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The following supplement accompanies the article

Site-specific environmental factors control bacterial and viral diversity in stormwater retention ponds Matthew A. Saxton, Nuha S. Naqvi, Faraz Rahman, Charleton P. Thompson, Randolph M. Chambers, James M. Kaste, Kurt E. Williamson*

*Corresponding author: kewilliamson@wm.edu

Aquatic Microbial Ecology 77: 23–36 (2016)

Table S1. Physical, geochemical and biological variables measured from coastal Virginia July 2012. Temp, Temperature; Cond, Conductivity; DO, dissolved oxygen; TDP, total dissolved phosphorus; DOC, dissolved organic carbon; chl a, chlorophyll a concentration; BacAB, bacterial abundance; VirusAB, virus abundance; viral richness via RAPD; bac richness, bacterial richness via tRFLP; BacProd, bacterial production. Bacterial and viral abundance values are the mean of duplicate samples ± standard deviation. Bacterial production values are the mean of triplicate incubations ± standard deviation. Pond Coordinates

Decimal Minutes

Drainage Area

hectare

Impervious Area

hectare

Percent Impervious

cover

Temp °C

Cond µS m-1

DO mg L-1

Salinity ppt

pH TDP µg L-

NOx µg L-1

NH4 µg L-1

DOC ppm

chl a µg L-1 ±SD

BacAB 105cells mL-1

±SD

VirusAB 106 VLP mL-1

±SD

viral richness

JT 37.259870°,  -76.737629°  

11.75 3.15 26.82 34.9 70.5 11.4 0 7.15 4.34 22.9 0.4 7.95 601.48±15.1 24.3±8.90 32.5±5.73 11

GD 37.271414°,  -76.716540°  

12.48 4.1 32.69 28 390 3.45 0.2 7 0.48 3.5 4.4 5.75 215.83±19.05 3.73±2.47 5.04±2.35 10

WM 37.274315°,   -76.757833°  

1.79 0.60 33.41 36.2 82 8.6 0 7.99 0.14 1.1 BDL 5.68 28.43±16.19 7.32±0.59 29.1±2.72 5

NT 37.277627°,  -76.743874°  

43.51 28.28 64.99 33 132.2 5.7 0.1 7.09 0.95 0.5 0.3 7.09 49.47±5.71 6.59±0.01 9.86±0.54 10

WL 37.347052°,  -76.736233°  

5.70 4.27 75.03 33.4 18.4 9.2 0 5.41 0.14 1.1 BDL 3.33 61.07±5.41 5.57±0.84 8.00±2.53 14

MT 37.342843°,  -76.741803°  

Data not available

Data not available

N/A 35 110.5 10.81 0 7.8 0.95 4.1 0.3 13.71 242.10±0 10.9±0.12 3.70±0.14 14

JHS 37.251069°,   -76.791905°  

14.14 1.96 13.82 37 71.8 6.35 0 7.35 2.62 17 BDL 4.54 716.65±19.83 7.33±1.33 16.5±2.39 11

GS 37.248892°,  -76.787857°  

6.46 0.69 10.62 35.5 55 11 0 7.66 0.48 2.6 BDL 4.82 118.88±0 3.12±2.07 7.76±7.67 5

PA 37.285761°,  -76.732202°  

2,79 1.27 45.50 29.8 35.5 4.1 0 6.11 3.48 27 BDL 3.71 73.08±22.50 3.06±0.03 4.37±0.31 9

CD 37.270661°,  -76.714076°  

5.70 2.14 37.59 29.1 254 6.59 0.1 7.8 1.57 3.2 11 5.57 744.85±317.69 3.35±0.32 14.0±0.60 16

HN 37.358448°,  -76.767256°  

6.79 1.40 20.59 32 43.5 11.5 0 7.18 0.19 BDL 18.8 4.12 163.54±7.83 5.58±0.59 9.25±0.96 11

SP 37.265934°,  -76.742532°  

1.44 0.97 67.51 27.7 43.5 3.95 0 7.26 1.76 2.9 BDL 5.67 59.46±11.99 5.68±0.10 5.17±0.15 16

MK 37.254448°,  -76.640455°  

6.67 5.10 90.00 33.1 17.5 9.36 0 7.32 0.24 BDL BDL 2.72 39.88±0.03 2.95±0.10 3.45±0.18 13

MB 37.256700°,  -76.642349°  

10.67 7.04 66.00 34 81.8 10.5 0 7.76 0.62 BDL BDL 4.86 200.03±19.83 3.21±0.54 4.00±0.21 14

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Pond Coordinates Decimal Minutes

Drainage Area

hectare

Impervious Area

hectare

Percent Impervious

cover

Temp °C

Cond µS m-1

DO mg L-1

Salinity ppt

pH TDP µg L-

NOx µg L-1

NH4 µg L-1

DOC ppm

chl a µg L-1 ±SD

BacAB 105cells mL-1

±SD

VirusAB 106 VLP mL-1

±SD

viral richness

RR 37.302226°,  -76.729164°  

62.62 Data not available

NA 31.7 66.1 10.1 0 7.85 0.76 1.7 0.3 4.12 297.46±39.66 3.92±4.33 3.28±3.75 8

KW 37.236458°,  -76.731598°  

4.38 1.72 39.21 30.2 48.5 6.52 0 7.5 0.57 2 0.4 5.87 56.86±0 4.26±0.27 6.96±0.38 10

PJ 37.241049°,  -76.778123°  

13.74 4.04 29.41 28.8 40.5 11.2 0 7.53 9.49 14.4 BDL 6.32 2060.72±254.04 5.37±0.86 10.1±1.15 11

FF 37.233691°,  -76.776248°  

5.47 1.28 23.41 33.4 63.8 6.9 0 7.67 0.48 BDL BDL 4.00 67.94±8.38 5.16±0.36 20.4±2.64 11

MP 37.311964°,  -76.786368°  

8.52 3.49 40.99 28.5 42.7 0.4 0 6.05 0.95 2.2 0.1 7.12 106.68±0.54 6.77±1.20 10.3±0.54 11

LG 37.315057°,  -76.786702°

6.79 2.59 38.09 35.6 72.5 9.5 0 8.02 3.58 15.8 BDL 7.03 137.22±3.04 3.75±0.01 16.4±0.97 17

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Table S2. Bioenv analysis results showing the set of environmental variables that best describe viral or bacterial community differences. VirusAB; viral abundance, BacAB; bacterial abundance, v-rich; viral richness, Temp; temperature, b-rich; bacterial richness, NOx; NO2 + NO3, Cond; conductivity, drainage area; total drainage area, imperv; impervious drainage area, % imperv, percent impervious drainage area, DO; dissolved oxygen

Data Set Variables r

Bacterial community via tRFLP vs environmental variables

VirusAB 0.4548 NOx, VirusAB 0.4467 Cond, BacAB, VirusAB 0.4858 Cond, NOx, BacAB, VirusAB 0.4749 Cond, NOx, BacAB, VirusAB, v-rich 0.4260

Viral community via RAPD vs environmental variables

imperv 0.1582 Drainage area, VirusAB 0.2092 Drainage area, % imperv, VirusAB 0.2563 Drainage area, % imperv, VirusAB, b-rich 0.2648 Drainage area, % imperv, VirusAB, b-rich, imperv 0.2587 Drainage area, % imperv, DO, VirusAB, b-rich, imperv 0.2539 Drainage area, % imper, DO, BacAB, VirusAB, b-rich, imperv 0.2398

Bacterial community via 16S sequence vs environmental variables

pH 0.5728 pH, b-rich 0.7619 pH, NH4, chla 0.7422 % imperv, pH, NH4, chla 0.7126 Drainage area, DO, pH, NH4, chla 0.7296 Drainage area, % imperv, DO, pH NH4, chla 0.7110 Drainage area, % imperv, DO, pH, NH4, BacAB, b-rich 0.6924 Drainage area, % imperv, DO, pH, NH4, chla, BacAB, b-rich 0.6617 Drainage area, % imperv, Temp, DO, pH, NH4, chla, BacAB, b-rich 0.6415 Drainage area, % imperv, Temp, DO, pH, NH4, chla, BacAB, vrich, b-rich 0.6360

Table S3. Sequencing results and alpha diversity measurements for bacterial community as determined via 16S rRNA amplicon sequencing. Alpha diversity measurements are rarefied to lowest read number. PD; phylogenetic diversity.

Pond Reads OTUs Chao1 InverseSimpson Faith’sPD GD 9411 353 394.2 21.8 20.0 CD 10904 435 505.8 29.8 24.4 MP 10959 272 304.3 22.1 15.7 GS 14005 451 512.0 65.0 23.5 MT 11041 409 472.5 47.2 22.4 JHS 12880 402 446.6 47.8 20.4 LG 12154 456 507.0 55.3 23.7 JT 10436 353 418.7 14.6 20.0

Table S4. Classification of reads affiliated with known virus sequences

Group Order Family CD (%) JHS (%) GS (%) JT (%) dsDNA viruses 23.44 64.06 79.37 27.36 Unassigned Adenoviridae 0.01 0.02 0.02 0.00 Ascoviridae 0.03 0.09 0.02 0.20 Baculoviridae 0.00 0.02 0.01 0.00 Caudovirales 20.01 56.52 66.64 22.44 Myoviridae 4.45 7.75 8.86 5.03 Podoviridae 8.85 28.07 37.72 6.87 Siphoviridae 6.27 19.24 18.90 10.02 unclassified 0.45 1.47 1.16 0.53 Herpesvirales Herpesviridae 0.00 0.10 0.01 0.03 Iridoviridae 0.09 0.05 0.12 0.04 Mimiviridae 0.01 0.01 0.00 0.01 Phycodnaviridae 0.15 0.43 0.58 0.41 Polydnaviridae 0.07 0.17 0.01 0.00 Poxviridae 0.01 0.01 0.00 0.01

unclassified dsDNA phages 2.84 6.16 11.29 3.71

unclassified dsDNA viruses 0.11 0.47 0.67 0.42

ssDNA viruses 75.85 34.10 16.65 70.82 Circoviridae 4.23 0.74 0.70 24.34 Geminiviridae 2.87 0.00 0.00 0.00 Microviridae 65.18 30.53 14.92 38.10 Nanoviridae 0.00 0.06 0.37 0.00

unclassified ssDNA viruses 3.55 2.78 0.66 8.39

ssRNA viruses 0.00 0.00 0.00 0.67 Satellites 0.00 0.00 2.73 0.00 unclassified viruses 0.59 1.00 1.24 0.75 unclassified virophages 0.13 0.84 0.03 0.39

Assignments computed via the GAAS tool (Angly et al., 2009), from a BLASTx comparison with NCBI refseq complete viral genomes proteins (E-value < 10-5). Bolded values indicate majority representations in each metagenomes; CD, Crim Dell; JHS, Jamestown High School; GS, Greensprings; JT, John Tyler.

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Table S5. Most represented viral genotypes within each metagenome according to GAAS

Metagenome Viral Genotype Relative abundance (%)

Host

CD Chlamydia phages 51 B Marine gokushovirus 10 B Bdellovibriophage phiMH2K 5 B Circovirus-like genome SAR-A 3 E Finch circovirus 2 E Acartia tonsa copepod circovirus 2 E African cassava mosaic virus 2 E Vibriophage VvAW1 1 B Myxococcus phage Mx8 1 B JHS Chlamydia phages 21 B Vibriophage VvAW1 7 B Marine gokushoviruses 6 B Thalassomonas phage BA3 5 B Bdellovibriophage phiMH2K 3 B Myxococcus phage Mx8 2 B Dragonfly-associated microphage 1 2 E Sulfitobacter phage pCB2047-A 2 B Brucella phage Pr 1 B Pelagibacter phage HTVC010P 1 B Rhodococcus phage RRH1 1 B GS Myxococcus phage Mx8 17 B Chlamydia phages 12 B Sulfitobacter phage pCB2047-A 6 B Pelagibacter phage HTVC010P 4 B Brucella phage Pr 3 B Verbesina encelioides leaf curl alphasatellite 3 E Bdellovibriophage phiMH2K 2 B Pseudomonas phage F10 2 B Bacillus phage TP21-L 2 B Synechococcus phage S-RIP1 1 B Burkholderia phage Bcep 22 1 B Brucella phage Tb 1 B Salmonella phage FSL SP-031 1 B Psychrobacter phage pOW20-A 1 B JT Chlamydia phages 23 B Bdellovibrio phage phiMH2K 15 B Cyclovirus Bat/USA/2009 8 E Circovirus-like genome RW-C 6 E Beak and feather disease virus 5 E Finch circovirus 4 E Starling circovirus 2 E Cyclovirus PKgoat21/PAK/2009 2 E

CD, Crim Dell; JHS, Jamestown High School; GS, Greensprings; JT, John Tyler; B, Bacteria; E, Eukaryote. Only viral genotypes with relative abundance ≥ 1% are shown

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Table S6. Classification of reads affiliated with prokayotic sequences

Phylum CD JHS GS JT Firmicutes 1.47 2.19 3.40 9.21 Proteobacteria 12.42 14.02 25.83 6.39 Bacteroidetes 1.10 1.08 2.16 2.00 Actinobacteria 0.78 0.84 0.96 1.12 Cyanobacteria 0.27 0.34 0.18 0.35 Euryarchaeota 0.37 0.26 0.19 0.24 Chloroflexi 3.27 2.37 0.79 0.10 Verrucomicrobia 0.20 0.17 0.04 0.16 Chlamydiae 0.33 0.09 0.10 0.08

Assignments made according to best BLASTx hit (E-value cutoff 10-5) using MG-RAST. Bolded values indicate majority representation within a metagenome; CD, Crim Dell; JHS, Jamestown High School; GS, Greensprings; JT, John Tyler.

Table S7. Assembly of Viral Contigs

Metagenome CD JHS GS JT Total contigs 1062 1097 894 702 Avg. contig size, bp 1,484 1,683 1665 1360 Largest contig, bp 36,026 35,771 33,169 16,048 Reads assembled (%) 32,704 (55.4) 23,573 (46.3) 41,149 (66.3) 1,983 (35.4) Singletons (%) 14,944 (25.3) 16,957 (33.3) 8,140 (13.1) 10,605(46.9) CD, Crim Dell; JH, Jamestown High School; GS, Greensprings; JT, John Tyler.

Table S8. Mapping of reads to fully-sequenced reference genomes

Metagenome CD JHS GS JT Most abundant reference genome

Chlamydia phage 2

Vibriophage VvAW1

Myxococcus phage Mx8

Bdellovibriophage phiMH2K

Genome type ssDNA, circular

dsDNA, circular dsDNA, circular ssDNA, circular

Genome size (bp) 4,563 38,682 49,534 4,594 Coverage (%) 76.70 27.14 18.17 65.30 CD, Crim Dell; JH, Jamestown High School; GS, Greensprings; JT, John Tyler

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Table S9. Annotation of longest contigs obtained from assembly

Metagenome Viral Genotype Relative abundance (%) Host CD Chlamydia phages 51 B Marine gokushovirus 10 B Bdellovibriophage phiMH2K 5 B Circovirus-like genome SAR-A 3 E Finch circovirus 2 E Acartia tonsa copepod circovirus 2 E African cassava mosaic virus 2 E Vibriophage VvAW1 1 B Myxococcus phage Mx8 1 B JHS Chlamydia phages 21 B Vibriophage VvAW1 7 B Marine gokushoviruses 6 B Thalassomonas phage BA3 5 B Bdellovibriophage phiMH2K 3 B Myxococcus phage Mx8 2 B Dragonfly-associated microphage 1 2 E Sulfitobacter phage pCB2047-A 2 B Brucella phage Pr 1 B Pelagibacter phage HTVC010P 1 B Rhodococcus phage RRH1 1 B GS Myxococcus phage Mx8 17 B Chlamydia phages 12 B Sulfitobacter phage pCB2047-A 6 B Pelagibacter phage HTVC010P 4 B Brucella phage Pr 3 B Verbesina encelioides leaf curl alphasatellite 3 E Bdellovibriophage phiMH2K 2 B Pseudomonas phage F10 2 B Bacillus phage TP21-L 2 B Synechococcus phage S-RIP1 1 B Burkholderia phage Bcep 22 1 B Brucella phage Tb 1 B Salmonella phage FSL SP-031 1 B Psychrobacter phage pOW20-A 1 B JT Chlamydia phages 23 B Bdellovibrio phage phiMH2K 15 B Cyclovirus Bat/USA/2009 8 E Circovirus-like genome RW-C 6 E Beak and feather disease virus 5 E Finch circovirus 4 E Starling circovirus 2 E Cyclovirus PKgoat21/PAK/2009 2 E

CD, Crim Dell; JHS, Jamestown High School; GS, Greensprings; JT, John Tyler; B, Bacteria; E, Eukaryote

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Table S10. Annotation of longest contigs obtained from assembly

Hit ID MGE type Host BitScore Evalue % ID Function CD – 70 ORFs protein:proph:180063 prophage Burkholderia pseudomallei 668 179 3.00E-45 35 hypothetical protein protein:vir:348 virus Myxococcus xanthus 305 4.00E-83 52 major virion structural protein protein:vir:100025 virus Prochlorococcus marinus 57 2.00E-08 26 T7-like tail fiber protein:vir:345 virus Myxococcus xanthus 245 3.00E-65 35 virion structural protein protein:proph:162200 prophage Janthinobacterium sp. Marseille 381 1.00E-106 51 hypothetical protein protein:vir:78415 virus Salmonella enteritidis 100 3.00E-21 42 hypothetical protein protein:proph:181793 prophage Acinetobacter baumannii 157 9.00E-39 63 site-specific DNA methylase protein:proph:170708 prophage Delftia acidovorans SPH-1 91 1.00E-18 41 hypothetical protein protein:proph:165361 prophage Silicibacter sp. TM1040 80 2.00E-15 44 hypothetical protein protein:proph:167404 prophage Acidovorax avenae subsp. citrulli 134 8.00E-32 33 hypothetical protein protein:vir:78682 virus Synechococcus sp. WH 8109 47 3.00E-05 22 tail fiber protein:vir:100025 virus Prochlorococcus marinus 83 3.00E-16 43 T7-like tail fiber protein:vir:364 virus Myxococcus xanthus 65 1.00E-10 53 hypothetical protein protein:proph:162208 prophage Janthinobacterium sp. Marseille 154 7.00E-38 50 hypothetical protein protein:proph:180017 prophage Burkholderia pseudomallei 668 295 3.00E-80 43 hypothetical protein GS – 53 ORFs protein:proph:175748 prophage Xanthobacter autotrophicus Py2 42 6.00E-04 28 alginate regulatory protein AlgP protein:proph:184451 prophage Vibrio vulnificus YJ016 88 1.00E-17 21 hypothetical protein protein:vir:5126 virus Sinorhizobium meliloti 166 2.00E-41 28 putative DNA methylase protein:proph:168641 prophage Desulfotomaculum reducens MI-1 70 2.00E-12 22 superfamily II helicase protein:vir:94976 virus Xanthomonas campestris 84 2.00E-16 31 hypothetical protein protein:vir:94974 virus Xanthomonas campestris 330 1.00E-90 39 possible DNA helicase protein:vir:94988 virus Xanthomonas campestris 54 2.00E-07 22 putative exonuclease protein:proph:169677 prophage Streptococcus pyogenes MGAS9429 80 3.00E-15 29 phage-related DNA polymerase protein:proph:167709 prophage Staphylococcus aureus MRSA252 88 9.00E-18 23 putative DNA polymerase protein:vir:95474 virus Pseudomonas aeruginosa 99 3.00E-21 62 hypothetical protein protein:vir:95474 virus Pseudomonas aeruginosa 52 7.00E-07 65 hypothetical protein protein:vir:95827 virus Escherichia coli K12 88 1.00E-17 26 gp25 protein:vir:95874 virus Escherichia coli K12 44 1.00E-04 19 gp24 JHS – 74 ORFs protein:proph:170952 prophage Delftia acidovorans SPH-1 100 1.00E-21 48 hypothetical protein protein:proph:162194 prophage Janthinobacterium sp. Marseille 93 4.00E-19 40 HNH endonuclease protein:proph:183030 prophage Escherichia coli HS 142 4.00E-34 25 hypothetical protein protein:proph:162359 prophage Novosphingobium aromaticivorans 52 9.00E-07 27 hypothetical protein protein:vir:108303 virus Thalassomonas loyana 172 4.00E-43 30 hypothetical protein

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Hit ID MGE type Host BitScore Evalue % ID Function protein:proph:183303 prophage Chromobacterium violaceum 74 2.00E-13 53 tail fiber-related protein protein:proph:166087 prophage Burkholderia thailandensis E264 65 8.00E-11 23 filamentous haemagglutinin protein:vir:2603 virus Roseobacter 59 7.00E-09 24 gp7 protein:vir:104473 virus Prochlorococcus marinus 82 6.00E-16 32 gp7 protein:vir:102996 virus Prochlorococcus marinus 101 1.00E-21 42 gp101 protein:proph:180718 prophage Bordetella bronchiseptica RB50 80 3.00E-15 28 hypothetical protein protein:proph:174766 prophage Nitrosomonas eutropha C91 61 1.00E-09 36 major tail protein protein:proph:171663 prophage Vibrio harveyi ATCC BAA-1116 54 2.00E-07 28 hypothetical protein protein:vir:104871 virus Prochlorococcus marinus 85 1.00E-16 50 phage tail fiber-like protein protein:proph:162082 prophage Methylococcus capsulatus str. Bath 73 3.00E-13 49 prophage LambdaMc01 protein:vir:99495 virus Lactobacillus johnsonii 64 2.00E-10 36 putative protein JT – 11 ORFs protein:proph:166746 prophage Listeria welshimeri serovar 6b 45 6.00E-05 29 phage capsid protein protein:proph:184121 prophage Gramella forsetii KT0803 59 4.00E-09 34 HNH endonuclease protein:vir:3922 virus Lactobacillus casei 105 3.00E-23 34 putative endodeoxyribonuclease

Number of ORFs in each contig predicted by GeneMark.hmm; BLASTx results obtained from ACLAME database (Evalue cutoff 10-3)

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Table S11. Comparison of viral community structure and diversity statistics according to PHACCS analysis

Metagenome CD JHS GS JT Mixedc 50kba GAASb 50kb GAAS 50kb GAAS 50kb GAAS Error 102.3 136.1 36.31 29.0 15.61 14.32 11.8 19.9 64.49 Richness 1618 5312 5763 3117 1098 756 4636 16847 7513 Evenness 0.914 0.929 0.956 0.951 0.935 0.932 0.957 0.965 0.954 Most abundant genotype (%)

3.407 1.800 1.002 1.412 2.866 3.487 1.052 0.481 0.953

H’ 6.75 8.04 8.27 7.65 6.55 6.17 8.07 9.39 8.51 Parameters: average shotgun sequence length 850 bp, overlap 20 bp, maximum 100,000 genotypes. CD, Crim Dell; JHS, Jamestown High School; GS, Greensprings; JT, John Tyler.

a – results of PHACCS analysis using assumed genome size of 50kb. b – results of PHACCS analysis using average genome size calculated by GAAS. c – based on average genome size across the four ponds

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Table S12. Spearman correlations between environmental and biological variables Virus Abund; viral abundance, Bac Abund; bacterial abundance, viral rich; viral richness, Temp = temperature, Bac Rich = bacterial richness, NOx; NO2 + NO3, Cond; conductivity, DOC; Dissolved Organic Carbon, drainage area = total drainage area, imperv area; impervious drainage area, % imperv, percent impervious drainage area, DO; dissolved oxygen.

* indicates p < 0.05, **indicates p<0.01

Cond DO pH TP NOx NH4 DOC

Bac Abund

Temp 0.241 .448* 0.439 -0.138 -0.015 -0.345 -0.024 0.226 Cond

-0.079 0.434 0.077 0.115 0.398 .480* 0.187

DO

0.394 -0.04 -0.099 0.036 -0.011 0.029 pH

0.044 -0.048 -0.129 0.113 -0.107

TP

.770** -0.036 .475* 0.168 NOx

0.008 0.398 0.162

NH4

0.346 0.182 DOC

.537*

Virus Abund

Bac Prod

DrainageArea

Imperv Area

% Impervious VBR

Bac Rich

Viral Rich

Temp 0.322 0.102 -0.309 0.147 0.424 -0.195 0.244 0.205 Cond 0.193 0.017 -0.169 -0.313 0.038 -0.045 -0.045 -0.039 DO 0.005 0.164 -0.136 0.101 0.15 -0.189 .456* 0.194 pH 0.063 -0.009 -0.089 -0.076 0.063 0.132 .477* 0.181 TP 0.204 -0.162 0.206 0.073 0.066 0.069 -0.222 0.336

NOx 0.187 -0.284 0.379 0.4 0.252 -0.197 -0.142 .513* NH4 -0.03 0.026 -0.167 -0.405 -0.119 -0.208 0.072 -0.053 DOC 0.277 0.179 0.085 0.127 0.022 0.138 -0.247 0.025 BacA

B .492* .563** -0.036 0.108 0.051 -0.105 -0.126 0.143

VirusAB

-0.114 0.187 -0.047 -0.092 -0.198 0.062 0.002 Bac Prod -0.08 0.163 0.03 0.115 -0.102 -0.12

Drain Area .492* -0.244 -0.035 -0.188 -0.184 Imprev Area 0.459 -0.023 -0.17 0.299

% Imperv -.577* -0.269 0.385 VBR 0.102 -0.063

Bac Rich 0.417

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Supplemental Figures

Figure S1. Bacterial OTUs in selected stormwater ponds observed at the phylum level on the x-axis with class affiliations faceted in bars as a percentage of total OTUs in each individual library. OTUs present at lower than 4 percent of the overall dataset were omitted.

Figure S2. a. Classification of reads according to best BLAST hit using MG-RAST (E-value cutoff of 10-5); b. Domain of known reads according to best BLAST hit using MG-RAST (E-value cutoff of 10-5). CD, Crim Dell; JHS, Jamestown High School; GS, Greensprings; JT, John Tyler.

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Figure S3. Relative abundance of reads assigned to functional subsystems using MG-RAST. Classification based on best BLAST hit (E-value cutoff of 10-5) using M5NR database. Asterisks indicate metabolic functions with significantly different (p < 0.05) representation across the lake metagenomes based on pairwise comparisons (t-tests); CD, Crim Dell; JHS, Jamestown High School; GS, Greensprings; JT, John Tyler.

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Figure S4. Rank-abundance curves obtained using the PHACCS analysis system on each metagenome; CD, Crim Dell; JHS, Jamestown High School; GS, Greensprings; JT, John Tyler.