Fungus-gardening ants (tribe Attini) have been subject to decades of research elucidating their major evolutionary transitions and radiations [1–5]. Despite these revolutionary advances, most attine research has occurred at higher taxonomic levels (species or higher). Consequently, we know very little about the microevolutionary processes that may underlying macroevolutionary (phylogenetic) patterns. This lack of understanding is partially technical since most genetic markers used to construct phylogenies typically exhibit little intraspecific variation, for both the ants and fungi [2, 6–8]. Mitochondrial DNA (mtDNA) markers, while useful for large scale geographic studies [9–11], may be generally problematic because some attines may have nuclear-incorporated mtDNA-like sequences or whose genomes contain pseudogenes [7, 12]. Moreover, mtDNA is maternally inherited, which may or may not be indicative of intraspecific patterns of gene flow since male and female ants are known to exhibit different patterns of gene flow [13–15]. Finer-level intraspecific examinations have typically required specific diploid markers such as microsatellites or single-nucleotide polymorphisms (SNPs) [16–19]. Both approaches represent co-dominant expression and genome-wide distributions, but microsatellites are short, non-coding, highly variable repetitive motifs , whereas SNPs are shorter and found throughout coding and non-coding genomic regions .
SNPs are popular in the genomics age because of the large quantities of potentially informative sites quickly generated in a single sequencing run, but they require considerable time and effort to identify and analyze, and are typically restricted to single studies. Conversely, microsatellite markers, once developed, can be used repeatedly in multiple studies, which may reduce their overall expense on a per-project basis . Moreover, recent comparative studies in insects, fungi, and birds reported no major population genetic structural differences between these two approaches [23–26], or that more SNPs were required to produce the same information as a single microsatellite marker .
Traditional microsatellite development techniques (i.e., constructing libraries, cloning) have yielded polymorphic microsatellite markers for both higher and lower attines [28–32]. However, because these markers were developed for tropical attines, they may or may not be useful for North American species (J.N. Seal, unpublished observations), which tend to belong to phylogenetically distinct lineages [3, 33]. Next-generation sequencing (NGS) techniques have revolutionized microsatellite marker development in terms of decreased time and cost, and increased quantity of potential markers [34–36], although only a few studies have utilized these techniques for attines. Extensive marker sets exist for Acromyrmex lundii , Atta laevigata , and two species in the lower attine genus Mycocepurus [18, 39].
Here, our aim was to use NGS techniques to develop microsatellite markers for two additional attine species, Trachymyrmex septentrionalis and Mycetomoellerius turrifex. Both are common and ecologically important fungus-gardening ants throughout southeastern North America [10, 40, 41]. We also explored the potential for cross-amplification of the T. septentrionalis markers in two closely related species: T. arizonensis and T. pomonae. These three species belong to Trachymyrmex sensu stricto, unlike M. turrifex which belongs to a new genus, Mycetomoellerius [3, 33, 42]. These markers will provide a new tool to study patterns of attine intraspecific genetic variation and phylogeographic structure.