Blanca Gallego-Tévar

Brenda J Grewell

Hélène Rousseau

Jean Keller

Abdelkader Ainouche

Oscar Lima

Stéphane Dréano

Armel Salmon

Enrique Figueroa

Malika Aïnouche

Jesús M Castillo

Genetic structure of Spartina hybrids between native Spartina maritima and invasive Spartina densiflora in Southwest Europe

Keywords: AMOVA, Analysis of Molecular Variance. ANOVA, Analysis of Variance. cpDNA, Chloroplast DNA. GB, Genbank. GD, Genetic distance. PCoA, Principal Coordinate Analysis. PD, Phenotypic Distance. SHZ, Spanish Hydrographic Zero. SSRs, Simple Sequence Repeats Hybridization, genetic diversity, microsatellite, genetic structure, heterosis, invasion biology

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Genetic structure of Spartina hybrids was determined by that of their parents.

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Hybrids were genetically more similar to S. maritima than to S. densiflora.

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Hybrids with greater genetic differentiation were more spatially separated from each other.

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Hybrids with greater genetic differentiation were present in contrasted environments.

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No correlation was found between the molecular markers and plant traits.

sterile Spartina F1 hybrid populations established at the Gulf of Cadiz (Southwest Iberian Peninsula) and that of their parental species native S. maritima and invasive S. densiflora using nuclear DNA (Simple Sequence Repeats) and chloroplast DNA sequences. We also analyzed the relationships between the spatial genetic structure of the hybrids, their phenotypic variability and their marsh environment. The studied populations of Spartina hybrids were establishing hybrid zones with a spatial genetic structure inherited from both parental species. The hybrids were genetically more similar to the native than to the invasive species. The hybrid populations with greater genetic differentiation were those more spatially separated from each other and that were present in more contrasted sedimentary environments, revealing respective isolation processes by distance and by environment. The [START_REF] Yakimowski | The role of homoploid hybridization in evolution: A century of studies synthesizing genetics and ecology[END_REF]) that promotes rapid genetic, epigenetic and phenotypic changes [START_REF] Cara | Epigenetic patterns newly established after interspecific hybridization in natural populations of Solanum[END_REF][START_REF] Chen | Mechanisms of genomic rearrangements and gene expression changes in plant polyploids[END_REF][START_REF] Salmon | Genetic and epigenetic consequences of recent hybridization and polyploidy in Spartina (Poaceae)[END_REF]. By resulting in novel genotypes, hybridization can lead to more rapid adaptive changes than through mutation [START_REF] Long | The genetic structure of admixed populations[END_REF][START_REF] Minder | Genetic structure of hybrid zones between Silene latifolia and Silene dioica (Caryophyllaceae): Evidence for introgressive hybridization[END_REF]. To identify the role of hybrids in the structuring of plant communities, it is necessary to analyze their underlying population genetics background, their dispersion mechanisms and the intensity of selection [START_REF] Sloop | Spatial and temporal genetic structure in a hybrid cordgrass invasion[END_REF]. Hybrid zones are considered important tools for the study of evolutionary processes between divergent populations and genetic structure at the community level [START_REF] Minder | Genetic structure of hybrid zones between Silene latifolia and Silene dioica (Caryophyllaceae): Evidence for introgressive hybridization[END_REF][START_REF] Sloop | Spatial and temporal genetic structure in a hybrid cordgrass invasion[END_REF][START_REF] Whitham | Plant hybrid zones affect biodiversity: tools for a genetic-based understanding of[END_REF]).

The regional-scale genetic structure that we observe today is determined by the differences generated between ancestral colonizers [START_REF] Slatkin | Gene flow and the geographic structure of natural populations[END_REF][START_REF] Sloop | Spatial and temporal genetic structure in a hybrid cordgrass invasion[END_REF]. Thus, according to the classical population genetic approach, genetic similarity among populations decreases with geographic distance, explained by the isolation by distance [START_REF] Malécot | The mathematics of heredity[END_REF][START_REF] Wright | Isolation by distance[END_REF] and the stepping-stone models (Kimura and Weiss, 1964). This has been directly related to the reduction of gene flow between more distant populations leading to reproductive isolation promoting genetic drift and selection [START_REF] Ellstrand | Is gene flow the most important evolutionary force in plants?[END_REF][START_REF] Slatkin | Gene flow and the geographic structure of natural populations[END_REF].

In the particular case of introduced species, genetic drift may be emphasized as a consequence of bottlenecks leading to a general reduction of genetic variability (founder effect) in the introduced population in relation to the genetic variability of the species in its native range [START_REF] Brzyski | Reproductive allocation between the sexes, across natural and novel habitats, and its impact on genetic diversity[END_REF][START_REF] Castillo | Low genetic diversity contrasts with high phenotypic variability in heptaploid Spartina densiflora populations invading the Pacific coast of North America[END_REF][START_REF] Dlugosch | Invading populations of an ornamental shrub show rapid life history evolution despite genetic bottlenecks[END_REF]. In addition, environmental (biotic and abiotic) heterogeneity may lead to genetic differentiation between populations, mainly by selective pressure that favors local adaptation [START_REF] Hübner | Strong correlation of wild barley (Hordeum spontaneum) population structure with temperature and precipitation variation[END_REF][START_REF] Linhart | Evolutionary significance of local genetic differentiation in plants[END_REF] as a consequence of isolation by environment [START_REF] Wang | Isolation by environment[END_REF]. The effect that hybridization may have on the established spatial genetic structure will depend on different factors such as the genetic variability of the parental species, the dispersion capacity of the hybrids, their fertility and backcrossing frequency with the parental species (introgression) and seedling recruitment (Abbott, 1992;[START_REF] Arnold | Evolution through genetic exchange[END_REF][START_REF] Ellstrand | Hybridization as a stimulus for the evolution of invasiveness in plants?[END_REF][START_REF] Graham | Narrow hybrid zone between two subspecies of big sagebruh (Artemisia tridentata: Asteraceae). II. Selection Gradients and Hybrid Fitness[END_REF][START_REF] Rieseberg | Major ecological transitions in wild sunflowers facilitated by hybridization[END_REF]. Thus, populations of hybrids with a marked genetic structure related to reproductive isolation and local adaptation can be found [START_REF] Sloop | Spatial and temporal genetic structure in a hybrid cordgrass invasion[END_REF], as well as others without genetic differences between populations in different geographic locations when introgression and sexual reproduction homogenize populations [START_REF] Yatabe | Genetic population structure of Osmunda japonica, rheophilous Osmunda lancea and their hybrids[END_REF].

The genomic changes that occur as a result of hybridization affect both genome structure and gene expression and consequently determine hybrid phenotypes [START_REF] Baack | A genomic view of introgression and hybrid speciation[END_REF]. In hybrid taxa, the phenotype is the combination of different types of gene expression that can be additive (equal to the mid-parent value), dominant (equal to the value of one parent) or overdominant (higher or lower than the values of both parents) [START_REF] Bassene | Non-additive phenotypic and transcriptomic inheritance in a citrus allotetraploid somatic hybrid between C. reticulata and C. limon: the case of pulp carotenoid biosynthesis pathway[END_REF]. Depending on these differences in gene expression, hybridization may affect the evolutionary process in different ways, being negative if low fitness hybrids with limited ecological range are produced that can lead to reproductive isolation, or having positive effects when the new genotypes hybrids with greater fitness and large ecological amplitudes [START_REF] Barton | The role of hybridization in evolution[END_REF]. The latter are the result of the predominance of transgressive traits (exceeding trait values of both parents) related to heterosis or hybrid vigor [START_REF] Chen | Molecular mechanisms of polyploidy and hybrid vigor[END_REF][START_REF] Lippman | Heterosis: revisiting the magic[END_REF]. In the last decades, there have been important advances in the knowledge of molecular mechanisms underlying gene expression of hybrids, although there is still lack of understanding of the genetic regulation of the phenotypes of hybrids [START_REF] Bird | The causes and consequences of subgenome dominance in hybrids and recent polyploids[END_REF][START_REF] Tirosh | A yeast hybrid provides insight into the evolution of gene expression regulation[END_REF][START_REF] Yoo | Nonadditive gene expression in polyploids[END_REF]. In hybrids deriving from at least one invasive species (crossed with a native or non-native species), it has been reported that heterosis frequently increases the invasiveness of the offspring [START_REF] Ellstrand | Evolution of invasiveness in plants following hybridization[END_REF][START_REF] Ellstrand | Hybridization as a stimulus for the evolution of invasiveness in plants?[END_REF]

; Hovick A C C E P T E D M A N U S C R I P T
and Whitney, 2014) such as the hybrids between the native Spartina foliosa Trin. and the invasive S. alterniflora Loisel in San Francisco Bay [START_REF] Ayres | Spread of exotic cordgrasses and hybrids (Spartina sp.) in the tidal marshes of San Francisco bay, California, USA[END_REF]. Also, hybridization involving an invasive species usually counteracts the decrease in population genetic variability as a consequence of bottlenecks in invasions derived from a single introduction event [START_REF] Ellstrand | Hybridization as a stimulus for the evolution of invasiveness in plants?[END_REF], increasing the success of the invasion in the offspring.

In the genus of polyploid grasses Spartina (syn. Sporobolus following [START_REF] Peterson | A molecular phylogeny and new subgeneric classification of Sporobolus (Poaceae: Chloridoideae: Sporobolinae)[END_REF]) (Poaceae), there are several examples of hybrids and allopolyploids between invasive and native species which exhibit transgressive traits that confer hybrid vigor [START_REF] Ainouche | Hybridization, polyploidy and invasion: Lessons from Spartina (Poaceae)[END_REF][START_REF] Ayres | Spread of exotic cordgrasses and hybrids (Spartina sp.) in the tidal marshes of San Francisco bay, California, USA[END_REF][START_REF] Castillo | The production of hybrids with high ecological amplitude between exotic Spartina densiflora and native S. maritima in the Iberian Peninsula[END_REF][START_REF] Lee | Responses to salinity of Spartina hybrids formed in San Francisco Bay, California (S. alterniflora × foliosa and S. densiflora × foliosa)[END_REF][START_REF] Pakenham-Walsh | Evolving invasibility of exotic Spartina hybrids in upper salt marsh zones of San Francisco Bay[END_REF].

Along the Atlantic coast of the Gulf of Cádiz (Southwest Iberian Peninsula), populations of hybrids between the native Spartina maritima (Curtis) Fernald. (2n = 6x = 60) and the invasive from the west coast of South America Spartina densiflora Brongn. (2n = 7x = 70) have been described in at least three different estuaries: the joint estuary of Tinto and Odiel Rivers, the estuary of Piedras River and the estuary of Guadiana River. Two different hybrids have been found: S. maritima x densiflora (2n = 9.5x = 95), product of the fecundation of unreduced ovules of S. maritima fertilized by S. densiflora pollen in low marshes, and S. densiflora x maritima (2n = 6.5x = 65) obtained by the fecundation of regular ovules of S. densiflora by S. maritima pollen in middle marshes [START_REF] Castillo | The production of hybrids with high ecological amplitude between exotic Spartina densiflora and native S. maritima in the Iberian Peninsula[END_REF]. These reciprocal hybrids are transgressive in their tiller height, lateral expansion of their tussocks, and survival along the intertidal gradient [START_REF] Castillo | The production of hybrids with high ecological amplitude between exotic Spartina densiflora and native S. maritima in the Iberian Peninsula[END_REF]. In addition, it has been observed that they play a disruptive role in the typical zonation pattern of invaded European salt marshes, showing a high invasive potential limited by their sterility (Gallego-Tévar et al., 2018a). [START_REF] Castillo | The production of hybrids with high ecological amplitude between exotic Spartina densiflora and native S. maritima in the Iberian Peninsula[END_REF] previously described the existance, maternal origin, chromosome number and potential habitat of Spartina hybrids in the Gulf of Cádiz. Here, we build from

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this foundation, and report novel insights derived from genetic analyses to provide greater insight on effects of the hybridization process. No previous study has analyzed the genetic structure of the invasion of the parental S. densiflora in the Gulf of Cádiz and whether this neophyte was introduced only once or several times. Following its abundance and distribution pattern, S. densiflora seems to be introduced firstly in the joint estuary of Odiel and Tinto rivers [START_REF] Nieva | Aspectos ecológicos en Spartina densiflora Brong[END_REF].

In this study, the spatial genetic structure of the Spartina hybrid populations established at the Gulf of Cadiz and that of their parental species S. maritima and S. densiflora was explored using nuclear DNA (Simple Sequence Repeats, SSRs). These co-dominant markers are potentially polymorphic and allow for genetic discrimination among populations [START_REF] Baisakh | Development and interspecific transferability of genic microsatellite markers in Spartina spp. with different genome size[END_REF][START_REF] Gedye | Advances towards a marker-assisted selection breeding program in prairie cordgrass, a biomass crop[END_REF][START_REF] Vieira | Microsatellite markers: what they mean and why they are so useful[END_REF] and are useful in detecting hybrids combining different parental allelic combinations [START_REF] Ayres | Hybridization between invasive Spartina densiflora (Poaceae) and native S. foliosa in San Francisco Bay, California, USA[END_REF][START_REF] Hogle | San Francisco Estuary Invasive Spartina Project Spartina Monitoring Program Approach[END_REF][START_REF] Sloop | Spatial and temporal genetic structure in a hybrid cordgrass invasion[END_REF]. Additionally, chloroplast (cp) DNA sequences were used to assess the maternal origin of hybrid taxa [START_REF] Ferris | Molecular evidence for the maternal parentage in the hybrid origin of Spartina anglica C.E. Hubbard[END_REF][START_REF] Baumel | Genetic evidence for hybridization between the native Spartina maritima and the introduced Spartina alterniflora (Poaceae) in South-West France: Spartina × neyrautii re-examined[END_REF]. We also analyzed the relationships between the spatial genetic structure of the hybrids, their phenotypic variability and their marsh environment. Hybrids and parental populations were phenotypically examined by measuring twelve vegetative morphological traits, and their sedimentary abiotic environment was characterized. This enabled us to study the genetic structure of populations of hybrids between the native S. maritima and the invasive S. densiflora in relation to their parental species. More specifically, we analyzed the relationship between genetic distances among populations and (1) their geographic distance to assess the relative contribution of gene flow and drift in the population structure and consequent isolation by distance, (2) their abiotic environmental distances to identify the influence of processes of local adaptation and isolation by environment, and (3) their phenotypic differentiation by using morphological markers to evaluate the relationship between genetic hypothesized that the genetic structure of these hybrids would be determined by the genetic structure of the parental populations, whose genetic differentiation would increase together with the geographical distance between estuaries and with the environmental distance due to the contrasted habitat of both parental species.

Material and methods

Study sites

This study was carried out in three estuaries along the Atlantic coast of Gulf of Cádiz (Southwest Iberian Peninsula): Tinto-Odiel Estuary (37º08'-37º20'N, 6º45'-7º02'W), Piedras Estuary (37º12'-37º18'N, 7º06'-7º12'W) and Guadiana Estuary (37º10'-37º16'N, 7º16'-7º28'W) (Figure 1). These estuaries are under Mediterranean climate with Atlantic influence presenting a mean sea level of +1.85 m relative to Spanish Hydrographic Zero (SHZ) and an average semi-diurnal tidal range of 2.10 m. Their coastal marshes are characterized by a welldefined plant zonation pattern in which low marshes (+2.44 to +2.91 m SHZ) are dominated by S. maritima, Salicornia ramosissima J. Woods and Sarcocornia perennis (Mill.) A.J.

Scott., whereas Atriplex portulacoides (L.) Allen, Sarcocornia perennis x fruticosa and S. densiflora are the most abundant taxa at middle marshes (+2.91 to +3.37 m SHZ), and high marshes are colonized by halophytes such as Arthrocnemum macrostachyum (Moric.) C. Koch, Limoniastrum monopetalum (L.) Boiss. and Atriplex halimus L. [START_REF] Castellanos | Nucleation and facilitation in saltmarsh succession : interactions between Spartina maritima and Arthrocnemum perenne[END_REF]Castillo et al., 2008a;[START_REF] Figueroa | Facilitated invasion by hybridization of Sarcocornia species in a salt-marsh succession[END_REF].

Plant collection

Before collecting the plant material for this study, we conducted field trips to locate tussocks of Spartina hybrids between S. maritima and S. densiflora following the first description by [START_REF] Castillo | The production of hybrids with high ecological amplitude between exotic Spartina densiflora and native S. maritima in the Iberian Peninsula[END_REF]. The hybrids were identified using the intermediate character of their adaxial crests (deeper than S. maritima and less than S. densiflora) and leaves (longer than S. maritima and shorter than S. densiflora) and the transgressive character of the tussock height [START_REF] Castillo | The production of hybrids with high ecological amplitude between exotic Spartina densiflora and native S. maritima in the Iberian Peninsula[END_REF]. During these field trips, we recorded that the presence of hybrids was abundant in the Guadiana Estuary, with some hundreds of hybrids in just one marsh location known locally as 'San Bruno', whereas only five tussocks of hybrids were located in the Piedras Estuary and eleven were documented in the Tinto-Odiel Estuary. The collection of plant material was carried out in June-July 2016 from all known hybrid tussocks in the joint estuary of Tinto and Odiel Rivers (5 hybrids from a middle marsh close to 'La Rábida' monastery (37° 13' 42" N, 6° 54' 39" W), 3 hybrids from a middle marsh close to the sandspit known locally 'La Cascajera' (37° 11' 2" N, 6° 57' 21" W), 2 hybrids from a middle marsh at 'Don Claudio II' marsh (37° 11' 10" N, 6° 56' 22" W), and 1 hybrid from a low marsh at 'Estero del Colmenar' (37° 14' 17" N, 6° 59' 21" W)). Collections also included the only five known hybrid tussocks in the estuary of Piedras River including 1 from a low marsh area close to the road from 'El Terrón' seaport to 'La Antilla' beach (37° 13' 2" N, 7° 10' 51" W) and 4 from two different middle marsh areas (37° 13' 4" N, 7° 10' 14" W). Collections were made of 10 hybrid tussocks from 'San Bruno' marsh in the estuary of Guadiana River, including 5 from a low marsh area (37° 11' 41" N, 7° 24' 22" W) and 5 from a middle marsh area (37° 11' 46" N, 7° 24' 10" W) (Fig. 1). The plant material of the parental species was sampled from the closest tussocks of S. maritima and S. densiflora to the sampled hybrids (N = 18 for S. maritima; N = 22 for S. densiflora). In total, 66 individuals were collected and analyzed in this study.

A C C E P T E D M A N U S C R I P T

Chloroplast DNA sequencing

DNA was extracted from the collected dried leaves of S. maritima, S. densiflora and their

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hybrids by employing a NucleoSpin Plant Extraction Kit (Macherey-Nagel GmbH & Co, Düren, Germany). The trnT-trnF chloroplast DNA (Cp-DNA) region was amplified using the primer pair a and b to recover the trnT-trnL segment, and primer pair c and f to recover the trnL-trnF segment (Taberlet et al. 1991). The Polymerase Chain Reaction (PCR) was performed in a Mastercycler thermal cycler (Eppendorf AG, Hamburg, Germany) and underwent denaturation for 2 min at 94 ºC, followed by 35 cycles at 94 ºC for 30 s, 54 ºC for 30 s, 72 ºC for 1 min 30 s, followed by an extension phase at 72 ºC for 7 min. PCR products were purified using a NucleoSpin Gel and PCR purification kit (Macherey-Nagel GmbH & Co), and sequenced directly (on both directions) by Sanger at Macrogen Europe sequencer (Amsterdam, The Netherlands).

Simple Sequence Repeats (SSRs)

Neutral molecular markers such as SSRs are commonly used for genotyping in studies of phenotypic variations since they provide independent and complementary assessment of genetic diversity to phenotypic variation most likely submitted to selective constraints (e.g. [START_REF] Chapman | A quantitative review of heterozygosity-fitness correlations in animal populations[END_REF]. Genetic diversity was analysed at eight microsatellite (SSR) loci, using eight primer pairs (MS2, MS7, MS13, MS14, MS15, MS16, MS17 and MS18) (Table 1) designed on genomic assemblies from Spartina maritima and successfully employed in S. densiflora [START_REF] Castillo | Low genetic diversity contrasts with high phenotypic variability in heptaploid Spartina densiflora populations invading the Pacific coast of North America[END_REF]. The protocol used for microsatellite locus amplification followed that of [START_REF] Baumel | Spartina versicolor Fabre: Another case of Spartina trans-Atlantic introduction?[END_REF] and [START_REF] Castillo | Low genetic diversity contrasts with high phenotypic variability in heptaploid Spartina densiflora populations invading the Pacific coast of North America[END_REF]. DNA extracts were diluted to a concentration of 10 ng µL -1 and amplified using the 8 microsatellite primer pairs selected.

We used FAM labelled reverse primers for PCR and amplification products were diluted to 1/30 before subsequent analysis. An aliquot of 2 µL of these diluted PCR products were mixed with 10 µL of formamide solution (975 µL formamide + 25 µL of Liz-500 size marker) and separated by electrophoresis in an ABI PRISM 16-capillary 3130xl Genetic Analyzer

Sedimentary variables and phenotypic plant traits

The sedimentary environment variables and phenotypic plant traits for the studied tussocks of S. maritima, S. densiflora and their hybrids were recorded at low tide during June and July 2016. Every sedimentary variable was measured at a depth of 0-5 cm in monospecific plots (ca. 50 x 50 cm) where discrete tussocks were located. Sediment redox potential (Eh) and elevation above SHZ were measured in situ in the field, being each measurement the mean of 3 sub-replicates. We used a Leica NA 820 theodolite (Singapore) with a resolution of 2 cm for recording elevation. Measurements were referred to m above SHZ by using tidal extremes as reference points [START_REF] Ranwell | Spartina salt marshes in southern england: C. tidal submergence and chlorinity in poole harbour[END_REF]. Sediment Eh was obtained by using a portable meter and an electrode system (Crison pH/mV p-506). On the other hand, pH of interstitial water, electrical conductivity (mS cm -1 ) and sediment water content (%) were determined in the laboratory after collecting sediment samples in 200 ml sealed recipients. Distilled water was added to the sediment (1:1, v/v) for the determination of pH (pH/redox Crison with the electrode M-506) and double water volume was used (1:2, v/v) for the measurement of electrical conductivity (conductivity meter, Crison-522) [START_REF] Curado | Plant zonation in Restored, Nonrestored, and Preserved Spartina maritima salt marshes[END_REF]. Sediment water content was measured as the difference between fresh weight (FW) and dry weight (DW) after drying ca. 100 g of sediment in a stove at 80 °C for 48 h.

The following vegetative plant traits were recorded at the same time as the collection of plant material for genetic analyses and the characterization of the sedimentary environment: number of live and dead leaves, length, base width and weight of the flag leaf (first adult leaf completely unfolded on a tiller), foliar area, the Specific Leaf Area (SLA), diameter and length of tillers and the number of leaves per tiller length, tiller density, and the

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Leaf Area Index (LAI). The number of live and dead leaves, and the length, base width and weight of the flag leaf were measured in 5 tillers randomly selected from each tussock. Foliar area was calculated as a triangle using leaves width at the base and length. Then, foliar areas were used to calculate SLA (m -2 g -1 ) [START_REF] Garnier | A standardized protocol for the determination of specific leaf area and leaf dry matter content[END_REF] dividing by the DW of the same leaves. The difference in leaf width between unfolded (manually) and folded (in the field) leave area was expressed in percentage to calculate leaf adaxial rolling [START_REF] Premachandra | Water stress and potassium fertilization in field grown maize (Zea mays L.): effects on leaf water relations and leaf rolling[END_REF]. In addition, diameter and length of tillers and the number of leaves per tiller length (leave cm -1 ) were recorded for the same 5 tillers per tussock. Also, the number of tillers in 10

x 10 cm plots (n = 5 plots) was counted for the determination of tillers density (tiller cm -2 ). LAI ( m 2 m-2 ) [START_REF] Jonckheere | Review of methods for in situ leaf area index determination: Part I. Theories, sensors and hemispherical photography[END_REF] was estimated as the product between the mean foliar area, the mean number of leaves per tiller and the mean tiller density of a given tussock divided by the soil occupied area by that tussock at the base of its tillers or by its aerial aboveground structures, including the area occupied by its tilted tillers.

Data analyses

Chloroplast DNA sequences of the hybrids were compared to those from S. maritima and S. densiflora collected in the three estuaries, in order to assess the maternal origin of the hybrids.

These sequences are deposited in Genbank under accession numbers MK314916 to MK314943. They were compared to those previously published for S. maritima (GB accessions KP176438, [START_REF] Rousseau-Gueutin | The chloroplast genome of the hexaploid Spartina maritima (Poaceae, Chloridoideae): Comparative analyses and molecular dating[END_REF], AF 275669, Baumel et al., 2001) and S. densiflora (GB accession AF372629, [START_REF] Baumel | Molecular phylogeny of hybridizing species from the genus Spartina Schreb. (Poaceae)[END_REF]. Sequences were aligned using Geneious 9.1.2 software [START_REF] Kearse | Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data[END_REF] and a phylogenetic (Maximum Parsimony) analysis was performed using MEGA X software [START_REF] Kumar | MEGA X: Molecular evolutionary genetics analysis across computing platforms[END_REF] SSR alleles were determined by comparison with the standard marker size (GeneScan-500 LIZ Size Standard), and the different "genotypes" (harbouring different allele

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combinations) were scored. Since the studied Spartina taxa are all polyploid, allelic dosage per individual cannot be ascertained. SSRs were then treated as "dominant markers", a convenient procedure for genetic analyses in polyploids [START_REF] Obbard | Simple allelic-phenotype diversity and differentiation statistics for allopolyploids[END_REF]. Alleles were recorded as either present or absent using the GenAlex 6.502 software (Peakall andSmouse, 2012, 2006). Up to 10 different alleles could be expected per locus since the taxa with the highest level of ploidy was S. maritima x densiflora (2n = 9.5x = 95 chromosomes) [START_REF] Castillo | The production of hybrids with high ecological amplitude between exotic Spartina densiflora and native S. maritima in the Iberian Peninsula[END_REF]. Genetic distances (GD) between individuals and populations of S. maritima, S. densiflora and their hybrids were calculated following [START_REF] Huff | RAPD variation within and among natural populations of outcrossing buffalograss [Buchlöe dactyloides (Nutt.) Engelm[END_REF] and [START_REF] Nei | Genetic distance and molecular phylogeny[END_REF], respectively. The intra-population genetic diversity parameters, effective number of alleles and Shannon Information Diversity Index [START_REF] Brown | Measuring genetic variability in plant populations[END_REF] were obtained for each studied population of the Spartina hybrids at the estuary level. In addition, Analysis of Molecular Variance (AMOVA) was used to determine the proportion of genetic differentiation within and among different hybrid populations at the estuary level. The fixation index (ΦPT), analogous to Wright's (FST), was obtained following the standardization of [START_REF] Meirmans | Using the AMOVA framework to estimate a standardized genetic differentiation measure[END_REF] as an indicator of population genetic differentiation for dominant data.

Two-way analysis of variance (ANOVA) were performed to compare mean sedimentary variables and vegetative plant traits (dependent variables) for the Spartina hybrids and their parental species among estuaries, using taxon and estuary as grouping factors. In case of significant ANOVA, Tukey's honestly significant difference (HSD) was used as post hoc analysis. Before using the parametric tests, dependent variables were assessed for homoscedasticity using Levene's test and for normality using the Shapiro-Wilk test. When these requirements for parametric analysis were not met, transformations of type inverse (for LAI) and square root (for number of dead leaves and tiller density) were carried out. These analyses were conducted using SigmaPlot (Systat Software, San Jose, CA) version 14 for Windows. Deviations of all data were calculated as standard error of the mean (SE) and

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a significance level (α) of 0.05 was applied for every analysis. Phenotypic (PD) and sedimentary (SD) distances between plants in the field were calculated as Gower's similarity index ranging between 0 and 1 [START_REF] Gower | A general coefficient of similarity and some of its properties[END_REF]) using the package vegan [START_REF] Oksanen | Vegan: community ecology package[END_REF] of R-software to obtain a pairwise dissimilarity matrix.

In order to compare genetic, phenotypic, sedimentary and geographic distance matrices, different Mantel's test for Matrix Correspondence [START_REF] Mantel | The detection of disease clustering and a generalized regression approach[END_REF] were conducted following the method of [START_REF] Smouse | Multiple regression and correlation extensions of the mantel test of matrix correspondence[END_REF] and [START_REF] Smouse | Matrix correlation analysis in anthropology and genetics[END_REF] using GenAlEx 6.502 software. Principal Coordinate Analysis (PCoA) with the SSR-based matrices of phenotypic and genetic distances, with the algorithm of [START_REF] Orlóci | Multivariate analysis in vegetation research[END_REF], was also carried out using GenAlEx 6.502 software. Canonical Correspondence Analyses (CCA) and the Monte-Carlo permutation test were performed using the vegan package of R-software (R Core Team 2016).

The CCA was conducted to test the significance of the relationship between the sedimentary characteristics measured at every monospecific hybrid stand and the presence/absence matrix of the different alleles of the 8 loci analyzed in individuals of each stand. The significance of the canonical correlation coefficients was evaluated with Monte-Carlo permutation tests (999 permutations).

Results

Genetic diversity

Chloroplast DNA sequences enabled the differentiation of S. maritima and S. densiflora plastomes and the determination of the maternal genome donor in hybrids. The trnL-trnT region exhibits diagnostic nucleotide substitutions and indels between S. maritima and S. densiflora, with a notable 384 bp deletion in S. densiflora (Fig. 2). There is almost no sequence variation among accessions from the same species. One S. maritima individual from Piedras Estuary displayed a unique 29 bp deletion, corresponding to a repeated region. This

A C C E P T E D M A N U S C R I P T
individual was not included in subsequent genetic comparisons, as it was not involved in the parentage of the detected hybrids (see below). Six hybrids from Tinto-Odiel, 1 from Guadiana (from low marshes) and 4 from Piedras Estuaries had S. maritima as maternal genome donor, whereas 5 hybrids from Tinto-Odiel, 1 from Guadiana (from middle marshes) and 1 from Piedras Estuaries had S. densiflora as maternal parent (Fig. 2). The analyses of one hybrid individual from low marsh and another one from middle marsh in Guadiana Estuary corroborated the maternal origin of both populations previously analysed by [START_REF] Castillo | The production of hybrids with high ecological amplitude between exotic Spartina densiflora and native S. maritima in the Iberian Peninsula[END_REF].

The eight SSR primer pairs amplified 1-7 different alleles per locus with an average of 5 alleles per locus and a total sum of 41 alleles. On average, the number of different alleles found per locus and individual for the parental species was 2 (1-4) and 2 (1-6) for S. densiflora and S. maritima, respectively. At the 8 analyzed loci, 31 different alleles were found for S. maritima and 24 for S. densiflora (Table 2). According to Nei's index, the genetic distance (GD) among populations of each parent was greater between Guadiana and Tinto-Odiel (S. densiflora: 0.045, S. maritima: 0.144) and Guadiana and Piedras Estuaries (S. densiflora: 0.063, S. maritima: 0.144) than between Tinto-Odiel and Piedras Estuaries (S. densiflora: 0.023, S. maritima: 0.005). The GD between populations of both parental species in the same estuary was higher in the Guadiana Estuary (0.684) than in Tinto-Odiel (0.449) and Piedras (0.428) Estuaries.

In hybrid plants, an average of 3 (0-6) different alleles per locus and individual was obtained, with a total of 36 different alleles for the 8 surveyed loci. All the alleles observed in the parental species were also present in different proportions in the hybrids, except one absent allele of S. densiflora (MS18-3) and three of S. maritima (MS14-4, MS15-7, MS18-7).

Thirteen alleles of the hybrids were unique in S. maritima, 8 alleles were exclusive of S. densiflora and 15 alleles of the hybrids were found in both parental species. This high number

A C C E P T E D M A N U S C R I P T
of S. maritima alleles compared to S. densiflora was maintained in both S. maritima x densiflora (13 unique alleles of S. maritima and 7 of S. densiflora) and S. densiflora x maritima (11 of S. maritima and 8 of S. densiflora) hybrids, although the maternal proportion increased for S. maritima x densiflora (Table 2). As for the parental species, the greatest GD for the hybrids according to the Nei's index, was found between the Guadiana and Tinto-Odiel (0.167) and between Guadiana and Piedras (0.125) Estuaries; while the GD between Tinto-Odiel and Piedras Estuaries was lower (0.028). Five unique alleles were found in Guadiana Estuary (MS14-1 and MS18-2 of S. maritima and the hybrids, MS15-7 of S. maritima and MS18-3 and MS18-4 of S. densiflora), 2 unique alleles were recorded in Tinto-Odiel Estuary (MS2-2 of S. maritima and the hybrids and MS18-7 of S. maritima), and 1 unique allele was found in Piedras Estuary (MS14-4 of S. maritima) (Table 2). Genetic differentiation of Spartina hybrids at the estuary level (ΦPT = 0.547) was higher among populations (55%) than within populations (45%) (Table 3). These genetic differences are represented in the PCoA of GD that allowed distinguishing the parental species along the PC-2 axis and positioning the hybrids in the center, closer to S. maritima than to S. densiflora. The PC-1 axis separated individuals from different estuaries, with Spartina populations from the Guadiana Estuary showing negative values and those of the Tinto-Odiel and Piedras Estuaries with positive values (Fig. 3A).

As expected, the hybrids showed higher genetic diversity (I) than their parental species at all estuaries, except at Piedras Estuary where it was similar for the hybrids and S. densiflora. In the Guadiana Estuary, the hybrids displayed a genetic diversity 2.00 times higher than S. densiflora and 2.50 times than S. maritima, while in both Tinto-Odiel and Piedras Estuaries it was 1.25 and 1.50 higher, respectively. The intrapopulation genetic diversity of the hybrids in the Guadiana Estuary was twice that of Tinto-Odiel and Piedras Estuaries. Despite having higher total number of alleles, S. maritima exhibited lower inter- 4).

Ecology and phenotypic diversity of the hybrids

The Guadiana Estuary had lower sediment salinity (17.1 ± 1.3 mS cm -1 ) and elevation (2.62 ±0.04 m) for every taxon than interstitial sediment salinity and elevation measured in Tinto-Odiel (24.8 ± 1.3 mS cm -1 and 2.96 ± 0.05 m, respectively) and Piedras Estuaries (22.9 ± 1.6 mS cm -1 and 3.08 ± 0.05 m, respectively). Moreover, sediments from the Tinto-Odiel Estuary exhibited lower redox potential (-9 ± 16 mV) than the other estuaries (Guadiana: 100 ± 16 mV, Piedras: 79 ± 19 mV). The Tinto-Odiel Estuary also had higher sediment pH (6.9 ± 0.1) than the Guadiana (6.7 ± 0.1) and Piedras (6.4 ± 0.1) sites. On the other hand, the recorded sedimentary variables did not change significantly between Spartina taxa within estuaries, except for sediment characteristics in the lower elevation occupied by S. maritima in the Tinto-Odiel Estuary as compared to areas occupied by the other studied taxa (Table 5; see Table 6 for statistical tests).

The vegetative phenotypes of the Spartina hybrids were characterized by some traits similar to one or to both parental species, others that were intermediate between the parental species, and some transgressive traits that were superior to both parents. Thus, SLA (except for hybrid individuals from the Piedras Estuary), the number of leaves and the leaf width for the hybrids were similar to those of S. maritima and greater than the values recorded for S. densiflora. In contrast, the tiller diameter of hybrids only for the Guadiana Estuary, and the number of dead leaves and the foliar area for all measured hybrids were similar to those of S. densiflora and greater than those of S. maritima. In addition, the number of leaves per tiller length for the hybrids was similar to this metric for S. densiflora and 2.5 times lower than data recorded for S. maritima. In contrast, leaf length, leaf rolling and tiller density for the hybrids were intermediate between the lower values of S. maritima and the higher values of S.

A C C E P T E D M

A N U S C R I P T densiflora. Tiller length for the hybrids at Guadiana Estuary (49.8 ± 2.6 cm) and almost LAI for every estuary (P-value = 0.072) exceeded those of both parental species (S. maritima: 13.5 ± 3.7 cm, S. densiflora: 29.6 ± 2.6 cm) (see Table 6 for statistical tests).

In general, Spartina plants of parents and hybrids from the Guadiana Estuary had 16% shorter leaves than leaves measured in the other estuaries. Plants from the Guadiana Estuary for every taxon showed also 71% higher tiller densities, 17% lower numbers of leaves and 16% smaller leaf areas than those of Piedras Estuary. Plants from the Piedras Estuary exhibited 34% lower LAI than Tinto-Odiel Estuary and 16% greater leaf width than the other two estuaries. Spartina maritima growing at Tinto-Odiel Estuary showed greater SLA than at Piedras, wider tillers than at Guadiana Estuary, and taller tillers and fewer leaves per tiller length than both estuaries (Table 5; see Table 6 for statistical tests).

The phenotypic difference between both parental species and their hybrids is shown in the graphic representation of the PD that separated S. maritima from the other taxa along the PC-2 axis. Spartina densiflora and the hybrids appeared mixed, with a slight tendency to separate S. densiflora in the opposite direction to S. maritima along the PC-2 axis. Instead of the difference reported above, the combination of the recorded phenotypic plant traits did not distinguish the populations from the three estuaries for any taxon (Fig. 3B).

The Mantel tests showed that the PD of the Spartina hybrids was independent of their sedimentary, genetic and geographic distances. PD between hybrids tended to increase with their GD, however this relationship was not significant (P-value = 0.060) (Fig. 4A). On the other hand, GD between hybrids increased with their sedimentary and geographic distances (Fig. 4B,C).

A total proportion of 85.8% of the total variance of the data linking sedimentary variables and alleles presence for every hybrid individual was explained by the first two axes of the CCA (Fig. 5). Axis 1 was strong and negatively correlated with sediment conductivity

A C C E P T E D M A N U S C R I P T
and marsh elevation, and Axes 2 was negatively correlated with sediment pH, and sediment water content correlated negatively with Axis 1 and positively with Axis 2 (Fig. 5; Supplementary material 1). Axes 1 and 2 were significant at P < 0.001, as reported by the Monte-Carlo permutation test (Supplementary material 1). All the hybrids from Guadiana Estuary and one from Piedras Estuary (H-P1) were positively correlated with Axis 1, while the rest of the hybrids from Piedras Estuary and every hybrid from Tinto-Odiel Estuary were negatively correlated with the primary axis. Alleles MS7.2, 7.4, 13.1 and 16.3 were associated negatively with Axis 1, whereas a group of 10 alleles were associated positively with Axis 1 (Fig. 5; Table S1, Supplementary material). Axis 2 was positively correlated with 2 hybrid individuals from Guadiana, 2 from Piedras and 1 from Tinto-Odiel Estuary, and negatively correlated with 1 hybrid from Guadiana, 1 from Piedras and 5 from Tinto-Odiel Estuary.

Three alleles (MS7.2, 7.3 and 13.1) were positively related to Axis 2, whereas the allele MS16.3 was negatively related to this axis (Fig. 5; Table S1, Supplementary material).

Discussion

Our results show that the genetic structure of sterile Spartina F1 hybrids between the native S. maritima and invasive S. densiflora along the coast of the Gulf of Cadiz (Southwest Iberian Peninsula) was mainly determined by the genetic structure of the parents, the hybrids being genetically more similar to the native than to the invasive species.

Although S. maritima individuals exhibited more different alleles (31) than S. densiflora (24) at the 8 investigated loci, their genetic diversity among tussocks from the same estuary was the lowest of the studied taxa. This low inter-individual variability of S. maritima is consistent with its low sexual reproduction [START_REF] Castellanos | Nucleation and facilitation in saltmarsh succession : interactions between Spartina maritima and Arthrocnemum perenne[END_REF][START_REF] Castellanos | Tiller dynamics of Spartina maritima in successional and non-successional mediterranean salt marsh[END_REF] and previous molecular investigations in Europe [START_REF] Yannic | Uniformity of the nuclear and chloroplast genomes of Spartina maritima (Poaceae), a salt-marsh species in decline along the Western European Coast[END_REF]. It also agrees with its high levels of local adaptation to stressful and stable low marshes [START_REF] Castellanos | Nucleation and facilitation in saltmarsh succession : interactions between Spartina maritima and Arthrocnemum perenne[END_REF] et al., 2008b;[START_REF] Contreras-Cruzado | Relationships between spatio-temporal changes in the sedimentary environment and halophytes zonation in salt marshes[END_REF]. S. densiflora, with hybrid origin [START_REF] Fortune | The enigmatic invasive Spartina densiflora: A history of hybridizations in a polyploidy context[END_REF], has a higher ploidy level (7x) than S. maritima (6x), which most likely increases genetic diversity [START_REF] Petit | Evolutionary consequences of diploid-polyploid hybrid zones in wild species[END_REF][START_REF] Soltis | The role of genetic and genomic attributes in the success of polyploids[END_REF]. In contrast, S. maritima exhibited greater interpopulation genetic differentiation between estuaries than S. densiflora. The weak population structure of S. densiflora agrees with its invasive status and associated bottlenecks resulting from founding effects [START_REF] Brzyski | Reproductive allocation between the sexes, across natural and novel habitats, and its impact on genetic diversity[END_REF][START_REF] Dlugosch | Invading populations of an ornamental shrub show rapid life history evolution despite genetic bottlenecks[END_REF].

; Castillo A C C E P T E D M A N U S C R I P T
Additionally, establishment of these invading populations is more recent than the native populations of S. maritima that can survive for decades, or even centuries, in non-successional marshes [START_REF] Castellanos | Tiller dynamics of Spartina maritima in successional and non-successional mediterranean salt marsh[END_REF], allowing natural selection, neutral mutations and genetic isolation to act longer on these native populations. Moreover, the genetic isolation between S. maritima populations may be stronger than between S. densiflora populations due to their lower sexual reproduction. S. densiflora produces many viable seeds [START_REF] Kittelson | Mechanisms of expasion for an introduces species of cordgrass Spartina densiflora, in Humboldt Bay, California[END_REF][START_REF] Nieva | Field variability of invading populations of Spartina densiflora Brong. in different habitats of the Odiel Marshes (SW Spain)[END_REF] that can be transported by tides and currents floating on water [START_REF] Xiao | Seed dispersal capacity and post-dispersal fate of the invasive Spartina alterniflora in saltmarshes of the Yangtze Estuary[END_REF] and by birds [START_REF] Vivian-Smith | Dispersal of salt marsh seeds on the feet and feathers of waterfowl[END_REF]. Invasive S. densiflora introduced along the Atlantic coast of North America also showed low genetic differentiation among populations [START_REF] Castillo | Low genetic diversity contrasts with high phenotypic variability in heptaploid Spartina densiflora populations invading the Pacific coast of North America[END_REF].

Our genetic analyses using cpDNA and nuclear SSR markers confirmed the hybrid origin of every tussock identified as such in the field and the reciprocal crosses that occurred in the Guadiana, Piedras and Tinto-Odiel Estuaries. In relation to the 8 SSR loci analyzed, most of the alleles present in the parental species were also present in their hybrids (27 out of 31 of S. maritima alleles and 22 out of 24 of S. densiflora alleles), while the 48% of the alleles of S. maritima and 63% of S. densiflora were shared between both parental species.

Consequently, the hybrids contained greater number of different alleles than both parental species separately. Hybridization is expected to increase the number of alleles at all loci [START_REF] Long | The genetic structure of admixed populations[END_REF] while non-inherited alleles may be associated to changes occurred during

A C C E P T E D M A N U S C R I P T
hybridization such as chromosomal rearrangement, gene loss or changes in genome size [START_REF] Baack | A genomic view of introgression and hybrid speciation[END_REF]), but we cannot reject possible technical bias in detecting some alleles. Thus, the hybrids showed higher inter-individual genetic diversity within populations than their parents, except in the Piedras Estuary population. The studied hybrids are sterile, each hybrid tussock being the product of independent hybridization events [START_REF] Castillo | The production of hybrids with high ecological amplitude between exotic Spartina densiflora and native S. maritima in the Iberian Peninsula[END_REF]. Gene flow within populations tends to homogenize the genetic differences between individuals [START_REF] Lenormand | Gene flow and the limits to natural selection[END_REF][START_REF] Slatkin | Gene flow and the geographic structure of natural populations[END_REF], which does not occur in these sterile hybrids as it could occur in the parental species, especially in S. densiflora as mentioned above.

Moreover, the fact that hybrids whose seed parent was S. maritima inherited all its genetic material from unreduced ovules [START_REF] Castillo | The production of hybrids with high ecological amplitude between exotic Spartina densiflora and native S. maritima in the Iberian Peninsula[END_REF] could explain that, globally, hybrids inherited more alleles from S. maritima than from S. densiflora. Nonetheless, the maternal origin of the hybrids has not induced a scenario in which all the individuals of the same hybrids would globally share more alleles with the seed parent than with the pollen parent.

Both parental species and their hybrids showed clear spatial genetic structure. The genetic structure of the hybrids was revealed by their higher genetic differentiation among populations (55%) than within populations (45%) at the estuary level, which was the consequence of the genetic structure of their parental species. Thus, the genetic structure of the hybrids reflected local crossing between parental species, which may be related to short distance dispersal of their pollen [START_REF] Davis | Pollen limitation causes an Allee effect in a wind-pollinated invasive grass (Spartina alterniflora)[END_REF], followed by restricted seed dispersal.

The tussocks from the Guadiana Estuary for every taxon were clearly differentiated from those sampled in the Piedras and Tinto-Odiel Estuaries, which are located closer to each other along the coast. Thus, the spatial genetic structure of the hybrids was directly related to geographic distances among populations. The total or partial reproductive isolation by distance predicts the increase of genetic differentiation with geographic distance due to reduction or lack of gene flow between populations [START_REF] Baack | The origins of reproductive isolation in plants[END_REF][START_REF] Wright | Isolation by distance[END_REF]. Sloop 2011) observed that hybrids between the native Spartina foliosa and the invasive Spartina alterniflora in San Francisco Bay also showed a marked genetic structure due to limitations in seeds and pollen dispersal. The GD of the hybrids analyzed in our study was also directly related to the differences in their sedimentary environments among marsh habitats that may have imposed selective pressures on the parental species, favoring isolation by environmental processes [START_REF] Wang | Isolation by environment[END_REF]). Thus, for example, S. maritima growing in low marshes starts its flowering earlier in the year than S. densiflora (B. Gallego-Tévar, personal observation). Similar observations were made in wild barley where the spatial population structure was determined not only by geographic isolation, but also by differences in environmental conditions such as elevation, temperature and rainfall [START_REF] Hübner | Strong correlation of wild barley (Hordeum spontaneum) population structure with temperature and precipitation variation[END_REF].

In our case, plants from Guadiana Estuary were colonizing in sediments with lower conductivities and at lower elevations than in Piedras and Tinto-Odiel Estuaries. Our results demonstrated a relationship between alleles and the sedimentary environment. Some alleles recorded only for hybrids from Guadiana Estuary were associated with relatively low sediment salinities. In contrast, specific alleles of hybrids from Tinto-Odiel and Piedras Estuary were associated with higher salinities, higher elevations, and with more acidic sediments.

The hybrids from Guadiana Estuary were the only hybrids showing transgressive tiller height, while the hybrids in Piedras and Tinto-Odiel Estuaries were taller than S. maritima but not different from S. densiflora. This was probably related to the recorded greater genetic differentiation between the parental species in the Guadiana Estuary, as greater genetic distances between parents have been frequently related to the development of heterosis leading hybrid vigor in their hybrids [START_REF] Ali | Relationship between genetic distance and heterosis for yield and morphological traits in winter canola (Brassica napus L.)[END_REF][START_REF] Krystkowiak | Relationship between phenotypic and genetic diversity of parental genotypes and the specific combining ability and heterosis effects in wheat (Triticum aestivum L.)[END_REF][START_REF] Pandey | Relationship of parental genetic distance with heterosis and specific combining ability in sesame (Sesamum indicum L.) based on phenotypic and molecular marker analysis[END_REF][START_REF] Reif | Genetic distance based on simple sequence repeats and heterosis in tropical maize populations[END_REF][START_REF] Stelkens | Genetic distance between species predicts novel trait expression in their hybrids[END_REF]. Along with differences in parental genotypes among populations, sedimentary environment differences in Guadiana Estuary Different studies in hybrids such as those on the genera Ipomopsis [START_REF] Campbell | Evolutionary dynamics of an Ipomopsis hybrid zone: confronting models with lifetime fitness data[END_REF], Artemisia [START_REF] Mcarthur | Narrow hybrid zone between two subspecies of big sagebrush (Artemisia tridentata: Asteraceae). VI. Respiration and water potential[END_REF] and Iris [START_REF] Arnold | Hybrid fitness, adaptation and evolutionary diversification: lessons learned from Louisiana Irises[END_REF] proved that the phenotype and the fitness of hybrids can be strongly influenced by the environment in which they develop (reviewed in [START_REF] Arnold | Hybrid fitness across time and habitats[END_REF]. Gallego-Tévar et al. (2018a) described the studied Spartina hybrids as showing very low spatial interaction with neighboring halophytes in the Guadiana Estuary. In this population, the hybrids grew taller than their parental species, and were displacing native species and even the invasive parental S. densiflora. These observations suggest the superior invasive behavior of these hybrids. Their high competitive ability coupled with sterility-limited ability to disperse, have resulted in a marked decrease in the spatial coincidence (two taxa growing together) of the Spartina hybrids with other halophytes from 62% in 2003 to 6% in 2016 though their relative cover has remained constant (Gallego-Tévar, unpublished field data).

The three taxa of Spartina had shorter leaves in Guadiana than in the other estuaries and higher tiller density and smaller number and area of leaves than in the Piedras Estuary.

But also, all taxa exhibited greater width of leaves in Piedras Estuary, where the lower pH was registered, with respect to the other estuaries and lower LAI than in Tinto-Odiel.

Although the influence of the genotype cannot be ruled out, these differences among the three estuaries could be marked by the environment since both Spartina maritima and S. densiflora are species with high reported phenotypic plasticity in contrasted environments (Castillo et al., 2016(Castillo et al., , 2014[START_REF] Castillo | Environmental determination of shoot height in populations of the cordgrass Spartina maritima[END_REF]. In fact, S. maritima presented taller tillers in Tinto-Odiel Estuary where the sediment redox potential was lower, which has been previously described in the species [START_REF] Castillo | Environmental determination of shoot height in populations of the cordgrass Spartina maritima[END_REF]. A common garden experiment including all sampled hybrid populations would help to resolve the extent to which the lack of correlation between GD and PD was due phenotypic plasticity related to clear environmental differences between estuaries When all phenotypical traits were taken into account, genetic, geographical and sedimentary environmental distances in hybrid populations among estuaries did not relate to their vegetative phenotypic distance, although PD and GD were close to positive correlation (P = 0.060). When polygenic inheritance of traits is operating, correlation between GD and PD decreases as the number of loci involved in the regulation of the phenotypic traits increases [START_REF] Burstin | Relationship between phenotypic and marker distances: Theoretical and experimental investigations[END_REF][START_REF] Lefebvre | Evaluation of genetic distances between pepper inbred lines for cultivar protection purposes: comparison of AFLP, RAPD and phenotypic data[END_REF]. In hybrid taxa genotypephenotype association is even more complex since gene expression can be non-additive [START_REF] Bassene | Non-additive phenotypic and transcriptomic inheritance in a citrus allotetraploid somatic hybrid between C. reticulata and C. limon: the case of pulp carotenoid biosynthesis pathway[END_REF][START_REF] Meyer | Heterosis associated gene expression in maize embryos 6 days after fertilization exhibits additive, dominant and overdominant pattern[END_REF] and the molecular mechanisms that regulate gene expression are not completely identified [START_REF] Bird | The causes and consequences of subgenome dominance in hybrids and recent polyploids[END_REF][START_REF] Tirosh | A yeast hybrid provides insight into the evolution of gene expression regulation[END_REF][START_REF] Yoo | Nonadditive gene expression in polyploids[END_REF]. In Spartina, hybridization and allopolyploidy were shown to be accompanied by substantial non-additive parental expression patterns in both controlled [START_REF] Chelaifa | Transcriptomic changes following recent natural hybridization and allopolyploidy in the salt marsh species Spartina × townsendii and Spartina anglica (Poaceae)[END_REF]) [START_REF] Chelaifa | Transcriptomic changes following recent natural hybridization and allopolyploidy in the salt marsh species Spartina × townsendii and Spartina anglica (Poaceae)[END_REF]) and natural conditions [START_REF] Ferreira De Carvalho | Gene expression variation in natural populations of hexaploid and allododecaploid Spartina species (Poaceae)[END_REF]. In the hybrids studied here, both our results and previous study [START_REF] Gallego-Tévar | Phenotypic plasticity of polyploid plant species promotes transgressive behaviour in their hybrids[END_REF] indicate phenotypic patterns reflecting parental dominance, additivity and over-dominance, making complex the genotype-phenotype relationship.

Conclusions

Populations of sterile Spartina hybrids between the native S. maritima and the invasive S. densiflora together with their parental species are establishing hybrid zones [START_REF] Hewitt | Speciation, hybrid zones and phylogeographyor seeing genes in space and time[END_REF], and are developing a spatial genetic structure inherited from both parental species in the Gulf of Cadiz (Southwest Iberian Peninsula). The hybrid populations with greater genetic differentiation are those more spatially separated from each other and that are present in more contrasted sedimentary environments, revealing respective isolation processes by distance and by environment likely originating from their parental species. The hybrids in the Guadiana Estuary were the most genetically differentiated and with the highest transgressive behavior in terms of tiller height. However, the relationship between the population genetic structure of these hybrids and their phenotype is complex and no correlation pattern was found between the molecular markers and the set of recorded vegetative morphological traits. This study provides new data on the dynamics of hybrid invasions, integrating effects of the genetic, phenotypic, geographical and environmental scenario with hybridization following invasion of a halophyte community by an exotic plant species.

A C C E P T E D

M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T

  and phenotypic distances. Considering the sterility of the studied Spartina hybrids, we

  Inc., Waltham, USA). Alleles were identified and binned using GeneMapper 4.1 software (Applied Biosystems Inc., Waltham, USA).

  than S. densiflora (Table

  and elevation) may also contribute to the heterosis shown by the hybrids.

Fig. 4 .Fig. 5 .

 45 Fig. 4. Mantel tests between genetic (GD), phenotypic (PD), geographic (GGD) and

Table 3

 3 Analysis of Molecular Variance (AMOVA) illustrating the proportion of genetic variation distributed within and among populations of hybrids between Spartina maritima and S. densiflora in three estuaries of the Gulf of Cadiz, Southwest Iberian Peninsula (ΦPT = 0.547, P < 0.001).

		U S C R I P
	N
	A
	M
	C C E P T E D
	A
	Source	df	SS	MS	Estimated variance	Percentage
	Among populations	2	48.884	24.442	2.687	55%
	Within populations	23	51.155	2.224	2.224	45%

Table 4

 4 Number of different alleles (Na), number of effective alleles (Ne), Shannon's Information Index (I) for populations of Spartina maritima, S. densiflora and their hybrids from three different estuaries of the Gulf of Cadiz (Southwest Iberian Peninsula). Values are mean ± SE. Hybrids Na 0.854 ± 0.129 0.780 ± 0.133 0.780 ± 0.128 0.585 ± 0.110 0.634 ± 0.125 0.780 ± 0.118 0.780 ± 0.113 0.707 ± 0.127 1.146 ± 0.124 Ne 1.075 ± 0.029 1.117 ± 0.041 1.174 ± 0.053 1.059 ± 0.031 1.122 ± 0.044 1.111 ± 0.040 1.102 ± 0.042 1.141 ± 0.047 1.296 ± 0.063 I 0.089 ± 0.026 0.114 ± 0.033 0.142 ± 0.041 0.057 ± 0.026 0.106 ± 0.035 0.102 ± 0.034 0.088 ± 0.032 0.120 ± 0.037 0.234 ± 0.048

				U S C R I P
				N
	S. maritima	Tinto-Odiel Estuary S. densiflora M Hybrids A S. maritima	Piedras Estuary S. densiflora	Hybrids	S. maritima	S. densiflora Guadiana Estuary
	N C C E P T E D 8 8	11	5	5	5	5	9	10
	A

Table 5

 5 Phenotypic traits and sedimentary variables for three populations of Spartina maritima, S. densiflora and their hybrids in three estuaries of the Gulf of Cadiz (Southwest Iberian Peninsula), TIOD, Tinto and Odiel Estuary; PI, Piedras Estuary; GU, Guadiana Estuary (values are mean ± SE).Leaves per tiller (cm -1 ) 0.17 ± 0.02 0.30 ± 0.02 0.31 ±0.02 0.08 ± 0.02 0.12 ± 0.02 0.11 ± 0.01 0.11 ± 0.02 0.11 ± 0.02 0.09 ± 0.01

					Spartina maritima		Spartina densiflora		Spartina hybrids
				TIOD	PI	GU	TIOD	PI	GU	TIOD	PI	GU
			Number of leaves	5 ± 0.3	6 ± 0.4	4 ± 0.4	3 ± 0.3	4 ± 0.4	3 ± 0.3	4 ± 0.3	5 ± 0.4	4 ± 0.3
			Number of dead leaves	0.3 ± 0.4	1 ± 0.5	0.3 ± 0.5	3 ± 0.4	2 ± 0.5	2 ± 0.4	2 ± 0.4	3 ± 0.5	4 ± 0.4
			Leaf width (cm)	0.6 ± 0.03	0.8 ± 0.04	0.6 ± 0.04	0.5 ± 0.03	0.6 ± 0.04	0.5 ± 0.03	0.6 ± 0.03	0.7 ± 0.04	0.7 ± 0.03
	Phenotypic traits	Leaf length (cm) Leaf area (cm 2 ) SLA (m 2 •g -1 ) Leaf Rolling (%)	17.4 ± 2.3 11.5 ± 1.9 0.013 ± 0.001 4 ± 5	19.3 ± 2.7 13.66 ± 2.7 15.4 ± 2.2 7.9 ± 2.2 0.009 ± 0.001 0.011 ± 0.001 0 ± 0 7 ± 5	39.1 ± 2.3 20.5 ± 1.9 0.007 ± 0.001 38 ± 5	38.8 ± 2.7 22.2 ± 2.2 0.009 ± 0.001 31 ± 6	29.7 ± 1.9 16.3 ± 1.6 0.008 ± 0.001 40 ± 4	27.7 ± 2.3 17.8 ± 1.9 0.012 ± 0.001 14 ± 5	27.8 ± 2.7 20.5 ± 2.2 0.010 ± 0.001 11 ± 6	28.8 ± 1.9 19.9 ± 1.6 0.010 ± 0.001 25 ± 4
			Tiller diameter (mm)	3.5 ± 0.2	3.8 ± 0.2	3.0 ± 0.2	3.5 ± 0.2	3.2 ± 0.2	3.2 ± 0.1	3.3 ± 0.2	3.7 ± 0.2	3.6 ± 0.1
			Tiller length (cm)	31.4 ± 3.1	19.6 ± 3.7	13.5 ± 3.7	37.7 ± 3.1	33.9 ± 3.7	29.6 ± 2.6	42.2 ± 3.1	43.5 ± 3.7	49.8 ± 2.6
			Tillers density (cm -2 )	0.15 ± 0.07 0.06 ± 0.08 0.19 ± 0.08 0.49 ± 0.07 0.37 ± 0.08 0.54 ± 0.06 0.27 ± 0.07 0.23 ± 0.08 0.38 ± 0.06
			LAI	4.72 ± 1.58 1.84 ± 1.58 3.04 ± 1.58 6.11 ± 1.340 5.26 ± 1.58 2.29 ± 1.12 7.71 ± 1.33 5.22 ± 1.58 6.29 ± 1.12
	Sedimentary	variables Conductivity (mS cm -1 ) 23.0 ± 2.3 pH 7.0 ± 0.1 Water content (%) 58 ± 5 Redox potential (mV) -65 ± 28	6.5 ± 0.2 19.5 ± 2.7 54 ± 7 109 ± 33	6.6 ± 0.1 13.8 ± 2.7 48 ± 7 85 ± 33	7.0 ± .1 27.2 ± 2.3 52 ± 5 16 ± 33	6.4 ± 0.2 22.9 ± 2.3 48 ± 7 59 ± 33	6.7 ± 0.2 19.2 ± 2.0 52 ± 5 143 ± 25	7.0 ± 0.1 24.2 ± 3.1 44 ± 5 21 ± 28	6.4 ± 0.2 26.3 ± 2.4 62 ± 6 69 ± 33	6.7 ± 0.1 18.2 ± 1.9 48 ± 5 73 ± 23

Acknowledgements

Blanca Gallego-Tévar thanks the Universidad de Sevilla for a research contract (Plan Propio de Investigación). Funding provided by École Doctorales Agro-Vie-Santé (VAS) of the University of Rennes 1 for international mobility and a cooperative agreement between the United State Department of Agriculture, Agricultural Research Service (USDA-ARS) Invasive Species and Pollinator Health Research Unit at Davis, California and the University of Seville facilitated this collaboration.

Funding: This work was supported by École Doctorales Agro-Vie-Santé (VAS) of the University of Rennes 1; the Plan Propio de Investigación of Universidad de Sevilla; and the United State Department of Agriculture, Agricultural Research Service (USDA-ARS) Invasive Species and Pollinator Health Research Unit [grant number 25-2030-6-043-F].

Declarations of interest: none.

Abbott, R.J., 1992. Plant invasions, interspecific hybridization and the evolution of new plant taxa. Trends Ecol. Evol. 7, 401-405. Nucleotide sequences, of the selected 8 microsatellite primer pairs and their corresponding repeated motifs (as detected in S. maritima genomic assemblies) analyzed in three populations of S. maritima, S. densiflora and their hybrids in Southwest Iberian Peninsula.

0.8 0.9 1 0.9 0.9 0.6 0.9

0.8 1 0.5 0.4 0.6 0.6 0 0.9 0.8 0.5 0.9 0 1 0.8 0

1 0.2 0.8 1 0 1 0.9 0 1 0.8 1 1 0.8 0.9 0.9 0 0.1 0.5 1 0

Elevation (m above SHZ) 2.41 ± 0.08 3.02 ± 0.09 2.52 ± 0.09 3.22 ± 0.08 3.20 ± 0.09 2.67 ± 0.07 3.23 ± 0.03 3.04 ± 0.08 2.68 ± 0.03

Table 6

F-statistic and P-values of two-way ANOVAs for phenotypic traits and sedimentary factors of populations from three different estuaries (Tinto-Odiel, Piedras and Guadiana Estuaries) and taxa (Spartina maritima, S. densiflora and their hybrids) as fixed factors, and their corresponding interactions. Significant differences are marked in bold.


Dr Nicolas Reina
email: reina.n@chu-toulouse.fr

Etienne Cavaignac

William H Trousdale

Jean-Michel Laffosse

Jos E Braga

Laterality and grip strength influence hand bone micro-architecture in modern humans, an HRpQCT study

Keywords: grip strength, human, laterality, trabecular bone, wrist

It is widely hypothesized that mechanical loading, specifically repetitive low-intensity tasks, influences the inner structure of cancellous bone. As such, there is likely a relationship between handedness and bone morphology. The aim of this study is to determine patterns in trabecular bone between dominant and non-dominant hands in modern humans. Seventeen healthy patients between 22 and 32 years old were included in the study. Radial carpal bones (lunate, capitate, scaphoid, trapezium, trapezoid, 1st, 2nd and 3rd metacarpals) were analyzed with high-resolution micro-computed tomography. Additionally, crush and pinch grip were recorded. Factorial analysis indicated that bone volume ratio, trabeculae number (Tb.N), bone surface to volume ratio (BS.BV), body weight, stature and crush grip were all positively correlated with principal components 1 and 2 explaining 78.7% of the variance. Volumetric and trabecular endostructural parameters (BV/TV, BS/BV or Tb.Th, Tb.N) explain the observed inter-individual variability better than anthropometric or clinical parameters. Factors analysis regressions showed correlations between these parameters and the dominant side for crush strength for the lunate (r 2 = 0.640, P < 0.0001), trapezium (r 2 = 0.836, P < 0.0001) and third metacarpal (r 2 = 0.763). However, despite a significant lateralization in grip strength for all patients, the endostructural variability between dominant and non-dominant sides was limited in perspective to inter-individual differences. In conclusion, handedness is unlikely to generate trabecular patterns of asymmetry. It appears, however, that crush strength can be considered for endostructural analysis in the modern human wrist.

Introduction Background

Frost's mechanostat theory first established the ability of bone to remodel in response to mechanical loading [START_REF] Frost | Bone "mass" and the "mechanostat": a proposal[END_REF]. Knowing that intrinsic factors such as bone mineral content, material properties, hormonal changes, age and sex all impact hand skeletal morphologythe effect of unique biomechanical signatures on bone architecture has been well documented [START_REF] Tocheri | A 3D quantitative comparison of trapezium and trapezoid relative articular and nonarticular surface areas in modern humans and great apes[END_REF][START_REF] Skinner | Human evolution. Human-like hand use in Australopithecus africanus[END_REF].

The potential effects of asymmetrical behaviors on the skeletal structure have long been documented. In particular, the tendency to preferentially use one hand in a variety of actions (laterality or 'handedness') has been argued to cause bone structural asymmetry [START_REF] Shaw | Is "hand preference" coded in the hominin skeleton? An in-vivo study of bilateral morphological variation[END_REF]). An important task is to determine whether the inner structure of bone reflects this behavior as suggested by endostructural patterns. Indeed, to investigate the origin of handedness in past populations, many researchers have relied on the inner bone morphology that can be traced in skeletal remains [START_REF] Macchiarelli | Hip bone trabecular architecture shows uniquely distinctive locomotor behaviour in South African australopithecines[END_REF]Lazenby et al. 2008b;[START_REF] Ubelaker | Can handedness be determined from skeletal remains? A chronological review of the literature[END_REF][START_REF] Barak | Trabecular evidence for a human-like gait in Australopithecus africanus[END_REF]. The effect of handedness on bone morphology in humans, however, remains uncertain. Further, most modern activities involve highly lateralized, repetitive, low-intensity mechanical loading. However, the effect of these behaviors on bone morphology is not well understood.

The lateralization of grip strength (GS) represents an important factor characterizing handedness, often had a standard posture, and three out of 17 were left-handed (Table 2). None had a lateralized occupation at work or in athletic activity, but they all had a self-reported handedness. Despite some patients having UCLA scores of 9 or 10, none practiced at competition level or could be considered as intensive practice. Only one patient was identified as a manual worker.

Micro-CT measurements and processing

Both the left and right wrists were analyzed. Patients sat during CT scan with their forearms placed in a rigid splint. A focus on the radial side of the hand is based on the assertion that the thumb column is the most likely to be impacted by prehensile and fine manipulation tasks [START_REF] Marzke | Evolution of the human hand: approaches to acquiring, analysing and interpreting the anatomical evidence[END_REF]. Moreover, muscles involved in prehension such as extensor carpi radialis longus and brevis are attached to the base of the 2nd and 3rd metacarpals. Thus, each participant had four carpal bones (lunate, scaphoid, capitate and trapezium) and three metacarpals (1st, 2nd and 3rd) analyzed on each side measured using an XTREMECT MICROCT (Scanco Medical, Switzerland). This machine produces isometric voxel size of 61 lm. Additionally, three-dimensional (3D) data analyses were performed using AMIRA 4.1 software (VSG, France). A 3D reconstruction of each bone (Fig. 1) was made initially, and volumes of interest (VOI) were placed. This technique was preferred to XTREMECT SCAN software in order to avoid any flaw due to potential motion during the exam on such small bones. Further, performing HRpQCT in vivo might generate images for which CT scan software would not assess precisely during acquisition. Our use of 3D reconstruction allowed for the verification of each slice, and to adjust gray-scales or correct artifacts. Finally, attenuation histograms were used to determine bone/soft tissue segmentation threshold in order to create 3D stacks of data in a DICOM format.

Endostructural parameters

Morphometric analyses were conducted with the CTAN software (SkyScan, www.bruker-microct.com). The following endostructural parameters were assessed using the VOI method: bone volume ratio (BV/TV), specific bone volume (BS/BV), trabecular pattern factor (Tb.Pf), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), degree of anisotropy (DA) and total 1: Wholly inactive, dependent on others, and cannot leave residence 2: Mostly inactive or restricted to minimum activities of daily living 3: Sometimes participates in mild activities, such as walking, limited housework and limited shopping 4: Regularly participates in mild activities 5: Sometimes participates in moderate activities such as swimming or could do unlimited housework or shopping 6: Regularly participates in moderate activities 7: Regularly participates in active events such as bicycling 8: Regularly participates in active events, such as golf or bowling 9: Sometimes participates in impact sports such as jogging, tennis, skiing, acrobatics, ballet, heavy labor or backpacking 10: Regularly participates in impact sports considered as a reflection of human adaptation to toolrelated behavior. GS lateralization has been described at a population level [START_REF] Mitsionis | Normative data on hand grip strength in a Greek adult population[END_REF]. Even though several factors may influence GS, a curvilinear relationship with age has been described with a peak between 25 and 50 years of age [START_REF] Mathiowetz | Grip and pinch strength: normative data for adults[END_REF]. In adults, GS is significantly higher (~10%) in men than in women, and this difference increases with age [START_REF] Kamide | Reference values for hand grip strength in Japanese community-dwelling elderly: a meta-analysis[END_REF]. However, this trend was reported only in right-handed individuals, whereas no GS asymmetry between genders was shown in left-handed people.

Few studies have examined the effects of these variations on bone architectureknowledge that would be useful in examining the reaction of trabecular bone to the infraliminar constraints of daily life. However, reference data for standard behaviors are missing. The present study uses high-resolution peripheral quantitative computed tomography (HRpQCT) in a sample of living individuals to identify endostructural features of the human wrist that correlate to anthropometric and clinical measurements of GS and handedness.

Objectives

This study assessed bone morphological factors influenced by lateralized activities of daily life in healthy, young, modern humans.

Methods

Ethical statement

The local Institutional Review Board approved the protocol. All participants received a verbal and written description of the protocol prior to participation. Following this, each participant provided written informed consent.

Study design

Criteria for inclusion were age between 20 and 40 years. Participants with a medical history of bone disease or wrist fracture were excluded. Twenty participants were included initially. Two with previous wrist fracture were excluded, and one with no self-reported handedness was excluded.

Our final study cohort included 17 patients (six female and 11 male). Sample size was decided after computing data from previous studies [START_REF] Tsegai | Trabecular bone structure correlates with hand posture and use in hominoids[END_REF].

Anthropometric and clinical measurements

Each participant completed a questionnaire to record their medical history, biometric data (height, weight and age), handedness and athletic activities (graded by UCLA score defined in Table 1). GS was recorded with a Jamar grip dynamometer (Jamar Plus+; Sammons Preston, Rolyon, Bolingbrook, IL, USA) for crush grip (fingers flexed on palm) and pinch grip (thumb and index finger). Each measurement was repeated three times, with averages reported. Patients porosity (Po.tot). For each bone (except the capitate), the VOI was placed centrally in the bone volume, using three orthoslices planes scaled at 50% of the largest diameter. For the capitate, due to its particular shape, a 50% scaled VOI was placed and centered in the head proximally. For metacarpals 1, 2 and 3, the VOIs were scaled at 50% of the largest diameter of the proximal epiphysis.

Statistical analyses

Data were analyzed using the R software (www.R-project.org). Values were normally distributed in the global sample, and subgroups were also examined.

Principal component analyses (PCA) were performed to investigate the relationships between variables. A factor map was used to identify the anthropometric and clinical measurements and the endostructural features that most influenced the variation in our cohort. The PCA highlighted the discriminating parameters for each bone taken separately or considered together. The influence of intrinsic patient-related variables [sex, body weight, stature, body mass index (BMI)] and extrinsic variables (UCLA activity level) on bone endostructure was assessed for each patient. Regressions were performed considering laterality and bone microstructure patterns as independent and dependent variables, respectively. The strongest statistical models were applied to GS and handedness. P-value significance was set at < 0.05.

Results

Parameters explaining the observed variation among our sample

Looking at all the bones from each patient, the factorial analysis indicated that bone volume ratio, number of trabeculae and bone surface per bone volume were positively correlated with principal component (PC)1 and accounted for 58.7% of total variance (Fig. 3). PC2 (20.0% of variance) was positively correlated with porosity and negatively with bone volume ratio, number of trabeculae, trabecular pattern factor and bone surface per bone volume. Therefore, some endostructural parameters appear to explain better the variation among our sample than anthropometric and clinical measurements. No sex effects were found (P = 0.488) in any bone. A clear separation appeared between each bone on PCA, and PC1 and PC2 were significant for bone specificity (P < 0.0001). However, on both principal components, overlapping was shown for lunate-scaphoid and trapezium-MC-3. Figure 3 highlights how the positive end of PC1 reflects a compact and dense trabecular architecture, with low porosity subsequently. The positive end of PC2 showed a more porous trabecular bone and less robustly built architecture. The PCA results for each bone after statistical rotation and extraction are summarized in Table 3. The highest percentages of variances explained by the two-first components were obtained for the capitate (97.5%) and the first metacarpal (96.5%). For both the capitate and the first metacarpal, the first PC was explained mainly by the bone volume ratio (BV/TV; Fig. 2) and Po.tot (Table 3). Overall correlations of significant patterns (i.e. estimation on PC1 or PC2 > 0.750) are presented in Fig. 3 for each bone. Capitate, MC-1 and MC-2 were the most consistent groups of bones within the sample. Capitate was found with a robust and specific architecture, while MC-1 and MC-2 showed higher porosity and thinner trabeculae.

GS

Although no extreme lateralized occupation (e.g. intensive lateralized sport or asymmetrical work) was recorded, we observed significant differences between dominant and non-dominant crush grip (36.0 AE 9.6 N; P = 0.002) and pinch grip (6.0 AE 2.1 N; P = 0.012). The differences observed for crushgripwereinfavor ofthedominantsideinallbut one patient (Table 4). The average difference was 7% (À15% to 15%) between both sides. Pinch grip had a different distributionwithnostatisticalrelationtosexor occupation.Four patients had a pinch grip in favor of the non-dominant side.

The average difference was 6% (À9% to 23%). The UCLA score was not correlated to the lateralization of pinch or crush strength, respectively, r 2 = 0.305, P = 0.769 and r 2 = 0.366, P = 0.646. The endostructural parameters for each bone were considered separately. Additionally, multiple regression analyses of GS indicated a highly significant correlation (P < 0.0001) between these parameters (PC1 and PC2) and the dominant side for the lunate (r 2 = 0.640), the trapezium (r 2 = 0.836, P < 0.0001) and the third metacarpal (r 2 = 0.763).

Laterality

We investigated whether handedness had an influence on the observed variability in endostructural values. It appeared that distribution is more related to bone than to side (Fig. 4). When considering each bone individually, we observed the distribution of dominant and non-dominant sides in PC1 vs. PC2 plots. As a general trend, we observed that the differences between dominant and non-dominant sides were limited and relatively less substantial than interindividual variability. Figure 4 shows the PCA analysis per side. PC1 was found not to be significant for laterality (P = 0.176) as well as PC2 (P = 0.796). The dominant and nondominant sides representing the same individual generally indicated similar endostructural values. For example, when we consider the endostructural values obtained for the lunate and the first metacarpal, patient number 15 appears relatively distinct from all the other individuals for both dominant and non-dominant sides. At the same time, lunate endostructural values obtained for dominant and non-dominant sides within several individuals are almost superimposed (e.g. patients 9 and 11). Importantly, no inter-individual difference due to sex, activity or GS explains the variability observed in this dataset. Further, these same trends are observed when examining the endostructural parameters of all bones together. Specifically for the capitate, the parameters BV/TV, BS/TV and Tb.N were higher in the dominant side whether Tb.Pf and Tb.Sp were lower. For the scaphoid, porosity was slightly higher in the dominant side but all the other parameters were comparable. For the lunate, BS/BV, Tb.Pf, Tb.Sp and porosity were higher in the dominant side whether BV/TV, BS/TV, Tb.N were lower. The trapezium, MC-1 and MC-2 had the same patterns, BV/TV, BS/TV, Tb.N were higher in the dominant side whether BS.BV, Tb.Pf, Tb.Sp and porosity were lower.

Discussion and conclusions

We sought to define the pattern of laterality in vivo on the carpal skeleton of the human hand. Specifically, we found bone volume ratio (BV/TV) as a specific parameter explaining variability in each bone. The number of trabeculae and the specific bone surface (BS/BV) appeared also recurrent indicators for variability. We observed a relationship between GS and trabeculae patterns for the lunate, MC-3 and trapezium. Conversely, pinch grip was inconsistent with hand preference and was not related to any specific variable. Despite these findings, the results of this study suggest that usual tasks (e.g. writing, eating) with a preferred hand do not stimulate sufficient asymmetric mechanostat biofeedback to influence bone modeling in the wrist. Indeed, a multifactorial analysis did not show any robust pattern. Further, endostructural parameters did not appear to be correlated with any anthropometric (body weight or stature), sex or clinical measurements. BMI was not involved in the variability in any bone. This is likely due to the fact that forelimbs are not used for locomotion [START_REF] Marzke | Upper-limb evolution and development[END_REF]. As such, the modern behaviors sampled in this study may Fig. 3 Variables correlations on principal component analysis (PCA) on PC1 (x-axis) and PC2 (y-axis) per bone studied. The explanation of the variance per PC (58% for PC1 and 20% for PC2) is meant for all the bones studied.

impact of handedness on bone in non-athletic individuals is necessary to fully understand the relationship between mechanostat biofeedback and bone modeling. Additionally, this study builds upon other studies that have reported the effect of age and sex on bone characteristics, but failed to investigate laterality [START_REF] Hasegawa | Age, sex, and grip strength determine architectural bone parameters assessed by peripheral quantitative computed tomography (pQCT) at the human radius[END_REF][START_REF] Edwards | Muscle size, strength, and physical performance and their associations with bone structure in the Hertfordshire Cohort Study[END_REF][START_REF] Szulc | Impaired bone microarchitecture at the distal radius in older men with low muscle mass and grip strength: the STRAMBO study[END_REF][START_REF] Crockett | Bone strength and muscle properties in postmenopausal women with and without a recent distal radius fracture[END_REF]. Finally, not create sufficient strains on the hand skeleton to influence bone modeling.

To our knowledge, this is the first study to assess the relationship between endostructure and strength in a living patient with standard activities. Unfortunately, the current literature is focused on athletes and extreme handedness [START_REF] Ozener | Extreme behavioral lateralization and the remodeling of the distal humerus[END_REF]). Yet, we believe an investigation into the our use of pQCT to analyze cross-sectional bone provides more detailed data than similar studies only focusing on bone mass density.

Limitations

This study has several limitations. First, the sample size is small. Though other well-designed studies using identical segmentation methods have been published with comparable sample sizes [START_REF] Stephens | Trabecular architecture in the thumb of Pan and Homo: implications for investigating hand use, loading, and hand preference in the fossil record[END_REF], we believe that further studies should be conducted using larger sample sizes to confirm the relationship between lateralized low-intensity activities on human bone morphology. Second, there are well-documented technical limitations with the collection of VOI data. Specifically, [START_REF] Kivell | Methodological considerations for analyzing trabecular architecture: an example from the primate hand[END_REF] expressed concerns about technical difficulties in collecting VOI data on wrist bones. Indeed, trabeculae distribution in such small bones is heterogeneous and choice of VOIs position is essential. As a consequence, assessments of handedness in fossil samples should be cautious, even in the case of observed side differences at the micro-architectural level. We used 61 lm voxels to limit time of acquisition, which can be considered a low resolution to assess endostructure on non-weight-bearing bones. The values reported, particularly the trabeculae thickness, appear to be higher than previously published data. That might be a limitation of our segmentation technique and the resolution used. Finally, we must consider some inaccuracy in determining the edges of the trabeculae due to marrow and fat content of the interstitial space, considering this is an in vivo study. [START_REF] Kivell | A review of trabecular bone functional adaptation: what have we learned from trabecular analyses in extant hominoids and what can we apply to fossils?[END_REF] detailed the difficulty of such an analysis in living samples and emphasized that current literature is focused only in high-activity athletes. Mice models have been developed with poor reliability and translational issues to human models. For these reasons, in vivo data must be considered with caution. As such, we reported all data as trends.

There are many data supporting the ability of high biomechanical loading to effect human bone structure. [START_REF] Erlandson | Higher premenarcheal bone mass in elite gymnasts is maintained into young adulthood after long-term retirement from sport: a 14-year follow-up[END_REF] used dual-energy X-rays absorptiometry analysis to show that premenarchal elite gymnasts had a higher bone mass in the femoral neck, when compared with non-gymnasts. Further, such a difference was proven to remain unchanged 14 years later when the loading stimulus had decreased. These results highlight the ability of bone modifications from repetitive biomechanical loading to persist long after the termination of the loading behavior. Similarly, [START_REF] Sone | Side-to-side differences in cortical bone mineral density of tibiae in young male athletes[END_REF] observed asymmetry in the tibia of individuals with a dominant leg for mobility and manipulation, and [START_REF] Sylvester | Factors influencing osteological changes in the hands and fingers of rock climbers[END_REF] used radiographs to show differences in bone morphology and pathology (e.g. osteoarthritis) based on human activity (specifically rock climbing). Therefore, there is little doubt that mechanical behavior affects bone structure. Yet, important questions regarding the relationship between mechanical behavior and bone morphology remainspecifically, the effect of repetitive biomechanical loads on cancellous and cortical bone. In this context, the impact of handedness on bone clearly represents one of the most challenging questions to address. This study sought to better understand the impact of handedness on bone modeling by investigating endostructural parameters in modern human carpal bones. Carpal modeling represents a hallmark of adaptation to tool manipulation [START_REF] Marzke | Upper-limb evolution and development[END_REF]. Our findings highlight that a level of activity or strength might be determined as a threshold to stimulate aptation to environment. Lazenby et al. (2008a) suggest that volumetric variables such as BV/TV and Tb.N are sensitive to mechanical regulation and handedness. Further, they found that the influence of age is felt more on the left rather than the right metacarpal given the human propensity for right-handedness. This remains not obvious in our results. Our data suggest that usual tasks (e.g. writing, eating) with a preferred hand may be important inter-individual differences in endostructural parameters (i.e. as trabecular thickness and bone volume ratio). These tasks, however, did not produce strong morphological differences between the two sides. It should be noted that contrary findings have been described in the literature. [START_REF] Stephens | Trabecular architecture in the thumb of Pan and Homo: implications for investigating hand use, loading, and hand preference in the fossil record[END_REF] assessed the laterality in Homo sapiens and Pan troglodytes and paniscus by micro-CT scan, identifying specific and significant patterns of laterality (BV/TV, degree of anisotropy and elastic modulus in the base of the first metacarpal). Our results suggest similar patterns for MC-1 and MC-2 bases and trapezium (BV/TV = 11/17 on MC-1, 9/17 on MC-2 and 11/17 on trapezium), but remain unclear for the rest of the thumb column. We did not find anisotropy as a significant factor. In that same study, however, despite a flawless method, the lateralization could not be reported as the bone analyzed came from individuals from the 1st and 3rd centuries. The authors assumed that asymmetry of the bone is explained by laterality, but this fact is not invariable. In our sample, one individual showed a higher crush grip in the right hand when he reported himself as left-handed. In a society where righthanded preference reported is over 90%, usual tasks are taught and tools are made to facilitate this lateralization. This is an interesting fact that should not be underestimated when analyzing fossils. Earlier studies have illustrated the effect of lateralized sport activity and bone morphology. Notably, [START_REF] Shaw | Is "hand preference" coded in the hominin skeleton? An in-vivo study of bilateral morphological variation[END_REF] documented an effect on humeral, ulnar and tibial shaft morphology based on specific lateralized biomechanical loading patterns in cricket and hockey players. However, no significant asymmetry was observed in their control groups with a non-lateralized activity. This finding based on pQCT of 0.5 mm slices (l-CT) suggested that bone modeling was not sufficient to produce a significant asymmetry in both non-professional athletes and runners and swimmers with no 'habitual, highly intense or repetitive, unilateral upper limb activities in the loading histories of these groups'. Shaw concluded that laterality can produce a significant asymmetry only in the case of a marked biomechanical contrast between right and left sidesas in the high repetitive biomechanical loading experienced by cricket and hockey players. It should be noted that Shaw's finding reports an estimate of the torsional and average bending rigidity of the diaphysis and cortical area, an indicator of a bone's mechanical performance under biomechanical loading [START_REF] Ruff | Who's afraid of the big bad Wolff?: 'Wolff's law' and bone functional adaptation[END_REF]). This method is limited in that it only concerns the diaphysis, and deemphasizes trabecular bone patterning. Moreover, no indication about patients' morphology, height, weight or BMI was analyzed as influencing factors. In our study, we assessed correlations between anthropometric variables and endostructure. As such, our study reinforces Shaw's finding that unilateral and repetitive stresses are required in low-intensity activities to mark bone structure in upper limb and carpal bones morphology.

Fig. 1

 1 Fig. 1 Three-dimensional (3D) segmentation of wrist bones prior to volume of interest (VOI) analysis.

Fig. 2

 2 Fig. 2 Volume of interest (VOI) in capitate (non-dominant side on left, dominant side in right) on patient 2. Trabeculae are thicker (Tb.Th 0.35 mm vs. 0.31 mm) on the right sample, and bone volume fraction BV/TV is higher (37.3% vs. 30.9%).

Fig. 4

 4 Fig. 4 Variables correlations on principal component analysis (PCA) on PC1 (x-axis) and PC2 (y-axis) for dominant and non-dominant sides. Dots indicate dominant and triangles indicate non-dominant. The explanation of the variance per PC (58% for PC1 and 20% for PC2) is meant for all the bones studied.

Table 1

 1 UCLA activity score.

Table 2

 2 Sample investigated in this study with individual parameters.

		Weight	Height	BMI	Sex	Laterality	UCLA
	1	67	172	22.6	M	Right	8
	2	48	162	18.3	F	Right	8
	3	62	167	22.2	F	Left	6
	4	68	176	22.0	M	Right	10
	5	75	169	26.3	M	Left	9
	6	60	157	24.3	F	Right	4
	7	63	183	18.8	M	Right	9
	8	60	174	19.8	M	Right	3
	9	60	169	21.0	F	Right	8
	10	66	180	20.4	M	Right	7
	11	75	179	23.4	M	Right	6
	12	74	182	22.3	M	Right	8
	13	74	183	22.1	M	Right	9
	14	67	168	23.7	F	Right	5
	15	81	180	25.0	M	Right	5
	16	67	182	20.2	M	Left	10
	17	60	169	21.0	F	Right	6
	Mean	66.3	173.6	22.0
	Min	48.0	157.0	18.3			3
	Max	81.0	183.0	26.3			10
	SD	8.0	7.8	2.2

BMI, body mass index.

Table 3

 3 Endostructural and anthropometric variables representation for each bone on principal component (PC 1 and 2).

			2	22.3			0.383		0.384		À0.416				À0.404
	MC-3		1	56.2				0.949	À0.880		0.845		À0.826		0.903
			2	19.0		0.785	0.905							0.839
	MC-2		1	67.7					À0.976	0.977		À0.905			0.976
			2	22.3
	MC-1		1	74.2					À0.986				À0.953		0.986
			2	39.0
	Capitate		1	58.5					0.965						À0.966
			2	20.2				0.365
	Trapezium		1	60.0				0.853	À0.993	À0.945	0.985				0.994
			2	32.3							À0.338	À0.837
	Lunate		1	41.1					À0.939		0.850	0.380			0.942
	All Scaphoid	Bone	P C 1 2 1 2	% Var 58.7 20.0 47.9 27.2	BMI	Height	Weight À0.410	BS/BV À0.933	BV/TV 0.917 À0.322	Tb.N 0.818 À0.328	Tb.Pf À0.890 0.943	Tb.Sp	Tb.Th 0.870	DA	Po.tot À0.916 0.330 0.980	BMI, body mass index; PC, principal component.

Table 4

 4 Patients strength for crush and pinch grips (N).

	Patients	Crush grip	Crush grip non-	Delta crush	Pinch grip	Pinch grip non-	Delta pinch
	#	dominant	dominant	(%)	dominant	dominant	(%)
	1	618.0	529.7	14	106.3	101.4	5
	2	255.1	215.8	15	93.2	78.5	16
	3	480.7	470.9	2	83.4	89.3	À7
	4	640.9	608.2	5	107.9	99.7	8
	5	506.9	582.1	À15	104.6	80.1	23
	6	320.5	294.3	8	81.8	85.0	À4
	7	608.2	542.8	11	139.0	147.2	À6
	8	539.6	519.9	4	99.7	99.7	0
	9	323.7	307.4	5	83.4	75.2	10
	10	503.6	500.3	1	75.2	81.8	À9
	11	670.4	572.5	15	106.3	101.4	5
	12	595.1	529.7	11	106.3	96.5	9
	13	565.7	497.0	12	103.0	93.2	10
	14	255.1	235.4	8	89.9	75.2	16
	15	588.6	526.5	11	106.3	103.0	3
	16	621.3	588.6	5	107.9	96.5	11
	17	467.6	425.1	9	91.6	80.1	13

Acknowledgements

No funding was required for this study. The authors declare that they have no conflict of interest. Informed consent was obtained from all individual participants included in the study. The authors would like to thank Dr. Tracy Kivell and Dr. Matthew Skinner from the University of Kent in UK for their great inputs in that study and their expertise in hand endostructure and evolutionary anthropology, Medes team -Institute for Space Medicine in Toulouse and especially Dr Arnaud Beck for technical support with micro-CT acquisition. The authors are also grateful to Professor Jean Michel Loubes and Laurent Maubisson for their help on statistics and methodology.

Author contributions

NR and JB designed the study and collected data. EC and JML contributed to statistical analysis and data collection. TK, WT and NR participated in writing discussion and revised the manuscript.