| Age determination is an important aspect of life history of amphibians and reptiles, but it’s difficult in wild. Most species were usually divided into different age groups based on their body size, since body size of amphibians and reptiles increase with growth of age, yet it is not always accurate. Skeletochronology has been turned out to be a rapid and reliable method for age determination, and widely used in amphibians and reptiles, but it is rarely used in sauria species are less. For the reason that development and tissue structure of bone varies among different species, improved skeletochronology method should be applied to the target species to get the accurate result.Toad-headed lizard (Phrynocephalus vlangalii) is an endemic species to China, and is widely distributed in the Qinghai-Tibet Plateau. Previous studies have tried to categorize P. vlangalii into adult, juvenile and hatching groups by snout-vent length (SVL). To further improve the age determination accuracy in this species, the present study adopted skeletochronology method, and did the folowing measurements including morphological traits of epiphysis and diaphysis in phalanx of P. vlangalii during development process, and lines of arrested growth (LAG) in various skeletons. Individual age was determined by skeletochronology, while age structure and growth rate were calculated. Maturity age of P. vlangalii was determined on individual age and reproductive traits. The phalanges growth rule was confirmed.Consecutive transverse and longitudinal sections were cut from the phalanx, and stained with Ehrlich’s hematoxylin. The two ends of the diaphysis cylinder insert into the epiphysises. the diaphysis is composed of periosteal bone and endosteal bone. But distribution of them in diaphysis cross-section are not equal, the proportion of endosteal bone is higher than periosteal bone in ventral phalanx, but in dorsal part is contrary. There is no endosteal bone in the diaphysis of newborn hatchling. Endosteal bone begin to deposit in the marrow cavity after1year old which leads to the diameter of the marrow cavity decrease with growth of individual age. Differentiation and development of epiphysises are distinct between distal and proximal of the phalanx. Distal epiphysis degenerates after born, hypertrophic zone and proliferation zone disappear gradually, but proximal epiphysis keeps proliferation until late3years old. Therefore, both periosteal bone and endosteal bone contribute to transverse growth of phalanx diaphysis, while Longitudinal growth primarily generates by proximal epiphysis.The bone of P. vlangalii grows fast during active season, and forms a circular band which is lightly stained by Ehrlich’s hematoxylin. While, the bone grows slowly or stagnantly during hibernation, and forms a deeper Ehrlich’s hematoxylin stained line called line of arrest growth (LAG). LAG could be found in phalange, metatarsus, tibia and coccygeal vertebra. Endosteal reabsorption is more severer in tibia and coccygeal vertebra, early LAGs in them are partly or entirely eroded. On the contrary, endosteal reabsorption is light in phalange and metatarsus. Embryo bone and born line could be observed in adult. Thus, the number of LAG in diaphysis of phalange and metatarsus equal to that of hibernations which individual experienced. Endosteal bone is dominate in growth of proximal diaphysis. and periosteal bone is thin or not present. However the LAGs that we concerned are on the periosteal bone. So only about1/3diaphysis in distal end of phalanx could observe all the LAGs.Individual age is determined by skeletochronology. From growth curve basing on age and SVL, we know that the growth rate of P. vlangalii decreased with increase of individual age, and SVL approached limit at4years old. There was no significant difference in mean age between the sexes (t=0.964, P>0.05). Tail length was significant difference between the sexes at age of3(t=4.2,P<0.001) and4(t=4.45,P <0.001) years old. But SVL and body mass have no difference at various age groups between the sexes.10.3%of the reproductive female were2years old, but all the male participated in breeding were3years old. Testis volume and diameter of seminiferous tubule were the lowest state at June, began to increase at July, reached the highest state in hibernation, and then degenerated after emerged from hibernation. There was no significant correlation for testis volume cycle of P. vlangalii with sunshine cycle (r=0.321, P>0.321), but testis volume has negative correlation with average temperature and precipitation (r=0.857, P<0.05; r=0.857, P<0.05).Bone histological study have shown that individual age determined by skeletochronology is feasible. we found that except tail length, SVL and body mass have no difference between the sexes at different age groups, so age determination by SVL is arguable. Determination of age at maturity and study of testis cycle provide important evidences for studies on life history of P. vlangalii. |
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