Allopatric instead of parapatric divergence in an ectomycorrhizal fungus (Laccaria trichodermophora) in tropical sky-islands

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.

Methods
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.