Molecular analysis of the diversity of terrestrial planarians (Platyhelminthes, Tricladida, Continenticola) in the Iberian Peninsula

This work is a prospective study to estimate the potential species diversity of terrestrial planarians in the Iberian Peninsula. Live specimens were collected from several Iberian localities and assigned to different morphotypes on the basis of their external morphological characteristics. From the same specimens, sequences from the mitochondrial Cytochrome Oxidasa subunit I (COI) and from the nuclear ribosomal gene 18S rRNA were obtained. Sequences from GenBank of the families Dendrocoelidae, Planariidae, Bipaliidae, Geoplanidae and Rhynchodemidae have been used as outgroups in the phylogenetic analysis. The results showed that terrestrial planarians have a wide distribution in the Peninsula, with all individuals found belonging to the Rhynchodemidae family. Morphological observations indicated the presence of 10 morphospecies confirmed by the molecular analyses. At the same time, COI sequences were successfully used as a molecular marker for species identification in the barcoding mode, which is of great use in groups like this with few external morphological characteristics. The combined data strongly suggest the presence of at least 15 species in the Iberian Peninsula, a number that nearly doubles previous estimates, indicating that terrestrial planarians are more diverse than expected in the region and, as proposed, may be a good biodiversity indicator and model for biogeographical studies.     

Occurrence and abundance of a mariner-like element in freshwater and terrestrial planarians (Platyhelminthes, Tricladida) from southern Brazil

Transposable elements are DNA sequences present in all the large phylogenetic groups, both capable of changing position within the genome and constituting a significant part of eukaryotic genomes. The mariner family of transposons is one of the few which occurs in a wide variety of taxonomic groups, including freshwater planarians. Nevertheless, so far only five planarian species have been reported to carry mariner-like elements (MLEs), although several different species have been investigated. Regarding the number of copies of MLEs, Girardia tigrina is the only planarian species in which this has been evaluated, with an estimation of 8,000 copies of the element per haploid genome. Preliminary results obtained in our laboratory demonstrated that MLE is found in a large number of different species of planarians, including terrestrial. With this in mind, the aim was to evaluate the occurrence and estimate the number of MLE copies in different planarian species collected in south Brazil. Twenty-eight individuals from 15 planarian species were analyzed. By using PCR and the hybridization of nucleic acids, it was found that MLE was present in all the analyzed species, the number of copies being high, probably over 10(3) per haploid genome.     

Repeated DNA elements in planarians of the Dugesia gonocephala group (Platyhelminthes,Tricladida)

The results of the isolation of repetitive DNA elements in the genome of Dugesia etrusca, a species of the Dugesia gonocephala group, are reported. These sequences, about 1.4 kb long, represent only a part of longer interspersed genomic units (De1 family) and appear to be limited to the genome of some planarians of this group, as indicated by a Southern blot analysis performed in different species and populations. The genomic relationships among different species and populations of the D. gonocephala group are discussed in relation to the results obtained in the present work. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Tracking and predation on earthworms by the invasive terrestrial planarian Bipalium adventitium (Tricladida, Platyhelminthes)

The potential ecological impact of exotic terrestrial planarians will be determined in part by their sensory abilities and predatory behavior. It has been suggested that these flatworms may only encounter their earthworm prey by chance, hence restricting the breadth of species they will feed upon and the number of microhabitats in which predator-prey interactions occur. We hypothesized that those flatworms that have already successfully invaded North America (genus Bipalium) actually detect and follow chemical trails of earthworms and possess the behavioral repertoire needed to feed on the prey in a range of microhabitats. We examined: (1) the tendency of Bipalium adventitium to follow chemical trails left by injured and un-injured earthworms; (2) the behavioral repertoire and predatory success of B. adventitium feeding on three earthworm species in subterranean tunnels; and (3) the response of flatworms to the reportedly defensive secretions of the earthworm Eisenia fetida in tunnels. B. adventitium detected and followed trails of earthworm mucus and secretions left by injured and un-injured earthworms. Flatworms followed trails on a range of substrates and pursued and captured three species of earthworms in subterranean tunnels, including individuals many times their mass. Although most behavior exhibited during underground attacks was similar to that reported for surface encounters, the flatworms also behaved in ways that blocked earthworm escape from tunnels. The flatworms were less successful at preying on E. fetida than on Lumbricus rubellus and Lumbricus terrestris in underground tunnels and showed some aversion to the secretions from E. fetida.     

Colonization and extinction of land planarians (Platyhelminthes,Tricladida) in a Brazilian Atlantic Forest regrowth remnant

Long-term assessments of species assemblages are valuable tools for detecting species ecological preferences and their dispersal tracks, as well as for assessing the possible effects of alien species on native communities. Here we report a 50-year-long study on population dynamics of the four species of land flatworms (Platyhelminthes, Tricladida, Terricola) that have colonized or become extinct in a 70-year-old Atlantic Forest regrowth remnant through the period 1955–2006. On the one hand, the two initially most abundant species, which are native to the study site, Notogynaphallia ernesti and Geoplana multicolor have declined over decades and at present do not exist in the forest remnant. The extinction of these species is most likely related with their preference for open vegetation areas, which presently do not exist in the forest remnant. On the other hand, the neotropical Geoplaninae 1 and the exotic Endeavouria septemlineata were detected in the forest only very recently. The long-term study allowed us to conclude that Geoplaninae 1 was introduced into the study area, although it is only known from the study site. Endeavouria septemlineata, an active predator of the exotic giant African snail, is originally known from Hawaii. This land flatworm species was observed repeatedly in Brazilian anthropogenic areas, and this is the first report of the species in relatively well preserved native forest, which may be evidence of an ongoing adaptive process. Monitoring of its geographic spread and its ecological role would be a good practice for preventing potential damaging effects, since it also feeds on native mollusk fauna, as we observed in lab conditions. Júlio Pedroni: Granted by CNPQ–Brazil.     

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.     

Global diversity of land planarians (Platyhelminthes, Tricladida, Terricola): a new indicator-taxon in biodiversity and conservation studies

Biodiversity conservation requires prioritization of areas for in situ conservation. In that perspective, the present study documents the global diversity of a component of the soil macrofauna, the land planarians, and concerns an exploratory analysis of their possible role as indicators of biodiversity. Diversity is described by three quantitative methods: (1) hotspots of species richness, selecting areas richest in species, (2) hotspots of range-size rarity, identifying areas richest in narrowly endemic species, and (3) complementarity, prioritizing areas according to their greatest combined species richness. The biodiversity measures of species richness and range-size rarity show a great correspondence in the identification of hotspots of diversity; both measures identify the following seven areas as the most biodiverse for land planarians: Sao Paulo, Florianopolis, western Java, Tasmania, Sri Lanka, North Island/New Zealand, and Sydney. It is discussed to what extent the results for the land planarians correspond with those obtained in other studies that assessed biodiversity hotspots for taxa on a global scale. It is noteworthy that land planarians identify a few global hotspots of diversity that generally do not feature, or only have low rankings, in other studies: New Zealand, southeastern Australia, and Tasmania.     

Phylogenetic relationships of the genus Dugesia (Platyhelminthes, Tricladida, Paludicola)

A phylogenetic analysis was performed on the genera and subgenera within the freshwater triclad family Dugesiidae, based on 19 terminal taxa and 17 morphological characters. The phylogenetic tree proposed has length of 27 steps and consistency index of 0.66. This phylogenetic hypothesis implies that the current genus Dugesia is paraphyletic and that its subgenera Girardia, Schmidiea and Dugesia S.S. should be elevated to the rank of genus. The genera Cura, Spathula and Neppia are presumed monophyletic by default because the database was unable to provide autapomorphies for any of these genera. The genera Dugesia S.S. and Neppia share sistergroup relationship. Several characters are discussed which were previously considered to be of phylogenetic importance but were not included in the present analysis. It is emphasized that sensory organs form potentially useful set of phylogenetic characters for the Dugesiidae.     

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.     

The aquatic triclads of the Grozet Islands (Platyhelminthes: Tricladida)

A new species of marine planarian, Obrimoposthia aparala sp. nov. , is described from the Crozet Islands and two other species are newly recorded for these islands, viz. 0. wandeli and Synsiphonium ernesti. The only known freshwater triclad from the Crozets, Dugesia seclusa , is redescribed from the type material. The morphology, ecology, and distribution of these four species is discussed.     

Chromosomal polymorphism in planarians (Turbellaria,Tricladida) and the plate tectonics of the western Mediterranean

In two populations of Dugesia (Schmidtea) mediterranea the third chromosome pair consists of two heteromorphous elements. This chromosomal polymorphism, which is common in other animal groups, was hitherto unknown in planarians.The evolutionary and biogeographical significance of this chromosomal polymorphism is discussed, for the geographical distribution of the two aberrant populations may have relevance for the theories of microplate dispersal involving the western Mediterrancan.     

Evidence that two types of 18S rDNA coexist in the genome of Dugesia (Schmidtea) mediterranea (Platyhelminthes, Turbellaria, Tricladida)

Sequences of 18S ribosomal DNA (rDNA) are increasingly being used to inferphylogenetic relationships among living taxa. Although the 18S rDNA belongsto a multigene family, all its copies are kept homogeneous by concertedevolution (Dover 1982; Hillis and Dixon 1991). To date, there is only onewell-characterized exception to this rule, the protozoan Plasmodium(Gunderson et al. 1987; Waters, Syin, and McCutchan 1989; Qari et al.1994). Here we report the 1st case of 18S rDNA polymorphism within ametazoan species. Two types (I and II) of 18S rDNA have been found andsequenced in the platyhelminth Dugesia (Schmidtea) mediterranea(Turbellaria, Seriata, Tricladida). Southern blot analysis suggested thatboth types of rDNA are present in the genome of this flatworm. This wasconfirmed through sequence comparisons and phylogenetic analysis using theneighbor-joining method and bootstrap test. Although secondary structureanalysis suggests that both types are functional, only type I seems to betranscribed to RNA, as demonstrated by Northern blot analysis. The findingof different types of 18S rDNAs in a single genome stresses the need foranalyzing a large number of clones whenever 18S sequences obtained by PCRamplification and cloning are being used in phylogenetic reconstruction.     

A new species of land planarian from Madeira (Platyhelminthes, Turbellaria, Tricladida)

A new species of terrestrial planarian from Madeira, Kontikia bulbosa sp. nov. , is described. The genus Kontikia is redefined and its scope and status reviewed.     

Notes on the biology of the freshwater planarian Dugesia bengalensis (Platyhelminthes Turbellaria Tricladida)

Dugesia bengalensis was described by Kawakatsu (Kawakatsu et al., 1983) from specimens collected in West Bengal. We have been studying populations from many different localities in Santiniketan and adjoining areas of West Bengal and can provide additional biological information.The species is hermaphroditic, and its breeding season was found to occur usually between October and March when the winter temperature falls below 25 °C. Outside of the breeding season, D. bengalensis is capable of asexual reproduction by binary fission (Mahapatra et al., 1987).Development of the reproductive organs appeared to be from neoblasts and other mesenchymal cells and, therefore, to be like that typical of other triclads with the exception that some of the neoblasts used for the reproductive tissue appeared to be derived from the intestinal region (Ghosh, 1988; cf. Teshirogi, 1986).During copulation, the partners were oriented in the same direction and not in a head-to-tail position as has been reported (Hyman, 1945) for some planarians.The oval, stalked cocoons were laid in marshy places, and during the period of summer (usually from April to June) they lay dormant in the sandy soil until the onset of the monsoon rains. Then, typically three or four months after they were laid, the cocoons hatched to yield three or four young, a remarkably low number for freshwater triclads (cf Ball & Reynoldson, 1981).     

A new and aberrant species of Dugesia (Platyhelminthes,Tricladida, Dugesiidae) from Madagascar

In this paper we report a new species of Dugesia of the family Dugesiidae from Madagascar, representing the fourth species of freshwater planarian known from this global biodiversity hotspot. In some respects the new species is aberrant, when compared with its congeners, being characterized by a head with smoothly rounded auricles, a peculiar course of the oviducts, including the presence of a common posterior extension, and by the asymmetrical openings of the vasa deferentia at about halfway along the seminal vesicle. Further, it is characterized by a ventral course of the ejaculatory duct with a terminal opening, very long spermiducal vesicles and unstalked cocoons. Its diploid chromosome complement with 18 chromosomes represents an uncommon feature among fissiparous species of Dugesia.     

Genetic divergence in Northamerican freshwater planarians of the Dugesia dorotocephala group (Turbellaria, Tricladida, Paludicola)

The genetic differentiation between the members of the Dugesia (Girardia) dorotocephala group was analyzed by means of multilocus electrophoresis, and comared to that of another planarian secies, D. tahitiensis, also belonging to the subgenus Girardia. The species examined were: D. dorotocephala s.s (2n = 16), D. arizonensis (2n = 8), D. jenkinsae (2n = 8), and the above mentioned D. tahitiensis (2n = 16). The former three species inhabit North America, and show different proportion of fissiparous and sexual individuals; the latter species inhabits Polynesia and is fully asexual. A total of 11 enzyme loci were genetically analyzed: Mdh-1, Mdh-2, Zdh-1, Idh-2, G3pdh, Got-1, Ck, Pgm-2, Ada, Mpi, and Gpi. Low values of observed mean heterozygosity per locus (Ho) were found in the populations studied, ranging from 0 to 0.18 (average 0.08. In asexual populations (except that of D. tahitiensis) fixed heterozygosity was observered in all the individuals for 1 or 2 loci. The genetic divergence between the species examined is very high, with many loci showing discriminating alleles in different taxa (Nei's genetic distance varies from 0.871 to 1.759). The populations of D. dorotocehala s.s., on the contrary, appear to be genetically quite homogenous average D= 0.019), and the genetic distance values are apparently unrelated to their geographic location and to their way of reproduction. The genetic distance between D. tahitiensis, a species not included in the D. dorotocephala group and D. dorotocephala s.s. is 1.314 and hence similar to the D value between two members of;he dorotocephala group: D. dorotocephala and D. jenkinsae (D = 1.303). The genetic relationships among the populations studied were established by UPGMA cluster analysis and multidimensional scaling. The descendence of the North American species with 2n = 8 from a dorotocephala-like ancestor with 2n = 16 is considered. It is suggested that the latter, as well as a tahitiensis-like line, also having 2n = 16, have originated from a common ancestor by geographic isolation.     

Telomeric DNA length and phylogenetic relationship of Baikal and Siberian planarians (Turbellaria, Tricladida)

Dynamics of the telomeric DNA (tDNA) length and the phylogeny of Baikal and Siberian planarians have been studied based on analysis of 18S rDNA and β-actin gene fragments. It was shown that there is a relationship between tDNA length and planarian size. Giant planarians, with a minor exception, have longer tDNA than do smaller planarians. Phylogenetic affinity between species that have stretched tracks of tDNA, large sizes, and similar habitats denotes the possible role of tDNA in the development of the high regenerative capacity of planarians.