Planarian (Platyhelminthes, Tricladida) Diversity and Molecular Markers: A New View of an Old Group

Planarians are a group of free-living platyhelminths (triclads) best-known largely due to long-standing regeneration and pattern formation research. However, the group"s diversity and evolutionary history has been mostly overlooked. A few taxonomists have focused on certain groups, resulting in the description of many species and the establishment of higher-level groups within the Tricladida. However, the scarcity of morphological features precludes inference of phylogenetic relationships among these taxa. The incorporation of molecular markers to study their diversity and phylogenetic relationships has facilitated disentangling many conundrums related to planarians and even allowed their use as phylogeographic model organisms. Here, we present some case examples ranging from delimiting species in an integrative style, and barcoding them, to analysing their evolutionary history on a lower scale to infer processes affecting biodiversity origin, or on a higher scale to understand the genus level or even higher relationships. In many cases, these studies have allowed proposing better classifications and resulted in taxonomical changes. We also explain shortcomings resulting in a lack of resolution or power to apply the most up-to-date data analyses. Next-generation sequencing methodologies may help improve this situation and accelerate their use as model organisms

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

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

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. © 2012 UBC Press

New morphological and molecular data reveal an important underestimation of species diversity and indicate evolutionary patterns in European Lepidocyrtus

The correct identification of morphological species is a key task for species richness estimation of any ecosystem. Although body colour is a widely used character identifying European Lepidocyrtus species, recent investigations using molecular data have revealed that species delineation using body colour can result in an underestimation of real species diversity because of the presence of cryptic species. Lepidocyrtus violaceus is a European species characterised by its dark violet body colour. Its wide distribution leads us to suspect that several cryptic species can be present within this morphospecies. Since traditional morphological characters have appeared insufficient for real diversity identification in Lepidocyrtus, new morphological characters were needed in order to describe the cryptic diversity detected by molecular data in this genus. Pseudopores are integumentary structures present in all Lepidocyrtus species, but the distribution of these structures has not been properly described in the genus, as well as in Entomobryioidea overall. In the present work we aimed to analyse whether L. violaceus is a monophyletic entity in Europe. Moreover, we aimed to determine if the position and number of pseudopores on the different parts of the body and appendages is a phylogenetically useful character in the identification of the species or superspecific entities. Fourteen populations of L. violaceus from five European countries, and another 25 Lepidocyrtus species from nine European countries have been studied. In total, 208 specimens have been analysed morphologically and half of them were studied molecularly using sequences of the genes COXII and EF-1α. Molecular data revealed that the widely distributed Lepidocyrtus violaceus morphospecies is a polyphyletic entity in Europe. Between 6 and 12 diferent cryptic species have been detected within this European morphospecies, and only the presence of pseudopores on the basal plate of the fourth abdominal segment has been found to be a promising diagnostic character between them. A common basal pattern of pseudopore distribution has been recognised in the European members of the genus, and also a diferential pattern within each European species group. As a general trend, an increase in the number of pseudopores has been detected from the most basal to the most derived species groups in the phylogeny of the genus in Europe

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 kewenseMoseley, 1878 had been cited till 2007. Fromthat 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 DistributionModeling (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, fromall 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 habitat

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

Assembling animals: trees, genomes, cells, and contrast to plants

The Animal Kingdom is an astonishingly diverse group. Together with plants and fungi is one of the three major lineages of multicellular eukaryotes. Due to anthropocentrism and/or genuine scientific interest, their origin and diversification are pivotal to modern evolutionary biology. In the last few decades, dramatic technological advances in molecular biology and computational power have generated new phylogenetic proposals, as well as new tools to compare genomes or study cell type evolution. These new approaches complement the insights from fields such as comparative morphology, evodevo, or palaeontology, which all together provide an integrative view of animal evolution, including major evolutionary transitions such as the origin of animals or the emergence of animals with bilateral symmetry. In this paper, we review recent developments in animal phylogenetics, comparative genomics, and cell type evolution related to these two transitions, and we compare animals to another major lineage of multicellular eukaryotes, plants

Evolutionary Analysis of Mitogenomes from Parasitic and Free-Living Flatworms

Copyright: © 2015 Solà et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The attached file is the published version of the article

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