Arbuscular mycorrhizal fungal diversity associated with Eleocharis ...

FEMS Microbiology Letters, 362, 2015, fnv081 doi: 10.1093/femsle/fnv081 Advance Access Publication Date: 19 May 2015 Research Letter

R E S E A R C H L E T T E R – Environmental Microbiology

Arbuscular mycorrhizal fungal diversity associated with Eleocharis obtusa and Panicum capillare growing in an extreme petroleum hydrocarbon-polluted sedimentation basin Ivan E. de la Providencia† , Franck O.P. Stefani† , Manuel Labridy, Marc St-Arnaud and Mohamed Hijri∗ ´ etale, ´ ´ and Jardin botanique de Montreal, ´ Institut de recherche en biologie veg Universite´ de Montreal 4101 rue ´ (Quebec) ´ Sherbrooke Est, Montreal H1X 2B2, Canada ∗ Corresponding author: IRBV, 4101 Rue Sherbrooke Est, Montreal ´ (QC) H1X 2B2, Canada. Tel: +1 514-343-2120; E-mail: [email protected] † These authors contributed equally to this work. One sentence summary: Remarkable diversity of arbuscular mycorrhizal fungi was found in an extreme petroleum hydrocarbon-polluted sedimentation basin. Editor: Stefan Olsson

ABSTRACT Arbuscular mycorrhizal fungi (AMF) have been extensively studied in natural and agricultural ecosystems, but little is known about their diversity and community structure in highly petroleum-polluted soils. In this study, we described an unexpected diversity of AMF in a sedimentation basin of a former petrochemical plant, in which petroleum hydrocarbon (PH) wastes were dumped for many decades. We used high-throughput PCR, cloning and sequencing of 18S rDNA to assess the molecular diversity of AMF associated with Eleocharis obtusa and Panicum capillare spontaneously inhabiting extremely PH-contaminated sediments. The analyses of rhizosphere and root samples over two years showed a remarkable AMF richness comparable with that found in temperate natural ecosystems. Twenty-one taxa, encompassing the major families within Glomeromycota, were detected. The most abundant OTUs belong to genera Claroideoglomus, Diversispora, Rhizophagus and Paraglomus. Both plants had very similar overall community structures and OTU abundances; however, AMF community structure differed when comparing the overall OTU distribution across the two years of sampling. This could be likely explained by variations in precipitations between 2011 and 2012. Our study provides the first view of AMF molecular diversity in soils extremely polluted by PH, and demonstrated the ability of AMF to colonize and establish in harsh environments. Keywords: Arbuscular mycorrhizal fungi; petroleum hydrocarbon pollution; symbiosis; virtual taxa; molecular diversity

INTRODUCTION The arbuscular mycorrhizal fungi (AMF) are a ubiquitous and important group of soil-inhabiting obligate biotrophic root fungi

(Smith and Read 2008) playing a major role in ecosystem functioning. During their interactions with plant roots, they are rewarded with fixed carbon in return for mineral and water uptake (Kiers et al. 2011). Beyond improving phosphorus (P) and

Received: 12 January 2015; Accepted: 13 May 2015 C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]

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FEMS Microbiology Letters, 2015, Vol. 362, No. 12

nitrogen (N) uptake (Smith and Read 2008), AM associations provide major ecological benefits to all terrestrial ecosystems since they play a key role in soil structure (Rillig 2004), plant community diversity and productivity (van der Heijden et al. 1998), plant tolerance to environmental stresses including drought, salinity, high concentration of trace metals, organic pollutants and other chemicals (Auge 2001; Ruiz-Lozano 2003; Vallino et al. 2006; Hassan et al. 2011; Debiane et al. 2012; Labidi et al. 2012, for review see: Schutzend ubel and Polle 2002), and also in protect¨ ¨ ing plants against pathogen attacks (Pozo and Azcon-Aguilar 2007; St-Arnaud and Vujanovic 2007; Ismail, McCormick and Hijri 2013). Because most terrestrial plants form AM associations (Wang and Qiu 2006), there is an increased interest to study AMF molecular diversity (Hiiesalu et al. 2014). Our knowledge on this aspect relies primarily in studies performed in natural ecosystems (Opik et al. 2010; Opik et al. 2013); however, little is known about AMF diversity in extreme human-impacted ecosystems such as highly petroleum hydrocarbon-polluted soils. As key players of ecosystem functioning, investigating AMF distribution in highly disturbed ecosystems is fundamental to understand natural process of land reclamation but also to understand plant-AMF relationships under such harsh conditions. While there is no evidence of direct hydrocarbon degradation by AMF, these fungi might stimulate soil microbial metabolic activity, leading to an acceleration of the immobilization and translocation of trace elements as well as degradation of organic pollutants (Joner et al. 2001; Liu and Dalpe´ 2009; Hassan, Hijri and St-Arnaud 2013), and their accumulation in plant tissues (Jing, He and Yang 2007). AMF capacity to improve soil resilience to organic pollution has been shown only under greenhouse or in vitro conditions Joner et al. 2001; Leyval et al. 2002; Joner and Leyval 2003; Liu et al. 2004; Volante et al. 2005; Verdin et al. 2006; Alarcon et al. 2008; Liang et al. 2009). So far, only one study reported AMF diversity in an artificial single-species plant community (i.e. Phragmites australis) from a plant-based bioremediation of groundwater (Fester 2013). Determination of the best approach for selecting competent microbes should be based on prior knowledge of the microbial communities inhabiting the target site (Tyagi, da Fonseca and de Carvalho 2011), since long-term exposure to a contaminant may have allowed different microbes to develop tolerance to highly polluted conditions. This will in turn facilitate the degradation processes occurring during remediation activities at the rhizosphere. Taking this step forward, the main objective of this study was to describe the AMF species richness and community structure associated with plants growing spontaneously in an open-air basin extremely polluted by petroleum hydrocarbons (PHs), using high throughput PCR, cloning and sequencing of the partial SSU rRNA gene. The site of study was chosen based on access and the extreme concentration levels of PHs already detected (Desjardin et al. 2014). Furthermore, we targeted two plant species E. obtusa and P. capillare because they were dominant in this basin according to Desjardin et al. (2014).

MATERIAL AND METHODS Site description and sampling The experimental site is a former sedimentation basin, located on the premises of a petrochemical plant at Varennes, on the south shore of the St. Lawrence River near Montreal (45◦ 41 56 N 73◦ 25 43 W). The site was in operation for over 40 years and definitely shut down in 2008, and left opened to spontaneous revegetation (Guidi, Kadri and Labrecque 2012).

The local mean monthly temperature from 2000 to 2012 is 15.3◦ C (±0.20 SE) from April to October and −4.1◦ C (±0.4 SE) from November to March. The mean annual precipitation is 650.4 mm (±29.4 SE) and 321.9 mm (±28.2 SE) over the ` same time periods (Environment Canada—Vercheres Weather Station; http://www.climate.weatheroffice.ec.gc.ca). The site is characterized by a patchy distribution of spontaneous vegetation mainly dominated by E. obtusa (Willd) and P. capillare L. (Desjardin et al. 2014) (Fig. S1, Supporting Information). Species belonging to these two families (Cyperaceae and Poaceae, respectively) have both mycorrhizal and non-mycorrhizal members, however no information is available about the formation of AM structures for both plants species in natural conditions (Wang and Qiu 2006). Samples of rhizospheric soil and roots from E. obtusa and P. capillare were harvested in October 2011 and 2012. Each sample of rhizospheric soil and roots was taken at 10 cm depth and was a composite from three individual plants. Three composite samples were harvested per plant species (Fig. S1, supporting information).

Sample preparation Soil was gently shaken off from the roots, and the remaining soil still attached was carefully harvested with a spatula and used for DNA extraction. Subsequently, roots were washed thoroughly with tap water, chopped into small pieces of about one cm long and homogenized. A composite sample prepared from all soil samples was analysed to determine PAHs and aliphatic hydrocarbon (C10-C50) concentrations (Maxxam Analytics, Montreal, QC) (Table S1, Supporting Information).

Estimation of root colonization Root staining was performed with 0.1% trypan blue following the protocol described in Phillips and Hayman (1970), and the percentage of root colonization was assessed independently for both AMF and dark septate endophytes (DSE) structures according to McGonigle et al. (1990). The Tukey’s HSD test was used to identify significant differences between plant species at P < 0.05.

DNA extraction, PCR amplification, cloning and sequencing Genomic DNA was isolated from 250 mg of sediment (wet weight) using the PowerSoil DNA Extraction Kit (Mo Bio Laboratories, Solana Beach, CA). Total genomic DNA from root samples was isolated from approximately 100 mg of root tissues using DNeasy Plant Mini Kit (Qiagen, Toronto, ON). The SSU rRNA gene was amplified using the AML1/AML2 primers (Lee, Lee and Young 2008) to build soil and root clone libraries. The PCR mixture was made up of 1× PCR buffer, 1.5 mM MgCl2 , 0.2 mM of each deoxynucleotide triphosphate, 1 mg of bovine serum albumin (Sigma, St. Louis, MO), 0.5 mM of each primer and 1 unit of HotStart Taq DNA polymerase (Qiagen, Toronto, ON) in a total volume of 25 ml. Thermal cycling conditions were as follows: initial denaturation at 95◦ C for 3 min; 30 cycles (pre-cloning PCR) to 38 cycles (post cloning PCR) at 94◦ C for 45 s, 55◦ C for 45 s and 72◦ C for 1 min and a final elongation at 72◦ C for 10 min. PCRs were done on a Eppendorf Mastercycler ProS thermocycler (Eppendorf, Mississauga, ON). PCR products were visualized on GelRed-stained 1.5% agarose gels using a Gel-Doc system (Bio-Rad, Mississauga, ON). The cloning procedure followed the protocol described in Stefani,

de la Providencia et al.

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Jones and May (2014) and Beaudet et al. (2015) with minor modifications: 2 μl of ligation product was used to transform 12.5 μl of competent cells. White bacterial colonies were amplified as described above except that the HotStart Taq was replaced with Kapa Taq (Kapa Biosystems, Boston, MA). Sequencing was commercially performed on an Applied Biosystems 3730xl DNA analyzer at the Genome Quebec Innovation Centre (McGill University, Montreal, Canada).

Bioinformatic and phylogenetic analyses Sequences were edited, cleaned and assembled in Geneious Pro v6.1.2. (Biomatters Ltd, Auckland, New Zealand). The similarity threshold for SSU sequences belonging to the same operational taxonomic unit (OTU) was set to 97% (uncorrected pairwise distance) to serve as a proxy for ‘species’. The presence of chimeras was inspected using BLASTN (Altschul et al. 1990). De novo chimera detection was performed as well using the command uchime˙denovo as implemented in USEARCH v7.0.595 (Edgar et al. 2011). The consensus sequences of each contig were combined with the sequences from Kruger et al. (2012) and with the closest sequences recovered in MaarjAM database (Opik et al. 2010). Alignments were done using MUSCLE v.3.5 (Edgar 2004). The DNA substitution model was determined using the Bayesian information criterion calculations implemented in jModelTest v2.1.3 (Darriba et al. 2012). Bayesian phylogenetic analyses were performed as described in Stefani, Jones and May (2014) with exception that the number of trees saved was set to 20 000 and the first 3000 trees were excluded before computing consensus trees with Bayesian posterior probabilities. Mothur v.1.31.2 (Schloss 2009) was used to calculate rarefaction curves and Venn diagrams. Nucleotide consensus sequences of each OTU were deposited at NCBI GenBank database and are registered under accession numbers: KF745192–KF745214. AMF communities from rhizosphere and roots samples were also investigated with a double principal coordinate analysis (DPCoA) (Pavoine, Dufour and Chessel 2004). DPCoA displays the first two orthogonal principal axes, based on the relation between an OTU dissimilarity matrix (one consensus sequence per OTU was used as input) and the corresponding abundance matrix.

RESULTS AMF molecular diversity and identity A total of 824 SSU Sanger sequences were analyzed, representing 21 OTUs at 97% of similarity. The Glomeromycota diversity was saturated in each library, suggesting that our sampling captured

Figure 1. Rarefaction curves showing the level of saturation of OTUs richness of Glomeromycota in field samples.

most of the AMF community diversity was recorded (Table 1 and Fig. 1). The Good’s non-parametric coverage estimator ranged between 96.9 and 100% for each library (Table 1). Phylogenetic inference using Bayesian phylogenetic analysis of 18S rDNA gene sequences based on Kruger’s reference data (Kruger et al. ¨ 2012) and on the closest matches found in MaarjAM database (Opik et al. 2010) showed that all the known families among the Glomeromycota with exception of Ambisporaceae, Archeosporaceae, Geosiphonaceae and Pacisporaceae were represented by the 21 OTUs (Fig. 2). Diversispora dominated the AMF community with a relative abundance of 46.7– 52.8% in rhizosphere samples associated with E. obtusa and P. capillare in 2011, respectively (Fig. 3). However, its relative abundance was only 0.8% in sediments associated with P. capillare and sampled in 2012. On the contrary, Paraglomus and VTX5 were the most abundant clades recorded in sediments sampled in 2012, with a relative abundance ranging from 16.7 to 27.6%, but they were not recorded in sediments sampled in 2011. Claroideoglomus was the only taxon recorded in 2011 and 2012 in sediments from the two plant species with a relative abundance ranging between 19 and 26.5%, respectively. It was also the OTU richest genus with seven OTUs out of 21. The genus Rhizophagus was detected in all libraries, and it was represented by only one OTU homologous to R. irregularis (average relative abundance of 19.2% with exception of sediments from P. capillare collected in 2012 where it represented only 0.8% of the AMF community). Surprisingly, the OTU

Table 1. Glomeromycota OTUs richness and diversity recovered in each library.

ES2011 ES2012 ER2011 ER2012 PS2011 PS2012 PR2011 PR2012 1 2

No. of sequences

Coverage (%)1

No. of OTUs

Inv Simpson index2

Shannon index

195 163 44 32 108 132 25 125

99.0 98.7 100 100 97.2 96.9 100 100

11 14 1 1 10 14 1 1

4.11 5.99 1 1 3.21 6.94 1 1

1.80 2.07 0 0 1.61 2.1 0 0

Good’s non-parametric coverage estimator. the inverse of the Simpson index.

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Figure 2. Bayesian phylogenetic tree based on nuclear small subunit (SSU) of rDNA consensus sequences showing the distribution of the 21 OTUs recorded at the Varennes field site (red labels) among the Glomeromycota tree. Sequence data were analyzed with the SSU sequences (black labels) from Kruger et al. (2012) and the closest match recovered from MaarjAM database. Bayesian posterior probabilities greater or equal than 0.95 are represented with black circles on nodes. Clades are identified to the genus level if they include SSU sequences from reference herbarium cultures or to virtual taxa if they only include specimen independent SSU sequences. The scale represents the branch length corresponding to expected substitutions per site.


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Figure 2. (Continued).

homologous to R. irregularis was the only one detected in root samples for both plant species across the two-years sampling (Figs 3 and 4). The genera Acaulospora, Funneliformis, Paraglomus, Scutellospora and Septoglomus were also represented by a single OTU. Finally, 12 OTUs (OTU 4, 6, 8, 9, 10, 12, 13, 16, 17, 19, 20, and 21) clustered only with virtual taxa from the MaarjAM database.

AMF community structure The OTU diversity recorded in rhizosphere samples from E. obtusa and P. capillare was very similar, with 20 and 18 OTUs, respectively, across the two years. Rhizosphere sediments asso-

ciated with E. obtusa and P. capillare shared 17 OTUs while one OTU was specific to P. capillare, represented by VTX 22 (OTU15), and three were specific to E. obtusa, represented by Diversispora (OTU11), Claroideoglomus (OTU20) and VTX 219–93 (OTU21). The AMF community structure was not influenced by the host plant since OTUs richness and abundance were highly similar in rhizosphere sediments associated with E. obtusa and P. capillare from each sampling year; however, the AMF community structure was totally different from one year to another (Figs 3 and 4). The AMF community structure in root samples was totally different since only one OTU belonging to the Rhizophagus genus was recorded in the roots of the two plant species during

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Root endophytic status of plants The microscopic examination of trypan blue-stained roots revealed the cooccurrence of AMF and DSE structures in roots from the field samples (Fig. S3, Supporting Information). Total AM colonization in these samples was low as it ranged between 4.84% ± 0.03 in P. capillare roots to 12.36% ± 0.05 in E. obtusa roots. DSE colonization was also low and ranged from 3.62 and 16.09%. Statistical analysis did not showed any significant differences for any of the categories examined for AM and/or DSE colonization (P > 0.05) (Table 2).

DISCUSSION Figure 3. Relative abundance of the 21 OTUs of Glomeromycota recorded in the rhizosphere soil and roots associated with E. obtusa in 2011 (ES2011, ER2011) and 2012 (ES2011, ER2011) and with P. capillare in 2011 (PS2011, PR2011) and 2012 (PS2012, PR2012). Each library represents the three samples collected per plant species each year.

Figure 4. Double principal coordinates analysis (DPCoA) performed on the Glomeromycotan communities recorded in the rhizosphere and roots associated with E. obtusa in 2011 (ES2011, ER2011) and 2012 (ES2011, ER2011) and P. capillare in 2011 (PS2011, PR2011) and 2012 (PS2012, PR2012). Each point is the combination of the three samples collected per plant species each year. Point size is proportional to the Rao diversity index computed for each library. Axis 1 explains 69.9% of the variation in of the community composition, while axis 2 explains 29.1 of the variation.

the two years of sampling. Despite the AMF community structure was different between the two years of sampling, its genetic diversity (based on Rao diversity index) was very similar in the four rhizosphere libraries (Fig. 4). The relative abundance recorded in the different libraries and the DPCoA (Figs 3 and 4) clearly showed that the AMF community structure recorded in the soil rhizosphere was highly similar within each year of sampling and not influenced by the plant species identity. The AMF community structure in the root samples was totally different since only one OTU belonging to the Rhizophagus genus was recorded in the roots of the two plant species during the two years of sampling. DPCoA showed as well that the genetic diversity (based on Rao diversity index) of the AMF communities was highly similar in the four rhizosphere libraries.

Exploring AMF diversity in a sedimentation basin dedicated to the storage of petroleum-hydrocarbon wastes from a former petrochemical plant offered a unique opportunity to document the ability of AMF to colonize and establish in the presence of PHs in extremely contaminated conditions. Chemical analyses of samples from the rhizosphere of E. obtusa and P. capillare revealed that the concentrations of many PAHs and C10-C50 were thousands of time above the recommendations of the Canadian environmental quality guidelines (Table S1, Supporting Information). Therefore, this sedimentation basin potentially represents an exceptional reservoir of AMF adapted to such level of PHs for future remediation activities. Although found at low levels, our study reports for the first time the mycorrhizal colonization of E. obtusa and P. capillare grown at high levels of hydrocarbon pollution. The taxonomic AMF diversity of the site was considerably high with 21 AMF taxa detected across two years, encompassing most of the described families within the Glomeromycota. Previous studies performed in situ in organic-polluted soils and aiming to describe the AM diversity, recorded the presence of Glomus aggregatum and G. mosseae (synonym Funnelifornis mosseae) (Cabello 1997), R. irregularis and F. mosseae (Fester 2013) and seven morpho-species (Franco-Ramirez et al. 2007). The diversity of AMF in trace metalcontaminated areas is generally reported to be less than 10 taxa (Weissenhorn, Leyval and Berthelin 1993; Bedini et al. 2010; Zarei et al. 2010; Hassan et al. 2011) with exception of 14 AM fungal 18S ribotypes recorded in the roots of Solidago gigantea (Vallino et al. 2006). Furthermore, F. mosseae is the most commonly reported AMF taxon in phytoremediation of trace-metal contaminated soils (Gaur and Adholeya 2004). In our study, Claroideoglomus, Diversispora and Rhizophagus taxa dominated the Glomeromycota community indicating that members from these genera may have greater capacity than others to tolerate the presence of oil-related organic pollutants. From a fungal point of view (i.e. mycocentric perspective) (Chagnon et al. 2013), the tolerance of these taxa is likely to be explained by the life history strategies developed by members of these genera (de la Providencia et al. 2005; de la Providencia, Fernandez and Declerck 2007; Chagnon et al. 2013). Members of Rhizophagus, Claroideglomus and Diversispora genera have been considered ruderal AM fungal species. These species have short life cycles, and they invest their energy mainly in the production of thousands of offspring, have a rapid development and evolved traits that are favored in instable environments. Within these genera, hyphal architecture is characterized by the interconnections of different hyphae by means of anastomosis and following hyphal damage, the hyphae are able to (1) colonize new roots, (2) explore new zone of substrates or (3) rarely recover the hyphal integrity (de la Providencia et al. 2007). All of these traits might confer


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Table 2. Arbuscular mycorrhizal (AM) and dark septate endophyte (DSE) colonization associated to P. capillare and E. obtusa. Different letters in a column indicated significant differences based on Tukey’s HSD (P < 0.05). AM colonization (%)1 Sampled plants

Year

AC2

VC3

TC4

DSE colonization (%)1

P. capillare E. obtusa P. capillare E. obtusa

2011

nr5 0.26 ± 0.001 nr nr

3.33 ± 0.02 a 0±0 2.95 ± 0.01 a 3.14 ± 0.02 a

4.84 ± 0.03 a 12.36 ± 0.05 a 12 ± 0.06 a 10.1 ± 0.05 a

16.09 ± 0.09 a 4.70 ± 0.02 a 5.31 ± 0.03 a 3.62 ± 0.02 a

–

0.11

0.91

0.96

P-value

2012

Mean ± SE (standard error). Arbuscular colonization. 3 Vesicular colonization. 4 AM total colonization. 5 The structure was not recorded. 1 2

an advantage to members of these genera to compete in harsh environment, like petroleum-polluted soils. Striking features of our results were the high dissimilarity in AMF diversity and community structure between rhizosphere soil and roots in both plant species and also from one year to the other, however the latter being very similar per plant species. The variation in AMF community structure observed between the two years of sampling might be explained by the large difference in precipitations between 2011 and 2012. The total precipitations from May to September were approximately 620 mm for 2011 and 400 mm for 2012 (Environmental Assessment Report of Montreal’s water bodies 2013). Sun et al. (2013) have reported that precipitation had a significant effect on AMF diversity, abundance and sporulation. Whatever the year of sampling and/or plant species considered, the AMF community structure was permanently characterized by the sole presence of a taxon related to R. irregularis in roots despite the high diversity associated to their respective rhizosphere. It is noteworthy that R. irregularis has been considered to be a generalist, opportunistic and an aggressive taxon colonizing perturbed ecosystems (Hassan et al. 2011). It has been proposed that if the interaction between plant and their symbiotic fungi increases the relative performance of the locally abundant plant species (i.e. positive feedback) that would lead to a loss of biodiversity at local scales. Conversely, if the interaction decreases the relative performance of the plant, then it would generate a negative feedback that could contribute to plant species coexistence (Bever et al. 2010). While AMF feedback has supported plant species coexistence, how reciprocal rewards strategies (Kiers et al. 2011) under highly stressful conditions could shape coexistence of their root symbionts remains to be studied. Generally, plants provide carbon as a reward for mineral nutrient exchanges with AMF isolates (Bever et al. 2009), thus we hypothesize that this type of interaction might increase AMF fitness resulting in competition and exclusion at the local scale (i.e. decrease or loss of one or several taxa). This might be considered as possible scenario explaining the dominance of R. irregularis in the roots of both plant species across our two years of sampling. This scenario can be reinforced under high environmental pressure, like highly hydrocarbon-polluted soils, where plants and their root fungal endophytes may develop strong cooperative interactions.

CONCLUSION This study provides the first detailed view of AMF diversity associated with naturally occurring plants in highly PH-polluted sed-

iments, and suggests that AMF can be potentially important microbial candidates in bioremediation of oil-contaminated soils. Although, further investigations are needed to demonstrate the potential of direct or indirect contributions of these AMF to the degradation of PH contaminants. Our results suggest that plant identity under such conditions does not shape AMF diversity. Further studies need to focus on determining the role of AMFassociated microbes on the AMF nutritional status and degradation of PH. These studies will undoubtedly clarify the mechanisms behind the persistence of AMF in disturbed ecosystems. A better understanding of the mechanisms involved is fundamental to isolate and cultivate the petroleum hydrocarbon-tolerant ecotypes with the most promise for future bioremediation efforts.

SUPPLEMENTARY DATA Supplementary data is available at FEMSLE online.

ACKNOWLEDGEMENTS We thank Petromont Inc. (ConocoPhillips Canada) for allowing us to access to the Varennes field site. We also thank David Denis and Youssouf Balde for technical support, Drs Terrence Bell and Karen Fisher Favret for comments on the manuscript.

FUNDING This project was supported by funds of Genome Canada and ´ Genome Quebec, which are greatly acknowledged. Conflict of interest. The GenoRem project contains several industrial partners, including ConocoPhillips, the company, which provided us with access to their site for this study. Our manuscript has in no way been modified by ConocoPhillips, nor has any industrial partner commented on, or influenced the analysis of the results.

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