Understanding the processes that shape the genetic diversity of freshwater planarians (Platyhelminthes, Tricladida, Dugesiidae) = Estudi dels processos que modelen la diversitat genètica en les planàries d'aigua dolça (Platyhelminthes, Tricladida, Dugesiidae)

[eng] The main goal of the present thesis has been to shed light into the processes that shape the genetic diversity of organisms, focussing in the following factors: the type of reproduction, the morphological diversity of genitalia, the karyological diversity, and the paleogeographic and paleoclimatic events. To do so, we have used as model organisms two different genera of freshwater planarians, viz. the genus Schmidtea and several Dugesia species from the Western Mediterranean region, focussing on the species D. subtentaculata. The analysis of the impact of the type of reproduction into the genetic characteristics of D. subtentaculata revealed that asexual fissiparous reproduction generates outstanding levels of intraindividual genetic diversity by the putative accumulation of mutations in the planarian stem cells. Importantly, the obtained results indicate that this intraindividual genetic diversity takes place in a mosaic context, something that has never been reported before. Thus, this genetic effect has been newly described as the mosaic Meselson effect, representing a variation of the well-known Meselson effect but at the mosaic level. Moreover, we have also found evidence that events of occasional sex in fissiparous populations of D. subtentaculata (facultative reproduction) allow the transmission to descendants of different combinations of the alleles generated by the mosaic Meselson effect through processes of segregation and outcrossing. This situation is not only crucial for increasing the genetic diversity between the individuals of fissiparous populations but also may allow fissiparous populations to get rid of the deleterious mutations that have not been eliminated at the intraindividual level. The integrative analysis of the molecular, morphological, and karyological characteristics within D. subtentaculata and between the different Schmidtea species has revealed that, in both groups, the degree of morphological variation in the anatomy of the copulatory apparatus is extremely low compared with the degree of molecular and karyological variation. These results indicate that the anatomy of the copulatory apparatus in planarians might be under a general state of morphological stasis, putatively due to hermaphroditism. Moreover, the high incidence of chromosomic rearrangements that has been found between the different populations of D. subtentaculata linked with their high genetic divergence, suggests that chromosomic rearrangements may play an important role driving speciation processes in planarians, principally in sexual populations showing a diploid karyotype. In agreement with this, the phylogenetic relationships inferred between the different Schmidtea species give support to a previous hypothesis of speciation between S. nova and S. lugubris due to chromosomic rearrangements. Importantly, these results have unveiled the existence of three new species within D. subtentaculata, viz. D. aurea, D. corbata, and D. vilafarrei, which are cryptic at the morphological level but can be perfectly diagnosed by molecular and karyological data. All these results point to the need of reconsidering the taxonomic framework that is currently being used to describe planarian species, which is principally based on morphological characters. Finally, we have found that the paleogeographic history of the Western Mediterranean during the last 30 million years have had a huge impact driving the genetic diversification of the different Dugesia species from this region, either by promoting vicariant events or by connecting previously isolated areas. Moreover, the divergence time estimation performed for Schmidtea points out that the low species richness of this genus together with the high genetic divergence between the different species could be a result of the harsh paleogeographic history of Europe during the Cretaceous period.[spa] El objetivo principal de esta tesis ha sido estudiar los procesos que modelan la diversidad genética de los organismos, centrándonos en el tipo de reproducción, la diversidad morfológica, la diversidad cariológica y los eventos paleogeográficos y paleoclimáticos. Para ello, hemos escogido como organismo modelo dos géneros diferentes de planarias de agua dulce, el género Schmidtea y varias especies de Dugesia de la región del Mediterráneo Occidental, trabajando principalmente con la especie Dugesia subtentaculata. Los resultados obtenidos en el análisis de los efectos genéticos del tipo de reproducción en D. subtentaculata han revelado que la reproducción asexual por fisión genera unos niveles muy elevados de diversidad genética intraindividual, posiblemente a causa de la acumulación de mutaciones en las células madre de las planarias. Es importante señalar que esta diversidad genética intraindividual ocurre en un contexto de mosaicismo, representando una variación del famoso "efecto Meselson". Por lo tanto, hemos descrito este nuevo fenómeno genético como el "efecto Meselson en mosaico". El análisis molecular, morfológico y cariológico de las diferentes poblaciones de D. subtentaculata y de las diferentes especies de Schmidtea ha revelado que, en ambos grupos, el grado de variación morfológica del aparato copulador (que es el principal carácter taxonómico en planarias) es extremadamente bajo en comparación con el grado de variación molecular y cariológica. Estos resultados no solo han respaldado una hipótesis previa de especiación cariológica en Schmidtea, sino que también han revelado la existencia de tres especies crípticas de Dugesia, las cuales han sido descritas bajo los nombres de D. aurea, D. corbata y D. vilafarrei. Todos estos resultados apuntan a la necesidad de reconsiderar el marco taxonómico que se está utilizando actualmente para describir las especies de planarias. Finalmente, los distintos análisis filogeográficos realizados indican que la historia paleogeográfica del Mediterráneo Occidental durante los últimos 30 millones de años ha tenido un gran impacto en la diversificación genética de las diferentes especies del género Dugesia en esta región. En el caso de Schmidtea, hemos visto que tanto la baja riqueza de especies de este género como la alta divergencia genética encontrada entre sus diferentes especies podrían ser el resultado de la historia paleogeográfica de Europa durante el período Cretácico

Outstanding intraindividual genetic diversity in fissiparous planarians (Dugesia, Platyhelminthes) with facultative sex

Background Predicted genetic consequences of asexuality include high intraindividual genetic diversity (i.e., the Meselson effect) and accumulation of deleterious mutations (i.e., Muller's Ratchet), among others. These consequences have been largely studied in parthenogenetic organisms, but studies on fissiparous species are scarce. Differing from parthenogens, fissiparous organisms inherit part of the soma of the progenitor, including somatic mutations. Thus, in the long term, fissiparous reproduction may also result in genetic mosaicism, besides the presence of the Meselson effect and Muller's Ratchet. Dugesiidae planarians show outstanding regeneration capabilities, allowing them to naturally reproduce by fission, either strictly or combined with sex (facultative). Therefore, they are an ideal model to analyze the genetic footprint of fissiparous reproduction, both when it is alternated with sex and when it is the only mode of reproduction

Phylotranscriptomics interrogation uncovers a complex evolutionary history for the planarian genus Dugesia (Platyhelminthes, Tricladida) in the Western Mediterranean

The Mediterranean is one of the most biodiverse areas of the Paleartic region. Here, basing on large data sets of single copy orthologs obtained from transcriptomic data, we investigated the evolutionary history of the genus Dugesia in the Western Mediterranean area. The results corroborated that the complex paleogeological history of the region was an important driver of diversification for the genus, speciating as microplates and islands were forming. These processes led to the differentiation of three main biogeographic clades: Iberia-Apennines-Alps, Corsica-Sardinia, and Iberia-Africa. The internal relationships of these major clades were analysed with several representative samples per species. The use of large data sets regarding the number of loci and samples, as well as state-of-the-art phylogenomic inference methods allowed us to answer different unresolved questions about the evolution of particular groups, such as the diversification path of D. subtentaculata in the Iberian Peninsula and its colonization of Africa. Additionally, our results support the differentiation of D. benazzii in two lineages which could represent two species. Finally, we analysed here for the first time a comprehensive number of samples from several asexual Iberian populations whose assignment at the species level has been an enigma through the years. The phylogenies obtained with different inference methods showed a branching topology of asexual individuals at the base of sexual clades. We hypothesize that this unexpected topology is related to long-term asexuality. This work represents the first phylotranscriptomic analysis of Tricladida, laying the first stone of the genomic era in phylogenetic studies on this taxonomic group

Three dispersal routes out of Africa: A puzzling biogeographical history in freshwater planarians

Aim Freshwater planarians may have a wide geographical range despite their assumed low vagility. Found across four continents, Dugesia may have either an ancient origin on a large palaeo landmass, followed by colonisation in different regions before continental fragmentation, or a more recent origin and subsequent transoceanic dispersal. We seek to resolve between these two hypotheses. Location Africa, Eurasia and Australasia. Taxon Genus Dugesia (Platyhelminthes: Tricladida: Dugesiidae). Methods We used data from the sequencing of six gene fragments and comprehensive taxonomic sampling of Dugesia from across its distribution range to reconstruct the phylogeny of this genus using maximum likelihood and bayesian inference methods. We conducted two phylogenetic dating analyses using Platyhelminthes fossils and palaeogeological events. Basing on the time-calibrated molecular phylogenetic framework we evaluated the contribution of vicariance and dispersal to the biogeographical evolution of Dugesia. By reconstructing the ancestral areas and present-day potential distribution using BioGeoBEARS and niche modelling, we elucidated the biogeographical history of the genus. Results The present-day distribution of Dugesia is a result of different vicariance and dispersal events. However, we also found evidence of transoceanic dispersal. Consistent with previous hypotheses, Dugesia dates to the Upper Jurassic in the Afro-Malagasy Gondwana region. We unveiled a novel biogeographical scenario for the genus, involving multiple events of colonisation in Eurasia from continental Africa via at least three dispersal routes. Main conclusions Dugesia is an ancient genus having reached its present distribution through a complex history of dispersal and vicariant events following its origin in southern Gondwana. Despite the low vagility of Dugesia, we found evidence of their overseas dispersal

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

Data from: Outstanding intraindividual genetic diversity in fissiparous planarians (Dugesia, Platyhelminthes) with facultative sex

Background: Predicted genetic consequences of asexuality include high intraindividual genetic diversity (i.e., the Meselson effect) and accumulation of deleterious mutations (i.e., Muller’s Ratchet), among others. These consequences have been largely studied in parthenogenetic organisms, but studies on fissiparous species are scarce. Differing from parthenogens, fissiparous organisms inherit part of the soma of the progenitor, including somatic mutations. Thus, in the long term, fissiparous reproduction may also result in genetic mosaicism, besides the presence of the Meselson effect and Muller’s Ratchet. Dugesiidae planarians show outstanding regeneration capabilities, allowing them to naturally reproduce by fission, either strictly or combined with sex (facultative). Therefore, they are an ideal model to analyze the genetic footprint of fissiparous reproduction, both when it is alternated with sex and when it is the only mode of reproduction. Results: In the present study, we generate and analyze intraindividual cloned data of a nuclear and a mitochondrial gene of sexual, fissiparous and facultative wild populations of the species Dugesia subtentaculata. We find that most individuals, independently of their reproductive strategy, are mosaics. However, the intraindividual haplotype and nucleotide diversity of fissiparous and facultative individuals is significantly higher than in sexual individuals, with no signs of Muller’s Ratchet. Finally, we also find that this high intraindividual genetic diversity of fissiparous and facultative individuals is composed by different combinations of ancestral and derived haplotypes of the species. Conclusions: The intraindividual analyses of genetic diversity point out that fissiparous reproduction leaves a very special genetic footprint in individuals, characterized by mosaicism combined with the Meselson effect (named in the present study as the mosaic Meselson effect). Interestingly, the different intraindividual combinations of ancestral and derivate genetic diversity indicate that haplotypes generated during periods of fissiparous reproduction can be also transmitted to the progeny through sexual events, resulting in offspring showing a wide range of genetic diversity and putatively allowing purifying selection to act at both intraindividual and individual level. Further investigations, using Dugesia planarians as model organisms, would be of great value to delve into this new model of genetic evolution by the combination of fission and sex

TMED9_Dryad

TMED9 alignment in fasta format used to infer the phylogenetic tre

Cox1_Dryad

Cox1 alignment in fasta format used to infer the phylogenetic tre

Data from: Outstanding intraindividual genetic diversity in fissiparous planarians (Dugesia, Platyhelminthes) with facultative sex

Background: Predicted genetic consequences of asexuality include high intraindividual genetic diversity (i.e., the Meselson effect) and accumulation of deleterious mutations (i.e., Muller’s Ratchet), among others. These consequences have been largely studied in parthenogenetic organisms, but studies on fissiparous species are scarce. Differing from parthenogens, fissiparous organisms inherit part of the soma of the progenitor, including somatic mutations. Thus, in the long term, fissiparous reproduction may also result in genetic mosaicism, besides the presence of the Meselson effect and Muller’s Ratchet. Dugesiidae planarians show outstanding regeneration capabilities, allowing them to naturally reproduce by fission, either strictly or combined with sex (facultative). Therefore, they are an ideal model to analyze the genetic footprint of fissiparous reproduction, both when it is alternated with sex and when it is the only mode of reproduction. Results: In the present study, we generate and analyze intraindividual cloned data of a nuclear and a mitochondrial gene of sexual, fissiparous and facultative wild populations of the species Dugesia subtentaculata. We find that most individuals, independently of their reproductive strategy, are mosaics. However, the intraindividual haplotype and nucleotide diversity of fissiparous and facultative individuals is significantly higher than in sexual individuals, with no signs of Muller’s Ratchet. Finally, we also find that this high intraindividual genetic diversity of fissiparous and facultative individuals is composed by different combinations of ancestral and derived haplotypes of the species. Conclusions: The intraindividual analyses of genetic diversity point out that fissiparous reproduction leaves a very special genetic footprint in individuals, characterized by mosaicism combined with the Meselson effect (named in the present study as the mosaic Meselson effect). Interestingly, the different intraindividual combinations of ancestral and derivate genetic diversity indicate that haplotypes generated during periods of fissiparous reproduction can be also transmitted to the progeny through sexual events, resulting in offspring showing a wide range of genetic diversity and putatively allowing purifying selection to act at both intraindividual and individual level. Further investigations, using Dugesia planarians as model organisms, would be of great value to delve into this new model of genetic evolution by the combination of fission and sex

Microplate tectonics and environmental factors as distribution drivers in Western Mediterranean freshwater planarians

Aim: Species biogeography mainly focuses on palaeogeographical events, while en-vironmental factors are generally overlooked despite their importance in species diversification. Here, we use an integrative approach to understand how palaeogeo-graphical and environmental processes shape species distribution and focus on fresh-water planarians as the model system. Location: Western Mediterranean. Taxon: Dugesia. Methods: We inferred the phylogenetic relationships of most known Dugesia species in the area using six molecular markers. We then estimated their divergence times and reconstructed their ancestral distribution ranges. We also performed environmental niche modelling analyses using Dugesia subtentaculata as a model to evaluate the ef-fects of several hydro-environmental variables and the likely existence of interspecific competition on Dugesia distributions. Results: Our results provide a new phylogenetic scheme for Dugesia from the Western Mediterranean and show that the time splits between the lineages and their putative ancestral distribution ranges are correlated with microplate tectonic dynamics within the region during the Oligocene-Miocene period. Our environmental niche modelling analyses indicate that the type of land cover and the slope of the terrain are the most important abiotic factors driving the distribution of Dugesia from this region. Finally, we found a partial niche overlap between D. subtentaculata and two other common planarian species from the Iberian Peninsula. Main conclusions: The microplate tectonic dynamics of the Western Mediterranean during the Oligocene-Miocene period, together with the position of the mountain ranges and posterior climate changes, may have played crucial roles in driving the biogeographical history of Dugesia in this region. Moreover, both interspecific com-petition and changes in fluvial characteristics driven by human activities may affect the current diversity and distribution of Dugesia in the Western Mediterranean. This study highlights the importance of integrating different types of information to study the biogeographical history of a species