American Society of Naturalists

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"Disentangling Positive Selection from Relaxed Selection in Animal Mitochondrial Genomes"

Posted on by Genrietta Yagudayeva

Kendra D. Zwonitzer,  Erik N. K. Iverson, Jess E. Sterling,Ryan J. Weaver, Bradley A. Maclaine, and Justin C. Havird. October 2023.

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DNA helix - public domain pictures
DNA helix - public domain pictures

Identifying the nature of adaptive selection and its tell-tale signs is not always as observable to the naked eye as the beaks of finches were to Darwin. More commonly, in today’s evolution studies, we explore molecular evolution for signatures of selection. A parameter that has been used extensively in predicting selection is the dN/dS ratio.

dN/dS is the ratio of non-synonymous to synonymous nucleotide substitution rates. Non-synonymous nucleotide changes alter the resulting protein’s amino acid sequence and function, whereas synonymous changes have no such effect. dN, or the rate of nonsynonymous substitutions, is often expected to be affected by selection and, thereby, is an indicator of adaptation. dS is mostly reflective of neutral, non-adaptive evolution. The dN/dS ratio identifies the likely selection type by its magnitude, where if the ratio is elevated, positive selection may be at play, indicating the spread of beneficial alleles within a population. If the ratio is relatively low, purifying selection likely dominates, where detrimental alleles are being removed within a population.

In this study, Justin C. Havird of the University of Texas at Austin and colleagues focused on using elevated dN/dS ratios and discussed their paradoxical causes. The paradox is that an elevated dN/dS ratio can result from both positive selection, an adaptive process, and relaxed purifying selection, a non-adaptive form of selection caused by a dramatic decrease in selection efficiency where detrimental alleles are fixed more often. Thus, elevated ratios can indicate polar opposite mechanisms when predicting the nature of selection, a clear source of confusion when interpreting molecular evolution.

To address this confusion, the authors reevaluated elevated ratio case studies of mitochondrial DNA (mtDNA) in various animals, including birds, bats, insects, snakes, electric fish, and primates with various bioinformatic tools beyond dN/dS. The cases they re-evaluated included comparing flighted (e.g., bats) and flightless lineages of mammals and evaluating metabolic innovations in certain ectothermic vertebrates (e.g., electrical generation in electric fish, venom production in certain snakes). mtDNA is vitally important in animal studies of molecular evolution because mitochondria have a central role in cellular respiration, and thus, genetic variation is often thought to reflect adaptive evolution. mtDNA is also a popular target in such studies, given the availability of complete mtDNA datasets for many animal groups.

Given the dichotomous nature of the dN/dS ratio, the researchers argue that it is vital to understand the mechanism generating elevated ratios. Otherwise, conclusions solely based on the dN/dS ratio may be widely misleading. Using mtDNAs from the NCBI database, mtDNA assembled from publicly available next-generation sequence data, and molecular evolution bioinformatics tools that can explicitly distinguish positive vs. relaxed selection, the authors concluded that the elevated dN/dS ratios reported were caused by both positive and relaxed purifying selection. However, many of the previously made conclusions might have resulted from misinterpretation, as none of the re-examined case studies consistently matched their predicted mechanism solely based on their elevated dN/dS ratios.

This article is a cautionary tale and warns not to jump to conclusions about the evolutionary mechanisms based solely on the molecular signature of dN/dS. Havird and colleagues present potential solutions to clarify other conclusions about future molecular evolution.


Disentangling different types of selection is a common goal in molecular evolution. Elevated dN/dS ratios (the ratio of non-synonymous to synonymous substitution rates) in focal lineages are often interpreted as signs of positive selection. Paradoxically, relaxed purifying selection can also result in elevated dN/dS ratios, but tests to distinguish these two causes are seldomly implemented. Here, we reevaluated seven case studies describing elevated dN/dS ratios in animal mitochondrial DNA (mtDNA) and their accompanying hypotheses regarding selection. They included flightless lineages versus flighted lineages in birds, bats, and insects and physiological adaptations in snakes, two groups of electric fishes, and primates. We found that elevated dN/dS ratios were often not caused by the predicted mechanism, and we sometimes found strong support for the opposite mechanism. We discuss reasons why energetic hypotheses may be confounded by other selective forces acting on mtDNA and caution against overinterpreting singular molecular signals, including elevated dN/dS ratios.

Genrietta in a sunflower patch. <br/>Photo credit: Genrietta Yagudayeva
Genrietta in a sunflower patch.
Photo credit: Genrietta Yagudayeva

Author Bio:

Genrietta is a 2nd year PhD student working at the Santos Lab in St. John's University, Queens, New York. The few projects she is involved in is the life-history of reptiles in relation to hemoglobin genes’ structure and function and its affinity to oxygen, E. anthonyi poison frogs as a model system to run transcriptomic analysis, and the poison frogs’ tadpole transport bioglue on a molecular and evolutionary basis. Outside of the lab, she is passionate about teaching and spreading science to diverse populations. In her free-time, she enjoys the beach, traveling, and visiting museums.