Over the past few decades, large-scale phylogenetic analyses of fungus-gardening ants and their symbiotic fungi have depicted strong concordance among major clades of ants and their symbiotic fungi, yet within clades, fungus sharing is widespread among unrelated ant lineages. Sharing has been explained using a diffuse coevolution model within major clades. Understanding horizontal exchange within clades has been limited by conventional genetic markers that lack both interspecific and geographic variation. To examine whether reports of horizontal exchange were indeed due to symbiont sharing or the result of employing relatively uninformative molecular markers, samples of Trachymyrmex arizonensis and Trachymyrmex pomonae and their fungi were collected from native populations in Arizona and genotyped using conventional marker genes and genome-wide single nucleotide polymorphisms (SNPs). Conventional markers of the fungal symbionts generally exhibited cophylogenetic patterns that were consistent with some symbiont sharing, but most fungal clades had low support. SNP analysis, in contrast, indicated that each ant species exhibited fidelity to its own fungal subclade with only one instance of a colony growing a fungus that was otherwise associated with a different ant species. This evidence supports a pattern of codivergence between Trachymyrmex species and their fungi, and thus a diffuse coevolutionary model may not accurately predict symbiont exchange. These results suggest that fungal sharing across host species in these symbioses may be less extensive than previously thought.

DNA was extracted from 14 Trachymyrmex arizonensis and T. pomonae workers and their Leucocoprinus sp. Symbionts that were collected from colonies in Arizona, USA. Three genes for ants and one gene for fungi were sequenced by Sanger (“conventional”) sequencing, and genotyping-by-sequencing was used to analyze SNPs. Alignments of each data type were used to estimate ant and fungus maximum likelihood trees in RAxML version 8.2.11. ParaFit and PACo were used to examine the congruence between ant and fungus trees, and the results from these analyses were compared to evaluate the differences in congruence between phylogenetic trees that were based on Sanger sequencing and those that were based on genotyping-by-sequencing methods.

The “SNP_RAxML_Pipeline.txt” file contains additional details on how to use the vcf_ref_extraction.py script (https://github.com/kbeigel/vcf_ref_extraction) and the raxml_ascbias script (https://github.com/btmartin721/raxml_ascbias), starting with VCF and Phylip files and ending with phylogenetic tree visualization. The vcf_ref_extraction script was written and run in PyCharm IDE Community Edition 2019.3.3 with the Python 3.8 interpreter. A tab-delimited text file (SampleID_VCFcode.txt) shows the ant and fungus samples used in the SNP analysis and their corresponding coded names in the VCF.