Supplementary material 2 from: Hill E, Milla L, Encinas-Viso F, O'Dwyer J, Gooden B, Hopper M, Roberts J (2025) Surface swabs outperform most traditional honeybee (Apis mellifera) hive samples for recovery of eDNA and eRNA. Metabarcoding and Metagenomics 9: e165436. https://doi.org/10.3897/mbmg.9.165436

OFFICIAL OFFICIAL Supplementary File 2: Supporting tables and figures Table S1 Total number of reads, mean reads per sample, total unique OTUs/taxa identified per sample type, and mean unique taxa per sample identified post cleaning for each substrate and marker. Values in parentheses represent standard deviations, and means are calculated across all individual samples. Bacteria (16S) Animal (COI) Fungi (ITS1) Plant (ITS2) Substrate Total reads Mean reads Total unique taxa Mean unique taxa Total reads Mean reads Total unique taxa Mean unique taxa Total reads Mean reads Total unique taxa Mean unique taxa Total reads Mean reads Total unique taxa Mean Unique taxa Hive Debris 22761 843 (901.46) 33 22.04 (4.91) 43502 1611.19 (2249.56) 4 2.59 (0.84) 329247 12194.33 (7281.02) 195 42.59 (12.54) 510953 18924.19 (10149.67) 56 18.74 (4.6) Hive Entrance 20685 795.58 (598.58) 32 21.85 (3.73) 52906 2300.26 (3999.39) 4 2.83 (0.83) 347253 13355.88 (6983.56) 232 56.58 (12.33) 512808 19723.38 (8964.55) 55 20.88 (4.71) Honey 5310 295 (202.87) 33 17.89 (4.56) 9815 613.44 (559.74) 4 1.81 (0.75) 42990 2528.82 (1537.82) 160 36.41 (13.19) 187170 11010 (9130.55) 51 23.76 (8.4) Internal Pollen 2969 70.69 (111.05) 27 5.4 (3.11) 937 58.56 (120) 4 1.5 (0.73) 280580 6680.48 (4605.41) 180 21.36 (8.12) 1038153 24717.93 (12286.53) 42 12.9 (3.16) Pollen Trap 2016 118.59 (62.37) 23 8.24 (2.97) 4629 420.82 (697.35) 4 1.64 (0.67) 178499 9916.61 (4110.64) 151 26.78 (7.46) 378011 21000.61 (14444.85) 43 15.33 (3.12) Return Forager Bees 105116 5839.78 (2378.66) 28 18.72 (1.93) 13140 730 (619.34) 4 2.56 (0.7) 168269 9348.28 (4433.22) 137 26.89 (6.92) 525715 29206.39 (13987.88) 33 13 (3.53) Top Box Bees 133221 7401.17 (3788.68) 27 18.5 (1.1) 13574 754.11 (573.6) 4 2.5 (0.51) 55944 3108 (2741.13) 90 13.33 (4.81) 286861 15936.72 (7693.5) 41 14.11 (3.76) Total 292078 1759.51 (3010.24) 35 15.19 (7.69) 138503 1073.67 (2121.86) 4 2.3 (0.87) 1402782 8450.49 (6301.94) 309 32.19 (16.8) 3439671 20720.91 (12107.32) 62 16.62 (5.87) OFFICIAL OFFICIAL Table S2 Significance comparisons between number of taxa observed between each sample type for markers ITS (fungi) and ITS2 (plants). HD = hive debris; HE = hive entrance; Ho = honey; IP = internal pollen; PT = pollen trap; RFB = return forager bees; TBB = top box bees. Significant differences between number of taxa observed based on t-test are shown in bold. ITS2 ITS HD HE Ho IP PT RFB TBB HD 1 1 0.168 0.098 0.026 0.061 HE 0.013 1 0.017 0.009 0.002 0.005 Ho 0.311 0.004 0.281 0.276 0.137 0.19 IP 0.0002 4.73E-06 0.238 1 1 1 PT 0.0004 5.83E-06 0.68 0.68 1 1 RFB 0.0002 7.85E-06 0.68 0.444 0.785 1 TBB 7.56E-08 1.63E-07 0.011 0.08 0.002 0.0002 Table S3 Significance comparisons between number of taxa observed between each sample type for markers 16S (bacteria) and COI (animal). HD = hive debris; HE = hive entrance; Ho = honey; IP = internal pollen; PT = pollen trap; RFB = return forager bees; TBB = top box bees. Significant differences between number of taxa observed based on t-test are shown in bold. COI 16S HD HE Ho IP PT RFB TBB HD 1 0.657 0.038 0.323 1 1 HE 1 0.56 0.032 0.261 1 1 Ho 0.088 0.038 0.53 1 0.56 0.536 IP 2.11E-06 7.43E-09 4.71E-07 1 0.029 0.053 PT 6.04E-06 9.38E-08 1.01E-05 0.088 0.232 0.207 RFB 0.018 0.001 0.693 4.71E-07 2.76E-05 1 TBB 0.011 0.0002 0.296 2.69E-06 6.59E-05 1 Table S4 Total number of reads assigned to contigs and unique viruses identified post-filtering for each substrate. Substrate Total reads Contig length range Average contig length Number of taxa Hive Entrance 4,243,239 535-7806 2833.81 29 Honey 130,845 503-5337 1298.83 42 Internal Pollen 11,846,161 573-9343 3865.82 10 Top Box Bees 1,099,671 594-8460 3544.36 24 Total 17,319,916 503-9343 3519.49 67 OFFICIAL OFFICIAL Fig.S1 Shannon diversity indices across each sample type for bacteria (16S), animal (COI), fungi (ITS) and plant (ITS2) metabarcoding markers. Hive Debris Swabs Hive Entrance Swabs Honey Internal Pollen Pollen Trap Return Forager Bees Top Box Bees 1 2 Alpha Diversity Measure Bacteria (16S) Hive Debris Swabs Hive Entrance Swabs Honey Internal Pollen Pollen Trap Return Forager Bees Top Box Bees 0.00 0.25 0.50 0.75 1.00 Alpha Diversity Measure Animal (COI) Hive Debris Swabs Hive Entrance Swabs Honey Internal Pollen Pollen Trap Return Forager Bees Top Box Bees 1.0 1.5 2.0 2.5 3.0 Alpha Diversity Measure Fungi (ITS1) Hive Debris Swabs Hive Entrance Swabs Honey Internal Pollen Pollen Trap Return Forager Bees Top Box Bees 0.5 1.0 1.5 2.0 Alpha Diversity Measure Plants (ITS2) OFFICIAL OFFICIAL Fig.S2 Species accumulation curves for bacteria (16S), fungi (ITS) and plant (ITS2) metabarcoding markers. 2 4 6 8 0 5 10 15 20 25 30 Bacteria (16S) Cumulative number of samples Number of taxa detected Hive Entrance Swabs Hive Debris Swabs Pollen Trap Internal Pollen Honey Return Forager Bees Top Box Bees 2 4 6 8 1 2 3 4 5 Animal (COI) Cumulative number of samples Number of taxa detected Hive Entrance Swabs Hive Debris Swabs Pollen Trap Internal Pollen Honey Return Forager Bees Top Box Bees 2 4 6 8 0 50 100 150 200 Fungi (ITS1) Cumulative number of samples Number of taxa detected Hive Entrance Swabs Hive Debris Swabs Pollen Trap Internal Pollen Honey Return Forager Bees Top Box Bees 2 4 6 8 0 10 20 30 40 50 Plants (ITS2) Cumulative number of samples Number of taxa detected Hive Entrance Swabs Hive Debris Swabs Pollen Trap Internal Pollen Honey Return Forager Bees Top Box Bees OFFICIAL OFFICIAL Fig.S3 Top 10 eukaryote species (excluding A. mellifera) identified in raw RNA sequencing outputs for each sample type. All species are plants except A. melanogenum which is a fungus. 0.00 0.25 0.50 0.75 1.00 Hive Entrance Swabs Honey Internal Pollen Top Box Bees Proportion of reads Aureobasidium melanogenum Camellia lanceoleosa Camellia sinensis Escallonia herrerae Escallonia rubra Eucalyptus grandis Heracleum sosnowskyi Macadamia integrifolia Telopea speciosissima Top 10 Eukaryote Species OFFICIAL OFFICIAL Fig.S4 Shannon diversity indices across each sample type for RNA viruses. Hive Entrance Swabs Honey Internal Pollen Top Box Bees 0.5 1.0 1.5 2.0 Alpha Diversity Measure Shannon diversity index for RNA data