Mario Barahona

Bernardo R Broitman

Sylvain Faugeron
email: sfaugeron@bio.puc.cl

Lucie Jaugeon

Andrés Ospina-Alvarez

David Véliz

Sergio A Navarrete

Environmental and demographic factors influence the spatial genetic structure of an intertidal barnacle in central-northern Chile

Keywords: Notochthamalus scabrosus, Seascape genetics, Larval dispersal, Coastal oceanography, Marine connectivity

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Population genetic diversity is important for a range of ecological and evolutionary processes. For example, genetic diversity can determine community structure and primary productivity [START_REF] Crutsinger | Plant genotypic diversity predicts community structure and governs an ecosystem process[END_REF]. It can be associated with the population growth rate of species [START_REF] Hughes | Ecological consequences of genetic diversity[END_REF], and it allows species to adapt to changing environments and fosters persistence over evolutionary time scales [START_REF] Reed | Correlation between fitness and genetic diversity[END_REF]. Therefore, a strong scientific understanding of the processes that influence spatial genetic variation, genetic diversity, and population structure in nature is of paramount importance to implement efficient conservation and management strategies.

In marine systems, an early paradigm assumed that most organisms were highly dispersive and presented large population sizes, and thus were able to resist genetic divergence at all but perhaps the largest spatial scales. This overly simplistic view was gradually replaced by an increased understanding of hydrographic heterogeneity in the coastal ocean and the advent of molecular genetics, which unveiled many potential causes for genetic structure and speciation in organisms with large dispersal potential [START_REF] Hellberg | Gene flow and isolation among populations of marine animals[END_REF][START_REF] Selkoe | A decade of seascape genetics: contributions to basic and applied marine connectivity[END_REF]. For example, due to the dependence of genetic diversity on population effective size [START_REF] Ellegren | Determinants of genetic diversity[END_REF], demographic changes along the geographic range of a species can leave a discernible footprint in its spatial genetic makeup. Also, a realistic oceanic environment, especially when there is strong topographic modulation, presents ample opportunities for variation and restriction in effective dispersal distances [START_REF] Largier | Considerations in estimating larval dispersal distances from oceanographic data[END_REF][START_REF] Pringle | Asymmetric dispersal allows an upstream region to control population structure throughout a species' range[END_REF][START_REF] Nickols | Marine population connectivity: reconciling large-scale dispersal and high self-retention[END_REF]. Geographic distance per se imposes a distance limitation to gene flow, driving increased genetic differentiation with increasing distance between populations, a pattern known as isolation by distance [START_REF] Wright | Isolation by distance[END_REF]. Besides the limitation imposed by dispersal between distant populations, phenotype-environment mismatches can impose biological barriers to gene flow [START_REF] Nosil | Reproductive isolation caused by natural selection against immigrants from divergent habitats[END_REF], producing an isolation by environment, where populations with greater environmental dissimilarity exhibit higher levels of genetic differentiation, blurring or reinforcing patterns generated by geographic distance alone [START_REF] Wang | Isolation by environment[END_REF].

High-resolution molecular and environmental data are now routinely used to assess the influence of landscape-scale environmental characteristics on genetic variation and spatial patterns in natural populations of species. Altogether, the mounting evidence suggests that considerable genetic structure occurs in marine populations around areas where environmental oceanographic factors exhibit strong spatial structure or geographic discontinuity.

The central-northern coast of Chile represents an interesting study system to evaluate the effects of environmental, demographic and geographic factors on genetic diversity and differentiation of marine organisms. Superimposed on what are smooth latitudinal trends in mean sea surface temperature (SST) along this highly productive upwelling ecosystem, there is a marked change in oceanographic regimes that takes place around 30° S. Such geographic discontinuity entails changes in upwelling-driven coastal circulation [START_REF] Hormazabal | Coastal transition zone off Chile[END_REF][START_REF] Aiken | Potential changes in larval dispersal and alongshore connectivity on the central Chilean coast due to an altered wind climate[END_REF], Aguirre et al. 2014), as well as prevailing hydrographic conditions, such as SST variability, surface chlorophyll, and nutrient availability [START_REF] Navarrete | Scales of benthic-pelagic coupling and the intensity of species interactions: from recruitment limitation to topdown control[END_REF], Tapia et al. 2014). Coincidentally, at this same latitude, several studies have reported the occurrence of geographic distribution endpoints of several intertidal and subtidal invertebrate species [START_REF] Lancellotti | Biogeographical patterns of benthic macroinvertebrates in the Southern Pacific littoral[END_REF][START_REF] Camus | Biogeografía marina de Chile continental[END_REF], phylogeographic breaks of several invertebrates and macroalgae [START_REF] Tellier | Phylogeographic analyses of the 30°S south-east Pacific biogeographic transition zone establish the occurrence of a sharp genetic discontinuity in the kelp Lessonia nigrescens: vicariance or parapatry?[END_REF][START_REF] Haye | Phylogeographic structure in benthic marine invertebrates of the Southeast Pacific coast of Chile with differing dispersal potential[END_REF], and large changes in population dynamics and abundance of dominant rocky shore species that otherwise extend far beyond this region [START_REF] Broitman | Geographic variation of southeastern Pacific intertidal communities[END_REF][START_REF] Navarrete | Scales of benthic-pelagic coupling and the intensity of species interactions: from recruitment limitation to topdown control[END_REF][START_REF] Rousset | GENEPOP'007: a complete re-implementation of the GENEPOP software for Windows and Linux[END_REF].

The geographic range of the intertidal barnacle Notochthamalus scabrosus [START_REF] Darwin | A monograph on the subclass Cirripedia with figures of all the species. The Balanidae, the Berrucidae, etc[END_REF]) spans the 30° S transition zone, and its complete larval development to settlement takes well over 1 mo at the water temperatures typically encountered in central Chile [START_REF] Venegas | Larval development of the intertidal barnacles Jehlius cirratus and Notochthamalus scabrosus (Cirripedia: Chthamalidae) under laboratory conditions[END_REF]. At the same time, the advective nature of coastal flow along central Chile [START_REF] Aiken | Along-shore larval dispersal kernels in a numerical ocean model of the central Chilean coast[END_REF]) sets the stage for a comparatively high potential for larval dispersal and genetic flow among distant populations of this species, as shown by biophysical models for other long-distance dispersers in the region [START_REF] Garavelli | Identifying appropriate spatial scales for marine conservation and management using a larval dispersal model: the case of Concholepas concholepas (loco) in Chile[END_REF].

Moreover, large variation in larval arrival rates and adult cover have been reported for the central-northern coast, which has been attributed to differences in the temporal regime of upwelling-favorable winds [START_REF] Navarrete | Scales of benthic-pelagic coupling and the intensity of species interactions: from recruitment limitation to topdown control[END_REF][START_REF] Lagos | Spatial environmental correlates of intertidal recruitment: a test using barnacles in northern Chile[END_REF]. A phylogenetic break in the N. scabrosus mitochondrial cytochrome oxidase I gene (mtCOI) around 30° S was reported by [START_REF] Zakas | Restricted gene flow in Chilean barnacles reflects an oceanographic and biogeographic transition zone[END_REF]. Although spatially stable, there are significant temporal changes in gene frequencies near the break (30° 55' S), presumably related to source-sink dynamics and/or low effective population sizes in this zone [START_REF] Laughlin | Mitochondrial lineages in Notochthamalus scabrosus as indicators of coastal recruitment and interactions[END_REF]. Based on a large-scale circulation model, [START_REF] Ewers-Saucedo | The oceanic concordance of phylogeography and biogeography: a case study in Notochthamalus[END_REF] suggested that the genetic break of N. scabrosus around 30° S requires differential performance of mtCOI lineages along the coast; in other words, it could not be maintained by dispersal limitation alone.

Therefore, the diversity and genetic structure of N. scabrosus may respond to multiple causes, such as phylogeography, demography, geographical isolation, and selection pressures driven by environmental variation along the coastline.

This study takes advantage of the genetic information gathered for N. scabrosus, based on mtCOI, as well as of a long-term database (5-13 yr) of monthly larval arrival (recruitment) of this species at multiple sites spanning the reported latitudinal break.

Together with surveys of adult abundance and satellite-based information of environmental (oceanographic) variables for the region (28-34° S), and the development of neutral microsatellite markers, we assessed the potential influence of nearshore environmental, demographic, and geographic factors on the genetic diversity and population structure patterns of N. scabrosus. Using neutral markers of gene flow allowed us to (1) characterize patterns of genetic diversity and the spatial genetic structure in N. scabrosus and (2) determine the relative importance of environmental, demographic, and geographic factors for genetic variation between and within populations of this widely distributed barnacle species.

MATERIALS AND METHODS

Hydrography of the study area

The coast of Chile between 18 and 42° S is under the broad influence of the northward flowing Humboldt Current (also called Chile-Peru Current). Close to shore, coastal hydrography is dominated by the dynamics of the Chilean Coastal Current (CCC), a predominantly northward surface stream forced by the prevailing south and southwest upwelling-favorable winds [START_REF] Aiken | A simulation of the Chilean Coastal Current and associated topographic upwelling near Valparaíso, Chile[END_REF][START_REF] Aiken | Potential changes in larval dispersal and alongshore connectivity on the central Chilean coast due to an altered wind climate[END_REF], which intensify during spring and early summer months, and around coastal topographic features [START_REF] Strub | Coastal ocean circulation off western South America[END_REF][START_REF] Tapia | Thermal indices of upwelling effects on inner-shelf habitats[END_REF][START_REF] Bravo | Seasonal variability of the Ekman transport and pumping in the upwelling system off central-northern Chile (~30° S) based on a high-resolution atmospheric regional model (WRF)[END_REF]. Our study area is located in a fairly straight shoreline stretch (Fig. 1) and is exposed to direct wave action [START_REF] Narváez | Onshore advection of warm water, larval invertebrate settlement, and relaxation of upwelling off central Chile[END_REF].

Within the study area, the main upwelling centers are Punta Talca, Punta Toro, Curaumilla, Pichilemu, and, to a lesser extent, Los Molles [START_REF] Silva | Evolución de un evento de surgencia frente a punta Curaumilla, Valparaíso[END_REF][START_REF] Wieters | Alongshore and temporal variability in chlorophyll a concentration in Chilean nearshore waters[END_REF][START_REF] Tapia | Thermal indices of upwelling effects on inner-shelf habitats[END_REF], 2014). In contrast, the bays of Cartagena, Valparaíso, and Coquimbo remain relatively protected from upwelling [START_REF] Kaplan | Large diurnal temperature fluctuations in the nearshore water column[END_REF][START_REF] Vargas | Internal tidal bore warm fronts and settlement of invertebrates in central Chile[END_REF][START_REF] Aiken | A simulation of the Chilean Coastal Current and associated topographic upwelling near Valparaíso, Chile[END_REF]. Four sampling sites (PTal, LMol, Cura, and Pich) were located in active upwelling centers, and 4 sites, namely Temb and Guan (Coquimbo Bay), Mont (Valparaíso Bay), and ECIM (Cartagena Bay), were located in places of weak upwelling.

For the 2 remaining sites (Apol and CBaj), records from in situ SST suggest that the hydrography of Apol may be similar to that of weak upwelling sites, while CBaj seems to be under the influence of active upwelling [START_REF] Valdivia | Disentangling the effects of propagule supply and environmental filtering on the spatial structure of a rocky shore metacommunity[END_REF].

An important geographic discontinuity in upwelling-favorable winds occurs around 30-32° S [START_REF] Strub | Coastal ocean circulation off western South America[END_REF][START_REF] Thomas | Seasonal distribution of satellite-measured phytoplankton pigment concentration along the Chilean coast[END_REF][START_REF] Hormazabal | Coastal transition zone off Chile[END_REF][START_REF] Navarrete | Scales of benthic-pelagic coupling and the intensity of species interactions: from recruitment limitation to topdown control[END_REF].

North of this latitude, equatorward winds are weaker but more persistent throughout the year, while to the south winds are stronger but temporarily more variable [START_REF] Hormazabal | Coastal transition zone off Chile[END_REF][START_REF] Navarrete | Scales of benthic-pelagic coupling and the intensity of species interactions: from recruitment limitation to topdown control[END_REF]. The change in oceanographic regimes determines or modulates the concentration and temporal variability of surface phytoplankton [START_REF] Thomas | Seasonal distribution of satellite-measured phytoplankton pigment concentration along the Chilean coast[END_REF], nutrient regimes (NO 3 ) of coastal waters [START_REF] Tapia | Latitudinal discontinuity in thermal conditions along the nearshore of central-northern Chile[END_REF], and functional structure of benthic communities [START_REF] Broitman | Geographic variation of southeastern Pacific intertidal communities[END_REF][START_REF] Navarrete | Scales of benthic-pelagic coupling and the intensity of species interactions: from recruitment limitation to topdown control[END_REF][START_REF] Wieters | Benthic community structure and spatiotemporal thermal regimes in two upwelling ecosystems: comparisons between South Africa and Chile[END_REF]).

Study species

Notochthamalus scabrosus is distributed along most of the rocky coasts of Ecuador, Peru, and Chile [START_REF] Brattström | Ecological and regional zoogeography of the marine benthic fauna of Chile[END_REF]. In the zone occupied by chthamalid barnacles, N. scabrosus inhabits the 3 intertidal elevations, with greater abundance in the middle and upper intertidal zones [START_REF] Paine | Perturbation and recovery patterns of starfish dominated intertidal assemblages in Chile, New Zealand and Washington State[END_REF][START_REF] Shinen | Coexistence and intertidal zonation of chthamalid barnacles along central Chile: interference competition or a lottery for space?[END_REF]). Adults are sessile filter feeding, hermaphroditic brooders. The life cycle of N. scabrosus includes a pelagic larval stage that lasts about 37 d at 15-18°C, with 6 naupliar stages with planktotrophic feeding and a cyprid stage competent for settlement [START_REF] Venegas | Larval development of the intertidal barnacles Jehlius cirratus and Notochthamalus scabrosus (Cirripedia: Chthamalidae) under laboratory conditions[END_REF].

Cyprid settlement occurs in pulses of larval arrival to the coast during a few days within the recruitment period, which is mainly concentrated in spring-summer [START_REF] Tapia | Spatial patterns of barnacle settlement in central Chile: persistence at daily to inter-annual scales relative to the spatial signature of physical variability[END_REF]).

Sampling of individuals, DNA extraction, and genotyping of microsatellites

At each of 10 study sites, 30 N. scabrosus adults of 3-6.4 mm rostrocranial length were collected from rocky platforms approximately 10-30 m long × 4-8 m wide.

Individual barnacles were identified as N. scabrosus in the field and were removed from the rock with a scalpel and immediately stored in tubes with 95% ethanol for preservation.

Total DNA was extracted using the salt/Proteinase K method [START_REF] Aljanabi | Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques[END_REF] and quantified in a spectrophotometer (Nanodrop).

Seven microsatellite loci were amplified by polymerase chain reaction (PCR). The microsatellite development procedure, the conditions under which the PCRs were performed, and the GenBank accession numbers can be found in Table S1 in the Supplement at www.int-res/articles/suppl/m123p456_supp.pdf. Alleles were identified by capillary electrophoresis in an ABI3130 Genetic Analyzer (Applied Biosystems), and the Excel FLEXIBIN macro [START_REF] Amos | Automated binning of microsatellite alleles: problems and solutions[END_REF]) was used to calibrate the reading and allele binning of each locus.

Genetic polymorphism

The total number of observed alleles (Na), number of private alleles (Pa), and observed (H o ) and expected (H e ) heterozygosity were calculated in GENALEX 6.5 [START_REF] Peakall | GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research -an update[END_REF]. Per locus gene diversity (Gd) and standardized allelic richness (Ar) were calculated in the FSTAT software version 2.9.3.2 [START_REF] Goudet | FSTAT, version 2.9.3.2, A program to estimate and test gene diversities and fixation indices[END_REF]). The Ar index was calculated using the rarefaction method to avoid bias due to differences in sample size [START_REF] Leberg | Estimating allelic richness: effects of sample size and bottlenecks[END_REF]. To evaluate deviations from the Hardy-Weinberg expectations (HWE), Fisher's exact tests were performed for heterozygote deficits at each site-locus combination, and U-score tests for global HWE per site through loci and per locus across sites (dememorization 10000; 100 batches; 10000 iterations) using the GENEPOP 4.2 software [START_REF] Rousset | GENEPOP'007: a complete re-implementation of the GENEPOP software for Windows and Linux[END_REF]. Linkage disequilibrium between all pairs of loci at each site and between each pair of loci across sites was assessed by Fisher's exact tests implemented in GENEPOP with this same parameter set. The inbreeding coefficient F IS by locus and site was quantified with GENETIX 4.05 [START_REF] Belkhir | GENETIX 4.04, logiciel sous Windows TM pour la génétique des populations. Laboratorie Génome, populations, interactions[END_REF], and departures from random expectations were assessed by 10000 permutations. For all multiple comparisons, the nominal level of significance of 5% was adjusted using the false discovery rate (FDR;

Benjamini & Hochberg 1995).

To test for large allele dropout and stuttering and to estimate the frequency of null alleles at each site-locus combination following Brookfield (1996: Eq. 4), data were analyzed with the MICROCHECKER software [START_REF] Van Oosterhout | MICRO-CHECKER: software for identifying and correcting genotyping errors in microsatellite data[END_REF]).

Population genetic structure

Global and pairwise genetic differentiation was evaluated calculating θ ST [START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF] and D EST (Jost 2008) indices, in GENALEX 6.5 [START_REF] Peakall | GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research -an update[END_REF] and running 10000 permutations to evaluate their significance. Jost's D EST outperforms G ST and its relatives (F ST ) over a range of sample sizes, including in situations where we have highly variable microsatellite loci with different numbers of alleles [START_REF] Heller | Relationship between three measures of genetic -2083[END_REF][START_REF] Gerlach | Calculations of population differentiation based on GST and D: Forget GST but not all of statistics![END_REF]), but it is recommended to compare results between differentiation indices [START_REF] Leng | Measuring population differentiation using GST or D? A simulation study with microsatellite DNA markers under a finite island model and nonequilibrium conditions[END_REF]. In all multiple comparisons, sites were used as population units, and the nominal level of significance, 5%, was adjusted using FDR. To identify population relationships in a 2-dimensional space, principal coordinate analyses (PCoAs) of the sites were computed and graphed in GENALEX 6.5 using the θ ST and D EST differentiation indices.

As null alleles can impose error in differentiation estimates [START_REF] Pompanon | Genotyping errors: causes, consequences and solutions[END_REF]), 2 approximations were conducted. First, using MICROCHECKER, we obtained a new database corrected for null alleles. MICROCHECKER adjusts the number of homozygote genotypes to reflect the estimated frequency of null alleles and the likely number of homozygotes given the adjusted allele frequencies and assuming random mating. We then repeated the previous differentiation analysis using the database adjusted by the frequency of null alleles. Second, pairwise F ST with and without the null allele correction was estimated using the expectation-maximization (EM) algorithm [START_REF] Dempster | Maximum likelihood from incomplete data via the EM algorithm[END_REF] with ENA correction to give an accurate estimate of F ST in the presence of null alleles using FREENA [START_REF] Chapuis | Microsatellite null alleles and estimation of population differentiation[END_REF]. The uncorrected and corrected pairwise F ST were then compared by means of a paired t-test.

To estimate the number of genetically differentiated groups, Bayesian-based clustering was used as implemented in STRUCTURE 2.3.4 [START_REF] Pritchard | Inference of population structure using multilocus genotype data[END_REF]. STRUCTURE was run using the admixture model, the assumption of correlated allelic frequencies between clusters, with and without the recessive alleles option that accounts for the null alleles [START_REF] Falush | Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies[END_REF][START_REF] Falush | Inference of population structure using multilocus genotype data: dominant markers and null alleles[END_REF], and considering sampling site information [START_REF] Hubisz | Inferring weak population structure with the assistance of sample group information[END_REF]). From [START_REF] Zakas | Restricted gene flow in Chilean barnacles reflects an oceanographic and biogeographic transition zone[END_REF] and [START_REF] Laughlin | Mitochondrial lineages in Notochthamalus scabrosus as indicators of coastal recruitment and interactions[END_REF], we know that k = 1 can be rejected, so all runs were made for k values between 2 and 10. Ten independent runs with 500000 Markov chain Monte Carlo replicates and a burn-in length of 50000 were used for each value of k. In order to select the k value that best captures the structure of the data, the statistic Δk, a measure of the second-order rate of change in the likelihood of k [START_REF] Evanno | Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study[END_REF] was implemented in STRUCTURE HARVESTER [START_REF] Earl | STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method[END_REF], and the values of Δk as a function of k were plotted. In CLUMPP 1.1 [START_REF] Jakobsson | CLUMPP: a cluster matching and permutation program for dealing with label switching and multimodality in analysis of population structure[END_REF], we merged the results of the 10 runs for each value of k, and DISTRUCT 1.1 [START_REF] Rosenberg | DISTRUCT: a program for the graphical display of population structure[END_REF]) was used to graphically visualize the results.

Demographic variables

Recruitment rates

At each site, an estimate of arrival rates of larval N. scabrosus was obtained by quantifying recruitment onto 10 × 10 cm Plexiglas plates covered with SafetyWalk TM (3M), an anti-slip surface that provides a heterogeneous substrate for larvae settlement and ensures homogeneity of conditions across plates and sites [START_REF] Menge | Recruitment vs. postrecruitment processes as determinants of barnacle population abundance[END_REF]. Five replicate collectors were fastened to the rocky substrate with stainless-steel bolts in the mid-upper intertidal zones of rocky platforms exposed to swell. Replicate collectors were replaced monthly, and recruitment rates were standardized to the number of ind. collector -1 d -1 . The monthly recruitment rates were then averaged to obtain the annual recruitment rates, and these in turn were averaged over the years to estimate the per site recruitment rate. At 8 of the 10 study sites, the collectors were initially deployed in late 1999 or early 2000, whereas at the 2 northernmost sites (CBaj and Apol), recruitment surveys began in mid-2009. The recruitment time series used here covered the period up to December 2013 for all sites.

Adult cover (abundance)

At each site, the benthic abundance of N. scabrosus was estimated using 7 to 10 quadrats of 0.25 m 2 , located along ca. 20-30 m alongshore transects. Transects were repeated at 3 intertidal elevations (low, mid-, and high intertidal zones) of the same rocky platforms where we deployed larval collectors. The 50 × 50 cm quadrat frame was divided into 25 equal squares with monofilament line, which was used to visually estimate adult abundance of N. scabrosus as percentage cover. Cover surveys were conducted approximately every 6 mo. For more details about the field methods, see [START_REF] Broitman | Geographic variation in diversity of wave exposed rocky intertidal communities along central Chile[END_REF].

Environmental and geographic variables

Environmental heterogeneity imposed by hydrographic conditions such as SST and productivity can directly or indirectly affect population genetic structure in marine organisms [START_REF] Bekkevold | Environmental correlates of population differentiation in Atlantic herring[END_REF][START_REF] Mendez | Isolation by environmental distance in mobile marine species: molecular ecology of franciscana dolphins at their southern range[END_REF][START_REF] Wei | Seascape genetics of the New Zealand greenshell mussel: sea surface temperature explains macrogeographic scale genetic variation[END_REF]. A multivariate indicator of environmental variability was constructed to test for correlation with the spatial genetic structure of N. scabrosus. To this end, spatio-temporal variations in chlorophyll a (chl a) concentration (mg m -3 ) and SST (°C) over a period of 10 yr (January 2003 to December 2013) were processed from monthly averages of Aqua MODIS satellite data with a 4 km spatial resolution using MatLab R2014a. Temporal variability in chl a and SST across the region was then decomposed by 2 separate principal component analyses (PCAs) of the respective time series, so that scores of sites on PC1 and PC2 (typically called empirical orthogonal function (EOF1 and EOF2) in the oceanographic literature, as they are carried out in the time domain), were used as multivariate representations of environmental conditions for either SST or chl a. In these analyses, the first axis (PC1) is dominated by the seasonal amplitude, with positive/negative values corresponding to sites with strong/weak seasonality. The second mode (PC2) is dominated by higher frequency variability, which in our system is chiefly synoptic variation corresponding to upwelling dynamics (see [START_REF] Wieters | Benthic community structure and spatiotemporal thermal regimes in two upwelling ecosystems: comparisons between South Africa and Chile[END_REF], Tapia et al. 2014[START_REF] Valdivia | Disentangling the effects of propagule supply and environmental filtering on the spatial structure of a rocky shore metacommunity[END_REF] for similar analyses). Latitudinal (Lat) and longitudinal (Lon) positions of each site were used as descriptors of geographic structure.

Environmental/demographic/geographic-genetic association analysis

Three complementary approaches, i.e. simple linear regressions, multi model selection, and matrix regression, were employed to test for associations among environmental, demographic, and geographic factors with the spatially structured genetic diversity of N. scabrosus. The linear regressions and model selection analyses used location (site)-specific data to evaluate the influence of our explicative variables on genetic diversity across sampling sites. The matrix regression analyses used the explicative variables as a proxy of 'seascape resistance' [START_REF] Mcrae | Isolation by resistance[END_REF] to evaluate their effects on among-site genetic differentiation. Our 8 predictive variables were the PC1 and PC2 of chl a and SST (hereafter CHLA1, CHLA2, SST1, and SST2), long-term averages of recruitment rates (Rec) and adult cover (Cov), and geographic location (Lat and Lon). The variables Rec and Cov were log 10 transformed before analyses to approximate normal distributions.

First, we conducted least squares (LS) linear multiple regression analyses between each metric of genetic diversity (Ar and Gd) and our 8 predictive variables. Second, a sensitivity analysis was performed using generalized linear models (GLMs) to select the best model of variables to explain spatial variation in our metrics of genetic diversity.

Because some predictor variables are highly correlated (see Table S3), we examined the impact of collinearity using the variance inflation factor (VIF) before running analyses. The variables Lat, Rec, and SST2 showed high (>10) VIF values, and were removed to minimize VIF values (<5). We then followed a stepwise approach for the sensitivity analysis, which was repeated for the 2 dependent variables (Ar and Gd) in R (R Core Development Team 2017): We (1) ran a full GLM that includes all predictive variables with VIF values <5; (2) examined the scatterplot of residuals versus predicted values (both in terms of the slope of the relationship and in the dispersion of the values) to check for the absence of trends; (3) sequentially removed (one by one) all predictive variables that were not significant (p > 0.05); (4) selected the most parsimonious 'suboptimal' GLM through a manual stepwise procedure according to the relative contribution of each factor to the variance explained by the model retaining predictive variables with a relative contribution 10%; (5) computed LS means of the dependent variable for each model parameter in order to assess the effect of specific variables on the dependent variables.

As an alternative way to obtain the best subset of explicative variables, we performed stepwise selection (both forward and backward) using the stepAIC function from the 'mass' package in R. This function uses the exact Akaike's information criterion (AIC) as the model selection criterion. Third, we used multiple regressions on distance matrices (MRDM; [START_REF] Manly | Randomization and regression methods for testing for associations with geographical, environmental and biological distances between populations[END_REF][START_REF] Legendre | Modeling brain evolution from behavior: a permutational regression approach[END_REF]) to estimate the independent effects of explicative variables on N. scabrosus among-site genetic differentiation. Briefly, partial regression slopes were estimated using standard multiple linear regression, but the significance of each term was determined by randomly permuting the explanatory variables one at a time while keeping the others constant [START_REF] Wang | Examining the full effects of landscape heterogeneity on spatial genetic variation: a multiple matrix regression approach for quantifying geographic and ecological isolation[END_REF]). This analysis was implemented using the 'ecodist' package [START_REF] Goslee | The ecodist package for dissimilarity-based analysis of ecological data[END_REF] in R, and significance was based on 10000 permutations using the genetic distance matrices (θ ST and D EST ) as response variables. Each of 8 matrices representing environmental, demographic, and geographic differences among sites were used as the predictor variables. Data were converted into matrices of pairwise distances calculating the absolute differences from sitespecific values of each variable following [START_REF] Amaral | Seascape genetics of a globally distributed, highly mobile marine mammal: the short-beaked common dolphin (genus Delphinus)[END_REF]. Due to its high VIF value (>10), the geographical variable Lat was removed, so a subset of 7 predictor variables was retained for the subsequent analysis.

RESULTS

Genetic polymorphism

The 7 microsatellite loci reached a total of 158 alleles in the 300 individuals of Notochthamalus scabrosus genotyped, which ranged from 79 in CBaj to 95 in Temb. In addition, we found 27 private alleles, with the highest number observed at Apol (Pa = 6). In contrast, Pich shared all of its alleles with most other sites. The Ar ranged from 11.0 in CBaj to 13.2 in Temb (mean Ar = 12.01 ± 0.72), while Gd ranged from 0.77 in ECIM to 0.83 in Apol (mean Gd = 0.81 ± 0.02). Both Ar and Gd indices showed a peak at Guan, Temb and Apol (located around Coquimbo Bay), while the lowest values were found at CBaj and ECIM for Ar, and at ECIM and Mont for Gd (Table S2).

All 10 populations exhibited significant heterozygote deficiency. Of the 70 sitelocus combinations, 58 showed a significant deviation from HWE based on Fisher's exact test and after FDR correction, while only 37 had significantly positive F IS -values based on a permutation test (Table S2). Using the corrected database for null alleles, 56 site-locus combinations remained significantly deviating from HWE with the exact test, and 30 sitelocus comparisons still had significant F IS >0 with permutation tests (Table S2).

Of the 210 linkage disequilibrium tests performed, none was significant after correcting for false positives (FDR), and none of the global tests for each pair of loci across sites was significant. The estimated frequency of null alleles by site-locus combination varied between 0 and 0.379, with an average frequency of 0.156 (SD = 0.089) across loci and sites (Table S2).

Population genetic structure

N. scabrosus showed statistically significant global genetic structure (θ ST = 0.013, p < 0.001; D EST = 0.040, p < 0.001). Pairwise θ ST and D EST were significant for 27 and 21 of the 45 comparisons at the nominal level ( = 0.05), of which 22 and 15 remained significant after corrections for multiple tests, respectively (Table 1). Significant pairwise comparisons were mostly between sites north of PTal versus sites south of LMol, and the sites with lowest and highest levels of differentiation were Guan vs. PTal (separated by 50.26 km) and Temb vs. Cura (separated by 419.88 km), respectively. CBaj had the highest number of significant pairwise comparisons for both θ ST and D EST (n = 9 and 8, respectively) followed by LMol (n = 7 and 6, respectively; Table 1). The first 2 coordinates of PCoAs with θ ST and D EST values explained 87.62 and 87.88% of total variation, respectively, and revealed similar structuring of sites (Fig. 2). The first axis of the PCoAs separated 2 principal groups, one composed of sites from LMol to the south, the other with the 3 northern sites (Temb, Apol, and CBaj), whilst Guan and PTal were between these 2 groups. Weak separation of sites within these regions was detected along the second PCoA axis, with Temb separated from Apol and CBaj, and LMol and Cura from Mont, ECIM, and Pich (Fig. 2).

Null alleles had some effect on our results: (1) the ENA method gave slightly, but significantly, lower F ST values (average F ST with ENA = 0.00815, SD = 0.00693) than those obtained without correction for the presence of null alleles (average F ST without ENA = 0.00951, SD = 0.00847; paired t = 3.74, p < 0.001); (2) global structure was lower but still significant with the adjusted database (θ ST = 0.012, p < 0.001; D EST = 0.018, p = 0.001); and (3) there were fewer significant pairwise comparisons after FDR corrections for D EST (only 1 significant comparison) and θ ST (15 of 22 comparisons still significant).

However, the main pattern of differentiation between sites north and south of PTal-LMol persisted with the adjusted database, as well as the most and least differentiated pairwise comparisons (Table 1).

The cluster analysis performed using STRUCTURE confirmed the existence of 2 clusters, one south of LMol and the other north of PTal (Fig. 3). For k = 3, a new cluster included CBaj, the northernmost site. According to Evanno's criteria, k = 4 was the most likely number of clusters (Fig. S1). However, no clear spatial pattern could be recovered from the assignment of individuals into these 4 clusters. This may be due to the correlated allele frequencies model, which tolerates differentiation of closely related populations, but is likely to overestimate k [START_REF] Pritchard | Inference of population structure using multilocus genotype data[END_REF]. The same trends were observed with the full or the adjusted databases (results not shown).

Demographic/geographic/environmental-genetic association analysis

Linear regressions showed that CHLA2 alone explained 48 and 61% of the total variance in Ar and Gd, respectively, having a significant positive linear relationship with both genetic diversity indices throughout the study region (Fig. 4). Additionally, Cov explained 33% of the variance of Ar, and SST2 and Lat explained 25 and 30% of the variance of Gd, respectively, but these relationships were not statistically significant (Fig. 4).

Statistical control of covariables using GLM model selection identified the variable CHLA2 as the most significant factor explaining variation in both Ar and Gd (Table 2). The second and third best models include the variables CHLA1 and Cov, which is consistent with results of the model selection based on AIC (Table 3), but the fraction of variance explained by these variables was minor in comparison to CHLA2 (see Table 2).

A different result was obtained from the MRDM analysis, which showed that the spatial structure (differences among sites) in SST2 and Cov had the strongest effects on genetic differentiation, as measured by θ ST and D EST . The overall model showed significant fit to the data (p < 0.05), and explained 55% of the total variance (Table 4).

DISCUSSION

The extent of effective dispersal and gene flow between populations in the coastal ocean can be much more complex than previously thought (e.g. [START_REF] Pringle | Going against the flow: maintenance of alongshore variation in allele frequencies in a coastal ocean[END_REF][START_REF] Teske | Oceanography promotes self-recruitment in a planktonic larval disperser[END_REF]). In the present study, we found subtle, yet significant levels of genetic differentiation in the intertidal barnacle Notochthamalus scabrosus, a species with high dispersal potential. Main differences occurred between sites located to the north and south of the reported phylogeographic latitudinal break at 30° S.

Our results suggest that population genetic diversity in N. scabrosus is influenced by environmental regimes manifested in patterns of temporal variability of surface chl a concentration, whereas among-site differences in SST fluctuations and benthic abundance of adults appear to be significant drivers of population genetic differentiation over space.

More broadly, the presence of sites that are both environmentally and genetically differentiated supports the idea of an ecological restriction to population connectivity, despite the long residence of larvae in the water column.

Larval arrival from the plankton can be responsible for local abundance and genetic variability patterns of benthic populations [START_REF] Iacchei | Combined analyses of kinship and FST suggest potential divers of chaotic genetic patchiness in high gene-flow populations[END_REF]. We found that the temporal variability in surface chl a was the most consistent covariable explaining spatial distribution of N. scabrosus genetic diversity. This general result is in line with studies showing that patterns of intraspecific genetic diversity of some mobile marine species are associated with variation in chlorophyll concentration [START_REF] Gaggiotti | Disentangling the effects of evolutionary, demographic, and environmental factors influencing genetic structure of natural populations: Atlantic herring as a case study[END_REF][START_REF] Mendez | Isolation by environmental distance in mobile marine species: molecular ecology of franciscana dolphins at their southern range[END_REF][START_REF] Amaral | Seascape genetics of a globally distributed, highly mobile marine mammal: the short-beaked common dolphin (genus Delphinus)[END_REF]. Variability in coastal chl a may be viewed as an integrated indicator of the environmental conditions to which invertebrate larvae and onshore adults are exposed, and likely determines both the feeding conditions (i.e. quantity and quality of food) and the larval transport to/off the shore. During upwelling, high food availability can translate into better larval condition and, at the same time, offshore and alongshore upwelling currents can promote the mixing of the offshore larval pool [START_REF] Barshis | Coastal upwelling is linked to temporal genetic variability in the acorn barnacle Balanus glandula[END_REF]. Then, during upwelling relaxation and downwelling events, this well fed/well mixed larval pool can reach local populations. In this manner, sites with constant strong upwelling have few possibilities of larval arrival due to increased larval waste [START_REF] Roughgarden | Recruitment dynamics in complex life cycles[END_REF][START_REF] Menge | Dynamics of coastal meta-ecosystems: the intermittent upwelling hypothesis and a test in rocky intertidal regions[END_REF], while on the other hand, sites with constant weak upwelling have more larval retention, therefore their recruitment comes from a poorly mixed larval pool. Other things being equal or homogeneous, high phytoplankton availability in coastal waters during larval development can therefore lead to higher recruitment (e.g. [START_REF] Olson | Food limitation of planktotrophic marine invertebrate larvae: Does it control recruitment success?[END_REF][START_REF] Cushing | Plankton production and year-class strength in fish populations:an update of the match /mismatch hypothesis[END_REF][START_REF] Menge | Recruitment vs. postrecruitment processes as determinants of barnacle population abundance[END_REF] and high larval physiological quality that should improve post-settlement survival [START_REF] Jarrett | Temporal variation in cyprid quality and juvenile growth capacity for an intertidal barnacle[END_REF][START_REF] Hentschel | Metamorphosis of barnacle nauplii: effects of food variability and a comparison with amphibian models[END_REF][START_REF] Phillips | Effects of nutrition-mediated larval condition on juvenile performance in a marine mussel[END_REF]) as well as overall juvenile condition [START_REF] Bertness | Extreme spatial variation in the growth and reproductive output of the acorn barnacle Semibalanus balanoides[END_REF][START_REF] Menge | Rocky intertidal oceanography: an association between community structure and nearshore phytoplankton concentration[END_REF][START_REF] Sanford | Spatial and temporal variation in barnacle growth in a coastal upwelling system[END_REF]. All of these factors may result in the maintenance of genetic diversity from the larval pool. Thus, variable upwelling will maximize larval condition and genetic diversity and, as predicted by the intermittent upwelling hypothesis [START_REF] Menge | Dynamics of coastal meta-ecosystems: the intermittent upwelling hypothesis and a test in rocky intertidal regions[END_REF], increase onshore recruitment. Further genetic studies should therefore intensify sampling of recently settled larvae across more diverse upwelling conditions.

Indirect evidence about the effect of upwelling/relaxation dynamics on barnacle recruitment [START_REF] Navarrete | Scales of benthic-pelagic coupling and the intensity of species interactions: from recruitment limitation to topdown control[END_REF][START_REF] Lagos | Spatial environmental correlates of intertidal recruitment: a test using barnacles in northern Chile[END_REF]) and the significant positive cross-correlations between mean chl a concentration and N. scabrosus recruitment and adult abundance (Table S3) suggest that it is a possible mechanism to explain the genetic pattern in N.

scabrosus.

Adult cover was used as a proxy of local abundance of N. scabrosus, a factor that in linear regressions explained 33% of total variance in allelic richness (although it was not statistically significant, Fig. 4). From examination of Fig. 4f, it seems clear that the site ECIM deviates largely from an otherwise good positive relationship formed by the other 9 sites. Indeed, removing ECIM from the analysis increases the relationship to r 2 = 0.57 (p = 0.019). The departure of ECIM from the general pattern illustrates well the complexity of determinants of genetic diversity in natural systems and why such univariate relationships between population size and genetic diversity are rarely found in marine environments (but see [START_REF] Mccusker | Positive relationships between genetic diversity and abundances in fishes[END_REF]. ECIM has some of the historically highest recruitment rates for N. scabrosus in the region [START_REF] Navarrete | Interhemispheric comparison of recruitment to intertidal communities: pattern persistence and scales of variation[END_REF]), yet it displays one of the lowest levels of genetic diversity (in both Ar and Gd indices, Table S2). Furthermore, only at ECIM did individuals have levels of relatedness significantly larger than expected from HWE (Fig. S2).

ECIM is located within Cartagena Bay, an open bay exposed to the southern swell, but in an 'upwelling shadow' where upwelling advection is largely reduced, apparently leading to high phytoplankton concentration [START_REF] Wieters | Alongshore and temporal variability in chlorophyll a concentration in Chilean nearshore waters[END_REF] and stronger stratification than other sites [START_REF] Kaplan | Large diurnal temperature fluctuations in the nearshore water column[END_REF][START_REF] Bonicelli | Wind-driven diurnal temperature variability across a small bay and the spatial pattern of intertidal barnacle settlement[END_REF]. On other shores of the world, low current velocities and water re-circulation, leading to increased local larval retention [START_REF] Mcshane | Recruitment processes in Haliotis rubra (Mollusca: Gastropoda) and regional hydrodynamics in southeastern Australia imply localized dispersal of larvae[END_REF], create distinctive patterns of genetic diversity in local populations (e.g. [START_REF] Dupont | Limits to gene flow in the slipper limpet Crepidula fornicata as revealed by microsatellite data and a larval dispersal model[END_REF][START_REF] Nicastro | Coastal topography drives genetic structure in marine mussels[END_REF][START_REF] Olivares-Bañuelos | Population structure of purple sea urchin Strongylocentrotus purpuratus along the Baja California peninsula[END_REF]. Thus, increased larval retention at ECIM, with comparatively low immigration from other populations, as suggested by numerical circulation models [START_REF] Aiken | Along-shore larval dispersal kernels in a numerical ocean model of the central Chilean coast[END_REF][START_REF] Ospina-Alvarez | Larval transport in the upwelling ecosystem of central Chile: the effects of vertical migration, developmental time and coastal topography on recruitment[END_REF]) and observational studies [START_REF] Bonicelli | Wind-driven diurnal temperature variability across a small bay and the spatial pattern of intertidal barnacle settlement[END_REF], may explain the higher genetic relatedness levels observed at this site. The reduced gains of genetic diversity from other sites (poorly mixed larval pool) may be the cause of reduced allelic richness, further supporting the relevance of connectivity patterns on adult population size and genetic diversity.

In natural populations, a genetic discontinuity along a continuously colonized range can arise as a consequence of an environmental discontinuity, either through selection against migrants or reduced fitness of interlineage hybrids [START_REF] Nosil | Reproductive isolation caused by natural selection against immigrants from divergent habitats[END_REF]. Both mechanisms involve local adaptation in response to selection imposed by divergent biotic or abiotic conditions [START_REF] Sanford | Local adaptation in marine invertebrates[END_REF][START_REF] Pflüger | A plea for simultaneously considering matrix quality and local environmental conditions when analyzing landscape impacts on effective dispersal[END_REF]. Our results support the idea that 'environmental distance,' imposed by among-site differences in SST, is a relevant factor to explain genetic differentiation among N. scabrosus populations.

Indeed, a similar effect has been observed in mammals [START_REF] Fullard | Population structure of long-finned pilot whales in the North Atlantic: a correlation with sea surface temperature?[END_REF][START_REF] Amaral | Seascape genetics of a globally distributed, highly mobile marine mammal: the short-beaked common dolphin (genus Delphinus)[END_REF], fishes [START_REF] Han | Phylogeography study of Ammodytes personatus in Northwestern Pacific: Pleistocene isolation, temperature and current conducted secondary contact[END_REF], Diopere et al. 2018), and intertidal and shallow (<5 m depth) coastal invertebrate species [START_REF] Banks | Genetic structure of a recent climate change-driven range extension[END_REF][START_REF] Wei | Seascape genetics of the New Zealand greenshell mussel: sea surface temperature explains macrogeographic scale genetic variation[END_REF]. Seawater temperature is also one of the most important factors controlling reproduction, development, and growth of ectothermic invertebrates [START_REF] Pechenik | Environmental influences on larval survival and development[END_REF][START_REF] O'connor | Temperature control of larval dispersal and the implications for marine ecology, evolution, and conservation[END_REF][START_REF] Byrne | Impact of ocean warming and ocean acidification on marine invertebrate life history stages: vulnerabilities and potential for persistence in a changing ocean[END_REF]). In the case of N. scabrosus, such adaptive divergence could be related to selective sorting of competent larvae and/or to post-settlement processes such as temperature requirements for metamorphosis and initial growth, or desiccation tolerance of recruits. Further studies combining genomic tools with high-resolution dispersal models and local experiments with settlers are necessary to discern among the possible mechanisms of population divergence.

CONCLUSION

Population genetic structure of Notochthamalus scabrosus, as assessed by neutral markers, is characterized by a sharp genetic discontinuity around 30° S, confirming previous conclusions based on mtCOI [START_REF] Zakas | Restricted gene flow in Chilean barnacles reflects an oceanographic and biogeographic transition zone[END_REF][START_REF] Laughlin | Mitochondrial lineages in Notochthamalus scabrosus as indicators of coastal recruitment and interactions[END_REF]. A modeling study by [START_REF] Ewers-Saucedo | The oceanic concordance of phylogeography and biogeography: a case study in Notochthamalus[END_REF] showed that dispersal alone could not generate such genetic discontinuity, and that differential lineage performance in adjacent but divergent environments must be considered. Our results strongly suggest that the environment is indeed influencing the spatial pattern of genetic diversity in N. scabrosus. Two main mechanisms could be hypothesized: temporal variability of the food (variation in phytoplankton abundance) and dispersive (upwelling-associated currents) coastal environments favor recruitment from a well fed/well mixed larval pool and therefore increase the allelic richness of benthic populations; and the ecological divergence in coastal ocean temperature may restrict effective dispersal across the 30° S boundary. Such patterns have not been observed in other barnacles, which are traditionally assumed to have large effective population sizes and large dispersal capacity, both of which could override the effects mentioned above. We interpret these results as suggestive that coastal circulation can limit larval connectivity among some populations, generating incomplete barriers to dispersal, which in turns facilitates effects of isolation by environment. Hence, the results of this work advance our understanding of how environmental seascapes can shape patterns of genetic diversity and population differentiation. In particular, our results highlight the importance of further defining the causal relationships between environmental variables and genetic diversity patterns of wild populations in order to guide future region-wide conservation and management efforts.

Acknowledgements. We thank to members of B.R.B.'s and S.A.N.'s groups for field assistance, as well as Gioconda Peralta for her guidance during DNA sequencing analyses. M.B. was supported by a CONICYT Doctoral Fellowship. The research was supported by the Center for Marine Conservation (grant ICM CCM RC130004) and the Center for Multiple Drivers of Marine Socio-Ecological Systems (grant ICM MUSELS NC120086), both of the Iniciativa Científica Milenio of the Ministerio de Economia, Fomento y Turismo. Further support for field work and oceanographic studies was provided by FONDECYT (grants 1160289 to S.A.N. and 1181300 to B.R.B.). LITERATURE CITED Aguirre C, Garreaud RD, Rutllant JA (2014) Surface ocean response to synoptic-scale variability in wind stress and heat fluxes off south-central Chile. Dyn Atmos Oceans

Table 1. Among-site genetic differentiation of Notochthamalus scabrosus at 7 microsatellite loci. Analyses were done with (A) the original database and (B) the database corrected for null alleles. Values of θ ST are above the diagonal and D EST values are below the diagonal. CBaj: Carrizal Bajo, Apol: Apolillado, Temb: Temblador, Guan: Guanaqueros, PTal: Punta Talca, LMol: Los Molles, Cura: Curaumilla, ECIM: Estación Costera de Investigaciones Marinas (Las Cruces), Pich: Pichilemu (see Fig. 1).


S Rizzolo
email: serena.rizzolo@univ-st-etienne.fr

A Boukenter

T Allanche

Y Ouerdane

S Girard

G Bouwmans

H El Hamzaoui

L Bigot

M Cannas

J Perisse

J-R Macé

S Bauer



Abstract-We investigated the capability of microstructured optical fibers to develop multi-functional, remotely-controlled, Optical Frequency Domain Reflectometry (OFDR) distributed fiber based sensors to monitor temperature in nuclear power plants or high energy physics facilities. As pure-silica-core fibers are amongst the most radiation resistant waveguides, we characterized the response of two fibers with the same microstructure, one possessing a core elaborated with F300 Heraeus rod representing the state-of-the art for such fiber technology and one innovative sample based on pure sol-gel silica. Our measurements reveal that the Xray radiations do not affect the capacity of the OFDR sensing using these fibers to monitor the temperature up to 1 MGy dose whereas the sensing distance remains affected by RIA phenomena. Index Terms-Optical fiber sensors, radiation, Rayleigh scattering.

I. INTRODUCTION

of Fiber Bragg Gratings (FBGs) [START_REF] Morana | Radiation tolerant fiber Bragg gratings for high temperature monitoring at MGy dose levels[END_REF] or with distributed measurements based on scattering mechanisms [START_REF] Phéron | High γ-ray dose radiation effects on the performances of Brillouin scattering based optical fiber sensors[END_REF]- [START_REF] Rizzolo | Radiation Effects on OFDR based sensors[END_REF].

In the panorama of fiber-based distributed sensing techniques several studies are devoted to investigate the employment of these sensors classes in harsh environments [START_REF] Phéron | High γ-ray dose radiation effects on the performances of Brillouin scattering based optical fiber sensors[END_REF]- [START_REF] Rizzolo | Radiation Effects on OFDR based sensors[END_REF]. In particular, X. Phéron et al. [START_REF] Phéron | High γ-ray dose radiation effects on the performances of Brillouin scattering based optical fiber sensors[END_REF] reported that Brillouin based sensors are affected by radiation because it induces an additional Brillouin frequency shift (BFS) that affects temperature and strain measurements. On the other hand, Radiation Induced Attenuation (RIA) degrades the response of Raman temperature based sensors by degrading differently the Stokes and Anti-Stokes signals [START_REF] Cangialosi | Hydrogen and radiation induced effects on performances of Raman fiber-based temperature sensors[END_REF], [START_REF] Cangialosi | Development of a Temperature Distributed Monitoring System Based On Raman Scattering in Harsh Environment[END_REF]. OFDR is one of the most promising techniques since it offers the best spatial resolution of few µm over 70 m of fiber length [START_REF] Soller | High resolution optical frequency domain reflectometry for characterization of components and assemblies[END_REF]. Moreover, our recent study showed that radiation does not affect Rayleigh mechanisms at the basis of this technique: indeed, temperature and strain coefficients remain unchanged, within the 5%, of error up to 10 MGy (SiO 2 ) for a large variety of standard fiber classes [START_REF] Rizzolo | Vulnerability of OFDR-based distributed sensors to high γ-ray doses[END_REF]. However, the potentialities of OFDR sensors are affected by RIA phenomena [START_REF] Rizzolo | Vulnerability of OFDR-based distributed sensors to high γ-ray doses[END_REF] which limits the sensing range. Therefore, before integration of the OFDR-based systems in harsh environments, their tolerance to the constraints associated with high levels of radiations have to be demonstrated.

The parameters having significant effects on RIA are well known, a very influential one concerns the nature of the dopants used to modulate the refractive index profile of the fiber core and cladding [START_REF] Girard | Gamma-rays and pulsed X-ray radiation responses of germanosilicate single-mode optical fibers: influence of cladding codopants[END_REF], [START_REF] Friebele | Compositional effects on the radiation response of Ge-doped silica-core optical fiber waveguides[END_REF]. Radiation also, can introduce structural changes in glass matrix by varying locally refractive index and modifying the scattering amplitude as a function of the length for a given fiber. In terms of optical fiber sensors (OFSs) performances, these changes can influence Rayleigh distributed measurements introducing errors in the measurement of controlled parameter, especially for in situ measurements.

The development of microstructured optical fibers (MOFs) is one of the most innovative progresses in the field of optical waveguides. These fibers present wavelength scale structures with a refractive index contrast that provides them unusual properties [START_REF] St | Photonic-crystal fibers[END_REF]. Two types of MOFs are usually distinguished: photonic bandgap (PBG) MOFs in which the light remains confined in a low index core thanks to a PBG cladding and high index core fibers in which the light is guided via modified total internal reflection (MTIR). D increased their demand for several applications such as sensors, diagnostics…, in particular for their employment in harsh environments associated with nuclear power plants, space or high energy physics facilities [START_REF] Berghmans | An Introduction to Radiation Effects on Optical Components and Fiber Optic Sensors[END_REF]. Thanks to some fibers advantages such as fast response, reduced volume and weight, several techniques are developed for monitoring different environmental parameters such as temperature, strain and radiation dose by replacing punctual sensor technologies by one sensor exploiting the whole optical fiber length sensitivity to external change [START_REF] Bao | Recent Progress in Distributed Fiber Optic Sensors[END_REF] in a discrete way as the case In literature, the transient radiation response of hollow core PBG (HC-PBG) fibers [START_REF] Girard | Radiation-induced effects in a new class of optical waveguides: the airguiding photonic crystal fibers[END_REF] is discussed and their steady state radiation responses have been presented by G. Cheymol et al. in [START_REF] Cheymol | High level gamma and neutron irradiation of silica optical fibers in CEA OSIRIS nuclear reactor[END_REF]- [START_REF] Olanterä | Gamma irradiation of minimal latency Hollow-Core Photonic Bandgap Fibres[END_REF]. From these studies, it appears that HC-PBG fibers are very promising for integration in steady state high dose level radiation environments where they exhibit RIA as low as 2.1 dB/km after 1 MGy at 1550 nm [START_REF] Olanterä | Gamma irradiation of minimal latency Hollow-Core Photonic Bandgap Fibres[END_REF]. However the results are more complex for transient -or pulsed-irradiation, as discussed in [START_REF] Girard | Radiation-induced effects in a new class of optical waveguides: the airguiding photonic crystal fibers[END_REF]. Furthermore, several issues limit the use of HC-PBG fiber such as the reliability, the splicing with other fibers, sources or detectors and their high costs. On the other hand, MTIR MOFs are easier to handle and present several useful properties that could be exploited to design new laser or plasma fiber-based diagnostics. Depending on their microstructuration, such fibers can be endlessly single-mode, present less sensitivity to bending loss, larger mode field diameters to limit non-linear phenomena occurring at high signal power. Another possible advantage, under irradiation and for fiber sensing, consists in their homogeneity in terms of glass properties. Such fibers can be made of a unique glass composition, without dopants or impurities to limit the fiber transmission degradation [START_REF] Girard | Gamma-rays and pulsed X-ray radiation responses of germanosilicate single-mode optical fibers: influence of cladding codopants[END_REF] or to limit the residual strain resulting from the different properties of core and cladding. To our knowledge, only a few studies have been devoted to the radiation response of this class of waveguides [START_REF] Girard | Gamma-radiation-induced attenuation in photonic crystal fibre[END_REF]- [START_REF] Girard | Transient radiation-induced effects on solid core microstructured optical fibers[END_REF]. In [START_REF] Girard | Gamma-radiation-induced attenuation in photonic crystal fibre[END_REF], a prototype MOF was tested and its high radiation sensitivity was attributed to the silica glass tubes used to design the fiber, the tested fiber being no more representative of today's advances. More complete comparative studies between MOFs and conventional fibers showed that puresilica or fluorine doped MOFs, which offers specific advantages compared to conventional fibers, are promising for use in harsh environments as the ones associated with Megajoule class lasers due to their radiation tolerance [18] [19].

In this work we study the steady state effects on Rayleigh radiation response of MOFs for distributed temperature and strain sensors up to 1 MGy dose levels. In particular, we explored the performances of Sol Gel originated MOFs, a promising technique that permits to obtain several classes of fibers with unique optical properties [20] [21]. Up to the best of our knowledge, this is the first study combining OFDR technique with MOFs, and for this reason we focused our attention on two pure silica core (PSC) samples that are known to be among the most radiation resistant classes of fibers, to compare our results with their standard counterpart. The goal of this work was to investigate on sensing capability of MOFs adapted for radiation applications. For this, we experimentally determined the temperature sensitivity coefficient of the two MOF samples before and after irradiation and we compared, during irradiation, the temperature changes monitored by thermocouple and through our OFDR sensor probing the added values of MOF fibers. A characterization of spectral RIA and its kinetic are also reported to evaluate the RIA related limitation that will lead to a comprehension on the work to be done for the integration of MOF distributed sensors in harsh environments.

II. MATERIAL AND METHODS

A. Investigated samples

The two investigated air/silica MOFs have been fabricated by the PhLAM-IRCICA laboratory in Lille (France) by the stack and draw technique. Both fibers present the same microstructured geometry, reported in Fig. 1 (a), with a hole diameter of 1.8 μm and a pitch Λ =3.9µm. The two fibers are made of different pure silica classes.

MOF1 is entirely made of pure-silica glass F300 tubes (Heraeus) with an adapted procedure to ensure a limited concentration of OH group at the tube surfaces that causes an excess of optical losses. The optical losses in this fiber (MOF1) are around 4 dB/km at 1550 nm.

MOF2 fiber core originates from Sol-Gel silica at low OH(<1ppm) [START_REF] Hamzaoui | From molecular precursors in solution to microstructured optical fiber: a Sol-gel polymeric route[END_REF] concentration and is combined to F300 Hereaus silica capillaries for the cladding region. Since no special cleaning procedure was applied to the sol-gel silica rod, the attenuation of this fiber (MOF2) is around 20 dB/km at 1550 nm. Fig. 1 (b) and Fig. 1 (c) report a 2D mapping of PL intensity around 400 nm under 325nm laser excitation in the microstructured cladding from -30 µm to 30 µm in both x and y direction with a step of 1.5 µm. This study highlights the presence of some emitting centers in the non-irradiated MOFs at the border of microstructured region due to processes fabrication. 1 (c) we can note that the presence of these centers is correlated with the presence of the most extern microstructured region: the centers appear in the region where the holes are not present. Likely, used tubes in the fabrication process presented some impurities inside that may cause the PL showed in Fig. 1 (b) and Fig. 1 (c). However, these emitting centers are far from the region of light propagation, thus not influencing the optical properties in the core.

B. Irradiation facilities

We investigate steady state as well as permanent radiation effect performing X-ray irradiations with the X-ray source facility (MOPERIX) in Laboratoire Hubert Curien (Saint Etienne, France). The dose-rate is evaluated with a probe in different points of the irradiation chamber, where cartography was performed. By a statistic done on obtained values in the chosen uniform region, we evaluate the dose-rate in our experiments that is (3.2±0.1) Gy(SiO 2 )/s and the reached dose is 1 MGy(SiO 2 ). Irradiations were performed at room temperature controlled by a K type thermocouple to register the fluctuation over time from day to night inside irradiation chamber.

C. Distributed temperature measurements

Distributed sensing measurements were done with an Optical Backscatter Reflectometer (OBR) 4600 from Luna Technologies. In all measurements described below the laser source was tuned over a range of 21 nm with a center wavelength around 1550 nm (with accuracy of 1.5 pm), yielding to a nominal spatial resolution of the Rayleigh scatter pattern of 40 µm along the investigated optical fiber propagation axis.

In situ OBR measurements were performed under X-ray irradiation on ~1 m -long fiber spooled on an aluminum plate in a circular zone of constant dose rate with a diameter of 8 cm. The acquisition is made every 5 sec during the first period of irradiation and then each 5 minutes; temperature is monitored by the comparison with a type K thermocouple data stuck on the same plate inside irradiation zone and close to the fiber.

Temperature calibration was done in an oven controlled by a type K thermocouple after irradiation; an example of recorded spectral shift as a function of optical fiber length is reported in Fig. 2 for non-irradiated and irradiated part in the different temperature steps. For such experiments, the samples of the same fiber type, both non-irradiated and irradiated one, were tested together (the total length of the sample was ~2m) to perform the temperature treatment at the same time. C T was deduced from the slope of the Rayleigh spectral shift with temperature in the range from 30 °C up to 70 °C with a ΔT of 5 °C.

D. Confocal micro-luminescence investigation

The permanent irradiation effects were studied via confocal micro-photoluminescence (PL) by using a LabRam Aramis spectrometer, equipped with a He-Cd ion laser probe emitting at 3.82 eV (325 nm), a x40 objective and micro-translation stages. The corresponding spatial resolution was about 1.5 μm.

The CML measurements are performed for both samples, before and after irradiation, the same experimental conditions (e.g. same length of the samples that were ~3 mm long, same days to assure not fluctuation of the laser source …), to compare the two samples as well as the non-irradiated irradiated fiber luminescence properties.

E. RIA measurements

In addition to the previously described tests, the same fibers were characterized with another setup allowing the measurement of the temporal (5 s time resolution) and spectral dependence (900-2200 nm) of the RIA during and after the Xray exposure. To this end, we used a laser-driven light source (EQ99 from Energetiq) and a near-IR spectrophotometer NIRQuest 512. For these measurements the samples are 20 m long and the temperature is monitored during the measurements with a type K thermocouple.

III. EXPERIMENTAL RESULTS

A. Evaluation of sensor performances

Temperature characterization on non-irradiated MOFs is shown in Fig. 3 where measured Rayleigh spectral shift as a function of temperature is reported together with linear fits for both samples. We observe a linear dependence in the two fibers, providing evidence for the potential of such fibers for temperature measurements. Extracted coefficients are obtained by making an average of the values obtained in each sensing point. We find close coefficients for the two fibers: (6.40±0.04) °C-1 for MOF1 and (6.44±0.01) °C-1 in MOF2, the errors being obtained from standard deviation on all the values. These values of C T are used to calculate the temperature of MOFs sensors during irradiation. To investigate the steady state radiation effect on OFDR measurements, we irradiated our samples up to a total dose of ~1 MGy(SiO 2 ) at a dose rate of (3.2±0.1) Gy/s through 87 hours run duration. In Fig. 4 are reported the obtained results for MOF1. We followed at the same time the temperature evolution with a thermocouple placed near the fiber in the irradiation zone. The temperature profiles obtained with the fiber and the thermocouple are shown in Fig. 4 (a), where fiber temperature was obtained by an average along the fiber segment under irradiation. We find that profiles are close and we are able to follow temperature increase during the first minutes of irradiation (due to the switch on of the X-ray tube) as well as temperature fluctuation from day to night inside the chamber. By comparing the temperatures measured by OFDR and the thermocouple we calculate the ΔT to highlight the possible differences caused by the irradiation. Results are shown in Fig. 4 (b) evidencing that radiation does not affect Rayleigh temperature measurements: indeed, except for a small decrease during the first minutes of irradiation, ΔT remains constant around the value of (-0.27±0.06) °C during the whole irradiation time. We found that temperature measured by OBR well agree with thermocouple one; however some differences are present between the two profiles. To better understand on the origin of this variation we show in Fig. 5 the temperature as a function of fiber length in MOF1 before the irradiation and for several doses up to 1 MGy. We can see that temperature is not homogeneous along the sample, with fluctuation from 0.7 °C at 10 kGy to 2 °C at 1 MGy. This profile can due to variation in the temperature in the irradiation chamber, since that temperature at the fiber level was not imposed by external devices, but also to the presence of some stressed parts in the spool that evolve with the time. Indeed, we note that temperature distribution remains more or less the same with the increasing of the doses. The same investigation was carried out for MOF2 and the results are reported in Fig. 6 and Fig. 7. As before, we see that OFDR and thermocouple temperature profiles match each other [Fig. 6 (a)] during the entire irradiation time, giving the possibility to detect temperature variation due to turning on of radiation as well as from day to night temperature changes. ΔT values in Fig. 6 (b) show that no radiation effects influence temperature measurements for MOF2 being variations scattered around 0 °C (with higher dispersion of 0.2 °C).

Temperature distribution along the fiber length is reported in Fig. 7 for non-irradiated MOF2 and sample irradiated at different doses. Here, we can see better what we already observe in MOF1. Indeed, temperature profiles along the fiber are the sample at the different doses. We can also note that between (13.40±0.01) m and (13.58±0.01) m the fiber temperature is substantially different from the other part of the sample. It is possible that when we stuck the fiber on the aluminum plate we apply some stress that evolves with time, thus leading to recorded variation. If we do not take in account this part of the sample, we obtain temperature variation along the fiber profile of 0.7 °C for all the doses. After irradiation we performed a new temperature calibration to evaluate possible changes in C T . Linear fits on Rayleigh spectral shift dependence on strain illustrated in Fig. 6. give C T values of (6.44±0.01) °C-1 in MOF1 and (6.44±0.02) °C-1 in MOF2 highlighting that radiation does not induce changes in temperature calibration curves.

B. Investigation on radiation induced MOF changes

Spatial distribution of radiation induced defects was studied over the transverse fiber section, thanks to the confocal microluminescence setup. Fig. 9 (a) shows typical PL spectra emitted from the core center and microstructured part of the cladding, in a region without holes, for non-irradiated and irradiated MOF1 at 1 MGy(SiO 2 ). Spectra exhibit a band peaked at 650 nm related to non-bridging hole oxygen centers (NBOHC) [START_REF] Skuja | The origin of the intrinsic 1.9 eV luminescence band in glassy SiO2[END_REF][START_REF] Vaccaro | Influence of fluorine on the fiber resistance studied through the non-bridging oxygen hole center related luminescence[END_REF] that is around 3 times more intense in the core center than in the cladding for irradiated sample whereas no PL luminescence in highlighted from non-irradiated one. The map of the PL intensity around 650 nm is reported in Fig. 9 (b), in the microstructured cladding and core of MOF1, showing that NBOHCs are concentrated in the core region even if a smaller amount of defects was induced by radiation also in the cladding. PL spectra emitted from the core center and microstructured part of the cladding in non-irradiated and irradiated at 1 MGy(SiO 2 ) samples, as well as the map of PL intensity around 650 nm for irradiated MOF2 are reported respectively in Fig. 10 (a) and Fig. 10 (b). Here again, we find the NBOHCs that are distributed in the microstuctured region with a maximum concentration in the solid pure silica core.

The maps of the PL intensity around 400 nm in the samples irradiated at 1 MGy(SiO 2 ) are not changed from those reported in pristine samples in Fig. 1 (a) and Fig. 1 (b) showing the presence of some blue emitting centers at the border of microstructured region due to stack and drawn fabrication processes that remain unaffected by radiations. It is known that radiation affects the OFDR-sensor response: a factor which limits their employment in presence of radiation is RIA by reducing the sensing range [START_REF] Rizzolo | Vulnerability of OFDR-based distributed sensors to high γ-ray doses[END_REF]. For our 70 m sensing range, considering that the maximum total losses permitted by the OFDR are 10 dB, we investigate the RIA in the infrared part of the spectrum to estimate the integration of such sensors in radiation environments. The results highlight that high level of online RIA are reached at 1 MGy(SiO2) for both fibers. We see that RIA increases with the wavelength, excluding that the tails of UV-Visible absorption defects-related bands can act as the main contributor to this attenuation. We note that all the curves, except the one corresponding to 1 MGy(SiO2), are below 140 dB/km. Looking better into the kinetics indeed, we see that, at 1550 nm (inset of Fig. 11), radiation induced losses increase with the irradiation time up to reach a value of ~ 150 dB/km at 1 MGy(SiO2), thus limiting the sensing length to ~ 65 m. In the case of MOF2 (see Fig. 12), we obtain RIA values of ~ 400 dB/km that drastically decrease the sensing range of the sensor to 25 m thus limiting the employment of such fiber types when higher tolerance is requested. We note that RIA results agree with CML ones, shown in Fig. 9 and Fig. 10, highlighting that Sol-Gel fiber (MOF2) is more sensitive to radiation than the F300based one.

V. CONCLUSION

In conclusion, obtained results show that OFDR-based sensors exploiting MOF fibers are not affected by radiationinduced errors as it is the case of FBGs, Brillouin, Raman sensors. It is known in literature that for FBGs with the radiation induces a Bragg Wavelength Shift (BWS) that depends on fiber composition [START_REF] Morana | Radiation tolerant fiber Bragg gratings for high temperature monitoring at MGy dose levels[END_REF], Brillouin sensors are affected by radiation with the Radiation Induced Brillouin Frequency Shifts (RI-BFS) [START_REF] Phéron | High γ-ray dose radiation effects on the performances of Brillouin scattering based optical fiber sensors[END_REF] and Raman sensors by the Δα affecting the ratio between Stokes and Anti-Stokes wavelengths [START_REF] Cangialosi | Hydrogen and radiation induced effects on performances of Raman fiber-based temperature sensors[END_REF], [START_REF] Cangialosi | Development of a Temperature Distributed Monitoring System Based On Raman Scattering in Harsh Environment[END_REF]. For OFDR measurements instead we find that temperature profiles are well determined in both samples with variation from thermocouple reference data giving an error on distributed temperature measurement of ~0.3 °C. More, temperature calibration curves show that C T value remains unchanged (within the experimental error) after the irradiation up to 1 MGy(SiO 2 ), results that are consistent with the previous one reported in [START_REF] Rizzolo | Radiation Response of OFDR Distributed Sensors Based on Microstructured Pure Silica Optical Fibers[END_REF] where the same samples were irradiated up to 50 kGy during a 3 hours long run. We see indeed that, distributed sensing is possible within an error the same incertitude when the temperature inside the irradiation chamber is not stable as well for longer irradiation period, permitting to appreciate the from day to night temperature variation.

The results highlighted by RIA measurements show, however, that employment of MOFs is limited by the attenuation. In particular, Sol-Gel microstructured optical fibers are more sensitive to radiation than their standard counterpart.

It is worth to mention that, an improvement in terms of optical losses and sensitivity to radiation, by applying an appropriate cleaning procedure to the sol-gel silica rod that constitute the fiber core, has to be achieved to extend the employment of MOFs to the majority of radiation applications but, even with reported performances, the development of OFDR sensing based on microstructured pure silica optical fibers in harsh environment that seems very promising up to the studied doses for fiber length of ~20 m.

Fig. 1

 1 Fig. 1 SEM images of the MOF1 fiber that is representative of the structure of both tested samples (a); A mapping of luminescence around 400 nm under 325nm excitation of in the microstructured part of the samples (highlighted by dotted square in (a)) is reported in (b) and (c) for MOF1 and MOF2 respectively, to evidence the presence of some emitting sites at the border of microstructured region. If we compare Fig. 1 (a) with Fig. 1 (b) and Fig.1 (c) we can note that the presence of these centers is correlated with the presence of the most extern microstructured region: the centers appear in the region where the holes are not present. Likely, used tubes in the fabrication process presented some impurities inside that may cause the PL showed in Fig.1 (b)and Fig.1 (c). However, these emitting centers are far from the region of light propagation, thus not influencing the optical properties in the core.

Fig. 2

 2 Fig. 2 Rayleigh spectral shift in the non-irradiated and 1 MGy irradiated MOF1 as a function of the path length at 9 different temperatures from 30 °C (reference trace) to 70 °C.

Fig. 3

 3 Fig. 3 Rayleigh spectral shift dependences on temperature for MOF1 (red circles) and MOF2 (blue circles) with respective linear fits (red and blue straight lines) before irradiation.

Fig. 4 (

 4 Fig. 4 (a) Temperature profiles obtained from MOF1 (red circles) and thermocouples (black circles) data and (b) differences between OFDR and thermocouple measurements as a function of time during irradiation (up to 1 MGy(SiO2) dose).

Fig. 5

 5 Fig. 5 Temperature distribution along the fiber length in MOF1 for nonirradiated sample (open black points) and sample irradiated at 10 kGy (black points), 50 kGy (red points), 100 kGy (green points), 500 kGy (blue points) and 1 MGy (orange points).

Fig. 6 (

 6 Fig. 6 (a) Temperature profiles obtained from MOF2 (blue circles) and thermocouples (black circles) data and (b) differences between OFDR and thermocouple measurements as a function of time during irradiation (up to 1 MGy(SiO2) dose).

Fig. 7

 7 Fig. 7 Temperature distribution along the fiber length in MOF2 for nonirradiated sample (open black points) and sample irradiated at 10 kGy (black points), 50 kGy (red points), 100 kGy (green points), 500 kGy (blue points) and 1 MGy (orange points).

Fig. 8

 8 Fig. 8 Rayleigh spectral shift dependences on temperature for MOF1 (red circles) and MOF2 (blue circles) with respective linear fits (red and blue straight lines) after irradiation.

Fig. 9 (

 9 Fig. 9 (a) PL spectrum emitted from 1 MGy(SiO2) MOF1 (solid line) and non-irradiated (dotted line) from the core center (black) and the cladding (red) under excitation at 325 nm. (b) Map of the PL intensity around 650 nm in the 1 MGy(SiO2) MOF1 shows that concentration of emitting centers around 650 nm is higher in the fiber core.

Fig. 10 (

 10 Fig. 10 (a) PL spectrum emitted from 1 MGy(SiO2) MOF1 (solid line) and non-irradiated (dotted line) from the core center (black) and the cladding (red) under excitation at 325 nm. (b) Map of the PL intensity around 650 nm in the 1 MGy(SiO2) MOF1 shows that concentration of emitting centers around 650 nm is higher in the fiber core. IV. DISCUSSION Confocal micro luminescence investigation revealed that Xrays induce point defects in our MOFs: NBOHCs are created during irradiation with distribution of these defects [as shown in Fig. 9 (b) and Fig. 10 (b)] being more concentrated in the core, phenomenon that can be due to a collapse of the core occurred during the fabrication of MOFs.

  Fig. 11 reports online spectral RIA measurements at different accumulated doses from 1.2 µm up to 2 µm, compared with the maximum values of RIA (highlighted with grey straight line) permitting to use OFDR-based sensors for MOF1.

Fig. 11

 11 Fig. 11 Online radiation induced attenuation spectra in the infrared region for MOF1 at different reached total doses from 10 kGy(SiO2) which is the lowest curve (black squares) up to 1 MGy(SiO2) that corresponds to the highest one (orange squares). The inset shows RIA as a function of time during irradiation up to 1 MGy(SiO2) where the losses reach the value of ~150 dB/km; the gray straight line at 140 dB/km highlights the maximum OFDR permitted optical losses at the operating wavelength (1550 nm) for 70 m of sensing length.

Fig. 12

 12 Fig.12Online radiation induced attenuation spectra in the infrared region for MOF2 at different reached total doses from 10 kGy(SiO2) which is the lowest curve (black squares) up to 1 MGy(SiO2) that corresponds to the highest one (orange squares). The inset shows RIA as a function of time during irradiation up to 1 MGy(SiO2) where the losses reach the value of ~400 dB/km; the gray straight line at 140 dB/km highlights the maximum OFDR permitted optical losses at the operating wavelength (1550 nm) for 70 m of sensing length.

  S. Rizzolo, Student Member, IEEE, A. Boukenter, T. Allanche, J. Périsse, G. Bouwmans, H. El Hamzaoui, L. Bigot, Y. Ouerdane, M. Cannas, M. Bouazaoui, J-R Macé, S. Bauer and S. Girard, Senior Member, IEEE

	Optical Frequency Domain Reflectometer
	Distributed Sensing Using Microstructured Pure
	Silica Optical Fibers under Radiations

URING the last decades the interest on fiber-based devices

VI. ACKNOWLEDGMENTS

The authors from PhLAM laboratory would like to thank K. Delplace for technical assistance. The authors acknowledge the financial support of the French national research agency through the NANOFIBER project (ANR-12-RMNP-0019). This work was supported by French National Research Agency through Labex CEMPI (ANR-11-LABX-0007) and Equipex FLUX (ANR-11-EQPX-0017), through Contrat de Projets Etat Région (CPER) "Photonics for Society"


Nadir Ali Birmani

Saima Naz

Gabriel Mouahid
email: mouahid@univ-perp.fr

First Record of Echinoparyphium recurvatum (Trematoda

Keywords: Charadriiformes, Charadriidae, Echinoparyphium recurvatum, Sindh Province, Southern Pakistan, Vanellus leucurus

come   L'archive ouverte pluridisciplinaire

Introduction

Genus Echinoparyphium is an important taxon in the family Echinostomatidae and has considerable importance in medical and veterinary sciences [3]. It has previously been described as Distomum by von Linstow [2] using specimens collected from the small intestine of Fuligula manila. Dietz [1] carried out a revision of Echinostomatidae and proposed different genus including the genus Echinoparyphium. Kanev [4] published a checklist with 151 species of Echinoparyphium using freshwater snails as first and second intermediate hosts, tadpoles and fish as a second intermediate hosts, and birds and mammals as definitive hosts. Since the creation of the genus, most of the published reports are based on the adequate descriptions of adult specimens. The more differentiating character is the morphometry of collar spines in adult stage, specifically the total number of spines (29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45), their sharply pointed shape and their arrangement in double row, with dorsal aboral spines noticeably longer than dorsal oral ones.

In the present paper, we report for the first time the presence of E. recurvatum recovered from the small intestine of the Whitetailed Lapwing Vanellus leucurus (Charadriiformes: Charadriidae) in the Sindh province (Southern Pakistan) and describe the mor-phology of the adult. The White-tailed Lapwing is inhabitant of freshwater marshy areas and lake shores. They more frequently seek food in shallow water. They are omnivorous and their food includes insects (chiefly beetles, grasshoppers, caterpillars,and fly larvae), mollusks, worms and crustaceans [5] which are potential sources of developmental stages of a variety of helminth parasites.

Materials And Methods

Four live White-tailed Lapwings Vanellus leucurus were collected from the Dadu city (GPS coordinates: 26°44'2.76"N; 67°46'46.20"E) located in the North-Western Sindh province, Pakistan. Birds were examined for the presence of trematode parasites in the Parasitology laboratory of the department of Zoology, University of Sindh, Jamshoro, in January 2016. A total of 64 trematodes belonging to the genus Echinoparyphium, were collected from the small intestine of two birds and were processed according to the method given by Garcia and Ash [6]. Line drawings were made with the help of a camera Lucida. All measurements were based on 13 mature specimens and are given in micrometer (µm) followed by range in parenthesis. For practical reasons, we have chosen to give the value of the minor axis and the major axis for any spherical or subspherical organ. Length and width are kept for elongated structures such as the body or its various parts, oesophagus, head collar spines, etc. Identification was made with the help of keys given by Jones et al. [START_REF]The Natural History Museum[END_REF]

Taxonomic Part

Description (based on 13 mature specimens; (Figures 123; Table 1)

Discussion

The genus Echinoparyphium is distinct from other genera of Echinostomatidae in possessing a well-developed head collar with 29 to 45 spines in a double row, with small spines covering the tegument extending to the testicular level ventrally and to the anterior margin of the ventral sucker dorsally. Large testes are tandem, elongated-oval, smooth or irregular, contiguous, post-equatorial and located in center of the hind body. Ovary small, spherical, median or submedian, pre-equatorial [9]. The genus Echinoparyphium counts many parasitic species of the intestine of many groups of birds and some species of mammals and the Type species is E. elegans [3,10,[START_REF] Echinoparyphium | Digenea: Echinostomatidae): the life cycle and redescription of the adult with a revision of the 43-spined members of the genus Echinoparyphium[END_REF]. Several species of genus Echinoparyphium have been found to infect the gastrointestinal tract of aquatic birds and mammals worldwide [12,13]. They are known to infect a large number of different taxa of birds (Anseriformes, Charadriiformes, Ciconiiformes, Columbiformes, Falconiformes, Galliformes, Gruiformes, Passeriformes, Podicipediformes, Strigiformes) and few taxa of mammals (Muridae (Hydromys chrysogaster, Rattus norvegicus, Rattus colletti and Rattus rattus), Cricetidae (Ondatra zibethicus), Canidae (Alopex lagopus) and Hominidae (Homo sapiens) [3,14].

According to the morphological description (Figure 123) and to the measurements (Table 1), our specimens belong to the genus Echinoparyphium and to the species recurvatum. It is the first time that this species is described in Pakistan and the first time that the avian host White-tailed Lapwing Vanellus leucurus is found to be a definitive host. Khan et al. [15] reported two species of Echinoparyphium in Pakistan: (i) Echinoparyphium ellisi [16] from Anas crecca in Nowshera (Khyber Pakhtunkhwa, one of the four provinces of Pakistan located in the northwestern Region) which belongs to the "recurvatum" group according to the phylogenetic study of Kostadinova et al. [17] and differs from our specimens in having a very long body (6200 x 1048 µm compared to our specimens 3868 x 684µm); (ii) Echinoparyphium recurvatum from Anas crecca in Peshawar (Khyber Pakhtunkhwa) which differs from our specimens in having 37 collar spines instead of 45 as it is well known for this species. The species known to have 37 collar spines is E. serratum described by Howell [18] and not E. recurvatum.

An updated list of natural avian hosts of E. recurvatum along with geographical distribution is given (Table 2). It shows that E. recurvatum is cosmopolitan species with a wide geographical distribution associated to a wide range of avian definitive host taxa including 7 orders and 10 families. The lack of specificity towards the definitive hosts is assigned to a low specificity towards the first and second intermediate hosts [19]

  andYamaguti [8]. Specimens Volume 3; Issue 01 Arch Vet Sci Tecnol,an open access journal ISSN : 2637-9988 are available at the Museum of Natural History in Paris under the voucher reference: MNHN HEL682.

Figure 1 :

 1 Figure 1: Morphology of Echinoparyphium recurvatum: Lateroventral view of entire adult worm.

Figure 2 :

 2 Figure 2: Morphology of Echinoparyphium recurvatum: anterior end of the adult with collar spined arrangement (45 spines), Oral Sucker (OS), Prepharynx (Pph), Pharynx (Ph) and Oesophagus (Oe).

Figure 3 :

 3 Figure 3: Morphology of Echinoparyphium recurvatum: genital pore region with Ventral Sucker (VS), Uterus (U) with Eggs (E), end part of the uterus or Metraterm (M), Genital Atrium (GA) and cirrus sac with Seminal Vesicle (SV), Pars Prostatica (PP), Ejaculatory Duct (ED) and Genital Pore (GP). Body small, spinose, elongate, tapering posteriorly, 3868 (3150-3905) length by 684 (521-690) width. Maximum width at level of ventral sucker. Forebody small, measuring 657 (600-682) length ventrally bent slightly. Hindbody measuring 2736 (2051-2818) length. Integument armed with very small spines which extend from the head crown to the testicular region. Head collar reniform, well developed, measuring 305 (288-310) by 263 (220-

Table 1 :

 1 . Volume 3; Issue 01 Comparative morphometrics (in µm) of adult worms of Echinoparyphium recurvatum obtained in different studies; length (L), width (W), major axis (Ma), minor axis (Mi), ng: not given and mean values given in parentheses after ranges.

	Arch Vet Sci Tecnol,an open access journal
	ISSN : 2637-9988

Table 2 :

 2 Natural avian hosts of Echinoparyphium recurvatum and their geographical location.