Impact of Long-Term Floods on Spatial Dynamics of Myrmica scabrinodis, a Host Ant of a Highly Threatened Scarce Large Blue (Phengaris teleius).

Extensively used wet meadows with high species diversity are under threat in Europe by anthropogenic pressure. The increasing frequency of prolonged flooding is emerging as an additional threat to this fragile environment. In our study, we investigated how prolonged flooding affects the spatial distribution and temporal dynamics (through mortality and recolonization process) of the host ant species Myrmica scabrinodis, which is essential for the survival of the endangered Scarce large blue (Phengaris teleius). The study was conducted in the flood-prone Ljubljansko barje plain situated on the southern edge of the species' global range. Prolonged flooding in the study area, possibly affecting the past and current distribution of the host ant M. scabrinodis, was recorded in 2010, 2013, and 2017. In 2020, we set 160 ant traps to estimate the distribution of host ants in a system of meadows covering the entire gradient of flood history. Results indicate that M. scabrinodis survives the flooding for up to three days, starting to disappear if flooding persists longer. After the flooding recedes, ants gradually recolonize empty habitats from the surrounding upland refugia. Our spatial analyses predict that the average recolonization speed was about 29 m per year and that in a year, ants compensate for the mortality effects of 1.8 days of flooding by recolonization in a year. These results show that flooding should be considered as an additional (in some areas, a major) threat to the endangered P. teleius through its deleterious effects on the host ant species.

First Detection of Honeybee Pathogenic Viruses in Butterflies.

Several pathogens are important causes of the observed pollinator decline, some of which could be transmitted between different pollinator species. To determine whether honeybee viruses can be transmitted to butterflies, a total of 120 butterflies were sampled at four locations in Slovenia. At each location, butterflies from three families (Pieridae, Nymphalidae, Hesperiidae/Lycenidae) and Carniolan honeybees (Apis mellifera carnica) were collected. The RNA of six honeybee viruses, i.e., acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus A (DWV-A), Sacbrood bee virus (SBV), and Lake Sinai virus 3 (LSV3), was detected by a specific quantitative method (RT-PCR). The presence of ABPV, BQCV, LSV3, and SBV was detected in both butterflies and honeybees. All butterfly and bee samples were negative for CBPV, while DWV-A was detected only in honeybees. The viral load in the positive butterfly samples was much lower than in the positive bee samples, which could indicate that butterflies are passive carriers of bee viruses. The percentage of positive butterfly samples was higher when the butterflies were collected at sampling sites with a higher density of apiaries. Therefore, we believe that infected bees are a necessary condition for the presence of viruses in cohabiting butterflies. This is the first study on the presence of pathogenic bee viruses in butterflies.

Five nuclear protein-coding markers for establishing a robust phylogenetic framework of niphargid crustaceans (Niphargidae: Amphipoda) and new molecular sequence data.

The data presented here includes selection of 5 successfully amplified protein-coding markers for inferring phylogenetic relationships of the family of amphipod crustaceans Niphargidae. These markers have been efficiently amplified from niphargid samples for the first time and present the framework for robust phylogenetic assessment of the family Niphargidae. They are useful for phylogenetic purposes among other amphipod genera as well. In detail, the data consists of two parts: 1. Information regarding markers, specific oligonucleotide primer pairs and conditions for PCR reaction that enables successful amplification of specific nucleotide fragments. Two pairs of novel oligonucleotide primers were constructed which enable partial sequence amplification of two housekeeping genes: arginine kinase (ArgKin) and glyceraldehyde phosphate dehydrogenase (GAPDH), respectively. Additionally, 3 existing combinations of oligonucleotide primer pairs for protein-coding loci for glutamyl-prolyl tRNA synthetase (EPRS), opsin (OP) and phosphoenolpyruvate carboxykinase (PEPCK) were proven to be suitable to amplify specific nucleotide fragments from selected amphipod specimens; 2. Information on novel nucleotide sequences from amphipod taxa of the family Niphagidae and related outgroup taxa. Unilocus phylogenetic trees were constructed using Bayesian analysis and show relationships among selected taxa. Altogether 299 new nucleotide sequences from 92 specimens of the family Niphargidae and related outgroup amphipod taxa are deposited in GenBank (NCBI) repository and available for further use in phylogenetic analyses.

Revised checklist of the butterflies of Serbia (Lepidoptera: Papilionoidea).

With studies spanning almost two centuries, butterflies are one of the best known insect groups in Serbia. However, there are still several inconsistencies regarding the number and selection of species included in national checklists published in the last decades. In order to overcome the confusing situation we provide a taxonomically up-to-date checklist of the butterflies for the country, including Kosovo, based on a comprehensive survey of the literature, inspection of available museum collections and from intensive field surveys over the last twenty years. Our aim is also to resolve some long standing problems with species potentially occurring in the country. For this purpose genitalia dissections and DNA barcoding have been used for identification where necessary. The annotated checklist includes 199 species of which Carcharodus orientalis is new for the country and the recently discovered white Anthocharis damone is listed for the first time. We also provide conclusive evidence of the presence of Melitaea ornata in Serbia. Among species listed in the previous species lists we excluded eight species due to inconclusive evidence, and provide a detailed explanation for their exclusion. We hope this publication will stimulate further studies of this important bioindicator group of insects and provide the basis for their conservation in the country.

An updated checklist of the European Butterflies (Lepidoptera, Papilionoidea).

This paper presents an updated checklist of the butterflies of Europe, together with their original name combinations, and their occurrence status in each European country. According to this checklist, 496 species of the superfamily Papilionoidea occur in Europe. Changes in comparison with the last version (2.6.2) of Fauna Europaea are discussed. Compared to that version, 16 species are new additions, either due to cryptic species most of which have been discovered by molecular methods (13 cases) or due to discoveries of Asian species on the eastern border of the European territory in the Ural mountains (three cases). On the other hand, nine species had to be removed from the list, because they either do not occur in Europe or lost their species status due to new evidence. In addition, three species names had to be changed and 30 species changed their combination due to new evidence on phylogenetic relationships. Furthermore, minor corrections were applied to some authors' names and years of publication. Finally, the name Polyommatusottomanus Lefèbvre, 1831, which is threatened by its senior synonym Lycaenalegeri Freyer, 1830, is declared a nomen protectum, thereby conserving its name in the current combination Lycaenaottomana.

Species delimitation in the Grayling genus Pseudochazara (Lepidoptera, Nymphalidae, Satyrinae) supported by DNA barcodes.

The Palaearctic Grayling genus Pseudochazara encompasses a number of petrophilous butterfly species, most of which are local endemics especially in their centre of radiation in SW Asia and the Balkans. Due to a lack of consistent morphological characters, coupled with habitat induced variability, their taxonomy is poorly understood and species delimitation is hampered. We employed a DNA barcoding approach to address the question of separate species status for several European taxa and provide first insight into the phylogeny of the genus. Unexpectedly we found conflicting patterns with deep divergences between presumably conspecific taxa and lack of divergence among well-defined species. We propose separate species status for Pseudochazara tisiphone, Pseudochazara amalthea, Pseudochazara amymone, and Pseudochazara kermana all of which have separate well supported clades, with the majority of them becoming local endemics. Lack of resolution in the 'Mamurra' species group with well-defined species (in terms of wing pattern and coloration) such as Pseudochazara geyeri, Pseudochazara daghestana and Pseudochazara alpina should be further explored using nuclear molecular markers with higher genetic resolution.

Annotated checklist of Albanian butterflies (Lepidoptera, Papilionoidea and Hesperioidea).

The Republic of Albania has a rich diversity of flora and fauna. However, due to its political isolation, it has never been studied in great depth, and consequently, the existing list of butterfly species is outdated and in need of radical amendment. In addition to our personal data, we have studied the available literature, and can report a total of 196 butterfly species recorded from the country. For some of the species in the list we have given explanations for their inclusion and made other annotations. Doubtful records have been removed from the list, and changes in taxonomy have been updated and discussed separately. The purpose of our paper is to remove confusion and conflict regarding published records. However, the revised checklist should not be considered complete: it represents a starting point for further research.

Genetic differentiation between two species of the medicinal leech, Hirudo medicinalis and the neglected H. verbana, based on random-amplified polymorphic DNA.

The medicinal leech is one of the few parasitic invertebrates widely used in medicine and as a scientific model object. Because of a dramatic decline in its natural populations, it is subject to considerable conservation effort. Despite all attention, there is confusion regarding the taxonomic status of different morphological forms. The prevailing view is that all varieties of medicinal leech in Europe represent the same species, Hirudo medicinalis. However, the present study based on RAPD molecular markers demonstrates that a second European taxon, H. verbana, forms a distinct species. Phenetic clustering and principal coordinate analysis of eight populations revealed the same basic structure, reflecting taxonomic rather than geographic subdivision. Variation between species explained 60% of the total molecular variance (phiCT=0.60, P<0.001). Both taxa displayed a significant number of specific RAPD markers. Conversely, no specific fragment supporting the geographic association of both taxa was found. Since the stronghold of commercially exploited medicinal leech populations in southeastern Europe and Turkey belongs to H. verbana, most medicinal and scientific applications probably use this species, not H. medicinalis. Appropriate taxonomic correction of international conservation conventions and legislation is essential.