Online Supplementary Information Diverse Soil RNA Viral Communities Have the Potential to Influence Grassland Ecosystems Across Multiple Trophic Levels

Online Supplementary Information Diverse soil RNA viral communities have the potential to influence grassland ecosystems across multiple trophic levels

Luke S. Hillary, Evelien M. Adriaenssens, David L. Jones and James E. McDonald Supplementary Table 1 – Sampling site descriptions.

Site Location Soil classification Soil description Altitude pH Electrical Total (m asl) conductivity carbon (µs cm-1) (%) A Upland 53° 13' 1.22" N Non-calcaric Very acid upland soil with a wet 431 4.27 39 29.1 peatland 4° 1' 8.78" W lithosol highly organic topsoil B Upland 53°13'33.00" N Typical podzolic Freely draining acid loamy soil 289 5.89 30 11.3 grassland 4° 0' 54.86" W brown soil over rock C Semi-improved 53° 13' 55.24" N Typical podzolic Freely draining slightly acid 77 4.61 27 11.2 grassland 4° 1’ 2.22” W brown soil loamy soil D Lowland 53° 14' 10.98" N Typical orthic Sandy clay loam, freely draining 19 5.78 42 3.62 grassland 4° 1' 1.74" W brown soil sheep-grazed soil E Coastal 53° 14' 34.17" N Saline alluvial Silt-textured, poorly draining soil 3 8.03 1810 2.89 grassland 4° 1' 18.78" W gley soil with periodic tidal inundation

Soils were classified according to Avery (1990). The major properties of the sites and soils are shown in Table S1 above, while a general description of the catena sequence is provided in Farrell et al. (2014) and Withers et al. (2020). The altitudinal gradient represents a primary productivity gradient with more intensive agricultural production at Site D which receives regular fertiliser and lime applications. The mean annual temperature at the bottom and top sites was 10.2 and 7.3 °C respectively, while the gradient in annual rainfall was 1065 to 1690 mm, respectively. All sites had a different vegetation cover (all dominated by grasses) and were grazed by Welsh mountain sheep (Ovis aries L.). Soil pH and electrical conductivity were measured in1:2.5 (w/v) soil-to-distilled water extracts using standard electrodes. Total C and N were determined on a TruSpec CN analyser (Leco Corp., St Joseph, MI). Site E is a soil developed on recent marine deposits and contains CaCO3 from shell deposits. Its pedogenic age is ca. 500 years. All other sites have a pedogenic age of ca. 10,000 years. Site A is developed on rhyolite, Sites B and C on Ordovician schist and shale, and Site D on mixed glacial till. Sites D and E rarely undergo freezing, while Sites A-C experience periodic freezing in winter with winter snow cover often present at Site A. The vegetation at Site A comprises Festuca ovina L., Juncus effusus L. and Trichophorum cespitosum (L.) Hartman. The vegetation at Site B is dominated by Agrostis canina L., Agrostis capillaris L., Anthoxanthum odoratum L. and Potentilla erecta (L.) Rauschel. The vegetation at Site C is dominated by Festuca ovina L. and Pteridium aquilinum L.. Site D is dominated by Lolium perenne L. and Trifolium repens L. while the vegetation at site E is dominated by Plantago maritima, Festuca sp. and Salicornia europaea. References Avery, B.W. (1990) Soils of the British Isles. CAB International, Wallingford, UK. Farrell, M., Macdonald, L.M., Hill, P.W., Wanniarachchi, S.D., Farrar, J., Bardgett, R.D., Jones, D.L. (2014) Amino acid dynamics across a grassland altitudinal gradient. Soil Biology and Biochemistry 76, 179-182 Withers, E., Hill, P.W., Chadwick, D.R., Jones, D.L. (2020) Use of untargeted metabolomics for assessing soil quality and microbial function. Soil Biology and Biochemistry 143, 107758.

a b

25% 50%

Upland peatland Upland peatland Upland grassland Upland grassland Semi-improved Semi-improved Lowland grassland Lowland grassland Coastal grassland Coastal grassland

c d

75% 95%

Upland peatland Upland peatland Upland grassland Upland grassland Semi-improved Semi-improved Lowland grassland Lowland grassland Coastal grassland Coastal grassland

Supplementary Figure 1 – Comparison of co-occurring vOTUs at (a) 25%, (b) 50%, (c) 75% and (d) 95% horizontal genome coverage thresholds. Similar conclusions on vOTU sharing between sampling sites when varying the alignment fraction required to judge a vOTU present within a sample. vOTUs are most commonly found within one site with the grassland sites sharing more common vOTUs than other habitats. The costal grassland site consistently shared the least number of vOTUs with the other habitats.

(a) Lenarviricota

(b) Pisuviricota

(d) Duplornaviricota

(e) Negarnaviricota

Supplementary Figure 2 – enlarged phylogenetic trees corresponding to those shown in figure 4 in the main mansucript. Branches with branch support ³0.6 are indicated by red circles. Upland peatland Upland grassland Semi-improved Lowland grassland Coastal grassland

Supplementary Figure 3 – distribution of putative ourmia-like viruses between sites. Within this study, these viruses are predominantly found in upland grassland/ peatland soils. (a)

YP 009337049.1 Tree scale: 1 YP 009333524.1 YP 009336613.1 YP 009333511.1 YP 009333273.1 YP 004063985.1 Mud crab virus YP 009333162.1 AAT81157.2 Taura syndrome virus Branch Support YP 006666503.2 Macrobrachium rosenbergii aTihu virus YP 009333197.1 YP 009333502.1 YP 009336690.1 YP 009333603.1 ≥ 0.6 YP 009329817.1 Bivalve RNA virus G1 YP 009333551.1 k127 944836 1 YP 009333180.1 k127 15261 1 YP 009329822.1 Bivalve RNA virus G5 YP 009336743.1 k127 9924 1 Study k127 904981 1 k127 999981 1 MN034545.1 1 k127 326780 1 This study k127 792793 1 k127 3091716 1 k127 1712756 1 k127 311490 1 k127 1566250 1 Starr et al. 2019 k127 1442436 1 YP 009337313.1 APG77328.1 MN034577.1 1 YP 009345032.1 Wolf et al. 2018 ABX74939.1 Rhopalosiphum padi virus APG78421.1 Aphid lethal paralysis virus k127 3053223 1 k127 1444601 1 k127 1699692 1 APG77985.1 k127 1975624 1 k127 2460238 1 Taxa k127 2538930 1 k127 2137431 1 k127 429463 3 k127 1633344 1 Dicistroviridae k127 442019 1 APG78622.1 YP 164440.1 Solenopsis invicta virus 1 YP 009336557.1 NP 066241.1 Acute bee paralysis virus AQY03950.1 Israeli acute paralysis virus YP 008888535.1 Formica exsecta virus 1 YP 009336838.1 YP 009336994.1 k127 369265 1 k127 2021044 2 k127 411627 1 APG78016.1 YP 009333547.1 YP 009333456.1 APG78061.1 k127 1500808 1 k127 564213 1 k127 1023714 1 k127 2559978 1 k127 249399 1 YP 009333200.1 YP 009333386.1 YP 009345054.1 k127 1118408 1 k127 2796739 1 k127 344793 1 k127 225184 1 k127 1660666 1 k127 1943093 1 k127 27502 2 k127 2044142 1 k127 1129322 1 YP 009336583.1 YP 009336571.1 AMO03208.1 Empeyrat virus YP 009252204.1 Anopheles C virus NP 647481.1 Cricket paralysis virus NP 044945.1 Drosophila C virus k127 3164641 1 k127 1196617 1 APG77930.1 k127 1320831 1 k127 2419857 1 k127 2536722 3 k127 2109415 1

(b)

Upland peatland Upland grassland Semi-improved Lowland grassland Coastal grassland

Supplementary Figure 4 – Pruned phylogenetic tree (a) and site distribution (b) of putative dicistro-like viruses. Viruses were predominantly found within upland/ semi-improved sites. Tree scale: 1 APG78228.1 APG78246.1 APG78273.1 APG78241.1 k127 2779331 1 AAP74192.1 YP 007419077.1 Rosellinia necatrix partitivirus 2 k127 1028089 1 YP 009273018.1 Arabidopsis halleri partitivirus 1 ALT00589.1 Raphanus sativus partitivirus 1 ANQ45203.1 Rose partitivirus k127 2976777 1 ABJ99996.1 Vicia faba partitivirus 1 Branch Support ALM62247.1 Soybean leaf-associated partitivirus 2 MN034289.1 1 ≥0.6 YP 009272944.1 Sophora japonica powdery mildew-associated partitivirus AFX73022.1 Grapevine partitivirus MN032724.1 1 Study MN032857.1 1 MN033957.1 1 MN035338.1 1 This study BAH08700.1 Flammulina velutipes isometric virus YP 003082248.1 Sclerotinia sclerotiorum partitivirus S Starr et al. 2019 MN036296.1 1 ANR02695.1 Rhizoctonia solani partitivirus 1 Wolf et al. 2018 MN033310.1 1 AND83003.1 Rhizoctonia solani partitivirus 1 (partial sequence) MN033699.1 2 BAT32942.1 Rosellinia necatrix partitivirus 7 Taxa Host association BAM36403.1 Rosellinia necatrix partitivirus 5 BAQ36631.1 Fusarium solani partitivirus 2 Dicistroviridae MN035912.1 1 Fungi associated k127 2706404 1 Plant associated k127 2604800 1 Partitiviridae k127 3120855 1 k127 393660 1 Picornaviridae k127 2586047 1 k127 2455837 1 Potyviridae k127 576665 1 k127 1289117 1 k127 85153 1

Supplementary Figure 5 – Pruned phylogenetic tree of putative partiti-like viruses. The majority of partiti-like vOTUs identified in this study are relatively closely related to Fusarium solani partitivirus 2 (indicated by short branch lengths at the bottom of the figure). APG76202.1 Tree scale: 1 YP 009173859.1 Orsay virus YP 004221742.1 Santeuil nodavirus k127 2771575 1 k127 542449 1 APG76216.1 NP 620109.1 Pariacoto virus APG76090.1 YP 009337883.1 Branch Support AIO11151.1 Mosinovirus APG76502.1 YP 009175040.1 Le Blanc nodavirus AQY59901.1 ≥ 0.6 YP 009342252.1 YP 009337285.1 APG76454.1 APG76507.1 APG76209.1 APG76200.1 Study k127 3249532 1 APG76117.1 APG76116.1 APG76104.1 YP 009329984.1 This study APG76593.1 APG76164.1 APG76103.1 YP 009333376.1 Starr et al. 2019 APG76642.1 NP 077730.1 Nodamura virus YP 009337806.1 k127 2458157 1 Wolf et al. 2018 k127 931303 1 APG76125.1 YP 004207810.1 Penaeus vannamei nodavirus NP 919036.1 Macrobrachium rosenbergii nodavirus APG76319.1 AKP18615.1 Lutzomyia nodavirus APG76281.1 k127 1820739 1 APG76300.1 Taxa APG76332.1 APG76600.1 APG76466.1 APG76486.1 Nodaviridae ABS29339.1 Flock House virus NP 689439.1 Boolarra virus k127 3393807 1 k127 724126 1 k127 1403543 1 k127 1044899 1 k127 2442395 1 k127 1031125 1 k127 2616941 1 APG76295.1 AII82234.1 ADK97710.1 k127 1669650 1 k127 3193470 1 k127 2831791 1 k127 2850814 2 k127 2466107 1 k127 384702 1 YP 009329958.1 YP 009330099.1 APG76285.1 APG76171.1 k127 240129 1 APG76124.1 APG76208.1 APG76582.1 APG76286.1 APG76289.1 APG76360.1 APG76585.1 APG76186.1 YP 009337844.1 APG76581.1 YP 009333380.1 AGQ56677.1 Dicentrarchus labrax betanodavirus YP 009333372.1 YP 009329890.1 k127 3291927 1 YP 009333611.1 k127 1468481 1 k127 1134143 1 k127 1844969 1 APG76338.1 ADI48250.1 APG76320.1 k127 2169659 1 YP 009336769.1 APG76388.1 AMO03241.1 AKH40302.1 APG76091.1 ADF97523.1 Alphanodavirus HB-2007/CHN YP 009337232.1 APG76311.1 YP 009336491.1 APG76471.1 sp|Q3KSM3.1|RDRP PRV YP 009337377.1 YP 009337881.1 YP 009337879.1 (a) (b)

Upland peatland Upland grassland Semi-improved Lowland grassland Coastal grassland

Supplementary Figure 6 – Enlarged phylogenetic tree (a) and site distribution (b) of noda-like viruses found in this study clustering near reference Nodaviridae RdRP sequences (a). Viral sequences were predominantly found in upland areas, with no noda-like viruses found in the coastal grassland site.