Directional Bilateral Asymmetry in Fish Otolith: A Potential Tool to Evaluate Stock Boundaries?

: The otolith, found in both inner ears of bony ﬁsh, has mainly been used to estimate ﬁsh age. Another application that has been developing signiﬁcantly in recent years, however, is the use of otolith shape as a tool for stock identiﬁcation. Often, studies have directly used the shape asymmetry between the right and left otoliths. We tested the magnitude of directional asymmetry between the sagittal otoliths (left vs. right) of 2991 individuals according to their catch locations, and we selected species to evaluate whether directional asymmetry may itself be a tool to evaluate stock boundaries. Elliptical Fourier descriptors were used to describe the otolith shape. We used a ﬂatﬁsh, the common sole ( Solea solea , n = 2431), from the eastern English Channel


Introduction
Otoliths are calcified structures, located in the inner ear cavity (left and right) of all teleost fish, that aid in hearing [1][2][3]. Otoliths show incremental structures with periodicity as they grow throughout the life of the fish and, unlike scales and bones, are metabolically inert (i.e., once deposited, otolith material is unlikely to be resorbed or altered) [4]. Consequently, otoliths have been primarily used as a tool for age determination in many fish species, thanks to the ability to track growth periodicity, from daily to annual growth increments. Moreover, otolith shape remains unaffected by short-term changes in fish condition [5] or environmental variation [1]. Accordingly, otolith shape has been used as a tool to identify species, reconstruct the composition of predator diet (fishes, seabirds, seals, etc.), and discriminate between fish stocks. Since Campana and Casselman in 1993 [5], many fishery scientists have developed this type of analysis for stock discrimination studies as a basis for understanding fish population dynamics and achieving reliable assessments for fishery management [6]. Several descriptors have been used to outline the external contour of otoliths: univariate descriptors (e.g., shape factors [7], geometric morphometric analyses [8][9][10], wavelet functions [11,12], growth markers [13], and the geodesic method [13]). However, elliptical Fourier analysis (EFA) remains the most widely used and robust method to describe otolith shape. As a result, 91 papers on the identification of marine fish populations or stock structure using otolith shape were published between 1993 and 2017.
The otolith shape of a fish depends on its genotype, the influence of environmental factors during its life (both biotic and abiotic), and on its stage of development (fish size, age, sex, and sexual maturation) [14][15][16][17][18][19][20][21][22][23][24][25][26]. Our review of the 91 published papers on marine fish stock identification based on otolith shape showed that only 20 of them estimated the asymmetry of both the right and the left otoliths (data from Web of Science with keywords "otolith" and "shape"; each publication was verified). Among 18 tested species, only 50% showed significant asymmetry of shape between otoliths.
In general, patterns of asymmetry in fish otoliths are classified into three types of asymmetries ( Figure 1). Fluctuating asymmetry is the result of random deviations from perfect bilateral symmetry [27,28] and is usually associated with stress and/or environmental heterogeneity [27][28][29][30]. Lemberget and McCormick [28] suggested that fluctuating asymmetry could be considered as a sensitive indicator of fish health and directly affect fish performance because otoliths are essential to balance and hearing. Antisymmetry occurs when there is a systematic but alternating deviation towards one side or the other in the population, thus generating a bimodal distribution with a mean of 0 in its extreme form. Asymmetry that consistently favours one side across an individual group is referred to as directional asymmetry. Directional asymmetry characterizes the consistently greater development of one side of the inner ear among individuals in a population. In this context, the objectives of the present study were to explore (1) if otolith directional asymmetry shape is observable within the species and (2) if there was spatial variation within the study area. Our study species were the common sole (Solea solea, Linnaeus, 1758) and the bogue (Boops boops, Linnaeus, 1758). The common sole is a flatfish species (Soleidae) of large economic interest and is highly exploited in the Mediterranean Sea and the eastern Atlantic Ocean from Senegal to Norway, especially in the North Sea and the English Channel. This species lives on fine sand and muddy substrates between 0 and 150 m in depth. In the English Channel, reproduction takes place between February and June, with a peak from April to May, mainly in the coastal areas of the Dover Strait and larger bays [31]. The bogue is a common roundfish species (Sparidae) of the northeast Atlantic Ocean and the Mediterranean Sea. The bogue is a gregarious, demersal species found at depths between 0 and 350 m over a variety of substrates. In the Mediterranean Sea, it is one of the most abundant fish species [32]. The aim of this study was to investigate whether the spatial variation of directional bilateral asymmetry in fish otoliths could be used as a potential tool to discriminate between stocks of fish as well as otolith shape.

Sample Collection
A total of 2991 pairs of sagittal otoliths (left and right) were extracted from bogues (n = 560) with a size range of 13 to 26 cm total length (TL) (18.38 ± 2.63 cm) and common soles (n = 2431) with a size range of 9 to 34 cm TL (27.43 ± 9.62 cm). Fishes were collected from 17 different locations ( Figure 2 and Table 1).
Bogue samples were collected from the Canary Islands to the Aegean Sea between 2013 and 2016, and common sole samples were collected from the western English Channel to the southern North Sea during 2017. Sampling was supported by 8 research institutes (Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), France; Centre for Environment, Fisheries and Aquaculture Science (CEFAS), UK; Institute for agricultural and fisheries research (ILVO), Belgium; Université Abderrahmane Mira, Algeria; APLYSIA institute, Italy; University of Tunis, Tunisia; Instituto Español de Oceanografía (IEO), Spain; and Hellenic Centre for Marine Research (HCMR), Greece) during four international surveys (Beam Trawl Survey (BTS), International Bottom Trawl Survey (IBTS), Channel Ground Fish Survey (CGFS), and International Bottom Trawl Survey in the Mediterranean Sea (MEDITS)). Samples were collected onboard fishing vessels and from fish markets. The age range of fish sampled was limited from 2 to 5 years to limit the bias of age on the otolith shape. Moreover, the macroscopic maturity stage was identified systematically to use only individuals beyond stage I (juvenile).

Otolith Shape Analysis
A calibrated high-resolution image (3200 dpi) of the proximal face of the whole left and right sagittal otolith was obtained using a scanner with reflected light. Within this process, a fixed single magnification was used to ensure as high a resolution as possible.
Images were processed using the image analysis system TNPC (Digital processing for calcified structures, version 7) with the sulcus acusticus facing up. In order to compare left and right otolith shapes, mirror images of left otoliths were used.
An elliptic Fourier analysis (e.g., [33]) was carried out on each otolith contour delineated and extracted after image binarization. All EFDs were obtained using TNPC 7 software. For each otolith, the first 99 elliptical Fourier harmonics (H) were extracted and normalised with respect to the first harmonic and were thus invariant to otolith size, rotation, and starting point of contour description [34]. To determine the number of harmonics required to reconstruct the otolith outline, the cumulated Fourier power (F) was calculated for each individual otolith as a measure of the precision of contour reconstruction obtained with n k harmonics (i.e., the proportion of variance in contour coordinates accounted for by the n k harmonics): where Ai, Bi, Ci, and Di are the coefficients of the H i harmonic. F(n k ) and n k were calculated for each individual otolith k in order to ensure that each individual otolith in the sample was reconstructed with a precision of 99.99% [33]. The maximum number of harmonics n = max(n k ) across all otoliths was then used to reconstruct each individual otolith of the sample.

Statistical Analyses
Asymmetry between left and right otolith shape was analysed as the effect of inner ear location (side, hereafter) on otolith shape. First, principal components analysis (PCA) was applied to a matrix of selected EFDs (EFDs as columns and individual otoliths as lines) of otolith contours [35], and a subset of the resulting principal components (PCs) was selected as otolith shape descriptors according to the broken stick model [36]. The matrix of selected PCs is referred to as 'shape matrix' hereafter. This procedure allowed us to decrease the number of variables used to describe otolith shape variability while ensuring that the main sources of shape variation were retained, and to avoid co-linearity between shape descriptors [35]. Second, partial redundancy analysis (pRDA) by species was performed on the shape matrix, using the side (left/right) as the potentially influential variable and the individual as the conditioning variable. This type of analysis was carried out for all individuals and then separately for each sampling location. pRDA is an extension of multiple regression to multivariate response data and an extension of PCA [36], combined with permutation tests (marginal effect, type II [37]) on the selected PCs matrix. To visualise differences in otolith shape between the right and left sides, an average otolith shape of each side group was rebuilt based on EFDs of the averaged shape. To evaluate DA amplitude, average left and right shapes were rebuilt based on EFDs averaged for each side at the level of all individuals and for each sampled location. DA amplitude was then computed as the percentage of non-overlapping surface between the reconstructed right and left otolith average shapes relative to the total area they covered after superposition.

Results
Among the 99 Fourier harmonics extracted to describe individual otolith contours, the first 26 harmonics for bogue and the first 28 harmonics for common sole explained at least 99.99% of the variation in the otolith contour of each individual and were used for further analysis. After PCA on the elliptic Fourier descriptors (EFDs), only the first six PCs for both species were kept for the shape matrix, according to the broken-stick model. The pRDA performed on data across all sampling sites detected a significant directional asymmetry between left and right otolith shape (p = 0.018 for bogue and p = 0.005 for the common sole). The amplitudes of directional asymmetry across all sampling sites, measured as the percentage of non-overlapping surface between the right and left otolith shape, were on average 2.77% for the bogue and 5.27% for the common sole ( Figure 3). These two mean values showed significant directional asymmetry for the bogue and common sole. In B. boops, only 5 out of 11 locations showed significant directional bilateral asymmetry, and these locations were in only two sectors: the Algerian coast (B2: Gulf of Oran, B3: Gulf of Bejaia, and B4: Gulf of Annaba) and the western part of the Italian coast (B8: Ligurian Sea and B9: Tyrrhenian Sea) (Figure 4). The main shape difference between left and right otoliths was located between the rostrum and the antirostrum. The right otolith was larger than the left otolith in the Ligurian and Tyrrhenian Seas, and this phenomenon was reversed in the Gulfs of Oran, Bejaia, and Annaba.
The mean level of bilateral asymmetry was higher in S. solea (5.27%) than in B. boops (2.77%) individuals. Moreover, all locations showed significant bilateral asymmetry on the same side, which characterized the directional asymmetry (p < 0.05 in pRDA for each location). The ventral part of the common sole otolith was the main area that showed asymmetry, with the left otolith consistently larger than the right. The asymmetry values varied in amplitude between 3.84% and 6.57%, depending on the sampling site ( Figure 5). These mean values of asymmetries could be separated into two large areas: the English part of the studied area (Thames Estuary, central area of the eastern English Channel, and English coast of the eastern English Channel), with asymmetry values of 3.84% to 4.72%, and the French part of the studied area (Mont Saint Michel Bay, Bay of Seine, Bay of Somme, and the French coast of the North Sea), with values of 5.76% to 6.57% ( Figure 5). The level of directional bilateral asymmetry between both parts of the eastern English Channel was significantly different (p < 0.05).

Discussion
Fish otolith shape is frequently used to discriminate between stock units within a species as a consequence of a number of factors, such as abiotic environmental parameters (e.g., temperature and salinity), biotic parameters (e.g., prey availability), and genetics [5,17,19,20,25,26,32]. Other ontogenetic factors, such as fish length [5,22,42,43], age [44], year class [5,7,45,46], sexual maturity [5,19], and sexual dimorphism [5,45] can also modify the otolith shape. However, the potential intra-individual factor between the right and left inner otolith as a source of variation in otolith shape has been very understudied. In biology, symmetry in bilaterally symmetrical organisms, including vertebrates, is so consistent as to be considered standard, and it is maintained by homeostatic processes [47]. However, numerous studies have documented a consistent asymmetry in human upper limb bones, with right side elements typically being larger, while lower limb bones tend to be more symmetric [48]. Humans preferentially use the right upper limb, and many skeletal studies have argued that lateralized behaviours among humans lead to differences in bone size and shape between sides, especially for the upper limbs [49]. For the otolith shape, fluctuating asymmetry, defined as the random deviations from perfect symmetry between the left and right otoliths, has been reported for some species of both roundfish and flatfish [28,[50][51][52]. Directional asymmetry has been measured for 18 fish species. Symmetry between the left and right otoliths, showing the two strictly similar parts of the vestibular system, has been observed in Gadus morhua [19,53], Coryphaena hippurus [54], Xiphias gladius [55], Scomber scombrus [14], Melanogrammus aeglefinus [56], Mullus barbatus [56], Clupea harengus [56], and Lutjanus kasmira [55]. Conversely, otolith directional asymmetry, showing lateralization, has been observed for the roundfish Liza ramada [57], Diplodus annularis [58], Scomberomorus niphonius [59], and Merlangius merlangus [56] as well as for the flatfish Solea solea [22,56], Pleuronectes platessa [56], Limanda limanda [56], and Lepidorhombus whiffiagonis [56]. This study corroborated the results observed in the otoliths of common sole, and identified otolith asymmetry in bogue. Directional bilateral asymmetry or antisymmetry in fish otoliths could be a result of differences in otolith biomineralisation between the left and right inner ears [56]. This asymmetry is an indicator of possible dysfunction, considering vestibular sensing [60]. Otolith directional asymmetry affects the acoustic functionality (sensitivity, temporal processing, and sound localization) [51,52] and kinetic swimming of fish (aberrant movement pattern or static space sickness) [60][61][62][63]. As in a previous study [56], our results showed that the observed level of otolith asymmetry in flatfish is higher than that in roundfish. For flatfish, the metamorphosis mechanism is an important source of shape asymmetry between the right and left otoliths because it causes a unique asymmetric body shape and lateralized behaviour due to adaptation to a bottom-living lifestyle, in addition to affecting the otolith shape of fish. In addition to the metamorphosis mechanism, other factors related to ontogeny (such as sexual maturity) could have potential effects on the level of directional bilateral asymmetry.
Otolith asymmetry could change during the life of a flatfish, especially after cranial deformation and the migration of one eye to the other side, caused by cell proliferation in suborbital tissue [64]. This lateralization process induces a difference in otolith biomineralisation (carbonate accretion rates), with the blind side generally growing faster in length and weight than the other side [56,[65][66][67]. Gravity is probably also a source of otolith asymmetry in flatfish [61]. Solea solea is a dextral species (right-eyed flatfish) with the right inner ear above the left inner ear, and the left otolith is larger than the right otolith. Consequently, this study corroborated that the blind side was the location of the widest otolith for flatfish [22,56]. In conclusion, if directional asymmetry is observed more frequently in flatfish than roundfish species, there might be environmental patterns other than gravity to explain the degree of otolith shape asymmetry in fish species.
No study has previously examined the fluctuations of directional bilateral asymmetry in fish otoliths according to geographic origin. The canalization process is defined as the tendency of a specific genotype to follow the same phenotypic trajectory under varied developmental and environmental influences (developmental stability), and it limits potential directional asymmetry. One study showed that the magnitude of asymmetry between scallop (Bivalvia: Pectinidae) valve shape could be linked to the type of ecomorph and, therefore, a difference in environmental pressures [68]. For fish species, molecular investigations have suggested that mandibular asymmetry corresponds to genomic loci [69]. The morphological asymmetry of scale-eating cichlids in Lake Tanganyika has a genetic basis in two individual groups [70]. The directional asymmetry of dental formulae and arch shape identified the hybrids and the parental species of the clonal fish Chrosomus eosneogaeus [71]. This experimental study showed that left-right asymmetry in the dentition and shape of the pharyngeal arches reflected phenotypic plasticity. Similarly, a study on Astyanax mexicanus highlighted the use of directional asymmetry of osteocranial shape to identify specimens from the cavefish population in comparison with those from the surface population. This difference was not directly associated with eye loss, but with cave-adapted fish [72]. Consequently, directional asymmetry concerning skeletal or calcified structures could be a genetic and/or phenotypic marker for studies on evolution, or it could be used as a tool to discriminate between populations.
For Boops boops, significant directional asymmetry associated with some geographical areas was observed, whereas no asymmetry was observed in other geographical areas. Fish in two large areas, the Algerian coast and the western part of the Italian coast, showed directional asymmetry, but these areas showed opposite differences between the left and the right otoliths. Consequently, this result suggests that the two specific individual groups are separated and that the Strait of Sicily is a mixed area influenced in the west by Atlantic Ocean currents and, conversely, in the east by the Levantine Intermediate Water current (LIW), which moves water masses from east to west [73]. There are no available data on the scale of the Mediterranean Sea on the stock distribution of Boops boops. On the Algerian coast, only one stock was identified for this area [74] as the result of asymmetry. For Solea solea, the otolith asymmetry values could be separated into two large areas: the English part and the French part of the English Channel and the southern North Sea. A previous study combined the life cycle stages, including (a) larval retention within spawning regions, (b) spatial segregation of juveniles inside separated coastal and estuarine nursery grounds, and (c) limited individual movement at the adult stages to identify three subpopulations of common sole in the eastern English Channel, with a separation between the English and the French parts [75]. Moreover, another study based on genotype and the otolith shape concluded that the structure of this species in the eastern English Channel was divided into several subunits, with noticeable isolation of the Seine River subunit [76]. The genetic and otolith shape approaches showed different discriminatory power. While these tools showed that the Seine River was isolated from the other parts of the eastern English Channel, the fish from the northeast and UK areas were not easily assigned according to the type of approach used (genetic or otolith shape). The discrimination difference explained by the otolith shape is related to a complex combination of genetic, ontogenetic, and environmental factors. As with otolith shape, the results of directional asymmetry identified two components in the fine-scale population structure of the common sole of the eastern English Channel. In conclusion, the directional bilateral asymmetry in fish otoliths confirms the conclusions of previous studies on stock discrimination using life history traits and otolith shape.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author.