Two new species of Simplicillium from the Lancang–Mekong River basin and a molecular reappraisal of Simplicillium formicae (Cordycipitaceae, Hypocreales)

The genus Simplicillium ( Cordycipitaceae , Hypocreales ) comprises fungi with diverse ecological lifestyles, ranging from arthropod pathogens to saprotrophs. However, species diversity and taxonomic relationships within this morphologically conserved genus remain incompletely understood. In this study, we combined six-locus phylogenetic analyses (nrSSU, ITS, nrLSU, tef-1α , rpb1 and rpb2 ), morphological characterization, and ecological information to investigate Simplicillium isolates obtained from soil and ant cadavers in the upper and middle reaches of the Lancang–Mekong River basin in China, Laos, and Thailand. Bayesian and maximum-likelihood analyses resolved three well-supported lineages, including two new species, Simplicillium mekongense and S. ovoideum . Both species form distinct clades within the S. subtropicum – S. pseudocercosporicola complex and can be distinguished by phialide morphology, conidial shape, and substrate association. Reassessment of publicly available sequence data revealed that the previously published rpb1 sequence attributed to the ex-type strain of S. formicae was misassigned, leading to an inaccurate representation of interspecific divergence and complicating species delimitation. The corrected molecular evidence, together with morphological comparisons, supports treating S. puwenense as a synonym of S. formicae . Furthermore, strain CBS 101267, previously identified as S. lanosoniveum , is reassigned to S. ovoideum based on phylogenetic and morphological evidence. This study clarifies species boundaries within Simplicillium , corrects erroneous sequence annotations, and refines the phylogenetic framework for future taxonomic studies of this genus. These findings contribute to a better understanding of fungal diversity in the Lancang–Mekong River basin and provide a basis for future investigations into the biocontrol potential of Simplicillium species.