Diversity and Assembling Processes of Bacterial Communities in Cryoconite

Transcription

1 Diversity and Assembling Processes of Bacterial Communities in Cryoconite Holes of a Karakoram Glacier Roberto Ambrosini 1, Federica Musitelli 1, Federico Navarra 1, Ilario Tagliaferri 1, Isabella Gandolfi 1, Giuseppina Bestetti 1, Christoph Mayer, Umberto Minora 3, Roberto Sergio Azzoni 3, Guglielmina Diolaiuti 3, Claudio Smiraglia 3, Andrea Franzetti 1 * 1 Dept. of Earth and Environmental Sciences (DISAT) - University of Milano-Bicocca, Milano, ITALY Bavarian Academy of Sciences and Humanities, Munich, GERMANY 3 A. Desio Dept. of Earth Sciences, Università degli Studi di Milano, Milano, ITALY *Corresponding author: Andrea Franzetti - Dept. of Earth and Environmental Sciences (DISAT) - University of Milano- Bicocca, Piazza della Scienza 1, 016 Milano ITALY Phone Fax: andrea.franzetti@unimib.it Keywords: cryoconite / cryosphere / Baltoro Glacier /

2 SUPPLEMENTARY MATERIAL Full details on PCR conditions Libraries for Illumina sequencing were built with a dual PCR protocol. The first PCR was performed in 75 µl volume reactions with GoTaq Green Master Mix (Promega Corporation, Madison, WI, USA) and 1 µm of each primer. The used primers were 783F and 1046R, which amplify a 8-bp fragment containing the hypervariable regions V5-V6 (783F: 5 -CAGGATTAGATACCC-3, Wang & Qian, 009; 1046R: 5 - CGACRRCCATGCANCACCT-3, Huber et al., 007). Both primers were added with Illumina adapters at 5 position. The cycling conditions were: initial denaturation at 98 C for 30 s; 0 cycles at 98 C for 10 s, 47 C for 30 s, and 7 C for 5 s and a final extension at 7 C for min. The second PCR was performed in 3 50 µl volume reactions by using 3 µl of the purified amplicons (Wizard SV Gel and PCR Clean-up System, Promega Corporation, Madison, WI, USA) from the first step as template and 0. µm of each primer. Primers contained regions complementary to the Illumina adapters and standard Nextera indexes (Illumina, Inc., San Diego, CA, USA). The cycling conditions were: initial denaturation at 98 C for 30 s; 15 cycles at 98 C for 10 s, 6 C for 30 s, and 7 C for 6 s and a final extension at 7 C for min. After the amplification DNA quality was evaluate spectrophotometrically and DNA was quantified using Qubit (Life Technologies, Carlsbad, CA, USA). The sequencing was carried out at Science for Life Sequencing facility (Stockholm, Sweden).

3 Table S1: Results from Indicator Species analysis aiming at identifying OTUs typical of different areas or group of areas. The classification of OTUs at class and order level (if available) is reported, as well as the value of the IndVal statistic and the significance of the test, corrected with the False Discovery Rate procedure.

4 1 Figure S1. Relative abundance of genera belonging to the order Burkholderiales. 100% Unclassified_Burkholderiaceae 90% Oxalicibacterium Limnobacter 80% Herminiimonas Burkholderia 70% Undibacterium Pelomonas 60% Unclassified_Alcaligenaceae Massilia 50% Ideonella Janthinobacterium 40% Unclassified_Oxalobacteraceae Curvibacter 30% Variovorax Aquabacterium 0% Unclassified_Burkholderiales 10% Unclassified_Burkholderiales_incertae_sedis Polaromonas 0% Area 1 Area Area 3 Area 4 Unclassified_Comamonadaceae Limnohabitans

5 Figure S. Boxplots of a) ph and d) Gini inequality index in different areas. Boxes enclose the first and the third quartiles of each measure, whiskers enclose 5th and 95th percentile, dots represent outliers. b): variation in the number of OTUs according to ph. c) variation in Shannon diversity index according to ph. 8

6 9 Figure S3. Relative abundance of OTUs belonging to Sphingobacteriales and Sphingomonadales at different ph value. Sphingobacteriales Sphingomonadales ph ph

7 1 Figure S4. Relative abundance of OTUs belonging to Sphingobacteriales and Sphingomonadales in different areas. 13 Sphingobacteriales Sphingomonadales Month Month 15 16