With the recent and continuing discovery of further cryptic bat species, it is essential to find morphological species discriminating characters. Pipistrellus pipistrellus (common pipistrelle) and Pipistrellus pygmaeus (soprano pipistrelle) have been recognized as separate species since 1997, but no reliable morphological species discriminating trait has yet been found. The most commonly used morphological species discrimination traits are ‘wing vein’ pattern and shape and color of the penis, but these have not been validated on sets of genetically identified specimens. The baculum (os penis) has long been used successfully in species discrimination in bats and other mammals. In this study, we tested the reliability of the established traits and demonstrated how to reliably separate the common pipistrelle and the soprano pipistrelle by simple baculum measurements. The bacula of museum specimens of these two species and of Pipistrellus hanaki were imaged with high-resolution microCT. Several measurements were taken on the size-calibrated volume images, and their value for species discrimination was tested by discriminant analysis with leave-one-out cross validation. We showed that P. pipistrellus and P. pygmaeus specimens can be discriminated by measuring the projected length, height, and width of the baculum (n = 48; all but one classified correctly). Geometric morphometrics was used to analyze and locate variations in baculum shape. Principal component analysis of baculum variation was not sufficient to separate these species. Most of the interspecific variation in baculum shape can be found in the proximal third (the base) of the baculum, and most individual variation can be observed in lateral view, especially in the dorsoventral curve. Quantitative details of morphology are becoming more important to distinguish cryptic species and understand their phylogeographic distributions. The simple baculum measurements can be used to classify single specimens and could be taken without microCT, on a resected baculum.
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