Normalizing gas‐chromatography–mass spectrometry data: method choice can alter biological inference

We demonstrate how different normalization techniques in GC‐MS analysis impart unique properties to the data, influencing any biological inference. Using simulations, and empirical data, we compare the most commonly used techniques (Total Sum Normalization ‘TSN’; Median Normalization ‘MN’; Probabilistic Quotient Normalization ‘PQN’; Internal Standard Normalization ‘ISN’; External Standard Normalization ‘ESN’; and a compositional data approach ‘CODA’). When differences between biological classes are pronounced, ESN and ISN provides good results, but are less reliable for more subtly differentiated groups. MN, TSN, and CODA approaches produced variable results dependent on the structure of the data, and are prone to false positive biomarker identification. In contrast, PQN exhibits the lowest false positive rate, though with occasionally poor model performance. Because ESN requires extensive pre‐planning, and offers only mixed reliability, and ISN, TSN, MN, and CODA approaches are prone to introducing artefactual differences, we recommend the use of PQN in GC–MS research

The social function of latrines: a hypothesis-driven research approach

The function of latrines is still debated in many mammals, and in most cases latrine use is likely multi-functional. Functional hypotheses can be broadly divided into five categories that are not all mutually exclusive: resource defence, information centre/advertisement, landmarks/orientation, parasite control, or predator–prey interactions. To standardise all investigations into latrine function across species, we present a hypothesis-driven research framework based on the following five key categories of data that can, and we argue should, be collected from latrines: (1) spatial distribution patterns; (2) temporal usage patterns; (3) individual visitation and contribution patterns (to individual latrines); (4) behaviour of visitors; and (5) scent signal content and longevity. We suggest that our proposed approach offers a much-needed standardised structure to guide investigation into the function of latrine use in mammals