Vocal divergence within species often corresponds to morphological, environmental, and genetic differences between populations. Wolf howls are long-range signals that encode individual, group, and subspecies differences, yet the factors that may drive this variation are poorly understood. Furthermore, the taxonomic division within the Canis genus remains contended and additional data are required to clarify the position of the Himalayan, North African, and Indian wolves within Canis lupus. We recorded 451 howls from the three most basal wolf lineages – Himalayan (Canis lupus chanco – Himalayan haplotype), North African C.l. lupaster, and Indian C.l. pallipes wolves – and present a howl acoustic description within each clade. With an additional 619 howls from seven Holarctic subspecies, we used a random forest classifier and principle component analysis on nine acoustic parameters to assess whether Himalayan, North African, and Indian wolf howls exhibit acoustic differences compared to each other and Holarctic wolf howls. Generally, both the North African and Indian wolf howls exhibited high mean fundamental frequency (F0) and short duration compared to the Holarctic clade. In contrast, the Himalayan wolf howls typically had lower mean F0, unmodulated frequencies, and short howls compared to Holarctic wolf howls. The Himalayan and North African wolves had the most acoustically distinct howls and differed significantly from each other and to the Holarctic wolves. Along with the influence of body size and environmental differences, these results suggest that genetic divergence and/or geographic distance may play an important role in understanding howl variation across subspecies.

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