New species of Lepidocyrtus (Collembola, Entomobryidae) from Italy with a discussion of characters defining European Lepidocyrtus lignorum-group.

Springtails are a group of hexapods whose true diversity is currently underestimated. This is because the morphological characters normally used in species diagnosis do not have the necessary resolution. This situation is especially evident in Entomobryidae, which is currently the most diverse family of springtails. The combination of morphological and molecular data has allowed us to describe a new species of the genus Lepidocyrtus, based on specimens from Northern Italy, as well as to redefine the diagnosis of the European Lepidocyrtus lignorum-group.

Phylogenetic relationships of the Geoplaninae land planarians (Platyhelminthes, Tricladida) assessed with a total evidence approach, with the description of a new species of Gigantea.

Several studies have focused on the phylogenetic relationships within the Geoplaninae land planarians (Tricladida). In those studies, ancient phylogenetic relationships remained obscure. In this work, the phylogeny of Geoplaninae is assessed through three different datasets, namely morphological, molecular, and both datasets combined, i.e, a total evidence approach (TE). The data matrix consisted of six DNA regions, including a newly developed marker (DOM5), and a morphological matrix with 37 characters. The study produced the best-resolved hypothesis so far for the phylogeny of Geoplaninae, although ancient clades still remain elusive. The effect of the morphological data on the TE tree topology and clade support is seemingly negligible. The phylogenetic tree also suggests that most of the diagnostic morphological characters of the genera are homoplastic, while unambiguous unique synapomorphies can characterize some supra-generic informal groupings.

A new species of alien terrestrial planarian in Spain: Caenoplana decolorata.

Terrestrial planarians found in a plant nursery in Spain in 2012 are described as a new species, Caenoplana decolorata. Dorsally they are mahogany brown with a cream median line. Ventrally they are pastel turquoise fading to brown laterally. Molecular data indicate that they are a member of the genus Caenoplana, but that they differ from other Caenoplana species found in Europe. One mature specimen has been partially sectioned, and the musculature and copulatory apparatus is described, confirming the generic placement but distinguishing the species from other members of the genus. It is probable that the species originates from Australia.

Five new pseudocryptic land planarian species of Cratera (Platyhelminthes: Tricladida) unveiled through integrative taxonomy.

BACKGROUND: Cratera is a genus of land planarians endemic to the Brazilian Atlantic forest. The species of this genus are distinguished from each other by a series of external and internal characters, nonetheless they represent a challenging taxonomic issue due to the extreme alikeness of the species analysed in the present work. To resolve these difficulties, we have performed morphological analyses and used three nuclear markers (ribosomal 18S and 28S, Elongation Factor, a new anonymous marker named Tnuc813) and two mitochondrial fragments (Cytochrome c oxidase subunit I gene, and a fragment encompasing NADH deshydrogenase subunit 4 gene, trnF and the beginning of the Cytochrome c oxidase I gene) in an integrative taxonomic study. METHODS: To unveil cryptic species, we applied a molecular species delimitation approach based on molecular discovery methods, followed by a validation method. The putative species so delimited were then validated on the basis of diagnostic morphological features. RESULTS: We discovered and described four new species, namely Cratera assu, C. tui, C. boja, and C. imbiri. A fifth new species, C. paraitinga was not highly supported by molecular evidence, but was described because its morphological attributes are unique. Our study documents for the genus Cratera the presence of a number of highly similar species, a situation that is present also in other genera of land planarians. The high number of poorly differentiated and presumably recent speciation events might be explained by the recent geological history of the area.

The identity of the invasive yellow-striped terrestrial planarian found recently in Europe: Caenoplana variegata (Fletcher amp; Hamilton, 1888) or Caenoplana bicolor (Graff, 1899)?

Terrestrial planarians with a dorsal yellow stripe and dark lateral surfaces and up to 15-20 cm long have been found in several countries in Europe, the earliest in 2008. They are similar to two species originally from Australia, Caenoplana variegata (Fletcher Hamilton, 1888) and C. bicolor (Graff, 1899), both described on external characters only, with no anatomical information. Careful reading suggests that there is no significant difference between the original descriptions. Further: observations on live specimens show considerable variation between individuals and in individuals over time and before and after feeding, negating any distinction between descriptions. Examination of three sectioned specimens shows considerable difference in sexual maturity, though one seems almost fully mature and the reproductive system is described. Molecular results show that specimens from the United Kingdom and Spain are of the same species. It is concluded that the planarians should be referred to as C. variegata, C. bicolor being a junior synonym.

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.

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.

Hidden diversity in forest soils: Characterization and comparison of terrestrial flatworm's communities in two national parks in Spain.

Terrestrial flatworms (Platyhelminthes, Tricladida, and Geoplanidae) belong to what is known as cryptic soil fauna of humid forests and are animals not easily found or captured in traps. Nonetheless, they have been demonstrated to be good indicators of the conservation status of their habitat as well as a good model to reconstruct the recent and old events affecting biodiversity. This is mainly due to their delicate constitution, their dependence on the integrity of their habitat, and their very low dispersal capacity. At present, little is known about their communities, except for some studies performed in Brazil. In this work, we analyze for the first time in Europe terrestrial flatworm communities. We have selected two protected areas belonging to the Red Española de Parques Nacionales. Our aims include performing a first study of the species richness and community structure for European terrestrial planarian species at regional and local scale. We evaluate the effect of type of forests in the community composition and flatworms' abundance, but also have into account the phylogenetic framework (never considered in previous studies) analyzed based on molecular data. We find differences in the species composition among parks, with an astonishingly high diversity of endemic species in the Parque Nacional de Picos de Europa and an extremely low diversity of species in the Parque Nacional de Ordesa y Monte Perdido. These divergent patterns cannot be attributed to differences in physical variables, and in addition, the analyses of their phylogenetic relationships and, for a few species, their genetic structure, point to a more probable historical explanation.

Evolutionary analysis of mitogenomes from parasitic and free-living flatworms.

Mitochondrial genomes (mitogenomes) are useful and relatively accessible sources of molecular data to explore and understand the evolutionary history and relationships of eukaryotic organisms across diverse taxonomic levels. The availability of complete mitogenomes from Platyhelminthes is limited; of the 40 or so published most are from parasitic flatworms (Neodermata). Here, we present the mitogenomes of two free-living flatworms (Tricladida): the complete genome of the freshwater species Crenobia alpina (Planariidae) and a nearly complete genome of the land planarian Obama sp. (Geoplanidae). Moreover, we have reanotated the published mitogenome of the species Dugesia japonica (Dugesiidae). This contribution almost doubles the total number of mtDNAs published for Tricladida, a species-rich group including model organisms and economically important invasive species. We took the opportunity to conduct comparative mitogenomic analyses between available free-living and selected parasitic flatworms in order to gain insights into the putative effect of life cycle on nucleotide composition through mutation and natural selection. Unexpectedly, we did not find any molecular hallmark of a selective relaxation in mitogenomes of parasitic flatworms; on the contrary, three out of the four studied free-living triclad mitogenomes exhibit higher A+T content and selective relaxation levels. Additionally, we provide new and valuable molecular data to develop markers for future phylogenetic studies on planariids and geoplanids.

Diversity of introduced terrestrial flatworms in the Iberian Peninsula: a cautionary tale.

Many tropical terrestrial planarians (Platyhelminthes, Geoplanidae) have been introduced around the globe. One of these species is known to cause significant decline in earthworm populations, resulting in a reduction of ecological functions that earthworms provide. Flatworms, additionally, are a potential risk to other species that have the same dietary needs. Hence, the planarian invasion might cause significant economic losses in agriculture and damage to the ecosystem. In the Iberian Peninsula only Bipalium kewense Moseley, 1878 had been cited till 2007. From that year on, four more species have been cited, and several reports of the presence of these animals in particular gardens have been received. In the present study we have: (1) analyzed the animals sent by non-specialists and also the presence of terrestrial planarians in plant nurseries and garden centers; (2) identified their species through morphological and phylogenetic molecular analyses, including representatives of their areas of origin; (3) revised their dietary sources and (4) used Species Distribution Modeling (SDM) for one species to evaluate the risk of its introduction to natural areas. The results have shown the presence of at least ten species of alien terrestrial planarians, from all its phylogenetic range. International plant trade is the source of these animals, and many garden centers are acting as reservoirs. Also, landscape restoration to reintroduce autochthonous plants has facilitated their introduction close to natural forests and agricultural fields. In conclusion, there is a need to take measures on plant trade and to have special care in the treatment of restored habitats.

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.

Evolutionary history of the Tricladida and the Platyhelminthes: an up-to-date phylogenetic and systematic account.

Within the free-living platyhelminths, the triclads, or planarians, are the best-known group, largely as a result of long-standing and intensive research on regeneration, pattern formation and Hox gene expression. However, the group's evolutionary history has been long debated, with controversies ranging from their phyletic structure and position within the Metazoa to the relationships among species within the Tricladida. Over the the last decade, with the advent of molecular phylogenies, some of these issues have begun to be resolved. Here, we present an up-to-date summary of the main phylogenetic changes and novelties with some comments on their evolutionary implications. The phylum has been split into two groups, and the position of the main group (the Rhabdithophora and the Catenulida), close to the Annelida and the Mollusca within the Lophotrochozoa, is now clear. Their internal relationships, although not totally resolved, have been clarified. Tricladida systematics has also experienced a revolution since the implementation of molecular data. The terrestrial planarians have been demonstrated to have emerged from one of the freshwater families, giving a different view of their evolution and greatly altering their classification. The use of molecular data is also facilitating the identification of Tricladida species by DNA barcoding, allowing better knowledge of their distribution and genetic diversity. Finally, molecular phylogenetic and phylogeographical analyses, taking advantage of recent data, are beginning to give a clear picture of the recent history of the Dugesia and Schmidtea species in the Mediterranean.

Molecular phylogeny of land and freshwater planarians (Tricladida, Platyhelminthes): from freshwater to land and back.

The suborder Tricladida (phylum Platyhelminthes) comprises the well-known free-living flatworms, taxonomically grouped into three infraorders according to their ecology: Maricola (marine planarians), Paludicola (freshwater planarians), and Terricola (land planarians). Molecular analyses have demonstrated that the Paludicola are paraphyletic, the Terricola being the sister group of one of the three paludicolan families, the Dugesiidae. However, neither 18S rDNA nor COI based trees have been able to resolve the relationships among species of Terricola and Dugesiidae, particularly the monophyly of Terricola. Here, we present new molecular data including sequences of nuclear genes (18S rDNA, 28S rDNA) and a mitochondrial gene (COI) of a wider sample of dugesiid and terricolan species. The new sequences have been analyzed, together with those previously obtained, in independent and concatenated analyses using maximum likelihood and bayesian methods. The results show that, although some parts of the trees remain poorly resolved, they support a monophyletic origin for Terricola followed by a likely return of some species to freshwater habitats. Relationships within the monophyletic group of Dugesiidae are clearly resolved, and relationships among some terricolan subfamilies are also clearly established and point to the need for a thorough revision of Terricola taxonomy.