In tropical sky-islands, cold-affinity populations tend to become isolated at highlands during the interglacial periods, and to expand into the lowlands where they become more connected during the glacial periods. Although this has been widely studied in trees, it is poorly understood how fungal symbionts can differentiate among mountains (allopatrically), or within a single mountain (parapatrically) due to climate fluctuations. Here, we conducted population genomic analyses on the ectomycorrhizal fungus Laccaria trichodermophora in three tropical sky-islands using Genotyping by Sequencing (GBS) at low DNA concentrations. There were no significant differences between altitudes within a single mountain, but we observed significant genetic differentiation among populations from different mountains, supporting the allopatric differentiation hypothesis. Our results indicate that L. trichodermophora populations are under a sky-island population dynamics that started during the Pleistocene climate fluctuations.

Samples were collected during the rainy season (July–September 2015) on three different and isolated volcanic peaks in the east-central portion of the TMVB (Sierra Negra, La Malinche and Nevado de Toluca). We collected Laccaria trichodermophora fruit bodies in a categorical sampling strategy: Pinus montezumae forests, from 2900 to 3200 masl (“low” hereafter); and sub-alpine grasslands and P. hartwegii forests, from 3900 to 4200 masl (“high”). All samples identified as L. trichodermophora  were genotyped with a reduced representation sequencing protocol (Genotyping by Sequencing, GBS). To do so, DNA was extracted anew from dried fruit bodies’ stipes (to avoid genetic recombination in spores DNA) using the DNeasy Plant mini kit (QIAGEN). After quantification with a Qubit V 3.0, DNA concentration was set to 5 ng/μl for all samples (with AE buffer; QIAGEN) for library preparation. For library preparation, the ApekI (G|CWGC) resticiton enzyme was used and, library preparation and sequencing was performed at the Genomic Analysis Platform at Laval University (Quebec, Canada) on an Ion Torrent Proton platform with single-end sequencing.

This repository contains the scripts and the processed data for the " Allopatric instead of parapatric divergence in an ectomycorrhizal fungus (Laccaria trichodermophora) in tropical sky-islands". GBS demultiplexed raw data is available at NCBI project PRJNA643251, samples SAMN15407898 - SAMN15407947. 

## Scripts
* `1_FastX_tools.sh`: demultiplexes the raw data
* `2_Trimmomatic.sh`: bardcode trimming and quality filters
* `3_bowtie.sh`: maping vs Laccaria bicolor V2.0 genome
* `4_stacks.sh`: SNP calling and population statics
* `5_PCA.R`: performs PCA
* `6_Pcadapt.R`: performs outlier tests with PCAdapt
* `6_bayescan.sh`: performs outliers test with Bayescan
* `7_SNAPP.sh`: bayesian analysis using SNP data
* `8_SnpEff.sh`: Variant annotation and prediction

## Data:
* `49ind.vcf`: *.vcf file using for all the subsequent analyses (conversion were done using PGDSpider)
* `sampling_49idnv_point.csv`: metada data of samples. Column names indicate the following: 
    - `ID`: numerical unique ID per sample.
    - `SampleIDFull`: name of the sample. The first letter (L) refers to Laccaria. The two second letter refer to mountain of origin (see Key). The next three letters refer to the sampling category (see Category), Alt=High and Baj=Low. Numbers at the end are individual samples ID within that mountain and altitude.
    - `Key`: mountain of origin, Ma= La Malinche, Ne = Sierra Negra, To = Nevado de Toluca.
    - `Longitude` and `Latitude` are given in decimal degrees and refer to sampled individual.
    - `Category`: sampling altidue High = 3,900 to 4,200 masl, `Low` = 2,900 to 3,200 masl.
* `Pop_map_Loc.txt`: Pop_map used in the SNP calling step in Stacks (4_stacks.sh)
* `Lac_snapp.xml`: SNAPP file (7_SNAPP.sh)
* `Barcodes_10.txt`: barcodes file used for demultiplexing (1_FastX_tools.sh)
* `Iontorrentlinker.fasta`: Adapters list used by Trimmomatic (2_Trimmomatic.sh)
* `Laccaria_FUNECO_alignment.nex`: ITS alignment nexus file 

## Software used

* FastX-Toolskit V 3.0
* Trimmomatic V 0.32 
* Bowtie2 V 2.2.9
* Stacks V 1.45
* R V 3.5.1 and Rstudio V 1.3.95
* Bayescan V 2.0 and PCAdapt V 1.0
* FastTree V 2.1
* MrBayes V 3.2.2
* SNAPP V 1.4.2
* SnpEff V 4.0
* Beast2 V 2.5.2

Additionally, some analyses were performed with GUI interface software, as follows:

* STRUCTURE V 2.3.4: for ancestry approach
* SplitTree V 4.14: network relationships between the samples
* Geneiuos V 10: ITS editing 
* Tassel V 5.2: SNP and samples filtering
* FastQC V 0.11.5: Quality control
* FigTree V 1.4.3: Tree visualization
* PGDspider V 2.1.1.3: for file conversion
* BioEdit V 7.2.5: Sequences editor
* Tracer V 1.7.1: Chains diagnosis
* DensiTree V 2.2.6: Tree visualization