动 物 学 研 究 2010，Oct. 31(5)：490−498 CN 53-1040/Q ISSN 0254-5853 Zoological Research DOI：10.3724/SP.J.1141.2010.05490 Embryonic development of the concave-eared torrent frog with its significance on taxonomy XIONG Rong-Chuan1,2, JIANG Jian-Ping1,* , FEI Liang1, WANG Bin1,2, YE Chang-Yuan1 (1. Chengdu Institute of Biology, the Chinese Academy of Sciences, Chengdu 610041, China; 2. Graduate University of the Chinese Academy of Sciences, Beijing 100049, China) Abstract: We investigated the early embryonic and larval development of the concave-eared torrent frogs, Odorrana tormota (Amphibia, Anura, Ranidae). Embryos were derived from artificial fertilization of frogs’ eggs, and the staging of development was based on morphological and physiological characteristics. Two major periods of development were designated: i) early embryonic period, from fertilization to operculum completion stage, lasted for 324 h at water temperature (WT) 18 −23℃; ii) larval period, from operculum completion stage to tail absorbed stage, took 1207 h at WT 20 − 24℃. Tadpoles of the concave-eared torrent frogs showed no evidence of abdominal sucker. Absence of this key characteristic supports the view from molecular systematics that concave-eared torrent frog does not belong to the genus Amolops. Two cleavage patterns were observed in embryos at 8-cell and 16-cell stages, with Pattern I - 2 (latitudinal cleavage at the 8-cell stage, and meridional cleavage at the 16-cell stage with two perpendicular meridional furrows) being the predominant pattern and only 1.5% belonging to Pattern II (meridional cleavage at the 8-cell stage and latitudinal cleavage at the 16-cell stage). The factors affecting cleavage and hatching ratios, developmental speed, and ecological adaptation were discussed. Key words: Odorrana tormota; Embryonic development; Artificial fertilization; Abdominal sucker; Embryonic cleavage 凹耳臭蛙胚胎发育及其分类学意义 熊荣川1, 2，江建平1，*，费 梁1，王 斌1, 2，叶昌媛1 （1. 中国科学院成都生物研究所，四川 成都 610041; 2. 中国科学院研究生院，北京 100049） 摘要：通过人工受精的方法获得的凹耳臭蛙（Odorrana tormota）的早期胚胎及胚后幼体的发育过程，根据胚 胎发育过程中的形态及生理特征变化规律进行分期。把凹耳臭蛙的发育过程分成两个阶段：1）早期胚胎发育阶段， 即从蛙卵受精到鳃盖完成期，在18～23℃水温下，凹耳臭蛙早期胚胎发育阶段历时324 h；2）蝌蚪发育阶段，即 从鳃盖完成期结束到尾部被完全吸收，本阶段在20～24℃水温条件下历时1 207 h。凹耳臭蛙蝌蚪未发现腹吸盘特 征，从形态特征上支持了分子系统分类学将之从湍蛙属划出的观点。实验中发现，多数胚胎在8细胞期为纬裂，16 细胞期为经裂，同时有小部分胚胎（1.5%）在8细胞期为经裂，16细胞期为纬裂。该文进一步讨论了影响卵裂率、 孵化率、发育速度，以及生态适应的因素。 关键词：凹耳臭蛙；胚胎发育；人工受精；腹吸盘；卵裂 中图分类号：Q959.53; Q133;Q954.4 文献标志码：A 文章编号：0254-5853-(2010)05-0490-09 The concave-eared torrent frog, found in two characteristics shared by other species in this group. isolated locations in China, was initially known as Rana There was no key identifying data from its tadpoles, i.e., tormotus (Wu , 1977). Fei et al (1990) classified this the presence of abdominal sucker. Li et al (2008) species as a member of genus Amolops and changed its subsequently reported that the tadpoles of R. tormotus do name to Amolops tormotus based on morphological not possess abdominal sucker (a key character for Received date: 2010-03-01; Accepted date: 2010-06-05 Foundation item: This study was supported by the National Natural Science Fundation of China (30730029) 收稿日期：2010-03-01；接受日期：2010-06-05 *通讯作者(Corresponding author)，E-mail：[email protected] No. 5 XIONG Rong-Chuan et al: Embryonic development of the concave-eared torrent frog 491 Amolops), and proposed a new genus Wurana, using R. two males), collected in May of 2008, were used in tormotus as its type species. Most recently, results of Experiment-I, and six (two females and four males), molecular systematics (Tang et al, 2007; Cai et al, 2007) collected in April of 2009, were used in Experiment-II showed that this species actually belongs to the genus and -III. Males and females were kept in separate plastic Odorrana, and renamed it as O. tormota — a aquaria in the laboratory. classification that is widely accepted to date (Stuart, 1.2 Artificial fertilization 2007; Frost, 2009; Fei et al, 2009b). Experiment-I: At 2:00 on May 10, 2008, one egg The concave-eared torrent frog is unique because clutch was found in the plastic aquarium housing the the tympanic membranes in adult males are highly female. The egg clutch was extracted and placed inside a unusual — they are recessed from the body surface, and glass Petri dish (12 cm dia); the egg clutch was then located at the far end of external auditory canals (Feng et artificially fertilized using a special testis liquid. The al, 2006). There are only two anuran species with this liquid was prepared fresh by first taking out the testis unique character in the family Ranidae: O. tormota that from two males — these were cut into pieces and mixed is distributed in eastern China, and Huia cavitympanum with 15 mL of de-chlorinated tap water. For fertilization, (Boulenger, 1893) that is distributed in Borneo. Both we poured the liquid onto the egg clutch. After 1 hour of species have been shown to use ultrasound to fertilization, at 8：45, we poured the liquid from the Petri communicate, to avoid being masked by the intense, dish and replaced with 100 mL of fresh water (21˚C) to predominantly low frequency ambient noise from local prevent dehydration. The Petri dish was placed in a streams in their breeding habitats (Feng et al, 2006; Arch constant-temperature room (maintained at −21˚C); the et al, 2009). water was changed daily to maintain adequate supply of Li et al (2006, 2008) were the first to study the oxygen. The development of the egg clutch was observed tadpoles of O. tormota. They collected tadpoles of every four hours. several species from Huangshan Hot Springs (Anhui Experiment-II: At 15：54 on April 21, 2009, we Province; one of the two habitats for O. tormota in China) injected gonadotropin-releasing hormone (GnRH-A6; and reared the tadpoles in the laboratory. Tadpoles of O. usage: 5 µg/kg BW) subcutaneously into the abdomen of tormota were identified retrogradely after they have two males and a female. Afterward, the frogs were metamorphosed into froglets. There is a caveat with the housed together in a plastic aquarium. At 18：50, we retrograde approach for identification of the species of found one egg clutch in the aquarium – this was retrieved tadpoles. Namely, at least four Odorrana species (O. and transferred into a glass Petri dish. The eggs were tormota, O. livida, O. schmackeri, O. exiliversabilis) are artificially fertilized in the same manner as in known to inhabit Huangshan Hot Springs. The Experiment-I. morphological appearances of tadpoles and froglets of Experiment-III: At 10：15 on April 24, 2009, one two of these species (O. tormota and O. exiliversabilis) female and two males received the same GnRH-A6 are so alike that they are essentially indistinguishable treatment as described for Experiment-II, and were (personal observation which will be reported in detail in housed in a plastic aquarium afterward. At 6：40 on April another paper). As such, the tenet that tadpoles of O. 25, 2009, we found one egg clutch in the aquarium — it tormota do not possess abdominal suckers is tenuous. So was retrieved and transferred into a Petri dish. The eggs it is critical to understand the tadpole of O. tormota from were artificially fertilized as described above for its embryonic development. Because obtaining fertilized Experiment-I. eggs of O. tormota in the frog’s natural habitat has All tadpoles developed from a single egg clutch proven to be very difficult (zero yield over the last were transferred into a plastic aquarium. Tadpoles were several years), we artificially fertilized frogs’ eggs in fed on chicken yolk. order to investigate the development of O. tormota 1.3 Observation of development embryos and tadpoles. The number of eggs in each egg clutch was counted. We used a vernier caliper to measure the diameter of 1 Materials and Methods individual eggs, and an optical microscope (Zeiss stemi 1.1 Specimens 2000-C) to observe the course of development. We collected a total of nine adult O. tormota from Developmental stages were determined using the staging Huangshan Hot Springs. Three of them (one female and criteria of Gosner (1960). The embryos were 492 Zoological Research Vol. 31 photographed using a camera (Canon PowerShot SD950 hatching, 91 hatchlings died, and 88 hatchlings stayed IS) attached to the microscope. From each stage, one to alive. The hatching rate was 24.87% (excluded the dead four specimens were taken out and fixed in glutaral- hatchlings). In Experiment-III, 18 embryos were fixed dehyde for further analysis. before hatching, 86 hatchlings died, and 43 hatchlings 1.4 Data analysis stayed alive. The hatching rate was 24.60% (excluded the We carried out the following analysis to depict the dead hatchlings). developmental pattern of O. tormota tadpoles: 1) 2.4 Metamorphosis ratio “Cleavage ratio” (defined as the ratio of the number of In Experiment-II, 16 tadpoles died before the eggs exhibiting the first cleavage to the total number metamorphosis. Thus, the metamorphism ratio was of eggs laid in one clutch); 2) “Hatching ratio” (defined 81.82%. In Experiment-III, 6 tadpoles died before as the ratio of the number of hatchlings to the number of metamorphosis. Thus, the metamorphism rate was eggs successfully fertilized). The eggs going through the 86.05%. first cleavage were treated as successfully fertilized; 3) 2.5 Developmental stages and their characteristics “Metamorphism ratio” (defined as the ratio of the For describing the developmental stages, we used number of froglets to the number of hatched tadpoles). data on development observations in Experiment-II (Tab. For Experiment-I, the fertilized eggs were divided 1). Stages in Experiment-I and III are similar to that in into two batches in the laboratory in Chengdu: i) A small Experiment-II except for a slight difference in the batch (~20 fertilized eggs) that was observed development time course.