Morphological data constitute fundamental information used within the life sciences, from descriptions of new species to studies of evolutionary questions (Cabral et al., 2012 Deans et al., 2015 Elgar et al., 2018 Thompson et al., 2011). 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. 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. Our results shed light on the fact that relying solely on the level of agreement between methods can be highly misleading. However, we found systematic bias in eight traits: apart from one trait where software measurements overestimated the microscopic measurements, the former underestimated the latter. Furthermore, the coefficients of variation did not differ between the methods. Our comparisons showed that agreement between the two methods was exceptionally high, without trait value dependence. The stereomicroscope measurements were extremely precise. 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 modeling-based statistical framework.

We employed a complex statistical framework to test several aspects of reproducibility and reliability between the methods. 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.

However, the reproducibility and reliability of morphometric data deduced from AntWeb compared to traditional microscope measurements has not yet been tested. A prime example is the database, which allows researchers from all over the world to study taxonomic, ecological, or evolutionary questions on the same ant specimens with ease.

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.

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