Organisers:

Supported by:

Abstract

The French Act of 11 February 2005 on equal rights and opportunities, participation and citizenship of people with disabilities highlights the need "for people with disabilities or reduced mobility" to have access to the complete mobility chain of the transport system. This law was initially planned to apply from 2015. Many efforts have been made to improve life for those with physical, visual and, to a lesser extent, hearing impairments. But there is a lot of room for improvement in situations regarding psychological, cognitive and mental impairment. This is largely due to a lack of knowledge on the difficulties experienced by the above when they travel.

A qualitative methodology called 'commented walks' (Thibaud, 2001) is employed by urbanists to investigate the quality of the urban environment using videos. This paper will show how this method is also useful to complete quantitative approaches and to point out the difficulties encountered by people with disabilities in the urban space.

The main recommendations from this research are: -need for a coherent and foolproof continuity of informational systems (staking, numbering of buildings in the street, bus destination displays...) -necessity to minimize the 'affordances' (Gibson, 1977) that might become highly disruptive cognitive attractors (Chevrier, Juguet, 2003) (i.e. advertising, illuminated signs that divert the focus of attention ...).

-need for a simplification and systematization of the cognitive procedures required to reach a destination.

-need for staff training : human assistance remains essential and can't be replaced by any automatons.

The videos resulting from the 'commented walk' methodology are important, firstly to explore the difficulties experienced, then to inform the necessary authorities and finally to feed into training programs designed for logistic officers or bus drivers.

The commented walk method as a way of highlighting precise daily mobility difficulties.

A case study focusing on cognitive or mental diseases.

Joël Meissonnier, CEREMA Dter Nord-Picardie, Lille, France. Virginie Dejoux, Université Paris 1, CRIDUP, Paris, France.

The commented walk method

A qualitative methodology called 'commented walks' (Thibaud, 2001) is used by urbanists to investigate the quality of urban environment using either photos or videos. This method was popularized in France, by the Research Center on Sonic space & Urban environment (CRESSON research laboratory -School of Architecture of Grenoble). It relies on the "thinking aloud" technique devised by Alan Newell and Herbert Simon (1972) in the US. «This is a journey with a real-time storytelling» (Miaux, 2008). The "commented walks" methodology leads to a very precise analysis of mobility choices process.

We were looking for a method to give us a better understanding of mobility behaviours, especially with people with disabilities. We were also looking more precisely for a method suitable for participants with verbal or non structured verbal difficulties. So, we carried out 4 'commented walks' in the city of Amiens in order to test the methodology: Marie and Etienne have trisomy 21 ; Fabien has an intellectual disability, he is illiterate but knows how to read figures ; Marion has a dyspraxia.

As the goal is for us to study mobility in urban environments we have adapted the methodological protocol according to Chevrier and Juguet's advices. As Marie, Fabien and Marion seemed relatively autonomous on public transport, we asked them to add slight challenges.

-In the morning Marie was going to her workplace with her father by car. She was returning alone, by bus. We suggested she tried an outward journey by public transport for the first time.

-Fabien seemed really capable of dealing with public transport in his daily routine. So we asked him to go to the zoo, a place he was not familiar with.

-Marion has excellent communication skills. One day she got lost in Amiens, so we asked her to try to find the path she had taken that day.

According to Chevrier & Juguet's work (2003), such a script is an in situ test. It aims to ask the travelers to face realistically a set of situations which forces them to adjust their action plans. Typically, people in public transport run on autopilot. Guided by their habits, they follow some procedures without questioning their practices. These practices become automatic. Indeed our films show clearly that a fictitious situation causes a break in the routine action chain. And they show how people face more unexpected events and less known routes.

Disruptive cognitive attractors : adverts

The presence of advertisements on the road contributed to Marion's confusion. They distracted her from looking for relevant informations.

An information discontinuity : one bus line and several final destinations.

Marie couldn't know the final destination because she had arrived from the back of the bus where there was only the bus number on display.

Too complex cognitive procedures to reach a destination

Marion doesn't bother with city maps because for her, most of the city maps require too many complex cognitive procedures to reach a destination. She tried to explain to us that the locations of city maps are badly placed. Usually located on the back of advertising hoardings, these geocentric local maps should permit a simple cognitive projection on the ground with north directly in front of the user thereby no longer requiring an additional cognitive operation such as turning in order to face north.

Human assistance : some needs for trainings

In Amiens's main interchange bus station, Fabien spontaneously questioned an employee of the transport company how to get to the zoo. The employee advised Fabien to take bus No. 1. Unfortunately he didn't think of indicating either the right direction and final destination or the location of the bus stop. This employee was probably lacking training.


A A T Weber
email: ale.weber@unibas.ch

L Abi-Rached

Nicolas Galtier

A Bernard

J I Montoya-Burgos

A Chenuil

A A -T . W E B E R

L A B I -R A C H E D

N G A L T I E R

A B E R N A R D

J I M O N T O Y A -B U R G O S

A C H E N U I L

-T Weber

Positive selection on sperm ion channels in a brooding brittle star: consequence of life-history traits evolution

Keywords: ion channel, marine invertebrate, reproductive isolation, sperm chemotaxis, sperm competition, transcriptome

Positive selection on sperm ion channels in a brooding brittle star: consequence of life-history traits evolution

Introduction

Understanding mechanisms leading to the formation of new species is a fundamental question in evolutionary biology. In particular, how reproductive barriers evolve, and what the genetic basis of barriers to gene flow is, remain enigmatic for many nonmodel organisms [START_REF] Noor | Speciation genetics: evolving approaches[END_REF][START_REF] Nosil | The genes underlying the process of speciation[END_REF][START_REF] Ellegren | Genome sequencing and population genomics in non-model organisms[END_REF]). Thanks to their recent divergence, closely related species are fascinating models to study the mechanisms leading to reproductive isolation and ultimately speciation [START_REF] Seehausen | Genomics and the origin of species[END_REF]. It is generally expected that while global genetic divergence between recently diverged species will be low, genes involved in reproductive isolation, also called 'speciation genes', will display higher levels of differentiation [START_REF] Nosil | The genes underlying the process of speciation[END_REF][START_REF] Wiley | Genomic divergence during speciation: causes and consequences[END_REF].

When different species live in the same geographic location (i.e. in sympatry), mechanisms preventing genetic exchange between species can occur before or after the fusion of gametes. Therefore, these mechanisms are classified as prezygotic or postzygotic factors of reproductive isolation, respectively. While postzygotic isolation leads to death or sterility of the newly formed embryos due to genomic incompatibilities (i.e. Bateson-Dobzhansky-Muller incompatibilities), prezygotic isolation can include, for instance, temporal prezygotic isolation, where reproduction periods are not overlapping. In marine invertebrates, where the sense of sight is limited to photoreception, mechanisms of species recognition are mostly mediated through chemical signals [START_REF] Palumbi | Genetic divergence, reproductive isolation, and marine speciation[END_REF]. For instance, there can be active avoidance of nonconspecific males and females, via chemical signals, to avoid interspecific mating [START_REF] Hendler | Reproduction of Marine Invertebrates[END_REF][START_REF] Bickford | Cryptic species as a window on diversity and conservation[END_REF][START_REF] Mercier | Advances in Marine Biology: Endogenous and Exogenous Control of Gametogenesis and Spawning in Echinoderms[END_REF]. Finally, when closely related species occur in sympatry, genes involved in gamete recognition can evolve under positive diversifying selection and display increased genetic differentiation, therefore preventing the recognition of gametes belonging to different species and their fusion [START_REF] Lessios | Speciation genes in free-spawning marine invertebrates[END_REF][START_REF] Vacquier | Selection in the rapid evolution of gamete recognition proteins in marine invertebrates[END_REF].

The most famous examples of gamete recognition proteins are the sperm protein bindin and its egg receptor EBR1 in sea urchins, and the sperm protein lysin and its egg receptor VERL in gastropods. The bindin is a protein involved in the recognition between the sperm and the egg vitelline envelope, as well as in the fusion of sperm and egg membranes. In contrast, the lysin is only involved in recognition and dissolution of the vitelline envelope, the fusion of sperm and egg membrane being mediated by the protein sp18 [START_REF] Lessios | Speciation genes in free-spawning marine invertebrates[END_REF][START_REF] Vacquier | Selection in the rapid evolution of gamete recognition proteins in marine invertebrates[END_REF]. Positive selection frequently targets these systems, as was shown for the bindin protein in sea urchins of the genus Strongylocentrotus, Paracentrotus, Heliocidaris and Echinometra [START_REF] Lessios | Speciation genes in free-spawning marine invertebrates[END_REF], as well as in sea stars [START_REF] Sunday | Sea star populations diverge by positive selection at a sperm-egg compatibility locus[END_REF][START_REF] Patiño | Sperm bindin divergence under sexual selection and concerted evolution in sea stars[END_REF], and for the egg receptor for sperm Obi1 [START_REF] Hart | Structure and evolution of the sea star egg receptor for sperm bindin[END_REF][START_REF] Hart | Incipient speciation of sea star populations by adaptive gamete recognition coevolution[END_REF]. Similarly, the proteins involved at the level of egg jelly and in particular their carbohydrate-recognition domains (suREJ) were found to be under positive diversifying selection in six species of sea urchins [START_REF] Mah | Positive selection in the carbohydrate recognition domains of sea urchin sperm receptor for egg jelly (suREJ) proteins[END_REF]. This receptor interacts with sulphated polysaccharides to induce the acrosome reaction, polysaccharides which also display structural changes between closely related species of Strongylocentrotus sea urchins [START_REF] Biermann | Carbohydrate-based species recognition in sea urchin fertilization: another avenue for speciation?[END_REF]. Prezygotic isolation through gamete recognition therefore seems to be the rule in sea urchins and likely sea stars, but whether this mechanism is general to echinoderms, and more generally to marine invertebrates, is still unclear.

With more than 2000 extant species, brittle stars (Ophiuroidea) form the largest class of echinoderms. Brittle stars occur in virtually every marine environment, from very shallow to abyssal depths, and from tropical to Arctic and Antarctic areas [START_REF] St€ Ohr | Global diversity of brittle stars (Echinodermata: Ophiuroidea)[END_REF]. Interestingly, this class includes numerous complexes of cryptic species, among which morphological characters are poorly or not (yet) differentiated [START_REF] Baric | Ecological parallelism and cryptic species in the genus Ophiothrix derived from mitochondrial DNA sequences[END_REF][START_REF] Sponer | Phylogeographic analysis of the brooding brittle star Amphipholis Squamata (Echinodermata) along the coast of New Zealand reveals high cryptic genetic variation and cryptic dispersal potential[END_REF][START_REF] O'hara | The Ophiocoma erinaceus complex: another case of cryptic speciation in echinoderms. Echinoderms: M€ unchen[END_REF][START_REF] Muths | Incomplete cryptic speciation between intertidal and subtidal morphs of Acrocnida brachiata (Echinodermata: Ophiuroidea) in the Northeast Atlantic[END_REF][START_REF] Muths | Large-scale genetic patchiness among NE Atlantic populations of the brittle star Ophiothrix fragilis[END_REF][START_REF] Boissin | Defining reproductively isolated units in a cryptic and syntopic species complex using mitochondrial and nuclear markers: the brooding brittle star, Amphipholis squamata (Ophiuroidea)[END_REF][START_REF] Boissin | Did vicariance and adaptation drive cryptic speciation and evolution of brooding in Ophioderma longicauda (Echinodermata: Ophiuroidea), a common Atlanto-Mediterranean ophiuroid?[END_REF][START_REF] Hunter | Evaluating connectivity in the brooding brittle star Astrotoma agassizii across the Drake passage in the Southern Ocean[END_REF][START_REF] St€ Ohr | Potential cryptic speciation in Mediterranean populations of Ophioderma (Echinodermata: Ophiuroidea)[END_REF][START_REF] Heimeier | Molecular species identification of Astrotoma agassizii from planktonic embryos: further evidence for a cryptic species complex[END_REF][START_REF] St€ Ohr | Morphological diagnosis of the two genetic lineages of Acrocnida brachiata (Echinodermata: Ophiuroidea), with description of a new species[END_REF][START_REF] Hoareau | The Southwestern Indian Ocean as a potential marine evolutionary hotspot: perspectives from comparative phylogeography of reef brittle-stars[END_REF][START_REF] Erez-Portela | Cryptic speciation and genetic structure of widely distributed brittle stars (Ophiuroidea) in Europe[END_REF][START_REF] Naughton | Antitropical distributions and species delimitation in a group of ophiocomid brittle stars (Echinodermata: Ophiuroidea: Ophiocomidae)[END_REF][START_REF] De Moura Barboza | Brittle stars from the Saint Peter and Saint Paul Archipelago: morphological and molecular data[END_REF][START_REF] Taboada | Contrasted phylogeographic patterns on mitochondrial DNA of shallow and deep brittle stars across the Atlantic-Mediterranean area[END_REF].

The brittle star cryptic species complex Ophioderma longicauda is composed of six divergent mitochondrial lineages displaying contrasting reproductive strategies (broadcast spawners and internal brooders; [START_REF] St€ Ohr | Potential cryptic speciation in Mediterranean populations of Ophioderma (Echinodermata: Ophiuroidea)[END_REF][START_REF] Boissin | Did vicariance and adaptation drive cryptic speciation and evolution of brooding in Ophioderma longicauda (Echinodermata: Ophiuroidea), a common Atlanto-Mediterranean ophiuroid?[END_REF]. This species complex, encompassing at least three distinct biological species, occurs in the East Atlantic Ocean and in the Mediterranean Sea. In the so-called broadcast spawners (the ancestral strategy), reproduction occurs once a year, fertilization is external and larvae experience a dispersive pelagic phase of about 6 days [START_REF] Fenaux | Le d eveloppement larvaire chez Ophioderma longicauda (Retzius)[END_REF][START_REF] Fenaux | Evolution saisonni ere des gonades chez l'Ophiure Ophioderma longicauda (Retzius), Ophiuroidea[END_REF]. In contrast, in the brooding species (the derived strategy), fertilization is presumably internal, embryos develop for several weeks in female-specific pouches (bursae; St€ ohr et al. 2009), and dispersal occurs only at the adult stage. The two reproductive systems presumably imply contrasted selective pressures at the gametic stage, which might impact the mechanism and evolution of prezygotic isolation. Here, we focused on two sympatric species from the O. longicauda complex, named temporarily C3 (broadcast spawner) and C5 (internal brooder) until formal description is completed (S. St€ ohr, A.A.-T. Weber, E. Boissin, A. Chenuil, in preparation). These two species, in addition to displaying different reproductive strategies, do not reproduce at the same time of the year and display genetic, ecological, physiological and morphological differences [START_REF] Weber | Thermotolerance and regeneration in the brittle star species complex Ophioderma longicauda: a preliminary study comparing lineages and Mediterranean basins[END_REF][START_REF] Weber | Genetic data, reproduction season and reproductive strategy data support the existence of biological species in Ophioderma longicauda[END_REF][START_REF] Weber | Influence of the larval phase on connectivity: strong differences in the genetic structure of brooders and broadcasters in the Ophioderma longicauda species complex[END_REF]. There is currently no gene flow between C3 and C5, and their divergence time is estimated to about 550 000 years [START_REF] Weber | Etude ecologique et g en etique du complexe d'esp eces cryptiques Ophioderma longicauda (Ophiuroidea: Echinodermata): Comparaison entre lign ees incubantes et lign ees produisant des larves planctoniques[END_REF]. Thus, this pair of closely related species provides an interesting model system to investigate reproductive isolation and genome differentiation. We used transcriptome sequencing and de novo assembly to compare the genetic diversity and investigate the role of natural selection in the reproductive isolation between O. longicauda C3 and C5. In contrast with sea urchins, we found that in O. longicauda, proteins associated to gamete recognition are not particularly fast evolving. Yet, we identified two genes associated to sperm motility, which were positively selected in the brooding species.

Material and methods

Individual sampling, lineage typing and RNA extraction

Sampling was performed in four localities across the Mediterranean and the Atlantic (Madeira, Greece, Portugal and France; Table S1, Supporting information). For each individual, gonads, embryos (when present) and a piece of arm were preserved in RNA later and later used for RNA extraction (Table S1, Supporting information); a piece of arm was also preserved in EtOH 95% for DNA extraction using a Chelex protocol [START_REF] Walsh | Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material[END_REF]. Species delimitation was performed according to the mitochondrial lineages described by [START_REF] Boissin | Did vicariance and adaptation drive cryptic speciation and evolution of brooding in Ophioderma longicauda (Echinodermata: Ophiuroidea), a common Atlanto-Mediterranean ophiuroid?[END_REF]. For each specimen, a COI gene fragment was PCR-amplified (forward primer: 5 0 CTT GCA GGA GGA GGA GAY CC 3 0 ; reverse primer: 5 0 AGT ATA AGC GTC TGG GTA GTC 3 0 ) and Sangersequenced (LGC Genomics, Germany). Individuals from Madeira, Sagres and Marseille belonged to the broadcasting species C3 (mitochondrial lineage L1 in [START_REF] Boissin | Did vicariance and adaptation drive cryptic speciation and evolution of brooding in Ophioderma longicauda (Echinodermata: Ophiuroidea), a common Atlanto-Mediterranean ophiuroid?[END_REF]), whereas individuals from Crete represented species C3 and C5 (mitochondrial lineages L1 and L3, respectively), in line with the species distribution described by [START_REF] Weber | Influence of the larval phase on connectivity: strong differences in the genetic structure of brooders and broadcasters in the Ophioderma longicauda species complex[END_REF]. To illustrate the presence of two distinct species, phylogenetic trees for COI and for the nuclear marker EF1a (Elongation factor 1a; retrieved from the transcriptomes generated in this study) are available in Fig. S1, Supporting information. RNA extractions were performed using a combination of standard guanidium thiocyanate-phenol-chloroform (GTPC) RNA extraction followed by an extra purification step with a RNeasy kit for RNA extractions (Qiagen). Both methods were used in combination with maximize quantity and purity of RNA, as recommended by [START_REF] Gayral | Next-generation sequencing of transcriptomes: a guide to RNA isolation in nonmodel animals[END_REF]. Twelve libraries for RNA sequencing were prepared (Table S1, Supporting information); seven with RNA from unique individuals (two from species C5 and five from species C3) and five with RNA from pooled individuals from four different localities (C5-Crete, C3-Crete, C3-France, C3-Madeira, C3-Portugal). Pools of individuals were performed using the same quantity of RNA per individual.

Sequencing and assembly

Library preparation and sequencing were performed by the genomic platform Genotoul (Toulouse). Libraries were constructed using TruSeq library preparation kit (Illumina) following manufacturer's instructions. Sequencing was performed with a Hiseq 2000 sequencer (Illumina). The complete bioinformatics pipeline used in this study is described in [START_REF] Gayral | Reference-free population genomics from next-generation transcriptome data and the vertebrate-invertebrate gap[END_REF]. For this assembly, only the individual libraries were used, to calculate the per-species nucleotide diversity and the individual heterozygosities. Briefly, raw reads were filtered for length (≥60 bp) and quality (Phred score ≥30). De novo assembly was performed using Abyss [START_REF] Simpson | ABySS: a parallel assembler for short read sequence data[END_REF]) and Cap3 [START_REF] Huang | CAP3: a DNA sequence assembly program[END_REF], according to the method B described in [START_REF] Cahais | Reference-free transcriptome assembly in non-model animals from nextgeneration sequencing data[END_REF]. The combination of Abyss and Cap3 was selected based on a combination of reliability and computational efficiency. In addition, this combination was recently shown to perform better than Trinity in the assembly of 26 species of plants [START_REF] Sarah | A large set of 26 new reference transcriptomes dedicated to comparative population genomics in crops and wild relatives[END_REF]. Reads were mapped to the contigs using BWA [START_REF] Li | Fast and accurate short read alignment with Burrows-Wheeler transform[END_REF]. Raw contigs were filtered for length (≥200 bp) and coverage (2.59 per individual). ORFs were predicted from the filtered contigs, and the longest ORF was kept. Genotypes were called for each position of each contig using read2SNPs and applying a filter for hidden paralogy as described in [START_REF] Gayral | Reference-free population genomics from next-generation transcriptome data and the vertebrate-invertebrate gap[END_REF]. Genotypes were only called if supported by a posterior probability above 0.95 and at least 10 reads. For each species, the per-site synonymous (p S ) and nonsynonymous (p N ) diversities were calculated, as well as the per-individual heterozygosity.

Research and annotation of orthologs

Orthologs between C3 and C5 were predicted using a reciprocal best BLAST hit approach, similarly to [START_REF] Gayral | Reference-free population genomics from next-generation transcriptome data and the vertebrate-invertebrate gap[END_REF]. BLASTP searches were performed using the 13 990 C3 ORFs as query and the 20 790 C5 ORFs as target, and vice versa with an e-value cut-off of 10e À5 . Results with no hit, with an overlap <60% or with an identity percentage <90% were discarded. In addition, results with a ratio of the score of the second best hit over the score of the best hit higher than 0.8 were removed, to avoid the inclusion of paralogs. This analysis yielded 4740 putative orthologs, a surprisingly low number for two species so closely related. A potential problem seemed to be the low number of ORFs retrieved for the broadcasting species C3: because we used the same methodology for sample preparation and sequencing, we hypothesized that this problem stemmed from a combination of high polymorphism in C3 and too much stringency in the coverage criterion.

To test this possibility, we attempted to recover C3 contigs using C5 contigs as references for mapping, and vice versa. To maximize the chances of recovering lost contigs, all the reads of each species were used (from single and pooled individuals) and coverage constraints were relaxed. C3 reads were mapped on the 11 405 C5 contigs displaying less than 80% identity with C3 contigs after the first BLAST search using BOWTIE2 [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF], and allowing a maximum of five differences (for 100-bp reads) with the reference contig to account for the fact the reads and the reference belong to two different species. C3 contigs were then reconstructed using MIRA [START_REF] Chevreux | Genome sequence assembly using trace signals and additional sequence information[END_REF]. Contigs with gaps in the coverage, or with an average coverage <109 or smaller than 250 bp, were discarded. A reciprocal best BLAST hit analysis was then performed to compare the newly reconstructed C3 contigs to the 20 790 C5 contigs and to verify whether these newly reconstructed C3 contigs indeed corresponded to some of the original C5 references. Using this method, 6156 additional C3 contigs were reconstructed, raising the total number of C3 contigs to 20 146. The equivalent analysis for C5 led to the reconstruction of 1333 additional C5 contigs, raising the total number of C5 contigs to 22 123. Comparison of the 20 146 C3 contigs to the 22 123 C5 contigs using a reciprocal best BLAST hit approach isolated a set of 12 229 orthologs. To ensure that the C3-C5 comparison only involved orthologous contigs, we used the ratio between the first and the second best BLAST hit score as a guide, and contigs with a ratio >0.8 were excluded.

Functional annotation was performed via BLASTP against the NR (nonredundant) database of NCBI using an e-value cut-off of 10e À5 . The top 20 hits were extracted and loaded in BLAST2GO for annotation [START_REF] Conesa | Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research[END_REF]. Mapping, annotation and slim ontology were performed with BLAST2GO using default parameters, except for the annotation cut-off parameter that was set to 45. GO categories were described using the level 3 of slim ontology. For the genes that did not display GO term annotations, BLASTP search was performed against the NR (nonredundant) database of NCBI using an e-value cut-off of 10e À5 .

Positively selected genes and gamete recognition genes

To characterize genes evolving under positive diversifying selection, we performed a first screen using the pipeline PSGfinder (Montoya-Burgos laboratory: https://genev.unige.ch/research/laboratory/Juan-Mon toya). This pipeline performs standard pairwise dN/dS calculation using a pair of aligned nucleotide sequences of orthologous protein-coding genes (one consensus sequence per species). The dN/dS ratio is calculated for alignment windows which size is determined by the presence of at least three nonsynonymous substitutions; this provides a sufficient dN starting signal that reduces the number of windows to be tested. The estimation of dN and dS values were obtained using the method 'yn00' (Yang & Nielsen 2000), including its assumptions, as implemented in PAML version 4.8 (Yang 2007). Windows with a dN/dS ratio > 1 were considered as indicative of gene parts displaying a signal of positive diversifying selection. The advantage of PSGfinder is that it uses pairwise comparison of orthologs, so positively selected candidate genes can be found even when only two species are available. Then, we confirmed that the most interesting candidate genes found with PSGfinder displayed signals of positive diversifying selection using a codon substitution model [modified branch-site model A; Bayes Empirical Bayes (BEB) analysis; O. longicauda C5 as foreground branch] of codeml implemented in PAML version 4.8 (Yang 2007), using outgroup sequences from public databases (sea urchin S. purpuratus) or kindly provided by Timothy O'Hara (brittle stars Ophiothrix angulata and Ophiophragmus wurdemani). Because of its change in reproductive mode, we expect the genes of the brooding species C5 to have evolved under positive selection. As control, we also performed the same analysis but using O. longicauda C3 as foreground branch (where we do not expect positive selection to occur). In addition, we used the recently developed BUSTED method which allows to identify genewide evidence of episodic positive selection [START_REF] Murrell | Gene-wide identification of episodic selection[END_REF], implemented in the HYPHY package [START_REF] Pond | HyPhy: hypothesis testing using In: Statistical Methods in Molecular Evolution[END_REF]. Furthermore, we used the branchsites random effects likelihood method which allows to detect lineages evolving under positive selection without an a priori hypothesis [START_REF] Pond | A random effects branch-site model for detecting episodic diversifying selection[END_REF], also implemented in the HYPHY package [START_REF] Pond | HyPhy: hypothesis testing using In: Statistical Methods in Molecular Evolution[END_REF]. Finally, we used ancestral sequence reconstruction methods (marginal reconstruction method; WAG amino acid replacement matrix) implemented in PAML version 4.8 (Yang 2007) to confirm which species was affected by natural selection.

Several proteins involved in reproduction (in sperm chemotaxis or in sperm-egg interaction) and whose evolution in other species involved diversifying selection were specifically isolated in our C3 and C5 contigs using BLASTP searches. These proteins were the bindin and its receptor EBR1, as well as the sperm receptor for egg jelly, suREJ, involved in activation of the acrosome reaction, sperm-activating peptides from S. purpuratus (speract) and from Asterias amurensis (asterosap) as well as their associated receptors (speract receptor and asterosap receptor), known to induce sperm chemotaxis. For the egg receptor for sperm Obi1, we used the sequence of the sea star Patiria miniata as query. We also searched for C3/C5 homologs of the sperm-specific calcium channels (Catsper 1-4) known to evolve under positive selection in rodents [START_REF] Podlaha | Positive selection for indel substitutions in the rodent sperm protein Catsper1[END_REF]) and primates

Results

Assembly statistics and annotation of orthologous genes

Illumina sequencing provided about 250 million raw reads, which were trimmed for length and quality-filtered. Assembly yielded 523 565 and 259 916 raw contigs for C3 and C5, respectively (Table 1). After filtering for length and coverage, 110 225 C3 and 118 043 C5 filtered contigs were obtained. Open reading frame prediction yielded 13 990 and 20 800 ORFs for C3 and C5, respectively. After ORF reconstruction based on 'unique' genes of each species, we obtained 20 146 and 22 123 ORFs for C3 and C5, respectively, of which 12 229 were clearly orthologous genes (Fig. 1). The average nucleotide divergence of the 12 229 orthologs at the nucleotide level was 0.93%. The higher number of genes in C5 might partly be due to the fact that developing embryos were included in the species C5 transcriptomes, contrary to the species C3 that was not sexually mature at the time of sampling.

Of the 12 229 identified orthologs, 5925 (48%) could be successfully annotated with GO terms. For biological process, primary metabolic process was the most abundant GO term (10.8%), followed by organic substance metabolic process (10.6%) and cellular metabolic process (8.1%) (Fig. S2, Supporting information). For molecular function, genes coding for binding were highly represented (67.8%) as well as transferase (10.5%) and hydrolase (12.9%) activities (Fig. S2, Supporting information).

Broadcast spawners display higher genetic diversity compared to brooders

To investigate genetic diversity, 1388 and 6700 genes were used for C3 and C5, respectively (Table 2). The smaller number of genes in C3 is most likely due to the stringent quality thresholds applied for genotype calling. Indeed, a position had to be covered with at least 10 reads per individual in order to be called. Given that the C3 individuals were not sampled at the same time of the year nor in the same localities (Table S1, Supporting information), they most likely displayed differences in gene expression levels and therefore differences in gene coverage. Using only C3 individuals from Crete (e.g. same locality as C5), we retrieved more than 5000 genes for the analysis, which gave the same diversity estimates as with the data set of 1388 genes (data available upon request). A total of 7607 and 18 408 SNPs were called for C3 and C5, respectively. The average per-site synonymous diversity (p S ) was about twice higher in C3 compared to C5, resulting in a higher p N /p S ratio in C5 (Table 2). In addition, the average heterozygosity was about 1.5 higher in C3 compared to C5.

Sperm-specific ion channels exhibit positive diversifying selection

Comparison of the 12 229 orthologs between the species C3 and C5 isolated 187 sequence windows displaying evidence of positive diversifying selection (PSGfinder, dNwindow/dSgene >1, P-value <0.001). These 187 windows were contained in 48 distinct genes, eleven of which could be successfully annotated with GO terms (Table 3). Another 17 of the 48 genes could be linked to biological process such as immunity, DNA transcription and phosphorylation through BLAST searches (Table S2, Supporting information); 45% (5/11) of the annotated genes encode proteins involved in ion transport, which is a significant enrichment compared to the global transcriptome annotation (3.6%; 215/5925). Interestingly, two genes involved in ion transport are well characterized functionally: the sperm-specific sodium-proton exchanger (NHE) and the tetrameric potassium-selective cyclic nucleotide gated channel (TetraKCNG) (Fig. 1, Table 3).

Positive selection analyses performed with PAML were concordant with the results of PSGfinder, with the majority of the positively selected sites retrieved with PAML were also observed in regions detected with PSGfinder (19/26; Figs 2 and3). In addition, ancestral sequence reconstructions revealed that positive diversifying selection particularly targeted the TetraKCNG and NHE genes in the brooding species C5 (Figs 2 and 3). Interestingly, for both genes, >75% (21/27) of the positively selected sites detected by PAML and divergent in the brooding species are conserved in all other species, even in the distantly related sea urchin Strongylocentrotus purpuratus (Figs 2 and3). Finally, the BUSTED method confirmed that both genes are evolving under positive selection (NHE: P-value = 0.00422; TetraKCNG: P-value = 0.0235), whereas only NHE was confirmed using the branch-sites random effects likelihood method (NHE: P-value = 0.016; TetraKCNG: P-value = 1).

NHE and TetraKCNG are well studied in the sea urchin S. purpuratus and are involved in sperm motility after activation of sperm with chemoattractants released by the eggs, such as speract [Fig. 4; see [START_REF] Darszon | Sperm-activating peptides in the regulation of ion fluxes, signal transduction and motility[END_REF] for the complete speract signalling model]. If sperm chemoattractants are species specific, they may be different between the brooding and the broadcasting species. To our knowledge, sperm chemoattractants are not described in brittle stars. Therefore, we attempted to find an ortholog of sperm chemoattractant in brittle stars based on the ones described in the literature, which are speract (in the sea urchin S. purpuratus) and asterosap (in the sea star A. amurensis). Unfortunately, we could not identify any ortholog, likely because of the small size of the peptides (ten amino acids for speract and 56 amino acids for asterosap; [START_REF] Hirohashi | Egg and sperm recognition systems during fertilization[END_REF][START_REF] Nakachi | Conserved sequences of sperm-activating peptide and its receptor throughout evolution, despite speciation in the sea star Asterias amurensis and closely related species[END_REF]. As ligand and receptor are likely to co-evolve, we isolated the sequences of the sperm chemoattractant receptor in the two O. longicauda species (guanylyl cyclase) but comparison of the two sequences did not reveal any evidence of positive diversifying selection (Table 4). Interestingly, however, of the ten nonsynonymous mutations found in the sperm chemoattractant receptor gene, nine occur in the extracellular (ligand-binding) part, whereas none are detected in the transmembrane part and only one is found in the intracellular part. In addition, a four-codon gap was found in the ligand-binding part of C5, suggesting that C3 and C5 may display different sperm chemoattractants. Therefore, mechanisms of species recognition could occur during sperm chemotaxis in the Ophioderma longicauda species complex. Finally, we found orthologs of the proteins involved in fertilization of Bindin, EBR1, suREJ1, Catsper 1-4, ARIS 1-3, Obi1 and Rendezvin. Yet, the evolution of these proteins did not reveal any evidence of positive diversifying selection in O. longicauda C3 and C5 (Table 4).

Discussion

Genetic diversity is lower in brooders

In this study, we used transcriptomes of two sympatric brittle stars belonging to the species complex Ophioderma longicauda to compare their level of genetic diversity and investigate how natural selection targeted genes potentially involved in their reproductive isolation. Genetic diversity, a central metric in species response to environmental disturbance [START_REF] Reusch | Ecosystem recovery after climatic extremes enhanced by genotypic diversity[END_REF][START_REF] Hughes | Ecological consequences of genetic diversity[END_REF], is about 1.5-2 times higher in the broadcasting species C3 compared to the brooding species C5, despite low average nucleotide divergence (<1%). These results are in line with a recent study investigating genetic diversity of 76 metazoan species, which showed that the main determinant of genetic diversity is parental investment [START_REF] Romiguier | Comparative population genomics in animals uncovers the determinants of genetic diversity[END_REF]. In particular, species categorized as K-strategists (high longevity, high parental investment, low number of large offspring) displayed lower genetic diversity than r-strategists (low longevity, low parental investment, high number of small offspring). These strategies highlight different responses of species to environmental disturbances [START_REF] Macarthur | The theory of island biogeography[END_REF]. In this context, the brooding O. longicauda C5 might be more

© 2017 John Wiley & Sons Ltd

Mechanisms of prezygotic isolation

We focused on genes displaying positive Darwinian selection involved in reproduction, because they may have played a role in reproductive isolation of O. longicauda brooding and broadcasting species. There are several steps leading to fertilization in free-spawning invertebrates: (i) sperm chemotaxis, which is the activation and attraction of sperm mediated by chemoattractants released by the egg, (ii) interaction of sperm with the egg jelly, which induces the acrosome reaction, (iii) binding of sperm to egg vitelline envelope, (iv) penetration of sperm through the egg envelope and (v) fusion of gamete membranes [START_REF] Vacquier | Evolution of gamete recognition proteins[END_REF]. So far, positively selected proteins contributing to prezygotic isolation in marine invertebrates were involved in sperm-egg interactions, either at the level of egg jelly (step ii) with the sperm receptors suREJ [START_REF] Mah | Positive selection in the carbohydrate recognition domains of sea urchin sperm receptor for egg jelly (suREJ) proteins[END_REF] or at the level of vitelline layer binding and penetration (steps iii-iv) with the sperm protein bindin [START_REF] Lessios | Speciation genes in free-spawning marine invertebrates[END_REF][START_REF] Vacquier | Selection in the rapid evolution of gamete recognition proteins in marine invertebrates[END_REF][START_REF] Evans | Sexual selection and the evolution of egg-sperm interactions in broadcast-spawning invertebrates[END_REF], as also shown recently for sea stars [START_REF] Patiño | Sperm bindin divergence under sexual selection and concerted evolution in sea stars[END_REF]. In Ophioderma longicauda, none of these proteins were found to evolve under positive selection. Interestingly, we could sequence and assemble, to our knowledge, the first bindin sequences of brittle stars, which displayed very low divergence between the brooding and the broadcasting species. Using domain prediction analyses, we did not detect any repetitive regions as found in sea stars [START_REF] Patiño | Sperm bindin divergence under sexual selection and concerted evolution in sea stars[END_REF]. It is therefore likely that the Ophioderma bindin sequences are incomplete, due to the difficulty of assembling repetitive regions using short read sequences. Further research on brittle star bindin should be performed to conclude on the role of bindin in species recognition and speciation.

In contrast, positive selection was found to act on two ion channels involved in sperm activation and motility. Interestingly, these genes have never been described as being positively selected between closely related species so far. The two sperm-specific ion channels (TetraKCNG and NHE) and their involvement in sperm activation have been extensively studied in sea urchins [START_REF] Beltr An | Signal transduction mechanisms regulating ion fluxes in the sea urchin sperm[END_REF][START_REF] Galindo | Sp-tetraKCNG: a novel cyclic nucleotide gated K+ channel[END_REF]Darszon ). In addition, the Na+/H+ exchanger (NHE) is also involved in the signalling of the acrosome reaction [START_REF] Beltr An | Signal transduction mechanisms regulating ion fluxes in the sea urchin sperm[END_REF]. NHE is essential for sperm motility, as shown with NHE-null male mice which displayed severely diminished sperm motility [START_REF] Wang | A new sperm-specific Na+/H+ Exchanger required for sperm motility and fertility[END_REF]). Yet, the exact mechanism explaining how mutations at the protein level influence the movement of ions remains to be investigated. Interestingly, positive selection was also found to occur in other sperm-specific ion channels, Catsper (1-4), which are involved in sperm motility in primates and rodents [START_REF] Podlaha | Positive selection on protein-length in the evolution of a primate sperm ion channel[END_REF][START_REF] Podlaha | Positive selection for indel substitutions in the rodent sperm protein Catsper1[END_REF]. These four channels, also found in O. longicauda, did not display signals of positive selection, contrary to TetraKCNG and NHE.

Positive selection on putative sperm motility genes: consequence of brooding evolution?

Although fertilization has not been directly observed in brooding O. longicauda, fertilization is very likely to occur internally, within the brooding pouches. Furthermore, all brooding females contained juveniles at a single stage of development [START_REF] Weber | Genetic data, reproduction season and reproductive strategy data support the existence of biological species in Ophioderma longicauda[END_REF], in favour of a single fertilization event. Several hypotheses were investigated to explain positive selection on ion channels TetraKCNG and NHE: (i) brooding conditions per se, (ii) male/female conflict, (iii) sexual selection via sperm competition and (iv) reinforcement, the most convincing hypotheses being (i) and (ii). environment possibly reduced in oxygen [START_REF] Strathmann | Constraints on egg masses. II. Effect of spacing, size, and number of eggs on ventilation of masses of embryos in jelly, adherent groups, or thin-walled capsules[END_REF]. According to [START_REF] Strathmann | Constraints on egg masses. II. Effect of spacing, size, and number of eggs on ventilation of masses of embryos in jelly, adherent groups, or thin-walled capsules[END_REF], oxygen is a limiting factor in many situations of brooding or in marine species providing brood protection, and the smaller size of such species with respect to their close relatives could be explained as an adaptation to oxygen availability (Strathmann et al. 1984). As a matter of fact, C3 individuals are on average larger than C5 individuals and reach also larger maximal sizes [START_REF] St€ Ohr | Potential cryptic speciation in Mediterranean populations of Ophioderma (Echinodermata: Ophiuroidea)[END_REF][START_REF] Weber | Genetic data, reproduction season and reproductive strategy data support the existence of biological species in Ophioderma longicauda[END_REF]. If oxygen levels are lower (or limiting) in the bursae, sperm with enhanced motility might have been positively selected to reach the eggs despite lower oxygen concentrations.

Second, another, indirect consequence of brooding may be an increase in the sexual conflict between males and females regarding the optimal ratio of egg/sperm encounters, due to the risk of polyspermy and its contrasted cost among sexes. This lethal event, occurring when several spermatozoa fertilize the same oocyte, might occur more often if they are present at high density [START_REF] Dale | How is polyspermy prevented?[END_REF][START_REF] Wong | Major components of a sea urchin block to polyspermy are structurally and functionally conserved[END_REF]. Natural selection would thus favour eggs emitting less (or less efficient) chemical attractants, thereby causing an increase in natural selection for sperm motility.

Third, sperm motility is an important component of sperm competition [START_REF] Birkhead | Sperm mobility determines the outcome of sperm competition in the domestic fowl[END_REF][START_REF] Levitan | Sperm velocity and longevity trade off each other and influence fertilization in the sea urchin Lytechinus variegatus[END_REF][START_REF] Gage | Spermatozoal traits and sperm competition in Atlantic salmon: relative sperm velocity Is the primary determinant of fertilization success[END_REF][START_REF] Snook | Sperm in competition: not playing by the numbers[END_REF]. In sea stars, patterns of bindin evolution are contrasted between gonochoric external fertilizers, which are subject to sperm competition, and selfing hermaphrodites (brooders or not), which are not [START_REF] Patiño | Sperm bindin divergence under sexual selection and concerted evolution in sea stars[END_REF]. Selfing hermaphrodites displayed no or low evidence of positive selection, whereas external fertilizers displayed numerous sites under positive selection. In O. longicauda, fertilization might occur as a single event within a given brooding chamber (cf above, and [START_REF] Weber | Genetic data, reproduction season and reproductive strategy data support the existence of biological species in Ophioderma longicauda[END_REF]. If adult behaviour ensures that sperm from a single male enters a brooding pouch, there will not be any sperm competition. However, without parentage analyses, the possibility that sperm from several males reach a brooding pouch cannot be ruled out. Indeed, multiple paternity was reported in several brooding sea urchins [START_REF] Chenuil | Paternity analysis in the Antarctic brooding sea urchin Abatus nimrodi. A pilot study[END_REF][START_REF] Maturana | Mating system and evidence of multiple paternity in the Antarctic brooding sea urchin Abatus agassizii[END_REF]. If sperm from several males enter a brooding chamber, competition among males may be increased due to higher gamete concentration, favouring positive selection on sperm motility genes.

Fourth, natural selection may have favoured mutations leading to prezygotic isolation between divergent populations or species to avoid wasting gametes in fertilizations producing unviable or sterile offspring, corresponding to the reinforcement hypothesis [START_REF] Lessios | Speciation genes in free-spawning marine invertebrates[END_REF]. According to this hypothesis, when two species diverge in allopatry, their genomes become incompatible whereas their fertilization systems may remain compatible. If hybridization occurs after secondary contact, hybrid embryos may have reduced fitness or may not develop at all, wasting reproductive effort. Consequently, divergent proteins preventing heterospecific fertilization would be positively selected by natural selection. In the case of Ophioderma, hybrids with reduced fitness may have occurred from the two possible types of crosses (C3 sperm with C5 oocytes and C5 sperm with C3 oocytes). Yet, as the cost of gamete loss is presumably higher in the brooding species (due to the higher parental investment observed in this species), selective pressures may have been higher in brooders (particularly in genes expressed in females and eggs, but by co-evolution, also on genes expressed in males and spermatozoa), explaining why positive selection was detected in this lineage. Why positive selection would be detected in sperm motility genes, and not other reproduction genes, has no conspicuous explanation under a strict reinforcement hypothesis. The reality may be a combination of several explanations. It is likely that selection due to the confined fertilizing environment (the first or second explanations) led to rapid evolution of some sperm proteins, which contributed to accelerate prezygotic isolation between brooders and broadcasters.

Sperm chemotaxis: a common mechanism of species recognition in brittle stars?

Our results suggest for the first time that sperm motility genes could have contributed to prezygotic isolation between closely related echinoderm species. Such an isolation could also have been favoured through chemotaxis differences, as sperm chemotaxis (through the occurrence of different sperm chemoattractants) plays for example an important role in sexual selection in the mussel Mytilus galloprovincialis [START_REF] Evans | Assessing the potential for egg chemoattractants to mediate sexual selection in a broadcast spawning marine invertebrate[END_REF]. Consistent with this, the brooding and broadcasting species likely display differences in their chemoattractants and related receptors. Indeed, we found that the sperm chemoattractant receptor of O. longicauda C5 displayed nine nonsynonymous mutations and a fourcodon gap in its extracellular (ligand-binding) domain. In contrast, the transmembrane and intracellular domains remained conserved between O. longicauda C3 and C5.

Previous research showed that sperm chemotaxis is generally not species specific in echinoderms, except for brittle stars. For example, sperm of the sea urchin Lytechinus pictus could successfully be activated by speract chemoattractants of the species Strongyloncentrotus purpuratus [START_REF] Guerrero | Tuning sperm chemotaxis by calcium burst timing[END_REF]. In addition, a recent study on speract and its receptor in five species of the sea urchin genus Diadema showed that these molecules evolve under strong purifying selection and not under diversifying selection [START_REF] Jagadeeshan | Evolution of gamete attraction molecules: evidence for purifying selection in speract and its receptor, in the pantropical sea urchin Diadema[END_REF]. Furthermore, sperm chemotaxis appeared to be mostly family specific in 19 species of holothurians [START_REF] Miller | Specificity of sperm chemotaxis among great barrier reef shallow-water Holothurians and Ophiuroids[END_REF], as well as in six sea star species [START_REF] Nakachi | Acrosome reaction is subfamily specific in sea star fertilization[END_REF]. In contrast, sperm chemotaxis was species specific in 10 of 15 species of brittle stars [START_REF] Miller | Specificity of sperm chemotaxis among great barrier reef shallow-water Holothurians and Ophiuroids[END_REF] suggesting that chemoattractants are mostly species specific in brittle stars. In particular, sperm chemotaxis specificity allowed separating two unidentified species belonging to the Macrophiothrix genus. Therefore, mechanisms of species recognition may occur during sperm chemotaxis in brittle stars, contrasting with previous research on marine invertebrates. Research on brittle star sperm-activating peptides, associated receptor and more generally, genes involved in sperm activation should be pursued, as they might be involved in numerous speciation events in this particular class of echinoderms as in other groups of animals.

Fig. 2

 2 Fig. 2 TetraKCNG protein alignment including Ophioderma longicauda C3 and C5, as well as outgroups Ophiothrix angular (brittle star) and Stronglylocentrotus purpuratus (sea urchin). Ancestral sequence reconstruction of Ophioderma was performed with PAML version 4.8. The region evolving under positive selection detected by PSGfinder and the positively selected sites detected by PAML are displayed. The alignment highlights that positive diversifying selection occurred in the brooding species Ophioderma longicauda C5. Levels of grey correspond to different amino acid families.

Fig. 3

 3 Fig. 3 NHE protein alignment including Ophioderma longicauda C3 and C5, as well as outgroups Ophiothrix angulata (brittle star), Ophiophragmus wurdemanii (brittle star) and Stronglylocentrotus purpuratus (sea urchin). Ancestral sequence reconstruction of Ophioderma was performed with PAML version 4.8. The region evolving under positive selection detected by PSGfinder and the positively selected sites detected by PAML are displayed. The alignment highlights that positive diversifying selection occurred in the brooding species Ophioderma longicauda C5. Levels of grey correspond to different amino acid families.

Fig. 4

 4 Fig.4Speract signalling model (modified from[START_REF] Darszon | Sperm-activating peptides in the regulation of ion fluxes, signal transduction and motility[END_REF]). In the sea urchin, Strongylocentrotus purpuratus, the sperm-activating peptides (SAP) speract, released by the eggs, bind to speract receptor (1) and activate a guanylyl cyclase (GC), which activates sperm motility through changes in intracellular cGMP (2), pH (3-5), cAMP (6-7) and calcium concentration (8-11). Sperm is attracted to the eggs along a gradient of chemoattractants (sperm chemotaxis). The positively selected genes (NHE and TetraKCNG) in O. longicauda C3 and C5 are highlighted. In the sea star Asterias amurensis, the SAP asterosap bind directly to the guanylyl cyclase to activate sperm motility. This is also likely the case in brittle stars, which are more closely related to sea stars compared to sea urchins. In O. longicauda C3 and C5, the guanylyl cyclase orthologs are divergent in the ligand-binding region (nine nonsynonymous mutations and one-four-codon gap), suggesting the existence of different chemoattractants in C3 and C5. Reproduced with permission from The International Journal of Developmental Biology (Int. J. Dev. Biol.) (2008) Vol: 52595-606.

Table 1

 1 Transcriptome assembly statistics generated in study

		C3	C5
		(5 individuals)	(2 individuals)
	Raw reads	164 464 891	87 959 550
	Raw contigs	523 565	259 916
	Filtered contigs	110 225	118 043
	ORFs	13 990	20 790
	Reconstructed ORFs	20 146	22 123
	Orthologous genes	12 229	12 229
	Annotated orthologous genes	5925	5925

Fig.

1

Summary of the gene number, orthologous gene number and positively selected genes between Ophioderma longicauda C3 and C5 found in this study.

Table 2

 2 Comparison of transcriptomic genetic diversity between broadcasting C3 and brooding C5 species

				Diversity
				ratio
		C3	C5	C3/C5
	Reproductive strategy	Broadcasters Brooders
	N individuals	5	2
	N contigs	1388	6700
	N SNPs	7607	18 408
	Average p S [910 À3 ]	17	8	2.12
	Average p N /p S ratio	0.181	0.269	0.67
	Average heterozygosity	6.27 (AE0.6)	4.16 (AE0.2) 1.51
	[910 À3 ](AESD)

SNPs, single nucleotide polymorphisms; Sd, standard deviation; p S , synonymous nucleotide diversity; p N , nonsynonymous nucleotide diversity.

© 2017 John Wiley & Sons Ltd

Table 3

 3 Positively selected genes between O. longicauda C3 and C5 that display GO term annotations. Gene name, matching organism after BLAST search and available GO

		Cellular component	Cell		Cell		Cell; plasma	membrane			Cell		Plasma membrane		No				No		No		Plasma membrane			Nucleus	Plasma membrane
		Molecular function	Ion channel activity		Ion channel activity		Transporter activity				Ion channel activity		Transporter activity		Transferase activity				Hydrolase activity		Binding; peptidase	activity	Phosphoprotein	phosphate activity		DNA binding	Protein binding
		Biological process	Ion transport		Ion transport		Ion transport;	cellular	component	organization	Ion transport		Ion transport		Nucleobase	containing	compound	metabolic process	Metabolic process		No		Response to	external stimulus;	signal	transduction	Transcription DNA	dependant	Multicellular	organismal	development; cell	differentiation
		Full gene name	Sperm-specific sodium-proton	exchanger	Tetrameric potassium-selective	cyclic nucleotide gated channel	PREDICTED: excitatory amino	acid transporter 3-like			PREDICTED: disrupted in renal	carcinoma protein 2 homolog	PREDICTED: solute carrier	family 13 member 5-like	Thymidine phosphorylase				PREDICTED: arylacetamide	deacetylase (esterase)-like	Tripartite motif-containing	protein 59	AChain A, Structural Studies Of	Protein Tyrosine Phosphatase	Beta Catalytic Domain In	Complex With Inhibitors	PREDICTED: zinc finger protein	167-like	PREDICTED: nephrin-like
	terms are displayed	Contig C3 Contig C5 Gene name Organism	Contig157183 Contig10800 NHE Strongylocentrotus	purpuratus	Contig5262 Contig45826 TetraKCNG Strongylocentrotus	purpuratus	Contig132964 Contig75158 LOC576432 Strongylocentrotus	purpuratus			Contig22861 Contig23039 LOC584316 Strongylocentrotus	purpuratus	Contig141406 Contig7832 LOC585510 Strongylocentrotus	purpuratus	Contig17797 Contig45476 / Crassostrea gigas				Contig168194 Contig2036 LOC100376819 Saccoglossus	kowalevskii	Contig_C3_26484 Contig26484 / Crassostrea gigas		Contig_C3_21879 Contig21879 / Homo sapiens			Contig_C3_32532 Contig32532 LOC100566073 Anolis carolinensis	Contig_C3_2308 Contig2308 LOC581462 Strongylocentrotus	purpuratus
																										© 2017 John Wiley & Sons Ltd

[START_REF] Podlaha | Positive selection on protein-length in the evolution of a primate sperm ion channel[END_REF].© 2017 John Wiley & Sons Ltd

#Sequence from the sea star Patiria miniata was used as query sequence.© 2017 John Wiley & Sons Ltd

© 2017 John Wiley & Sons Ltd

Acknowledgements

We are very grateful to Christos Arvanitidis from the Hellenic Center for Marine Research (HCMR, Crete) who hosted us in his laboratory, brought pertinent advice for general organization and organized the sampling sessions. We are also grateful to João Reis and João Rodrigues for logistical help and participation to sampling sessions. We would like to thank the CCMAR Institute of Faro and the Assemble European programme for providing funding for the sampling in Portugal (ASSEMBLE grant agreement no. 227799). JIMB was supported by SNSF grant (3100A0-104005). Many thanks to Thanos Dailianis, Elena Sarropoulou, Magdalini Christodoulou, Fr ed eric Zuberer, Laurent Vanbostal, Didier Weber and Thi Weber for providing samples or for helping during the sampling sessions. We thank the genomic platform Genotoul (INRA, Toulouse) for transcriptome sequencing. We are very grateful to Pierre Pontarotti who provided computing infrastructure to analyse the data and for fruitful discussions. We also would like to thank Chlo e Jeanne Hot for helping using PSGfinder and Walter Salzburger for providing useful comments to improve this manuscript. Finally, we would like to thank two anonymous reviewers for their comments on a previous version of the manuscript.

Data accessibility

The raw transcriptome reads were deposited on the Sequence Read Archive (SRA) (Accession no.: SRP096095) under the BioProject PRJNA360063. The assembled transcriptomes of Ophioderma longicauda C3 and C5 and all the alignments listed in Tables 3 and4 are available from the Dryad Digital Repository: http:// dx.doi.org/10.5061/dryad.713q3.

First, positive selection may be a direct consequence of brooding. Indeed, the evolution of brooding causes important changes in the environmental conditions surrounding the eggs. The eggs are gathered into pouches (the bursae) and are therefore present at higher densities than in the open water, and in an

Supporting information

Additional supporting information may be found in the online version of this article.

Table S1 Details of sampling localities, dates and individuals used in this study.

Table S2 BLASTP search for 37 positively selected genes between O. longicauda C3 and C5 that did not display GO term annotations.


C Birck

I Epaillard

M.-F Leccia

C Crassous

A Morand

C Miaud

C Bertrand

L Cavalli

S Jacquet

P Moullec

R Bonnet

C Sagot

E Franquet

Y M Nellier

M E Perga

N Cottin

C Pignol

E Malet

E Naffrechoux

C Giguet-Covex

I Jouffroy-Bapicot

D Etienne

L Millet

P Sabatier

B Wilhelm

B Perren

F Arnaud

Sentinel lakes: a network for the study and management of mountain lakes in the French Alps and in Corsica

High-altitude lakes are vulnerable ecosystems that require protection and sustainability management, although their overall functioning is still poorly understood. In France protected area managers and scientists are cooperating to address this problem. Their results show the huge diversity of these altitude lakes and imply specificities in their functioning and in the way they respond to stressors. Multidisciplinary studies on these ecosystems, on their individual history and the stressors they face, need to be developed in a number of lakes, alongside long-term monitoring surveys. This is the objective of the Sentinel lakes network.

Introduction

Mountain lakes of the French Alps and Corsica have been created by outstanding natural conditions and events and shaped by a long running interaction with humans. At elevations between 1 800 and 2 800 m, in some cases ice-covered for eight months of the year, they constitute unique ecosystems in Europe. As they are distant from large urban areas and rather difficult to reach, mountain lakes are usually considered pieces of natural land preserved from the impacts of human activities. However, recent international studies have challenged this view, showing that they have been subjected to severe human stresses, on both local and global scales, sometimes for ages.

The objective of this article is to present a panel of ongoing studies, carried out within the framework of the French network Sentinel lakes, which investigates how mountain lakes respond to local or global stressors.

Mountain lakes are important for their biodiversity and the ecosystem services they provide. They also have great cultural and socioeconomic value, being the place for numerous human activities, such as tourism and outdoor activities, fishing, hydroelectricity pro-duction… Lake managers face a dilemma since they are required to preserve the lakes' natural state while maintaining traditional human activities linked to these areas. Each of these activities is likely to trigger, in the short or longer term, substantial changes in the ecological state of these mountain lakes [START_REF] Schindler | Alteration of nutrient cycles and algal production resulting from fish introductions into mountain lakes[END_REF]Knapp 2001), especially since their peculiar properties (small size, extreme climate conditions, remoteness, small number of species) make them highly sensitive to any biological or chemical disturbances in the environment. Besides, these lakes have usually been subjected to severe and old anthropogenic pressures, the memory of which the lakes have kept within their sediments. The mountain catchment basins have been modified by human presence since Neolithic times and especially from the Bronze Age onwards through deforestation, pasturing practices [START_REF] Sturm | Sédiments lacustres: des archives révélatrices d'origine naturelle et anthropiques[END_REF][START_REF] Giguet-Covex | Observatoire et rétro-observatoire de l'état écologique des plans d'eau d'altitude : réflexions sur la notion d'état de référence et évaluation des facteurs de dégradation des lacs d'altitude. Rapport final[END_REF], mining activities [START_REF] Brancelj | Effects of land use and fish stocking on a mountain lake -evidence from the sediment[END_REF], and through fish stocking and fishing, for which documentation exists going back to the Middle Ages [START_REF] Brancelj | Effects of land use and fish stocking on a mountain lake -evidence from the sediment[END_REF][START_REF] Pister | Wilderness fish stocking: History and perspective[END_REF]). More recently (second half of the 20 th century), tourism development could have also affected lake ecosystem functioning.

Among managers there is also a growing concern about the impact of global changes on the ecological state and future trajectories of these lakes. Indeed, some European mountain lakes have been shown to be contaminated by atmospheric inputs of nutrients (nitrogen fertilizer and phosphorus) or pollutants, such as metals (lead, mercury [START_REF] Arnaud | A year history of lead contamination in northern French Alps reconstructed from distant lake sediment records[END_REF]Thévenon et al. 2011)) or persistent organic pollutants (PAH or pesticide residues, DDE), carried over long distances [START_REF] Battarbee | Mountain lakes, pristine or polluted?[END_REF]. They were also repeatedly shown as affected by climate change (EMERGE project, 2000, EU FP5).

Rangers and scientists surveying mountain lakes in the French Alps and in Corsica have observed recent changes in their status, such as an increase in the algal production and the appearance of cyanobacteria [START_REF] Cazaubon | Changes in erosion patterns during the Holocene in a currently treeless subalpine catchment inferred from lake sediment geochemistry (Lake Anterne, 2063 m asl, NW French Alps): the role of climate and human activities[END_REF], reduced water quality, hypoxic con- These inventories focus on aquatic invertebrates, amphibians and fish, macrophytes and algae, and have already been set up on more than 30 lakes. On completion of this programme, the park aims to study one or several lakes in each valley that will be representative of the park's lakes in terms of altitude, surface area and trophic level. At the same time, major efforts have been made to collect, validate and format all available biological, but also mesological and socio-economic data for each lake. This work has been planned to enable coordinated management between stakeholders and users (fishermen, shepherds, hikers, etc.) and their federation or representatives.

Inventory results, which still need to be completed, have already given the park authorities an idea of the specific richness and composition of the living community with the registration of several patrimonial species (Somatochlora alpestris, Alytes obstetricans and others). The plan for the next few years is to continue developing studies and inventories on these taxa with the aim of monitoring lakes to a greater extent and establishing certain land management operations (e. g. reducing organic waste from cattle and mountain huts and a stop on stocking lakes with young fish) to assess their impact on biodiversity. Some comparative approaches are also being set up to involve all of the lakes and new investigation methods (barcoding) will be tested.

Biodiversity in the lakes

Planktonic diversity and impacts of fish introduction in high-altitude lakes (French Alps)

To improve their management Ecrins National Park has launched studies for a better knowledge of alpine lakes functions and impacts of fish introduction. A collaboration with IMBE (Institut Méditerranéen de Biodiversité et d'Ecologie) initiated in 1992 highlighted that in certain lakes, as food resources are limited, two years are generally necessary for the maturation of the gonads of Salvelinus alpinus (Figure 4, [START_REF] Cavalli | Diet, growth and reproduction of the artic charr (Salvelinus alpinus (L.)) in a high alpine lake: Muzelle (Oisans, France)[END_REF]. This was demonstrated by the presence of Savoie, Asters has implemented a monitoring on 5 of the mountain lakes on the territory that started 15 years ago. Chemical, physical and biological parameters are measured each year by the rangers to assess the evolution of the ecosystem. One of these lakes is more closely surveyed by the scientific partners with the aim of better understanding the ecosystem and how it works. As a natural consequence the partners of the network have given Asters the mission to encourage exchanges and to coordinate activities like the development of a standardized protocol. Contact carole.birck@asters.asso.fr two female fish groups, one with high gonadosomatic index (GSI = Gonad weight / fish weight) and another one with low gonadosomatic index. Moreover, this approach revealed the impact of introduced fishes (topdown control / predation effect) on zooplankton communities (planktonic crustaceans and rotifers). In cold lakes, when food resources are scarce, fish predation had a direct impact on the large microcrustacean zooplankton; in contrast, in warmer lakes, food resources are more abundant and the lower fish predation on crustaceans leads to an important crustacean predation on rotifers, with an effect on some rotifer species form [START_REF] Cavalli | Combined effects of environmental factors and predatorprey interactions on zooplankton assemblages in five high alpine lakes[END_REF].

Management & Policy Issues

-PREPRINT -PREPRINT -PRPRINT -PREPRINT -PREPRINT -PREPRINT -PREPRINT -PREPRINT -PREPRINT This collaboration allowed adopting valuable management measures (dates, species, sizes) for fish stocking. Since 2005 a long-term survey of planktonic trophic food webs has been conducted in collaboration with the park rangers and ONEMA. The structural and functional diversities were characterized for the French Alps. The study was based on a large variety of highly constrained ecosystems (different landscape and internal characteristics) and revealed high planktonic heterogeneity. Recently flow cytometric analyses of viral, bacterial and phytoplanktonic components have allowed observing planktonic biodiversity and confirmed the heterogeneity between lakes (Figure 5).

On the left panel, based on chlorophyll a, fluorescence and side scatter (related mainly to shape and size), at least five different dot populations corresponding to different phytoplanktonic groups (referred to as phyto-x) can be identified. On the right panel, following nucleic acid staining, and based on DNA-dye fluorescence and side scatter, three groups of viruses (referred to as virus-like particle or VLP-x) and two groups of heterotrophic prokaryotes (mainly bacteria named bact-1 & bact-2) can be identified. This figure illustrates the importance of the abundance and the diversity of some key planktonic groups in the functioning of mountain lakes.

Figure 4 -Relationship between gonadosomatic index and length for female mature (•) and immature (■) Arctic char in

Muzelle lake in October. Reprint from [START_REF] Cavalli | Diet, growth and reproduction of the artic charr (Salvelinus alpinus (L.)) in a high alpine lake: Muzelle (Oisans, France)[END_REF], published in Journal of Fish Biology The long-term survey revealed the complex interactions between global (climatic change, atmospheric deposition) and local stressors (pastures, mountain huts) and their impacts on the structure of food webs and in turn on the ecosystem services. Water chemical analysis (N, P, Carbon), temperature records and atmospheric deposition analyses (Ga, In, Sb, Tl, Ge, Ag, Se, Pb, Cu, Ni, Cd, As...) were conducted in certain lakes.

Ecology of amphibians in high-altitude lakes and interactions with introduced fishes

The introduction of non-native fishes into ponds and lakes is a common practice throughout the world, even in protected areas like national parks. In the Alps many lakes have been stocked with different species of carnivorous fish such as salmonids. The objective of the study is to understand the potential impact of introduced fishes on the Alpine amphibian community. The first step is to describe the ecology of Alpine amphibians, by applying a standardized sampling protocol to infer species occurrence according to aquatic and terrestrial habitat characteristics in a significant number of aquatic sites. The knowledge of stocking practices will allow comparing areas differing historically in the presence of fishes. The presence of at least two amphibian species greatly varying in their life history traits (frog and newt) will allow comparing the causal effect of fish predation on amphibian life stages and ultimately population dynamics. The understanding of the complex relations between fish presence and amphibian population dynamics will help to propose management actions for both amphibian conservation and sport fishing practices. The standard protocol will also permit to infer temporal amphibian population trends that can be connected with egg recovery plans and more global environmental changes such as climate. present several properties that still make them major ecological issues. First, PCBs are highly stable, resistant to degradation. As a consequence, they persist in the environment even 35 years after interdiction and can still cause local sanitary problems. Second, their semivolatility is responsible for their atmospheric longrange transport and widespread distribution, even in remote ecosystems such as Polar regions and highaltitude lakes. Lastly, PCB hydrophobicity promotes their transfer and bioaccumulation in food webs, up to fish. Besides, PCBs represent a wide range of congeners with specific physico-chemical properties and can then be regarded as model compounds for understanding the general risks related to other persistent organic pollutants.

While the atmospheric transport of PCBs, basically driven by physical rules of distillation / deposition, is well understood, the processes that occur between PCB deposition and fish contamination have been so far poorly studied. As a matter of fact, recent studies performed on European lakes exposed to similar atmospheric deposition highlighted, but failed to explain, substantial differences in the PCB contamination of their fish communities. Our hypothesis in this ongoing work is that this variability arises from the biological processes and pathways by which deposited PCBs are transferred within the food web to the fish compartment. Hence, two lakes submitted to similar atmospheric inputs of PCBs but different food web composition might ultimately exhibit different fish contamination, because of different abilities of their food web to transfer PCBs.

The study sites are two lakes in Ecrins National Park (Lake La Muzelle, Lake Plan Vianney). These two lakes are located close to each other at similar altitudes but are characterized by different trophic status and food webs. PCBs are measured seasonally in major geo chemical and biological compartments, ranging from dissolved, size-fractioned organic matter, sediment up to zooplankton, macro-invertebrates and fishes.

Results obtained so far confirmed that in spring fish from Plan Vianney have PCB levels that are twice those in La Muzelle. Fish contamination with PCBs occurs essentially during snowmelt, suggesting that the interaction between the release of PCBs stocked in the winter snow and the start of algal growth is a crucial step for the transfer of the PCBs into the lake food web. Indeed, in this particular season, PCBs are distributed differently in the organic matter size fractions between the two lakes, confirming the role played by the ecological processes in the mobilization of PCBs. Current and future research aims at a stronger sampling effort during snowmelt in order to refine our understanding of the ongoing interaction between released PCBs and biological components, ex-situ experiments on lake sediments to apprehend PCB bioavailability and a paleolimnological reconstruction of the historical PCB contamination of these lakes.

Past evolution of three high-altitude lake ecosystems (French Alps)

Reconstructing and understanding past ecological changes of high-altitude Alpine lakes could be helpful for improving their management. Towards this goal lake sediments represent interesting archives as they record past changes in their biological, biogeochemical and physical states. They also allow accessing the factors driving such changes.

Sediment cores from three different lakes across the French Alps (Lake Anterne, Lauzanier and Allos, at 2 063 m, 2 285 m and 2 228 m, respectively) were studied by multiproxy approach with sedimentological, geochemical (mineral and organic) and biological analyses (Figure 6). Results showed that all three lakes underwent changes in their productivity and in bottom oxygen concentrations. However, these changes occurred at different periods and were triggered by factors that varied between lakes.

For example, the productivity of Lake Lauzanier increased from the Little Ice Age (LIA, 1350-1900 AD), first as a result of the combined effects of an intense pasturing activity and a relatively high hydrological activity (Figure 6), increasing the transfer of nutrients from the watershed to the lake. However, atmospheric nitrogen deposits are thought to be responsible of the lake's high trophic level and the degradation of oxygenation conditions, recorded in this lake from the late 1950s (Figure 6).

In the case of Lake Anterne, the main change in lake productivity has been recorded from 1980 and is probably due to the acceleration of global warming in the Alps, in particular through the increase in the icefree period (Figure 6).

Primary productivity in Lake Allos increased around 1920, probably in response to the fish introduction. Since the 1980s lake water quality and oxygenation conditions have been improving (Figure 6). Although the pasturing pressure and hydrological activity were high during the LIA at Allos, they did not encourage lake productivity as observed in Lake Lauzanier. An explanation for such different responses to similar environmental factors might lie in the fact that the watershed of Lake Allos is more sensitive to erosion than that of lake Lauzanier. It is thus possible that, beyond a turbidity threshold, productivity is limited by the lack of light in realizing photosynthesis.

In conclusion, this study underlines a recent decrease of local pressures on lake ecosystems. In contrast, we observe changes attributed to the impact of global forcing factors on these remote lakes.

Conclusion

There is evidence that, in spite of their remote position, high-altitude Alpine lakes have undergone modifications triggered by human pressure. The studies undertaken in lakes of the French Alps have dem- Multidisciplinary research can help to understand how the ecosystem responds to these multi-stressors conditions. Monitoring of a large number of mountain lakes and sharing the results within the network will also enable better protection and management of these habitats.

Other scientists and / or protected areas managers working on or interested in the subject are kindly invited to contact us.

Figure 1 -

 1 Figure 1 -Biological inventory on Lake Long Inférieur, Roya Valley. © P. Richaud

From

  knowledge to management The restoration of the lakes' natural function is one of the aims outlined in the Charter of the Mercantour National Park. In this framework and in relation to the ALCOTRA 2007 -2013 transboundary project Mercantour -Alpi Marittime Generalised Biological Inventory, special attention has been paid to these hydrosystems in order to provide a picture of the initial state of their biodiversity.

Figure 3 -

 3 Figure 3 -Example of lake documentation sheet

Figure 5 -

 5 Figure 5 -An example of flow cytometry cytograms revealing different phytoplankton, bacterial and viral groups

  PCB contamination in high-altitude lakes Polychlorobiphenyls (PCBs) are synthetic organic pollutants the production and use of which have been banned for over 35 years. However, these compounds

  Figure 6 -Representation of catchments, lakes and coring sites. The different curves correspond to indicators reflecting past lake ecosystem evolutions (lake productivity through indices of organic matter quality (IH, hydrogen index; IO, oxygen index; C/N, carbon-to-nitrogen ratio); oxygenation conditions are also presented in comparison with forcing factors affecting them (temperature, erosion, grazing activity and atmospheric nitrogen deposits, per lake) onstrated the impacts on biodiversity of local activities such as fish stocking but also the consequences of atmospheric pollution (PCBs, Nitrogen…) on the characteristics of the lakes.Multidisciplinary research can help to understand how the ecosystem responds to these multi-stressors conditions. Monitoring of a large number of mountain lakes and sharing the results within the network will also enable better protection and management of these habitats.

Authors

Birck, C. & I. Epaillard: Asters, Conservatoire des espaces naturels de Haute-Savoie Asters is the manager of the nine nature reserves located in Haute-Savoie, France. Its other missions are to develop environmental expertise, protect and manage habitats and species, and to inform and raise awareness of the general public.