Is shape in the eye of the beholder? The reproducibility of geometric morphometric analyses on live animals

Geometric morphometrics is widely used to quantify morphological variation between biological specimens, but the fundamental influence of observer bias on data reproducibility is rarely considered, particularly in studies using photographs of live animals taken under field conditions. We examined this using four independent observers that applied an identical landmarking protocol to replicate photographs of 291 live Atlantic salmon (Salmo salar L.) from two rivers. Using Hotelling’s T2 analyses, we found significant inter-observer differences in mean body shape, suggesting that the observers introduced a systematic error despite following the same landmarking protocol. No significant differences were detected when the landmarking process was repeated by the same observer on a random subset of photographs. Importantly, in spite of significant observer bias, small but statistically significant morphological differences between fish from the two rivers were found consistently by all observers and combined datasets of two separate observers. Pairwise tests of angles of vectors of shape change showed that these between-river differences in body shape were analogous across observer datasets suggesting a general reproducibility of findings obtained by geometric morphometric studies. Overall, we show that, even when significant inter-observer error is introduced during digitisation, following an identical landmarking protocol can identify biological differences between populations. This study indicates that data sharing across research groups can yield reliable results and therefore has important implications for the future development of data sharing in geometric morphometrics.