Current Herpetology 22 (2): 73-90, December 2003 (C) 2003by The Herpetological Society of Japan Normal Development of Microhyla ornata: The First Description of the Complete Embryonic and Larval Stages for the Microhylid Frogs (Amphibia: Anura) SATOSHI SHIMIZU AND HIDETOSHI OTA* Tropical Biosphere Research Center, University of the Ryukyus, Senbaru 1, Nishihara, Okinawa 903-0213, JAPAN Abstract: Developmental changes in external morphological characters were examined for the whole embryonic-larval period of Microhyla ornata from Okinawajima Island, Ryukyu Archipelago, Japan. By considering the extent of morphological changes observed and compatibility with standard develop- mental tables hitherto proposed for other anuran groups, a developmental table consisting of 45 developmental stages was proposed for this species. Eight developmental terms, each consisting of several successive stages, were also defined-Cleavage-Blastula [stages 1-10], Gastrula [stages 11-14], Neurula [stages 15-18], Tail bud [stages 19-21], External gill [stages 22-28], Hind limb bud [stages 29-33], Hind limb formation [stages 34-41], and Metamorphosis [stages 42-45]). From previously described conspecific samples from other localities, our sample exhibited appreciable differences in the egg diameter, timing of pigmentaion at the position of the stomodium, and a few other tadpole characters. Key words: Developmental table; External characters; Microhyla ornata; Micro- hylidae; Anuran amphibians; Okinawajima Island INTRODUCTION matic morphological changes (e.g., tail forma- tion, perforation and closure of the spiracle, One of the prominent life history character- limb formation, tail reduction). Appropriate istics common to most living amphibians is staging of the larval period is, therefore, the presence of an aquatic larval period, which fundamental to various life history studies of immediately follows the initial embryonic amphibians. Indeed, tables of developmental development after fertilization and ends with stages that describe morphological changes the completion of metamorphosis (Duellman during the larval period have been proposed and Trueb, 1994; Altig and McDiarmid, for quite a few amphibian taxa (see Gosner, 1999a). During the larval period, amphibians, [1960], Nieuwkoop and Faber [1967], Fox anurans in particular, exhibit a series of dra- [1983], Iwasawa and Futagami [1992], McDi- armid and Altig [1999], and other works cited *Correspondingauthor. Tel: +81-98-895-8937; therein). Nevertheless, such developmental Fax: +81-98-895-8966. tables are not yet available at all for several E-mail:[email protected] major groups of anurans. 74 Current Herpetol. 22 (2) 2003 The family Microhylidae is a well defined ings depicting morphological features of the monophyletic group of anurans (Ford and embryonic and larval stages given therein suf- Cannatella, 1993; Haas, 2003), and is com- fered extensive simplification and thus were posed of more than 60 genera and 310 species not very informative. from tropical, subtropical, and partially tem- perate regions of Asia, northern Australia, MATERIALS AND METHODS sub-Saharan Africa (including Madagascar), and the New World (Frost, 1985; Duellman, Three pairs of Microhyla ornata in amplexus 1993). Because of the presence of several were collected from Kita-nakagusuku Village prominent morphological and ecological on Okinawajima Island in June 2003. These features (Altig and Johnston, 1989; Altig and pairs were brought back to our laboratory McDiarmid, 1999a, b; Haas, 2003), tadpoles (also located on Okinawajima), where they of the Microhylidae have been a subject of were separately housed in a plastic container intensive evolutionary, systematic, and ana- (20×35cm in area, 26cm in height) filled with tomical studies (e.g., Starrett, 1973; Wasser- 101 of tap water (25±1C, left for a few days sug, 1980, 1984, 1989; Wassersug and Pyburn, in advance). Eggs deposited were also kept in 1987; Khan, 2000). Nevertheless, complete the same container under the same conditions tables of normal larval development are not until they had grown to larvae with complete yet available for any member of this family spiracles. Then they were divided into several (Fox, 1983; McDiarmid and Altig, 1999). groups, each consisting of 100 or fewer individ- Therefore, we have prepared a developmen- uals. Each group was housed separately in a tal table, covering all embryonic and larval container of the same size and amount of periods, for Microhyla ornata, an Oriental water as above. All containers with larvae microhylid species broadly distributed from were left outside in shade so as to let the larvae South and Southeast Asia to southern China grow under natural temperature. and the Ryukyu Archipelago (Frost, 1985; For each container, 2 of water was removed Zhao and Adler, 1993: but see Kuramoto and the same amount of tap water (prepared [1976, 1987], Dubois [1987], Maeda and and left for a few days in advance) was added Matsui [1989], and Khan [2000] for the possi- every second day. A mixture of minced and bility of its being a composite of more than dried shrimps and vegetables, sold as diet for one species). For this species, a few previous aquarium fish, was used to feed the tadpoles. studies yielded data on some aspects of repro- One gram of the mixture, further milled into duction and life history, such as those on the powder, was scattered over the surface of water reproductive season (Kuramoto, 1973; Schleich in each container after every change of water. and Kastle, 2002), fecundity parameters Water temperature was measured to nearest (Matsui and Ota, 1984; Schleich and Kastle, 0.1C with a digital thermometer between 2002), dietary effect on larval growth (Dash 16:00 and 17:00 every day. and Dei, 1998), larval morphology at certain Embryonic and larval developmental stages developmental stages (Chou and Lin, 1997; were defined so as to be as compatible as Khan, 2000; Schleich and Kastle, 2002; etc.), possible with those proposed by previous and size at metamorphosis (Maeda and Mat- authors (Gosner [1961] for anurans in general, sui, 1989; Dash and Dei, 1998). With respect and Iwasawa and Futagami [1992] for Hyla to its embryonic and larval development, Liu japonica). For the description of each of the et al. (1996) provided some descriptions on the stages from fertilization (stage 1) to the basis of materials from continental China. completion of the spiracle (stage 27), five These descriptions were, however, very brief developing eggs or embryos were sampled at and were limited to the period from fertiliza- random. They were measured for the total tion to spiracle completion. Moreover, draw- length, fixed in 8% formalin, and subjected to SHIMIZU & OTA-DEVELOPMENT OF MICROHYLA ORNATA 75 detailed observations under a dissecting micro- pairs in two times. In the latter case, the scope. For each of the stages subsequent to second oviposition commenced approximately stage 27, at least five larvae were also ten minutes after the initial oviposition. The randomly sampled. Each larva was put in a total number of eggs laid by the three pairs was shallow dish filled with water, cooled until 220, 386, and 910. At deposition, the eggs motionless by adding a piece of ice to the formed a single layer on the water surface like water, measured for the total length (TOL), many other species of the Microhylidae (Haas, body length (BL) and tail length (TAL), and 2003). From fertilization to the completion of subjected to detailed observations. metamorphosis took 40 days. All measurements were taken to nearest To document the series of changes in 0.01mm by a micrometer attached to a dis- external morphological characters during the secting microscope. Drawings were prepared embryonic and larval development, a total of using camera-lucida. 45 developmental stages were defined (Table 1, Fig. 1). Following Iwasawa and Futagami RESULTS (1992), these stages were grouped into eight terms as follows: Cleavage-blastula, consisting One of the male-female pairs deposited all of stages 1-10; Gastrula, stages 11-14; Neu- eggs at one time after being housed in a rula, stages 15-18; Tail bud, stages 19-21; container, whereas each of the remaining two External gill stages 22-28; Hind limb bud, TABLE 1. Stages of normal development of Microhyla ornata. Stages 1-28 were obtained during devel- opment at 25±1C; Stages 29-45 at 19-26C (natural outdoor temperatures of Okinawajima Island). Numerals with or without lower case letters in parentheses and brackets following stage numbers indicate numbers of grossly corresponding stages of Gosner (1960) and Iwasawa and Futagami (1992), respectively. 76 Current Herpetol. 22 (2) 2003 TABLE 1. -Extended. SHIMIZU & OTA-DEVELOPMENT OF MICROHYLA ORNATA 77 TABLE 1. -Extended. 78 Current Herpetol. 22 (2) 2003 TABLE 1. -Extended. SHIMIZU & OTA-DEVELOPMENT OF MICROHYLA ORNATA 79 FIG. 1. Sketches of embryos (including developing eggs) and larvae of Microhyla ornata from Okinawajima. Numeral beneath each drawing or each group of drawings corresponds to that of the develop- mental stage defined in Table 1. 80 Current Herpetol. 22 (2) 2003 FIG. 1. -Extended. SHIMIZU & OTA-DEVELOPMENT OF MICROHYLA ORNATA 81 FIG. 1. -Extended. 82 Current Herpetol. 22 (2) 2003 FIG. 1. -Extended. SHIMIZU & OTA-DEVELOPMENT OF MICROHYLA ORNATA 83 FIG. 1. -Extended. 84 Current Herpetol. 22 (2) 2003 FIG. 1. -Extended. SHIMIZU & OTA-DEVELOPMENT OF MICROITIYLA ORNATA 85 FIG. 1. -Extended. 86 Current Herpetol. 22 (2) 2003 FIG. 1. -Extended. SHIMIZU & OTA-DEVELOPMENT OF MICROHYLA ORNATA 87 FIG. 1. -Extended. 88 Current Herpetol. 22 (2) 2003 stages 29-33; Hind limb formation, stages 34- continental Chinese sample (Liu et al., 1996). 41; and Metamorphosis, stages 42-45. With respect to tadpole morphology, our Throughout the embryonic and larval devel- sample differs from the Thailand sample in opment, TOL varied considerably (Table 1). having a silvery sheen in the belly as a result of The value was quite stable from fertilization the appearance of iridescent cells at the Com- (stage 1) to the end of the Gastrula (stage 14). pletion of spiracle stage (stage 28) (Khan, Then it started to increase gradually with the 2000).