Recent analyses of mass segregation diagnostics in star forming regions invite a comparison with the output of hydrodynamic simulations of star formation. In this work we investigate the state of mass segregation of stars (i.e. sink particles in the simulations) in the case of hydrodynamical simulations which omit feedback. We first discuss methods to quantify mass segregation in substructured regions, either based on the minimum spanning tree (Allisons Lambda), or through analysis of correlations between stellar mass and local stellar surface number densities. We find that the presence of even a single outlier (i.e. a massive object far from other stars) can cause the Allison Lambda method to describe the system as inversely mass segregated, even where in reality the most massive sink particles are overwhelmingly in the centres of the subclusters. We demonstrate that a variant of the Lambda method is less susceptible to this tendency but also argue for an alternative representation of the data in the plane of stellar mass versus local surface number density. The hydrodynamical simulations show global mass segregation from very early times which continues throughout the simulation, being only mildly influenced during sub-cluster merging. We find that up to approx. 2-3% of the massive sink particles (m > 2.5 Msun) are in relative isolation because they have formed there, although other sink particles can form later in their vicinity. Ejections of massive sinks from subclusters do not contribute to the number of isolated massive sink particles, as the gravitational softening in the calculation suppresses this process.
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