Dynamic skeletogenesis in fishes: Insight of ... - Wiley Online Library

DEVELOPMENTAL DYNAMICS 241:1507–1524, 2012

RESEARCH ARTICLE

a

Dynamic Skeletogenesis in Fishes: Insight of Exercise Training on Developmental Plasticity

Developmental Dynamics

¨ nbaum,1,2 Richard Cloutier,1* and Bruno Vincent3 Thomas Gru

Background: Through developmental and evolutionary time, organisms respond variably to their environment not only in terms of size and shape but also in terms of timing. Developmental plasticity can potentially act on various aspects of the timing of developmental events (i.e., appearance, cessation, duration, sequence). In this study, we address the developmental plasticity of median fin endoskeleton by using exercise training on newly-hatched Arctic charr (Salvelinus alpinus). Results: Developmental progress of cartilage formation (i.e., chondrification) in all fins is less influenced than ossification by an increase of water velocity. The most responsive elements, meaning those elements with greater onset plasticity owing to a water velocity increase, differ in terms of early versus late developmental events. The most responsive elements are those that chondrify and to a greater extent ossify later in the development. Conclusions: Plasticity is documented for the timing of appearance (i.e., onset) and the timing of transition from cartilage to bone (i.e., transitions of skeletal states) rather than the order of events within a sequence. Similarities of plastic response in developmental patterns could be used as a powerful criterion to strengthen the identification of phenotypic modules. Developmental Dynamics 241:1507–1524, 2012. V 2012 Wiley Periodicals, Inc. C

Key words: phenotypic/developmental plasticity; fish median fins; endoskeleton; cartilage/bone; developmental events; developmental sequences; water velocity; Salvelinus Key findings: Different properties of developmental events (i.e., onset, offset, relative sequences, and trajectories) might be influenced epigenetically by changes in environmental conditions (e.g., differential water velocity). Developmental plasticity of skeletal events is shown by changes in their timing of onset and in their developmental progress with respect to environmental conditions. The onset of cartilage formation (chondrification) is less influenced than the onset of bone formation (ossification) by differential water velocity. Changes in environmental conditions have only minor effects on the relative order of skeletal events (developmental sequences) and their cumulative addition through time (developmental trajectories). Accepted 4 July 2012

INTRODUCTION From a micro-evolutionary perspective, a major task of Evo-Devo is to understand how development structures, limits, and expresses the underlying genotypic architecture

(and variation) to generate specific phenotypes and to produce phenotypic variation (Corley, 2002). Even if natural selection is a major driving evolutionary mechanism that acts upon phenotypes, the environment

(and its changes) serves not only as a selective filter for a specific phenotype within a spectrum of trait variability (Hall, 1999; Newman and Mu¨ller, 2000; Balon, 2003). Developmental processes by means of the epigenetic

´ volutive, Universit Rimouski, Rimouski, Qu Laboratoire de Biologie E e du Qu ebec a ebec, Canada ´ glise, Nouvelle-E ´ cosse, Canada Departement des Sciences, Universit e Sainte-Anne, Pointe-de-l’E 3 Rimouski, Rimouski, Qu Departement de Biologie, Chimie et G eographie, Universit e du Qu ebec a ebec, Canada Grant sponsor: Natural Sciences and Engineering Research Council of Canada (NSERC); Grant number: Discovery grant 238612. ´ Rimouski, 300 all *Correspondence to: Richard Cloutier, Laboratoire de Biologie Evolutive, Universit e du Qu ebec a ee des Ursulines, Rimouski, Qu ebec, Canada, G5L 3A1. E-mail: [email protected] 1 2

DOI 10.1002/dvdy.23837 Published online 4 September 2012 in Wiley Online Library (wileyonlinelibrary.com).

C 2012 Wiley Periodicals, Inc. V


¨ NBAUM ET AL. 1508 GRU

control [defined as the sum of the genetic and non-genetic factors that control selectively the gene expression to produce and to organize cells that further increase phenotypic complexity during development (Hall, 1999; Newman and Mu¨ller, 2000)] transform the genetic variation and the environmental changes in phenotypic variation (Hallgrı´msson et al., 2005). Thus, studies on developmental processes can address two classes of mechanisms: (1) those responsible for the production of specific/defined phenotypes (i.e., developmental stability or canalization) despite genotypic variation and/or changes in environmental conditions; and (2) those that induce alternative phenotypes (i.e., developmental plasticity, polyphenism) from a single genotype owing to changes in environmental conditions (Sholtis and Weiss, 2005). In this study, our aim was to address the latter class and specifically to provide an empirical examination of the plasticity of skeletal events through development. Phenotypic plasticity among wild populations of numerous teleost species has been repeatedly documented, mainly in terms of adult body shape differences likely associated with trophic and/or habitat differences. Although the plasticity is unquestioned, it is frequently difficult to identify precise links between the environmental factors in a complex environment and the induced phenotypic changes. However, developmental plasticity has been tested experimentally on a variety of species, phenotypes, and environmental factors. Phenotypic plasticity has been primarily studied in teleosts [e.g., adrianichthyid (Kawajiri et al., 2011), anarhichadids (Pavlov and Moksness, 1994), cichlids (Crispo and Chapman, 2010), clupeids (Fuiman et al., 1998), cyprinids (Mabee et al., 2000), gasterosteids (Garduno-Paz et al., 2010), moronids (Georgakopoulou et al., 2007), salmonids (Pakkasmaa and Piironen, 2001; Peres-Neto and Magnan, 2004; Gru¨nbaum et al., 2007, 2008; Fischer-Rousseau et al., 2009; Cloutier et al., 2010; Totland et al., 2011)] most likely owing to the facility of rearing experiences; however, there is no a priori reason to think that it is limited to teleosts.

Although thermally induced plasticity has been highly documented (Pavlov and Moksness, 1994, 1997; Fuiman et al., 1998; Mabee et al., 2000; Georgakopoulou et al., 2007; Schmidt and Starck, 2010; Kawajiri et al., 2011), numerous environmental/experimental factors have also been investigated such as dissolved oxygen (Schmidt and Starck, 2010), salinity (Schmidt and Starck, 2010), diet (Meyer, 1987; Huysseune et al., 1994; Muschick et al., 2011), predator odors (Frommen et al., 2011), hormonal conditions (Shkil et al., 2010), habitat heterogeneity (Garduno-Paz et al., 2010), and water velocity (Pakkasmaa and Piironen, 2001; PeresNeto and Magnan, 2004; Gru¨nbaum et al., 2007, 2008; Cloutier et al., 2010; Totland et al., 2011). Water velocity is of primary interest for at least two reasons. First, water velocity is a factor ubiquitous to all fish activities such as locomotion, feeding, reproduction, and predator avoidance (Azuma et al., 2002; Pakkasmaa and Piironen, 2001). Second, the main loads on tissue (muscles, bones) arise from the forces generated by the animal and the reactions of the surrounding medium (van der Meulen et al., 2006). Therefore, many species of fish control buoyancy via their swim bladder, the effects of gravitational forces are then limited compared with those of terrestrial animals. The plastic responses themselves vary according to the factor, the range of variation of the factor, the developmental period at which the factor is applied, and the responsiveness of the species. A wide range of plastic responses has been reported in the literature. Focusing on anatomical plasticity, the following traits exemplify the types of responses: size [e.g., gills (Crispo and Chapman, 2010), brain (Crispo and Chapman, 2010), head (Meyer, 1987), body (Pakkasmaa and Piironen, 2001; Peres-Neto and Magnan, 2004; Georgakopoulou et al., 2007; Gru¨nbaum et al., 2008; Schmidt and Starck, 2010; Frommen et al., 2011; Kawajiri et al., 2011), fins (Fischer-Rousseau et al., 2009)] and shape changes [e.g., head (Meyer, 1987), pharyngeal (Muschnick et al., 2011) and oral jaws (Meyer, 1987), body (Pakkasmaa and Piironen, 2001;

Peres-Neto and Magnan, 2004; Georgakopoulou et al., 2007; Gru¨nbaum et al., 2007; Fischer-Rousseau et al., 2009; Garduno-Paz et al., 2010; Frommen et al., 2011)], bone matrix (Totland et al., 2011), number of serially repeated elements [e.g., pharyngeal teeth, vertebrae, fin rays, spines (Arratia and Schultze, 1992; Mabee et al., 2000; Georgakopoulou et al., 2007; Shkil et al., 2010; Kawajiri et al., 2011)], and timing of ossification (Pavlov and Moksness, 1994, 1997; Fuiman et al., 1998; Mabee et al., 2000; Cloutier et al., 2010, Fiaz et al., 2012). It is also known that fin positioning is plastic with respect to changes in hydrodynamic conditions (Fischer-Rousseau et al., 2009, 2010). The functional recruitment of median fins is known to vary during fish ontogeny (Osse and van den Boogaart, 1995) as well as with water velocity (Drucker and Lauder, 2005). From a functional perspective, the dorsal and anal fins act primary as keels to stabilize the body, whereas the caudal fin participates in the generation of thrust for locomotion (Webb and Fairchild, 2001). The correlative link between plasticity in developing endoskeletal structures of median fins in relation to exercise training remains to be further addressed. Evidently, most morphological and physiological systems are potentially responsive to experimental and environmental factors. Independently of the combination of species, phenotypes, and environmental factors, numerous studies suggest that the phenotypic response results from a modification of either the metabolism and/or developmental rates (Meyer, 1987; Fuiman et al., 1998; Mabee et al., 2000; Schmidt and Starck, 2010; Kawajiri et al., 2011). In theory, developmental plasticity could be observed on (1) the onset (i.e., timing of occurrence of a developmental event), (2) the offset (i.e., timing of cessation), and (3) the duration of an event (i.e., difference between timing of onset and offset), as well as on (4) the sequences and (5) trajectories of developmental events. In this study, our aim is to provide and to discuss an empirical examination of exercise training effect on the developing median fin endoskeleton of Salvelinus alpinus. Among salmonids, the Arctic

DEVELOPMENTAL PLASTICITY ON FISH SKELETON 1509


charr (Salvelinus alpinus) is one of the most promising species for coldwater aquaculture in eastern Canada (Le Franc¸ois et al., 2002; Gru¨nbaum et al., 2008). Its strong potential for aquaculture lies in high growth rate, efficiency of food conversion ratio (Le Franc¸ois et al., 2002) and well-known rearing practices (Johnston, 2002). We were especially interested in using endoskeletal data to define developmental events for comparative studies on the plasticity of developmental timing. Four issues have been addressed: (1) developmental progress, (2) relative developmental sequence of events, (3) transition of skeletal states (i.e., transition from cartilage to bone), and (4) developmental trajectory.

RESULTS General Morphology of Median Fins The dorsal fin is generally composed of 13 (12–14) elongated proximal radials (PR) and 12 (10–13) rounded distal radials (DR) that support lepidotrichia. The PR and DR are organized in a one-to-one relationship (Fig. 1A–C) except for the first proximal radial (PR1) that bears no distal radial. PR and DR are cartilaginous elements that eventually ossify perichondrally. In newly-hatched specimens (ca. 13-mm SL), no distal radial are formed and eight to nine proximal radials are already present, six being the lowest number (PR5–PR10) found in a specimen of 12.9-mm SL (Fig. 1A). Chondrification occurs initially in central proximal radials and proceeds bidirectionally in an alternate manner (Fig. 1A, B). Ossification starts at PR3–4 and proceeds posteriorly. Ossification of distal radials was not initiated even in the larger specimens (ca. 45-mm SL) (Fig. 1C). Morphology, relationships, direction of chondrification and ossification of PR and DR of the anal fin are highly similar to that of the dorsal fin. Generally, 12 (10–13) PR and 11 (9–12) DR are present (Fig. 1D–F). As in the dorsal fin, PR1 is never associated with a distal radial. In newly-hatched specimens, five to seven PR are already present although no radials were found in specimens under 13.5-mm SL

(Fig. 1D). Central proximal radials are the first to form and chondrification proceeds bidirectionally as in the dorsal fin (Fig. 1D, E). Ossification starts at PR3–4 and proceeds from anterior to posterior; no ossified distal radials were present in the larger specimens (ca. 45-mm SL) (Fig. 1F). The caudal fin elements analyzed belong to vertebrae known as preural (PU1–5) and ural centra (U2, U4). The anatomical distinction between these two types of centra (vertebrae) depends on their relative position in the body axis from the branching point of the caudal artery and from the parahypural; centra located anteriorly are referred to as preural, whereas centra located posteriorly are referred to ural (de Pinna, 1996). Ventrally to the notochord, the elements include haemal arches (HA) and spines (HS) of PU2–5, a parahypural (PH) (i.e., ventral component of PU1), and six hypurals (H1–6). Dorsally, two epurals (E1–2) and three uroneurals (i.e., modified neural arches) referred to as the stegural (ST) and uroneurals 2 and 3 (UN2–3) are present; the remaining dorsal components are neural arches (NA) and spines (NS) of PU2–5 (Fig. 1G–I). Generally, 28 caudal elements are present although a seventh hypural (H7) and a fourth uroneural (UN4) were found in some specimens (see Gru¨nbaum and Cloutier, 2010). UN4 and H7 were not considered in the analyses because of their seldom occurrence. In newly-hatched specimens of 13.5-mm SL, several cartilaginous ventral elements are already present including HA of PU2–5, HS of PU2, PH, and H1–4; no dorsal elements are formed posteriorly to NA of PU3 (NA3) at this size (Fig. 1G). H5– 6, E1, and ST are the next elements to form in 15-mm-SL specimens. E2, UN2, and NA of PU2-5 and NS of PU2–5 are formed in 16-mm-SL specimens; at this size, all hypurals are ossified (Fig. 1H). The last cartilaginous element to form in the caudal fin is UN3 in 17-mm-SL specimens. Most caudal elements are ossified at 20mm SL (Fig. 1I). No clear pattern in the direction of chondrification and ossification was found in caudal elements. Even if some variations occur in the number of elements composing the three fins, especially in the caudal

fin, it only reflects intraspecific variation as independent from the treatment (Gru¨nbaum and Cloutier, 2010). The velocity treatment consisted of exposing during 100 days post-hatching, newly-hatched fish, to four different water velocities kept constant throughout the experiment (see Experimental Procedures section). The velocity treatments, given in absolute value, correspond to water velocity of 3.2 cm s-1 (A ¼ fast treatment), 1.6 cm s-1 (B ¼ medium treatment), 0.8 cm s1 (C ¼ slow treatment), and 0.4 cm s-1 (D ¼ still treatment, normal condition/reference treatment).

Developmental Progress Developmental progress refers to a cumulative occurrence of specific developmental events extrapolated from a series of sampled individuals. In terms of dynamic skeletogenesis, developmental progress refers to the progressive modeled onset of the cartilaginous state (chondrification) and the ossified state (ossification) for a given element through development. Modeling of onset was performed by using logistic regressions (for the description of the statistical analysis see Experimental Procedures section). In the dorsal fin, out of 25 elements generally found (27 in maximum), the logistic model was significant for 17, 16, 18, and 16 cartilaginous elements in treatments A, B, C, and D, respectively. Elements common to all treatments (i.e., 16) are proximal radials PR1–4 and distal radials DR2–13. Independently of treatments, most elements chondrified between 13.5 and 19 mm SL except for DR2 and DR13, which chondrified between 20 and 25 mm SL. Chondrification onset is little influenced by the water velocity increase (Fig. 2A); the latest elements to form (i.e., DR2 and DR13) were slightly affected by the treatments. The logistic model was significant for 13, 12, 13, and 11 ossified elements in treatments A, B, C, and D, respectively. The elements common to all treatments (i.e., 11) are proximal radials PR1–11 (Fig. 3A). Proximal radials begin to ossify at approximately 22.5-mm SL, all of them being ossified at approximately 50-mm SL. A general trend is that ossification



Fig. 1. Skeletal anatomy of dorsal (A–C), anal (D–F), and caudal (G–I) fins in S. alpinus. Characteristic steps of skeletal elements formation in each fin are shown from alevin and juvenile specimens. Size and age of specimens are as follows: (A) 14.20-mm SL (0 dph), (B) 15.54-mm SL (12 dph), (C) 23.07-mm SL (64 dph), (D) 15.61-mm SL (8 dph), (E) 15.15-mm SL (20 dph), (F) 31.57-mm SL (78 dph), (G) 12.87-mm SL (0 dph), (H) 16.66-mm SL (26 dph), and (I) 31.57-mm SL (78 dph). Cartilaginous elements are stained blue (Alcian blue) and bones are stained red (Alizarin Red S) following the method described in the Experimental Procedures section. DR, distal radial; E, epural; HA, haemal arch; HS, haemal spine; H, hypural; L, lepidotrichia; NA, neural arch; NS, neural spine; PH, parahypural; PR, proximal radial; PU, preural centrum; ST, stegural; U, ural centrum; UN, uroneural. Anterior is to the left. The representation of the median fin elements schematized the skeleton without the vertebrae and their associated elements (i.e., NA, NS, HA, HS).

onset is more influenced than chondrification by the water velocity increase even if the number of PR and DR found to be significant by the logistic model differs between chon-

drification progress and ossification progress. Similarly to chondrification, ossification onsets for central elements are little influenced by the differential water velocity; the most re-

sponsive elements are the later ossified PR1 and PR9–11 in all treatments, PR12 in treatments A, B, and C, and PR13 in treatments A and C (Fig. 3A).



Fig. 2. Developmental progress of chondrification in S. alpinus. Modeled chondrification onsets (SL50) of elements common to all treatments are shown for dorsal (A), anal (B), and caudal (C) fins. (A) PR1–4, proximal radials 1–4, DR2-DR13, distal radials 2–13; (B) PR1–5, PR10– 12, proximal radials 1–5 and 10–12, DR2–12, distal radials 2–12; (C) E1–2, epurals 1–2; HSPU3– 5, haemal spine of preural centra 3–5; H5–6, hypurals 5–6; NSPU2–5, neural spine of preural centra 2–5; ST, stegural; UN2, uroneural 2. Dotted lines in A and B schematized the antero-posterior morphological organization of PR elements in dorsal and anal fins, respectively. In C, elements were organized in relation to SL50 from the slower treatment D.

In the anal fin, out of 23 elements generally found (25 in maximum), the logistic model was significant for 20, 24, 23, and 23 cartilaginous elements in treatments A, B, C, and D, respectively. The elements common to all treatments (i.e., 19) are proximal radials PR1–5 and PR10–12, and distal radials DR2–12. Independently of treatments, most elements chondrified between 12.5 and 20-mm SL, except for PR13, DR2, and DR12, which chondrified between approximately 24 and 55-mm SL. Chondrification onset is little influenced by the treatments (Fig. 2B) although compared to the dorsal fin, the later chondrified elements (i.e., PR12, PR13, DR2 and DR12) are highly responsive to the water velocity increase. The logistic model was significant for 11, 11, 10, and 9 ossified elements in treatments A, B, C, and D, respectively (Fig. 3B). The elements common to all treatments (i.e., nine) are proximal radials PR1–9. Proximal radials begin to ossify at approximately 25-mm SL; all of them are ossified at approximately 45-mm SL. As in the dorsal fin, ossification onset is more influenced by an increase of water velocity than chondrification although the number of significant PR and DR differs between chondrification and ossification. Similarly to chondrification, the most responsive elements for which ossification onsets are highly influenced by the water velocity increase are the later ossified PR1, PR2, and PR8–9 in all treatments, PR10 in treatments A, B, and C, and PR11 in treatments A and B (Fig. 3B). In the caudal fin, out of 28 elements, 13, 14, 15, and 14 cartilaginous elements were found to be significant by the logistic model in treatments A, B, C, and D, respectively. The elements common to all treatments (i.e., 13) are E1–2, H5–6, HS of PU3–5, NS of PU2–PU5, and UN2. Most elements chondrified between approximately 14 and 18mm SL with the exception of HS of PU3 and NS of PU2, which form respectively at approximately 12.5 and 13-mm SL. As for the dorsal and anal fins, water velocity increase has little effect on the chondrification onset (Fig. 2C). However, compared to



Fig. 3. Developmental progress of ossification in S. alpinus. Modeled ossification onsets (SL50) of elements common to all treatments are shown for dorsal (A), anal (B), and caudal (C) fins. (A) PR1-11, proximal radials 1–11; (B) PR1–9, proximal radials 1–9; (C) E1–2, epurals 1–2; HAPU2– 5, haemal arch of preural centra 2–5; HSPU2–5, haemal spine of preural centra 2–5; H1–6, hypurals 1–6; NAPU2–5, neural arch of preural centra 2–5; NSPU2–5, neural spine of preural centra 2–5; PH, parahypural; ST, stegural; UN2–3, uroneurals 2–3. Dotted lines in A and B schematized the antero-posterior morphological organization of PR elements in dorsal and anal fins, respectively. In C, elements were organized in relation to SL50 from the slower treatment D.

the dorsal and anal fins, most caudal elements display chondrification onset plasticity among treatments. HS of PU3 and NS of PU2 have greater onset plasticity among treatments; compared to other treatments, onsets values are lower in treatment C for HS of PU3 and in treatments B and C for NS of PU2. The logistic model was significant for all ossified elements (i.e., 28) analyzed, independently of treatments (Fig. 3C). Caudal elements begin to ossify at approximately 15.5-mm SL, all of them being ossified at approximately 21.5-mm SL. As in the dorsal and anal fins, ossification onset of caudal elements is more influenced by an increase of water velocity than chondrification. Similarly to the dorsal and anal fins, elements that ossify late in the sequence (all elements with the exception of ST, PH, H1–6, and the anteriormost uroneural UN2) are the most responsive. These elements display greater onset plasticity among treatments (Fig. 3C). Interestingly, onsets of late ossified elements show an inversion of response between treatments C and D; late ossified elements in treatment D have smaller SL50 compared to treatment C. These results reveal four main patterns of developmental progress. First, developmental progress of chondrification is less influenced than ossification by an increase of water velocity in all fins. Second, the most responsive elements, meaning those elements with greater onset plasticity owing to a water velocity increase, are the elements that chondrify (i.e., especially peripheral proximal and distal radials in the dorsal and anal fins) and to a greater extent the elements that ossify late. Third, responsive elements show a smaller SL50 in faster velocity (treatment A) for all fins; they chondrify and to a greater extent ossify at a comparatively smaller size with a water velocity increase. These elements correspond to the hypurals in the caudal fin as well as the central proximal and distal radials in the dorsal and anal fins. Fourth, for all treatments, the onset of chondrification of the various elements show overlapping values among fins, whereas the onset of ossification has


Comparison of Relative Developmental Sequences


The effect of water velocity on the changes in terms of the relative timing of endoskeletal elements of the median fins was analyzed by comparing congruence of chondrification and ossification sequences (i.e., relative order of events) by using Spearman correlation coefficients (Table 1). In all fins, patterns of chondrification and ossification are strongly congruent among treatments as shown by the highly significant correlation coefficients (e.g., in all fins for chondrification and ossification P < 0.0005 for all treatment comparisons, with the exception of the anal fin ossification where P < 0.001 for comparison between treatments A versus D, Table 1). Thus, the relative timing of chondrification and ossification among endoskeletal elements within each one of the fins is fairly consistent among treatments, suggesting a high stability of median fin skeletal development (e.g., Spearman correlation coefficients are for chondrification 0.859 < rs < 0.996, and for ossification 0.928 < rs 1.000, Table 1). An increase of water velocity has induced only minor changes in the temporal order of sequences of the endoskeletal elements.

Transition of Skeletal States

Fig. 4. Transition of skeletal states between chondrification and ossification onsets in S. alpinus. Treatment comparisons are reported for endoskeletal elements of dorsal (A), anal (B), and caudal (C) fins. States transitions are expressed on Y-axis by ossification minus chondrification onsets (mm) in function to chondrification onsets (mm) in X-axis. MCP’s represent the amount of variation among treatments in transition of states for each element. All possible treatment comparisons are shown for those elements that are comparable between each state and common to all treatments.

distinctly smaller values for all caudal elements than those in the dorsal and anal fins. Caudal elements are

all ossified before the elements of the dorsal and anal fins initiate their ossification.

In chondral bones (bony elements preformed of cartilage), the inherent switch of states from cartilage to bone provides a characteristic referred to as transition of states that can be derived from differential onsets. The transition of states is a duration calculated as the difference between the onset of ossification and the onset of chondrification for a specific developmental event. In order to evaluate changes in the transition of skeletal states with respect to differential water velocity, we used the minimum convex polygons (MCP) method to quantify the variation among the four treatments for each significant element (see details in Experimental Procedures section). At the level of element, the MCP display plasticity in the transition of skeletal states among treatments for each median fin (Fig. 4). In relation to


TABLE 1. Spearman Rank Correlation Coefficients (rs) Comparisons of Developmental Sequences in S. alpinusa Chondrification

Treatment Fins

comparisons

rs

P

Dorsal

A A A B B C A A A B B C A A A B B C

0.929 0.965 0.931 0.984 0.988 0.986 0.860 0.859 0.862 0.993 0.996 0.996 0.930 0.949 0.993 0.967 0.929 0.945

< < < < < < < < < < < < < < < < <