon 4, a – fruit elliptical in shape, no. NM-F 3188, b – young fruit with reminisce of free styles at top and showing central placentation of seeds, no. NM-F 3188, c – pentacarpellate capsules in apical view, no. NM-F 3188, d – fruit with five locules, no. NM-F 3235; e, f: Taxon 6, e – elongated fruit in lateral view, the persistent perianth at the base of the fruit (arrowhead), no. NM-F 3194, f – fruit in apical view, no. NM-F 3194; g, h: Taxon 5, g – elongated fruit in lateral view, no. NM-F 3193, h – fruit showing remains of a persistent calyx in the basal part (arrowhead), no. NM-F 3193; i–k: Taxon 7, i – pentacarpellate capsules of broadly elliptical shape, no. NM-F 4091, j – fruit, apical view, no. NM-F 4091, k – fruit with five seeds (arrowheads) ellipsoidal or triangular in outline and with a thick seed coat, no. NM-F 4091. in Plant Mesofossils From The Late Cretaceous Klikov Formation, The Czech Republic

Text-fig. 4. Scanning electron micrographs (a, c, e–k) and X-ray microtomographic orthoslices (b, d) of capsular fruits compose of five carpels from Zliv-Řídká Blana locality. a–d: Taxon 4, a – fruit elliptical in shape, no. NM-F 3188, b – young fruit with reminisce of free styles at top and showing central placentation of seeds, no. NM-F 3188, c – pentacarpellate capsules in apical view, no. NM-F 3188, d – fruit with five locules, no. NM-F 3235; e, f: Taxon 6, e – elongated fruit in lateral view, the persistent perianth at the base of the fruit (arrowhead), no. NM-F 3194, f – fruit in apical view, no. NM-F 3194; g, h: Taxon 5, g – elongated fruit in lateral view, no. NM-F 3193, h – fruit showing remains of a persistent calyx in the basal part (arrowhead), no. NM-F 3193; i–k: Taxon 7, i – pentacarpellate capsules of broadly elliptical shape, no. NM-F 4091, j – fruit, apical view, no. NM-F 4091, k – fruit with five seeds (arrowheads) ellipsoidal or triangular in outline and with a thick seed coat, no. NM-F 4091.

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Text-fig. 2. Salicaceae (a–g), Cannabaceae (h–n), cf. Betulaceae (o–r). a–g: Saxifragispermum, USNM PAL 772341. Scale bar = 5 mm except as indicated. a–b: Lateral, c: apical, and d: basal views of fruit, reflected light, palladium coated; apex at top of (a, b). e: Equatorial transverse section reflected light; arrows indicate presumed seeds, scale bar = 2 mm. f: Detail of locule contents extracted from (e), transmitted light, scale bar = 200 Μm. g: Interwoven trichomes or fibers from locule, transmitted light, scale bar = 5 Μm. h–j: Celtis. h, i: USNM PAL 772342, reflected light, palladium coated, scale bar = 5 mm. h: Lateral view parallel with plane of dehiscence. i: Lateral view perpendicular to plane of dehiscence. j: DMNH EPI.47809, Celtis in lateral view; showing reticulate sculpture and the vertically-oriented, plane of dehiscence (arrow), scale bar = 5 mm. k–m: Aphananthe. USNM PAL 772344, reflected light, palladium coated, scale bar = 5 mm. k: Apical view, note triangular cross section and apical plug (arrow). l: Lateral view, apex up. m: Lateral view at 90° to (l). n: Detail of cellular pattern at surface of endocarp, scale bar = 0.5 mm. o–r: in The Early Middle Eocene Wagon Bed Carpoflora Of Central Wyoming, U.S.A.

Text-fig. 2. Salicaceae (a–g), Cannabaceae (h–n), cf. Betulaceae (o–r). a–g: Saxifragispermum, USNM PAL 772341. Scale bar = 5 mm except as indicated. a–b: Lateral, c: apical, and d: basal views of fruit, reflected light, palladium coated; apex at top of (a, b). e: Equatorial transverse section reflected light; arrows indicate presumed seeds, scale bar = 2 mm. f: Detail of locule contents extracted from (e), transmitted light, scale bar = 200 Μm. g: Interwoven trichomes or fibers from locule, transmitted light, scale bar = 5 Μm. h–j: Celtis. h, i: USNM PAL 772342, reflected light, palladium coated, scale bar = 5 mm. h: Lateral view parallel with plane of dehiscence. i: Lateral view perpendicular to plane of dehiscence. j: DMNH EPI.47809, Celtis in lateral view; showing reticulate sculpture and the vertically-oriented, plane of dehiscence (arrow), scale bar = 5 mm. k–m: Aphananthe. USNM PAL 772344, reflected light, palladium coated, scale bar = 5 mm. k: Apical view, note triangular cross section and apical plug (arrow). l: Lateral view, apex up. m: Lateral view at 90° to (l). n: Detail of cellular pattern at surface of endocarp, scale bar = 0.5 mm. o–r:

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Text-fig. 9. Carpolithes (a–r). a–d: Carpolithes sp. 5. USNM PAL 772370. Scale bar = 5 mm, reflected light, palladium coated. a: Lateral view of seed, apex up, possible raphe descending from apex toward viewer. b: Lateral view of seed, apex up, possible raphe on right. c: Lateral view, opposite side, apex up, possible raphe on left. d: Apical view, note central pit with raphe descending towards bottom margin. e–h: Carpolithes sp. 6. USNM PAL 772371. Scale bar = 5 mm. e: Basal view illustrating depression and keel in plane of bisymmetry, reflected light, palladium coated. f–h: Micro-CT scan surface rendering. f: Lateral view showing relatively smooth rounded surface. g: Specimen rotated 180° from (f), surface partially eroded. h: Longitudinal view, showing median keel. i–m: Carpolithes sp. 7 USNM PAL 772372. Scale bar = 5 mm. i: View of intact face of globose fruit, possible apical constriction at top. j: Lateral view, intact surface to right, possible apical constriction at top, both micro-CT scan surface renderings. k: Apical view. l: Face view illustrating the mineral filling and the fine, radiating structure of the fruit wall on the left and right margins, both reflected light, palladium coated. m: Closeup of the cellular layer on the left of (l), micro-CT scan surface rendering. n–p: Carpolithes sp. 8. USNM PAL 772373. Scale bar = 3 mm, reflected light, palladium coated. n: Lateral view of pyrene-like structure, one ridge running vertically in the center of view, the other two forming the left and right margins. o: Lateral view of pyrene-like structure, ridge in (n) on the left. p: End-on view illustrating one convex, one concave, and one relatively flat to very slightly concave face. q, r: Carpolithes sp. 9 USNM PAL 772374. Scale bar = 5 mm, reflected light, palladium coated. q: Exterior of the smooth broken half-sphere. r: Interior of the broken half-sphere. in The Early Middle Eocene Wagon Bed Carpoflora Of Central Wyoming, U.S.A.

Text-fig. 9. Carpolithes (a–r). a–d: Carpolithes sp. 5. USNM PAL 772370. Scale bar = 5 mm, reflected light, palladium coated. a: Lateral view of seed, apex up, possible raphe descending from apex toward viewer. b: Lateral view of seed, apex up, possible raphe on right. c: Lateral view, opposite side, apex up, possible raphe on left. d: Apical view, note central pit with raphe descending towards bottom margin. e–h: Carpolithes sp. 6. USNM PAL 772371. Scale bar = 5 mm. e: Basal view illustrating depression and keel in plane of bisymmetry, reflected light, palladium coated. f–h: Micro-CT scan surface rendering. f: Lateral view showing relatively smooth rounded surface. g: Specimen rotated 180° from (f), surface partially eroded. h: Longitudinal view, showing median keel. i–m: Carpolithes sp. 7 USNM PAL 772372. Scale bar = 5 mm. i: View of intact face of globose fruit, possible apical constriction at top. j: Lateral view, intact surface to right, possible apical constriction at top, both micro-CT scan surface renderings. k: Apical view. l: Face view illustrating the mineral filling and the fine, radiating structure of the fruit wall on the left and right margins, both reflected light, palladium coated. m: Closeup of the cellular layer on the left of (l), micro-CT scan surface rendering. n–p: Carpolithes sp. 8. USNM PAL 772373. Scale bar = 3 mm, reflected light, palladium coated. n: Lateral view of pyrene-like structure, one ridge running vertically in the center of view, the other two forming the left and right margins. o: Lateral view of pyrene-like structure, ridge in (n) on the left. p: End-on view illustrating one convex, one concave, and one relatively flat to very slightly concave face. q, r: Carpolithes sp. 9 USNM PAL 772374. Scale bar = 5 mm, reflected light, palladium coated. q: Exterior of the smooth broken half-sphere. r: Interior of the broken half-sphere.

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Text-fig. 54. Scanning electron microscope (SEM; a–e, g, h) and synchrotron x-ray microscopy (SRXTM; f, i) images of unnamed angiosperm fruits and seeds; Torres Vedras locality, Portugal. a) Follicle sp. 1, narrow elongate follicle with sessile and decurrent stigma; b) Follicle sp. 2, broad, dehisced follicle with elongate and transverse fibers lining the locule; c) Angiosperm seed sp. 1 with thin, smooth seed coat; d) Angiosperm seed sp. 2 with verrucate seed coat; e) Angiosperm seed sp. 3 with exotestal and foveolate seed coat; f) Angiosperm seed sp. 4 with smooth seed coat; g, h) Angiosperm seed sp. 5 with raised epidermal cells forming a reticulate pattern; i) Wedge-shaped angiosperm fruit with remains of floral parts near apex. Specimens, TV43-S136726 (a), TV44-S148144 (b), TV43-S170074 (c), TV43-S136747 (d), TV43-S170073 (e), TV38-S174615 (f), TV44-S148003 (g), TV44-S148004 (h), TV43-S174685 (i). Scale bars 300 Μm (a–i). in The Early Cretaceous Mesofossil Flora Of Torres Vedras (Ne Of Forte Da Forca), Portugal: A Palaeofloristic Analysis Of An Early Angiosperm Community

Text-fig. 54. Scanning electron microscope (SEM; a–e, g, h) and synchrotron x-ray microscopy (SRXTM; f, i) images of unnamed angiosperm fruits and seeds; Torres Vedras locality, Portugal. a) Follicle sp. 1, narrow elongate follicle with sessile and decurrent stigma; b) Follicle sp. 2, broad, dehisced follicle with elongate and transverse fibers lining the locule; c) Angiosperm seed sp. 1 with thin, smooth seed coat; d) Angiosperm seed sp. 2 with verrucate seed coat; e) Angiosperm seed sp. 3 with exotestal and foveolate seed coat; f) Angiosperm seed sp. 4 with smooth seed coat; g, h) Angiosperm seed sp. 5 with raised epidermal cells forming a reticulate pattern; i) Wedge-shaped angiosperm fruit with remains of floral parts near apex. Specimens, TV43-S136726 (a), TV44-S148144 (b), TV43-S170074 (c), TV43-S136747 (d), TV43-S170073 (e), TV38-S174615 (f), TV44-S148003 (g), TV44-S148004 (h), TV43-S174685 (i). Scale bars 300 Μm (a–i).

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Text-fig. 1. Gastonispermum portugallicum gen. et sp. nov. seeds from the Early Cretaceous Famalicão locality (sample 025), Portugal; Synchrotron radiation X-ray tomographic microscopy (SRXTM, volume renderings). Note remains of mounting media on several seeds (¤). a) Seed in oblique view showing seed shape, the slightly raised raphal ridge and the position of hilum (hi) and micropyle (mi) on the raphal side of the seed (S170218). b, c) Seeds in lateral view (b, S170234; c, S175095). d–f) Holotype (S174820); seed in lateral view (d) and cut volume rendering (e, f) through the median plane of the seed showing palisade-shaped sclerenchyma cells of exotesta and remains of embryo (emb) and surrounding nutritive tissue (e, cut between yz0440-0530; f, cut between slices yz440-480). g) Hilum (hi) and micropyle (mi) of seed in (1a) showing the Y-shaped micropylar slit in the outer integument. h) Cut volume rendering through the median plane of the seed (cut at yz0492) showing seed coat mainly composed of palisade-shaped cells of the exotesta (S174435). i) Seed surface showing the raised outlines of the undulate anticlinal walls of the exotestal cells (S175045). Scale bars = 500 µm (a–e); 250 µm (g); 125 µm (f, i). in Extinct Taxa Of Exotestal Seeds Close To Austrobaileyales And Nymphaeales From The Early Cretaceous Of Portugal

Text-fig. 1. Gastonispermum portugallicum gen. et sp. nov. seeds from the Early Cretaceous Famalicão locality (sample 025), Portugal; Synchrotron radiation X-ray tomographic microscopy (SRXTM, volume renderings). Note remains of mounting media on several seeds (¤). a) Seed in oblique view showing seed shape, the slightly raised raphal ridge and the position of hilum (hi) and micropyle (mi) on the raphal side of the seed (S170218). b, c) Seeds in lateral view (b, S170234; c, S175095). d–f) Holotype (S174820); seed in lateral view (d) and cut volume rendering (e, f) through the median plane of the seed showing palisade-shaped sclerenchyma cells of exotesta and remains of embryo (emb) and surrounding nutritive tissue (e, cut between yz0440-0530; f, cut between slices yz440-480). g) Hilum (hi) and micropyle (mi) of seed in (1a) showing the Y-shaped micropylar slit in the outer integument. h) Cut volume rendering through the median plane of the seed (cut at yz0492) showing seed coat mainly composed of palisade-shaped cells of the exotesta (S174435). i) Seed surface showing the raised outlines of the undulate anticlinal walls of the exotestal cells (S175045). Scale bars = 500 µm (a–e); 250 µm (g); 125 µm (f, i).

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Text-fig. 6. Reyispermum parvum gen. et sp. nov. seeds from the Early Cretaceous Vale de Água locality, Portugal (a, b, d, e: sample 141, c: sample 300); Synchrotron radiation X-ray tomographic microscopy (SRXTM, orthoslices). a) Longitudinal orthoslice (yz0563) through the median plane of the seed (holotype, S174178) showing the palisade-shaped cells of exotesta and collapsed inner parts of seed coat, raised exotestal tissue (arrow head) immediately adjacent to the lower edge of the hilum (hi), micropyle (mi) and remains of cellular nutritive tissue. b) Longitudinal orthoslice (xz0659) through the micropylar region perpendicular to the median plane showing the exotestal tissue surrounding the transverse micropyle slit (mi). c) Longitudinal orthoslice (yz0500) through the median plane of the seed (S175095) showing raised tissue (arrow head) immediately adjacent to the lower edge of the hilum (hi), micropyle (mi) and poorly preserved cells of the seed coat. d) Longitudinal orthoslice (xz0810) through middle part of seed perpendicular to the median plane (S174178) showing seed coat and remains of cellular nutritive tissue. e) Tangential and longitudinal orthoslice (xz0162) through the seed coat of holotype (S174178) showing thickened, undulate cell walls of exotesta (ex) and the short, thin-walled cells of tegmen (te) with a finely striate wrinkled surface. Scale bars = 250 µm (a, c, d); 125 µm (b, e). in Extinct Taxa Of Exotestal Seeds Close To Austrobaileyales And Nymphaeales From The Early Cretaceous Of Portugal

Text-fig. 6. Reyispermum parvum gen. et sp. nov. seeds from the Early Cretaceous Vale de Água locality, Portugal (a, b, d, e: sample 141, c: sample 300); Synchrotron radiation X-ray tomographic microscopy (SRXTM, orthoslices). a) Longitudinal orthoslice (yz0563) through the median plane of the seed (holotype, S174178) showing the palisade-shaped cells of exotesta and collapsed inner parts of seed coat, raised exotestal tissue (arrow head) immediately adjacent to the lower edge of the hilum (hi), micropyle (mi) and remains of cellular nutritive tissue. b) Longitudinal orthoslice (xz0659) through the micropylar region perpendicular to the median plane showing the exotestal tissue surrounding the transverse micropyle slit (mi). c) Longitudinal orthoslice (yz0500) through the median plane of the seed (S175095) showing raised tissue (arrow head) immediately adjacent to the lower edge of the hilum (hi), micropyle (mi) and poorly preserved cells of the seed coat. d) Longitudinal orthoslice (xz0810) through middle part of seed perpendicular to the median plane (S174178) showing seed coat and remains of cellular nutritive tissue. e) Tangential and longitudinal orthoslice (xz0162) through the seed coat of holotype (S174178) showing thickened, undulate cell walls of exotesta (ex) and the short, thin-walled cells of tegmen (te) with a finely striate wrinkled surface. Scale bars = 250 µm (a, c, d); 125 µm (b, e).

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Text-fig. 7. Lusitanispermum choffatii gen. et sp. nov. seeds from the Early Cretaceous Famalicão locality (sample 025), Portugal; Synchrotron radiation X-ray tomographic microscopy (SRXTM, volume renderings). a) Holotype; seed in lateral view showing seed shape; note that the seed is broken near the lower surface of the hilum (S174345). b) Oblique apical view of micropylar-hilar region of holotype showing slightly ruptured micropylar slit (mi) in the outer integument and two bulging and abraded areas (arrow heads) close to hilum. c) Seed in oblique lateral-raphal view showing the two bulging structures (arrow heads) immediately adjacent to the lower edge of the hilum (S174472). d) Tangential, longitudinal cut (cut at yz0131) through the seed coat of seed in (7c) showing the undulate anticlinal cell walls of the exotesta cells that are thickest towards the outside and thinner towards the inside. Scale bars = 500 µm (a–c); 250 µm (d). in Extinct Taxa Of Exotestal Seeds Close To Austrobaileyales And Nymphaeales From The Early Cretaceous Of Portugal

Text-fig. 7. Lusitanispermum choffatii gen. et sp. nov. seeds from the Early Cretaceous Famalicão locality (sample 025), Portugal; Synchrotron radiation X-ray tomographic microscopy (SRXTM, volume renderings). a) Holotype; seed in lateral view showing seed shape; note that the seed is broken near the lower surface of the hilum (S174345). b) Oblique apical view of micropylar-hilar region of holotype showing slightly ruptured micropylar slit (mi) in the outer integument and two bulging and abraded areas (arrow heads) close to hilum. c) Seed in oblique lateral-raphal view showing the two bulging structures (arrow heads) immediately adjacent to the lower edge of the hilum (S174472). d) Tangential, longitudinal cut (cut at yz0131) through the seed coat of seed in (7c) showing the undulate anticlinal cell walls of the exotesta cells that are thickest towards the outside and thinner towards the inside. Scale bars = 500 µm (a–c); 250 µm (d).

Text-fig. 6. Scanning electron microscope (SEM, a, g, h) and synchrotron radiation X-ray tomographic microscopy (SRXTM, b–f) images of fruits and pollen grains of Canrightiopsis crassitesta (a–c, g, h) and fruit of Canrightiopsis intermedia (d–f); Catefica locality, Portugal. a) Dorsal view of fruit showing rim of hypanthium (arrowheads); b) Surface rendering of longitudinal section in the median plane of fruit (cut between orthoslices yz0440-0510) showing the thin fruit wall, thick endotesta of the seed coat (en, dark blue) and the orthotropous, pendent seed with the chalaza (ch) near the fruit apex and the micropyle (mi) at the fruit base; note the tiny embryo (emb) adjacent to the micropyle at the base of the fruit; c) Longitudinal section (orthoslice xz0511) through the seed wall showing the thick, finely crystalliferous endotesta (en) surrounding the nutritive tissue of the seed; d) Surface rendering of fruit in dorsal view showing rim of the hypanthium (arrowheads) and apical stigmatic region (st); e) Surface rendering of longitudinal section of fruit in (d) (cut at orthoslice xz0560) showing the crystalliferous endotesta (en, dark blue) and the inner tissues of the seed; f) Longitudinal section (orthoslice xz0560) through fruit and seed showing the finely crystalliferous endotesta (en) and thin fruit wall (fr); g, h) Pollen grains from apical region of fruit showing poorly defined margin of the single colpus, reticulate tectum and muri ornamented by minute verrucae. Specimens, Catefica 343-S174311 (a), Catefica 49-S174159 (b, c), Catefica 50-S174905 (d–f), Catefica 342-S122089 (g, h). Scale bars = 300 Μm (a, b, d, e), 150 Μm (c, f), 6 Μm (g), 3 Μm (h). g, h published with permission from Grana. in The Early Cretaceous Mesofossil Flora Of Catefica, Portugal: Angiosperms

Text-fig. 6. Scanning electron microscope (SEM, a, g, h) and synchrotron radiation X-ray tomographic microscopy (SRXTM, b–f) images of fruits and pollen grains of Canrightiopsis crassitesta (a–c, g, h) and fruit of Canrightiopsis intermedia (d–f); Catefica locality, Portugal. a) Dorsal view of fruit showing rim of hypanthium (arrowheads); b) Surface rendering of longitudinal section in the median plane of fruit (cut between orthoslices yz0440-0510) showing the thin fruit wall, thick endotesta of the seed coat (en, dark blue) and the orthotropous, pendent seed with the chalaza (ch) near the fruit apex and the micropyle (mi) at the fruit base; note the tiny embryo (emb) adjacent to the micropyle at the base of the fruit; c) Longitudinal section (orthoslice xz0511) through the seed wall showing the thick, finely crystalliferous endotesta (en) surrounding the nutritive tissue of the seed; d) Surface rendering of fruit in dorsal view showing rim of the hypanthium (arrowheads) and apical stigmatic region (st); e) Surface rendering of longitudinal section of fruit in (d) (cut at orthoslice xz0560) showing the crystalliferous endotesta (en, dark blue) and the inner tissues of the seed; f) Longitudinal section (orthoslice xz0560) through fruit and seed showing the finely crystalliferous endotesta (en) and thin fruit wall (fr); g, h) Pollen grains from apical region of fruit showing poorly defined margin of the single colpus, reticulate tectum and muri ornamented by minute verrucae. Specimens, Catefica 343-S174311 (a), Catefica 49-S174159 (b, c), Catefica 50-S174905 (d–f), Catefica 342-S122089 (g, h). Scale bars = 300 Μm (a, b, d, e), 150 Μm (c, f), 6 Μm (g), 3 Μm (h). g, h published with permission from Grana.

Text-fig. 3. Synchrotron radiation X-ray tomographic microscopy (SRXTM) images of fruits of Canrightia foveolata sp. nov.; Catefica locality, Portugal. a) Volume rendering of fruit showing prominent rim around the middle of the fruit with reduced tepals (arrowheads) and partly abraded fruit wall exposing the pitted endotesta surface of one of two seeds (arrow); note two of the vascular bundles (vb) extending from the base of the fruit to the tepals; b) Voltex of fruit showing prominent rim around the fruit (arrowhead) and dense precipitation of crystals in the endothelium cells of one of the two seeds in the fruit; c) Longitudinal section of fruit (orthoslice yz0520) showing the inferred hypanthium rim (arrow head) and two seeds, one with a dense precipitation of crystals; note the prominent endothelium cells (asterisks) of the inner integument and the well-developed fruit wall above the seeds; d) Transverse section through basal part of fruit and seeds close to the micropyle (orthoslice xy0312) showing partly abraded fruit wall with five vascular bundles (vb) and details of the seed coat with endotesta (oi-end) surrounding the tegmen consisting of an outer epidermis (ii-o), middle layer (ii-m) and a distinct inner epidermis (endothelium) consisting of radially elongated cells (asterisk); e) Transverse section (orthoslice xy1680) through apical part of the fruit close to chalaza showing the tips of two seeds; note the endotesta (oi-end) surrounded by thick-walled cells of the exotesta (oi-o); f) Transverse section (orthoslice xy1485) through fruit in the region of the hypanthium rim showing sections through the two seeds close to the chalazal region; note endotesta (oi-end) surrounded by larger cells of exotesta (oi-o) and fruit wall (fr). Specimen, Catefica 49-S174249 (holotype, a–f). Scale bars = 300 Μm (a–c, e, f), 100 Μm (d). in The Early Cretaceous Mesofossil Flora Of Catefica, Portugal: Angiosperms

Text-fig. 3. Synchrotron radiation X-ray tomographic microscopy (SRXTM) images of fruits of Canrightia foveolata sp. nov.; Catefica locality, Portugal. a) Volume rendering of fruit showing prominent rim around the middle of the fruit with reduced tepals (arrowheads) and partly abraded fruit wall exposing the pitted endotesta surface of one of two seeds (arrow); note two of the vascular bundles (vb) extending from the base of the fruit to the tepals; b) Voltex of fruit showing prominent rim around the fruit (arrowhead) and dense precipitation of crystals in the endothelium cells of one of the two seeds in the fruit; c) Longitudinal section of fruit (orthoslice yz0520) showing the inferred hypanthium rim (arrow head) and two seeds, one with a dense precipitation of crystals; note the prominent endothelium cells (asterisks) of the inner integument and the well-developed fruit wall above the seeds; d) Transverse section through basal part of fruit and seeds close to the micropyle (orthoslice xy0312) showing partly abraded fruit wall with five vascular bundles (vb) and details of the seed coat with endotesta (oi-end) surrounding the tegmen consisting of an outer epidermis (ii-o), middle layer (ii-m) and a distinct inner epidermis (endothelium) consisting of radially elongated cells (asterisk); e) Transverse section (orthoslice xy1680) through apical part of the fruit close to chalaza showing the tips of two seeds; note the endotesta (oi-end) surrounded by thick-walled cells of the exotesta (oi-o); f) Transverse section (orthoslice xy1485) through fruit in the region of the hypanthium rim showing sections through the two seeds close to the chalazal region; note endotesta (oi-end) surrounded by larger cells of exotesta (oi-o) and fruit wall (fr). Specimen, Catefica 49-S174249 (holotype, a–f). Scale bars = 300 Μm (a–c, e, f), 100 Μm (d).

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Ant handling changes myrmecochore seed coat microbiomes and alters diversity of seed-borne plant pathogenic fungi

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DATASET OF "Evolutionary Pathways to Thin-Coated Seeds in Crops: Insights from Allometric Scaling" by Milla et al.

<table> <tbody> <tr> <td>This database is composed of four spreadsheets:</td> </tr> <tr> <td>data</td> <td>Unpublished data used in the manuscript. Data that was published earlier is not included to avoid duplicates, but reference to their source is given in the other two spreadsheets</td> </tr> <tr> <td>species_in_sources</td> <td>Key to the sources of data for the project, for each species, and each observation within species</td> </tr> <tr> <td>list_of_sources</td> <td>Key to sources plus full reference of each key</td> </tr> <tr> <td>list_accessions</td> <td>list of accessions identities for each crop and domestication status</td> </tr> <tr> <td> </td> <td> </td> </tr> <tr> <td>Meaning of colums in each spreadsheet</td> </tr> <tr> <td> </td> <td> </td> </tr> <tr> <td>data:</td> <td> </td> </tr> <tr> <td>id_data</td> <td>id of row in this spreadsheet</td> </tr> <tr> <td>id_sources</td> <td>id of row in the species_in_sources spreadsheet</td> </tr> <tr> <td>species</td> <td>Botanical name of the species</td> </tr> <tr> <td>family</td> <td>Family adscription of the species</td> </tr> <tr> <td>genus</td> <td>genus</td> </tr> <tr> <td>species_epith</td> <td>species epithet</td> </tr> <tr> <td>cultivar_subsp_type</td> <td>for domesticated genotypes, cultivar, subspecies, or type</td> </tr> <tr> <td>accesion_id</td> <td>accesion id from seedbanks</td> </tr> <tr> <td>crop</td> <td>for domesticated genotypes and their direct wild progenitors: common name of the crop</td> </tr> <tr> <td>dom_status_c_w</td> <td>whether an entry comes from a domesticated or a wild genotype or species</td> </tr> <tr> <td>dom_status_im_lr_wp_wnowp</td> <td>domestication status of the entry, either an improved cultivar of a crop (im), a landrace (lr), a wild progenitor (wp) or a wild species not closely related to any crop (wnowp)</td> </tr> <tr> <td>seed_mass_mg</td> <td>seed mass, in mg</td> </tr> <tr> <td>seed_coat_mass_mg</td> <td>seed coat mass, in mg</td> </tr> <tr> <td>kernel_mass_mg</td> <td>kernel mass, in mg</td> </tr> <tr> <td>key_to_source</td> <td>code to link with the references listed in the list_of_sources spreadsheet</td> </tr> <tr> <td> </td> <td> </td> </tr> <tr> <td> </td> <td> </td> </tr> <tr> <td>species_in_sources:</td> <td> </td> </tr> <tr> <td>id_sources</td> <td>see above for data spreadsheet</td> </tr> <tr> <td>species</td> <td>see above for data spreadsheet</td> </tr> <tr> <td>family</td> <td>see above for data spreadsheet</td> </tr> <tr> <td>genus</td> <td>see above for data spreadsheet</td> </tr> <tr> <td>species_epith</td> <td>see above for data spreadsheet</td> </tr> <tr> <td>cultivar_subsp_type</td> <td>see above for data spreadsheet</td> </tr> <tr> <td>accesion_id</td> <td>see above for data spreadsheet</td> </tr> <tr> <td>crop</td> <td>see above for data spreadsheet</td> </tr> <tr> <td>dom_status_c_w</td> <td>see above for data spreadsheet</td> </tr> <tr> <td>dom_status_im_lr_wp_wnowp</td> <td>see above for data spreadsheet</td> </tr> <tr> <td>key_to_source</td> <td>see above for data spreadsheet</td> </tr> <tr> <td> </td> <td> </td> </tr> <tr> <td>list_of_sources:</td> <td> </td> </tr> <tr> <td>key_to_source</td> <td>code to link with the references</td> </tr> <tr> <td>source</td> <td>full reference</td> </tr> <tr> <td> </td> <td> </td> </tr> <tr> <td>list_accessions:</td> <td>see "data" sheet</td> </tr> </tbody> </table>

Figure 6 in Potential neuroprotective of trans-resveratrol a promising agent tempeh and soybean seed coats-derived against beta-amyloid neurotoxicity on primary culture of nerve cells induced by 2-methoxyethanol

Figure 6. Treatment Group with resveratrol standard after induced by Beta-Amyloid (10 x10).

Figure 3 in Potential neuroprotective of trans-resveratrol a promising agent tempeh and soybean seed coats-derived against beta-amyloid neurotoxicity on primary culture of nerve cells induced by 2-methoxyethanol

Figure 3. Treatment group: 2- ME + Resveratrol isolated from Tempeh (10 x10).

Figure 8 in Potential neuroprotective of trans-resveratrol a promising agent tempeh and soybean seed coats-derived against beta-amyloid neurotoxicity on primary culture of nerve cells induced by 2-methoxyethanol

Figure 8. Treatment Group with resveratrol tempeh + 2-ME after induced by Beta-Amyloid (10 x10).

Figure 5 in Potential neuroprotective of trans-resveratrol a promising agent tempeh and soybean seed coats-derived against beta-amyloid neurotoxicity on primary culture of nerve cells induced by 2-methoxyethanol

Figure 5. Treatment group: 2- ME (10 x10).

Figure 9. Treatment Group with 2 in Potential neuroprotective of trans-resveratrol a promising agent tempeh and soybean seed coats-derived against beta-amyloid neurotoxicity on primary culture of nerve cells induced by 2-methoxyethanol

Figure 9. Treatment Group with 2-ME after induced by Beta-Amyloid (10 x10).