Micromorphology of Pine Needle Primordia and Young Needles after Bud Dormancy Breaking.

Using a scanning electron microscope, the micromorphologies of needle primordia and the young needles of seven pine species (Pinus cembra, P. mugo, P. nigra, P. rigida, P. sylvestris, P. strobus, and P. uncinata) were analyzed at phenological stages B2 and B3 (according to Debazac). In B2, needle tips were rounded or pointed, depending on the species. In P. cembra and P. strobus, teeth were noted on the tips. Teeth were also visible on the margins in P. mugo, P. cembra, and P. strobus. Stomata became visible in the late B2 phase (P. sylvestris, P. mugo, and P. nigra) near the needle tips and were arranged in rows. In the B3 phase, needle tips were pointed. Only in P. strobus was the needle tip slightly rounded. The teeth on the margin in all the species were pointed. In P. strobus, their size and density along the margin decreased basipetally. In B3 for all the species, numerous stomata were visible. In P. sylvestris, P. cembra, and P. strobus, Florin rings were also observed. These observations could be useful in pine systematics but also in palaeobotanical or physiological studies. To the best of our knowledge, this is the first study on the micromorphology of very young needles in representatives of the genus Pinus.

Integrative taxonomy reveals new, widely distributed tardigrade species of the genus Paramacrobiotus (Eutardigrada: Macrobiotidae).

In a moss sample collected in Ribeiro Frio, Madeira, Paramacrobiotus gadabouti sp. nov. was found and described using the integrative taxonomy approach. The new species is described based on morphological and morphometric data from both phase-contrast light microscopy (PCM), as well as scanning electron microscopy (SEM). Moreover, four DNA markers, three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI) markers, were used to elucidate the phylogenetic position of the new species within the family Macrobiotidae. The new species has a microplacoid that placed it within Parmacrobiotus richtersi group and exhibit richtersi-type eggs having processes terminated with cap-like structures. Paramacrobiotus gadabouti sp. nov. is most similar to Pam. alekseevi, Pam. filipi and Pam. garynahi, but differs from them mainly in details of egg morphology and morphometrics. Unlike other species from this group, which were confirmed as bisexual and showed limited distribution, Paramacrobiotus gadabouti sp. nov. is yet another parthenogenetic species with a wide distribution, demonstrating that at least some tardigrades confirm to the hypothesis of 'everything is everywhere'.

Morphological diversification of alien and native aquatic snails of the genus Physa and Aplexa (Gastropoda: Physidae) of Western and Central European range.

This is the first comparative study on alien and native Physidae (Gastropoda: Heterobranchia) from Western and Central Europe. Morphology, ecological features and distribution are presented for each physid species. We analysed taxonomical features of physid snails from Europe in great detail. Scanning electron microscopy (SEM) methods were used to elucidate the external morphology of the shells of physid gastropods that occur in Western and Central Europe. On comparison we found significant differences in the external morphology among the species. Morphological analyses facilitate the recognition of variations of physid shells. An interspecific similarities were noted within Physidae while interspecific differences were identified in the morphology of apex and spires. The lowest intraspecific variability in shells was noted between particular individuals of P. fontinalis and A. hypnorum. The most characteristic features and differences of representatives of Physidae are presented and discussed. This is especially important for the identification of Physa gyrina and the worldwide invasive species Physa acuta which resemble each other in shape and are difficult to distinguish. We also present a new identification key for physid species, including the results of ecological assessment and discuss current distribution of these species in Europe.

Reflectance spectroscopy and machine learning as a tool for the categorization of twin species based on the example of the Diachrysia genus.

In our work we used noninvasive point reflectance spectroscopy in the range from 400 to 2100 nm coupled with machine learning to study scales on the brown and golden iridescent areas on the dorsal side of the forewing of Diachrysia chrysitis and D. stenochrysis. We used our approach to distinguish between these species of moths. The basis for the study was a statistically significant collection of 95 specimens identified based on morphological feature and gathered during 23 years in Poland. The numerical part of an experiment included two independent discriminant analyses: stochastic and deterministic. The more sensitive stochastic approach achieved average compliance with the species identification made by entomologists at the level of 99-100%. It demonstrated high stability against the different configurations of training and validation sets, hence strong predictors of Diachrysia siblings distinctiveness. Both methods resulted in the same small set of relevant features, where minimal fully discriminating subsets of wavelengths were three for glass scales on the golden area and four for the brown. The differences between species in scales primarily concern their major components and ultrastructure. In melanin-absent glass scales, this is mainly chitin configuration, while in melanin-present brown scales, melanin reveals as an additional factor.

An integrative description of Diploechiniscus oihonnae/ (Richters, 1903) population from near the original type locality in Merok (Norway).

In the present study, we used integrative taxonomy to describe a population of Diploechiniscus oihonnae (Richters, 1903) from the neotype locality in Merok (Norway). We found no differences in the chaetotaxy formula between the life stages and sex of Dpl. oihonnae. The presence of filaments at Bd in some specimens of Dpl. oihonnae appears to be random and most likely represents the morphological variability of this species. We also obtained DNA sequences of 18S rRNA, 28S rRNA, ITS-2, and COI of Dpl. oihonnae from the neotype locality for comparison with the sequences available in GenBank, which showed low genetic differences between the neotypic population and specimens from other localities. Therefore, we decided to establish our specimens from Merok as neotype and neoparatypes of Dpl. oihonnae. Additionally, based on morphological characters, Dpl. horningi (Schuster Grigarick, 1971) was synonymised with Dpl. oihonnae.

Taxonomic Significance of Seed Morphology in Veronica L. (Plantaginaceae) Species from Central Europe.

In this study, 1159 seeds of 29 Central European species of the genus Veronica were analyzed based on scanning electron microscopy images. The species belonged to nine subgenera: Beccabunga, Chamaedrys, Cochlidiosperma, Pellidosperma, Pentasepalae, Pocilla, Pseudolysimachium, Stenocarpon and Veronica, following the newest phylogenetic classification of the genus. Nine measured characteristics of seeds and nine ratios were analyzed statistically using ANOVA followed by post hoc testing, cluster analysis and discriminant analysis. In most cases, the results were not congruent with the contemporary classification of the genus. Examinations of qualitative seed features by scanning electron microscopy included the cochlidiospermous or discoid seed type, the seed shape, the general sculpture of the seed coat surface, the sculpture of anticlinal and periclinal walls and some species-specific traits such as the presence of the epidermal appendix. All these features, apart from seed shape, were useful to distinguish all subgenera and some species within subgenera: Beccabunga, Chamaedrys, Pellidosperma, Pocilla (only V. filiformis) and Veronica. The identification key based on the seed micromorphological features was prepared.

Integrative description of five Pseudechiniscus species (Heterotardigrada: Echiniscidae: the suillus-facettalis complex).

Pseudechiniscus is a morphologically homogeneous heterotardigrade genus with a relatively low number of morphological features useful for the species discrimination. The species of the Pseudechiniscus suillus-facettalis complex are some of the most challenging tardigrades to identify. Here, we examine several populations from Antarctica, Italy, Madagascar and Norway that would have most likely been attributed to Pse. suillus prior to the recent redescription of the species. Populations were analysed using integrative taxonomy-a combination of classical morphology and morphometry, as well as genetic data. Besides minute differences in dorsal sculpture and morphometry, we found clear, species-specific differences in ventral sculpture which are very useful in discrimination of Pseudechiniscus species. Based on morphology (mainly ventral sculpture) and significant genetic distances in COI and ITS-2 sequences, we describe five new species: Pse. angelusalas sp. nov. from Madagascar, Pse. dastychi sp. nov. from Antarctica, Pse. ehrenbergi sp. nov. from Italy as well as Pse. indistinctus sp. nov. and Pse. lacyformis sp. nov. from Norway. Finally, we provide an updated phylogenetic tree of the genus Pseudechiniscus based on COI sequences.

Preliminary evaluation of application of a 3-dimensional network structure of siloxanes Dergall preparation on chick embryo development and microbiological status of eggshells.

The spatial network structure of Dergall is based on substances nontoxic to humans and the environment which, when applied on solid surfaces, creates a coating that reduces bacterial cell adhesion. The bacteriostatic properties of siloxanes are based on a purely physical action mechanism which excludes development of drug-resistant microorganisms. The aims of the present study were to 1) evaluate a Dergall layer formed on the eggshell surface regarding the potential harmful effects on the chick embryo; 2) evaluate antimicrobial activity and estimate the prolongation time of Dergall's potential antimicrobial activity. Dergall at a concentration of 0.6% formed a layer on the eggshell surface. In vitro testing of the potential harmful effects of Dergall by means of a hen embryo test of the chorioallantoic membrane showed no irritation reaction at a concentration of 3% and lower. The hatchability of the groups sprayed with a Dergall water solution with a concentration of 0 to 5% was 89.1 to 93.8% for fertilized eggs (P > 0.05) but decreased to 63.7% (P < 0.05) in the group sprayed with a 6% concentration of the solution. This phenomenon was caused by embryo mortality in the first week of incubation. At the concentration of 0.6%, Dergall exhibited strong antibacterial properties against bacteria such as Staphylococcus aureus, Escherichia coli, Shigella dysenteriae, Shigella flexneri, and Salmonella typhimurium. For Streptococcus pyogenes, the highest antibacterial activity of Dergall was reported in the concentrations of 100 and 50%. For Pseudomonas aeruginosa, no antibacterial activity of Dergall was generally observed, but in vivo testing showed a strong decrease of all gram-negative bacteria growth. Moreover, a prolonged antimicrobial effect lasting until 3 D after disinfection was observed, which makes Dergall a safe and efficient disinfectant.

Integrative description of bisexual Paramacrobiotus experimentalis sp. nov. (Macrobiotidae) from republic of Madagascar (Africa) with microbiome analysis.

In a moss samples collected on Madagascar two populations of Paramacrobiotus experimentalis sp. nov. were found. Paramacrobiotus experimentalis sp. nov. with the presence of a microplacoid and areolatus type of eggs is similar to Pam. danielae, Pam. garynahi, Pam. hapukuensis, Pam. peteri, Pam. rioplatensis and Pam. savai, but it differs from them by some morphological and morphometric characters of the eggs. The p-distance between two COI haplotypes of Pam. experimentalis sp. nov. was 0.17%. In turn, the ranges of uncorrected genetic p-distances of all Paramacrobiotus species available in GenBank was from 18.27% (for Pam. lachowskae) to 25.26% (for Pam. arduus) with an average distance of 20.67%. We also found that Pam. experimentalis sp. nov. is bisexual. This observation was congruent on three levels: (i) morphological - specimen size dimorphism; (ii) structural (primary sexual characteristics) - females have an unpaired ovary while males have an unpaired testis and (iii) molecular - heterozygous and homozygous strains of the ITS-2 marker. Although symbiotic associations of hosts with bacteria (including endosymbiotic bacteria) are common in nature and these interactions exert various effects on the evolution, biology and reproductive ecology of hosts, there is still very little information on the bacterial community associated with tardigrades. To fill this gap and characterise the bacterial community of Pam. experimentalis sp. nov. populations and microbiome of its microhabitat, high throughput sequencing of the V3-V4 hypervariable regions in the bacterial 16S rRNA gene fragment was performed. The obtained 16S rRNA gene sequences ranged from 92,665 to 131,163. In total, 135 operational taxonomic units (OTUs) were identified across the rarefied dataset. Overall, both Pam. experimentalis sp. nov. populations were dominated by OTUs ascribed to the phylum Proteobacteria (89-92%) and Firmicutes (6-7%). In the case of samples from tardigrades' laboratory habitat, the most abundant bacterial phylum was Proteobacteria (51-90%) and Bacteroides (9-48%). In all compared microbiome profiles, only 16 of 137 OTUs were shared. We found also significant differences in beta diversity between the partly species-specific microbiome of Pam. experimentalis sp. nov. and its culturing environment. Two OTUs belonging to a putative bacterial endosymbiont were identified - Rickettsiales and Polynucleobacter. We also demonstrated that each bacterial community was rich in genes involved in membrane transport, amino acid metabolism, and carbohydrate metabolism.

Two new species of Tardigrada from Ecuador (South America).

In two moss samples collected in El Cajas National Park (Azuay Province, Ecuador), 33 specimens and 19 eggs of a new species of Macrobiotus and 56 heterotardigrades belonging to a new Echiniscus species were extracted. Macrobiotus dulciporus sp. nov. belongs to the hufelandi group and is most similar to Macrobiotus maculatus Iharos, 1973. It differs from M. maculatus mainly based on the presence of an oral cavity armature of the patagonicus type, smooth egg chorion surface, lower number of processes on the egg circumference and some morphometric characters of the egg processes. Echiniscus hannae sp. nov. belongs to the bigranulatus group, but differs from all species in this group due to the presence of large, smooth areas on its dorsal plate.

Variability of Echiniscus tristis Gasiorek & Kristensen, 2018-is morphology sufficient for taxonomic differentiation of Echiniscidae?

The majority of species in the genus Echiniscus (Heterotardigrada) have been described based on differences in the chaetotaxy or dorsal sculpture. Dorsal sculpture is, in general, considered to be species-specific and not very variable; however, many problems have arisen due to various interpretations of microscope images, which has led to taxonomic confusion in the genus Echiniscus. Conversely, chaetotaxy is generally much easier to interpret, even using low-quality microscope optics. In this study, we emended the description of Madagascan population of Echiniscus tristis Gasiorek & Kristensen, 2018 that exhibits several different types of chaetotaxy and dorsal sculpture. The analysed specimens were characterised by two types of chaetotaxy, A-C-Dd-E and A-Dd-E, but we also found a wide range of variation in appendage number, shape and length. The observed differences are partly correlated with life stages. Additionally, we analysed DNA sequences of 28S rRNA, ITS-2 and COI of the two main morphotypes, and did not find significant genetic differentiation of the two morphotypes. This highlights the importance of analysing the morphology of both immature stages and adults, as well as of DNA markers in tardigrade species identification.

An integrative description of a new tardigrade species Mesobiotus romani sp. nov. (Macrobiotidae: harmsworthi group) from the Ecuadorian Pacific coast.

In a mixed moss and lichen sample collected in Esmeraldas Province in north-western Ecuador, 20 tardigrades and 11 eggs, belonging to a new species of the genus Mesobiotus, were found. In addition to the traditional taxonomic description with morphometrics, light and scanning microscopy imaging, we also provide nucleotide sequences of three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI) DNA fragments of the new species. Based on the egg chorion morphology, Mesobiotus romani sp. nov. is the most similar to: M. binieki, M. coronatus, M. dimentmani, M. patiens, M. perfidus, M. philippinicusi, M. pseudoblocki, M. pseudocoronatus, M. pseudopatiens, M. radiatus, M. rigidus, M. simulans and M. wuzhishanensis, but differs mainly by some specific characters of both egg and adult morphology, and morphometrics.

An integrative redescription of the nominal taxon for the Mesobiotus harmsworthi group (Tardigrada: Macrobiotidae) leads to descriptions of two new Mesobiotus species from Arctic.

The Mesobiotus harmsworthi group has a global distribution, with localities in polar, temperate and tropical zones. Since the first species of the harmsworthi group was described in the beginning of the 20th century, tens of new species within the group were found and named. However, the diagnosis of the nominal Mesobiotus harmsworthi is insufficient and enigmatic, thus it can be is a serious obstacle in solving the taxonomy of this group. Here, we integratively redescribe the nominal species for the genus Mesobiotus, i.e., Mesobiotus harmsworthi and clarify taxonomic statuses of the two subspecies: M. harmsworthi harmsworthi and M. harmsworthi obscurus that have been recognised as distinct taxa for more than three decades. Traditionally, egg chorion in M. harmsworthi was considered almost smooth and without any traces of areolation, however here we report many misunderstandings that accumulated across decades and we show that, in fact, the chorion in this species exhibits a partially developed areolation. We present an integrative (morphological, morphometric and molecular) diagnosis of the nominal taxon and we confirm that it differs from other species of the harmsworthi group by morphological characters of both animals and eggs. Additionally, we describe two new species of the genus Mesobiotus: M. skorackii sp. nov. from the Kyrgyz Republic (using classical morphological description) and M. occultatus sp. nov. from Svalbard Archipelago (by means of integrative taxonomy). Finally, we also provide the first genetic phylogeny of the genus Mesobiotus based on COI sequences which, together with molecular species delimitation, independently confirms the validity of the analysed taxa.

Cylapofulvidius Chérot & Gorczyca, 2000-a synonym of Fulvidius Poppius, 1909 (Hemiptera: Heteroptera: Miridae: Cylapinae).

The genus Cylapofulvidius Chérot & Gorczyca, 2000 is proposed as a junior subjective synonym of Fulvidius Poppius, 1909 syn. nov. and all members of Cylapofulvidius are transferred to the genus Fulvidius. The genus Fulvidius and its type species F. punctatus Poppius, 1909 are redescribed, and diagnoses of each Fulvidius species, habitus picture of the adult and illustration of male genital structures of most of the treated species are provided. Scanning electron micrographs of selected structures of Fulvidius webbi (Chérot & Gorczyca, 2000) are presented for the first time. A key to species of Fulvidius is provided.