Virome of riverside phytocommunity ecosystem of an ancient canal. Shixing Yang1,2§, Tongling Shan3§, Yan Wang1, Jie Yang1, Xu Chen1, Yuqing Xiao1, Zhenqiang You4, Yumin He1, Min Zhao1, Juan Lu1, Zijun Yang1, Ziyuan Dai1, Qi Liu1, Yuxin Yao1, Xiang Lu1, Hong Li1, Rui Zhou1, Wang Li5, Chenglin Zhou5, Xiaochun Wang1, Quan Shen1, Hui Xu6, Xutao Deng7, Eric Delwart7,8, Wen Zhang1* 1. Department of Microbiology, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, China. 2. International Center for genomics research, Jiangsu University, Zhenjiang, Jiangsu 212013, China. 3. Chinese Academy of Agricultural Sciences, Shanghai, 200241, China. 4. Zhejiang Academy of Medical Sciences, Hangzhou Medical College, Hangzhou, Zhejiang 310013, China. 5. Department of Laboratory Medicine, Jiangsu Taizhou People’s Hospital, Taizhou, Jiangsu 225300, China 6.The affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001, China. 7. Vitalant Research Institute, San Francisco, California 94118, United States of America. 8. Department of Laboratory Medicine, University of California San Francisco, California 94118, United States of America. §These authors contribute equally to this work. *corresponding author: Wen Zhang, address: School of Medicine, Jiangsu University, 310 Xuefu Road, Zhenjiang, Jiangsu 202103, China, E-mail: [email protected]

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TABLE OF CONTENTS Supplementary Figures………………………………………………………………………………………….…………………….……4

Supplementary Figure 1. Pictures of plant strains or leaves collected in the present study…………………………………….….….4

Supplementary Figure 2. The map for sampling site of plant samples in this study…………………………………………………...12

Supplementary Figure 3. Comparison of the percentage of number of viral sequence reads against total unique reads

in individual library between cultivated and wild plants...... 13

Supplementary Figure 4. Comparison of the numbers of virus type detected in cultivated and wild plants………………………..14

Supplementary Figure 5. Phylogenetic tree of pant-associated viruses belonging in the family Dicistroviridae……………………..15

Supplementary Figure 6. Viral genome organizations of pant-associated viruses belonging in the family Dicistroviridae…………16

Supplementary Figure 7. Phylogenetic tree of pant-associated viruses belonging in the family Iflaviridae……………………….…17

Supplementary Figure 8. Viral genome organizations of pant-associated viruses belonging in the family Marnaviridae…………..18

Supplementary Figure 9. The phylogenetic tree of pant-associated viruses belonging in the family Marnaviridae…………...…….19

Supplementary Figure 10. The phylogenetic tree of plant-associated parvovirus-like viruses…………………….………………….20

Supplementary Figure 11. Viral genome organizations of the plant-associated parvovirus-like viruses………………………….…21

Supplementary Figure 12. The phylogenetic tree of plant-associated Noda-like viruses………………………………………….…..22

Supplementary Figure 13. The phylogenetic tree of plant-associated Permutotetra-like viruses…………………………………….23

Supplementary Figure 14. The phylogenetic tree of plant-associated Yanvirus-like viruses……………………………………….…24

Supplementary Figure 15. The phylogenetic tree and viral genome organization of plant-associated Chuvirus-like viruses……...25

Supplementary Figure 16. The phylogenetic tree and viral genome organization of plant-associated Bastro-like viruses……..…..26

Supplementary Figure 17. The phylogenetic tree and viral genome organization of plant-associated Hepe-like viruses…………..27

Supplementary Figure 18. The phylogenetic tree of plant-associated Botybirnavirus viruses………………………………………...28

Supplementary Figure 19. The phylogenetic tree and viral genome organization of plant-associated Narna-like viruses……..…..29

Supplementary Figure 20. The phylogenetic tree and viral genome organization of plant-associated Picorna-like viruses………..30

Supplementary Figure 21. The phylogenetic tree and viral genome organization of plant viruses belonging in the family

Tombusviridae………………………………………………………………………………………………………………………….31

Supplementary Figure 22. The phylogenetic tree of plant-associated letuo-like virus and viruses belonging in the family

Luteoviridae……………………………………………………………………………………………………………………………32

Supplementary Figure 23. The phylogenetic tree of plant-associated parti-like virus and viruses belonging in the family

Partitiviridae…………………………………………………………………………………………………………………………...33

Supplementary Figure 24. The phylogenetic tree and viral genome organization of plant-associated Sobemo-like viruses…….….34

Supplementary Figure 25. The phylogenetic tree of plant-associated Rhabdo-like viruses……………………………………….…..35

Supplementary Figure 26. The phylogenetic tree and viral genome organization of plant-associated Bufivirus-like viruses……...36

Supplementary Figure 27. The phylogenetic tree and genome organization of plant-associated viruses showing significant

sequence similarity to viruses belonging in the family Geminiviridae…………………………………………………….……….37

Supplementary Figure 28. The phylogenetic tree and genome organization of plant-associated viruses belonging in the family

Genomoviridae…………………………………………………………………………………………………………………………38

Supplementary Figure 29. The phylogenetic tree of plant-associated Alphasatellitidae viruses………………………………………39

Supplementary Figure 30. The phylogenetic tree and genome organization of plant-associated CRESSV Group1 viruses………..40

Supplementary Figure 31. The phylogenetic tree and genome organization of plant-associated CRESSV Group2 viruses………..41

Supplementary Figure 32. The phylogenetic tree and genome organization of plant-associated CRESSV Group3 viruses………..42

Supplementary Figure 33. The phylogenetic tree and genome organization of plant-associated CRESSV Group4 viruses………..43

Supplementary Figure 34. The phylogenetic tree and genome organization of plant-associated CRESSV Group5 viruses………..44

Supplementary Figure 35. The phylogenetic tree and genome organization of plant-associated CRESSV Group6 viruses………...45

Supplementary Figure 36. The phylogenetic tree of plant-associated viruses belonging in the family Microviridae………………..46

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Supplementary Figure 37. The phylogenetic tree of Potyviridae…………………………………………………………………………47

Supplementary Figure 38. Potential cross-species infection of viruses among different species of plants…………………………...48

Supplementary Figure 39. The phylogenetic tree of plant viruses belonging in the family Bromoviridae…………………………...49

Supplementary Figure 40. The phylogenetic tree of plant viruses belonging in the family Closteroviridae………………………….50

Supplementary Figure 41. The phylogenetic tree of plant viruses belonging in the family Comovirinae……………………………51

Supplementary Figure 42. The phylogenetic tree of plant viruses belonging in the family Botourmiaviridae………………………52

Supplementary Figure 43. The phylogenetic tree of plant viruses belonging in the family Tymovirales viruses……………………53

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Supplementary Figure 1.Pictures of plant strains or leaves collected in the present study

plant004, Cynodon plant007, Photinia plant008, Morus alba L. plant009, Cirsium setosum plant010, Cirsium plant011, Metaplexis plant012, Solanum nigrum

dactylon (L.) Pers. serrulata Lindl. (Willd.) MB. japonicum Fisch. ex DC. japonica (Thunb.) Makino L.

plant013, Erigeron annuus plant014, Osmanthus plant015, Oxalis corniculata plant017, Humulus plant018, Commelina plant020, Conyza plant021, Buxus sinica

(L.) Pers. fragrans (Thunb.) Lour. L. scandens communis canadensis (L.) Cronq. (Rehd. et Wils.) Cheng

plant022, Rhododendron plant023, Magnolia plant024, Perilla frutescens plant025, Humifuse plant026, Duchesnea indica plant027, Hydrocotyle plant028, Ophiopogon

simsii Planch. grandiflora L. (L.) Britt. Euphorbia Willd. ex (Andr.) Focke sibthorpioides Lam. bodinieri

4

Schlecht.

plant029, Albizia julibrissin plant030, Broussonetia plant031, Polygonum plant032, Koelreuteria plant033, Bidens pilosa L. plant034, Aster subulatus plant037, Cinnamomum

Durazz. papyrifera (Linn.) ex Vent. persicaria L. paniculata Laxm. Michx. camphora (L.) presl

plant038, Phyllostachys plant039, Nandina plant040, Galium odoratum plant041, Sapium plant042, Jasminum plant043, Pontederia plant044, Boehmeria nivea sulphurea (Carr.) A. et C. domestica Thunb. (L.) Scop. sebiferum (L.) Roxb. nudiflorum Lindl. cordata L. (L.) Gaudich.

Riv.

5 plant045, Oenothera plant046, Typha orientalis plant047, Lindernia plant048, Pharbitis nil (L.) plant049, Chenopodium plant050, Taxodium plant051, Commelina speciosa crustacea (L.) F. Muell Choisy album L. distichum (L.) Rich. bengalensis

plant052, Melia azedarach plant053, Celtis sinensis plant054, Cotoneaster plant055, plant056, Ipomoea batatas plant057, Calystegia plant058, Dendranthema

L. Pers. microphyllus Lindl. Trachelospermum (L.) Lam. hederacea Wall.ex.Roxb. indicum (L.) Des Moul.

jasminoides (Lindl.) Lem.

plant059, Amygdalus plant060, Zea mays L. plant061, Achyranthes plant062, Vigna plant063, Cucurbita plant064, Cucumis sativus plant065, Solanum persica L. bidentata Blume unguiculata (Linn.) Walp. moschata Duch. ex Poiret L. melongena L.

6 plant066, Capsicum plant067, Pittosporum plant068, Aucuba japonica plant069, Artemisia argyi plant070, Amaranthus plant071, Abutilon plant072, Glycine max annuum L. tobira (Thunb.) Ait. var. variegata Levl. et Van. tricolor theophrasti Medicus (Linn.) Merr.

plant073, Sesamum plant074, Cassia tora Linn. plant075, Luffa cylindrica plant076, Allium fistulosum plant077, Lycopersicon plant078, Ipomoea aquatica plant079, Brassica indicum L. (L.) Roem. esculentum Mill. Forsk. caulorapa Pasq.

plant080, Myriophyllum plant081, Zizania latifolia plant084, Fatsia japonica plant085, Hydrocharis plant086, Spirodela plant087, Azolla imbricata plant088, Trapa natans L. verticillatum L. (Griseb. ) Stapf (Thunb.) Decne. et Planch. dubia polyrrhiza (L.) Schleid. (Roxb.) Nakai

7 plant096, Canna indica L. plant097, Mahonia fortunei plant098, Rubus plant099, Clerodendrum plant100, Lophatherum plant101, Phytolacca plant102, Alangium

(Lindl. ) Fedde lambertianus Ser. trichotomum Thunb. gracile acinosa Roxb. chinense (Lour.) Harms

plant103, Cyclosorus plant104, Eriobotrya plant106, Sonchus arvensis plant107, Zehneria plant108, Punica granatum plant109, Toona sinensis (A. plant110, Forsythia interruptus (Willd. ) H. Ito japonica (Thunb.) Lindl. L. japonica (Thunberg) H. Y. L. Juss.) Roem. suspensab (Thunb.) Vahl

Liu）

plant111, Trichosanthes plant112, Ginkgo biloba L. plant113, Pueraria lobata plant114, Pinellia ternata plant118, Melilotus plant119, Kummerowia plant120, Populus kirilowii Maxim. (Willd.) Ohwi officinalis (L.) Pall. striata (Thunb.) Schindl. tomentosa

8 plant121, Cnidium plant122, Celosia argentea plant123, Brassica plant124, Arachis hypogaea plant125, Basella alba L. plant126, Portulaca plant127, Hydrocotyle monnieri (L.) Cuss. L. chinensis L. Linn. oleracea L. vulgaris

plant128, Sorghum bicolor plant129, Gerbera anandria plant130, Eucommia plant131, Lagerstroemia plant132, Prunus cerasifera plant133, Raphanus sativus plant134, Ocimum

(L.) Moench (L.) Sch.-Bip. ulmoides Oliver indica L. Ehrhar (Jacq.) Rehd. L. basilicum L.

plant135, Cucumis melo L. plant136, Abelmoschus plant137, Allium plant139, Cucumis melo L. plant140, Citrullus lanatus plant142, Echinochloa plant143, Digitaria

manihot (Linn.) Medicus tuberosum var. agrestis Naud. (Thunb.) Matsum. et Nakai crusgalli (L.) Beauv. sanguinalis (L. ) Scop.

9 plant144, Miscanthus plant146, Viburnum plant147, Citrus reticulata plant148, Potentilla supina plant149, Pyrus communis plant150, Sedum plant151, Lactuca sativa L. sinensis Anderss. Odoratissimum Ker-Gawl. Blanco Blanco L. sarmentosum Bunge var. ramosa Hort.

plant153, Cyperus plant155, Salvia plebeia R. plant156, Eclipta prostrata plant157, Eleusine indica plant158, Vigna radiata plant159, Artemisia plant160, Spiraea salicifolia rotundus L. Br. (L. ) L. (L.) Gaertn. (Linn.) Wilczek carvifolia

plant161, Ilex cornuta plant162, Ulmus pumila L. plant163, Cerasus plant164, Sapindus plant165, Cycas revoluta plant166, Loropetalum plant167, Paederia foetida

Lindl. et Paxt. yedoensis (Matsum.) Yu et mukorossi Gaertn. Thunb. chinense var.rubrum Yieh

Li

10 plant168, Euonymus alatus plant169, Lygodium plant170, Salvia japonica plant171, Elephantopus plant172, Reineckia plant173, Ludwigia plant174, Juncus

(Thunb.) Sieb. japonicum (Thunb.) Sw. Thunb. scaber L. carnea (Andr.) Kunth prostrata Roxb. effusus L.

plant175, Viola philippica plant176, Phyllanthus plant177, Clinopodium plant178, Coreopsis plant179, Emilia sonchifolia plant180, Sonchus plant181, Teucrium

urinaria L. chinense (Benth.) O. Ktze. drummondii Torr. et Gray (L.) DC. oleraceus L. fruticans

plant182, Verbena plant183, Lonicera japonica plant184, Lysimachia plant185, Centella asiatica plant186, Acer plant187, Pterocarya plant188, Hypericum officinalis L. christinae Hance (L.) Urban buergerianum Miq. stenoptera monogynum L.

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Supplementary Figure 2. The map for sampling site of plant samples in this study. The numbers on the map show the sampling sites where the corresponding numbers of plant sample were collected.

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Supplementary Figure 3. Comparison of the percentage of number of viral sequence reads against total unique reads in individual library between cultivated and wild plants. Each column shows the percentage of the number of viral sequence reads against the total number of unique reads in a single library. The library IDs are showed under the histogram, the red columns (n=63) are results of cultivated plants, while the black columns (n=82) show results of wild plants. The remaining 16 plant samples in which we did not detect viral reads are not included here.

13

Supplementary Figure 4. Comparison of the numbers of virus type detected in cultivated and wild plants. Each column shows the number of virus types in a single library. The library IDs are shown under the histogram, the red columns (n=63) are results of cultivated plants, while the black columns (n=82) show results of wild plants. The remaining 16 plant samples in which we did not detect viral reads are not included here.

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Supplementary Figure 5. Phylogenetic tree of pant-associated viruses belonging in the family Dicistroviridae. Bayesian inference trees were constructed using MrBayes v3.2 based on amino acid sequence of RdRp domain of RNA viruses, NS protein of parvovirus-like viruses, Rep protein of CRESS DNA virus, or Major capsid protein of microvirus. The names of virus strains which were newly identified are shadowed with different colors based on their host species. The silhouetted organism denotes host taxa of viruses identified in the present study and their best matched relatives based on BLASTx searching in GenBank. Virus family or genus within which the certain cluster of viruses belong is shown. Red dots represent 100% posterior probability support for MrBayes.

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Supplementary Figure 6. Viral genome organizations of pant-associated viruses belonging in the family Dicistroviridae. The genome structure of the reference strain and those viruses identified in this study are drawn. Within each genome, the upper gray boxes are ORFs, while the underneath boxes define regions with blast matches to a viral protein or a protein domain whose detailed information is provided under each box. Different protein or domain is marked with different color.

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Supplementary Figure 7. Phylogenetic tree of pant-associated viruses belonging in the family Iflaviridae. Figure legend is the same as Supplementary Figure 5.

17

Supplementary Figure 8. Viral genome organizations of pant-associated viruses belonging in the family Marnaviridae. Figure legend follows Supplementary Figure 6.

18

Supplementary Figure 9. The phylogenetic tree of pant-associated viruses belonging in the family Marnaviridae. Figure legend is the same as Supplementary Figure 5.

19

Supplementary Figure 10. The phylogenetic tree of plant-associated parvovirus-like viruses. Figure legend is same as Supplementary Figure 5.

20

Supplementary Figure 11. Viral genome organizations of the plant-associated parvovirus-like viruses. Figure legend follows Supplementary Figure 6.

21

Supplementary Figure 12. The phylogenetic tree of plant-associated Noda-like viruses. Figure legend is the same as Supplementary Figure 5.

22

Supplementary Figure 13. The phylogenetic tree of plant-associated Permutotetra-like viruses. Figure legend is the same as Supplementary Figure 5.

23

Supplementary Figure 14. The phylogenetic tree of plant-associated Yanvirus-like viruses. Figure legend is the same as Supplementary Figure 5.

24

Supplementary Figure 15. The phylogenetic tree and viral genome organization of plant- associated Chuvirus-like viruses. Figure legend is the same as Supplementary Figure 5 and 6.

25

Supplementary Figure 16. The phylogenetic tree and viral genome organization of plant- associated Bastro-like viruses. Figure legend is the same as Supplementary Figure 5 and 6.

26

Supplementary Figure 17. The phylogenetic tree and viral genome organization of plant- associated Hepe-like viruses. Figure legend is the same as Supplementary Figure 5 and 6.

27

Supplementary Figure 18. The phylogenetic tree of plant-associated Botybirnavirus viruses. Figure legend is the same as Supplementary Figure 5.

28

Supplementary Figure 19. The phylogenetic tree and viral genome organization of plant- associated Narna-like viruses. Figure legend is the same as Supplementary Figure 5 and 6.

29

Supplementary Figure 20. The phylogenetic tree and viral genome organization of plant- associated Picorna-like viruses. Figure legend is the same as Supplementary Figure 5 and 6.

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Supplementary Figure 21. The phylogenetic tree and viral genome organization of plant viruses belonging in the family Tombusviridae. Figure legend is the same as Supplementary Figure 5 and 6.

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Supplementary Figure 22. The phylogenetic tree of plant-associated letuo-like virus and viruses belonging in the family Luteoviridae. Figure legend is the same as Supplementary Figure 5.

32

Supplementary Figure 23. The phylogenetic tree of plant-associated parti-like virus and viruses belonging in the family Partitiviridae. Figure legend is the same as Supplementary Figure 5.

33

Supplementary Figure 24. The phylogenetic tree and viral genome organization of plant- associated Sobemo-like viruses. Figure legend is the same as Supplementary Figure 5 and 6.

34

Supplementary Figure 25. The phylogenetic tree of plant-associated Rhabdo-like viruses. Figure legend is the same as Supplementary Figure 5.

35

Supplementary Figure 26. The phylogenetic tree and viral genome organization of plant- associated Bufivirus-like viruses. Figure legend is the same as Supplementary Figure 5 and 6.

36

Supplementary Figure 27. The phylogenetic tree and genome organization of plant-associated viruses showing significant sequence similarity to viruses belonging in the family Geminiviridae. Figure legend is the same as Supplementary Figure 5.

Replication protein Capsid protein Hypothetical protein Direction of ORF

37

Supplementary Figure 28. The phylogenetic tree and genome organization of plant-associated viruses belonging in the family Genomoviridae. Figure legend is the same as Supplementary Figure 5.

Replication protein Capsid protein Direction of ORF

38

Supplementary Figure 29. The phylogenetic tree of plant-associated Alphasatellitidae viruses. Figure legend is the same as Supplementary Figure 5.

39

Supplementary Figure 30. The phylogenetic tree and genome organization of plant-associated CRESSV Group1 viruses. Figure legend is the same as Supplementary Figure 5.

Replication protein Capsid protein Direction of ORF

40

Supplementary Figure 31. The phylogenetic tree and genome organization of plant-associated CRESSV Group2 viruses. Figure legend is the same as Supplementary Figure 5.

Replication protein Capsid protein Direction of ORF

41

Supplementary Figure 32. The phylogenetic tree and genome organization of plant-associated CRESSV Group3 viruses. Figure legend is the same as Supplementary Figure 5.

Replication protein Capsid protein Direction of ORF

42

Supplementary Figure 33. The phylogenetic tree and genome organization of plant-associated CRESSV Group4 viruses. Figure legend is the same as Supplementary Figure 5.

Replication protein Capsid protein Direction of ORF

43

Supplementary Figure 34. The phylogenetic tree and genome organization of plant-associated CRESSV Group5 viruses. Figure legend is the same as Supplementary Figure 5.

Replication protein Capsid protein Direction of ORF

44

Supplementary Figure 35. The phylogenetic tree and genome organization of plant-associated CRESSV Group6 viruses. Figure legend is the same as Supplementary Figure 5.

Replication protein Capsid protein Direction of ORF

45

Supplementary Figure 36. The phylogenetic tree of plant-associated viruses belonging in the family Microviridae. Figure legend is the same as Supplementary Figure 5.

46

Supplementary Figure 37. The phylogenetic tree of Potyviridae. Figure legend is the same as Supplementary Figure 5.

47

Supplementary Figure 38. Potential cross-species infection of viruses among different species of plants. Neighbor-joining trees based on nucleotide sequence of complete genomes of viruses belonging in Marnaviridae, Geminiviridae, Genomoviridae, Luteoviridae, Parvoviridae, Sobemo-like virus or Unclassified CRESS virus are shown here. Virus strain names are labeled on each branch followed by percentage of viral reads number against total unique reads number in corresponding library. Virus host plant species name are shown, dotted lines are used here to indicate the sampling sites of different species of plant. The collecting plants and its geographical location are connected by dotted lines of different colors. In the same phylogenetic cluster, the color of dotted lines behind virus hosts are the same. Sequence identity based on complete genome between neighboring sequences in the same cluster are labeled between branches in phylogenetic tree.

48

Supplementary Figure 39. The phylogenetic tree of plant viruses belonging in the family Bromoviridae. Figure legend is the same as Supplementary Figure 5.

49

Supplementary Figure 40. The phylogenetic tree of plant viruses belonging in the family Closteroviridae. Figure legend is the same as Supplementary Figure 5.

50

Supplementary Figure 41. The phylogenetic tree of plant viruses belonging in the family Comovirinae. Figure legend is the same as Supplementary Figure 5.

51

Supplementary Figure 42. The phylogenetic tree of plant viruses belonging in the family Botourmiaviridae. Figure legend is the same as Supplementary Figure 5.

52

Supplementary Figure 43. The phylogenetic tree of plant viruses belonging in the family Tymovirales viruses. Figure legend is the same as Supplementary Figure 5.

53