Summary1. The generalized mixed Yule-coalescent (GMYC) model has become one of the most popular approaches for species delimitation based on single-locus data, and it is widely used in biodiversity assessments and phylogenetic community ecology. We here examine an array of factors affecting GMYC resolution (tree reconstruction method, taxon sampling coverage/taxon richness and geographic sampling intensity/geographic scale). 2. We test GMYC performance based on empirical data (DNA barcoding of the Romanian butterflies) on a solid taxonomic framework (i.e. all species are thought to be described and can be determined with independent sources of evidence). The data set is comprehensive (176 species), and intensely and homogeneously sampled (1303 samples representing the main populations of butterflies in this country). Taxonomy was assessed based on morphology, including linear and geometric morphometry when needed. 3. The number of GMYC entities obtained constantly exceeds the total number of morphospecies in the data set. We show that c. 80% of the species studied are recognized as entities by GMYC. Interestingly, we show that this percentage is practically the maximum that a single-threshold method can provide for this data set. Thus, the c. 20% of failures are attributable to intrinsic properties of the COI polymorphism: overlap in inter-and intraspecific divergences and non-monophyly of the species likely because of introgression or lack of independent lineage sorting. 4. Our results demonstrate that this method is remarkably stable under a wide array of circumstances, including most phylogenetic reconstruction methods, high singleton presence (up to 95%), taxon richness (above five species) and the presence of gaps in intraspecific sampling coverage (removal of intermediate haplotypes). Hence, the method is useful to designate an optimal divergence threshold in an objective manner and to pinpoint potential cryptic species that are worth being studied in detail. However, the existence of a substantial percentage of species wrongly delimited indicates that GMYC cannot be used as sufficient evidence for evaluating the specific status of particular cases without additional data. 5. Finally, we provide a set of guidelines to maximize efficiency in GMYC analyses and discuss the range of studies that can take advantage of the method.

uncovering cryptic biodiversity is essential for understanding evolutionary processes and patterns of ecosystem functioning, as well as for nature conservation. As European butterflies are arguably the best-studied group of invertebrates in the world, the discovery of a cryptic species, twenty years ago, within the common wood white Leptidea sinapis was a significant event, and these butterflies have become a model to study speciation. Here we show that the so-called 'sibling' Leptidea actually consist of three species. The new species can be discriminated on the basis of either DnA or karyological data. such an unexpected discovery challenges our current knowledge on biodiversity, exemplifying how a widespread species can remain unnoticed even within an intensely studied natural model system for speciation. G iven the global biodiversity crisis [1][2][3] , cataloguing the earth's species has become a race against time. Several studies have highlighted the presence and importance of cryptic biodiversity, which might represent a substantial proportion of Earth's natural capital. An estimate of cryptic species diversity is important to better understand evolutionary processes and patterns of ecosystem functioning, while also having deep implications for nature conservation 4,5 . The recent increase in the number of reported cryptic species is, in large part, owing to an increasing amount of studies incorporating DNA-based techniques, including large-scale approaches such as DNA barcoding 6 , which often provide resolution beyond the boundaries of morphological information. However, documenting cryptic diversity based on DNA data alone is generally not sufficient, prompting calls for integrative morphological, ecological and molecular approaches 7,8 . Recent estimates on the distribution of cryptic diversity are contradictory, and are based on a thin empirical foundation 4,9 . In any case, it is to be expected that the highest number of yet-to-be-discovered cryptic species is concentrated in the most biodiverse and least explored regions of our planet (that is, tropical areas). In temperate regions such as Europe, it is assumed that the level of unrecognized diversity is low, not only because of lower species richness, but also because taxonomic research has been intense for many groups of organisms. Such a case is represented by butterflies, probably the best-studied group of invertebrates, which have become a flagship for insect conservation efforts in Europe 10,11 .The discovery of a new European species of wood white (Leptidea sp.) at the end of the twentieth century was an important event in butterfly systematics. Leptidea sinapis (Linnaeus, 1758), a common butterfly with Palaearctic distribution was found to 'hide' a cryptic species, Leptidea reali (Reissinger, 1989) 12,13 . After the two species were shown to be separable based on their genitalia-but not on their wing morphology 13 -several studies revealed that L. reali is often sympatric with L. sinapis and that its distribution is almost as wide as that ...