Resolving the stonefly phylogeny puzzle via mitochondrial genomes

Stoneflies (order Plecoptera) rank among the most ancient lineages of winged insects. Beyond their role as key bioindicators for evaluating freshwater ecosystem health, they carry ecological value and evolutionary significance.

For decades, however, constructing a robust phylogenetic framework for stoneflies has posed a persistent challenge—hampered by an incomplete fossil record, complex morphological evolution, and limited genomic sampling.

To address this challenge, a collaborative research team has analyzed mitochondrial genome data from 97 stonefly species—representing all 17 of the order’s extant families—and applied an integrative analytical framework combining multiple evolutionary models.

The team, led by Prof. Cai Chenyang from the Nanjing Institute of Geology and Paleontology of the Chinese Academy of Sciences, and Prof. Du Yuzhou from Yangzhou University, published their findings in the journal iScience.

The team successfully reconstructed the backbone phylogeny for stoneflies. They clarified evolutionary relationships across major lineages, resolved critical divergence events, and established a refined temporal timeline for stonefly evolution.

These findings provide pivotal molecular and chronological evidence to understand the origin, adaptation, and radiation of this ancient insect group.

While approximately 4,000 extant stonefly species have been identified and their morphology extensively studied, two core questions have long fueled debate in entomology: stoneflies’ precise placement within the broader insect tree of life, and the evolutionary relationships between their families.

A key unresolved issue has been the basal relationships between Euholognatha and Systellognatha—the two major suborders of Plecoptera—due to limitations in gene sampling and evolutionary modeling in prior research.

To overcome these limitations, the researchers newly sequenced the mitochondrial genomes of 29 extant stonefly species, then combined these with 68 publicly available datasets. This effort marked the first time that a complete mitochondrial genomic representation has been achieved for all 17 stonefly families.

The team then employed both maximum likelihood and Bayesian inference approaches—with a focus on advanced models like CAT-GTR, which effectively accounts for site heterogeneity in genomic data—to conduct a comprehensive phylogenetic reconstruction of global stonefly fauna.

The analyses resolved the early diversification of Euholognatha and supported Scopuridae as the earliest-diverging lineage within this suborder. This finding aligns with existing morphological evidence and suggests that the loss of courtship behavior in Scopuridae is likely a secondary evolutionary reduction, rather than an ancestral trait, the researchers noted.

The study sheds new light on controversial taxa, identifying Taeniopterygidae as the sister group to Leuctridae. It also clarifies evolutionary relationships within Systellognatha, supporting Styloperlidae as the suborder’s earliest-diverging lineage.

By integrating the latest paleontological and stratigraphic data, the team further reconstructed the temporal framework of stonefly evolution. Their results indicate that crown-group stoneflies originated during the Pennsylvanian period (approximately 323–299 million years ago).

Major diversification events among extant stonefly families, meanwhile, occurred between the Cisuralian period (299–272 million years ago) and the Early Triassic (252–247 million years ago).

This study highlights the potential of mitochondrial genomic data, when analyzed under optimized evolutionary models, to resolve higher-level phylogenetic relationships in insects.