During the present study, DNA sequence data, gross morphology and scanning electron microscopy (SEM) were used to examine cryptic species boundaries in the velvet worm, Peripatopsis clavigera species complex, from the southern Cape Afrotemperate forest belt in South Africa. Sequence data were generated for the mitochondrial COI and the nuclear 18S rRNA loci and phylogenetically analysed using both a Bayesian inference and a maximum-likelihood approach. Both the COI data and the combined DNA sequence topology (COI+18S) revealed the presence of five clades within the Peripatopsis clavigera species complex, and revealed that specimens from Tulbagh were distantly related and represented a sixth clade. The evolutionary distinction of the five clades was corroborated to varying degrees by the four species-delimitation methods (ABGD, PTP, GMYC and STACEY); however, both the gross morphological data and the SEM provided limited diagnostic differences between the five clades. Furthermore, the COI haplotype network and phylogeographic analyses provided evidence of genetic isolation between lineages that are currently syntopic. The distribution of genealogically exclusive and widespread maternal lineages was atypical among velvet worms and did not reflect the general trend of genetic and geographical isolation. Instead, lineages exhibited admixture among localities, a result most likely due to fluctuations in climatic conditions affecting the southern Cape Afrotemperate forest during the Pliocene–Pleistocene period as evident from our divergence time estimations. Four novel, narrow-range endemic species – P. ferox, sp. nov., P. mellaria, sp. nov., P. edenensis, sp. nov. and P. mira, sp. nov. – are described within the P. clavigera species complex, whereas the Tulbagh specimens are described as P. tulbaghensis, sp. nov. Collectively, these results demonstrate that Peripatopsis likely contains several undescribed species.
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