Unprecedented technological advances in digitization and the steadily expanding open-access digital repositories are yielding new opportunities to quickly and efficiently measure morphological traits without transportation and advanced/expensive microscope machinery. A prime example is the AntWeb.org database, which allows researchers from all over the world to study taxonomic, ecological, or evolutionary questions on the same ant specimens with ease. However, the reproducibility and reliability of morphometric data deduced from AntWeb compared to traditional microscope measurements has not yet been tested.

Here, we compared 12 morphological traits of 46 Temnothorax ant specimens measured either directly by stereomicroscope on physical specimens or via the widely used open access software tpsDig utilizing AntWeb digital images. We employed a complex statistical framework to test several aspects of reproducibility and reliability between the methods. We estimated (i) the agreement between the measurement methods and (ii) the trait value dependence of the agreement, then (iii) compared the coefficients of variation produced by the different methods, and finally, (iv) tested for systematic bias between the methods in a mixed modelling-based statistical framework.

The stereomicroscope measurements were extremely precise. Our comparisons showed that agreement between the two methods was exceptionally high, without trait value dependence. Further, the coefficients of variation did not differ between the methods. However, we found systematic bias in eight traits: apart from one trait where software measurements overestimated the microscopic measurements, the former underestimated the latter.

Our results shed light on the fact that relying solely on the level of agreement between methods can be highly misleading. In our case, even though the software measurements predicted microscope measurements very well, replacing traditional microscope measurements with software measurements, and especially mixing data collected by the different methods, might result in erroneous conclusions. We provide guidance on the best way to utilize virtual specimens (2D z-stacked images) as a source of morphometric data, emphasizing the method’s limitations in certain fields and applications.