Clarifying the phylogenetic relationships and taxonomy of Stenonema, Stenacron and Maccaffertium, three common eastern North American mayfly genera

Stenonema, Stenacron, and Maccaffertium are three closely related genera of mayflies (Ephemeroptera:Heptageniidae) commonly found across North America. Due to their primarily aquatic life history and sensitivity to aquatic pollutants, these mayflies are often used as water quality indicators. However, there is little morphological variation within these genera, leading to difficulties in identification and rampant taxonomic confusion, limiting their utility as bioindicators. In an attempt to resolve the phylogenetic relationships of Stenonema, Stenacron, and Maccaffertium, and to clarify their higher-level classifications, we sequenced regions of two mitochondrial genes (cytochrome oxidase subunit 1 (cox1) and 16S ribosomal RNA (rrnl)) and two nuclear genes (Wingless (Wg) and histone H3) from 60 individuals representing most of the described species in these genera and included data from representatives of three heptageniid genera (Kageronia, Macdunnoa and Pseudiron) proposed in previous studies to be closely related to our focal taxa as well as two more distantly related heptageniid genera (Epeorus and Heptagenia) to root the phylogenies. Maximum likelihood and Bayesian analysis were conducted on single-gene and concatenated multi-gene data sets and species tree methods were utilized to resolve relationships. These analyses resolved Stenacron as a monophyletic group sister to a clade comprising Macdunnoa, Maccaffertium and Stenonema. Maccaffertium was found to be paraphyletic, with Stenonema femoratum resolved within Maccaffertium as sister to M. mexicanum. Many relationships remained unresolved or varied across analyses, making revision of the classification based on phylogenetic considerations challenging. To minimize confusion while naming clades and acknowledging uncertainty in our phylogenetic conclusions, we redefine Stenonema to include Maccaffertium and propose three subgenera-Stenonema, Maccaffertium and Lewisa- for key well-supported clades.