Genome size, measured as the haploid nuclear DNA content (C-value), is extremely variable among eukaryotes. This variation has long puzzled biologists, because it could not be accounted by organismal complexity or the total number of genes (C-value paradox). In the last decades it has become evident that the observed genome size variation is largely caused by differences in the content of non-coding and/or repetitive DNA, such as introns, pseudogenes, or transposable elements [1, 2]. Nevertheless, there are still many unanswered questions about genome size diversity, such as the actual causes driving the differences in DNA content, speed and mode of changes in genome size over population genetic and longer evolutionary time scales, or the cellular and organismal consequences of large vs. small genome size .
So far, most genome size comparisons in animals have been done at high taxonomic levels, e.g., between classes, families, or genera (e.g.[4–7]). Studies on genome size variation among strains or closely related species are still very scarce (but see, [8, 9]). Yet, variation at these lower taxonomic levels might provide important clues on the tempo and mode of genome size evolution. Whenever two species have become separated in their evolutionary trajectories, their genome sizes might diverge neutrally, due to independently occurring processes of genome expansion and genomic deletion, unless there are factors constraining the evolution of genome size. In asexually reproducing organisms such processes might even occur on the population level. Such accumulated differences can become important, because they may contribute to genomic incompatibility, hybrid inviability, or reproductive isolation. For instance, a causal role of genome changes in speciation has been suspected for gen(om)e duplications [10, 11] and transposable elements (see , and references therein). In this study we focus on the evolution of genome size in Brachionus plicatilis, a cryptic species complex consisting of at least 14 closely related species [13–15].
Cryptic species complexes have been described in many animal groups (e.g., [16–18]). In particular, small microscopic invertebrates seem to harbor large amounts of hidden genetic diversity within morphotypes that had been traditionally classified as a single species. The rotifer B. plicatilis is one of the most striking examples of such hidden diversity: initially described as a single species, it has experienced an enormous taxonomic inflation to currently 14-22 postulated species, based on molecular markers [14, 15]. Morphological discrimination among some species of this complex is possible, however difficult, as it involves tight experimental control over environmental and developmental variation  or sophisticated analysis methods combined with high sample sizes . Despite the morphological similarity among members of the B. plicatilis complex, recent studies have demonstrated extensive ecological diversification in terms of temperature or salinity preferences  and prezygotic and postzygotic reproductive isolation among members in this species complex [14, 22, 23]. In addition there are large differences in body size, with more or less continuous variation across species [14, 24]. In the last decade, B. plicatilis has emerged as an important model organism for studies on speciation, sexual signaling [25–27], mate choice [23, 28], biogeography [29, 30], or selection in the wild [31, 32]. B. plicatilis is thus also a promising future candidate for genome sequencing.
In this study we use the flow cytometry method to estimate genome size variation in the B. plicatilis complex. We measured the genome sizes of 33 different clones, representative of 12 cryptic species and analyze these data in a phylogenetic context. In addition, we examined body size and egg size variation in a subset of our clones and tested whether genome size is significantly correlated with any of these variables. Finally we discuss our results in the light of studies on reproductive isolation among members of the Brachionus plicatilis species complex.