Physiology, gene expression, and epiphenotype of two Dianthus broteri polyploid cytotypes under temperature stress.

Increasing evidence supports a major role for abiotic stress response in the success of plant polyploids, which usually thrive in harsh environments. However, understanding the ecophysiology of polyploids is challenging due to interactions between genome doubling and natural selection. Here, we investigated physiological responses, gene expression, and the epiphenotype of two related Dianthus broteri cytotypes-with different genome duplications (4× and 12×) and evolutionary trajectories-to short extreme temperature events (42/28 °C and 9/5 °C). The 12× cytotype showed higher expression of stress-responsive genes (SWEET1, PP2C16, AI5L3, and ATHB7) and enhanced gas exchange compared with 4×. Under heat stress, both ploidies had greatly impaired physiological performance and altered gene expression, with reduced cytosine methylation. However, the 12× cytotype exhibited remarkable physiological tolerance (maintaining gas exchange and water status via greater photochemical integrity and probably enhanced water storage) while down-regulating PP2C16 expression. Conversely, 4× D. broteri was susceptible to thermal stress despite prioritizing water conservation, showing signs of non-stomatal photosynthetic limitations and irreversible photochemical damage. This cytotype also presented gene-specific expression patterns under heat, up-regulating ATHB7. These findings provide insights into divergent stress response strategies and physiological resistance resulting from polyploidy, highlighting its widespread influence on plant function.

Recurrent allopolyploidizations diversify ecophysiological traits in marsh orchids (Dactylorhiza majalis s.l.).

Whole-genome duplication has shaped the evolution of angiosperms and other organisms, and is important for many crops. Structural reorganization of chromosomes and repatterning of gene expression are frequently observed in allopolyploids, with physiological and ecological consequences. Recurrent origins from different parental populations are widespread among polyploids, resulting in an array of lineages that provide excellent models to uncover mechanisms of adaptation to divergent environments in early phases of polyploid evolution. We integrate here transcriptomic and ecophysiological comparative studies to show that sibling allopolyploid marsh orchid species (Dactylorhiza, Orchidaceae) occur in different habitats (low nutrient fens vs. meadows with mesic soils) and are characterized by a complex suite of intertwined, pronounced ecophysiological differences between them. We uncover distinct features in leaf elemental chemistry, light-harvesting, photoprotection, nutrient transport and stomata activity of the two sibling allopolyploids, which appear to match their specific ecologies, in particular soil chemistry differences at their native sites. We argue that the phenotypic divergence between the sibling allopolyploids has a clear genetic basis, generating ecological barriers that maintain distinct, independent lineages, despite pervasive interspecific gene flow. This suggests that recurrent origins of polyploids bring about a long-term potential to trigger and maintain functional and ecological diversity in marsh orchids and other groups.

Spatially explicit assessment of genetic variation to inform conservation effort for an endangered Mediterranean conifer, Cedrus atlantica.

Preserving the genetic diversity of forest species is critical for maintaining their adaptive potential and allowing for generation turnover in forest ecosystems. Considering an uncertain future, it is necessary to establish reliable genetic conservation strategies to optimize the genetic variation preserved within populations in a spatially explicit context to assist decision-makers. Hence, we aimed to incorporate genetic information into spatially designed conservation actions. Cedrus atlantica is a large, long-lived conifer native to the mountains of North Africa, threatened by extinction. The relevant genetic units for conservation were selected using Bayesian analysis. The relative contribution of the populations to the genetic pool that maximized the species' genetic diversity was calculated to design an optimal seed bank. Finally, the relationship between the genetic composition and bioclimatic variables was estimated and projected throughout the study area under current and future climatic conditions. Three relevant genetic units were found for C. atlantica conservation that maximizes genetic diversity in a spatial context. Bioclimatic variables with the highest influence on genetic composition were closely related to climate warming and decreased soil water availability. We identified the role of genetic markers in designing a reliable conservation strategy for forest trees considering climate change, increased deforestation, and aridity. Projections of genetic composition due to the climate in the study region of North Africa provide spatially explicit guidance for optimizing the selection and preservation of seed banks.

Evaluation of Reference Genes in the Polyploid Complex Dianthus broteri (Caryophyllaceae) Using qPCR.

Dianthus broteri is an endemic complex which is considered the largest polyploid series within the Dianthus genus. This polyploid species involves four cytotypes (2×, 4×, 6× and 12×) with spatial and ecological segregation. The study of gene expression in polyploid species must be very rigorous because of the effects of duplications on gene regulation. In these cases, real-time polymerase chain reaction (qPCR) is the most appropriate technique for determining the gene expression profile because of its high sensitivity. The relative quantification strategy using qPCR requires genes with stable expression, known as reference genes, for normalization. In this work, we evaluated the stability of 13 candidate genes to be considered reference genes in leaf and petal tissues in Dianthus broteri. Several statistical analyses were used to determine the most stable candidate genes: Bayesian analysis, network analysis based on equivalence tests, geNorm and BestKeeper algorithms. In the leaf tissue, the most stable candidate genes were TIP41, TIF5A, PP2A and SAMDC. Similarly, the most adequate reference genes were H3.1, TIP41, TIF5A and ACT7 in the petal tissue. Therefore, we suggest that the best reference genes to compare different ploidy levels for both tissues in D. broteri are TIP41 and TIF5A.

Phenotypic diploidization in plant functional traits uncovered by synthetic neopolyploids in Dianthus broteri.

Whole-genome duplication and post-polyploidization genome downsizing play key roles in the evolution of land plants; however, the impact of genomic diploidization on functional traits still remains poorly understood. Using Dianthus broteri as a model, we compared the ecophysiological behaviour of colchicine-induced neotetraploids (4xNeo) to diploids (2x) and naturally occurring tetraploids (4xNat). Leaf gas-exchange and chlorophyll fluorescence analyses were performed in order to asses to what extent post-polyploidization evolutionary processes have affected 4xNat. Genomic diploidization and phenotypic novelty were evident. Distinct patterns of variation revealed that post-polyploidization processes altered the phenotypic shifts directly mediated by genome doubling. The photosynthetic phenotype was affected in several ways but the main effect was phenotypic diploidization (i.e. 2x and 4xNat were closer to each other than to 4xNeo). Overall, our results show the potential benefits of considering experimentally synthetized versus naturally established polyploids when exploring the role of polyploidization in promoting functional divergence.

Plant Volatile Organic Compounds Evolution: Transcriptional Regulation, Epigenetics and Polyploidy.

Volatile organic compounds (VOCs) are emitted by plants as a consequence of their interaction with biotic and abiotic factors, and have a very important role in plant evolution. Floral VOCs are often involved in defense and pollinator attraction. These interactions often change rapidly over time, so a quick response to those changes is required. Epigenetic factors, such as DNA methylation and histone modification, which regulate both genes and transcription factors, might trigger adaptive responses to these evolutionary pressures as well as regulating the rhythmic emission of VOCs through circadian clock regulation. In addition, transgenerational epigenetic effects and whole genome polyploidy could modify the generation of VOCs' profiles of offspring, contributing to long-term evolutionary shifts. In this article, we review the available knowledge about the mechanisms that may act as epigenetic regulators of the main VOC biosynthetic pathways, and their importance in plant evolution.

Diving in uncharted waters: An updated genetics toolkit highlights the challenges of polyploidy in landscape genomics analyses.

As molecular ecologists, we have by necessity become adept at working across computational platforms. A diverse community of scientists has developed a broad array of analytical resources spanning command line to graphical user interface across Linux, Mac, and Windows environments and a dizzying array of program-specific input formats. In light of this, we often explore our data like free divers - filling our lungs with air and descending for a short period of time into one part of our data set before resurfacing, reformatting, and preparing for our next analysis. In this issue of Molecular Ecology Resources, Meirmans (2020) presents an updated version of GenoDive, a program with a toolkit that provides users with the opportunity to stay a while and delve deeper into the diverse portfolio of information provided by a genomic data set. The comprehensive nature of GenoDive coupled with its unique capability to handle both diploid and polyploid data also provides an opportunity to reflect on the unevenness of resources available for the analysis of polyploid versus diploid data. Since new updates include the addition of plug-ins for genotype-environment association analyses, we limit the observations presented here to the common tools used for landscape genomics analyses.

Early diversification and permeable species boundaries in the Mediterranean firs.

BACKGROUND AND AIMS: Inferring the evolutionary relationships of species and their boundaries is critical in order to understand patterns of diversification and their historical drivers. Despite Abies (Pinaceae) being the second most diverse group of conifers, the evolutionary history of Circum-Mediterranean firs (CMFs) remains under debate. METHODS: We used restriction site-associated DNA sequencing (RAD-seq) on all proposed CMF taxa to investigate their phylogenetic relationships and taxonomic status. KEY RESULTS: Based on thousands of genome-wide single nucleotide polymorphisms (SNPs), we present here the first formal test of species delimitation, and the first fully resolved, complete species tree for CMFs. We discovered that all previously recognized taxa in the Mediterranean should be treated as independent species, with the exception of Abies tazaotana and Abies marocana. An unexpectedly early pulse of speciation in the Oligocene-Miocene boundary is here documented for the group, pre-dating previous hypotheses by millions of years, revealing a complex evolutionary history encompassing both ancient and recent gene flow between distant lineages. CONCLUSIONS: Our phylogenomic results contribute to shed light on conifers' diversification. Our efforts to resolve the CMF phylogenetic relationships help refine their taxonomy and our knowledge of their evolution.

Niche divergence and limits to expansion in the high polyploid Dianthus broteri complex.

Niche evolution in plant polyploids remains controversial and evidence for alternative patterns has been reported. Using the autopolyploid Dianthus broteri complex (2×, 4×, 6× and 12×) as a model, we aimed to integrate three scenarios - competitive exclusion, recurrent origins of cytotypes and niche filling - into a single framework of polyploid niche evolution. We hypothesized that high polyploids would tend to evolve towards extreme niches when low ploidy cytotypes have nearly filled the niche space. We used several ecoinformatics and phylogenetic comparative analyses to quantify differences in the ecological niche of each cytotype and to evaluate alternative models of niche evolution. Each cytotype in this complex occupied a distinct ecological niche. The distributions were mainly constrained by soil characteristics, temperature and drought stress imposed by the Mediterranean climate. Tetraploids had the highest niche breadth and overlap due to their multiple origins, whereas the higher ploidy cytotypes were found in different, restricted, nonoverlapping niches. Niche evolution analyses suggested a scenario with one niche optimum for each ploidy, including the two independent tetraploid lineages. Our results suggest that the fate of nascent polyploids could not be predicted without accounting for phylogenetic relatedness, recurrent origins or the niche occupied by ancestors.

Adaptive sequence evolution is driven by biotic stress in a pair of orchid species (Dactylorhiza) with distinct ecological optima.

The orchid family is the largest in the angiosperms, but little is known about the molecular basis of the significant variation they exhibit. We investigate here the transcriptomic divergence between two European terrestrial orchids, Dactylorhiza incarnata and Dactylorhiza fuchsii, and integrate these results in the context of their distinct ecologies that we also document. Clear signals of lineage-specific adaptive evolution of protein-coding sequences are identified, notably targeting elements of biotic defence, including both physical and chemical adaptations in the context of divergent pools of pathogens and herbivores. In turn, a substantial regulatory divergence between the two species appears linked to adaptation/acclimation to abiotic conditions. Several of the pathways affected by differential expression are also targeted by deviating post-transcriptional regulation via sRNAs. Finally, D. incarnata appears to suffer from insufficient sRNA control over the activity of RNA-dependent DNA polymerase, resulting in increased activity of class I transposable elements and, over time, in larger genome size than that of D. fuchsii. The extensive molecular divergence between the two species suggests significant genomic and transcriptomic shock in their hybrids and offers insights into the difficulty of coexistence at the homoploid level. Altogether, biological response to selection, accumulated during the history of these orchids, appears governed by their microenvironmental context, in which biotic and abiotic pressures act synergistically to shape transcriptome structure, expression and regulation.

Deciphering the ecophysiological traits involved during water stress acclimation and recovery of the threatened wild carnation, Dianthus inoxianus.

Dianthus inoxianus is an endangered species endemic from a small littoral area in the SW Spain, with an unusual flowering season under the adverse conditions of dry Mediterranean summer. A greenhouse experiment was designed to assess the physiological traits involved in drought acclimation and recovery of 3-month-old plants. The evolution of plant water status, leaf gas exchange, chlorophyll fluorescence, photosynthetic pigments concentrations and a quantitative analysis of photosynthesis limitations were followed during water stress and re-watering. Our results indicated that the plant water status, Ψw and RWC, only decreased at the end of the drought period (18th day), together with the net photosynthetic rate, AN. Photosynthetic impair was mainly caused by diffusional limitations (SL and MCL) of CO2, as indicated the joint and marked decrease of gs, gm and Ci during drought period, while Vc,max did not vary. After rewatering, leaf water status recovered faster than photosynthetic one, reaching control values on day 1 after recovery, while AN, gm and Ci took 7 days. Additionally, gs showed the slowest recovery taking 15 days, but gs decrease was enough to keep Ψw and RWC at constant values throughout the experiment. Results suggest a high tolerance and recovery of D. inoxianus from severe drought periods. This drought tolerance was also reflected in the stability of its photochemical apparatus and pigments concentrations, as indicated the constant values of Fv/Fm, ФPSII and pigments concentrations through experimental period. However, prolonged drought events due to global climate change could negatively affect the physiological mechanisms of this species.

Epigenetic contribution to successful polyploidizations: variation in global cytosine methylation along an extensive ploidy series in Dianthus broteri (Caryophyllaceae).

Polyploidization is a significant evolutionary force in plants which involves major genomic and genetic changes, frequently regulated by epigenetic factors. We explored whether natural polyploidization in Dianthus broteri complex resulted in substantial changes in global DNA cytosine methylation associated to ploidy. Global cytosine methylation was estimated by high-performance liquid chromatography (HPLC) in 12 monocytotypic populations with different ploidies (2×, 4×, 6×, 12×) broadly distributed within D. broteri distribution range. The effects of ploidy level and local variation on methylation were assessed by generalized linear mixed models (GLMMs). Dianthus broteri exhibited a higher methylation percent (˜33%) than expected by its monoploid genome size and a large variation among study populations (range: 29.3-35.3%). Global methylation tended to increase with ploidy but did not significantly differ across levels due to increased variation within the highest-order polyploidy categories. Methylation varied more among hexaploid and dodecaploid populations, despite such cytotypes showing more restricted geographic location and increased genetic relatedness than diploids and tetraploids. In this study, we demonstrate the usefulness of an HPLC method in providing precise and genome reference-free global measure of DNA cytosine methylation, suitable to advance current knowledge of the roles of this epigenetic mechanism in polyploidization processes.

Phylogeography above the species level for perennial species in a composite genus.

In phylogeography, DNA sequence and fingerprint data at the population level are used to infer evolutionary histories of species. Phylogeography above the species level is concerned with the genealogical aspects of divergent lineages. Here, we present a phylogeographic study to examine the evolutionary history of a western Mediterranean composite, focusing on the perennial species of Helminthotheca (Asteraceae, Cichorieae). We used molecular markers (amplified fragment length polymorphism (AFLP), internal transcribed spacer and plastid DNA sequences) to infer relationships among populations throughout the distributional range of the group. Interpretation is aided by biogeographic and molecular clock analyses. Four coherent entities are revealed by Bayesian mixture clustering of AFLP data, which correspond to taxa previously recognized at the rank of subspecies. The origin of the group was in western North Africa, from where it expanded across the Strait of Gibraltar to the Iberian Peninsula and across the Strait of Sicily to Sicily. Pleistocene lineage divergence is inferred within western North Africa as well as within the western Iberian region. The existence of the four entities as discrete evolutionary lineages suggests that they should be elevated to the rank of species, yielding H. aculeata, H. comosa, H. maroccana and H. spinosa, whereby the latter two necessitate new combinations.

Big thistle eats the little thistle: does unidirectional introgressive hybridization endanger the conservation of Onopordum hinojense?

Hybridization is known to have a creative role in plant evolution. However, it can also have negative effects on parental species. Onopordum is a large genus whose species frequently hybridize. In the Southwest Iberian Peninsula, the rare O. hinojense co-occurs with the widely distributed O. nervosum, and hybrids between these two taxa have been described as O. × onubense. In this study we determine the extinction risk in a hybrid zone, both for hybrids and parentals, using analyses of morphological and cytogenetic traits as well as genetic markers and demographic models. To investigate the introgression process we used amplified fragment length polymorphism (AFLP) markers, Bayesian analyses and genome scan methods. Morphology, genome size and molecular markers confirmed homoploid hybridization and also indicated unidirectional backcrossing of F1 hybrids with O. nervosum, which is likely to swamp O. hinojense, the parental with lower pollen size and a very low fruit set (8%). Genome scan methods revealed several loci significantly deviating from neutrality. Finally, our demographic modeling indicated that the higher fitness of O. nervosum threats the survival of O. hinojense by demographic swamping. Our study provides strong new evidence for a scenario of rapid extinction by unidirectional introgression and demographic swamping. The multifaceted approach used here sheds new light on the role of introgression in plant extinctions.

Effects of tree architecture on pollen dispersal and mating patterns in Abies pinsapo Boiss. (Pinaceae).

Plant architecture is crucial to pollination and mating in wind-pollinated species. We investigated the effect of crown architecture on pollen dispersal, mating system and offspring quality, combining phenotypic and genotypic analyses in a low-density population of the endangered species Abies pinsapo. A total of 598 embryos from three relative crown height levels (bottom, middle and top) in five mother plants were genotyped using eleven nuclear microsatellite markers (nSSRs). Paternity analysis and mating system models were used to infer mating and pollen dispersal parameters. In addition, seeds were weighed (N = 16 110) and germinated (N = 736), and seedling vigour was measured to assess inbreeding depression. Overall, A. pinsapo shows a fat-tailed dispersal kernel, with an average pollen dispersal distance of 113-227 m, an immigration rate of 0.84-26.92%, and a number of effective pollen donors (Nep ) ranging between 3.5 and 11.9. We found an effect of tree height and relative crown height levels on mating parameters. A higher proportion of seeds with embryo (about 50%) and a higher rate of self-fertilization (about 60%) were found at the bottom level in comparison with the top level. Seed weight and seedling vigour are positively related. Nevertheless, no differences were found in seed weight or in seedling-related variables such as weight and length of aerial and subterranean parts among the different relative crown height levels, suggesting that seeds from the more strongly inbred bottom level are not affected by inbreeding depression. Our results point to vertical isotropy for outcross-pollen and they suggest that self-pollen may ensure fertilization when outcross-pollen is not available in low-density population.

Do chromosome numbers reflect phylogeny? New counts for Bombacoideae and a review of Malvaceae s.l.

UNLABELLED: • PREMISE OF THE STUDY: Whole genome duplication (WGD) and specific polyploidy events marked turning points for angiosperm genome structure and evolution. Therefore, cytogenetic studies of polyploidy-prone groups such as the tropical Malvaceae and plant formations such as as the Brazilian Cerrado have gained further importance. We present new chromosome counts for Cerrado Bombacoideae and revised chromosome numbers for the Malvaceae s.l., compare these between subfamilies, and relate them to phylogenetic signal.• METHODS: We studied the chromosome number of Eriotheca candolleana, E. gracilipes, E. pubescens, Pachira glabra, Pseudobombax longiflorum, and P. tomentosum. We also compared Eriotheca species ploidy levels using flow cytometry. We compiled chromosome numbers for 557 species of Malvaceae s.l., including 37 Bombacoideae species. We included this information in a phylogenetic reconstruction based on chloroplast matK-trnK DNA to evaluate chromosome evolution of the Malvaceae s.l. and the Bombacoideae in particular.• KEY RESULTS: The Cerrado Bombacoideae presented consistently high chromosome numbers. Numbers for Eriotheca species were among the highest and varied among populations. Flow cytometry analyses showed similar 1Cx DNA for all cytotypes and indicated neopolyploidy. Chromosome numbers differed between subfamilies, with the lowest numbers in the Malvoideae and Byttnerioideae and the highest in Tilioideae. Chromosome numbers had significant phylogenetic signal for Bombacoideae but not for Malvoideae or Malvaceae s.l.• CONCLUSIONS: Clearly distinct chromosome numbers allied to monophyly provide some support for a circumscription of the Bombacoideae and distinction within the Malvaceae. The phylogenetic signal for chromosome number supports the idea of an ancient WGD and further neopolyploidy events as important evolutionary trends for the Bombacoideae.

Phylogeography of SW Mediterranean firs: different European origins for the North African Abies species.

The current distribution of Western Mediterranean Abies species is a result of complex geodynamic processes and climatic oscillations that occurred in the past. Abies sect. Piceaster offers a good study model to explore how geo-climatic oscillations might have influenced its expansion and diversification on both sides of the W Mediterranean basin. We investigated the genetic variation within and among nine populations from five Abies species by molecular markers with high and low mutation rates and contrasting inheritance (AFLP and cpSSR). Analyses revealed the opening of the Strait of Gibraltar as an effective barrier against gene flow between the Southern Iberian (A. pinsapo) and North African (A. marocana and A. tazaotana) firs. The A. pinsapo populations in Spain and likewise those of the A. marocana - A. tazaotana population complex were not differentiated, and no evidence was found to distinguish A. tazaotana at the species level. Diversification of Abies across North Africa could occur by way of at least two vicariant events from Europe, in the west, giving rise to the A. marocana - A. tazaotana complex, and in the east, giving A. numidica. Secondary contacts among species from Abies sect. Piceaster (A. pinsapo and A. numidica), and with A. alba (Abies sect. Abies) are also indicated. However, there is a closer relationship between the Algerian fir (A. numidica) and the North Mediterranean widespread A. alba, than with the Moroccan firs (A. marocana and A. tazaotana) or the Southern Iberian (A. pinsapo). We also discuss the distribution range of these taxa in its paleogeological and paleoclimatic context, and propose that part of the modern geography of the South-Western Mediterranean firs might be traced back to the Tertiary.

AFLPsim: an R package to simulate and detect dominant markers under selection in hybridizing populations.

BACKGROUND: In spite of a large diversity of approaches to investigate loci under selection from a population genetic perspective, very few programs have been specifically designed to date to test selection in hybrids using dominant markers. In addition, simulators of dominant markers are very scarce and they do not usually take into account hybridization. RESULTS: Here, we present a new, multifunctional, R package for dominant genetic markers, AFLPsim. This package can simulate dominant markers in hybridizing populations and implements genome scan methods for detecting outlier dominant loci in hybrids. In addition, it includes tools for further manipulating the results, plotting them and other tasks. We describe and tabulate the major functions implemented in AFLPsim. In addition, we provide some demonstration of its use and we perform a comparative study with other software. Finally, we conclude by briefly describing the input and output formats. CONCLUSIONS: The R package AFLPsim application provides several useful tools in the context of hybridization studies. It can simulate dominant markers in hybridizing populations and predict their demographic evolution. In addition, we implement a new genome scan method for detecting outlier dominant loci in hybrids, which shows a rather high sensitivity and is very conservative in comparison with Gagnaire et al.'s, Bayescan and introgress. The application is downloadable at http://cran.r-project.org/web/packages/AFLPsim/.

Nuclear microsatellite primers for the endangered relict fir, Abies pinsapo (Pinaceae) and cross-amplification in related Mediterranean Species.

Twelve nuclear microsatellite primers (nSSR) were developed for the endangered species Abies pinsapo Boiss. to enable the study of gene flow and genetic structure in the remaining distribution areas. Microsatellite primers were developed using next-generation sequencing (454) data from a single Abies pinsapo individual. Primers were applied to thirty individuals from the three extant localities. The number of alleles per locus ranged from one to four. Cross-amplification was tested for other Abies species from the Mediterranean Basin, and most of the loci showed higher polymorphisms in the Mediterranean species than in A. pinsapo. These microsatellite markers provide tools for conservation genetic studies in Abies pinsapo as well other Abies species from the Mediterranean Basin.

Phylogeography of Trichuris populations isolated from different Cricetidae rodents.

The phylogeography of Trichuris populations (Nematoda) collected from Cricetidae rodents (Muroidea) from different geographical regions was studied. Ribosomal DNA (Internal Transcribed Spacers 1 and 2, and mitochondrial DNA (cytochrome c- oxidase subunit 1 partial gene) have been used as molecular markers. The nuclear internal transcribed spacers (ITSs) 1 and 2 showed 2 clear-cut geographical and genetic lineages: one of the Nearctic region (Oregon), although the second was widespread throughout the Palaearctic region and appeared as a star-like structure in the minimum spanning network. The mitochondrial results revealed that T. arvicolae populations from the Palaearctic region were separated into 3 clear-cut geographical and genetic lineages: populations from Northern Europe, populations from Southern (Spain) and Eastern Europe (Croatia, Belarus, Kazahstan), and populations from Italy and France (Eastern Pyrénean Mountains). Phylogenetic analysis obtained on the basis of ITS1-5·8S-ITS2 rDNA sequences did not show a differential geographical structure; however, these markers suggest a new Trichuris species parasitizing Chionomys roberti and Cricetulus barabensis. The mitochondrial results revealed that Trichuris populations from arvicolinae rodents show signals of a post-glacial northward population expansion starting from the Pyrenees and Italy. Apparently, the Pyrenees and the Alps were not barriers to the dispersal of Trichuris populations.