Sara Usandizaga

Paula Valenzuela

Juan Diego Gaitán-Espitia

Christophe Destombe

Marie Laure Guillemin
email: marielaure.guillemin@gmail.com

Marie-Laure Guillemin

Reproductive effort in the domesticated red alga Agarophyton chilense: differences between farms and natural populations

Keywords: Aquaculture, Gracilariales, cystocarp production, reproductive effort, investment, trade-off, vegetative growth

Macroalgal domestication and farming can induce significant ecological and biological changes in exploited species. In the red macroalga, Agarophyton chilense, marine farming is based on clonal propagation by cuttings of the largest plants. This type of mass selection by farmers can have a considerable impact on the life history characteristics of cultivated strains. In this study, we investigated the potential effect of this type of selection on the reproduction in A. chilense, comparing female gametophytes from the natural and farmed populations. Our results showed strong differences between these two types of populations. As expected, individuals were larger in the farm than in the wild population. On the other hand, the number of cystocarps per centimeters of fronds was ten times lower in the farm than in the natural population. These differences suggest that artificial selection and cultivation environment significantly modify life-history traits in this macroalga.

Moreover, the positive relationship between female size and number of cystocarps per centimeters of fronds observed in Dichato point out to the possible existence of cost of reproduction in A. chilense, with bigger females allocating more resources or energy to reproductive structures in natural populations.

Introduction

New macroalgal cultivation methods, most often adapted from the agricultural practices of terrestrial plants, are being developed in marine aquaculture [START_REF] Robinson | Genetic improvement of macroalgae: status to date and needs for the future[END_REF][START_REF] Hafting | Prospects and challenges for industrial production of seaweed bioactives[END_REF]. These new cultivation practices in human-controlled environments are associated with significant ecological changes [START_REF] Smith | Neo-Darwinism, niche construction theory, and the initial domestication of plants and animals[END_REF], as well as alterations in the biology and reproduction of cultivated species [START_REF] Zeder | Core questions in domestication research[END_REF][START_REF] Valero | Perspectives on domestication research for sustainable seaweed aquaculture[END_REF]. In macroalgae, these domestication processes are still poorly understood and further research, integrating knowledge on the evolutionary biology and ecology of natural populations and on the selective pressures exerted by cultivators, is therefore necessary to promote sustainable macroalgal aquaculture (for review [START_REF] Valero | Perspectives on domestication research for sustainable seaweed aquaculture[END_REF]. In some species of red algae, cultivation is based on the clonal propagation of rapidly growing algal strains on farms. This type of clonal multiplication can generate an important impact in the characteristics of the biological cycle and the genetic diversity of cultivated species [START_REF] Guillemin | Genetic variation in wild and cultivated populations of the haploid-diploid red alga Gracilaria chilensis: How farming practices favor asexual reproduction and heterozigosity[END_REF][START_REF] Valero | Perspectives on domestication research for sustainable seaweed aquaculture[END_REF]. One example of this is the Rhodophyta Agarophyton chilense (C.J.Bird, McLachlan et E.C.Oliveira) Gurgel, J.N.Norris et Fredericq previously referred as Gracilaria chilensis. This species has been domesticated and cultivated in Chile since the early 1970s, mainly for agar production [START_REF] Buschmann | Seaweed future cultivation in Chile: perspectives and challenges[END_REF].

It has been hypothesized that farmers have unconsciously selected for fast growth rate genotypes in farms, potentially simultaneously counter-selecting against high reproductive investment [START_REF] Guillemin | Genetic variation in wild and cultivated populations of the haploid-diploid red alga Gracilaria chilensis: How farming practices favor asexual reproduction and heterozigosity[END_REF][START_REF] Usandizaga | Better off alone? Compared performance of monoclonal and polyclonal stands of a cultivated red alga growth[END_REF]. Indeed, in controlled laboratory conditions, trade-offs have been reported between growth and reproduction for this species [START_REF] Guillemin | Evidence of reproductive cost in the triphasic life history of the red alga Gracilaria chilensis (Gracilariales, Rhodophyta)[END_REF].

Agarophyton chilense presents a typical isomorphic haplodiplontic life cycle with two free-living isomorphic generations: haploid gametophytes (males and females) and diploid tetrasporophyte. When released, the haploid tetraspores attach to the substrate to give new gametophyte individuals consisting of perennial holdfasts from which spaghetti-like thalli grow. Male gametes (spermatia) are released in the water column. Female thalli bear uniformly distributed gametangia, which consist of three cells and can be observed only on thallus sections under microscope [START_REF] Kling | Morphogenesis of the frond of Gracilaria verrucosa (Hudson) Papenfuss: the differentiation of vegetative and sexual areas[END_REF]. Fertilization occurs on the female individual and involves complex cytological events, resulting in the formation of a cystocarp on the female [START_REF] Hommersand | Sexual reproduction and cystocarp development[END_REF]. The development of a haploid pericarp is induced, within which the zygote divides mitotically (gonimoblast), protected and nurtured by the female. After a few weeks of maturation, the cystocarp liberates thousands of diploid spores named carpospores [START_REF] Lefebvre | Le fonctionnement du carposporophyte de Gracilaria verrucosa et ses repercussions sur la strategic de reproduction[END_REF][START_REF] Fredericq | Proposal of the Gracilariales Ord. Nov. (Rhodophyta) based on an analysis of the reproductive development of Gracilaria verrucosa[END_REF][START_REF] Kain | A review of the life history, reproduction and phenology of Gracilaria[END_REF]. These carpospores, after their release, give rise to new tetrasporophyte individuals.

Two types of populations, potentially presenting strong difference in reproductive investment [START_REF] Guillemin | Genetic variation in wild and cultivated populations of the haploid-diploid red alga Gracilaria chilensis: How farming practices favor asexual reproduction and heterozigosity[END_REF], are found in Chile: (1) natural populations formed by individuals growing from a perennial holdfast and fixed to hard substratum and (2) floating thalli that grow and propagate by self-replication in sandy and muddy bays and estuaries (i.e., farms). The natural populations are maintained by sexual reproduction and spores recruitment [START_REF] Guillemin | Genetic variation in wild and cultivated populations of the haploid-diploid red alga Gracilaria chilensis: How farming practices favor asexual reproduction and heterozigosity[END_REF]. In these populations fertile tetrasporophytes, male and female gametophytes are frequently encountered [START_REF] Meneses | Sources of morphological variation in populations of Gracilaria chilensis Bird, McLachlan & Oliveira of Chile[END_REF]Vieira et al. 2018a). On the other hand, farmed populations -growing on muddy or sandy beds -are composed by a high proportion of genetically identical units [START_REF] Guillemin | Genetic variation in wild and cultivated populations of the haploid-diploid red alga Gracilaria chilensis: How farming practices favor asexual reproduction and heterozigosity[END_REF], mainly maintained through human-assisted thallus breakage and embedding. Most of the Chilean crops of A. chilense are composed of clonal tetrasporophytes, except the farmed population in Lenga (36° 45' S, 73° 11' W), where growing thalli are mainly female gametophytes [START_REF] Guillemin | Genetic variation in wild and cultivated populations of the haploid-diploid red alga Gracilaria chilensis: How farming practices favor asexual reproduction and heterozigosity[END_REF].

Farmed thalli are generally recognized to be poorly reproductive sexually [START_REF] Prieto | Variation of phenophases of Gracilaria chilensis Bird, Mclachlan and Oliveira (Rhodophyta, Gigartinales), in laboratory and field culture conditions. Presence of mixed phases[END_REF].

However, even if slightly less reproductive individuals have been observed in farms, no clear significant differences have been reported in terms of frequency of reproductive thalli when compared to natural populations [START_REF] Guillemin | Genetic variation in wild and cultivated populations of the haploid-diploid red alga Gracilaria chilensis: How farming practices favor asexual reproduction and heterozigosity[END_REF].

In haplodiplontic red macroalgae, the quantification of cystocarps production is simple since these reproductive structures are visible with the naked eye and have hemispherical fruiting bodies which develop on the female gametophyte after fertilization (Richerd et al. 1993, Kamiya and[START_REF] Kamiya | Investigations on reproductive affinities in red algae[END_REF]. In A. chilense (as G. chilensis) and Gracilaria domingensis (Kützing) Sonder, the vegetative growth of female gametophytes decreases after fertilization during the development of cystocarps [START_REF] Santelices | Regenerative capacity of Gracilaria fragments: effects of size, reproductive state and position along the axis[END_REF][START_REF] Guimarães | Life history, reproduction and growth of Gracilaria domingensis (Gracilariales, Rhodophyta) from Brazil[END_REF]). These observations are consistent with resource allocation theory which predicts that reproduction imposes a cost on an organism (for reviews see DeWreede and Klinger 1988;[START_REF] Santelices | Patterns of reproduction, dispersal and recruitment in seaweeds[END_REF]). The number and size of cystocarps produced by a female has been used to measure red macroalgae reproductive output [START_REF] Edyvean | Population biology of the crustose red alga Lithophyllum incrustans Phil. 3. The effects of local environmental variables[END_REF]. The reproductive effort, classically defined as the relative amount of resources allocated to reproduction over a defined period of time, is much more complex to measure. The ratio of the reproductive biomass to that of the whole plant or the vegetative biomass has been used as a proxy of reproductive effort in Fucales [START_REF] Cousens | Quantitative reproduction and reproductive effort by stands of the brown alga Ascophyllum nodosum (L.) Le Jolis in south-eastern Canada[END_REF][START_REF] Ang | Cost of reproduction in Fucus distichus[END_REF][START_REF] Åberg | Patterns of reproductive effort in the brown alga Ascophyllum nodosum[END_REF] and crustose macroalgae [START_REF] Edyvean | Population biology of the crustose red alga Lithophyllum incrustans Phil. 3. The effects of local environmental variables[END_REF]. In red macroalgae, reproductive output will depend on the female genotype and the number of gametangia it produces (Engel and Destombe 2002), the quantity and quality of male gametes (Engel and Destombe 2002;Engel et al. 2002) and the female investment in cystocarps [START_REF] Kamiya | Dependence of the carposporophyte on the maternal gametophyte in three ceramiacean algae (Rhodophyta), with respect to carposporophyte development, spore production and germination success[END_REF]. However, reproductive output and reproductive effort are sometimes difficult to distinguish, and cystocarp production per unit area or frond length has been used as an indicators or proxy for reproductive effort [START_REF] Krueger-Hadfield | Reproductive effort of Mastocarpus papillatus (Rhodophyta) along the California coast[END_REF]. In this study, we examined the relationship between thallus size and reproductive output in natural versus crop populations.

We hypothesize that clonal propagation and artificial selection in farmed populations have induced reproductive costs in A. chilense evidenced by the trade-off between thallus size and cystocarps production.

Materials and methods

. Collection and maintenance of algal material

In A. chilense, gametophytes and tetrasporophytes share the same gross morphology and the phase and sex can only be recognized during the reproduction period. Even if reproductive individuals could be encountered all year round and reproductive season is not clearly defined for A. chilense, the highest percentage of reproductive individuals is encountered during summer (December to April in the southern hemisphere, Guillemin M-L. unpublished data) and the highest spore production occur at the end of summer / beginning of autumn (after February, Vieira et al. 2018b).

Reproductive female gametophytes were collected in March 2009 in the natural population of Dichato (36° 32' S, 72° 56' W) and in the crop of Lenga (36° 45' S, 72° 11' W) separated geographically by about 25 km (see Figure 1). We sampled different genets (i.e. females with distinct holdfasts) in Dichato and different ramets (i.e. thalli developing from an under-ground thallus system) in the farm of Lenga. In Dichato, females with cystocarps were selected directly in the field. In Lenga, a farm where much less reproductive structures are generally encountered [START_REF] Guillemin | Genetic variation in wild and cultivated populations of the haploid-diploid red alga Gracilaria chilensis: How farming practices favor asexual reproduction and heterozigosity[END_REF], only well-isolated thalli (i.e., a well-defined group of fronds surrounded by at least 10 cm of sand from any other A. chilense) were randomly selected (N=100) and the above-ground biomass was collected. In each site, individuals were collected in an area no larger than 20 m 2 . The collected individuals were placed separately in plastic bags and transported in a cooler to the "Calfuco Water Resources Coastal Laboratory", in Valdivia. Once at the laboratory, the presence of cystocarps was revised thoroughly under a stereoscope microscope (Zeiss Stemi DV4/DR) and speciesspecific molecular sex markers were amplified in vegetative individuals from Lenga using the protocol defined by the authors [START_REF] Guillemin | Characterization of genetic markers linked to sex determination in the haploid-diploid red alga Gracilaria chilensis[END_REF]. In total, 20 females were selected in each population for further analyses.

Biomass and reproductive investment estimators

In the natural population of Dichato, all females were attached to the substratum and primary fronds were defined as thalli directly growing from the holdfast. In the farm of Lenga, after cutting individuals at sand level, the independent thalli forming the clump were defined as primary fronds. In both populations all primary fronds and up to seven secondary fronds per female were measured. For each female, we measured four estimators of biomass: i) the dry weight (g), ii) the volume of the thallus (i.e., the volume of a cylinder of the maximum length and maximum diameter of the thallus in cm 3 ), and iii) the length of primary fronds (cm) and iv) the length of secondary fronds (cm). As reproductive output estimators, we measured: i) the number of cystocarps observed per centimeter of primary fronds (no. of cystocarps • cm -1 ) and, ii) the number of cystocarps observed per centimeter of secondary fronds (no. of cystocarps • cm -1 ). Hereafter, the diameter of the cystocarps was measured under a stereoscope microscope in the lab (Zeiss Stemi DV4/DR). Up to 60 cystocarps were measured per female in Dichato and up to 45 in Lenga. In order to verify for the presence of viable carpospores in the cystocarps, six fragments of fronds carrying cystocarps were taken haphazardly in each population and fixed in 10% formalin-seawater. Material embedded in paraffin was sectioned in a microtome (Leica RM 2035) and stained with dilute methylene blue or haematoxylin-eosin for observation of general morphology. Photomicrographs were taken with an Olympus BX41 microscope.

Statistical analysis

Data were tested for homogeneity of variances and normal distribution using Levene's and Shapiro-Wilk tests, respectively. When existence of non-normal residuals and/or heteroscedasticity was detected, data were logarithmically transformed prior analyses.

Student T tests were performed to evaluate differences between Dichato and Lenga whenever homogeneity of variances was achieved (i.e., dry weight, volume of the thallus and primary and secondary fronds length), while the non-parametric Mann-Whitney U-tests were used when homogeneity of variances was not fulfilled after data transformations (i.e., number of cystocarps per centimeter of primary and secondary frond). Correlation between length of primary fronds and cystocarps production in females were tested using Spearman rank correlation, performed independently in Dichato and Lenga. All differences were considered significant at p-value < 0.05 and correction for multiple testing was carried out using the Bonferroni method. All analyses were performed in R (3.6.3 version) (R Core Team 2016).

Results

In the farm of Lenga, cystocarps were observed in only 80% of the females (16 out of 20), whereas in the natural population of Dichato, all 20 females studied bear cystocarps.

Strong differences were observed between Dichato and the crop of Lenga for female size (length of primary and secondary fronds, volume of thallus and dry weight, p < 0.05 for all Student T tests, Table 1) and production of cystocarps (number of cystocarps observed per centimetre of primary and secondary frond, p < 0.05 for both Mann-Whitney U-tests, Table 1). Females from Dichato were much smaller than the ones from Lenga (dry weight, volume of thallus and length of primary fronds, Table 1). The females sampled in the natural population of Dichato were characterized by a higher production of cystocarps with a number of cystocarps per centimeter of frond ten times higher for primary fronds and twenty times higher for secondary fronds than the females from Lenga (Table 1). No significant difference in cystocarp size was observed between populations (Table 1). The number of cystocarps observed along the primary fronds was positively correlated with their length in Dichato (Figure 2) but not in Lenga (Figure 2). The number of cystocarps along the secondary fronds was not correlated with their length irrespective of the locality (rs = -0.234, p = 0.306 and rs = -0.137, p = 0.575 in Dichato and Lenga, respectively, data not shown). Finally, carpospores were observed in cross-section of cystocarps in both localities (Figure 3).

Discussion

Our results demonstrated that female gametophytes of Agarophyton chilense are bigger and less sexually reproductive in farms than in natural populations. These differences suggest that cultivation significantly modified life-history traits of this macro alga. In this species, cultivation techniques have usually relied only on replanting of thallus cuttings. This practice allows farmers to selectively multiply (consciously or unconsciously) the phenotypes with the highest biomass production but also the ones best adapted to these new growing conditions. We propose that the ecological modification of the environment and the clonal selection oriented towards the production of biomass operated by farmers (i.e., the first step in the domestication process of the species; [START_REF] Valero | Perspectives on domestication research for sustainable seaweed aquaculture[END_REF], had the indirect consequence of reduced investment in sexual reproduction leading to a much lower reproductive output. Observations of the effect of domestication on the reproduction on plants suggest that human selection for increased vegetative propagation led to architectural changes that resulted in reduced sexual fecundity (in cassava, [START_REF] Mckey | The evolutionary ecology of clonally propagated domesticated plants[END_REF]) and fertility (e.g. in potato, [START_REF] Simmonds | A review of potato propagation by means of seed, as distinct from clonal propagation by tubers[END_REF]and yams, Segnou et al. 1992). In cultivated seaweeds, strong evidence for domestication exist (Agarophyton, [START_REF] Guillemin | Genetic variation in wild and cultivated populations of the haploid-diploid red alga Gracilaria chilensis: How farming practices favor asexual reproduction and heterozigosity[END_REF]Kappaphycus, Ask and[START_REF] Ask | Advances in cultivation technology of commercial eucheumatoid species: a review with suggestions for future research[END_REF]. Nevertheless, no clear results have been obtained regarding the diminished reproductive output as a consequence of domestication process in macroalgae yet.

Field studies focused on natural populations of A. chilense have shown that the probability of a frond to become fecund is size-dependent (Vieira et al. 2018a and b). In the present study, a positive relationship between the size of the primary fronds and the number of cystocarps produced was observed in the natural population (Dichato). This could be explained by the fact that the external part of this organ (the pericarp) is produced directly by the female. Indeed, in the Florideophyceae, the size of the cystocarp and the number of spores produced by cystocarp are related to the thallus biomass supporting the reproductive structures (in Antithamnion nipponicum, Ceramium boydenii and C. Japonicum; [START_REF] Kamiya | Dependence of the carposporophyte on the maternal gametophyte in three ceramiacean algae (Rhodophyta), with respect to carposporophyte development, spore production and germination success[END_REF]. In the same way, in brown algae, positive correlations between thallus size and the proportion of biomass allocated to reproductive tissue have been reported in various species (Ascophyllum nodosum, Åberg 1996; Hizikia fusiformis, [START_REF] Zou | Seasonal pattern of reproduction of Hizikia fusiformis (Sargassaceae, Phaeophyta) from Nanao Island, Shantou, China[END_REF]Sargassum thunbergii, Zhang et al. 2009) and were related to the higher number of reproductive meristems available for receptacle production present along the thalli of larger individuals [START_REF] Zhang | Size-dependence of reproductive allocation of Sargassum thunbergii (Sargassaceae, Phaeophyta) in Bohai Bay, China[END_REF]). On the other hand, the positive relationship between the size of the frond and the production of cystocarps was not observed in females from the farmed population (Lenga). In addition, these females had on average a production of cystocarps ten times lower than in the natural population. As the development of cystocarps occurs after the fertilization [START_REF] Hommersand | Sexual reproduction and cystocarp development[END_REF], this result could be linked either to a low number of carpogonium (female reproductive structure) per female or to a general low availability of male gametes in this farm. Indeed, contrary to what is observed in natural populations where the sex ratio is generally balanced [START_REF] Guillemin | Genetic variation in wild and cultivated populations of the haploid-diploid red alga Gracilaria chilensis: How farming practices favor asexual reproduction and heterozigosity[END_REF], the number of males in Lenga is reduced (data not shown). Farmers seem to avoid males in A. chilense crops probably due to their lower vegetative growth rate, when compared to females or tetrasporophytes [START_REF] Guillemin | Evidence of reproductive cost in the triphasic life history of the red alga Gracilaria chilensis (Gracilariales, Rhodophyta)[END_REF]. Despite these differences between the two types of populations, we assume that the few reproductive males observed in the farmed population can actively participate in fertilization. Furthermore, we cannot rule out the hypothesis that the male gametes could come from neighboring natural populations, maintaining some degree of sexual reproduction in the farm. It has been shown in Gracilaria gracilis that spermatia have a lifespan of more than 6 hours and can be carried by currents [START_REF] Destombe | Viability and dissemination of spermatia of Gracilaria verrucosa (Gracilariales, Rhodophyta)[END_REF]).

According to life-history theory, reproductive cost implies trade-offs in resource distribution, expressed as changes in future growth, fecundity, and/or survival [START_REF] Stearns | The evolution of life histories[END_REF][START_REF] Álvarez-Cansino | Gender-specific costs of reproduction on vegetative growth and physiological performance in the dioecious shrub Corema album[END_REF]Vieira et al. 2018a;Vieira et al. 2018b). In this context, some empirical studies in plants have documented trade-offs in terms of allocation of energy and limited resources, particularly between reproduction and vegetative growth [START_REF] Ashman | A dynamic perspective on the physiological cost of reproduction in plants[END_REF][START_REF] Campbell | Experimental test of sex allocation theory in plants[END_REF][START_REF] Ehrlén | Storage and the delayed cost of reproduction in the understorey perennial Lathyrus vernus[END_REF][START_REF] Henriksson | Differential shading of branches or whole trees: survival, growth, and reproduction[END_REF][START_REF] Obeso | The cost of reproduction in plants[END_REF][START_REF] Obeso | A hierarchical perspective in allocation to reproduction from whole plant to fruit and seed level[END_REF][START_REF] Bañuelos | Resource allocation in the dioecious shrub Rhamnus alpinus: the hidden costs of reproduction[END_REF][START_REF] Álvarez-Cansino | Gender-specific costs of reproduction on vegetative growth and physiological performance in the dioecious shrub Corema album[END_REF]. In some Rhodophyta, gametophytic females have been shown to present a clear decrease in vegetative growth when reproductive [START_REF] Santelices | Regenerative capacity of Gracilaria fragments: effects of size, reproductive state and position along the axis[END_REF][START_REF] Guimarães | Life history, reproduction and growth of Gracilaria domingensis (Gracilariales, Rhodophyta) from Brazil[END_REF] as resources are diverted from the adjacent thallus to allow the development of cystocarps [START_REF] Kamiya | Dependence of the carposporophyte on the maternal gametophyte in three ceramiacean algae (Rhodophyta), with respect to carposporophyte development, spore production and germination success[END_REF]. In A. chilense, vegetative fronds show a higher survival and a better growth rate than reproductive ones irrespective of the type of individual analyzed (male gametophyte, female gametophyte, or tetrasporophyte) [START_REF] Guillemin | Evidence of reproductive cost in the triphasic life history of the red alga Gracilaria chilensis (Gracilariales, Rhodophyta)[END_REF]. Studies monitoring A. chilense in natural populations also support the existence of a trade-off between survival and reproduction (Vieira et al. 2018a and b). A study developed in central Chile focusing on gametophytes of the two intertidal red algae Ahnfeltia durvillaei and Gymnogongrus furcellatus, suggests the existence of reproductive costs and trade-offs between growth and fecundity [START_REF] Camus | Size-specific reproductive parameters in red algae: a comparative analysis for two sympatric species from Central Chile[END_REF].

The study documented a size-independent threshold in reproductive effort for each species.

In the same way, field studies in brown algae support the existence of a reproductive cost, evidenced by decrease in growth or survival rates in reproductive individuals [START_REF] Ang | Cost of reproduction in Fucus distichus[END_REF][START_REF] Åberg | Patterns of reproductive effort in the brown alga Ascophyllum nodosum[END_REF].

In this study, despite the lower production of cystocarps in females from the farmed population, these structures seem to be fertile and produce carpospores. However, it is very unlikely that these spores will succeed in germinating in this type of sandy environment, which could explain the low frequency of tetrasporophytes in this farm. The evolution of functionally sterile clonal thalli in farms has been previously hypothesized in A. chilense as a result of farming techniques mostly/only based on clonal multiplication of vegetative thalli [START_REF] Guillemin | Genetic variation in wild and cultivated populations of the haploid-diploid red alga Gracilaria chilensis: How farming practices favor asexual reproduction and heterozigosity[END_REF]. The theoretical framework behind this evolutionary hypothesis suggests that organisms maintained only by asexual reproduction for a number of generations could lose their ability of reproduce sexually due to the accumulation of sterility mutations in populations where sexual recruitment is impaired by the environment ("use it or lose it" hypothesis; [START_REF] Eckert | The loss of sex in clonal plants[END_REF]. In these cases, sterility arises through the accumulation of mutations in genes necessary for sexual reproduction (i.e. genes involved in meiosis, gamete production or necessary for gametes encounter and fusion), these becoming non-functional.

The lack of sexual reproduction during various generations impedes counter selection and the purge of the mutation load accumulated along these genes [START_REF] Eckert | The loss of sex in clonal plants[END_REF]. Our results do not, however, support the idea of a sterilization of the Lenga farm clonal female thalli. This result may be explained by the relatively recent domestication of A. chilense [START_REF] Buschmann | Cultivation of Gracilaria in the seabottom in southern Chile: A review[END_REF][START_REF] Valero | Perspectives on domestication research for sustainable seaweed aquaculture[END_REF]) and evolutionary pathway to sterilization could still be ongoing in this species. However, measurements made during the present study include only the cystocarps formation while other important steps in the sexual life cycle have not been considered (e.g., the capacity of the carpospores to settle, germinate and grow) [START_REF] Fletcher | The settlement, attachment and establishment of marine algal spores[END_REF]. Complementary common garden experiments and genomic screening are now needed to test for differences in survival and growth of carpospores produced by females from natural and farmed populations and study the potential load of accumulated mutation in genes necessary for sexual reproduction.

Acknowledgments

  This research was funded by Fondo Nacional de Desarrollo Científico y Tecnológico, Gobierno de Chile (FONDECYT #1090360 and #1170541) awarded to M-L. Guillemin. JDGE was supported by the Research Grants Council (ECS 27124318) of Hong Kong. M-L G and CD benefited from the support of the French National Research Agency with regard to an investment expenditure programme IDEALG (ANR-10-BTBR-04), and from the international research network BioDiversity, Evolution and Biotechnology of Marine Algae (GDRI No.0803). We thank P. Antileo, K. Contreras and O. Huanel for their help in the field.

Fig. 1 .

 1 Fig. 1. Map of the Chilean coast (A) and of the Concepción region, where Dichato and Lenga

Figure 2 :

 2 Figure 2: Relation between the primary frond length (cm) and the number of cystocarps

Figure 3 .

 3 Figure 3. Photograph of female thalli from Dichato -details of cystocarps growing on the

Fig. 1

 1 Fig. 1

Fig. 2

 2 Fig. 2

Fig. 3

 3 Fig. 3

Table 1 :

 1 Differences in dry weight, thallus volume, length of primary and secondary fronds, number of cystocarps observed per centimetre of primary and secondary fronds and size of cystocarps between females from the natural population of Dichato and the crop of Lenga. Bold characters represent significant values (*Mann-Whitney U Test; $ Student T test) after a Bonferroni correction for multiple tests; 20 individual measured per locality; values are means ± SE. n.a.: not applicable.

		Natural
			Crop Population
	Variables	Population		df	t-value	p-value
			Lenga
		Dichato
	Dry weight (g)	8.39 ± 5.82	50.09 ± 39.54	40	-6.55	( $ ) < 0.001
	Volume of the thallus (cm 3 )	82.84 ± 70.96	291.01 ± 145.65	30	-5.84	( $ ) < 0.001
	Length of primary fronds (cm)	8.93 ± 4.88	27.54 ± 7.57	30	-9.89	( $ ) < 0.001
	Number of cystocarps per centimetre of primary frond	2.09 ± 1.27	0.26 ± 0.28	n.a.	n.a.	( * ) < 0.001
	Length of secondary fronds (cm)	5.29 ± 2.08	7.94 ± 2.66	38	-4.00	( $ ) = 0.001
	Number of cystocarps per centimetre of secondary frond	2.00 ± 0.93	0.17 ± 0.22	n.a.	n.a.	( * ) < 0.001
	Average size of cystocarps (mm)	1.05 ± 0.34	1.66 ± 0.57	22	-1.45	( $ ) = 0.140


Marine Cacioppo
email: marine.cacioppo@sfr.fr

Antoine Marin

Hélène Rauscent

Estelle Le Pabic

Florence Gaillard

Sylvain Brochard

Ronan Garlantézec

Armel Crétual

Isabelle Bonan

Ronan Garlantezec

Armel Cretual

A new child-friendly 3D bimanual protocol to assess upper limb movement in children with unilateral cerebral palsy: Development and validation

Keywords: Unilateral cerebral palsy, three-dimensional motion analysis, upper limb, bimanual task, movement quality NCT03888443

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INTRODUCTION

3D motion analysis (3DMA) is increasingly used to objectively quantify UL movement in children with uCP [START_REF] Jaspers | Review of quantitative measurements of upper limb movements in hemiplegic cerebral palsy[END_REF], both to increase understanding of movement disorders and to evaluate treatment effectiveness. However, there is currently no consensus regarding the most appropriate tasks or variables to evaluate in order to provide a global description of UL movement. Unimanual reaching or grasping tasks are frequently used because they are simpler to analyse than bimanual tasks, however they do not reflect reallife since most daily life activities involve the collaborative use of both hands (Butler et al., 2010b;[START_REF] Hung | Planning and coordination of a reach-grasp-eat task in children with hemiplegia[END_REF]Jaspers et al., 2011a). To date, only a few studies have proposed bimanual tasks for 3DMA, but their psychometric properties have not been analysed [START_REF] Hung | Bimanual coordination during a goal-directed task in children with hemiplegic cerebral palsy[END_REF][START_REF] Hung | Whole body organization during a symmetric bimanual pick up task for children with unilateral cerebral palsy[END_REF][START_REF] Klotz | Motion analysis of the upper extremity in children with unilateral cerebral palsy-An assessment of six daily tasks[END_REF][START_REF] Rudisch | Kinematic parameters of hand movement during a disparate bimanual movement task in children with unilateral Cerebral Palsy[END_REF]. Furthermore, although many studies of children with uCP have evaluated joint angles and spatiotemporal parameters (i.e. velocity, movement duration, etc.) (Butler et al., 2010a;[START_REF] Hung | Bimanual coordination during a goal-directed task in children with hemiplegic cerebral palsy[END_REF]Jaspers et al., 2011a;[START_REF] Mailleux | Clinical assessment and three-dimensional movement analysis: An integrated approach for upper limb evaluation in children with unilateral cerebral palsy[END_REF][START_REF] Rudisch | Kinematic parameters of hand movement during a disparate bimanual movement task in children with unilateral Cerebral Palsy[END_REF], few have evaluated smoothness (movement regularity) and trajectory straightness (the extent to which the movement is performed directly in a straight line without deviations), particularly during bimanual tasks, despite the fact these variables are important indicators of movement quality (Butler et al., 2010a;Jaspers et al., 2011a;[START_REF] Mailleux | Clinical assessment and three-dimensional movement analysis: An integrated approach for upper limb evaluation in children with unilateral cerebral palsy[END_REF].

In order to provide an overall assessment of UL movement in children with uCP, a 3D bimanual protocol called 'Be An Airplane Pilot' (BE-API) was developed with standardized tasks integrated into a game scenario [START_REF] Bouvier | Proposal of a new 3D bimanual protocol for children with unilateral cerebral palsy: Reliability in typically developing children[END_REF][START_REF] Gaillard | Assessment of bimanual performance in 3-D movement analysis: Validation of a new clinical protocol in children with unilateral cerebral palsy[END_REF]. The protocol aimed to explore all the degrees of freedom (DoF) of the impaired UL known to be limited in children with uCP by encouraging and analysing movements in functional amplitudes. The protocol was found to be reliable for the evaluation of some kinematic parameters but it had several drawbacks: (1) none of the tasks specifically explored shoulder elevation or wrist extension; (2) the protocol was assessed in a few children with severe motor deficits and was thus not representative of all the children who could perform the protocol; and (3) there was no evaluation of variables relating to movement quality. An updated version of the BE-API, the BE-API 2.0, was thus developed to address these limits, with new tasks to ensure that all UL DoF were evaluated.

The aims of this study were therefore to assess the validity and reliability of the BE-API 2.0, including (1) content validity by verifying that the DoF of interest for each task were largely mobilized compared to the other DoF, (2) discriminative ability through comparison with typically developing children (TDC), (3) construct validity, through analysis of the relationship between 3DMA variables and a clinical evaluation of bimanual performance and (4) reliability including within and between-session reliability.

METHODS

Participants

In this prospective, case-control study, twenty children with uCP were recruited from the Physical Medicine and Rehabilitation Department of Rennes University Hospital (France) between February and June 2019.

Inclusion criteria: children aged from 6 to 17 years, with sufficient grasp ability to perform the protocol tasks (MACS (Manual Ability Classification System) level I to III [START_REF] Eliasson | The Manual Ability Classification System (MACS) for children with cerebral palsy: scale development and evidence of validity and reliability[END_REF]). Exclusion criteria: severe cognitive or visual disturbances, UL pain, previous UL surgery, and botulinum toxin injections less than three months prior to participation. Twenty age matched, typically developing children (TDC) were included for comparison (Supplementary Material 1). One child with uCP did not complete the protocol and his data were excluded from the analysis (Supplementary Material 2).

Approval for this study was obtained from the ethical committee Ouest II of Angers (accepted 04/02/2019, N°2019/05). All parents and children received oral and written information about the study before participating.

BE-API 2.0 protocol

The BE-API 2.0 protocol consisted of 3D motion analysis of UL movements during five "flying missions" using a game set-up (2-handed joystick, turbo, shifter, dashboard, box and buzzer) placed on a table. Each "flying mission" involved a bimanual task specifically designed to explore either one or two DoF that are typically limited in children with uCP [START_REF] Coluccini | Kinematic characterization of functional reach to grasp in normal and in motor disabled children[END_REF][START_REF] Fitoussi | Kinematic analysis of the upper limb: a useful tool in children with cerebral palsy[END_REF][START_REF] Mackey | Deficits in Upper-Limb Task Performance in Children With Hemiplegic Cerebral Palsy as Defined by 3-Dimensional Kinematics[END_REF], named DoF of interest: task 1 "flying over mountains" = elbow extension and wrist adduction (Video 1), task 2 "slaloming" = shoulder rotations (video 2), task 3 "hooking the luggage" = shoulder elevation and humeral plane of elevation (Video 3), task 4 "opening the door" = wrist extension (Video 4) and task 5 "refuelling" = elbow supination (Video 5). Of these 5 tasks, Task 3 "hooking the luggage on dashboard" and Task 4 "opening the door" were developed for this new version of BE-API, with two new objects (the dashboard and the shifter), in order to explore the DoF that were not specifically mobilized in the previous version.

Tasks 2, 3, 4 and 5 were asymmetrical, i.e. the movement of interest was only performed by the impaired (non-dominant) UL while the dominant UL was involved in a different, simultaneous movement. The BE-API 2.0 protocol is fully detailed in Table 1.

The starting position for each task was upright sitting on an adjustable chair with 90° of hip, knee and elbow flexion. The forearm and hand were positioned on the table.

In order to facilitate immersion in the playful scenario, the decor was improved compared to the first version of BE API with the projection of a slide show (images or photographs according to the participant's age) for each "mission" on a screen in front of the participant.

Motion capture and data processing

Data were collected with a 10-camera VICON® system at a sampling frequency of 100 Hz (Oxford Metrics, UK). Twenty-six 9mm reflective markers were applied to the trunk, arms, forearms and hands according to the International Society of Biomechanics (ISB) recommendations [START_REF] Wu | International Society of Biomechanics. ISB recommendation on definitions of joint coordinate systems of various joints for the reporting of human joint motion--Part II: shoulder, elbow, wrist and hand[END_REF]. Trunk (lateral-flexion, flexion-extension and rotations), shoulder (rotations, elevation and humeral plane of elevation), elbow (flexion-extension and pronation-supination), and wrist (flexion-extension and abduction-adduction) angles were calculated using the Euler sequences recommended by the ISB [START_REF] Wu | International Society of Biomechanics. ISB recommendation on definitions of joint coordinate systems of various joints for the reporting of human joint motion--Part II: shoulder, elbow, wrist and hand[END_REF]. The shoulder joint was defined as the "thoracohumeral joint" and its centre was estimated using a functional method [START_REF] Lempereur | In vivo estimation of the glenohumeral joint centre by functional methods: accuracy and repeatability assessment[END_REF]. All data were processed with Matlab® (MathWorks, Natick, MA, USA).

Participants performed 5 consecutive movement repetitions (5 trials) each of tasks 1-4 at their own selfselected speed. At the end of the 5 trials of a given task, 1 trial of task 5 "refuelling" was performed in order to maintain a playful link between tasks. Task 5 was thus performed four times, at the end of each of the other tasks. The chronological order of a session was: Task 1 (four trials) -Task 5 (one trial) -Task 2 (four trials) -Task 5 (one trial) -Task 3 (four trials) -Task 5 (one trial) -Task 4 (four trials) -Task 5 (one trial).

For tasks 1, 2, 3 and 4, the first trial was ignored as a training trial, thus four trials of each task (4 trials x 5 tasks) were analysed. For each DoF and each task, the mean value of the 4 trials was calculated for each variable.

Sessions lasted around 30 minutes: positioning of markers and set-up (10 minutes), explanation (5 minutes), 3D acquisition (10 minutes) and removal of markers (5 minutes). All sessions were performed under exactly the same conditions by each participant.

Study schedule

For the uCP group, the inclusion visit was either scheduled during a routine consultation or was carried out by telephone. For the TDC group, it was carried out by telephone. Visits 1 and 2 (uCP group only) were scheduled 2 to 4 weeks apart with the same assessor for the evaluation of between-session reliability.

Kinematic variables and clinical assessment

The use of each variable for the evaluation of validity or reliability is shown in Supplementary Material 8.

Kinematic variables

The following angle-related variables were calculated for each DoF for each task:

 Maximum angle value (MAX)

 Range of motion (RoM) (maximum-minimum angle value during a trial)  The Arm Profile Score (APS): a kinematic index that reflects the total deviation of the UL movement during each task (angular values) developed by Jaspers et al. (Jaspers et al., 2011d). It was calculated as the root mean square error average of the 10 joint movements (thorax, shoulder, elbow, wrist) during each task and compared to the TDC group (see Supplementary Material 9). The global-APS was calculated by averaging the APS values for the five tasks.

Two movement quality variables were evaluated from the displacement of the wrist joint centre:  Smoothness was calculated by the spectral arc length (SPARC), from the measurement of changes on the Fourrier spectrum, providing a negative value [START_REF] Balasubramanian | On the analysis of movement smoothness[END_REF][START_REF] Balasubramanian | A robust and sensitive metric for quantifying movement smoothness[END_REF]. This method was chosen for its high accuracy. Higher SPARC values indicate smoother movement.

 Trajectory straightness was evaluated using the index of curvature (IoC) (Butler et al., 2010a;Jaspers et al., 2011c). The IoC corresponds to the distance travelled by the hand divided by the linear distance between the start and stop position; therefore, an IoC of 1 indicates a straight trajectory. The IoC was only calculated for the two unconstrained tasks (tasks 3 and 5) during which participants did not hold a fixed object (e.g. the 2-handed joystick or shifter).

Clinical assessment

The Assisting Hand Assessment (AHA) [START_REF] Krumlinde-Sundholm | The Assisting Hand Assessment: current evidence of validity, reliability, and responsiveness to change[END_REF], a performance-based measure of bimanual activities, was only assessed in the uCP group on Visit 1. Children first performed a video-recorded play session and then a trained occupational therapist scored their performance on the 22 items using a 4-point criterion-referenced rating scale. The total score was then converted to AHAunits, where 100 corresponds to a high level of spontaneous use of the impaired hand. The items describe different object-related actions of the assisting hand (Supplementary Material 9). Two items were considered relevant for the evaluation of construct validity in this study: "move your forearm" (pronation-supination) and "flow in bimanual task performance" (movement smoothness).

Statistical analysis

All statistical tests were performed with a two-tailed significance level of 5% using MATLAB® (MathWorks, Natick, MA, USA) and SAS, v.9.4® (SAS Institute, Cary, NC, USA).

Validity

Content validity: Each task was specifically designed to evaluate one or two DoF of interest, by inducing large amplitude movements of those DoF. Tasks were considered to have good content validity if the DoF targeted by the task was one of the 3 DoF with the greatest RoM values during the task.

Discriminant validity: MAX, RoM, SPARC and IoC were compared between the uCP and TDC groups using a repeated measures ANOVA (mixed model). A rank-based model was used when the distribution was not normal.

Construct validity: Relationships between AHA score and APS (APS for each task and global-APS) were calculated for the uCP group. Two specific relationships were analysed: (1) mean MAX and RoM of forearm pronation-supination (task 5) and the "move your forearm" item of the AHA, (2) mean SPARC and IoC for each task and the "smoothness of movement" item of the AHA. Bivariate correlations were calculated using Spearman or Pearson's rank correlation coefficients according to the distribution of the data. A correlation coefficient >0.90 was considered 'very high', 0.70-0.89 'high', 0.50-0.69 'moderate', 0.30-0.49 'low' and <0.30 'little or no correlation' [START_REF] Hinkle | Applied Statistics for the Behavioral Sciences[END_REF].

Reliability

Within-session reliability was evaluated in the uCP and TDC groups during Visit 1 by comparing the values of the variables for each movement cycle of each task; between-session reliability was only evaluated for the uCP group (Visits 1 and 2).

The reliability of RoM, MAX, APS, SPARC and IoC data were assessed using the intraclass correlation coefficient (ICC(2,k)) and classified as 'excellent' (≥0.80), 'good' (0.60-0.79), 'moderate' (0.40-0.59), and 'poor' (<0.40). The standard error of measurement (SEM) [START_REF] Engdahl | Reliability of upper limb movement quality metrics during everyday tasks[END_REF]Jaspers et al., 2011b) was calculated and used to calculate the minimal detectable change (MDC) which is the minimal amount of change that a measurement must show to reflect true change (MDC = SEM X 1.96 X √2) [START_REF] Engdahl | Reliability of upper limb movement quality metrics during everyday tasks[END_REF].

RESULTS

Validity

Content validity (Fig. 2)

Five of the seven DoF of interest had the largest amplitudes during the respective tasks. Content validity was thus confirmed for tasks 3, 4, 5; partially for task 1 "Flying over mountains" but not for task 2 "Slaloming". See Fig. 2 for details.

Discriminant validity (Table 2)

Joint angles: MAX and RoM for the DoF of interest in each task

Significantly lower values of elbow extension MAX and RoM (task 1, P<0.05), wrist abduction-adduction RoM (task 1, P<0.05), humeral plane of elevation RoM (task 3, P=0.005), wrist extension MAX and RoM (task 4, P<0.005) and pronation-supination MAX and RoM (task 5, P<0.005) were found in the uCP group compared with the TDC group.

Smoothness and trajectory: SPARC and IoC

SPARC values were significantly lower in the uCP group than the TDC group for task 3 "Hooking the luggage" (P<0.05) but there were no differences for the other tasks. There was a significant between-group difference in the IoC for task 3 (P<0.05) with less straight movement in the uCP group.

Construct validity

Mean AHA score was 66.95 AHA-units (SD 17.03). AHA score was significantly correlated with APS for each task (r=-0.78 in task 1; r=-0.84 in task 2; r=-0.76 in task 3; r=-0.78 in task 4 and r=-0.86 in task 5; P≤0.001), and with Global-APS (r=-0.84; P<0.001).

Significantly moderate correlations were found between forearm pronation-supination MAX and RoM (task 5 "Refuelling") and the AHA "move your forearm" item (respectively r=0.68 and r=-0.65; P<0.005). The SPARC was moderately and significantly correlated with the AHA "smoothness of movement" item in task 3 "Hooking the luggage" (r=0.47; P<0.05) and task 4 "Opening the door" (r=-0.53; P<0.05). The IoC and AHA "smoothness of movement" item in task 3 "Hooking the luggage" were significantly, moderately correlated (r=-0.53; P<0.05).

Within and between-session reliability

Joint angles (Table 3)

MAX and RoM

For all the DoF of interest, within-session reliability was excellent for both MAX (0.88<ICC<0.99) and RoM (0.85<ICC<0.98) in both groups. The mean MDC for RoM was 5.8° (2.48<MDC<9.81°) in the TDC group and 5.94° (2.19-10.23°) in the uCP group.

In the uCP group, between-session reliability was excellent for both MAX and RoM for elbow extension, wrist abduction-adduction, wrist extension, pronation-supination, and MAX shoulder elevation (0.84<ICC<0.97), and was good for the other MAX and RoM values (0.64<ICC<0.78). The between-session MDC for RoM was 12° (5.35°<MDC<19.46°).

Arm Profile Score

Between-session reliability of the APS was excellent for each task (0.93<ICC<0.95) and the mean MDC was 5.57° (5.24°<MDC<5.79°).

Smoothness and trajectory (Table 4)

SPARC

Within-session reliability of the SPARC was moderate-to-good for tasks 1 and 2 for the TDC group (ICC=0.72 and ICC=0.53, respectively) and for tasks 1, 3 and 4 for the uCP group (0.61<ICC<0.76). MDC values were higher for the uCP than the TDC group for all tasks (0.33-1.08 and 0.17-0.80, respectively).

The between-session reliability of the SPARC was also moderate-to-good in tasks 3 and 4 for the uCP group (0.57<ICC<0.84). The between-session MDC values were below 0.36, except for task 2 (MDC=0.50).

IoC

Within-session reliability of the IoC was good for tasks 3 and 5 in both groups (0.62<ICC<0.77). MDC values were lower in task 3 (0.22 and 0.69) than in task 5 (4.52 and 2.44) in both groups.

Between-session reliability of the IoC was good for tasks 3 and 5 for the uCP group (0.61 and 0.73, respectively). The MDC values were 0.44 for task 3 and 1.22 for task 5.

DISCUSSION

The results of this study showed that the BE-API 2.0 protocol, an innovative, quantitative evaluation of bimanual function, that included movement quality variables had content, discriminative and construct validity, and within-and between-session reliability in a representative sample of children with uCP.

Content validity

Most of the tasks of the BE API 2.0 protocol successfully met the challenge of inducing large amplitude movements of the DoF of interest. Moreover, the child-friendly task design allowed more movement spontaneity than more experimental designs. Importantly, although the BE-API is not composed of activities of daily living, it measured ranges of motion that are functionally relevant to everyday life [START_REF] Gates | Range of Motion Requirements for Upper-Limb Activities of Daily Living[END_REF]. Moreover, the tasks that were newly introduced into the BE-API 2.0 induced larger amplitudes of movement in the DoF of interest than the previous version (e.g. MAX shoulder elevation was 112° versus 68.7° previously) [START_REF] Gaillard | Assessment of bimanual performance in 3-D movement analysis: Validation of a new clinical protocol in children with unilateral cerebral palsy[END_REF]. Thus the conception of tasks that specifically focused on one or two DoF allowed functional joint amplitudes to be fully explored (Butler et al., 2010b;[START_REF] Hung | Planning and coordination of a reach-grasp-eat task in children with hemiplegia[END_REF]Jaspers et al., 2011a). Task 2 "Slaloming" did not induce the expected amplitude of shoulder rotation, probably because of the high proportion of children with MACS level III included in the study. However, task 2 "slaloming" required turning the wheel, which was the most difficult movement of the protocol and thus is useful for exploring joint synergies and compensatory movements (Butler et al., 2010b;[START_REF] Gaillard | Kinematic motion abnormalities and bimanual performance in children with unilateral cerebral palsy[END_REF]Jaspers et al., 2011a;[START_REF] Mailleux | Clinical assessment and three-dimensional movement analysis: An integrated approach for upper limb evaluation in children with unilateral cerebral palsy[END_REF].

Discriminant validity

Both RoM and MAX joint angle variables successfully discriminated between the children with uCP and the TDC. The new tasks confirmed that humeral plane of elevation and wrist extension amplitudes were significantly lower in the children with uCP compared to the TDC (Jaspers et al., 2011a;[START_REF] Klotz | Motion analysis of the upper extremity in children with unilateral cerebral palsy-An assessment of six daily tasks[END_REF][START_REF] Rudisch | Kinematic parameters of hand movement during a disparate bimanual movement task in children with unilateral Cerebral Palsy[END_REF]. The lack of between group differences in shoulder elevation was likely due to scapular compensation for deficits in gleno-humeral elevation [START_REF] Brochard | The role of the scapulo-thoracic and glenohumeral joints in upper-limb motion in children with hemiplegic cerebral palsy[END_REF].

The SPARC and IoC movement quality variables both successfully differentiated between the uCP and TDC groups for task 3 "Hooking the luggage" only. In that task, the hand trajectory was less straight and smooth in the uCP group, as has been found previously for unimanual reaching tasks (Butler et al., 2010a;Jaspers et al., 2011a;[START_REF] Mailleux | Clinical assessment and three-dimensional movement analysis: An integrated approach for upper limb evaluation in children with unilateral cerebral palsy[END_REF]. It has been demonstrated that during the simultaneous use of both hands, non-symmetrical movement interference could impact on performance [START_REF] Rudisch | Kinematic parameters of hand movement during a disparate bimanual movement task in children with unilateral Cerebral Palsy[END_REF]. The movement performed in task 3 differed from that in the other tasks because it: 1) was a simple, large amplitude movement, 2) was unconstrained, and thus required more motor control mechanisms (to provide both strength and stability against gravity); and 3) required precision at the start and end of the movement (to grasp the object, then to stabilize and fix it to the top of the dashboard). These results suggest that the SPARC and IoC are useful tools to identify differences between children with uCP and TDC, provided that an appropriate task is chosen, i.e. one which involves an unconstrained, large amplitude and challenging movement. The addition of such movement quality variables to the usual evaluation of kinematic variables provides a global assessment of upper limb function.

Construct validity

The BE-API 2.0 protocol was found to have good construct validity, as demonstrated by the strong relationship between the UL kinematic variables and the widely used clinical assessment of bimanual activities, the AHA. This finding was reinforced by the fact that correlations between the AHA and APS were stronger than those reported for unimanual tasks [START_REF] Gaillard | Kinematic motion abnormalities and bimanual performance in children with unilateral cerebral palsy[END_REF][START_REF] Mailleux | Clinical assessment and three-dimensional movement analysis: An integrated approach for upper limb evaluation in children with unilateral cerebral palsy[END_REF] (respectively r=-0.75 and r=-0.49), suggesting that the BE-API 2.0 truly measured bimanual performance.

Previous 3DMA studies only explored correlations between the AHA and joint angle variables (Gaillard et al., 2019;[START_REF] Mailleux | Clinical assessment and three-dimensional movement analysis: An integrated approach for upper limb evaluation in children with unilateral cerebral palsy[END_REF], this is the first study to integrate movement quality variables. The results of the AHA, a semi-quantitative evaluator-dependent measure, and BE-API 2.0, an objective evaluation, were convergent for both joint angle and movement quality variables. Similarly to the combination of clinical and 3DMA data that is common practice in gait analysis, the AHA and BE-API 2.0 thus appear to be complementary for the assessment of bimanual performance.

Reliability

Reliability of joint angle variables was high for all the DoF of interest (within-session reliability for both groups and between-session reliability for the uCP group). The ICC values calculated were higher than those reported for previous unimanual protocols (Jaspers et al., 2011a(Jaspers et al., , 2011b)). With regards to the movement quality variables, the SPARC provided a highly precise calculation of movement smoothness [START_REF] Balasubramanian | On the analysis of movement smoothness[END_REF][START_REF] Balasubramanian | A robust and sensitive metric for quantifying movement smoothness[END_REF], but it appeared to be very sensitive to the type of movement analysed.

To date, the reliability of the SPARC had only been approximately evaluated in children with uCP in unimanual conditions using inertial sensors [START_REF] Newman | Measuring upper limb function in children with hemiparesis with 3D inertial sensors[END_REF]. A study of healthy adults performing daily activities found similar within-session reliability (ICC 0.49-0.64) but higher between-session reliability (ICC 0.81-0.84) than the present study [START_REF] Engdahl | Reliability of upper limb movement quality metrics during everyday tasks[END_REF]. Until now, the reliability of the IoC had only been evaluated in children with uCP performing unimanual tasks (Butler et al., 2010a;[START_REF] Schneiberg | Reliability of kinematic measures of functional reaching in children with cerebral palsy[END_REF].

This study thus provides reference data and MDC values for all kinematic variables that can be used in clinical practice and research. Most of the differences in joint angle values between the uCP and TDC groups were higher than the MDCs for the uCP group, demonstrating the clinical pertinence of these measures.

Although the reliability of the APS had already been evaluated, no MDC value had been determined (Jaspers et al., 2011d). The low MDC value (<6°) found for the APS in the present study supports its utility for the assessment of UL movement deviations.

Limitations

The number of subjects to be included was not based on a statistical calculation but was defined according to the feasibility of inclusion. However, this was also the case in similar studies published in the literature [START_REF] Gaillard | Assessment of bimanual performance in 3-D movement analysis: Validation of a new clinical protocol in children with unilateral cerebral palsy[END_REF]Jaspers et al., 2011bJaspers et al., , 2011c;;[START_REF] Schneiberg | Reliability of kinematic measures of functional reaching in children with cerebral palsy[END_REF]. The relatively small number of participants might have led to the study being underpowered, which could explain some of the low values found. This study was carried out in a single centre with assessors who were welltrained in the use of the BE API 2.0 protocol. A period of training might be necessary to obtain the same level of validity and reliability in other centres, and before it is used in routine practice. In this study, the shoulder girdle was considered as a thoraco-humeral joint, the lack of evaluation of scapular movement is an inherent deficiency of the model.

Conclusion

The results of this large, methodologically rigorous study showed that the BE-API protocol is valid and reliable for the bimanual assessment of UL motor deficits in children with uCP aged from 6 to 18 years and with different levels of impairment. The evaluation of both joint angle and movement quality variables in this protocol could increase understanding of pathological UL movement patterns as well as support clinical decisions and help to determine well-targeted treatment plans. Further studies are now needed to determine the sensitivity to change of the protocol for the assessment of the effectiveness of therapeutic interventions.

Biography

Marine Cacioppo is a physical medicine and rehabilitation pediatric physician and researcher. She has her Master's Degree from Rennes University (France) on movement sport and health. Her researches focuses on understanding of impaired upper limb function in children with physical disabilities using motion analysis. She is currently working on assessment of bimanual performance in the context of her PhD, within the research team BE a CHILD ((BrEizh Center for technological innovation And research for CHILDren with Disabilities). Taking a magnet (one per trial) placed on the table on the paretic (non-dominant) side (10 cm lateral to the hand), and hanging it on the magnetic dashboard (placed at 45° to a line emanating from the sternum on the paretic side), as high as possible. At the same time, the other hand keeps the wheel horizontal.

"Take a piece of luggage and place it as high as possible on the dashboard, without standing up. Keep the wheel straight with your other hand"

One cycle= -Start: grasping the magnet on the table -End: magnet hung on the dashboard.

The magnet can be stabilized on the table by the evaluator to help the child to grasp it. The child must press the buzzer with the dominant hand. He/she must then supinate the affected (non-dominant) hand to catch a gas coin dropped and place it on the gas tank.

"Press the buzzer with the palm of your hand and keep holding it down. I will drop a coin into your other hand so that you can refuel the airplane -you will have to turn your other hand palm-up to catch the coin and place it on the gas tank.."

One cycle= -Start: forearm and hand flat on the table -End: hand turned over with gas coin in palm If full supination is not possible, the child turns his/her hand as much as possible and the coin is placed in the hand by the assessor.

The BE-API 2.0 protocol is composed of five bimanual tasks performed successively: task 1 "Flying over mountain summits", task 2 "Slaloming", task 3 "Hooking the luggage on dashboard" and task 4 "Opening the door". Each task represents a flying mission in order to become an airplane pilot. Between each flying mission, task 5 "Refuelling" is carried out to "refuel the airplane".

The starting position for each task was upright sitting on an adjustable chair with 90° of hip, knee and elbow flexion. The forearm and hand were placed on the table.

  with the affected hand (non-dominant) 90° upwards then 90° downwards while pressing the turbo with the dominant hand."Press the turbo with one hand and turn the wheel right up and right down with the other hand, don't let go of the wheel

  Parachutists are on the back of the plane ready to jump, the pilot has to open the plane door to drop them.The child has to pull a shifter towards him/her and then bring it forward with the affected (non-dominant) hand, while the other hand keeps the wheel horizontal. To refuel the airplane, the pilot has to fill the gas tank.

Figure 1 :

 1 Figure 1: All range of motion values for children with uCP and TDC DoF of interest defined before the study are framed. AA: Abduction-adduction; DoF: Degree of Freedom; EL: Elevation; FE: Flexion-Extension; IER: Internal-External Rotation; PoE: Plane of Elevation; PS: Pronation-Supination; TDC: typically developing children; uCP: unilateral Cerebral Palsy

ACKNOWLEDGMENT

We wish to thank the participants and their families, the team of the Paediatric Physical Medicine and Rehabilitation department of Rennes University Hospital and M2S laboratory. We also thank Johanna Robertson for language assistance and constructive criticism.

This work was supported by the IPSEN Company. They provided financial support for the conduct of the research. They were not involved in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

CONFLICT OF INTEREST STATEMENT


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Shaleen Vira

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Célia Amabile

Thomas Errico

Frank Schwab

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Spino-femoral muscles affect sagittal alignment and compensatory recruitment: a new look into soft tissues in adult spinal deformity

Keywords: Spino-femoral muscles, Sagittal malalignment, Compensatory recruitment, Adult spinal deformity

Introduction

Abnormal pelvic anatomical shape has been implicated in the development of sagittal malalignment in adult spinal deformity (ASD), as does the lack of compensatory mechanism recruitment [START_REF] Ferrero | Analysis of an unexplored group of sagittal deformity patients: large sagittal malalignment but low pelvic tilt[END_REF]. However, muscles have been an under-appreciated component that cannot be overlooked in the investigation of ASD. Prior studies on lumbar degenerative kyphosis reveal that kyphotic patients have significantly smaller lumbar muscle and higher proportion of fat deposits in multifidus and erector spinae muscles [START_REF] Kang | MRI of paraspinal muscles in lumbar degenerative kyphosis patients and control patients with chronic low back pain[END_REF]. Indeed, muscle degeneration requires more attention due to the close relationship with sagittal malalignment. Previous studies demonstrated that musculoskeletal fragility associated with sarcopenia can contribute to falls in patients since muscle performance might be altered by the loss of muscle mass and strength [START_REF] Helbostad | Consequences of lower extremity and trunk muscle fatigue on balance and functional tasks in older people: a systematic literature review[END_REF][START_REF] Hsu | Balance control in elderly people with osteoporosis[END_REF]. In addition to muscles located in the lumbar spine region, other parts of musculoskeletal system may also contribute to sagittal alignment regulation and postural control. On a higher-level point of view, postural control is a continuous process during which the neuro-central system analyzed input from the vestibular, visual and proprioceptive systems and then responds through muscular efforts.

The alteration of muscle architecture and activation in lower limbs may contribute to the maintenance of upright stance in ASD patients. The compensatory mechanisms for malalignment have been well addressed, including the role of lower limbs, with increased understanding of the contribution in the recruitment chain [START_REF] Diebo | Musculoskeletal compensatory mechanisms for spinal malalignment: an age-related study[END_REF][START_REF] Ferrero | Role of pelvic translation and lower-extremity compensation to maintain gravity line position in spinal deformity[END_REF]. However, the role of various muscles in this compensatory cascade remains to be investigated. A better understanding of lower limb compensation and how the muscular system affects compensation would benefit clinical evaluation and targeted therapy. Therefore, the current study aims to investigate the contribution or correlation between muscle degeneration and sagittal malalignment as well as compensatory mechanism recruitment with a novel MRI 3D reconstruction technique.

Methods

Subjects

This is a prospective pilot study on muscle profiles of ASD patients with institutional review board approval and written informed consent. Inclusion criteria were as follows: female patients older than 35 years old, meeting at least one of the following ASD radiographic criteria: coronal curve larger than 30°, sagittal pelvic tilt (PT) larger than 20°, pelvic incidence (PI) and lumbar lordosis (LL) mismatch (PI-LL) larger than 10°, or C7-S1 sagittal vertical axis (SVA) larger than 4 cm [START_REF] Schwab | Scoliosis research society-schwab adult spinal deformity classification[END_REF]. Patients with previous spinal surgery history, existing hip/thigh/knee instrumentation or presenting contraindication for MRI, were excluded from this study. Only women were recruited due to higher prevalence of ASD in women. The quality of life of the patients was evaluated by SRS-22 questionnaires and Oswestry Disability Index (ODI).

Radiographic analysis

All patients underwent standing full spine films. Radiographs were measured using validated software (SpineView, Laboratory of Biomechanics ENSAM ParisTech, France) [START_REF] Champain | Validation of new clinical quantitative analysis software applicable in spine orthopaedic studies[END_REF], and the following sagittal radiographic parameters were obtained: PI, PI-LL, PT, SVA. After calculating the PI-LL/ PT ratio, patients were stratified based on PI-LL/PT as two groups using 40% and 60% percentile as threshold to distinguish different pelvic compensation recruitment. The PI-LL/ PT ratio stands for the level of pelvic compensation involvement in the setting of lumbosacral malalignment.

MRI acquisition

MRI was performed on a 3 T whole-body scanner (Magnetom Verio, Siemens Healthcare, Erlangen, Germany) using a 24-channel spine matrix coil and three 4-channel flex coils from the same vendor. The imaging protocol included a T1-weighted turbo spin echo (T1w TSE) sequence for applying the two points Dixon method (TR/ TE = 829/15.7 ms, acquisition matrix = 512 × 384, in plane resolution = 0.98 × 0.98 mm 2 , slice thickness = 5 mm, slice gap = 5 mm, iPat = 2, 40 slices, flip angle = 150°, bandwidth = 315 Hz/pixel, turbo factor = 3, echo spacing = 15.7, acquisition time = 4:38 min). Water and fat images were automatically generated by the scanner. Image volume covered the proximal tibia to the lumbar spine (until inferior endplate of T12 vertebra) and was acquired in four stages. Total acquisition time was 25 min.

3D Muscle Reconstruction

The 3D reconstruction of individual muscles, listed in Table 1, was performed using Muscl'X software (ENSAM, Laboratory of Biomechanics, Paris, France), a validated software [START_REF] Moal | Validation of 3D spinopelvic muscle reconstructions based on dedicated MRI sequences for fat-water quantification[END_REF] already employed in previous literature [START_REF] Moal | Volume and fat infiltration of spino-pelvic musculature in adults with spinal deformity[END_REF]. Some muscles were combined, since the low contrast made an accurate separation of the individual muscles difficult. The lumbar part of the psoas was reconstructed separately, but at a point where the distinction with the iliacus was not possible, it was then integrated into the iliacus. The external obturator, adductor longus, brevis and magnus and pectineus were reconstructed into a single group named "Adductor." The vastus lateralis and intermedius were reconstructed together. The muscle reconstructions were done on the fat images. Figure 1 (reprinted with permission from the authors of previous literature [START_REF] Moal | Volume and fat infiltration of spino-pelvic musculature in adults with spinal deformity[END_REF]) presents the 3D reconstruction of the left muscles for one patient. The femurs were also reconstructed on the water images; the contrast between the cortical and cancellous bones was greater on water images.

Right and left muscles were grouped according to the joint (spine, hip and knee) and by mechanical action Flexor Extensor of spine, hip and knee (Table 1). The function of muscles is summarized in Table 1, while the function of gluteus medius and minimus was left empty due to their complexity.

Quantification of fat and contractile components and muscle's parameters

The fat-water ratio by voxel was calculated as previously reported [START_REF] Moal | Volume and fat infiltration of spino-pelvic musculature in adults with spinal deformity[END_REF]. From the 3D reconstructions, muscular volumes were calculated (Vmuscle) for each muscle, and the

Statistical analysis

Muscle volume and fat infiltration were characterized by mean and standard deviation. Correlations between each muscle/muscle group and radiographic parameters were performed. Stepwise linear regressions of PT and SVA using all muscle parameters were performed with stepwise at the first, and then, linear regression of PT and SVA using volume and fat infiltration of significantly related muscle was again performed. In addition, muscle parameters were compared between patients with well alignment and malalignment using nonparametric t test. For each statistical analysis, the level of significance was set at 0.05.

Results

Demographic

A total of 22 ASD patients were enrolled in this study with a mean age of 61.05 ± 12.55 years old and mean BMI of 23.38 (Range: 20.08-32.45). All radiographic parameters are summarized in Table 2, as well as the mean fat infiltration. The spine extensor possessed the largest fat infiltration (31.73%), followed by spine flexor (25.80%), hip extensor (15.27%) and knee flexor (13.14%).

In all patients, age was positively correlated with Pfat of almost all muscle groups (p < 0.05, Online Appendix 1), indicating larger muscle degeneration as age increased.

Meanwhile, BMI was barely correlated with volume and fat

lumborum Q.L x - - - - - Erectus spinae E.S x - - - - - Iliopsoas - - x - x - - Biceps femoris short B.F.S - - X - - x Biceps femoris long B.F.L - - X - - x Semi-membranosus S.M - - X - - x Semi-tendinosus S.T - - X - - x Gluteus maximus G.max - - X - - - Rectus femoris R.F - - - x x - Gracilis G - - - x - x Sartorius S - - - x - x Adductor A - - - x - - Tensor fascia lata T.F.L - - - x - - Vastus lateralis and intermedius V.L - - - - x - Vastus medialis V.M - - - - x - Gluteus medius G.med - - - - - - Gluteus minimus G.min - - - - - -

Correlation between radiographic parameters and muscle parameters

Significant correlations were observed between sagittal alignment radiographic parameters and those describing the muscle groups (Table 3).

For regional alignment, PI-LL mismatch correlated with a decrease in spine flexor and extensors volumes (r = -0.445 and -0.508, p = 0.038 and 0.016, respectively); no correlations were observed between PI-LL and the fat infiltration of the different muscle groups (all p > 0.05). Lumbar lordosis negatively correlated with the volume and fat infiltration ratios of

Spine Flexor

Spine Extensor (r = -0.481 and -0.443, p = 0.024 and 0.039, respectively).

For global alignment, SVA correlated with all muscle groups except spine flexors (Table 3). In terms of each specific muscle, SVA moderately to strongly correlated with an increase in fat infiltration of Hamstrings, G.Med and G.Min (r = 0.46-0.63, all p < 0.05, Online Appendix 2). However, volume and fat infiltration of specific spinal muscles showed no correlation with SVA; also SVA showed no correlation to standardized muscle volumes. For compensation recruitment, PT showed moderate to strong negative correlations with the volume of spinal flexor and extensor (r = -0.531 and -0.509, both p < 0.05), knee flexor and extensor (r = -0.433, -0.483, both p < 0.05) and hip extensor (r = -0.465, p = 0.029). In terms of each specific muscle, volume of muscles was significantly correlated with PT including volume of G.Med, vastus lateralis and intermedius, vastus medialis, biceps femoris long, semimembranosus, iliopsoas and erectus spinae (r = 0.42-0.58, all p < 0.05, Online Appendix 3).

The PT/PI ratio, evaluating percentage of pelvic compensation recruitment, demonstrated strong correlation with the volume of spine extensor (r = -0.62, p = 0.003), suggesting that larger PT recruitment was correlated with smaller muscle volume of spine extensors.

Regression to predict SVA and PT

Stepwise linear regression for both SVA and PT was first performed based on muscle parameters. Results revealed that muscle volume of biceps femoris long was only predictor for PT (R 2 = 0.34, p = 0.005) and fat infiltration of G.Min as only predictor for SVA (R 2 = 0.45, p = 0.001, Table 4). Considering strong correlation between fat infiltration of B.F Long and of G.Med as well as between fat infiltration of B.F Long and of G.Min, both volume and fat infiltration parameters for G.Med, G.Min, and BF Long were forced to be included in the second model, increasing predictive power for PT (R 2 = 0.44) and SVA (R 2 = 0.61, Table 4).

Comparison between patients with different compensation recruitments

As summarized in Table 5, 2 subgroups were described based on PI-LL/PT to distinguish patients with pelvic compensation recruitment. Patients with larger PT recruitment were found with statistically significant smaller volume of G.med, more fat infiltration of G.min and B.F.L. Also, this subgroup was also found with a trend of smaller volume of G. min, B.F.L and a trend of increased fat infiltration of G.med.

Correlation between muscle parameters and HRQOL

In the 19 patients with HRQOL measurement, only SRS-22 self-image domain was significantly correlated with the volume of spinal extensor (r = 0.514, p = 0.035). SRS-22 function domain showed trend of correlation to (Pfat of hip flexor)/(Pfat of hip extensor) (p = 0.070), while SRS-22 mental, pain domain and total score showed no correlation to muscle parameters. Regarding ODI score, the fat infiltration of knee extensor showed trend of correlation to ODI without statistical significance (p = 0.056, Online Appendix 4).

Discussion

In sagittal malalignment scenario, in order to maintain a erect posture, the compensatory mechanism is recruited through the involvement of muscles and the corresponding radiographic presentations such as pelvic retroversion and knee flexion are the consequences [START_REF] Diebo | Sagittal alignment of the spine: what do you need to know?[END_REF]. A better understanding of the role of muscles in sagittal malalignment and compensation may lead to a more thorough understanding of the underlying pathoanatomical cascade that leads to ASD and would thus represent a potential therapeutic target upstream of bony pathology that may only represent the symptoms of the underlying etiology [START_REF] Schwab | Fine-tuned surgical planning in adult spinal deformity: determining the lumbar lordosis necessary by accounting for both thoracic kyphosis and pelvic incidence[END_REF]. With the use of a MRI based 3D reconstruction of the muscles [START_REF] Moal | Validation of 3D spinopelvic muscle reconstructions based on dedicated MRI sequences for fat-water quantification[END_REF][START_REF] Moal | Volume and fat infiltration of spino-pelvic musculature in adults with spinal deformity[END_REF], the current study has the possibility to quantitatively evaluate the volume and fat infiltration of muscles and brings more accurate description of how the muscle impacts or correlates with sagittal malalignment.

Several previously reported studies have examined the relationship between spinal and the spinal extensors with cross-sectional area (CSA), which has the limitations of the position-related change of muscle CSA and the inaccuracy to represent the volume of entire muscle [START_REF] Kang | MRI of paraspinal muscles in lumbar degenerative kyphosis patients and control patients with chronic low back pain[END_REF][START_REF] Meakin | The relationship between sagittal curvature and extensor muscle volume in the lumbar spine[END_REF]. The result of the current study, demonstrating that larger volume of spinal extensor and smaller fat infiltration of spinal flexor both correlated with lumbar lordosis, provided more solid evidence with 3D muscle reconstruction. Spinal muscle strength could dictate the degree of lordotic curve from a biomechanical perspective. Recent modeling suggests that a spine with large lordosis requires a greater follower load (concept proposed by Patwardhan, [START_REF] Patwardhan | A follower load increases the load-carrying capacity of the lumbar spine in compression[END_REF] defined as internal compressive load passing through the centers of rotation of the spinal segments) in the standing position than one with minimal lordosis [START_REF] Meakin | Modeling the effect of variation in sagittal curvature on the force required to produce a follower load in the lumbar spine[END_REF]. Thus, increased lumbar lordosis requires larger extensor musculature and more extension strength over flexion to provide sufficient follower loads, as shown in the results that both volume and fat infiltration of Spine Flexor Spine Extensor correlated with LL.

The current study demonstrated that pelvic retroversion was associated with and could be predicted from gluteus medius, gluteus minimus and hamstring muscle degeneration. This is the first description regarding G. Med/Min and an interesting one since previously stronger gluteus maximus, the hip extensor, was regarded as the responsible muscle for pelvic retroversion [START_REF] Alvim | Influences of the extensor portion of the gluteus maximus muscle on pelvic tilt before and after the performance of a fatigue protocol[END_REF]. The function of G.Med and G.Min is both difficult to determine. Anatomically, G.Med and G.Min are both primary hip abductors with rotation and flexion being possible. Instead of treating G.Med as a whole muscle, Soderberg et al. divided G.Med into 3 parts [START_REF] Soderberg | Electromyographic study of three parts of the gluteus medius muscle during functional activities[END_REF]. Gottschalk et al. further described that anterior and middle parts act as abductor and hip flexor [START_REF] Gottschalk | The functional anatomy of tensor fasciae latae and gluteus medius and minimus[END_REF]. Beck et al. reported that G.Min acts as a flexor and an abductor of the hip and also as either an internal or external rotator [START_REF] Beck | The anatomy and function of the gluteus minimus muscle[END_REF]. Due to the complex and ever-changing function of both G.Med and G.Min, two speculations are proposed here to serve as possible explanations for how the G.Med and G.Min affect pelvic retroversion.

One speculation is that the muscle imbalance between hip extensors and flexors leads to pelvic retroversion. In the current study, G.Max, the main hip extensor, was found to be similar between patients with and without pelvic retroversion in terms of both volume and fat infiltration. Considering the weaker G.Med and G.Min which may potentially act as hip flexor, the similar G.Max would be relatively stronger due to the imbalance between flexor and extensor in patients with more PT recruitment. The imbalance between flexor and extensors was also observed in cerebral palsy patients (significantly smaller PT) with overactivated G.Med/Min and weakened G.Max, just the opposite to the ASD patients studied here [START_REF] Wolf | Which functional impairments are the main contributors to pelvic anterior tilt during gait in individuals with cerebral palsy?[END_REF]. Another explanation could be the reverse pathologic cascade: pelvic retroversion would lead to possible degeneration of G.Med and G.Min. Ross et al. confirmed that more pelvic retroversion would result in acetabular anteversion and cranial version and thus result in more internal rotation [START_REF] Ross | Effect of changes in pelvic tilt on range of motion to impingement and radiographic parameters of acetabular morphologic characteristics[END_REF][START_REF] Ross | Functional acetabular orientation varies between supine and standing radiographs: implications for treatment of femoroacetabular impingement[END_REF]. A recent study on hip range of motion also reported that increased pelvic retroversion may cause decreased femoral neck rotation and hip external rotation [START_REF] Shimizu | A cohort study of adult spinal deformity and its relation with hip-knee range of motion[END_REF]. If the pelvis responded to spinal malalignment with pelvic retroversion, then femoral head tends to rotated medially spontaneously and muscles responsible for internal rotation, G.Med and G.Min, could be de-activated which in turn lead to their fatty degenerative atrophy.

In addition to gluteal muscles, the hamstring was also found to be associated with pelvic retroversion as well as sagittal global alignment. Also, it is the volume of biceps femoris long that showed negative correlation with PT. Previous studies have reported similar observations in the relationship between hamstring shortness and retroverted pelvis as well as higher trunk flexion [START_REF] Carregaro | Does reduced hamstring flexibility affect trunk and pelvic movement strategies during manual handling?[END_REF]. In patients with low back pain, tight hamstring was correlated with limit hip motion [START_REF] Esola | Analysis of lumbar spine and hip motion during forward bending in subjects with and without a history of low back pain[END_REF]. Hamstring shortness was also extensively reported in spondylolisthesis patients with restricted hip flexion. The hamstring tightness in Scheuermann kyphosis was also reported and defined as a definite sign or etiological factor. Hosman et al. found that tight hamstrings were linked to limit lumbar and pelvic range of motion and risk of postoperative sagittal negative imbalance indicating overretroverted pelvis [START_REF] Hosman | Scheuermann kyphosis: the importance of tight hamstrings in the surgical correction[END_REF]. In the current study, the volume of B.F.L was correlated with and could be used to predict PT, while larger SVA was correlated with more fat infiltration of B.F.L, which implied less contribution to compensatory mechanisms such as hip extension and knee flexion.

The current study also reported the feasibility to predict sagittal malalignment with only G.Med, G.Min and B.F.L. Actually, predicting sagittal alignment from muscle parameters is challenging in common clinical settings that are typically not equipped to obtain the appropriate advanced imaging and subsequently analyze the data rapidly. Nevertheless, the strong influence of soft tissue alterations on sagittal alignment calls for future studies to develop targeted treatments toward muscle degeneration in the setting of sagittal malalignment. The corresponding conservative treatments toward G.Med, G.Min and B.F.L may have the potential to help maintain sagittal alignment and relieve low back pain prior surgical intervention. In addition, the evaluation of patient's soft tissue profile could also aid in the understanding of patient-specific compensatory mechanisms and thus a better surgical planning. For example, perhaps sagittal malalignment patients tight hamstrings may require less aggressive osteotomy to prevent potential postoperative negative SVA.

The limitations of the current study mainly lie in the small sample size. Full-body radiographic evaluation with femoral version, knee varus/valgus is not available at the time of patient recruitment and should be further analyzed in the future study.

Conclusion

Using novel MRI 3D reconstruction techniques, this study is the first to quantify the relationship between degeneration of spino-femoral muscle and sagittal malalignment. This pathoanatomical study identifies the close relationship between gluteal, hamstring muscles and PT, SVA, which deepens our understanding of the underlying etiology that contributes to adult spinal deformity. It is hoped that the detailed insights gained could help to devise targeted strategies and incorporate patients' soft tissue profiles for the prevention and treatment of adult spinal deformity.

  model of repartition was used to calculate the volumes of fat components (Vfat) and contractile components (Vcont) (Vmuscle = Vfat + Vcont). The volumes were then normalized between individuals based upon the volume of the right femur (Pmuscle = Vmuscle/Vfemur). The fat infiltration was evaluated via the percentage of fat component (Pfat = 100*Vfat/Vmuscle). Lower volume (smaller Vmuscle and Pmuscle) and more fat infiltration (larger Pfat) indicated a greater muscle degeneration.

Fig. 1

 1 Fig. 1 Medial and frontal view of all the left muscles reconstructed for one patient. (Reprint with permission from [10])

Table 1

 1 Muscles reconstructed in this study and then grouped by function and joints for further analysis

	Abbreviations in	Spine	Spine flexor	Hip extensor	Hip flexor	Knee	Knee flexor
	this article	extensor				extensor
	Quadratus

Table 2

 2 Summary of radiographic characteristics and muscle of the cohort

		N	Mean	SD
	PI-LL (°)	22	1.38	19.24
	C7-S1 SVA (mm)	22	16.06	56.60
	T1PA (°)	22	17.12	12.55
	SS (°)	22	31.18	10.56
	PT (°)	22	22.69	11.54
	PI (°)	22	53.87	13.41
	T4-T12 kyphosis (°)	22	45.04	18.84
	Thoracic Cobb angle (°)	14	36.77	20.32
	Thoracolumbar/lumbar Cobb	19	29.95	16.78
	angle (°)
	Fat infiltration of muscles
	Spine flexor (%)	22	25.80	11.54
	Spine extensor (%)	22	31.73	13.23
	Hip flexor (%)	22	11.60	4.26
	Hip extensor (%)	22	15.27	6.87
	Knee flexor (%)	22	13.14	4.39
	Knee extensor (%)	22	8.82	3.98
	Table 3 Correlations between
	radiographic parameters and
	muscle parameters

Table 4

 4 Linear regression for SVA and PT with muscle parameters

		Dependent variable	Independent variables	Regression method	R 2
		C7-S1 SVA	Pfat of gluteus minimus	Stepwise	0.45
			Volume and Pfat of biceps femoris	Enter	0.61
				long, gluteus medius and minimus
		PT	Volume of biceps femoris long	Stepwise	0.34
			Volume and Pfat of biceps femoris	Enter	0.44
				long, gluteus medius and minimus
	Table 5 Comparison between patients with different pelvic compensation	Number		Less PT recruitment 9	More PT recruitment 9	p
		PI-LL		-11.34 ± 9.84	16.65 ± 26.12	0.008
		PT		17.18 ± 8.03	26.83 ± 14.14	0.090
		Standardized volume of G.max	2.66 ± 0.31	2.71 ± 0.69	0.848
		Standardized volume of G.med	1.24 ± 0.16	1.08 ± 0.15	0.040
		Standardized volume of G.min	0.40 ± 0.05	0.35 ± 0.05	0.056
		Standardized volume of B.F.L		0.65 ± 0.11	0.53 ± 0.14	0.056
		Pfat of G.max		12.89 ± 4.07	20.22 ± 10.97	0.078
		Pfat of G.med		9.12 ± 3.88	13.67 ± 6.89	0.104
		Pfat of G.min		14.92 ± 5.53	23.77 ± 9.28	0.026
		Pfat of B.F.L		8.74 ± 2.57	13.28 ± 5.53	0.040
		Bold values indicate significant correlations

Acknowledgements

The manuscript submitted does not contain information about medical device(s)/drug(s). This work received funding from Youth Fund of Natural Science Foundation of Jiangsu Province (BK20180122). This work received funding from Key Project supported by Medical Science and Technology Development Foundation, Nanjing Department of Health (YKK18092).

Compliance with ethical standards

Conflict of interest The authors declared that they have no conflict of interest.