Mottled ducks Anas fulvigula lay their eggs in grass nests near to the ground, usually close by water. Though this nest looks quite full with nine eggs, a clutch of mottled duck eggs can contain as many as twelve or thirteen eggs. Organisms are genetically diverse because of sexual reproduction. Sexual reproduction begins with sperm and egg cells, which are produced through a process called meiosis.
Metrics details. Schmidtea mediterranea Platyhelminthes, Tricladida, Continenticola is found in scattered localities on a few islands and in coastal areas of the western Mediterranean. Although S. Its present distribution has been proposed to stem from the fragmentation and migration of the Corsica-Sardinia microplate during the formation of the western Mediterranean basin, which implies an ancient origin for the species.
To test this hypothesis, we obtained a large of samples from across its distribution area. Using known and new molecular markers and, for the first time in planarians, a molecular clock, we analysed the genetic variability and demographic parameters within the species and between its sexual and asexual populations to estimate when they diverged.
Molecular data clustered the studied populations into three groups that correspond to the west, central and southeastern geographical locations of the current distribution of S. Mitochondrial genes show low haplotype and nucleotide diversity within populations but demonstrate higher values when all individuals are considered.
The nuclear marker shows higher values of genetic diversity than the mitochondrial genes at the population level, but asexual populations present lower variability than the sexual ones. Neutrality tests are ificant for some populations. Phylogenetic and dating analyses show the three groups to be monophyletic, with the west group being the basal group. This species probably adapted to temperate climates in the middle of a changing Mediterranean climate that eventually became dry and hot. These data also suggest that in the mainland localities of Europe and Africa, sexual individuals of S.
Schmidtea mediterranea Benazzi et al. Nonetheless, little is known about its evolutionary history and demographics, although it possesses several intriguing features.
The genus Schmidtea includes only four species: S. With the exception of S. Although fissiparity is also common in other genera of planarians, such as Girardia [ 9 ] and Dugesia [ 10 ], most asexual fissiparous populations of these genera are triploid.
The asexual strain occurs only in a few locations, in Catalonia and the Balearic Islands, where sexual populations have not yet been found. In the remaining distribution area, only sexual diploids are found, with the only exceptions being a triploid sexual population in Sardinia [ 13 ] and a triploid asexual fissiparous population in Menorca, the latter bearing a translocation between the 1 st and 3 rd chromosomes only one of each triplet [ 7 ].
Distribution of the species and haplotype distribution in populations. Pie charts indicate the proportion of the different haplotypes in each of the eleven populations.
Colours and names of the haplotypes correspond to those indicated in the haplotype networks Figure 2. Indeed, the fragmentation of continental microplates the Corsica-Sardinia block, CSb and their clockwise and counterclockwise migration gave rise, during the Oligocene and early Miocene periods, to the Balearic Islands, Corsica, Sardinia, Calabria and the Kabilies in Algeria [ 15 — 18 ]. If this hypothesis is true, it implies that this species, or a close ancestor, was already present on the microplate before it first broke away, approximately 30 mya, splitting the CSb from the Spanish coast.
The poor dispersion capability of freshwater planarians they require permanent freshwater courses or ponds, and no forms resistant to desiccation have been described and their impossibility to cross large marine masses, make other explanations e. Although a clear link between CSb fragmentation and migration and the successive speciation events has been demonstrated for other groups of animals [ 19 — 21 ], the case of S. Furthermore, when and how the asexual populations of S.
This situation suggests multiple possible explanations for when and where they originated.
Asexual S. Altogether, these ideas pose several interesting questions: 1 Is the microplate fragmentation the origin of the present distribution of S. To answer these questions and to widen our knowledge on the evolutionary history of S. Molecular phylogenies are currently the best option to provide solid evidence on the origin, relationships and divergence in time among populations, particularly for organisms such as planarians, which are lacking reliable morphological characteristics. Furthermore, if these events can be correlated to some geological phenomena, a molecular clock can be set and calibrated, and the evolutionary history can be put into a temporal context.
Finally, a comparative analysis of the genetic nucleotide variability within and among populations may allow us to identify the evolutionary factors responsible for the levels and patterns of genetic variation and therefore for the biological diversity [ 22 ].
We have sequenced fragments of two mitochondrial genes one for the first time in planariansand taking into the genomic information available for S. Because nuclear and mitochondrial markers accumulate changes at different rates, they provide complementary information on the effects of different types of factors, namely, those affecting different historical times. Using these markers, we measured the levels of genetic variation among and within all populations sampled and inferred the phylogenetic tree for these species.
In addition, we have calibrated, for the first time in planarians, a molecular clock to estimate the age of the species and the divergence among populations. The obtained show the usefulness of the new markers and attest that the phylogeography and evolutionary history of S. Samples covering all known distributions of S. Described populations from Mallorca [ 23 ] and north of Catalonia [ 7 ] could not be recovered.
Sampling in Morocco and Algeria retrieved a population of S. The populations from Spain, Corsica and Tunisia are the only populations described in the literature for those regions and were found during the present project sampling; no new populations have been found.
Altogether, a total of specimens were collected from 11 localities. Specific primers Table 2some deed for this study, were used to amplify partial fragments of the mitochondrial genes cytochrome oxidase I COI and cytochrome b CYB. We also searched for new nuclear markers, and based on sequences provided to us by other researchers Abril, J. Amplification products were sequenced directly with the same primers used in the PCR reaction using Big Dye 3.
DNA sequences were aligned with ClustalW [ 25 ] and optimised by sight using the amino acidic sequence as a guide for mitochondrial genes. Some individuals were heterozygous for the nuclear gene N13 Table 3. Eight colonies from each individual were amplified and sequenced using the T3 and T7 primers included in the kit following the procedure described in the section. We used the program DnaSP v5.
Genotype diversity for N13 was calculated by using the H D formula [ 29 ] and replacing haplotypes with genotypes. This method is based on a simulated annealing procedure aimed at identifying groups of populations that are geographically homogeneous and maximally differentiated in terms of an among-group component of the overall genetic variance without the prior assumption of group composition that is necessary for AMOVA. The levels of divergence between S. The non-coding region of N13 presented high sequence variability between S.
A genetic differentiation statistic, S nnwas estimated, and its statistical ificance was determined using a permutation test 10, replicates. To determine if the pattern of the polymorphism for the N13 fragment conforms to that expected under the neutral hypothesis, we applied two neutrality tests to detect the specific fingerprint of recent population expansions, heavy bottlenecks or other selective and demographic scenarios: Tajima's D [ 32 ] and R 2 [ 33 ].
These statistics were selected because they are not sensitive to the haplotype phase reconstruction and R 2 has the advantage of behaving well for small sample sizes.
Their statistical ificance was estimated using coalescent computer simulations 10, replicates. Bayesian inference BI was estimated using either with MrBayes 3. Because the fossil record of freshwater planarians is non-existent, no molecular clock for the Tricladida is available. Hence, to calibrate the molecular clock and to infer the approximate age of Schmidtea mediterraneawe used two-step dating.
First, an analysis with a partial sequence of the COI gene bpwhich belongs to 19 species of the families Dugesiidae and Planariidae Additional file 1was performed. We calibrated the data based on the biogeographical hypothesis proposed by Ball [ 38 ].
Analysing the present day distribution of the Dugesidae genera, Ball suggested that the center of the origin of this family would have been situated in southern Gondwanaland. The breakage of this supercontinent, thus separating from what would become America to the west, would have resulted in the origin of the Girardia genus exclusively American in that region, while the rest of the genera or their ancestors would have appeared in the eastern block; Dugesia and Schmidtea currently present in Africa, Asia and Europe would have originated in Africa and Europe, respectively, after their ancestors moved northward.
Hence, we have used the split between the continents of Africa and South America ending around mya as a calibration point to determine the maximum age of the separation of the genera Dugesia and Schmidtea from the genus Girardia. For this second analysis, which consisted of a comparison of populations from the same species, it was expected that the rates of evolution would not vary among populations.
In addition, a phylogenetic analysis with ML was performed to obtain boostrap support for the nodes.
For the COI and CYB sequences, we created two datasets for each gene: one for phylogenetic analyses and the other for population genetic studies. For phylogenetic analyses, the COI alignment contained sequences of bp. CYB was more difficult to amplify, so the dataset contained only sequences of bp.
However, the ends of some sequences, for both COI and CYBwere shorter because of sequencing difficulties and contained a series of Ns. In both cases, these sites were removed from the datasets for the rest of analyses.
The nuclear fragment N13a non-coding region was used in population genetic analyses but not in phylogenetic inference analyses because its levels of polymorphism were high within the populations studied but low among them and, consequently, there was not enough information for the analyses at this level. Additionally, there were difficulties with amplification of N13 in some individuals and, hence, a single dataset was obtained with sequences of bp.
All nucleotide polymorphism are shown in Table 3. For both genes, a single haplotype was found in 7 of the 11 populations studied. We used the PHASE algorithm to reconstruct the alleles of N13 that were present in the heterozygous individuals in these populations.
The conflicting between the PHASE outcome and the cloning experiments were most likely derived from the assumption of PHASE in which all of the individuals analysed belong to a single panmictic population in Hardy-Weinberg equilibrium, a situation that posterior analyses see below showed not to be the case here.