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In 1923, Robert Feulgen designed a quantitative approach to measure the relative DNA content of a species (its so-called “C-value”, which is defined as half of the DNA content of a somatic cell and is expressed in pg). The Feulgen approach has several advantages: it can be used on ethanol-preserved samples, it is relatively inexpensive, and it does not require sophisticated equipment ( Hardie et al. 2002 ,  Jeffery and Gregory 2014 ,  Kasten 2003 ). Gathering DNA content information is a key preliminary step for whole-genome sequencing of non-model species and also yield important information for ecological and evolutionary studies as well as for conservation ( Jeffery et al. 2013 ), since genome size has been shown to be strongly correlated to extinction risk in animals and  plants ( Vinogradov 2004 ,  Vinogradov 2003 ).Among invertebrates, Niphargidae is among the largest families of freshwater subterranean amphipods in the world, and more than 450 niphargid species have been described ( Horton et al. 2021 ). Thus, it is considered an ideal model for evolutionary and ecological studies. Using a new, improved Feulgen protocol, we generated novel genome size estimates for 26 samples of  Niphargus   schellenbergi   collected from central and western European niphargids. The obtained C-values ranged from 2.75 to 5.25 pg. Such a two-fold intraspecific variation is unusual but not unheard of: it occurs also in the monogonont rotifer  Brachionus asplanchnoidis  ( Stelzer et al. 2021 ). Further research will be required to find out the mechanisms responsible for this variation, which may result from copy-number variation, polyploidy and/or various amounts of transposable elements.

A Feulgen glimpse into genome evolution during range expansion: a case study of the subterranean amphipod Niphargus schellenbergi