Two new pseudocryptic species in the medium-sized common European land snails, Fruticicola Held, 1838; as a result of phylogeographic analysis of Fruticicola fruticum (O. F. Müller, 1774) (Gastropoda: Helicoidea: Camaenidae).

Fruticicola fruticum (O. F. Müller, 1774), a medium-sized helicoid snail in the Camaenidae, has a wide range in Europe, reaching from the Urals and the Caucasus to the Balkans, and from the southern part of Scandinavia, through Central Europe to eastern and central France and northern Italy. There are numerous studies on its distribution, biology, life cycle, etc., but little is known about the genetic diversity of this taxon. Here, we studied the phylogeny and phylogeography of F. fruticum using two mitochondrial markers: cytochrome oxidase subunit I (COI) and 16S ribosomal RNA (16S); and four nuclear markers: 18S ribosomal RNA (18S), 28S ribosomal RNA (28S), internal transcribed spacer (ITS-2), and histone 3 (H3). The study was based on 59 populations sampled across the range. Whereas nuclear markers showed little differentiation, phylogenetic analysis of COI sequences clearly confirmed the distinctness of the European Fruticicola and Asian Bradybaena (p-distance 0.229). Within Fruticicola 54 haplotypes were detected, haplotype diversity (Hd) = 0.973 ± 0.006, nucleotide diversity (π) = 0.137 ± 0.005. ABGD and PTP delimitation analyzes distinguished eight mOTUs. Two sequences (our mOTU C) from Russia were published in the GenBank as two distinct species: F. schrenckii and F. transbaicalia. Seven further mOTUs identified in our study formed three distinct lineages, regarded as species. The first (mOTU A and mOTU B), represented by 40 populations, occupies a wide range across northern and central Europe, extending east to Ukraine and south to northern Croatia (mOTU B). It encompasses the type locality of F. fruticum, and can be recognized as F. fruticum sensu stricto. Another lineage (mOTU D and mOTU E), represented by six populations in central Romania, appears to form another species. Both mOTUs were found together in one population. A third lineage, containing mOTUs F, G and H, represented by 14 populations, was distributed across the Balkans from N.E. Croatia to Bulgaria. p-distances between the three species ranged from 0.172 to 0.219, and between all the mOTUs, pooled together, from 0.172 to 0.258. The highest genetic diversity was found in species 3 (0.112) and the lowest in species 1 (0.025), despite its largest geographic distribution. Pairwise p-distances, Tamura 3-parameter distances, composite likelihood distances, as well as the coancestry coefficient FST, calculated for all populations pooled together were significantly associated with geographic distance, but this was not the case within each of these three species. The significant association for all populations reflected high diversity between the species coupled with high geographic distances between their populations, not the character of intraspecies diversity. With a few exceptions, there hold a rather infinite island model with low migration. AMOVA detected 78% of the variance between the three species, 18% among populations within the species, and only 3.6% within the populations. The low genetic diversity of widespread F. fruticum s. stricto, compared with much higher diversity of two narrowly distributed newly found species of Fruticicola, may reflect the rapid spread of the former into previously uninhabitable regions, while the latter were able to maintain populations in glacial refugia. The estimated time of divergence between the three species, 1.7-2.19 mya, suggests their ancestors' isolation in southern European refugia during the lower Pleistocene, the Gelasian/Calabrian. There was no clear association of variation in shell morphology and lineage or mOTU identity; on external characters, these species are semicryptic, subtle differences in reproductive anatomy among them were found.

Divergence preceding island formation among Aegean insular populations of the freshwater snail genus Pseudorientalia (Caenogastropoda: Truncatelloidea).

Freshwater snails that inhabit islands are excellent model organisms for testing relationships between geological events and phylogeography, especially in the Aegean region. Although many Aegean islands were searched in the present study, species of the genus Pseudorientalia were only found on Lesvos, Samos, and Chios. Phylogenetic relationships between specimens living on these three islands were analysed using COI and 16S rRNA molecular markers and morphological data. A high level of diversity was found between islands. Genetic distances between clades showed differences high enough for the samples from different islands to be considered distinct species (p-distance: 0.105-0.133). These results are also supported by obvious morphological differences in shell morphology between islands. The mean divergence time between the Lesvos clade and Samos/Chios clade was 24.13 ± 3.30 Mya; between the Samos and Chios clades the divergence time was 14.80 ± 1.11 Mya. Our data suggest that high divergence may have occurred between Pseudorientalia populations during the Upper and Middle Miocene, when the Aegean region was part of a united landmass. It is possible that the observed highly divergent Pseudorientalia clades are relicts of high regional diversity that existed in the past.

Cloacal anatomy of the male Carpathian newt, Lissotriton montandoni (Amphibia, Salamandridae), in the breeding season.

This study presents the first light microscopy-based description of the cloacal anatomy of male Carpathian newts (Lissotriton montandoni). This European newt species hybridizes with its sister species, the smooth newt (L. vulgaris), despite a high level of prezygotic isolation. The goal of the study was to ascertain possible anatomical differences in cloacal anatomy, especially pheromone and spermatophore producing glands, which might potentially affect reproductive isolation between L. montandoni and L. vulgaris. The cloaca of L. montandoni males consists of the cloacal tube, cloacal chamber, pseudopenis, and aggregations of cloacal glands. Four main types of cloacal glands were recognized. Pheromone-producing dorsal glands are of two types due to differences in their secretory epithelium: cuboidal and high prismatic. The remaining glands: ventral, pelvic, and Kingsbury's glands, are most likely involved in the synthesis of spermatophore components. Two distinct groups of ventral glands were identified: posterior and anterior ventral glands. In comparison with L. vulgaris, no evident differences were found that could potentially affect courtship pheromone synthesis by dorsal glands or the base and the cap of the spermatophore produced by the remaining cloacal glands.

Asymmetric female preferences for courtship pheromones in two closely-related newt species, the smooth newt (Lissotriton vulgaris) and the Carpathian newt (L. montandoni) (Salamandridae).

The smooth (Lissotriton vulgaris) and Carpathian (L. montandoni) newts are sister species. These are separated by a moderate genetic distance, but exhibit striking morphological differences, especially in male epigamic traits. In the areas where they co-occur, they readily mate with each other and produce viable hybrids. However, a high level of pre-zygotic isolation with an unknown behavioral basis has been reported. The complex courtship of newts consists of at least three types of modality: chemical, visual, and tactile. The relative significance of these in mate choice is unclear, but it is commonly accepted that pheromones are an important communication channel. The goal of this study was to determine whether the females of L. vulgaris and L. montandoni exhibit preferences for conspecific extracts from the pheromone-producing abdominal (dorsal) glands. Females of both species spent more time in proximity to the source of the abdominal gland extracts of their own species when a liver extract was presented as an alternative. In a second trial, females were simultaneously confronted with conspecific and heterospecific abdominal gland extracts. Asymmetric preferences were found. Lissotriton vulgaris females were not selective, whereas L. montandoni females preferred the conspecific abdominal gland extract. This finding is consistent with the results of earlier experiments on mate choice in these species. The results strongly indicate that pheromones play a crucial role in courtship and species recognition in this pair of closely related, hybridizing species.

Two new species of the Balkan genus Paladilhiopsis Pavlovic, 1913 (Caenogastropoda, Moitessieriidae).

The Balkan Peninsula is inhabited by the worldwide most diverse subterranean gastropod fauna. This fauna is still poorly studied, since its habitats are not easily accessible, and its sampled populations are mostly not rich in specimens' numbers. Often only empty shells are known, but the shell is hardly useful, not only in phylogeny reconstruction, but even in species determination. The exclusively obligatory subterranean family Moitessieriidae is especially poorly studied. Representatives of the genus Paladilhiopsis Pavlovic, 1913 (Moitessieriidae) collected at three localities, distributed in Croatia and Bosnia & Herzegovina, were studied. The pigmentation of their shells and soft parts, as well as the female and male reproductive organs in one taxon, are presented. The partial sequences of the molecular markers mitochondrial cytochrome oxidase subunit I (COI) and nuclear histone 3 (H3) were used to infer their systematic status and phylogenetic relationships. Two species new to science are described. For one of them, also studied anatomically, 15 specimens were sequenced for COI, and all show the same haplotype.

A new genus and species of Hydrobiidae Stimpson, 1865 (Caenogastropoda, Truncatelloidea) from Peloponnese, Greece.

Minute caenogastropod brackish-water gastropods, formerly classified as Hydrobia, are important elements of the brackish-water fauna and were objects of intensive study for many years. Until now, five genera have been distinguished, most of them represented by a number of species, but rather indistinguishable without molecular data (cytochrome oxidase subunit I - COI). In the eastern Mediterranean region, they are still poorly studied. In this paper, we present a new species of "Hydrobia" from the brackish Moustos spring, Arkadia, eastern Peloponnese, Greece. The shell, protoconch, radula, female reproductive organs, and penis are described and illustrated, together with the molecular (COI) relationships with other hydrobiids. All data confirm that these snails represent a distinct taxon, which must be classified as a new species belonging to a new genus. The formal descriptions are given. The closest, sister taxon is Salenthydrobia Wilke, 2003. The molecularly estimated time of divergence, 5.75 ± 0.49 Mya, coincides with 5.33 Mya, which is the time of the Oligocene flooding that terminated the Messinian salinity crisis. During the latter period, brackish "Lago-Mare" habitats were most probably suitable for the last common ancestor of Salenthydrobia and the newly described genus. Later, the Pliocene flooding isolated the Apennine and Peloponnese populations, promoting speciation.

Crenobiont, stygophile and stygobiont molluscs in the hydrographic area of the Trebisnjica River Basin.

In the paper the crenobiont, stygophile and stygobiont malacofauna of the karst area of Popovo Polje around Trebinje (Eastern Herzegovina, BiH) is presented. The materials were collected from springs, caves and interstitial habitats (with a Bou-Rouch pump) at 23 localities. The following species were found: Pisidium cf. personatum A.W. Malm, 1855, Theodoxus callosus (Deshayes, 1833), Sadleriana fluminensis (Küster, 1852), Radomaniola curta (Küster, 1852), Radomaniola cf. bosniaca (Radoman, 1973), Kerkia briani Rysiewska & Osikowski, 2020, Montenegrospeum bogici (Pesic & Glöer, 2012), Litthabitella chilodia (Westerlund, 1886), Travunijana vruljakensis Grego & Glöer, 2019, a new genus and species of the Sadlerianinae, Emmericia ventricosa Brusina, 1870, Iglica cf. absoloni (A.J. Wagner, 1914), Plagigeyeria tribunicae Schütt, 1963, Paladilhiopsis arion Rysiewska & Osikowski, 2021, Valvata montenegrina Glöer & Pesic, 2008, Radix labiata (Rossmässler, 1835), Galba truncatula (O. F. Müller, 1774), Ancylus recurvus Martens, 1783, Ancylus sp. and the amphibiotic Succinea cf. putris (Linnaeus, 1758). The redescription of the genus Travunijana Grego & Glöer, 2019, applying the characteristics of shell, female reproductive organs and penis, is also presented. The new genus and species are described, based on the shell, penis, radula and fragmentary data on the female reproductive organs. For all species, the mitochondrial cytochrome oxidase subunit I (COI) is applied to confirm the determination; in the case of Travunijana and the new genus, the nuclear histone H3 locus is also used, in order to infer both their distinctiveness and phylogenetic relationships.

New data on the valvatiform-shelled Hydrobiidae (Caenogastropoda, Truncatelloidea) from southern Greece.

The minute valvatiform-shelled Hydrobiidae are less studied than other hydrobiid gastropods. In this paper, new data on these snails are presented, which have been collected at twelve springs in southern Greece: one in Boeotia, one on Evvoia Island, and ten on the Peloponnese Peninsula. Mitochondrial cytochrome oxidase subunit I (COI) and nuclear histone (H3) have been used to confirm the determinations and infer the relationships of the studied gastropods. They represent the genera Daphniola, Graecoarganiella and Isimerope. New localities, expanding the known geographic ranges, have been presented for Daphniolahadei and Daphniolalouisi. A species of Daphniola found at two localities has been identified as a species new to science, and its description, including the shell, penis, and female reproductive organs is given. Possible relationships between Graecoarganiella and Isimerope are discussed; their representatives are possibly new species. At one locality a single specimen likely represents a new genus: it was found to be most closely related with Islamia, but genetically (p-distance) too distant to be congeneric with Islamia.

Effects of embryonic exposure to chromium (VI) on blood parameters and liver microstructure of 1-day-old chickens.

Hexavalent chromium (Cr(VI)) has carcinogenic, nephrotoxic, hepatotoxic, and neurotoxic effects. Exposure to Cr(VI) can also lead to hematological alterations and blood biochemical changes. The literature on Cr(VI) toxicity concerns mostly adult forms of vertebrates. In this study, an attempt was made to determine the effect on the developing chicken embryo of Cr(VI) in ovo administration. It was observed that chromium affected the hatchability of chicks in a dose-dependent manner. At a dose from 25 to 250 µg per egg, Cr(VI) resulted in a statistically significant reduction of hatchability. Chromium administrated at lower doses (1.56 and 2.5 µg per egg) caused a statistically insignificant increase of hatchability. However, chromium at a level of LD50 (15.6 µg per egg) or 1/10 LD50 (1.56 per egg) did not cause major changes in hematological parameters or plasma biochemical indices in newly hatched chicks. The same doses did not lead to any histopathological changes in the liver.

A new species of Kerkia Radoman, 1978 (Caenogastropoda, Hydrobiidae) from Bosnia and Herzegovina.

A new species of Kerkia, K. briani Rysiewska & Osikowski, sp. nov. is described from the spring Policki Studenac Vrelo (Crkvina), adjacent to the Trebisnjica River (Bosnia and Herzegovina) collected with Bou-Rouch technique, pumped from an interstitial habitat 50 cm below the bottom of the spring. The shell, female reproductive organs, and the penis are described and illustrated. Mitochondrial cytochrome oxidase subunit I (COI) and nuclear histone H3 partial sequences confirm the distinctness of the new species, and molecularly based phylogenetic relationships of Kerkia are briefly presented.

Impairment of reproductive capabilities in three subsequent generations of asymmetric hybrids between Eisenia andrei and E. fetida from French, Hungarian and Polish laboratory colonies.

Eisenia andrei (Ea) and E. fetida (Ef) lumbricid earthworms are simultaneous hermaphrodites potentially capable of self-fertilization and hybridization. We have shown previously that reproductive isolation in these species is incomplete in Ea and Ef earthworms of French provenance, as viable offspring appeared in inter-specific pairs. Fertile asymmetric hybrids developed from Ea-derived ova fertilized by Ef-derived spermatozoa, as well as Ea or Ef specimens derived after self-fertilization (resulting from admixture of endogenously produced spermatozoa with sperm from a partner), but never Ef-hybrids from Ef-ova fertilized by Ea-spermatozoa. The latter appeared only in backcrosses of Ea-hybrids with the Ef. Here we show that these phenomena are not unique for French Ea/Ef earthworms, but are shared by earthworms from French, Hungarian, and Polish laboratory cultures. Semi-quantitative studies on fertility of Ea-derived hybrids revealed gradually decreasing numbers of offspring in three successive generations, more rapid in backcrosses with Ef than with Ea, and the absence of progeny in pairs of hybrids, despite the presence of cocoons in almost all pairs. Based on species specific mitochondrial and nuclear DNA sequences, we provide the first examples of two unique sterile hybrids with mitonuclear mismatch and potential mitonuclear incompatibility among offspring of one of the hybrid+Ef pairs. Earthworms from the investigated populations did not reproduce when kept from hatching in isolation or with representatives of Dendrobaena veneta but started reproducing upon recognition of a related partner, such as Ea, Ef or their hybrids. The existence of Ea or Ef specimens among offspring of hybrid+Ea/Ef pairs might be explained either by partner-induced self-fertilization of Ea/Ef or hybrid-derived ova, or by cross-fertilization of Ea/Ef /hybrid ova by partner-derived spermatozoa; the latter might contribute to interspecific gene introgression.

Revealing the stygobiotic and crenobiotic molluscan biodiversity hotspot in Caucasus: Part I. The phylogeny of stygobiotic Sadlerianinae Szarowska, 2006 (Mollusca, Gastropoda, Hydrobiidae) from Georgia with descriptions of five new genera and twenty-one new species.

The position of the southwestern Caucasus as a stygobiotic Mollusca hotspot is confirmed. Molecular data of stygobiotic gastropods revealed the diversity of subfamily Sadlerianinae Szarowska, 2006, inhabiting the subterranean environment of Georgia. In addition to the well-known endemic genera Pontohoratia Vinarski, Palatov & Glöer, 2014 and Motsametia Vinarski, Palatov & Glöer, 2014, five more genera were identified in northwestern Georgia as new to the science: Kartvelobia gen. nov., Imeretiopsis gen. nov., Caucasopsis gen. nov., Caucasogeyeria gen. nov., and Hausdorfenia gen. nov. Additionally, 21 new species were found to inhabit the studied area (Samegrelo, Imereti, Racha regions in Georgia).

Phylogenetic relationships of the Balkan Moitessieriidae (Caenogastropoda: Truncatelloidea).

The family Moitessieriidae includes minute dioecious gastropods exclusively inhabiting subterranean waters, including thermal ones. Only empty shells were collected in most species, the vast majority of them are described from their gross shell morphology alone. Several visits to a site are usually required to obtain at least some living individuals. High variability in shell morphology and the lack of diagnostic features, coupled with anticipated high levels of endemism, has resulted in a long list of nominal moitessierid species. Type specimens stored as empty shells omit unambiguous identification and delimitation of species boundaries. Due to inaccessibility of cave animals and consequent lack of material suitable for molecular analysis, the phylogenetic relationships, as well as the taxonomy of the family at genus/species level, are far from being understood. The anatomy of the family is also poorly known and provided only for a few taxa. The distinctness of the Moitessieriidae has sometimes been questioned, and their monophyly not proved. Twelve species of the Balkan Moitessieriidae are considered: two species of Paladilhiopsis, two species of Bythiospeum, six species of Iglica, Costellina turrita and Lanzaia bosnica. The shell morphology of each species, as well as the reproductive system of Paladilhiopsis and Iglica, were analysed. DNA sequences of nuclear histone H3, ribosomal 18S, ribosomal 28S and mitochondrial cytochrome oxidase subunit I (COI) were applied to infer phylogenetic relationships among the taxa. The sequences of Bythiospeum from GenBank have been used to infer relationships between Bythiospeum and Paladilhiopsis that were recently synonymized. Paladilhiopsis and Iglica are distinct, but closely related genera, as is the genus Bythiospeum, which does not occur in the Balkans. Its relationships with both former taxa remain unresolved. The Moitessieriidae are clearly distinct from all other families of the Truncatelloidea, however, their monophyly remains doubtful.

The existence of fertile hybrids of closely related model earthworm species, Eisenia andrei and E. fetida.

Lumbricid earthworms Eisenia andrei (Ea) and E. fetida (Ef) are simultaneous hermaphrodites with reciprocal insemination capable of self-fertilization while the existence of hybridization of these two species was still debatable. During the present investigation fertile hybrids of Ea and Ef were detected. Virgin specimens of Ea and Ef were laboratory crossed (Ea+Ef) and their progeny was doubly identified. 1 -identified by species-specific maternally derived haploid mitochondrial DNA sequences of the COI gene being either 'a' for worms hatched from Ea ova or 'f' for worms hatched from Ef ova. 2 -identified by the diploid maternal/paternal nuclear DNA sequences of 28s rRNA gene being either 'AA' for Ea, 'FF' for Ef, or AF/FA for their hybrids derived either from the 'aA' or 'fF' ova, respectively. Among offspring of Ea+Ef pairs in F1 generation there were mainly aAA and fFF earthworms resulted from the facilitated self-fertilization and some aAF hybrids from aA ova but none fFA hybrids from fF ova. In F2 generation resulting from aAF hybrids mated with aAA a new generations of aAA and aAF hybrids were noticed, while aAF hybrids mated with fFF gave fFF and both aAF and fFA hybrids. Hybrids intercrossed together produced plenty of cocoons but no hatchlings independently whether aAF+aAF or aAF+fFA were mated. These results indicated that Ea and Ef species, easy to maintain in laboratory and commonly used as convenient models in biomedicine and ecotoxicology, may also serve in studies on molecular basis of interspecific barriers and mechanisms of introgression and speciation. Hypothetically, their asymmetrical hybridization can be modified by some external factors.

Sperm transport after insemination in the Alpine newt (Triturus alpestris, Caudata, Salamandridae).

The goal of this study was to test if sperm transport to the spermathecae in the Alpine newt (Triturus alpestris) requires active co-operation of the female. Artificial insemination of anaesthetised female newts was conducted using spermatophores collected from courting males and with sperm duct contents collected from sacrificed males. Sperm was present in the spermathecae of 9 out of 10 females inseminated with the spermatophores but in only 1 out of 8 females inseminated with sperm duct contents. The females of both groups laid some eggs after insemination, and a portion of these eggs in group of females inseminated with spermatophores were fertilized. However, the number of eggs produced by the females was much lower than typical egg-production in newts. The presence of sperm in the spermathecae of females inseminated with spermatophores and lack of sperm in the spermathecae of females inseminated with sperm duct contents suggests that sperm transport is either induced by the substances present in spermatophores and/or that sperm from the sperm duct is not fully mobile in comparison with sperm from the spermatophores.

Complete mitochondrial genome of the Greek marsh frog Pelophylax cretensis (Anura, Ranidae).

We sequenced the complete mitochondrial genome of the Greek marsh frog Pelophylax cretensis, a water frog species endemic to the island of Crete. The genome sequence was 17,829 bp in size, and the gene order and contents were identical to those of previously reported mitochondrial genomes of other water frog species. This is the first complete mitogenome (i.e. including control region) described for western Palaearctic water frogs.

Phylogenetic relationships among four new complete mitogenome sequences of Pelophylax (Amphibia: Anura) from the Balkans and Cyprus.

We present the full-length mitogenome sequences of four European water frog species: Pelophylax cypriensis, P. epeiroticus, P. kurtmuelleri and P. shqipericus. The mtDNA size varied from 17,363 to 17,895 bp, and its organization with the LPTF tRNA gene cluster preceding the 12 S rRNA gene displayed the typical Neobatrachian arrangement. Maximum likelihood and Bayesian inference revealed a well-resolved mtDNA phylogeny of seven European Pelophylax species. The uncorrected p-distance for among Pelophylax mitogenomes was 9.6 (range 0.01-0.13). Most divergent was the P. shqipericus mitogenome, clustering with the "P. lessonae" group, in contrast to the other three new Pelophylax mitogenomes related to the "P. bedriagae/ridibundus" lineage. The new mitogenomes resolve ambiguities of the phylogenetic placement of P. cretensis and P. epeiroticus.

Does the genetic structure of spring snail Bythinella (Caenogastropoda, Truncatelloidea) in Bulgaria reflect geological history?

Bythinella is a minute dioecious caenogastropod that inhabits springs in central and southern Europe. In the Balkans, previous studies have addressed its morphological and genetic differentiation within Greece and Romania while the Bulgarian species have remained poorly known. The aim of the present paper has been to expand the knowledge on the subject in Bulgaria. Shell morphology and anatomy of the reproductive organs were examined, and a fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene and the nuclear ribosomal Internal Transcribed Spacer 1 (ITS-1) were sequenced from 15 populations. Additional sequences from eight previously studied populations were included in our analyses. Phylogenetic analyses revealed five main mitochondrial DNA clades, which were partly confirmed by analyses of the ITS-1 sequences. The genetic differentiation between the clades was found to be in the range p=2.4-11.8%. Most of the populations belonged to clade I, representing Bythinella hansboetersi, and were distributed in SW Bulgaria. Clades II and III inhabit areas adjacent to clade I and were most closely related with the latter clade. Much more distinct were clade V, found at one locality in NW Bulgaria, and clade IV, found at one locality in SE Bulgaria, close to the sea. Four populations were found in caves, but only one of these represented a distinct clade. Considering the observed pattern of interpopulation differentiation of Bythinella in Bulgaria, we can suppose that isolation between clades I, II and III may have been caused by glaciations during the Pleistocene. The time of isolation between the above three clades and clade IV coincides with the Messinian Salinity Crisis, and the time of isolation between the clade V and the other four most probably reflects the isolation of the Rhodopes from western Balkan Mts by the seawater of the Dacic Basin.