the egg batches of h. nigrescens were collected in niigata prefecture , japan , in april 2012 .
arrows indicate the level of the line between epaxial muscles and hypaxial muscles . a : st 38 , b : st 50 , c : st 58 , d : st 63a , e : st 66 , f : st 68 .
scale bar=5 mm . and table 1table 1.specimens of hynobius nigrescens used in this studydevelopmental stagehabitatsvl*(mm)st38aquatic7.27.37.5st50aquatic9.910.510.8st58aquatic12.913.514.1st63aaquatic16.817.117.2st66aquatic21.121.622.2st68terrestrial24.925.225.7*snout - vent length . ) , as described by iwasawa and yamashita .
the earliest stage used in this study was st 38 , which is gill formation iii when the gills bud and balancers elongate .
the larvae of st 38 swim in water by lateral undulation of trunk . the next developmental stage used in this study was st 50 , which is digital differentiation iii when the balancers disappear and first and second finger primordials clearly develop .
the third developmental stage used was st 58 , which is digital differentiation vi when the fourth toe is clearly recognized .
they swim by lateral undulation of trunk and sometimes hold the bottom by forelimbs to stabilize their body .
the fourth developmental stage used was st 63a , which is full - grown larva i when the membrane between each toe disappears .
the larvae of st 63a swim using lateral undulation of trunk in addition to crawl on the bottom .
the fifth developmental stage 
 was st 66 , which marks the disappearance of fin ii when the dorsal fin regresses as far back as the hind limbs and small gill pieces remain .
the last developmental stage used was st 68 , which is completion of metamorphosis when the gills and tail fin have completely disappeared and the eyeballs protrude .
samples were fixed in a straight body position in 10% formalin and were then transferred to 70% ethanol solution .
arrows indicate the level of the line between epaxial muscles and hypaxial muscles . a : st 38 , b : st 50 , c : st 58 , d : st 63a , e : st 66 , f : st 68 .
the following groups of the trunk muscles were examined in this study : dorsal muscles , lateral hypaxial muscles and abdominal muscles . during dissection , specimens were kept wet by moistening with water to avoid drying and subsequent measurement error .
when the muscle was dried , the weights of the muscle would be measured lighter than in actual , and thus , the assumed measurement error was delivered from the drying of muscles . each trunk muscle group was weighed using an electronic balance auw220 ( shimadzu co. , ltd . ,
the weight ratio of each muscle group against the total weight of all measured trunk muscles was calculated .
observation of trunk muscles : the components of the trunk muscles developed and changed morphologically with growth ( fig . 2fig .
2.lateral views of ontogenetic changes of the trunk muscles in hynobius nigrescens . a : st 38 , b : st 50 , c : st 58 , d-1 : st 63a after skinned , d-2 : st 63a after removed m. obliquus externus , e-1 : st 66 after skinned , e-2 ; st 66 after removed m. obliquus externus , f-1 : st 68 after skinned , f-2 : st 68 after removed m. obliquus externus .
at st 38 , they possessed a single thick dorsal muscle and a single thick m. ventralis ( fig .
at st 50 , when the first and second finger primordia had developed patently , a thin m. transversus abdominis with fibers extending craniodorsally developed from m. ventralis and became ventrally enlarged ( fig .
the dorsal muscles were segmented by myosepta , as also observed at st 38 . when the hind limbs were revealed and forelimbs were developed at st 58 , a thin m. obliquus externus with fibers running caudoventrally developed along the edge of the abdominal contour line of m. transversus abdominis . from the ventral edge of m. obliquus externus , the muscle fibers of m. obliquus externus became parallel to the 
 sagittal line ( fig . 2c ) . at st 63a , when they crawled on the bottom in water and swam by undulation , m. obliquus externus dorsally developed to the level of the lateral line between epaxial muscles and hypaxial muscles ( fig .
2d ) . a thin m. rectus abdominis occurred at the ventral edge of the trunk ( fig .
at st 66 , when they crawled on the bottom in water in addition to swimming , m. rectus abdominis expanded and increased in thickness and was obviously separated from the fibers of the lateral hypaxial layers ( fig .
2e ) . at st 68 , after metamorphosis and movement to land for walking on ground , m. rectus abdominis became enlarged ( fig .
lateral views of ontogenetic changes of the trunk muscles in hynobius nigrescens . a : st 38 , b : st 50 , c : st 58 , d-1 : st 63a after skinned , d-2 : st 63a after removed m. obliquus externus , e-1 : st 66 after skinned , e-2 ; st 66 after removed m. obliquus externus , f-1 : st 68 after skinned , f-2 : st 68 after removed m. obliquus externus .
trunk muscle weight ratios : the muscle group weight ratios are represented in table 2table 2.ontogenetic changes of weight ratios of trunk muscles in h. nigrescensdevelopmental stageaverages of svl * ( mm)weight ratio ( % ) dorsal muscleslateral hypaxial musclesabdominal musclest387.33  0.252.2  2.347.7  2.30.0st5010.4  0.355.1  2.344.8  2.30.0st5813.5  1.155.3  1.841.7  2.23.0  0.4st63a17.0  0.656.7  2.638.3  2.94.9  0.3st6621.6  1.360.0  1.229.9  2.410.0  1.3st6825.2  1.261.4  2.322.1  3.516.5  1.4*snout - vent length , mean  s.e.m .. ontogenetic changes were identified in the muscle group ratios among stages .
the muscle weight ratio of the dorsal muscles increased with growth from 52.2% at st 38 to 61.4% at st 68 ( the averages of three samples of weight ratios of the trunk muscles : table 2 ) .
in contrast , the weight ratio of the lateral hypaxial muscles decreased with growth from > 40% at st 38 , 50 and 58 to < 30% at st 66 ( table 2 ) .
the weight ratio of the abdominal muscles increased with growth ( table 2 ) . at st 38 and 50
, the salamander did not possess abdominal muscles , with the percentage of abdominal muscles being recorded as zero . from st 58 to 68 ,
observation of trunk muscles : ontogenetic changes were recognized in the trunk muscles in h. nigrescens .
maurer   described that m. rectus abdominis arose from the ventral edges of m. obliquus internus on the timing of start of development of m. obliquus externus . in h. nebulosus
, m. rectus abdominis develops from the ventral edges of m. obliquus externus and m. obliquus internus when the development of m. obliquus externus starts . in this study of h. nigrescens , m. rectus abdominis developed and enlarged from the ventral line when m. obliquus externus developed .
the timing of formation of m. rectus abdominis on the ventral line in this study coincided with 
 maurer   and fujimoto .
the number of lateral hypaxial muscles differed between h. nigrescens in this study and h. nebulosus in fujimoto .
h. nigrescens has two layers , m. obliquus externus and m. transversus abdominis , as lateral hypaxial muscles , except for m. ventralis .
in contrast , h. nebulosus has three layers : m. obliquus externus , m. obliquus internus and m. transversus abdominis , except for m. ventralis .
the developmental sequence of the hypaxial trunk muscles of h. nebulosus is reported as follows : 1 ) m. ventralis ( the ventral muscle ) , 2 ) m. obliquus internus ( the inner lateral hypaxial layer ) from the ventral muscle , 3 ) m. obliquus externus ( the outer lateral 
 hypaxial layer ) and m. rectus abdominis and 4)m .
transversus abdominis , which sequence was similarly observed in h. nigrescens , but m. obliquus internus does not appear and m. transversus abominis appears before m. obliquus externus and m. rectus abdominis in h. nigrescens .
simons and brainerd   discussed that the habitat and predominant locomotor mode of salamanders do not appear to be strong associated with the number of lateral hypaxial layers .
it was suggested that the differences in the number of lateral hypaxial layers in these phylogenetically very close species , h. nebulosus and h. nigrescens suggest the presence of interspecific variation in this genus and further studies on other congeneric species are required . in 
 this study , we observed only from lateral view .
at st 38 , immediate hatching , the number of lateral hypaxial layer was only one , and the layer was thick ( fig .
a typical fish possesses a thick trunk muscle divided into epaxial and hypaxial segments by a myosepta , but it does not show a layered structure .
because the larvae of h. nigrescens locomote by swimming and they do not possess limbs at st 38 , they have a single thick m. ventralis as the lateral hypaxial muscle for undulatory swimming . during later developmental stages , the thickness of m. ventralis decreased , and a thin layer of m. transversus abdominis developed from the ventral edge of m. ventralis ( fig .
2b ) . at st 58 , a thin layer of m. obliquus externus developed as one of the lateral hypaxial muscles ( fig .
the two lateral hypaxial muscles at this stage were thinner than m. ventralis present during early developmental stages ( fig . 2c ) .
when urodelians move to land , they need to resist both torsion and lateral bending .
since resisting torsion can be absorbed by the two lateral hypaxial layers which fibers run in a cross direction with each other , the number of the lateral hypaxial muscles possibly increased from one to two with growth .
furthermore , muscle fibers running across each other in the two lateral hypaxial layers strengthen the body in a manner similar to the lamination of chipboard . at st 58 ,
when the fourth toe is clearly recognized , the muscle fibers of the lateral hypaxial muscle become longitudinal at the ventral edge of the trunk ( fig .
m. 
 rectus abdominis developed and enlarged at st 58 , 63a , 66 and 68 ( fig .
because m. rectus abdominis generally contributes to maintaining posture   and sustaining the animals own weight , evolutionary acquisition of this muscle was possibly essential for terrestrial locomotion .
it has been argued that typical fish do not have m. rectus abdominis   because their basic trunk muscle structure is composed of epaxial and hypaxial muscles that facilitate lateral bending . because of buoyancy , the need of sustaining inward organs weight decreases in fish .
then , fish does not have m. rectus abdominis which function is sustaining own weight . when adult salamanders are compared , terrestrial species and semi - aquatic species possess a separated and larger m. rectus abdominis , whereas aquatic species possess a smaller and unseparated m. rectus abdominis 
 [ 14 , 15 ] .
[ 14 , 15 ] suggested that a separated and larger m. rectus abdominis facilitated terrestrial locomotion by resisting gravity and that m. rectus abdominis is not essential for an aquatic lifestyle .
separated m. rectus abdominis more specializes its function of sustaining own weight than unseparated ones from lateral hypaxial muscles [ 14 , 15 ] .
thus , the interspecific difference of m. rectus abdominis between aquatic and terrestrial species is parallel to the muscular 
 differences found between the aquatic and terrestrial stages of h. nigrescens .
trunk muscle weight ratios : ontogenetic changes in the weight and weight ratios of the muscle groups are given in table 2 . though the actual mass of all muscle groups increased according to growth , the degree of growth was different among muscle groups
. the weight ratios of the dorsal and abdominal muscles increased with growth , hence the decrease of the weight ratios of the lateral hypaxial muscles .
m. dorsalis trunci , which is the largest epaxial muscle , stabilizes the trunk against sagging and torsion and increases the stiffness of the trunk during walking . 
 because the need for stabilizing the trunk and resisting gravity on land occurs only after metamorphosis
, we assume that the increase in the weight ratio of the dorsal muscles is related to the transition from water to land .
the weight ratio of the lateral hypaxial muscles decreased with growth ( table 2 ) .
lateral hypaxial muscles function to control torsion and lateral bending   and to stabilize the body [ 1 , 3 , 4 ] .
because lateral hypaxial muscles are necessary for undulatory swimming , we suggest that larvae possess larger lateral hypaxial muscles than terrestrial juveniles . after the limbs develop , the role of the lateral hypaxial muscles possibly decreases .
therefore , we consider that the decreased importance of the lateral hypaxial muscles results in the decreased weight ratios of the lateral hypaxial muscles . after they are equipped with limbs , urodelians mainly locomote by undulatory swimming in addition to aquatic walking .
this study suggests that they gradually modify the trunk muscles to 
 prepare for movement on land .
abdominal muscles function to prevent sagittal extension of the trunk by the action of the epaxial muscles   and to sustain the body weight against gravity .
abdominal muscles appeared and increased in size after the appearance of the interdigital processes in the hind limb anlage .
it is possible that such growth of the abdominal muscles facilitates terrestrial life . during middle developmental stages from st 50 to st 63a , salamanders start swimming in water using their limbs ( personal observations ) . at this stage , they depend less on the lateral hypaxial muscles and more on the dorsal and abdominal muscles . after metamorphosis , they start adapting to terrestrial life by enlarging the dorsal and abdominal muscles . in conclusion ,
the ontogenetic changes in the trunk muscles of h. nigrescens are linked with habitat transition from water to land , with the muscle construction changing in adaptation from aquatic swimming to resisting gravity .
 we investigated ontogenetic changes in the trunk muscles of the japanese black salamander ( hynobius nigrescens ) before , during and after metamorphosis . given that amphibians change their locomotive patterns with metamorphosis , we hypothesized that they may also change the structure of their trunk muscles . 
 the trunk muscles were macroscopically observed , and the weight ratios of each trunk muscle group were quantified at six different developmental stages . immediately after hatching , we found that the lateral hypaxial muscle was composed of one thick m. ventralis , from ventral edge of which m. transversus abdominis arose later , followed by m. obliquus externus and m. rectus abdominis . 
 the weight ratios of the dorsal and abdominal muscles to the trunk muscles increased with growth . 
 we suggest that a single thick and large lateral hypaxial muscle facilitates swimming during early developmental 
 stages . 
 the increase in the weight ratios of the dorsal and abdominal muscles with growth possibly assists with gravity resistance necessary for terrestrial life . 
MATERIALS AND METHODS
RESULTS
DISCUSSION