Isolation and characterization of 8 microsatellite loci for the ‘‘killer shrimp’’, an invasive Ponto-Caspian amphipod Dikerogammarus villosus (Crustacea: Amphipoda)

Dikerogammarus villosus is a freshwater amphipod of the Ponto-Caspian origin recognized as one of the 100 worst alien species in Europe, having negative impact on biodiversity and functioning of the invaded aquatic ecosystems. The species has a wide ecophysiological tolerance and during the last 20 years it has rapidly spread throughout European inland waters. In consequence, it presents a major conservation management problem. We describe eight polymorphic microsatellite loci developed for D. villosus by combining a biotin-enrichment protocol and new generation 454GS-FLX Titanium pyrosequencing technology. When genotyped in 64 individuals from two locations, the loci exhibited a mean diversity of 4.87 alleles per locus (2–13). The mean observed and expected heterozygosities were, respectively, 0.439 (0.091–0.844) and 0.468 (0.089–0.843). Gametic disequilibrium was not detected for any pair of loci. The microsatellite markers will be a valuable tool in assessing the demographic processes associated with invasion of the killer shrimp from a genetic point of view

Out of the Black Sea: Phylogeography of the Invasive Killer Shrimp Dikerogammarus villosus across Europe

The amphipod Dikerogammarus villosus has colonized most of the European main inland water bodies in less than 20 years, having deteriorating effect on the local benthic communities. Our aim was to reveal the species phylogeography in the native Black Sea area, to define the source populations for the colonization routes in continental Europe and for the newly established UK populations. We tested for the loss of genetic diversity between source and invasive populations as well as along invasion route. We tested also for isolation by distance. Thirty three native and invasive populations were genotyped for mtDNA (COI, 16S) and seven polymorphic nuclear microsatellites to assess cryptic diversity (presence of deeply divergent lineages), historical demography, level of diversity within lineage (e.g., number of alleles), and population structure. A wide range of methods was used, including minimum spanning network, molecular clock, Bayesian clustering and Mantel test. Our results identified that sea level and salinity changes during Pleistocene impacted the species phylogeography in the Black Sea native region with four differentiated populations inhabiting, respectively, the Dnieper, Dniester, Danube deltas and Durungol liman. The invasion of continental Europe is associated with two sources, i.e., the Danube and Dnieper deltas, which gave origin to two independent invasion routes (Western and Eastern) for which no loss of diversity and no isolation by distance were observed. The UK population has originated in the Western Route and, despite very recent colonization, no drastic loss of diversity was observed. The results show that the invasion of the killer shrimp is not associated with the costs of loosing genetic diversity, which may contribute to the success of this invader in the newly colonized areas. Additionally, while it has not yet occurred, it might be expected that future interbreeding between the genetically diversified populations from two independent invasion routes will potentially even enhance this success

Tiger beetle fauna (Coleoptera: Carabidae, Cicindelinae) of Morocco: distribution, phenology and list of taxa

The diversity and distribution of Cicindelinae in Morocco, including new unpublished data, is summarized and discussed. In total 17 species are reported from the country. Cicindela campestris campestris is excluded from the Moroccan fauna while the occurrence of Myriochila mirei is doubtful and should be confirmed by new data. The area adjacent to the Mediterranean Sea and Atlantic Ocean coastlines holds the highest species diversity, while mountainous regions are characterized by lower diversity but also by high level of species endemism. Grouped on the basis of their chorotypes, Moroccan Cicindelinae fall into six different groups: West Mediterranean (44% of Cicindelinae species), Maghreb endemics (22%), Mediterranean (11%), Saharian (11%), Mediterranean-Westturanian (6%) and Afrotropico-Indo-Mediterranean (6%). According to their phenology, the Moroccan tiger beetles can be divided into three groups: 1) spring active species, 2) spring-summer active species, and 3) summer active species

First record of Echinogammarus pungens (H. Milne Edwards, 1840) (Crustacea, Amphipoda) from Africa with the checklist of North African freshwater gammarids

Taking into account the vast area of North Africa, the freshwater gammarid fauna of this area remains rather poorly studied. So far, 20 species of the three following genera: Echinogammarus (11 species), Gammarus (8 spp.) and Chaetogammarus (1 sp.) have been reported from the area in literature. Another species, Echinogammarus pungens (H. Milne Edwards, 1840) was found in the river Titria (Oued Titria), north-western Tunisia, in April 2010. Individuals of both sexes including ovigerous females and juveniles were recorded at the site. This is a first report of this species from Tunisia and from North Africa extending the checklist of freshwater gammarid fauna of North Africa to 21 species. Interestingly the species has apparently replaced Echinogammarus tacapensis (Chevreux et Gauthier, 1924), reported from the same section of the Titria River in 1981. Such radical exchange of gammarid fauna and sudden appearance of the species that has never before been recorded from Africa remains mysterious. Two tentative explanations of that phenomenon are taken into account. First, that E. pungens is in fact a circum-Mediterranean species and was simply overlooked in this area before. Second, that the species is a recent migrant to North African inland waters introduced accidentally either by humans or by waterfowl

Morphology and genome size of Epipactis helleborine (L.) Crantz (Orchidaceae) growing in anthropogenic and natural habitats

Background The process of apophytism or spreading native species to human-made habitats is one of the main elements in the creation of plant cover in anthropogenic areas. Lately, an increase of anthropogenic localities with valuable flora has been observed. Apophytes are also members of the family Orchidaceae, especially from the genus Epipactis. The aim of the study was to (i) determine and compare the phenotypic variation of E. helleborine (L.) Crantz plants in anthropogenic and natural habitats, (ii) compare the genome size of plants growing in natural and anthropogenic habitats. The results reported in this study may indicate that a habitat influences morphological characteristics of plant species. Methods Field studies were conducted on four native stands and four stands in anthropogenic areas of E. helleborine in Poland in years 2011–2013. Biometrical analyses were performed on shoots and flowers. The flowers were characterised by 25 biometric features and measured using a Nikon SMZ 800 binocular, microscopic Moticam-1SP cameras and the MIPlus07 programme (Conbest Co.). The nuclear DNA content was determined in fresh and young leaves of E. helleborine, collected from four natural and four anthropogenic populations. Results We observed that in anthropogenic populations: (i) shoots were higher than shoots from natural populations, (ii) flowers differed significantly in terms of ten biometric features between habitats, (iii) the genome size of some population differed significantly between plants growing in natural and anthropogenic habitats. Discussion According to some researchers, the presence of phenotypic variability and the occurrence of ecotypes are adaptation strategies of plants to environmental changes. In our opinion, in the case of the studied anthropogenic habitats (roadside) in which the E. helleborine populations grew, we can talk about ecofen due to the often repeated set of characteristic features, i.e., high shoots, long inflorescence and long, broad leaves. We agree, however, that it is difficult to isolate a taxonomic unit for ecofen due to the lack of experimental research

Catch fast and kill quickly: do tiger beetles use the same strategies when hunting different types of prey?

Background Tiger beetles (Coleoptera: Cicindelidae) are fast running predatory insects preying on different small insects and other terrestrial arthropods. Prey is located by sight and captured after short and fast pursuit interspersed with pause-and-look behaviour. At least some tiger beetle species can recognise the size and location of prey using memory, which probably allows them to achieve greater hunting success. Material and Methods Two eurytopic tiger beetle species known to occur in different types of habitat were used in the study: Cicindela hybrida hybrida, a very common central European beetle found even in artificial habitats such as sandy roads or gravel pits, and Calomera littoralis nemoralis, a species widely distributed in southern European countries and occurring on sandy sea beaches, in salt marshes, as well as on sandy banks of rivers and lakes. Both species are very similar in body size. Specimens used in the study were collected in the field and later tested in the laboratory. We checked whether tiger beetles use different hunting strategies when attacking prey of different sizes and abilities to escape as well as whether the sex of the studied species makes a difference in its hunting behaviour. Results The hunting strategies of both tiger beetle species consist of the following main phases: identification, pursuit (often with stops), attack, and optional release of the prey, and then the secondary attack, abandonment of the prey, or consumption of the prey. Considerable differences were noticed in hunting behaviour depending on the type of prey, its movement ability and escape potential. Caterpillars were attacked without pursuit, in the head or directly behind the head where a concentration of nerves and main muscles responsible for walking are located. Effective attacks on beetles were executed at the connection between the thorax and the abdomen. Calomera littoralis strongly preferred slow moving prey, while Cicindela hybrida preferred in equal measure slow moving prey and medium-sized fast moving prey. The experiment on the preferred size of prey indicated small beetles and small caterpillars as favoured by Calomera littoralis, while Cicindela hybrida preferred medium-sized fast moving prey and large caterpillars. Discussion The hunting behaviour of Calomera littoralis and Cicindela hybrida is complicated and includes a number of phases allowing to locate, capture and kill the prey. Beetles are able to discriminate between different types of prey and apply different behavioural tactics to hunt it. As the particular strategies are used to increase hunting success, and as a result allow to accumulate energy for future activity of the predator, it can be expected that such a type of hunting behaviour is characteristic also of other tiger beetle species

SEM differences in sporophyte micromorphology of Plagiothecium nemorale and P. longisetum (Plagiotheciaceae, Bryophyta)

Plagiothecium nemorale sensu lato is described as a variable Eurasian taxon. Recent studies indicate that this variability is reflected in both qualitative and quantitative features of its gametophyte and its genetic variation. This taxon comprises at least two separate species: P. nemorale (Mitt.) A. Jaeger and P. longisetum Lindb. The present paper examines whether the gametophyte variability of these species is reflected in the micromorphological variation of the sporophyte. The analysis revealed that the seta of P. nemorale is shorter than that of P. longisetum. In addition, regarding the exostome teeth, the lower portion of the outer surface is characterised by loosely-arranged, moniliform cristae in P. nemorale, with conical papillae on the upper side; however, in P. longisetum it possesses tightly arranged, epapillose cristae, with clearly flattened papillae on the upper side. In addition, the inner surface is more gently papillose in P. nemorale than in the latter. These findings not only describe hitherto unknown features of the sporophyte of these species, but also justify the resurrection of P. longisetus as separate from P. nemorale

Mitochondrial Genomes, Phylogenetic Associations, and SNP Recovery for the Key Invasive Ponto-Caspian Amphipods in Europe

The Ponto-Caspian region is the main donor of invasive amphipods to freshwater ecosystems, with at least 13 species successfully established in European inland waters. Dikerogammarus spp. and Pontogammarus robustoides are among the most successful, due to their strong invasive impact on local biota. However, genomic knowledge about these invaders is scarce, while phylogeography and population genetics have been based on short fragments of mitochondrial markers or nuclear microsatellites. In this study, we provide: (i) a reconstruction of six mitogenomes for four invasive gammarids (D. villosus, D. haemobaphes, D. bispinosus, and P. robustoides); (ii) a comparison between the structure of the newly obtained mitogenomes and those from the literature; (iii) SNP calling rates for individual D. villosus and D. haemobaphes from different invasion sites across Europe; and (iv) the first time-calibrated full mitogenome phylogeny reconstruction of several Ponto-Caspian taxa. We found that, in comparison to other gammarids, the mitogenomes of Ponto-Caspian species show a translocation between the tRNA-E and tRNA-R positions. Phylogenetic reconstruction using the mitogenomes identified that Ponto-Caspian gammarids form a well-supported group that originated in the Miocene. Our study supports paraphyly in the family Gammaridae. These provided mitogenomes will serve as vital genetic resources for the development of new markers for PCR-based identification methods and demographic studies

Cenozoic origins of the genus Calliarcys (Insecta, Ephemeroptera) revealed by Micro‑CT, with DNA barcode gap analysis of Leptophlebiinae and Habrophlebiinae

We are grateful to Michel Sartori (Lausanne, Switzerland) and José Ángel Martín del Arco (Salamanca, Spain) for donating the specimens of C. humilis to SMNS, BC CAS, and UŁ collections. We are also grateful to Christel and Hans-Werner Hoffeins (Hamburg, Germany), Mike Reich (BSPG, Munich, Germany), and Evgeny Perkovsky (Schmalhausen Institute of Zoology, Kyiv, Ukraine) for access to their collections of fossil mayflies from the Eocene Rovno amber. We would like to thank Kateřina Bláhová (IE, BC CAS) and Milan Pallmann (SMNS) for technical assistance with line drawings and the preparation of a set of macro photographs. Tomasz Mamos (UniLodz, Poland) is acknowledged for his help in the Bayesian reconstruction of phylogeny, and Łukasz Trębicki (UniLodz, Poland) for help in molecular laboratory. Comments from reviewers helped to improve the manuscript. JA-T thank the staff of Bruker SkyScan in Kontich (Belgium) for their effectiveness and fast support, for their constant improvements to the software, and for implementing the new options we requested. In this respect, we are especially indebted to Alexander Sasov (now at NeoScan, https:// neosc an. com), Stephan Boons, Xuan Liu, Phil Salmon, and Vladimir Kharitonov. We would like to thank the reviewers for their thoughtful comments and efforts towards improving our manuscript. LSID urn:lsid:zoobank. org:pub:C58BEE82-0EC6-4C59-A02D-1E5F796179B6Additional information Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1038/ s41598- 022- 18234-4.Funding Open access funding enabled and organized by the University of Łódź (Poland). RJG acknowledges the financial support of the Grant Agency of the Czech Republic (No. 21-05216S) and institutional support of the Institute of Entomology (Biology Centre of the Czech Academy of Sciences) RVO: 60077344. Acquisition of research equipment used in this study has been carried out within equipment subsidy granted by Alexander von Humboldt Foundation [Georg Forster Research Fellowship for Experienced Researchers] for RJG.Mayflies (Ephemeroptera) are among the oldest pterygote insects, with the earliest fossils dating back to the Late Carboniferous. Within mayflies, Leptophlebiidae are a highly diverse and widespread group, with approximately 140 genera and 640 species. Whereas taxonomy, systematics, and phylogeny of extant Leptophlebiidae are in the focus of extensive studies, little is known about leptophlebiid fossil taxa. Because fossil remains of Ephemeroptera in sedimentary rocks are relatively rare, inclusions of mayflies in amber are a unique source of information on their evolution and diversity in the past. Leptophlebiidae found in Cenozoic resins mostly belong to the subfamilies Leptophlebiinae (in Eocene Baltic amber) and Atalophlebiinae (in Miocene Dominican and Mexican ambers). In the present contribution, we confirm the first finding of the genus Calliarcys from Eocene Baltic amber by using Micro-CT, which allowed confirming its generic placement by visualizing diagnostic key characters otherwise hidden by a cloud of turbidity. Additionally, we present first molecular data on the extant species Calliarcys humilis Eaton, 1881 from the Iberian Peninsula and the barcode gap analysis for Leptophlebiinae and Habrophlebiinae.Funding Open access funding enabled and organized by the University of Łódź (Poland)Grant Agency of the Czech Republic (No. 21-05216S)Institute of Entomology (Biology Centre of the Czech Academy of Sciences) RVO: 60077344Alexander von Humboldt Foundation [Georg Forster Research Fellowship for Experienced Researchers

Cities and Disturbed Areas as Man-made Shelters for Orchid Communities

Many species from the family Orchidaceae spread in anthropogenic habitats and numerous studies documenting this process are known. Unfortunately, such data are scattered throughout various papers and reports, sometimes fragmentary and hard to reach (the ‘grey literature’). Scientific elaboration on this topic still lacks a comprehensive review and summary of the scale of this phenomenon. Therefore, the main aim of this study was to gather, review and analyse such data, seeking the answer to the question whether the man-made habitats can be considered as refugee for orchids. The paper summarises data on the occurrence of orchid species in man-made habitats in Europe originating from published and unpublished sources. The particular emphasis was placed in urban habitats. For this purpose, the floristically data from 42 European cities were used. The conducted studies showed that the apohytism phenomenon in the family Orchidaceae was more widespread than had been previously reported. As a result, 70 species of orchids in the distinguished man-made habitats were found. The majority of the species grow on sand and clay pits. The most common species were Epipactis helleborine and Dactylorhiza majalis. The gathered data have confirmed that man-made habitats become refugee for many orchid species in the aftermath of the loss of their natural habitats. Thus, protection of sites transformed by man with orchid occurrence should be taken into consideration. These sites can become a source of very useful information for biogeographically and phylo-geographically analyses of many valuable and endangered species