Abstract. The aim of this paper is to describe morphological characteristics of fresh- water triclads in Herzegovina and to provide a key for their. 1 Introduction. The polyclads are the most primitive free living flatworms of the phylum Platyhelminthes. These worms are dorso-ventrally. -Eight new species belonging to the genus Cycloporus (Platyhelminthes. Polycladida,. Euryleptidae) are described from Australasian waters including eastern.

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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 regenerationmost 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.

Phylum Platyhelminthes

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.

Our aim was to determine the centre of colonisation origin, whether the populations are recent, and whether the species is expanding. The species presents 3 most frequent haplotypes, differing in a maximum of 11 platyhelminthee 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 juenal the central Mediterranean region.

Platyhhelminthes potential distribution predicted by climatic variables based modelling shows a preference for coastal areas and fits well with the observed data. The distribution and frequency of the most frequent haplotypes and the presence of heteroplasmic individuals allow platyhemlinthes to gain an understanding of the recent history of the species, together with previous knowledge on its phylogenetic relationships platyyhelminthes 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 Jurnsl 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 platyhelminhtes predicts a likely future expansion to northern and more interior areas following the increase in temperatures due to climate change.

Dugesia sicula Lepori, is a freshwater planarian phylum Platyhelminthes, order Tricladida, suborder Continenticola typically found in ponds, streams and springs close to the Mediterranean coast. The first record of D.

Fissiparity is an asexual form of reproduction that involves transversal division of the individuals into two fragments and subsequent regeneration of the absent structures. Fissiparous planarians do not develop a copulatory apparatus, which precludes the assignment of these populations to any species since this structure provides most of the defining characters for species description in planarians.

However, the morphological characters of the copulatory apparatus of the sexual specimens allowed the species to be described. None of the originally described sexual populations pkatyhelminthes be currently found [ 4 ]; but, some sexual populations in northern Africa Tunisia and Algeria have been recently reported [ 5 – 7 platynelminthes.

Moreover, a molecular-based study [ 8 ] showed that most of the numerous fissiparous populations of genus Dugesia present in the western Mediterranean belong to D. Under certain conditions mostly in the laboratory triploid fissiparous planarians can develop hyperplasic ovaries and a copulatory apparatus [ 4910 ], these individuals are known as ex-fissiparous. The literature suggests that D.

Contrasting with this wide distribution the species presents extremely low variability for the COI gene platyhelmintthes 8 ] among populations located hundreds of kilometres apart.

These two features wide distribution and low genetic variability could be a consequence of a recent expansion promoted by human activities, as has been shown for other organisms [ 13 ]. Alternatively, the low levels of variability could be exclusively a consequence of their fissiparous reproduction, as described in Schmidtea mediterranea [ 14 ] and other organisms [ 15 ], and its wide distribution may be a consequence of its active spread over the years.


In both cases, the hypotheses contrast with the poor dispersion capability assumed for freshwater planarians [ 1617 ]. Freshwater planarians do not exhibit larval dispersal stages or forms resistant to desiccation; these individuals thus require contiguous freshwater bodies to survive and disperse [ 1617 ].

However, introductions due to human activities have been documented in other freshwater planarians, such as Girardia tigrina [ 18 ] and Schmidtea polychroa [ 19 ] and more recently have also been proposed for other Dugesia species [ 20 ], but in all these cases the introduced animals either have a restricted distribution Dugesia or its introduction and progression in the new areas has been followed by scientist.

Fissiparous reproduction can have effects on the distribution of genetic variability within the populations and even on individuals that could perhaps help disentangle this situation. Regeneration after fission is driven by neoblasts [ 2122 ], which are pluripotent stem cells. A new mutation in a neoblast would expand in the individual when this neoblast generates new tissues after the fission of the animal, thus increasing frequency of the mutation in the individual.

In this manner, we would expect to observe cells with different sequences within one individual. This could be considered a special case of heteroplasmy that differs from cases in which heteroplasmy is transmitted sexually or parthenogenetically through the mitochondria present in the oocyte and the spermatozoon in some cases where all cells from a new individual will present the same haplotypes.

Heteroplasmy has been detected in many species [ 23 ], and some studies in humans have shown it increases with age in somatic cells and is more frequent in certain tissues, such as muscle tissue, most likely because of increases in the number of mutations caused by the presence of oxidative radicals [ 2324 ].

Heteroplasmy has also been detected in COI sequences of fissiparous populations of D. Due to their characteristics, in fissiparous planarians the somatic heteroplasmy would be maintained in populations and increased over time.

If this situation holds in fissiparous populations of D.

The absence or low frequency of heteroplasmic specimens in a specific locality of fissiparous reproducing individuals could be interpreted as a consequence of a recent bottleneck, potentially because of recent colonisation by a few individuals or recovery from a recent population-wide decrease. The alternate condition, the presence of many heteroplasmic individuals, particularly those exhibiting a high number of different low-frequency haplotypes, could then potentially be related to aged individuals and populations, although a recent colonisation by highly heteroplasmic individuals could also explain this outcome.

Following the same reasoning, one would not expect to find heteroplasmy, or different copies of mitochondrial genes within an individual in sexual organisms. In the present study, we analysed the genetic diversity and structure using COI sequences of a broad sampling of D. With this data we expect to be able to answer questions on where the centre of origin of D. Individuals from the Boussadia and Soukra populations s39 and s36 in Tunisia reproduce sexually [ 56 ].

Individuals collected from Crete s55Greece, also possess a copulatory apparatus. Approximately 15 individuals between 1 and 20, Additional file 2: Table S1 were analysed from each location. In some individuals, the sequence had to be obtained in two non-overlapping fragments since we were not able to obtain amplifications in a single fragment.

Sequencing was performed using Big Dye 3. All populations were identified as D. Sampling sites for D. Specimens from s39 and s36 exhibited sexual reproduction. The individuals from s55 possessed a copulatory apparatus.

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

We cloned the PCR products from sexual 5 individuals and fissiparous individuals, within the latter group we included both specimens who showed double peaks in the sequences obtained directly from the PCR product 11 and some that did not show this pattern 5.

Approximately ten colonies from each individual were amplified and sequenced using the T3 and T7 primers included in the kit. Alignment was performed by sight based on the amino acid sequence, and the sequences were submitted to GenBank [GenBank: We tested whether the number of substitutions observed in the cloned sequences agreed with the expected random accumulation of mutations promoted by the polymerase errors to determine whether the variation observed in the sequences was caused by errors in the PCR process.


Therefore, we assumed that errors in the PCR process followed a Poisson distribution, and the expected mean was computed using the platyhelminnthes error platyhe,minthes 6. In a functional protein coding sequence, more changes are expected in the 1st and 3rd codon positions. To test the functionality of the sequences obtained in this study we calculated the expected number of changes per codon site in the case of a non-functional sequence 1: Those populations for which only one sequence was available were not included.

Haplotype and nucleotide diversity were also calculated for each of the cloned individuals.

Two haplotype networks were constructed to study the relationships among haplotypes using TCS v1. To study the possible expansion of D. The independence of 19 climatic variables were tested based on the R 2 statistic: Finally four independent climatic variables were used: From 61 known localities 58 analysed in this study and 3 selected from the literature [ 7 ] —Algeria— and personal communications of L. Leria —Mallorca— and E. Table S1 of these sequences showed double peaks in the chromatogram and were discarded for the population analyses.

Because some populations had only one sequenced specimen s04, s18, and s30 or all of their individuals exhibited polymorphic positions, only individuals from 51 populations could be analysed. Only 16 populations showed more than one haplotype, and the majority of these showed two haplotypes. The haplotype network was based on an alignment of sequences and a length of base pairs.

Haplotype B was present in individuals from 18 populations, the majority of which were found northwest of the Mediterranean. The other 12 haplotypes were unique or observed in only two individuals. Haplotypes A and B were observed simultaneously in 6 populations from the centre of the Mediterranean. Haplotype network and distribution in the studied populations. Red corresponds to haplotype A, dark blue corresponds to haplotype B, and brown corresponds to haplotype C. Only haplotypes from non-polymorphic individuals are represented.

The grey dots on the map show populations in which all of the specimens showed polymorphic sites. Table S2 were sequenced to determine the haplotypes of those individuals.

Locations with polymorphic individuals. The black dots represent locations with polymorphic individuals. The white dots iurnal populations in which all of the individuals are non-polymorphic. Polymerase mistake versus heteroplasmy test.

Expected number of point mutations for each individual based on a polymerase error rate of 6. In all cases we found multiple haplotypes within each individual even for those not showing polymorphic sites in the direct PCR sequence.

All of the individuals with polymorphic sites exhibited significant differences between the two values except for the s None of the sexual individuals analysed had a significant result. Moreover, the cloning results showed that haplotypes A, B and C were present simultaneously in some of the specimens Additional file 2: This result suggests that, in these individuals, despite that some point mutations could be caused by polymerase error, there are most likely low-frequency haplotypes that we cannot detect in the direct sequences using PCR.

In the subsequent analyses, we considered all of the individuals that showed a significant result to be heteroplasmic and the remaining individuals to be non-heteroplasmic. Figure S2the single haplotypes sequenced from the non-heteroplasmic individuals were discarded since those must most probably be the result of a polymerase error as shown in the previous tests.

The final network contained sequences and 57 different haplotypes were observed; haplotypes A, B and C were the most frequent and appeared 46, 43 and 16 times, respectively. The remaining haplotypes were unique in the majority of the cases and showed a single difference from the A, B or C haplotypes. All of the analysed individuals, except specimen s Table S2exhibited at least one of the most frequent jurrnal A or Band all of the individuals showed at least one unique haplotype Additional file 1: Cloned specimen haplotype network and distribution.

The pie charts represent the haplotypes observed plafyhelminthes each cloned heteroplasmic individual Additional file 2: The cloned individuals that were not heteroplasmic do not have pie charts.