DOI: 10.5061/dryad.kkwh70s1d
title: Allopatric instead of parapatric divergence in an ectomycorrhizal fungus (Laccaria trichodermophora) in tropical sky-islands

description: 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.
description: 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 <em>Laccaria trichodermophora</em> fruit bodies in a
 categorical sampling strategy: <em>Pinus
 montezumae </em>forests, from 2900 to 3200 masl (“low” hereafter);
 and sub-alpine grasslands and <em>P. hartwegii </em>forests,
 from 3900 to 4200 masl (“high”). All samples identified
 as <em>L. trichodermophora</em>  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.
description: This repository contains the scripts and the processed data for
 the " Allopatric instead of parapatric divergence in an
 ectomycorrhizal fungus (<em>Laccaria trichodermophora</em>) in
 tropical sky-islands". GBS demultiplexed raw data is available at
 NCBI project PRJNA643251, samples SAMN15407898 - SAMN15407947. 
 <br> ## Scripts<br> * `1_FastX_tools.sh`:
 demultiplexes the raw data<br> * `2_Trimmomatic.sh`: bardcode
 trimming and quality filters<br> * `3_bowtie.sh`: maping vs Laccaria
 bicolor V2.0 genome<br> * `4_stacks.sh`: SNP calling and population
 statics<br> * `5_PCA.R`: performs PCA<br> * `6_Pcadapt.R`:
 performs outlier tests with PCAdapt<br> * `6_bayescan.sh`: performs
 outliers test with Bayescan<br> * `7_SNAPP.sh`: bayesian analysis
 using SNP data<br> * `8_SnpEff.sh`: Variant annotation and
 prediction ## Data:<br> * `49ind.vcf`: *.vcf file
 using for all the subsequent analyses (conversion were done using
 PGDSpider)<br> * `sampling_49idnv_point.csv`: metada data of
 samples. Column names indicate the following: <br>     - `ID`:
 numerical unique ID per sample.<br>     - `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.<br>   
  - `Key`: mountain of origin, Ma= La Malinche, Ne = Sierra Negra, To =
 Nevado de Toluca.<br>     - `Longitude` and `Latitude` are given in
 decimal degrees and refer to sampled individual.<br>     -
 `Category`: sampling altidue High = 3,900 to 4,200 masl, `Low` = 2,900 to
 3,200 masl.<br> * `Pop_map_Loc.txt`: Pop_map used in the SNP calling
 step in Stacks (4_stacks.sh)<br> * `Lac_snapp.xml`: SNAPP file
 (7_SNAPP.sh)<br> * `Barcodes_10.txt`: barcodes file used for
 demultiplexing (1_FastX_tools.sh)<br> * `Iontorrentlinker.fasta`:
 Adapters list used by Trimmomatic (2_Trimmomatic.sh)<br> *
 `Laccaria_FUNECO_alignment.nex`: ITS alignment nexus file 
 ## Software used * FastX-Toolskit V
 3.0<br> * Trimmomatic V 0.32 <br> * Bowtie2 V 2.2.9<br>
 * Stacks V 1.45<br> * R V 3.5.1 and Rstudio V 1.3.95<br> *
 Bayescan V 2.0 and PCAdapt V 1.0<br> * FastTree V 2.1<br> *
 MrBayes V 3.2.2<br> * SNAPP V 1.4.2<br> * SnpEff V
 4.0<br> * Beast2 V 2.5.2 Additionally, some
 analyses were performed with GUI interface software, as follows:
 * STRUCTURE V 2.3.4: for ancestry approach<br> * SplitTree
 V 4.14: network relationships between the samples<br> * Geneiuos V
 10: ITS editing <br> * Tassel V 5.2: SNP and samples
 filtering<br> * FastQC V 0.11.5: Quality control<br> * FigTree
 V 1.4.3: Tree visualization<br> * PGDspider V 2.1.1.3: for
 file conversion<br> * BioEdit V 7.2.5: Sequences editor<br> *
 Tracer V 1.7.1: Chains diagnosis<br> * DensiTree V 2.2.6: Tree
 visualization