1. Zingiberales Zingiber officinialis (Zingiberaceae) Calathea lancifolia (Marantaceae) Ctenanthe oppenheiminiana (Marantaceae) Musa sp (Musaceae) Caryota mitis (Arecaceae) Arecales Rhapis excelsa (Arecaceae) Asparagales Ludisia discolor (Orchidaceae) Iris germanica (Iridaceae) Alismatales Elodea canadensis (Araceae) Clivia miniata (Amaryllidaceae) Asparagales Figure S1 P4MP4M P5MP5M P6MP6M P7MP7M P5UP5U P6UP6U 781 759 877 1009 1045 1023 891 913 1176 1155 929 1061 m/z 700120010008009001100 758 780 745 877 891 913 1009 1023 1045 P4MP4M P5MP5M P6MP6M P4UP4U P5UP5U P6UP6U m/z 700120010008009001100 759 781 745 877 891 1009 1045 913 1023 1177 1155 1141 P4MP4M P5MP5M P6MP6M P7MP7M P4UP4U P5UP5U P6UP6U P7UP7U m/z 700120010008009001100 P4MP4M P5MP5M P5P5 759 781 797 891 913 930 1046 1024 701 P6MP6M m/z 700120010008009001100 P4MP4M P5MP5M P5UP5U P4UP4U 701 832 965 1097 759 745 891 877 P7P7 P8P8 P6P6 P5P5 m/z 700120010008009001100 P4MP4M P5MP5M P5P5 P7P7 701 833 965 1097 759 891 P6P6 P8P8 m/z 700120010008009001100 P4MP4M P5MP5M P5P5 P6P6 759 745 701 913 891 877 833 929 781 797 P4UP4U m/z 700120010008009001100 P4MP4M P5MP5M P5P5 P7P7 P6P6 P8P8 701 833 965 1097 759 745 891 877 1009 1023 P6MP6M P5UP5U m/z 700120010008009001100 P4MP4M P5MP5M P5P5 759 781 797 891 913 930 1046 1024 701 P6MP6M m/z 700120010008009001100 781 759 745 913 891 877 1045 1023 1009 P4MP4M P5MP5M P6MP6M P6UP6U P4UP4U P5UP5U m/z 700120010008009001100 Supplementary Fig. S1. MALDI-TOF mass spectrum of the isolated xylo-oligosaccharides generated by xylanase treatment of the 1 M KOH extracts of AIR from selected monocots. The [M+Na] + and [M-H+2Na] + ions corresponding to the most abundant xylo-oligosaccharides in each mixture are labeled as described in Figure 2 (main text).

2. Figure S2 Brachypodium distachyon (Poaceae) Setaria italica (Poaceae) Miscanthus x giganteus (Poaceae) 4.24.34.44.54.64.75.15.25.35.45.55.6 Chemical Shift (ppm) Panicum virgatum (Poaceae) Oryza sativa (Poaceae) Reducing α-Xylp Reducing β-Xylp Terminal α-GlcpA Terminal 4Me-α-GlcpA 2,4-β-Xylp (MG) Terminal α-Araf 2-α-Araf 3,4-β-Xylp (Araf) Cyperus alternifolius (Cyperaceae) Terminal α-GlcpA Terminal 4Me-α-GlcpA 2,4-β-Xylp (MG) Tillandsia usneoides (Bromeliaceae) Ananus comosus (Bromeliaceae) α-GalpA α-Rhap β-Xylp (Rhap) 2-α-Araf 3,4-β-Xylp (Araf) Poales Supplementary Fig. S2. Partial 600-MHz 1D 1 H NMR spectra of xylo-oligosaccharides of selected commelinid species.

3. Figure S3 Zingiberales 4.24.34.44.54.64.75.15.25.35.45.55.6 Chemical Shift (ppm) Strelitzia alba (Streliziaceae) Hedychium coronarium (Zingiberaceae) Amomum costatum (Zingiberaceae) Tradescantia virginiana (Commelinaceae Reducing α-Xylp Reducing β-Xylp Terminal α-GlcpA Terminal 4Me-α-GlcpA 2,4-β-Xylp (MG) Starch α-GalpA α-Rhap β-Xylp (Rhap) Terminal α-Araf Cocos nucifera (Arecaceae) Sabal etonia (Arecaceae) Howea forsteriana (Arecaceae) Terminal α-Araf 2-α-MeGlcpA (Arap) 3,4-β-Xylp (Araf) Commelinales Arecales Supplementary Fig. S3. Partial 600-MHz 1D 1 H NMR spectra of xylo-oligosaccharides of selected Zingiberales, Commelinales and Arecales species.

4. Figure S4 4.24.34.44.54.64.75.15.25.35.45.55.6 Chemical Shift (ppm) Allium cepa (Amarylidaceae) Crinum americanum (Amarylidaceae) Agapanthus africanus (Amarylidaceae) Agave americana (Asparagaceae) Reducing α-Xylp Reducing β-Xylp 2-α-GlcpA (Arap) Terminal α-GlcpA Terminal 4Me-α-GlcpA 2,4-β-Xylp (Arap) Terminal α-Arap 2-α-MeGlcpA (Arap) Asparagus officinalis (Asparagaceae) Tip Asparagus officinalis (Asparagaceae) Stem α-GalpA α-Rhap β-Xylp (Rha) Asparagales Supplementary Fig. S4 Partial 600-MHz 1D 1 H NMR spectra of xylo-oligosaccharides isolated from selected Asparagales (non-commelinid) species.

5. Figure S5 Dioscoreales Liliales Pandales Alismatales Acorales Spirodela polyrhiza (Araceae) Lemna minor (Araceae) 4.24.34.44.54.64.75.15.25.35.45.55.6 Chemical Shift (ppm) Orontium aquaticum (Araceae) Reducing α-Xylp Reducing β-Xylp 2-α-GlcpA (Arap) Terminal α-GlcpA Terminal 4Me-α-GlcpA 2,4-β-Xylp (Arap) Terminal α-Arap Alstroemeria sp. (Alstroemeriaceae) Tulip sp. (Liliaceae) Reducing α-Xylp Reducing β-Xylp α-GalpA H1-Terminal α-GlcpA H1-Terminal 4Me-α-GlcpA α-Rhap 2,4-β-Xylp (GlcA) β-Xylp (Rha) 2,4-β-Xylp (MeGlcA) Pandanus utilis (Pandanaceae) Acorus americanus (Acoraceae) Dioscorea alata (Dioscoreaceae) Starch Supplementary Fig. S5 Partial 600-MHz 1D 1 H NMR spectra of xylo-oligosaccharides isolated from selected Liliales, Pandales, Discoreales Alismatales, and Acorales species.

6. 2-α-MeGlcpA (Arap) 2,4-β-Xylp (MeGlcpA-Arap) 2,4-β-Xylp (MeGlcpA-Galp) Terminal α-Arap (MeGlcpA) Terminal β-Galp (MeGlcpA) 2-α-MeGlcpA (Galp) Reducing α-Xylp Terminal α-GlcpA Terminal 4Me-α-GlcpA Figure S6 Supplementary Fig. S6 Partial 600-MHz 1D 1 H gCOSY NMR spectra of xylo-oligosaccharides generated from Eucalyptus grandis wood GX. The labeled cross-peaks correspond to correlations between vicinal protons of the glycosyl residues in the sidechains containing GlcA/MeGlcA substituted with α-l- Arap or β-d-Galp residues.

7. 7008009001000110012001300 m/z 915 929 1075 1089 959 753 945 769 1119 1249 P 3 M-2AB P 4 M-2AB P 5 M-2AB P 2 M-2AB Figure S7 a 7008009001000110012001300 m/z 1031 711 1003 1191 871 1089 1163 1249 1061 929857 P 5 - 2AB P 6 - 2AB P 4 M-2AB P 3 M-2AB P 5 M-2AB P 3 - 2AB b P 4 - 2AB Supplementary Fig. S7. ESI-MS spectra of the per-O-methylated and 2AB-labeled xylo- oligosaccharides from Setaria italica. Xylo-oligosaccharides were generated by selectively labeling the reducing ends of polysaccharides in Setaria italica AIR with 2AB, extracting the labeled polysaccharides with 1 M KOH, fragmenting the resulting polysaccharides with endo-xylanase, separating the resulting products into fractions enriched in acidic and neutral oligosaccharides, and per- O-methylating the oligosaccharides in each fraction. a Spectrum of the fraction enriched in acidic oligosaccharides P 2 M, P 3 M, P 4 M, and P 5 M, which correspond to [M + Na] + ions at m/z 769, 929, 1089, and 1249, respectively. b Spectrum of the fraction enriched in neutral oligosaccharides P 3, P 4, P 5, and P 6, which correspond to [M + Na] + ions at m/z 711, 871, 1031, and 1191,, respectively. Comparable oligosaccharides were generated from Miscanthus giganteus, Panicum virgatum, Oryza sativa, and Brachypodium distachyon.

8. 929 755 697 595 503 363 m/z X  X  X  2AB 697 MM 755 595 MS 2 m/z 929 MS 3 m/z 929→ 755 755 595 523 363 581 m/z Figure S8 X  X  2AB MM 595 PP 755 697 Supplementary Fig. S8. ESI-MS n indicates that the reducing-end xylose of Setaria italica GAX is frequently substituted with GlcA. The precursor ion at m/z 929 (i.e., P 3 M-2AB) was selected from the MS 1 spectrum (see supplementary Fig. S7a) of the fraction enriched in acidic oligosaccharides and subjected to ESI-MS n. Possible precursor ion structures and their fragmentation leading to the generation of Y-ions are shown in each spectrum. The fragmentation pathway (929 – 755 – 595/523) is consistent with the sequence Xyl-Xyl-(MeGlcA)-Xyl-2AB. The data do not rule out the possibility that an oligosaccharide with the sequence (Pentose)-Xyl-(MeGlcA)-Xyl-2AB, which corresponds to an oligosaccharide bearing a pentosyl sidechain, may also be present. 697 595 755 929  X  X  2AB 523 MM 595 X  X  2AB  M 595 523

9. 915 741 683 755 595 363 m/z MS 3 m/z 1089 → 915 1089 915 857 755 897 595 363 MS 2 m/z 1089 m/z 523 755 595 363 m/z MS 4 m/z 1089 → 915 → 755 581 Figure S9 X→X→X→X→2AB 857 MM 915755595 X→X→X→2AB 857 MM 915 PP 595 X  X  X  2AB 683 MM  741595 →X→X→2AB 683 MM PP 741 595  X  X  X  2AB 683 MM 755595  X  X  2AB 523 MM 595 Supplementary Fig. S9. Further evidence that the reducing-end xylose of Setaria italica GAX is frequently substituted with GlcA. The precursor ion at m/z 1089 (i.e., P 4 M-2AB) was selected from the MS 1 spectrum (see supplementary Fig. S7a) of the fraction enriched in acidic oligosaccharides and subjected to ESI-MS n. Fragmentation events leading to the generation of Y ions are indicated in each spectrum.