Methods
Study sites and sample selection
Map-1 Map of six study sites among five provinces (From left to right: Alberta-Slave lake forest, Saskatchewan-Old channel river, Ontario-Twist lake, Quebec-Dasserat, Quebec-Cimon, New Brunswick- Upper green river)
Six locations among five provinces across Canada are selected as the study sites, and map-1 shows the locations of all study sites on the map. As shown in table-1, information including province ID, location, latitude, longitude, and elevation are recorded by the Alberta Forest Service after the study sites are confirmed.
Table-1 Site information table (Adapted from Alberta Forest Service,1993)
Data collection
Experimental design
In each study site, five 20-30 years old mature white spruce trees with a diameter at breast height (DBH) larger than 20 cm are selected as samples to collect foliar and phloem tissue for the later chemical and genetic analysis. All selected trees grow on clay-loam soil, and they have a distance of 2.5 m between each other.
Sample collection
Foliar tissue was collected from the current-year branches of each spruce tree via a pole pruner in July 2020 (shown in photo-3). The collected foliar samples are stored at -40℃ with dry ice until DNA extraction and analysis. Five phloem samples were collected from the stem of each spruce tree via a hollow leather punch with a 20mm diameter (shown in photo-4). Each phloem sample was ground into a fine powder with liquid nitrogen in a grinding bowl and stored in a 15 ml falcon tube at -20℃ until chemical analysis.
Photo-3 Collecting foliar tissue from each spruce tree via a pole pruner (photo taken by Aziz Ullah, 2020)
Photo-4 Hollow leather punch and phloem samples
Measurement and analyze protocol
Foliar DNA extraction and analysis
Foliar DNA was extracted by the EZNA ® Fungal DNA Kit (Omega Bio-tek, Norcross, GA, USA) and then amplified via a two steps polymerase chain reaction (PCR) using primers internal transcribed spacer (ITS) 1F and 4 (Bullington & Larkin, 2015). The end product of PCR was transferred to the Molecular Biology Facility at the University of Alberta for Illumina sequencing. The raw data will be the numbers of amplicon sequence variants (ASVs) in each endophyte genera in each spruce tree. Higher ASV numbers indicate higher endophyte abundance.
Measuring terpene compounds in phloem samples
Phloem extraction protocols were established by Ullah et al. in 2021. We collected a 100 mg ground phloem sample from each spruce tree and extracted it with 0.5 ml hexane two times (Ullah et al., 2021). The concentration of terpenes was measured by sending extracted phloem samples to a Gas Chromatograph/Mass Spectrometer (GC/MS, Agilent 7890A/5062C, Agilent Tech., Santa Clara, CA) (shown in photo 5).
Photo-5 Photo of Gas Chromatograph/Mass Spectrometer to measure terpene concentration.
Statistical analysis
All statistical analyses were finished by R-studio (version RStudio 2022.12.0+353 "Elsbeth Geranium" Release (7d165dcfc1b6d300eb247738db2c7076234f6ef0, 2022-12-03) for Windows Mozilla/5.0 (Windows NT 10.0; Win64; x64) AppleWebKit/537.36 (KHTML, like Gecko) RStudio/2022.12.0+353 Chrome/102.0.5005.167 Electron/19.1.3 Safari/537.36) based on basic R (version 4.2.1).
In the data exploration section, boxplots of total/each endophyte abundance, total/each monoterpene concentration, and total/each sesquiterpene concentration were performed to evaluate the distribution and variation in the data. Principle coordinate analysis (PCoA) was made to check the overall correlations among fungal endophytes abundance, terpene concentration, and different study sites.
In the result part, a grouped bar chart of fungal endophyte abundance across six study sites was created to give a direct feeling of the abundance variations. Another grouped bar chart was made to test the variation of terpene concentration among six study sites. A perMANOVA followed by a univariate pairwise comparison is done for both fungal endophyte abundance and terpene concentration to show the variation among six study sites. To test the correlations between fungal endophytes abundance and terpene concentration, two gradient analyses were made.