Planarian cell number depends on blitzschnell, a novel gene family that balances cell proliferation and cell death.

Control of cell number is crucial to define body size during animal development and to restrict tumoral transformation. The cell number is determined by the balance between cell proliferation and cell death. Although many genes are known to regulate those processes, the molecular mechanisms underlying the relationship between cell number and body size remain poorly understood. This relationship can be better understood by studying planarians, flatworms that continuously change their body size according to nutrient availability. We identified a novel gene family, blitzschnell (bls), that consists of de novo and taxonomically restricted genes that control cell proliferation:cell death ratio. Their silencing promotes faster regeneration and increases cell number during homeostasis. Importantly, this increase in cell number leads to an increase in body size only in a nutrient-rich environment; in starved planarians, silencing results in a decrease in cell size and cell accumulation that ultimately produces overgrowths. bls expression is downregulated after feeding and is related to activity of the insulin/Akt/mTOR network, suggesting that the bls family evolved in planarians as an additional mechanism for restricting cell number in nutrient-fluctuating environments.

Cryptic species delineation in freshwater planarians of the genus Dugesia (Platyhelminthes, Tricladida): Extreme intraindividual genetic diversity, morphological stasis, and karyological variability.

The keystone of planarian taxonomy traditionally has been the anatomy of the copulatory apparatus. However, many planarian species comprise asexual fissiparous populations, with the fissiparous animals not developing a copulatory apparatus, thus precluding their morphological identification. Incorporation of molecular data into planarian systematics has been of great value, not only in the identification of fissiparous individuals but also as an additional source of information for determining species boundaries. Nevertheless, the discrepancy between morphological and molecular data has highlighted the need for extra sources of taxonomic information. Moreover, a recent study has pointed out that fissiparous reproduction may lead to high levels of intraindividual genetic diversity in planarians, which may mislead molecular analyses. In the present study we aim to test a new up-to-date integrative taxonomic procedure for planarians, including intraindividual genetic data and additional sources of taxonomic information, besides morphology and DNA, using Dugesia subtentaculata sensu lato as a model organism, a species with an intricate taxonomic history. First, we used three different methods for molecular species delimitation on single locus datasets, both with and without intraindividual information, for formulating Primary Species Hypotheses (PSHs). Subsequently, Secondary Species Hypotheses (SSHs) were formulated on the basis of three types of information: (1) a coalescent-based species delimitation method applied to multilocus data, (2) morphology of the copulatory apparatus, and (3) karyological metrics. This resulted in the delimitation of four morphologically cryptic species within the nominal species D. subtentaculata. Our results provide evidence that the analysis of intraindividual genetic data is essential for properly developing PSHs in planarians. Our study reveals also that karyological differentiation, rather than morphological differentiation, may play an important role in speciation processes in planarians, thus suggesting that the currently known diversity of the group could be highly underestimated.

Multi-gene phylogeny of Tetrahymena refreshed with three new histophagous species invading freshwater planarians.

Planarians represent an insufficiently explored group of aquatic invertebrates that might serve as hosts of histophagous ciliates belonging to the hymenostome genus Tetrahymena. During our extensive research on freshwater planarians, parasitic tetrahymenas were detected in two of the eight planarian species investigated, namely, in Dugesia gonocephala and Girardia tigrina. Using the 16S and 18S rRNA genes as well as the barcoding cytochrome oxidase subunit I, one ciliate species was identified as T. scolopax and three species were recognized as new forms: T. acanthophora, T. dugesiae, and T. nigricans. Thus, 25% of the examined planarian taxa are positive for Tetrahymena species and three of them represent new taxa, indicating a large undescribed ciliate diversity in freshwater planarians. According to phylogenetic analyses, histophagous tetrahymenas show a low phylogenetic host specificity. Although T. acanthophora, T. dugesiae, and T. scolopax clustered together within the "borealis" clade, the former species has been detected exclusively in G. tigrina, while the two latter species only in D. gonocephala. Tetrahymena nigricans, which has been isolated only from G. tigrina, was classified within the "paravorax" clade along with T. glochidiophila which feeds on glochidia. The present phylogenetic reconstruction of ancestral life strategies suggested that the last common ancestor of the family Tetrahymenidae was free-living, unlike the progenitor of the subclass Hymenostomatia which was very likely parasitic. Consequently, there were at least seven independent shifts back to parasitism/histophagy within Tetrahymena: one each in the "paravorax" and "australis" clades and at least five transfers back to parasitism in the "borealis" clade.

Gap Junctional Blockade Stochastically Induces Different Species-Specific Head Anatomies in Genetically Wild-Type Girardia dorotocephala Flatworms.

The shape of an animal body plan is constructed from protein components encoded by the genome. However, bioelectric networks composed of many cell types have their own intrinsic dynamics, and can drive distinct morphological outcomes during embryogenesis and regeneration. Planarian flatworms are a popular system for exploring body plan patterning due to their regenerative capacity, but despite considerable molecular information regarding stem cell differentiation and basic axial patterning, very little is known about how distinct head shapes are produced. Here, we show that after decapitation in G. dorotocephala, a transient perturbation of physiological connectivity among cells (using the gap junction blocker octanol) can result in regenerated heads with quite different shapes, stochastically matching other known species of planaria (S. mediterranea, D. japonica, and P. felina). We use morphometric analysis to quantify the ability of physiological network perturbations to induce different species-specific head shapes from the same genome. Moreover, we present a computational agent-based model of cell and physical dynamics during regeneration that quantitatively reproduces the observed shape changes. Morphological alterations induced in a genomically wild-type G. dorotocephala during regeneration include not only the shape of the head but also the morphology of the brain, the characteristic distribution of adult stem cells (neoblasts), and the bioelectric gradients of resting potential within the anterior tissues. Interestingly, the shape change is not permanent; after regeneration is complete, intact animals remodel back to G. dorotocephala-appropriate head shape within several weeks in a secondary phase of remodeling following initial complete regeneration. We present a conceptual model to guide future work to delineate the molecular mechanisms by which bioelectric networks stochastically select among a small set of discrete head morphologies. Taken together, these data and analyses shed light on important physiological modifiers of morphological information in dictating species-specific shape, and reveal them to be a novel instructive input into head patterning in regenerating planaria.

Dugesia sicula (Platyhelminthes, Tricladida): the colonizing success of an asexual Planarian.

BACKGROUND: Dugesia sicula is the only species of its genus not presenting an endemic or restricted distribution within the Mediterranean area. It mostly comprises fissiparous populations (asexual reproduction by body division and regeneration), most likely sexually sterile, and characterized by an extremely low genetic diversity interpreted as the consequence of a recent anthropic expansion. However, its fissiparous reproduction can result in an apparent lack of diversity within the species, since genetic variation within individuals can be as large as between them because most individuals within a population are clones. We have estimated haplotype and nucleotide diversity of cytochrome oxidase I within and among individuals along the species distribution of a broad sample of D. sicula, including asexual and the two only sexual populations known today; and predicted its potential distribution based on climatic variables. Our aim was to determine the centre of colonisation origin, whether the populations are recent, and whether the species is expanding. RESULTS: The species presents 3 most frequent haplotypes, differing in a maximum of 11 base pairs. As expected from their fissiparous mode of reproduction, in half of all the analysed localities many individuals have multiple heteroplasmic haplotypes. The distribution of haplotypes is not geographically structured; however, the distribution of haplotypes and heteroplasmic populations shows higher diversity in the central Mediterranean region. The potential distribution predicted by climatic variables based modelling shows a preference for coastal areas and fits well with the observed data. CONCLUSIONS: The distribution and frequency of the most frequent haplotypes and the presence of heteroplasmic individuals allow us to gain an understanding of the recent history of the species, together with previous knowledge on its phylogenetic relationships and age: The species most probably originated in Africa and dispersed through the central Mediterranean. After one or multiple populations became triploid and fissiparous, the species colonized the Mediterranean basin, likely both by its own means and helped by human activities. Its present distribution practically fulfils its potential distribution as modelled with climatic variables. Its prevalence in coastal regions with higher water temperatures predicts a likely future expansion to northern and more interior areas following the increase in temperatures due to climate change.

Stem cell systems and regeneration in planaria.

Planarians are members of the Platyhelminthes (flatworms). These animals have evolved a remarkable stem cell system. A single pluripotent adult stem cell type ("neoblast") gives rise to the entire range of cell types and organs in the planarian body plan, including a brain, digestive-, excretory-, sensory- and reproductive systems. Neoblasts are abundantly present throughout the mesenchyme and divide continuously. The resulting stream of progenitors and turnover of differentiated cells drive the rapid self-renewal of the entire animal within a matter of weeks. Planarians grow and literally de-grow ("shrink") by the food supply-dependent adjustment of organismal turnover rates, scaling body plan proportions over as much as a 50-fold size range. Their dynamic body architecture further allows astonishing regenerative abilities, including the regeneration of complete and perfectly proportioned animals even from tiny tissue remnants. Planarians as an experimental system, therefore, provide unique opportunities for addressing a spectrum of current problems in stem cell research, including the evolutionary conservation of pluripotency, the dynamic organization of differentiation lineages and the mechanisms underlying organismal stem cell homeostasis. The first part of this review focuses on the molecular biology of neoblasts as pluripotent stem cells. The second part examines the fascinating mechanistic and conceptual challenges posed by a stem cell system that epitomizes a universal design principle of biological systems: the dynamic steady state.

Fluvial basin history in the northeastern Mediterranean region underlies dispersal and speciation patterns in the genus Dugesia (Platyhelminthes, Tricladida, Dugesiidae).

In this study we analyzed the phylogenetic relationships of eastern Mediterranean freshwater planarians of the genus Dugesia, estimated divergence times for the various clades, and correlated their phylogeographic patterns with geological and paleoclimatic events, in order to discover which evolutionary processes have shaped the present-day distribution of these animals. Specimens were collected from freshwater courses and lakes in continental and insular Greece. Genetic divergences and phylogenetic relationships were inferred by using the mitochondrial gene subunit I of cytochrome oxidase (COI) and the nuclear ribosomal internal transcribed spacer-1 (ITS-1) from 74 newly collected individuals from Greece. Divergence time estimates were obtained under a Bayesian framework, using the COI sequences. Two alternative geological dates for the isolation of Crete from the mainland were tested as calibration points. A clear phylogeographic pattern was present for Dugesia lineages in the Eastern Mediterranean. Morphological data, combined with information on genetic divergences, revealed that eight out of the nine known species were represented in the samples, while additional new, and still undescribed species were detected. Divergence time analyses suggested that Dugesia species became isolated in Crete after the first geological isolation of the island, and that their present distribution in the Eastern Mediterranean has been shaped mainly by vicariant events but also by dispersal. During the Messinian salinity crisis these freshwater planarians apparently were not able to cross the sea barrier between Crete and the mainland, while they probably did disperse between islands in the Aegean Sea. Their dependence on freshwater to survive suggests the presence of contiguous freshwater bodies in those regions. Our results also suggest a major extinction of freshwater planarians on the Peloponnese at the end of the Pliocene, while about 2Mya ago, when the current Mediterranean climate was established, these Peloponnese populations probably began to disperse again. At the end of the Pliocene or during the Pleistocene, mainland populations of Dugesia colonized the western coast, including the Ionian Islands, which were then part of the continent.

[Telomere length and phylogenetic relationship of Baikal and Siberian planarians (Turbellaria, Tricladida)].

Dynamics of the telomeric DNA (tDNA) and the phylogeny of the Baikal and Siberian planarians have been studied based on the analysis of the 18S rDNA and beta-actin gene fragments. A relationship between tDNA and the planarians size has been demonstrated. Giant planarians with a minor exception have longer tDNA than little planarians. Phylogenetic affinity between the species that have the stretched tracks of tDNA, big size and similar habitats may indicate possible role of tDNA in the development of the indefinite regenerative capacity of planarians.

Evidence for the persistence of the land planarian species Microplana terrestris (Müller, 1774) (Platyhelminthes, Tricladida) in microrefugia during the Last Glacial Maximum in the northern section of the Iberian Peninsula.

The land planarian species Microplana terrestris (Müller, 1774), shows a wide distribution in the north of the Iberian Peninsula, where mature humid forests can be found. Since most terrestrial planarians require the presence and good condition of wet forests to survive, a parallel evolution of the taxon and its habitat might be expected. Performing molecular analyses (mitochondrial cytochrome oxidase I and nuclear ITS-1 genes) we estimated the demography and biogeographic history of the species in that region. Our results show the species to present levels of genetic diversity likely originating before the Pleistocene. However, it presents a genetic structure that presumably resulted from its survival in various refugees during the Pleistocene glacial cycles. The two main genetic groups, present on the Iberian Peninsula, seem to have different origins: the western one being of Iberian origin, while the eastern group may have been the result of a re-colonization from the north. In both cases, their biogeographical history mirrors their habitat range movements, reinforcing the phylogeographical hypothesis put forward for its preferred habitat, i.e. humid forests.

Existence of two sexual races in the planarian species switching between asexual and sexual reproduction.

In certain planarian species that are able to switch between asexual and sexual reproduction, determining whether a sexual has the ability to switch to the asexual state is problematic, which renders the definition of sexuals controversial. We experimentally show the existence of two sexual races, acquired and innate, in the planarian Dugesia ryukyuensis. Acquired sexuals used in this study were experimentally switched from asexuals. Inbreeding of acquired sexuals produced both innate sexuals and asexuals, but inbreeding of innate sexuals produced innate sexuals only and no asexuals. Acquired sexuals, but not innate sexuals, were forced to become asexuals by ablation and regeneration (asexual induction). This suggests that acquired sexuals somehow retain asexual potential, while innate sexuals do not. We also found that acquired sexuals have the potential to develop hyperplastic and supernumerary ovaries, while innate sexuals do not. In this regard, acquired sexuals were more prolific than innate sexuals. The differences between acquired and innate sexuals will provide a structure for examining the mechanism underlying asexual and sexual reproduction in planarians.

The genus Phagocata Leidy (Platyhelminthes, Tricladida) in Israel, a new species of Phagocata from Lake Kinneret, and an emended description of Dugesia salina.

The genus Phagocata (Platyhelminthes, Tricladida) is represented in Israel by two pigmented species, namely, Phagocata armeniaca (Komárek, 1916), reported previously from Armenia, the Caucasus and eastern Turkey, and more recently known from the constantly cold headwaters of the River Jordan in northern Israel, and Phagocata punctata sp. nov., which was found inhabiting the littoral of the south-eastern shore of Lake Kinneret where there are large seasonal temperature fluctuations. Several samples of both species were collected in different seasons and raised in the laboratory at various temperatures and their habitats, morphology, karyology (2n = 34) and breeding behaviour were compared. The results of cross-breeding experiments are given, with most of the F1 offspring showing a range of patterns of colouration, gut branching and fecundity which were intermediate to those of the parent species. However, crossbreeding also resulted in a small proportion of abnormalities among the F1, and especially the F2, generations, indicating an incomplete reproductive compatibility, but supporting the hypothesis of their common origin. An emended description of Dugesia salina (Whitehouse, 1914), is also given together with karyological data (2n = 16) from material collected from a saline spring, En Sheva (Tabgha), situated on the north-western shore of Lake Kinneret.

A new species of terrestrial planarian of the genus Microplana (Platyhelminthes: Turbellaria; Tricladida: Continenticola) from Yorkshire, United Kingdom; with a discussion of Microplana humicola Vejdovsky 1890.

Two mostly white terrestrial planarians, about 7 mm long, found in a garden in Yorkshire, UK, are described. They have a single pair of eyes and both specimens are fully mature, with a single pair of ovaries, several testes, a conical penis papilla and a genito-intestinal duct, characters of the genus Microplana, but differ in color and size from other species of the genus and are described as Microplana edwardsi sp. nov.

Molecular barcoding and phylogeography of sexual and asexual freshwater planarians of the genus Dugesia in the Western Mediterranean (Platyhelminthes, Tricladida, Dugesiidae).

Planarians of the genus Dugesia have a worldwide distribution with high species diversity in the Mediterranean area. In this area, populations with a triploid karyotype that reproduce by fissiparity are exceptionally frequent, outnumbering the sexual populations. This situation poses interesting questions, such as the age of these asexual lineages, whether they all belong to the same species or whether the triploidization event is recurrent, and what factors (climatic, geographical, historical...) explain the prevalence of these asexual forms. However, asexual populations cannot be assigned to a species due to the lack of copulatory apparatus--the main structure used in species identification. In this study, we have developed a DNA barcoding method, based on COI and ITS-1 sequences, which allows the assignment of the fissiparous forms to sexual species. At the same time, phylogenetic analysis from species of the western Mediterranean have unveiled the presence of species with highly differentiated populations alongside species with a wide distribution and almost no genetic variation. The roles of habitat instability, dispersal capacity and human activities are briefly discussed.

Diet assessment of two land planarian species using high-throughput sequencing data.

Geoplanidae (Platyhelminthes: Tricladida) feed on soil invertebrates. Observations of their predatory behavior in nature are scarce, and most of the information has been obtained from food preference experiments. Although these experiments are based on a wide variety of prey, this catalog is often far from being representative of the fauna present in the natural habitat of planarians. As some geoplanid species have recently become invasive, obtaining accurate knowledge about their feeding habits is crucial for the development of plans to control and prevent their expansion. Using high throughput sequencing data, we perform a metagenomic analysis to identify the in situ diet of two endemic and codistributed species of geoplanids from the Brazilian Atlantic Forest: Imbira marcusi and Cephaloflexa bergi. We have tested four different methods of taxonomic assignment and find that phylogenetic-based assignment methods outperform those based on similarity. The results show that the diet of I. marcusi is restricted to earthworms, whereas C. bergi preys on spiders, harvestmen, woodlice, grasshoppers, Hymenoptera, Lepidoptera and possibly other geoplanids. Furthermore, both species change their feeding habits among the different sample locations. In conclusion, the integration of metagenomics with phylogenetics should be considered when establishing studies on the feeding habits of invertebrates.

Planarian Diversity and Phylogeny.

Hundreds of planarian species exist worldwide, representing a rich phenotypic diversity. This chapter presents an overview of the morphology and anatomy of various taxonomic groups of planarian flatworms, focusing on features enabling recognition and identification of the animals. The most recent view on the phylogenetic relationships of the planarians is presented, together with geographic distribution patterns of major groups of triclads. The chapter concludes with a brief methodological section outlining species identification on basis of anatomical features. In conjunction with the established laboratory model species, the phenotypic diversity of planarians provides rich opportunities for comparative studies, which this chapter aims to inspire.

Biogeography and karyotypes of freshwater planarians (Platyhelminthes, Tricladida, Paludicola) in southern Brazil.

In the Tricladida (Platyhelminthes), the incidence of different biotypes identified by several ploidy levels is very common. Planarians collected in the State of Rio Grande do Sul were identified using cytogenetics. Different species distributions were observed with respect to Rio Grande do Sul's geomorphology, which could have been caused by their different microhabitats. Girardia tigrina and G. anderlani consisted of diploid and triploid individuals, whereas G. schubarti showed diploids, triploids, and mixoploids; for all these species, individuals of different ploidies were sympatric. Only for diploid G. anderlani were B chromosomes observed. These B chromosomes seem to have an irregular segregational behavior during mitosis, and possibly also during meiosis. However the processes (e.g., selection, mutation) of maintaining 2n, 3n, and 2n/3n individuals within natural populations of G. schubarti remain to be clarified.

First report of the land planarian Diversibipalium multilineatum (Makino & Shirasawa, 1983) (Platyhelminthes, Tricladida, Continenticola) in Europe.

Introduction of alien species may significantly affect soil ecosystems, through predation or disruption of components of native ecosystems (Winsor et al. 2004; Álvarez-Presas et al. 2014; Justine et al. 2014). Land planarians have been reported as alien species in soils throughout the world and, among those, some species are considered to be successful invaders, e.g. Platydemus manokwari de Beauchamp, 1963, Arthurdendyus triangulatus (Dendy, 1894), Bipalium adventitium Hyman, 1943, Bipalium kewense Moseley, 1878 and Dolichoplana striata Moseley, 1877 (Winsor et al. 2004; Álvarez-Presas et al. 2014; Justine et al. 2014, 2015). Soil moisture status seems to be an important element for their successful invasion (Fraser & Boag 1998). In Europe at least 18 species of alien land planarians have been recorded since now and some of them are considered as invasive ones, e.g. P. manokwari (cf. Justine et al. 2014). Although the alien land planarian B. kewense has been reported to occur in many greenhouses in Italy (Bello et al. 1995), no data are available on its establishment and/or impact on natural environments. On 28th September 2014, 20 specimens (~1 individual/m2) of the land planarian Diversibipalium multilineatum (Makino & Shirasawa, 1983) (Fig. 1), native to Japan, were collected under pots, branches and plastic materials in a private garden located in the center of Bologna (Emilia Romagna, Central Italy), near the urban park Giardini Margherita (44°29' N, 11°21' E; WGS84). Thirty plant species (both indigenous and alien), mainly cultivated as bonsai (e.g. Lagerstroemia indica L., Juniperus procumbens (Siebold ex Endl.) Miquel), were present in this shady, wet garden (25 m2). Between March 2014 and June 2015, 70 more specimens of D. multilineatum were collected at the same site, mainly at dusk and dawn after rain. Reproduction by fission and regeneration processes were observed in several of those specimens, which were kept for some time in captivity. A specimen of D. multilineatum was also collected in a garden in Léguevin (Haute-Garonne, France), which will be described in a forthcoming paper by Justine et al. (in prep.) (see also Kawakatsu et al. 2014). Specimens without a genital pore were initially ascribed to D. multilineatum on the basis of their external appearance: the dorsal surface was brownish yellow and presented five longitudinal stripes at the head plate and the neck, showing the typical appearance of the species. The middorsal stripe was widened at its anterior end, on the head plate, and at the pharynx level. The ventral pattern of the animals at the pharyngeal region was also characteristic, with the middorsal stripe widened at this level. The Italian Diversibipalium specimens used for the molecular analysis were fixed and preserved in absolute ethanol. Fragments of the mitochondrial gene COI and 28S ribosomal RNA nuclear gene (GenBank Acc. Numbers KU245358 and KU245357, respectively) were obtained using the procedure and COI primers described in Álvarez-Presas et al. (2008) and Solà et al. (2013). The French specimen's COI (Specimen MNHN JL177, GenBank Acc. Number KT922162) was obtained as described in Justine et al. (2015). 28S sequences of 14 Bipaliinae specimens and four Microplana species (outgroup) retrieved from GenBank were included in the phylogenetic analyses (Fig. 2). Sequence alignment was obtained by using the online software MAFFT version 7 (Katoh & Standley 2013), while ambiguously aligned positions were removed using the program Gblocks (Talavera & Castresana 2007) with default settings, excepting the minimum number of sequences for a flank position at the minimum value (set at 10) and with half of the allowed gap positions. The final alignment had a length of 1589 bp. We used two phylogenetic inference approaches: maximum likelihood (ML), using the RaxML 8.2.3 software (Stamatakis 2014), and Bayesian inferences (BI), using MrBayes 3.2.4 (Ronquist et al. 2012). The evolutionary model used, GTR+I+G, was estimated to be the best with the software jModeltest 2.1.7 (Darriba et al. 2012; Guindon & Gascuel 2003), using the Akaike Information Criterion (AIC). MrBayes analyses were performed for 10-milion generation with sampling parameters every 103 and a 25% default burn-in value for the final trees. Convergence of the two runs (average standard deviation of split frequencies << 0.01) and likelihood stationarity were checked. The maximum likelihood analyses were performed under 1000 bootstrap pseudoreplicates. The phylogenetic results show a close and highly supported relationship of the Italian Diversibipalium specimens with those from Japan and South Korea that have been identified as D. multilineatum (Fig. 2). Diversibipalium multilineatum is the sister-group of B. nobile Kawakatsu & Makino, 1982, but with low support. The COI sequences of the French (MNHN JL177) and the Italian Diversibipalium specimens were compared in Geneious v. 8.0.5 (http://www.geneious.com, Kearse et al. 2012) and were found to be identical. These results indicate that the species introduced in both countries is the same, and most probably concerns the species D. multilineatum. The pathways of introduction of D. multilineatum are currently unknown, although a relationship between the horticultural trade and the introduction of alien land planarians is well known (Álvarez-Presas et al. 2014 and references therein). Here we report the first occurrence of individuals of D. multilineatum outside Asia. The GenBank sequence of D. multilineatum from South Korea is not yet supported by a published description of the specimen, while it is debatable whether South Korea should be considered part of the natural range of D. multilineatum, which only seems to include Japan. In the present paper, we consider the South Korean animal to be an introduced specimen. Soil moisture status, temperature, and food availability are considered to be the main factors determining the presence of terrestrial planarians (Boag et al. 1998); the microclimatic conditions of the Italian garden were similar to plant nurseries and greenhouses, while an abundance of food was available, such as isopods [Porcellionides pruinosus (Brandt, 1833)], oligochaetes [Dendrobaena attemsi (Michaelsen, 1902) and several juveniles of Lumbricus spp.] and gastropods [Cernuella cisalpina (Rossmassler, 1837), Cornu aspersum (O.F. Müller 1774), Deroceras reticulatum (O.F. Müller, 1774), Discus rotundatus (O.F. Müller, 1774), Limacus flavus (Linnaeus, 1758), Milax nigricans (Philippi, 1836), Papillifera papillaris (Linnaeus, 1758), Pomatias elegans (O.F. Müller, 1774)]. Moreover, winter 2014 reached the highest temperatures and rainfall of the last two decades (source: CNR-ISAC, Bologna), thus favouring establishment and spread of D. multilineatum. The potential environmental impacts of some invasive flatworms are well documented (Álvarez-Presas et al. 2014; Justine et al. 2014) and, even if these effects have not yet been assessed for D. multilineatum, the adoption of precautionary measures and of early intervention is here strongly recommended (Genovesi & Shine 2004). Finally, knowledge of the introduction pathway(s), together with the analysis of prey preference and possible impact on the invertebrate fauna, will be essential to halt or at least to limit the spread of this introduced land flatworm.

Anatomical deviation of male organs of land planarians from Rio de Janeiro, Brazil, with description of two new species of Cratera (Platyhelminthes, Tricladida).

Two new land planarian species, collected in the State of Rio de Janeiro, Brazil, are described. Their external aspect is similar to that of Imbira marcusi Carbayo et al., 2013 and Pseudogeoplana theresopolitana (Schirch, 1929), respectively. The analysis of the internal organs, however, revealed they belong to the genus Cratera. The male copulatory organs of one species is very different from any other geoplaninid, for the penis papilla holds a large, distal cavity receiving the ejaculatory duct and, furthermore, the papilla projects vertically downwards from the roof of the male atrium. Thus we consider it as a new species, Cratera cuarassu sp. nov. The second species differs from its congeners in that the dorsal insertion of the penis papilla is anterior to the ventral one, and in that the female atrium is narrowed in the anterior portion. The species was found in the type locality of Pseudogeoplana theresopolitana (Schirch, 1929) and compares well with it in the external features. However, since its internal organs are unknown and the type material of the species is seemingly lost, we describe it as Cratera anamariae Carbayo, sp. nov.

Phylogeography of competing sexual and parthenogenetic forms of a freshwater flatworm: patterns and explanations.

BACKGROUND: Models of the maintenance of sex predict that one reproductive strategy, sexual or parthenogenetic, should outcompete the other. Distribution patterns may reflect the outcome of this competition as well as the effect of chance and historical events. We review the distribution data of sexual and parthenogenetic biotypes of the planarian Schmidtea polychroa. RESULTS: S. polychroa lives in allopatry or sympatry across Europe except for Central and North-Western Europe, where sexual individuals have never been reported. A phylogenetic relationship between 36 populations based on a 385 bp fragment of the mitochondrial cytochrome oxidase I gene revealed that haplotypes were often similar over large geographic distances. In North Italian lakes, however, diversity was extreme, with sequence differences of up to 5% within the same lake in both sexuals and parthenogens. Mixed populations showed "endemic" parthenogenetic lineages that presumably originated from coexisting sexuals, and distantly related ones that probably result from colonization by parthenogens independent from sexuals. CONCLUSIONS: Parthenogens originated repeatedly from sexuals, mainly in Italy, but the same may apply to other Mediterranean regions (Spain, Greece). The degree of divergence between populations suggests that S. polychroa survived the ice ages in separate ice-free areas in Central, Eastern and Southern Europe and re-colonised Europe after the retreat of the major glaciers. Combining these results with those based on nuclear markers, the data suggest that repeated hybridisation between sexuals and parthenogenetic lineages in mixed populations maintains high levels of genetic diversity in parthenogens. This can explain why parthenogens persist in populations that were originally sexual. Exclusive parthenogenesis in central and western populations suggests better colonisation capacity, possibly because of inbreeding costs as well as hybridisation of sexuals with parthenogens.

A robust molecular phylogeny of the Tricladida (Platyhelminthes: Seriata) with a discussion on morphological synapomorphies.

The suborder Tricladida (Platyhelminthes: Turbellaria, Seriata) comprises most well-known species of free-living flatworms. Four infraorders are recognized: (i) the Maricola (marine planarians); (ii) the Cavernicola (a group of primarily cavernicolan planarians); (iii) the Paludicola (freshwater planarians); and (iv) the Terricola (land planarians). The phylogenetic relationships among these infraorders have been analysed using morphological characters, but they remain uncertain. Here we analyse the phylogeny and classification of the Tricladida, with additional, independent, molecular data from complete sequences of 18S rDNA and 18S rRNA. We use maximum parsimony and neighbour-joining methods and the characterization of a unique gene duplication event involving the Terricola and the dugesiids to reconstruct the phylogeny. The results show that the Maricola is monophyletic and is the primitive sister group to the rest of the Tricladida (the Paludicola plus the Terricola). The Paludicola are paraphyletic since the Terricola and one paludicolan family, the Dugesiidae, share a more recent common ancestor than the dugesiids with other paludicolans (dendrocoelids and planariids). A reassessment of morphological evidence may confirm the apparent redundancy of the existing infraorders Paludicola and Terricola. In the meantime, we suggest replacing the Paludicola and Terricola with a new clade, the Continenticola, which comprises the families Dugesiidae, Planariidae, Dendrocoelidae and the Terricola.