Research

I work in evolutionary ecology to identify and understand selection pressures on populations. Much of my research focuses on the roles of phenotypic plasticity and local adaptation in explaining life history traits variations according diverse habitats, using amphibians as models (Pelophylax complexes, Rana temporaria), but also nematode parasites used to tack host movements (Ashworthius sidemi, Spiculopteragia spiculoptera, Oesophagostomum sp). Hence, I use an ecological and experimental approaches combined with molecular biology on wild populations in order to study intra-specific variations along a latitudinal gradient, and to compare life history traits among populations originating from different habitats to dissociate local adaptation from phenotypic plasticity.  

Post-Doctoral Project at the University of Reims Champagne-Ardenne (URCA), France: Parasites used as a management tool: tracking nematodes in wild populations of roe deer to infer contacts and map the social network. 

Currently, I’m working in VECPAR team at the University of Reims Champagne-Ardenne (URCA) under the supervision of Hubert Ferté and in collaboration with the team URCA-CERFE on “IN SITU” project, which aim is to study the ecological connectivity of mammals from Champagne-Ardenne region. In “IN SITU”, my mission is to study population structure of parasite community of roe deer (Capreolus capreolus) in order to evaluate direct and indirect (exploitation of the same area at different time) contacts between populations.

Hence, we are particularly interested in two species of parasitic nematodes located in strongyles wild deer and belonging to different taxonomic groups, Spiculoptéragia spiculoptera (Trichostrongylidae) and Oesophagostomum venulosum (Strongylidae). This innovative approach that uses the parasites as biological tracers will be coupled with the study of the population structure of mammalian hosts, and spatial modeling performed by partners of  SITU project, to highlight the existence of biological corridors for each species of mammals in the region.

In parallel, I am involved in a second project led by Hubert Ferté studying parasite prevalence of Alaria alata (Trematode) in water frogs (Pelophylax sp.) and “brown” frogs (Rana sp.). The objective here is to observe the prevalence of this parasite in different species of amphibians infected (taxonomic identification based on morphological and molecular analyzes), but also to assess which stages of these amphibians are the more sensitive to parasitic infestation.

Collaborators: Hubert Ferté (URCA), Damien Jouet (URCA), Rémi Helder (CERFE)

Post-Doctoral Project at the University of Helsinki, Finland: Sex-reversals in the common frog (Rana temporaria) from Lapland.

The common frog (Rana temporaria) is a small amphibian which is widespread in Europe, and it is the only amphibian species that has managed to adapt to subarctic climatic conditions. As such, the northern populations of common frogs – genetically distinct from their southern conspecifics (e.g. Laugen et al. 2001; Palo et al. 2003; Matsuba & Merilä 2006) – can viewed to represent a unique intraspecific level of biodiversity that we should strive to preserve.

Previous studies in EGRU of common frogs in Kilpisjärvi (Lapland, Finland) have revealed peculiar sex ratio dynamics: there are many more adult females than males in these northern populations (Alho 2004, 2008, Merilä, Herczeg & Alho, 2008). Possible explanations for this sex ratio bias include sex differences in early life mortality, and/or sex-reversals caused by some environmental factors. Utilizing sex-linked genetic markers, Alho et al. (2010) compared the genetic-sex with the phenotypic-sex of Kilpisjärvi frogs and observed 9% mis-matches. This result – which evoked great international interest (e.g. Perrin 2010) – supports the hypothesis that sex-reversals are occurring in Kilpisjärvi.

Figure 1: Histological section of 

intersexed gonads. The large 

testicular mass (top) appears 

to be a mature testis, but 

contains scattered  oocytes. 

The ovarian tissue contains 

pre-vitellogenic oocytes and

 scattered testicular tissue 

(Olmstead et al. 2009).

Sex determination mechanisms in vertebrates range from genetic sex determination (GSD; e.g. mammals, birds) involving sex chromosomes, to environmental sex determination (ESD; e.g. many reptiles, few amphibians; Wallace et al. 1999, Sarre et al. 2011). In ESD, the sex of the individual is determined by environmental conditions experienced during early development. However, sex determination of some organisms is under the influence of both genotypic and environmental effects, and their phenotypic sex is determined by the interaction of these two factors (Baroiller et al. 2009). For example, an individual’s genetically determined sex can be environmentally reversed during ontogeny, a phenomenon known as environmental sex reversal (ESR; occurs in many fishes and amphibians; Stelkens & Wedekind 2010).

Environmental factors capable of influencing sex determination include temperature, pH, and environmental contaminants (Figure 1) (Wallace et al. 1999, Hayes et al., 2002, Petterson & Berg, 2007, Olmstead et al. 2009). Even social conditions experienced as juveniles are known to influence sex-determination (Wallace et al. 1999), but clearly, more experiments exploring the occurrence of environmental sex determination in amphibians is needed. 

I joined in 2011 the Ecological Genetics Research Unit (EGRU) at the University of Helsinki to follow this research project on sex-reversal phenomenon of common frogs under the supervision of Pr. Juha Merilä. First, I led a field expedition at the Biological Station of Kilpisjärvi in Northern Lapland (Finland) to monitor the isolated population of common frogs and to collect DNA and tissues samples, in order to evaluate the population situation which was observed between 1998 and 2004, and assess the persistence of sex-reversed individuals, phenomenon already observed in this region. In a second step, I observed age structure, body size and growth range of these common frogs to assess the effect of extreme environmental conditions on the activity of this species. Then, I work on the development of sex-linked molecular markers using reads from Next-Generation-Sequencing analysis to identify the genotypical sex of common frogs. Finally, I contributed to RACE project (Risk Assessment of Chytridiomycosis to European Amphibian Biodiversity), proceeding to the screening of chytrid fungus of these isolated common frogs from Northern Finnish Lapland. 

Collaborators: Juha Merilä (University of Helsinki), Jussi Alho (University of Helsinki), Anssi Laurila (University of Uppsala), Mårten Hjernquist (University of Uppsala), Fredrik Soderman (University of Uppsala).

Articles n°3 and n°4  

PhD thesis at the University of Angers, France: "Hybridization complexes in European water frogs: genetic identification, ecological constraints, and adaptation abilities"

Hybridization is receiving renewed attention as hybrid zones are known to be places where important evolutionary events occur. (Arnold, 1997; Rieseberg, 1997; Barton, 2001; Baack & Rieseberg, 2007). Defined as interbreeding between differentiated lineages or taxa, interspecific hybridization events occur in almost all sexually reproducing groups of organisms (Seehausen 2004, Mallet 2005), and are regular and probably important in nature {Seehausen, 2004). Long-term consequences of hybridization depends on the genetic basis of hybrid fitness, include fusion of previously distinct lineages, extinction of one or both lineages, evolution of reproductive isolation via reinforcement, and production of novel, highly fit hybrid phenotypes (Lippman, 2007). This hybrid vigour is due to the heterosis effect (because of higher heterozygotie rate), which confers advantages to increased genetic diversity (Moore 1977, Tunner and Nopp 1979), and can permit hybrid lineages to colonize new niches or allow speciation through parallel selection pressures and divergent natural selection (Parris et al. 1999, Rieseberg et al. 2003, Rhode and Cruzan 2005, Fitzpatrick and Bradley Shaffer 2007); (Gross and Rieseberg 2005, Robbins et al. 2010). Thus, natural hybridization event is an admixture which can create new phenotypes allowing a great phenotypic plasticity and facilitates adaptive evolution in modified or degraded habitats (Rieseberg et al. 2003, Johansen-Morris and Latta 2008).

Among hybridization events, unisexual hybrids constitute a peculiar case in evolutionary ecology especially interesting to study the adaptation processes. These hybrid lineages are resulting from crosses between differentiated bisexual species, and stabilising through a clonal (parthenogenesis, gynogenesis) or hemiclonal (hybridogenesis) reproductive mode (Vrijenhoek 1993). They are generally thought to be evolutionary ‘dead-ends’ (Maynard Smith 1978, Simon et al. 2003), but the persistence of stable unisexual lineages in numerous independent taxa, sometimes ancient, contradicts this generality (Judson & Normarck, 1996; Normack et al., 2003). As an example, stable unisexual hybridogenetic/bisexual complexes have been observed in fishes (Poeciilidae and Cyprinidae), insect (Phasmatodae and Chironomidae, and amphibians (Ranidae) (Schultz 1969, Berger 1973, Tinti et al. 1995, Collares-Pereira et al. 1999). In these hybridogenetic complexes, gametogenesis is characterized by distortion prior to meiosis leading to genome exclusion of one parental species in the hybrid germ line, and by the production of clonal gametes which only contain the genome from the other parental species without recombination (Schultz 1969). The hybridogen generally reproduce, as a sexual parasite, with the parental species from which the genome is discarded, for maintaining at each generation its lineage (Graf and Polls Pelaz 1989, Beukeboom and Vrijenhoek 1998, Holsbeek et al. 2008). Mating between hybrids produce non-hybrid offspring possessing two hemiclonal genomes, and often die at an early larval stage, because they express recessive deleterious mutations (Binkert et al. 1982, Semlitsch and Reyer 1992, Guex et al. 2002).

  European water frogs (Amphibia: Ranidae) are characterized by several hybridogenetic complexes (Graf and Polls Pelaz 1989). “Pelophylax esculentus complex” is the most widespread and abundant complex, occurs in Central and Eastern Europe, and involve:

            -  the parental species P. ridibundus (genome RR) Pallas, 1771,

            - P. lessonae (genome LL) Camerano, 1882,

            - and the hybridogen P. kl. esculentus (genome RL) Linnaeus, 1758.

These hybrids are characterized by a hybridogenetic  reproduction mode (Schultz 1969):  the genome of P. lessonae is eliminated from the germline prior to meiosis, and the genome of the other parental species, P. ridibundus, is clonally transmitted through gametes to the next hybrid generation. Indeed, the hybridogens, considered as a unisexual organisms, usually coexist with P. lessonae, the sexual parental host species, on which depends the restoration of hybrid lineages (named L-E system) (Uzzell and Berger 1975).

Another hybridogenetic complex occurs in Southern France and Northern Spain, “P. grafi complex” (Graf et al. 1977, Graf and Polls Pelaz 1989, Pagano et al. 2001c, Daf et al. 2006). In the P-G system, analogous to the L-E system:

            - P.kl. grafi (genome RP) is the hybrid between

            - P. perezi Seoane, 1885 (genome PP),

            - and P. ridibundus.

Hybridogenetic lineages produce only haploid gametes with ridibundus genome, and restore at each generation through matings with P. perezi (Graf and Polls Pelaz 1989).

Strongly interesting in evolutive ecology and by the complexity of this group of amphibians, I began my PhD in 2007 in collaboration with two research units, the “Centre de Recherche et de Formation en Eco-Ethologie” (CERFE, France) and “Laboratoire d’études des Systèmes Anthropisés”, Université d’Angers (LEESA, France). I was co-supervised by Alain Pagano, Professor Associated at the University of Angers, and Remi Helder, Research Engineer at CERFE, University of Reims. My PhD focused on these two hybridogenetic complexes of water frogs in the Ardennes (Northeast part of France), in Bretagne and Pays de la Loire (Northwest part of France) and in Northeast part of Spain). Aims of this project were:

            i) to identify the different taxa of these complexes using new molecular tools (Allozymes, PCR-RFLP method on ITS2, microsatellites),

            ii) to analyse of environmental effects on taxonomic distribution (aquatic and terrestrial parameters) and ecological constraints

            iii) and to evaluate habitat effect on life-history traits and larval performances of tadpoles through experimental breeding.

A new PCR-RFLP-based method for an easier systematic affiliation of European water frogs

We developped a non-invasive, PCR- RFLP based method that allows reliable determination of the European water frog species Pelophylax lessonae and P. ridibundus and the hybrid form P. esculentus. Maximum likelihood analysis of ITS2 sequences revealed two robust monophyletic clades corresponding to parental species. Three restriction enzymes (KpnI, HaeII, SmaI) were used to digest three conserved ITS2 domains. Taxonomic identification was unambiguous, the three restriction enymes gave the same results. The protein electrophoretic method of identification was carried out on a French reference sample to test its efficiency. Our new method confirmed circa 83% of identification of the old one. We concluded that difference between identifications was due to introgression.

Collaborators:  Torsten Ohst (Museum für Naturkunde), Jörg Plötner (Museum für Naturkunde)

Article n°1, Mol Ecol Res

Heterosis and clonal genome effects in water frog hybrid complexes: A preliminary attempt at measuring their respective implication.

Unisexual hybrids may combine heterosis and accumulation of deleterious mutations. This contrasting advantages and disadvantages question the fate of these lineages. As waterfrogs constitute hybridogenetic complexes where hemiclonal hybrids occur, we aim to compare the performances of distinct tadpole lineages (hybrid or non hybrid but bearing a clonal genome). In a first step, we compared the fitness-related larval life-history traits (survival and growth rates, time to metamorphosis and mass at metamorphosis) evidencing an intermediate performance of hybridogenetic tadpoles P. kl. grafi in comparison with the parental species. In a second step, we compared the performances of P. ridibundus tadpoles with different genomes: either containing 100% sexual genomes or 50%. No strong differences were observed among lineages, suggesting few effect of mutation accumulation except for the RRg lineage that exhibited a bad performance, may be due to a more ancient clonal history. Our data suggest that P. ridibundus tadpoles are more competitive than other southern water frog, and represent a risk for the diversity of indigenous water frog assemblages in France and Spain. 

Collaborators: Gaston-Denis Guex (University of Zürich), Thomas Uzzell (Academy of Natural Science)

Article n° 6  (Submitted)

Distribution and niche partition

Water frogs from the complex P. esculentus was investigated in the Ardennes department (14 sites) in 2007 and the Maine-et-Loire in 2008 (12 sites) to refine our understanding of the ecological requirements of different taxa (Morand & Joly 1995). The observed pond were located in different habitats and in areas influenced differently (from pond directly connected to the river to the pond in meadow at the top of hill). Each site has been characterized (physicochemical parameters, land use), and frogs were caught momentary to study species composition. Thus, among the 759 individuals captured, the composition was significantly different between two regions, the Ardennes containing essentially P. lessonae and hybrids, while Maine-et-Loire was mainly composed of P. ridibundus and hybrids. We confirmed a differential use of habitat between the parental species of frogs of the complex P. esculentus, supporting the hypothesis of ecological niche partition (Pagano et al., 2001, Plénet et al. 2000), and observed correlations with new factors influencing their abundance: P. ridibundus is present in grassland habitats with a strong river influence, then a short distance from the river, at low altitudes, in water few turbid and low in nitrates; P. lessonae is more present in forest environments with low impact river, at a greater distance from the river and at higher altitude, rather basic pH. In contrast, the hybrid is present in all ponds, sometimes in sympatry with P. ridibundus, sometimes with P. lessonae, or with both, revealing the large ecological valence of this ubiquitous group. Its abundance is correlated to the same parameters as P. lessonae, but it seems to support environments containing nitrates.

Evolutionary success of hybrids: Larval performance variations of water frogs Pelophylax klepton esculentus tadpoles from different habitats

Hybrid zones are known to be places where important evolutionary events occur. Having observed a very broad distribution of hybrids, we investigated intra-specific variability of water frog hybrids in response to their environmental modifications. We examined through six crosses (forest, meadow, or mixed origins) the potential effect of habitat of origin on larval development of Pelophylax klepton esculentus tadpoles. We thus exposed them to 20°C and 26°C rearing conditions, temperatures similar, respectively, to those recorded in a field survey of forest and meadow habitats. Tadpoles raised at 26°C had a better survival and growth rates, a smaller size and quicker metamorphosis than those raised at 20°C. Larval performances varied regarding habitat of origin of tadpoles : forest tadpoles survived better, were smaller and metamorphosed earlier than those from meadow at 20°C. However meadow and mixed tadpoles had a better proportion of survivors at 26°C. P. kl. esculentus tadpoles had different larval performances according to their habitat of origin. Because they exhibited a better survival rate, forest tadpoles had a higher phenotypic plasticity than meadow ones. It suggests a local adaptation of meadow tadpoles to their habitat. Therefore, the evolutionary success of these widespread hybrids might be due to the existence of several different lineages. 

Collaborators: Audrey Chaput-Bary (MNHN Paris), Olivier Pays (University of Angers)

Article n°5 (Submitted)