2008

Asiatic Herpetological Research, Vol. 11

pp.105–109

Sperm Morphology of Five Rhacophorus (Amphibia: Anura:Rhacophoridae) Species from China
LI MEI QIN1,2, ZHONG HUA ZHENG1,*, JIAN PING JIANG1, FENG XIE1 AND YUN MING MO3
1Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, 610041,China,
2
Postgraduate university of Chinese academy of sciences, Beijing, 100049, China,
3Natural History Museum of Guangxi, Nanning, 530023, China.
*Corresponding author E-mail: zhengzh@cib.ac.cn

Abstract.- Sperm shape and size of five species of the genus Rhacophorus from China were investigated in the presentstudy. Our results reveal the presence of two possible monophyletic lineages: the first is composed of R. chenfui, R.
dugritei and R. omeimontis, has relatively small spermatozoa with a with a coiled head and a thin tail, while the second, composed of R. mutus and R. megacephalus, have spermatozoa that are longer with a straight head and filiform
shape.
Keywords.- Amphibian, Rhacophoridae, Rhacophorus, Polypedates, spermatozoa, morphology.

Introduction
The genus Rhacophorus (sensu lato) is a member of thefamily Rhacophoridae and contains approximately 80species worldwide, 24 of which are found in China (Feiet al., 2005). Rhacophorids are predominantly arboreal,
sharing with basal ranids expanded digital pads andmantellids the intercalary phalangeal elements (Frost etal., 2006). These frogs are distributed in the tropical andsub-tropical regions of eastern and southern Asia; inChina, they inhabit southern areas north to Qinling.
Phylogenetic and taxonomic relationships within thisgenus are still conjectural and controversial. For
instance, the genus Polypedates is valid according to
Frost et al. (2007), but it is absent in Fei et al. (2005).
Furthermore, Frost et al. (2007) placed R. chenfui, R.
dugritei, R. omeimontis in Rhacohporus, while R. mutus
and R. megacephalus were placed in the genus
Polypedates by Fei et al. (2005).
Previous studies have revealed that some morphological characters of the spermatozoa were unique togiven taxa in the Anura (Kuramoto and Joshy, 2000;
Kuramoto, 1996; Zheng et al., 2000a, 2000b, 2002). For
example, the spermatozoa of most Rana species arecharacterized by a cylindrical head and a thin tail, whilespiral and corkscrew-shaped sperm head and waved tailare typical of spermatozoa in the family Megophryidae(Zheng et al., 2002), and fusiform-shaped spermatozoa
are unique to the family Bombinatoridae (Zheng et al.,2000a). These studies make it evident that sperm morphology is not only variable between taxa, but can be
useful for elucidating taxonomic relationships(Kuramoto and Joshy, 2000). In this study, the shape andsize of spermatozoa in Rhacophorus chenfui, R.

© 2008 by Asiatic Herpetological Researchdugritei, R. omeimontis, R. megacephalus and R. mutus
were examined for the purpose of resolving their cryptic
phylogenetic relationships.

Materials and Methods
Collecting localities are listed in Table 1. All specimens
were collected during their breeding seasons, (i.e., Mayto July) from 1998 to 2005.
Frogs were euthanized by inserting a medical needle through the occipital ostium to destroy the spinal
cord. Next, the testes were removed and immediately
fixed with 10% formaldehyde, squashed and macerated
with a clean toothpick; sperm were suspended on slides,
air-dried and stained with acid carmine for 40 seconds.
Slides were examined on a ZEISS Axioplan2 lightmicroscope (LM). Other testes were fixed with 3% glutaraldehyde for about two hours and centrifuged at 3000
rpm for 30 s; the supernatant was discarded and the pellet rinsed with double distilled water. A drop of theresulting sperm suspension was placed on a cover slip,
air-dried, coated with gold, and observed on a JEOL
JSM-5900LV scanning electron microscope (SEM).
Spermatozoan pictures were shot by ZEISSAxioVision 4.0. Sperm length was measured by software ArcView GIS 3.2. Sperm length data were analyzed with SPSS 11.5. The length of sperm head ofRhacophorus chenfui, R. dugritei, R. omeimontis werecalculated by the formulate L=Nlπ (L: the length ofsperm head, π = 3.14, l = the diameter of helix, N = thenumber of turns in the helix).

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Figure 1. Spermatozoa of five tree frogs. a: Rhacophorus chenfui; b: R. dugritei; c: R. omeimontis; d: R. mutus; e: R.
megacephalus; f: detail of the sperm head of R. mutus. A: acrosome; H: head; T: tail.

235

260

230

248.6

225

228.84250
240

220215230.65210230222.04

205

220

202.9

213.1

200
195

210200190
185190Sichuan
YunnanR. mutus

Guangxi

TaipeiR. megacephalus

Figure 2. The total length of spermatozoa in different populations of Rhacophorus mutus and R. megacephalus.
Units: µm.

Results
The spermatozoa of all five species consisted of two portions, the head and the tail. The sperm head ofRhacophorus chenfui (Fig. 1a) was shaped like a coiledspiral, the tail was thin and wavy (shared with characteristic shared with R. dugritei (Fig. 1b) and R. omeimontis(Fig. 1c)), and the slightly-coiled apical section waslikely the acrosome. The shape of the sperm in R. mutus(Fig. 1d) was filiform, consisting of a straight, thickhead and a thin wavy tail. A thinner section (Fig. 1f, seearrow) at the tip of sperm head was likely the acrosome(Fig. 1f, a). Bends and waves could usually be observed
from the midpoint of the sperm.
Spermatozoa measurements are shown in Table 1.
Total sperm length in Rhacophorus chenfui and R.
dugritei is much shorter than those measured for R.
megacephalus and R. mutus; the sperm of R. omeimontis
was of intermediate length.
The sperm head of Rhacophorus dugritei was theshortest and thinnest, and the head of R. omeimontis waslonger than that of R. dugritei, and thinner than those of
all other four species. Head length in R. chenfui was also
relatively short, and it was also slightly thicker than thatof R. dugritei. R. megacephalus had the largest sperm
head among all five species, with the head of R. mutus
being only slightly smaller.
The coiled head of Rhacophorus chenfui, R. omeimontis and R. dugritei were largely similar. However,the sperm head of R. chenfui was longer than the tail ,with the ratio of sperm head to total sperm length being
1:0.86.
Sperm size of Rhacophorus megacephalus and R.
mutus differed remarkably between populations. The
spermatozoa of R. megacephalus in Sichuan population
were longer than those in the Guangxi population, andthose of R. mutus were longer than those in the Yunnan
population compared to the Guangxi population (Table1; Fig. 2).

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Table 1. Sperm measurements of Rhacophorus species.
SpeciesR. chenfui

LocalityE`mei,
SichuanR. dugritei
R. omeimontis

Hongya,SichuanHongya,Sichuan
Hejiang,
SichuanR. megacephalus

N

Tengchong,
YunnanShangsi,Guangxi

20202020

2020

Tengchong,R. mutus

YunnanShangsi,Guangxi

2020

Head length

Tail length

66.35±7.05

57.10±4.36(53.75 %)(46.25 %)59.97±10.67

63.60±16.73(47.59 %)(50.47 %)75.43±8.17107.99±14.12(41.12 %)(58.88 %)104.31±12.36

136.85±9.54(43.25 %)(56.75 %)94.05±15.30

136.60±14.38(40.78 %)(59.22 %)80.33±12.08

141.71±12.00(36.18 %)(63.82 %)95.32±8.96133.52±8.66(41.65 %)(58.35 %)78.22±10.66

124.68±9.81(38.55 %)(61.45 %)Total length

Headwidth

Turns ofhead coil

123.45±8.60

0.76±0.1

8.15±0.59

126.02±10.97

0.52±0.1

5.90±0.72

183.42±17.00 0.50±0.12 6.80±0.62241.16±11.61

0.89±0.15—

230.65±20.20 0.85±0.04—

222.04±11.01

0.83±0.09—

228.84±14.01 0.79±0.08—

202.90±11.02—

0.83±0.09

Unit: µm. N: number of spermatozoa;

Discussion
Spermatozoa morphology.- The morphology of thesperm observed here for Rhacophorus chenfui, R.
dugritei and R. omeimontis appeared to be very similar
to that reported by Mizuhira et al. (1986) for R. arboreus
and R. schlegelii, as well as that reported by Kuramoto(1996) for R. viridis amamiensis, R. owstoni and R.
moltrechti. The sperm heads of latter species, however,while coiled, did not appear to have the two twisted subcoils characteristic of the former three species.
Conversely, the shape of the spermatozoa in R. mutusand R. megacephalus were very similar to those seen in
Polypedates leucomystax, P. megacephalus (Kuramoto,1996) and P. maculatus (Kuramoto and Joshy, 2000),
where the head is linear (not coiled) and no fibers in thetail. Based on the variation of sperm morphology
observed here, the five species examined can be divided
into two groups: tthe first group (consisting of R. chenfui, R. dugritei and R. omeimontis) is characterized by ahelical sperm head and a thin sperm tail, and the second
group (consisting of R. mutus and R. megacephalus) ischaracterized by a thread-like sperm head and a thin tail.
Sperm size differ remarkably between species, also
between populations. The average length of spermato-zoa in Rhacophorus chenfui was 123.45±8.60µm, whilethat of R. megacephalus was 230.25±19.81 µm, a difference of statistical significance (P < 0.01, n = 20, oneway analysis of variance). In the different populations ofR. megacephalus, total spermatozoa length in theSichuan, Yunnan, Guangxi and Taipei populations was
241.16 µm, 230.65 µm, 222.04 µm, 213.1 µm(Kuramoto, 1996), respectively, illustrating a reductionin length towards the Pacific Ocean (Fig. 3). This phenomenon was also observed in R. mutus (average length
228.84 µm in the Yunnan population and 202.9 µm in
the Guangxi population).
The implications for taxonomy from spermatological
data.- On the basis of skeletal characters, Liem (1970)
placed Rhacophorus dugritei and R. omeimontis into thegenus Polypedates. Uncertain as to the limits of these
two genera, Fei (1999) and Fei et al. (2005) tentatively
treated all Polypedates as Rhacophorus. Based onmolecular evidence, however, Frost et al. (2007) placed
R. chenfui, R. dugritei and R. omeimontis in
Rhacohporus, and placed R. mutus and R. megacephalus
in Polypedates. In view of the spermatological data presented here, the latter hypothesis presented by Frost etal. (2007) appears to be best supported.

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Figure 3. Map of collection localities.

Acknowledgments
This research was funded by grants from National
Natural Science Foundation of China, No.30570195

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Submitted: 07 December 2006
Accepted: 13 March 2007