Mitonuclear discordance across taxa is increasingly recognized as posing a major challenge to species delimitation based on DNA sequence data. Integrative taxonomy has been proposed as a promising framework to help address this problem. However, we still lack compelling empirical evidence scrutinizing the efficacy of integrative taxonomy in relation to, for instance, complex introgression scenarios involving many species. Here, we report remarkably widespread mitonuclear discordance between about 15 mitochondrial and 4 nuclear Brachionus calyciflorus groups identified using different species delimitation approaches. Using coalescent-, Bayesian admixture-, and allele sharing-based methods with DNA sequence or microsatellite data, we provide strong evidence in support of hybridization as a driver of the observed discordance. We then describe our combined molecular, morphological, and ecological approaches to resolving phylogenetic conflict and inferring species boundaries. Species delimitations based on the ITS1 and 28S nuclear DNA markers proved a more reliable predictor of morphological variation than delimitations using the mitochondrial COI gene. A short-term competition experiment further revealed systematic differences in the competitive ability between two of the nuclear-delimited species under six different growth conditions, independent of COI delimitations; hybrids were also observed. In light of these findings, we discuss the failure of the COI marker to estimate morphological stasis and morphological plasticity in the B. calyciflorus complex. By using B. calyciflorus as a representative case, we demonstrate the potential of integrative taxonomy to guide species delimitation in the presence of mitonuclear phylogenetic conflicts.

The discovery and exploration of cryptic species have been profoundly expedited thanks to developments in molecular biology and phylogenetics. In this study, we apply a reverse taxonomy approach to the Brachionus calyciflorus species complex, a commonly studied freshwater monogonont rotifer. By combining phylogenetic, morphometric and morphological analyses, we confirm the existence of four cryptic species that have been recently suggested by a molecular study. Based on these results and according to an exhaustive review of the taxonomic literature, we name each of these four species and provide their taxonomic description alongside a diagnostic key.

Greece is situated in the East Mediterranean region and in the Balkan peninsula, i.e., a European biodiversity hotspot with high endemism in subterranean and freshwater fauna, highlighting the need to understand its biodiversity. A literature search was undertaken to present a checklist of cladocerans and copepods based on a compilation of published and current data, from 1892 up to 2022 from inland surfaces and subterranean water bodies from different regions of Greece. For Cladocera, 80 species were recorded (9 families with 35 genera). The most diverse families were Chydoridae (20 genera with 33 species) and Daphniidae (5 genera with 27 species). For copepoda, 134 taxa were recorded, in surface water bodies (12 families with 34 genera), subterranean water bodies (7 families with 27 genera), and parasitic copepods (3 families with 3 genera). The most diverse families in surface waters were Cyclopidae (15 genera with 41 taxa) and Diaptomidae (5 genera with 17 species), while those in subterranean waters were Cyclopidae (11 genera with 35 taxa) and Canthocamptidae (6 genera with 17 taxa). More species are expected to be discovered after sampling understudied regions, especially islands, as well as water bodies such as temporary pools, swamps, ditches, puddles, and the littoral parts of lakes, while molecular studies are needed to clarify various cases of complex taxonomy.

Grazing potential (GP, in % day⁻¹) was estimated for the plankton communities of 13 Greek lakes covering the trophic spectrum, in order to examine its sensitiveness in discriminating different classes of ecological water quality. Lakes with high GP values exhibited high zooplankton biomass dominated by large cladocerans or/and calanoids while lakes with low GP values had increased phytoplankton biomass and/or domination of small-bodied zooplankton indicating intensive fish predation. GP successfully distinguished among ecological water quality classes (estimated using the phytoplankton water quality index PhyCoI) indicating its potential use as a metric for ecological water quality assessment. As a next step, PhyCoI index was modified to include GP as a metric in order to enhance the phytoplankton-based ecological status classification of lakes incorporating zooplankton as a supporting factor. The PhyCoI_GP successfully assessed the ecological water quality in accordance with PhyCoI classification whereas it was significantly correlated with the eutrophication proxy TSI_SD based on Secchi Depth. Thus, we propose to use the modified phytoplankton index PhyCoI_GP for monitoring the ecological water quality of lakes.