Molecular species delimitation methods recover most song-delimited cicada species in the European Cicadetta montana complex.

Molecular species delimitation is increasingly being used to discover and illuminate species level diversity, and a number of methods have been developed. Here, we compare the ability of two molecular species delimitation methods to recover song-delimited species in the Cicadetta montana cryptic species complex throughout Europe. Recent bioacoustics studies of male calling songs (premating reproductive barriers) have revealed cryptic species diversity in this complex. Maximum likelihood and Bayesian phylogenetic analyses were used to analyse the mitochondrial genes COI and COII and the nuclear genes EF1α and period for thirteen European Cicadetta species as well as the closely related monotypic genus Euboeana. Two molecular species delimitation methods, general mixed Yule-coalescent (GMYC) and Bayesian phylogenetics and phylogeography, identified the majority of song-delimited species and were largely congruent with each other. None of the molecular delimitation methods were able to fully recover a recent radiation of four Greek species.

Histochemical detection of anionic components in the cephalopod brain.

Brains obtained from three species of mediterranean cephalopods (Loligo, Sepia, Octopus) were fixed in Bouin's solution. Paraffin sections were cut sequentially at the frontal plane and used for Alcian blue staining (critical electrolyte concentration method), colloidal iron hydroxide staining procedure, the periodic acid Schiff's reagent method, and the lead tetra-acetate-Schiff method. The stained sections were evaluated at 2 regions of different histological composition: the palliovisceral ganglion and the optic lobe. A high concentration of anionic components was found in synaptic regions of the neuropil whereas neuronal cell bodies showed a relatively weak staining of these constituents. There was a significant reaction of the perineuronal glia nets in the cellular rind of the palliovisceral ganglion. From the comparison of staining patterns obtained with the 4 methods in this study and literature data it can be concluded that the detected anionic sites are mainly carboxyl groups of acidic proteins and/or glycoproteins. Sulphate groups may be present in lower concentrations. Their distribution reveals that the role of anionic components other than sialic acids in the invertebrate brain might be discussed in the context of synaptic transmission similar to that in vertebrates. The possible involvement of the glia cell population has to be taken into consideration.