65 (1): 117 – 130 © Senckenberg Gesellschaft für Naturforschung, 2015. 4.5.2015 Development of the bony skeleton in the Taiwan salamander, Hynobius formosanus Maki, 1922 (Caudata: Hynobiidae): Heterochronies and reductions Anna B. Vassilieva 1 *, June-Shiang Lai 2, Shang-Fang Yang 2, Yu-Hao Chang 1 & Nikolay A. Poyarkov, Jr. 1 1 Department of Vertebrate Zoology, Biological Faculty, Lomonosov Moscow State University, Leninskiye Gory, GSP-1, Moscow 119991, Russia — 2 Department of Life Science, National Taiwan Normal University, 88, Sec. 4 Tingchou Rd., Taipei 11677, Taiwan, R.O.C. — *Cor- responding author; vassil.anna(at)gmail.com Accepted 19.ii.2015. Published online at www.senckenberg.de / vertebrate-zoology on 4.v.2015. Abstract The development of the bony skeleton in a partially embryonized lotic-breeding salamander Hynobius formosanus is studied using the ontogenetic series from late embryos to postmetamorphic juveniles and adult specimen. Early stages of skull development in this spe- cies are compared with the early cranial ontogeny in two non-embryonized lentic-breeding species H. lichenatus and H. nigrescens. The obtained results show that skeletal development distinguishes H. formosanus from other hynobiids by a set of important features: 1) the reduction of provisory ossifications (complete absence of palatine and reduced state of coronoid), 2) alteration of a typical sequence of ossification appearance, namely, the delayed formation of vomer and coronoid, and 3) the absence of a separate ossification center of a lacrimal and formation of a single prefrontolacrimal. These unique osteological characters in H. formosanus are admittedly connected with specific traits of its life history, including partial embryonization, endogenous feeding until the end of metamorphosis and relatively short larval period. Key words Hynobiid salamanders, osteology, cranial ontogeny, embryonization, endogenous feeding, evolution. Introduction The development of the skeleton, especially the skull, (Ple tho dontidae) lacking larval period (WAKE, 1966; in urodelan amphibians is strongly affected by the life ALBERCH, 1987; WAKE & HANKEN, 1996; EHMCKE & CLE­ history. Thus, it is widely known that paedomorphic MEN, 2000; MARKS, 2000; DULCEY CALA et al., 2009). The salamanders which retain larval morphology in the adult influence of life history on skull development is studied state do not complete metamorphic remodeling of the in more detail in the plethodontids which embrace pae- skull. They display diverse juvenile traits in their cranial domorphic, metamorphosing and highly embryonized di- bone composition and arrangement, as well as in their rect-developing species (WAKE, 1966; MUTZ & CLEMEN, dentition and hyobranchium; the latest stages of skele- 1992; ROSE, 1995; 1996; MARKS, 2000). Although direct tal development are usually omitted in these urodelans developers are not encountered among other urode- (see ROSE, 2003 for review; IvanovIć et al., 2014). In lan families, many salamander species display a cer- contrast, some features of early skeletal ontogeny are tain degree of the embryonization of their ontogeny. omitted in the direct-developing lungless salamanders Embryonization implies the incorporation of larval stag- ISSN 1864-5755 117 Vassilieva, A.B. et al.: Development of the bony skeleton in Hynobius formosanus Maki, 1922 es into the intraoval development (MATSUDA, 1987) and streams along the road from Lushan to Songyuan and may lead to the direct development without free larval Tienchi mountain shelter on the slopes of Nenggao- stage. Caudate amphibians with partial embryoniza- Shan Mt., Renai Township, Nantou County, Taiwan tion have relatively larger eggs with abundant yolk re- (24.0471° N; 121.2564° E; 2576 m a.s.l., egg sacs col- serves, longer embryonic period and their larvae hatch at lected on 13 February 2008). The other group of clutches more advanced stages compared with non-embryonized was collected on 15 June 2009 by Nikolay A. Poyarkov, species. Apart from some plethodontids, partially em- Yu-Hao Chang, Yong-Cheng Huang and Li-Yi Chang bryonized ontogeny is characteristic, for example, for in a small intermittent mountain stream on the slopes Amphiuma means (Amphiumidae) (GUNZBUrgER, 2003), of Pintian-Shan near the mountain trail from Wuling Rhya cotriton spp. (Rhyacotritonidae) (WORTHINGTON & to Shyue-Shan in Jianshi Township, Hsinchu County, WAKE, 1971; KArrAKER, 1999), Chioglossa lusita ni ca Taiwan (24.4324° N; 121.2754° E; 3130 m a.s.l.). After and Mertensiella caucasica (Salamandridae) (TAR K HNI­ collection and transportation, the egg sacs were placed SHVILI & SERBINOVA, 1993; 1997; SEQUEIRA et al., 2003). under laboratory conditions in Moscow State University The Hy nobiidae also include species, which fit cer- (2008) and in National Taiwan Normal University (2009). tain crite ria of embryonization, for example, the Asian Eggs in all egg sacs collected were counted, dia meters clawed sa la manders Onychodactylus spp. (REGEL & EP­ of eggs at the earliest stages of cleavage (zygote / two STEIN, 1975; IWASAWA & KERA, 1980; AKITA & MIYAZAKI, blastomeres) were measured with a digital caliper to the 1991; SER BI NOVA & SOLKIN, 1992; PARK, 2005; POYARKOV nearest 0.01 mm. In total, 12 and 7 clutches were collect- et al., 2012) and some lotic-breeding species of the ed in 2008 and 2009, respectively. Embryos and larvae genus Hyno bius, for example H. kimurae (MISAWA & were reared in the aquaria with conditioned highly aer- MATSUI, 1997a; AKITA, 2001) and especially H. formosa­ ated tap water at the temperature 10 °C (NTNU labora- nus and H. sonani inhabiting the mountain streams in the tory) or 14 – 16 °C (MSU laboratory) until the completion highlands of Taiwan (KAKEGAWA et al., 1989). Previous of metamorphosis. Metamorphosed juveniles from the studies revealed that skeletal ontogeny in embryonized clutches obtained in 2009 were kept for one year in plas- hyno biids displays, in particular, certain heterochronic tic containers (15 × 10 cm) with wet sponges and shelters shifts in the development of the skull bones and the re- on the bottom and fed ad libitum with Dro so phila sp. duction of those bones which normally develop in larvae (NTNU laboratory). An adult specimen of H. for mo sanus and disappear during metamorphosis (MISAWA & MATSUI, (female, TWN-09 H-005) used in the study was collected 1997b; SMIRNOV & VASSILIEVA, 2002; VASSILIEVA et al., in the same site as the clutches in February 2009 and pre- 2013). Among other embryonized Hynobius species the served in 75 % ethanol. The late embryos (with differenti- Tai wan salamander H. formosanus is of special interest ated limbs), larvae, and posmetamorphic ju ve niles were for the morphological study because this species displays regularly euthanized at different stages and fixed in 10% certain unique life history traits. Namely, H. formosanus neutral-buffered formalin. In total, 50 specimens of H. has a remarkably short larval period (LUE et al., 1982; formosanus were used in the present study. KAKEGAWA et al., 1989; LUE & CHUANG, 1992) and is the Additionally, for comparison, we examined series of only hynobiid known to date which may complete the early larvae of two lentic Hynobius species, the Tohoku metamor phosis relying only on its massive yolk reserves salamander H. lichenatus and the Japanese black sala- without exogenous feeding (KAKEGAWA et al., 1989; our mander H. nigrescens (11 and 8 specimens, respective- data). Some preliminary data on H. formosanus develop- ly), kindly provided by Dr. Tadashi Nakazato (Nakadai ment reported by us (CHANG et al., 2009) indicate that the Junior High School, Tokyo, Japan). The egg sacs of both specific life history traits in this species apparently affect species were collected in a small natural pond on slopes its cranial ontogeny. Herein we present the detailed de- of Fuji-san Mt., Yumotoshiobara, Tochigi Prefecture scription of the skeletal development in H. for mo sanus (36.9555° N; 139.7871° E; 1120 m a.s.l.). After hatch- and compare it with available data on the development in ing, larvae were reared in the laboratory of Nakadai other hynobiids. Junior High School (Itabashi, Itabashi-ku, Tokyo) in plastic containers 15 × 10 cm filled with aerated tap wa- ter until several posthatching days and preserved in 75% Materials and Methods ethanol prior to the beginning of exogenous feeding. In all specimens, the snout-vent length (SVL) and to- tal length (TL) were measured using a digital caliper to Skeletal morphology and development of Hynobius the nearest 0.1 mm; all numeric parameters are given as formosanus were examined in an ontogenetic series of mean ± SD. To facilitate the interspecific comparison of late embryos, larvae, and postmetamorphic individuals the developmental features, H. formosanus embryos and obtained from the eggs collected in the natural breeding larvae were staged according to the tables of normal de- sites. The clutches (egg sacs) were collected in two lo- velopment of both Onychodactylus japonicus (IWASAWA calities in central Taiwan. In January and February 2008, & KERA, 1980) and Hynobius nigrescens (IWASAWA & H. formosanus reproduction was observed by Nikolay A. YAMASHITA, 1991), omitting features connected with the Poyarkov and Yu-Hao Chang in road-side ditches made development of V toe, claws, and balancers. In the chap- of stones and filled with water from small springs and ter “Results” stages of H. formosanus are given in a 118 VERTEBRATE ZOOLOGY — 65 (1) 2015 Table 1. Specimens