Species within the eutardigrade superfamily Macrobiotoidea are usually differentiated through their eggshell morphology. However, this trait can show very high intraspecific variability and, as consequence, species delimitation is challenging without molecular support. Although eggshell variability usually consists of small details, cases of species with very different egg morphotypes are known. Paramacrobiotus bifrons sp. nov. here described with an integrative approach, represents one of the species with extreme variability in eggshell morphology and it belongs to the areolatus group. The production by this gonochoric species of both areolatus-type and csotiensis-type eggs, the latter being rare in the group but most abundant in this species, was assessed by DNA barcoding, culturing, and inter-morphotype crosses. The reason underlying the production of two different eggshells resulted not related to tested culturing conditions, seasonality, or male’s influence. Confocal (with and without staining), Light, and Scanning Electron Microscopies analyses of the two egg morphotypes laid by P. bifrons sp. nov. allowed to redescribe the csotiensis-type egg, shed light on its composition (excluding chitin as a component), and to find shared details supporting the belonging of the two egg morphotypes to the same species.

Microorganisms (sensu lato, i.e., including micrometazoans) are thought to have cosmopolitan geographic distributions due to their theoretically unlimited dispersal capabilities, a consequence of their tiny size, population dynamics, and resistant forms. However, several molecular studies of microorganisms have identified biogeographic patterns indicating cryptic speciation and/or weak species definitions. Using a multi-locus approach with the genus Milnesium (Tardigrada), we aimed to determine the genetic structure of populations worldwide and the effects of long distance dispersal (LDD) on genetic connectivity and relationships across the six continents. Our results on this micrometazoan's genetic structure and LDD at global and micro-local scales indicate contrasting patterns not easily explained by a unique or simple phenomenon. Overall, we report three key findings: (i) confirmation of long distance dispersal for tardigrades, (ii) populations with globally-shared or endemic micro local haplotypes, and (iii) a supported genetic structure instead of the homogeneous genetic distribution hypothesized for microorganisms with LDD capabilities. Moreover, incongruences between our morphological and molecular results suggest that species delimitation within the genus Milnesium could be problematic due to homoplasy. Duality found for Milnesium populations at the global scale, namely, a molecular phylogenetic structure mixed with widely distributed haplotypes (but without any apparent biogeographic structure), is similar to patterns observed for some unicellular, prokaryotic and eukaryotic, microorganisms. Factors influencing these patterns are discussed within an evolutionary framework.

The species of the genus Ramazzottius (Ramazzottiidae, Eutardigrada) are among the most common and widespread tardigrade species in the world. Most of the 28 Ramazzottius species have been described only with morphological characters which were most of the time represented only with drawings. The discovery of a new species of this genus in the Black Forest (Germany) provided the opportunity to compare this species with the type specimens of ten Ramazzottius species, to propose the status of species dubia for Ramazzottius edmondabouti, and through new photographs to elucidate the anatomy of animals and eggs (in particular of the head sensory regions, eye spots, buccal tube, ornamentations of the dorsal posterior cuticle, and morphology of egg processes). These thorough observations led to a better understanding of the diversity and evolution, not only of this cosmopolitan genus, but also of other eutardigrade genera. The new species Ramazzottius kretschmanni is described with an integrative approach integrating morphological (light and electron microscopy observations and morphometric data) and molecular (cox1 and ITS2 genes) data. The PTP and ASAP analyses confirmed the validity of the new species from a molecular point of view. The new species is morphologically similar to Ramazzottius oberhaeuseri, but is distinguishable by the smooth cuticle, the presence of a “cheek-like” area on the head, and the size of egg processes as well as different sequences of the molecular markers.

Due to the difficulties associated with detecting and correctly identifying mirids, developing an accurate species identification approach is crucial, especially for potential harmful species. Accurate identification is often hampered by inadequate morphological key characters, invalid and/or outdated systematics, and biases in the molecular data available in public databases. This study aimed to verify whether molecular characterization (i.e. DNA barcoding) is able to identify mirid species of economic relevance and if species delimitation approaches are reliable tools for species discrimination. Cytochrome c oxydase 1 (cox1) data from public genetic databases were compared with new data obtained from mirids sampled in different Italian localities, including an old specimen from private collection, showing contrasting results. Based on the DNA barcoding approach, for the genusOrthops, all sequences were unambiguously assigned to the same species, while inAdelphocoris, LygusandTrigonotylusthere were over-descriptions and/or misidentifications of species. On the other hand, inPolymerusandDeraeocoristhere was an underestimation of the taxonomic diversity. The present study highlighted an important methodological problem: DNA barcoding can be a good tool for pest identification and discrimination, but the taxonomic unreliability of public DNA databases can make this method useless or even misleading.

This study considers in a new light a population already studied and tentatively attributed to Macrobiotus cf.terminalis (Bertolani, Rebecchi, 1993), by joining molecular approaches to the traditional, but indispensable, light microscopy, and to scanning electron microscopy.Differences in animal and, above all, egg shell morphology, and the peculiar cox1 sequence indicate that this population clearly pertains to a new species, M. vladimiri sp.n., which is here described.The results obtained can be considered as an example of how a modern taxonomical and biogeographical research can be carried out on this animal phylum and in general on the animals belonging to the so called meiofauna, where characters bound to morphology are often very few.This is the first tardigrade species to be described and barcoded contemporarily.

Notostraca taxonomy is controversial, mainly owing to the high intraspecific variability of morphological characters; other complexity factors are represented by the consistent rate of reproductive variation. We undertook the molecular analyses of Italian Lepidurus taxa through the characterization of 12S, 16S and COI mitochondrial genes in new L. apus demes and in the L. couesii populations recently discovered in Southern Italy. For L. apus, the analysis of a wider data set (both in terms of molecular markers and of analysed populations) confirms the previously suggested specific rank of differentiation between its so far recognized subspecies, the Italian L. apus lubbocki and the European L. apus apus. The Italian L. couesii samples pertain to a well‐defined taxonomic entity, strictly related to Canadian L. couesii. In 12S dendrograms, this taxon strongly diverges from the other North American Lepidurus species, but the relationships among nearctic taxa are unresolved. On the other hand, a high affinity between Italian/Canadian L. couesii and the circumpolar species L. arcticus is scored and a possible descent from a common ancestor is hypothesized.

SUMMARY Anhydrobiosis is a highly stable state of suspended animation in an organism due to its desiccation, which is followed by recovery after rehydration. Changes occurring during drying could damage molecules, including DNA. Using the anhydrobiotic tardigrade Paramacrobiotus richtersi as a model organism, we have evaluated the effects of environmental factors, such as temperature and air humidity level (RH), on the survival of desiccated animals and on the degradation of their DNA. Tardigrades naturally desiccated in leaf litter and tardigrades experimentally desiccated on blotting paper were considered. Replicates were kept at 37°C and at different levels of RH for 21 days. RH values and temperature, as well as time of exposure to these environmental factors, have a negative effect on tardigrade survival and on the time required by animals to recover active life after desiccation. DNA damages (revealed as single strand breaks) occurred only in desiccated tardigrades kept for a long time at high RH values. These results indicate that during the anhydrobiotic state, damages take place and accumulate with time. Two hypotheses can be formulated to explain the results: (i) oxidative damages occur in desiccated specimens of P. richtersi, and (ii) high temperatures and high RH values change the state of the disaccharide trehalose, reducing its protective role.

The living fossil Triops cancriformis comprises bisexual (either gonochoric or hermaphroditic) and unisexual populations. Genetic surveys have recently revealed a general trend of low differentiation of 12S and 16S mitochondrial genes. We, therefore, surveyed further mitochondrial (COI gene and control region) and nuclear markers (dinucleotide microsatellites) to assess the genetic variability and to establish any relationship with the different reproductive modes found in European populations. The mitochondrial analyses confirmed the pattern of low variability. Hence, the low mitochondrial genetic variability appears as a common feature of the genus Triops . The microsatellite analysis found that Italian populations are monomorphic or exhibit little polymorphism, while other European samples display a higher degree of polymorphism and private alleles. Spanish, Austrian and Italian populations show patterns of Hardy–Weinberg disequilibrium that could be explained by the mode of reproduction, or by a higher frequency of null alleles in these populations. The low diversity and differentiation among Italian populations lead us to question the Monopolization Hypothesis. One microsatellite locus appears to be sex-linked, with heterozygotes detected only in males and hermaphrodites.

. The ultrastructure of the male gonad of Eulimnadia texana (Branchiopoda, Spinicaudata) has been observed for the first time to investigate the sexuality of a well-studied case of androdioecy in the animal kingdom. The male gonad is a double structure located in the hemocoel throughout the entire body length on each side of the midgut. Male gametes originate from the wall and mature centripetally toward the lumen; the proliferative activity is very high and continuous and therefore the mature gonad is full of numerous germ cells. Inside the lumen several degenerative stages are found mixed with sperm cells and spermatids, the latter two being not easily distinguishable because of the slight differences between them. The evolutionary meaning of the degenerative process in E. texana male gametes is difficult to explain, and we propose some hypotheses about its possible role or cause in the studied population: (a) to help build spermatophores, (b) to act as a trophic component for viable sperm, (c) as a manifestation of inbreeding depression, and/or (d) to regulate the number of sperm cells.

Abstract Androdioecy (mixtures of males and hermaphrodites) is a rare mating system in both the plant and animal kingdoms. Androdioecy has been described in three branchiopod species, and is best known from the clam shrimp Eulimnadia texana Packard. Herein we describe sex ratio, genetic and histological evidence from the clam shrimp Eulimnadia agassizii Packard that suggest androdioecy is also found in this species. The E. agassizii population sampled had all-females, and when these females were isolated and allowed to produce eggs, those eggs yielded 100% female offspring in 15 out of 15 cases. Additionally, the originally isolated females proved to be completely homozygous at each of the six allozyme loci scored. The offspring from these isolated females also proved to be homozygous for the same alleles as their parent. Tissue sectioning of the gonad found that the “females” actually produced testicular tissue in the posterior portion of the gonad. Taken together, these data are entirely consistent with those of the androdioecious E. texana, and thus indicate that E. agassizii is also an androdioecious species, bringing the total number of branchiopod species with this form of reproduction to four.

The worldwide distributed genus Monochamus Megerle, 1821 (Coleoptera Cerambicydae) comprises beetles that may become pests of economic importance in conifer stands in the Nearctic and Palearctic Regions. Besides direct damage due to the larval tunnelling habits, they have also been recognized as main vectors of the phytoparasitic nematode Bursaphelenchus xylophilus (Steiner & Buhrer, 1934) (Nematoda Aphelenchoididae). We analysed the complete mitochondrial cytochrome oxidase I gene and a fragment of the small subunit RNA gene sequences (1536 base pairs) in the five European species. These are: Monochamus galloprovincialis (Olivier, 1795), morphologically distinguished in two subspecies M. galloprovincialis galloprovincialis (Olivier, 1795) and M. galloprovincialis pistor (Germar, 1818); Monochamus sutor (Linneus 1758); Monochamus saltuarius (Gebler 1830); Monochamus sartor (Fabricius, 1787) and Monochamus urussovi (Fischer, 1806). For appropriate comparisons, also the Asiatic Monochamus alternatus Hope, 1842 and a Japanese M. saltuarius sample have been analysed. Both genes show an absolute identity between the two subspecies of M. galloprovincialis and a strong affinity between M. sartor and M. urussovi: the morphological subdivisions of the former taxon in two subspecies and of the latter in two entities of specific level are therefore not supported genetically. On the other hand, the Italian and the Japanese samples of M. saltuarius always cluster together in all trees, and for the remaining taxa, no doubt about their rank of specific differentiation emerges from present analyses. From a phyletic point of view, tree topology indicates the Japanese M. alternatus as the most differentiated taxon and the Euroasiatic M. saltuarius as basal to all other strictly European entities. Chromosome analyses show that the diploid autosomal complement ranges from 18 in M. saltuarius to 20 in M. galloprovincialis, and 22 in M. sartor, but a XX-Xy(p) sex determining system is shared by all analysed taxa. The M. saltuarius karyotype appears as the most primitive from which the others may be derived through Robertsonian fissions. Karyological data therefore agree with molecular analyses in indicating a basal position of Euroasiatic M. saltuarius with respect to the group of European Monochamus taxa; among these, M. galloprovincialis and M. sartor represent two clearly diverging evolutionary units. Furthermore, karyotype analyses substantiate molecular conclusions about the identity between M. galloprovincialis galloprovincialis and M. galloprovincialis pistor.

Branchiopods reproductive mechanisms range from gonochorism to unisexuality, passing through androdioecy. In order to contribute to still lacking or controversial basic knowledges, we analyzed the karyotype of the main Italian taxa: the Notostracan living fossils Triops cancriformis (parthenogenetic) and Lepidurus apus lubbocki (bisexual), and the Conchostracan Eoleptestberia ticinensis (bisexual). Also one male obtained from a supposedly hermaphroditic Austrian population of T cancriformis was checked. In T cancriformis a diploid number of 12 chromosomes is observed in both females and male; this is in line with previous results on other Italian populations, but contrasts with observations on European samples. The richness of normal meiotic pictures indicates that the male specimen is able to produce sperms. L. apus lubbocki shows a diploid number of 10 chromosomes; in male specimens the mispairings during meiosis I could explain the haploid number of 6 chromosomes obtained from diakinetic plates in Palestinian samples of the same subspecies found in literature. Irregular meiotic divisions also highlight the ultrastructurally observed abortive spermatogenesis. Finally, in the presently analyzed population of E. ticinensis, the adult females show 10 chromosomes as diploid number, while males present 10 or 11 elements; in the nauplii, chromosome numbers range from 8 to 12, differing among unrelated individuals, between nauplii produced by the same female and even within the same nauplius. B elements are taken into account.

. Recent reports of occasional males in Austrian populations of Triops cancriformis have promoted interest in an analysis of the ultrastructure of the male gonad. It appears as a double thick and well-defined tubular structure running along the midgut, inside the hemocoel. It is composed of two portions: the wall and the lumen. The former is made up of germ cells that are widespread and intermixed with somatic cells. The lumen is ∼200 μm wide and acts as the collecting site of spermatids and sperm. The germ cells are recognizable by their rounded appearance and by the occurrence of exceptionally long synaptonemal complexes in their nuclei. Their maturation implies a volume elongation and an aggregation in a characteristic “cyst” arrangement. The cysts migrate towards the lumen, where they break open, releasing spermatids and sperm. At the end of this process the lumen is filled with maturing gametes. The sperm have very condensed chromatin nuclei and partially reduced cytoplasm where the most striking features are the axoneme and strewn microtubules together with evident pseudopodia. Our morphological data support the full functionality of males in T. cancriformis. However, further work on gametogenesis, distribution of sexes, and genetic analysis of breeding experiments are needed to reach a fuller understanding of the role of males in middle and northern European populations of this species.

Ten fragments of a wood-inhabiting polypore were found in the early Neolithic village of ‘La Marmotta’ (Anguillara Sabazia, Rome), formerly located on the shore of the Bracciano Lake. Five of these were found in three different huts, and two outside near other structures. Some fragments of the partly degraded specimens were used for DNA sequencing in order to the identity of the fungus, which proved to be Daedaleopsis tricolor. Pharmacological aspects of this and other previously discovered prehistoric polypores are also noted.

Five dinucleotide markers were obtained from a microsatellite genomic library of the tadpole shrimp Triops cancriformis . The number of alleles at each locus was low (maximum three alleles) but all loci were polymorphic. The presence of null alleles could not be disregarded. Heterozygosity levels were indicative of selfing in the studied populations whose real sexuality will be further tested using these markers.

Polymerase chain reaction amplifications of genomic DNA in 17 individuals of bisexual and parthenogenetic populations of three subspecies of Bacillus rossius (Insecta Phasmatodea) revealed that the species still harbours the whole variability of the ancestral Bag320 satellite family, since monomers of all non-hybrid Bacillus taxa plus private sequences occur in it. Bag320 monomers had not been rescued as a major satellite component in B. rossius, but possibly represent the remnant of a set of diverging sequences present in the Bacillus ancestor. Following the library hypothesis, these monomer variants have been differently amplified along the evolutionary pathways leading to present taxa in agreement with the mitochondrial phylogeny of the genus. The putative converted tracts observed are explained as the results of past gene conversion events.