Asian Herpetological Research 2017, 8(1): 22–26 ORIGINAL ARTICLE DOI: 10.16373/j.cnki.ahr.160036

Intraspecific Polymorphism of Rice , multistriata (Anura: Ranidae), in Lingnan, Huangshan, China

Diancheng YANG1,2#, Lifang PENG1,2, Jingcheng XU1 and Song HUANG1#*

1 College of Life and Environment Sciences, Huangshan University, Huangshan 245021, China 2 School of Sciences, Tibet University, Lahsa 850000, China

Abstract Many anuran exhibit striking color and/or pattern polymorphisms. We sampled 599 adult rice , Fejervarya multistriata, from a rice paddy in the periphery of the Lingnan Nature Reserve (Huangshan, Anhui). We recognized ten morphs based on three relatively stable and distinct morphological characters: dorsal ground color (green or gray), middorsal stripe (absence, wide, or narrow), occipital rusty spot (absence or presence). The grey individuals are more common and larger than the green individuals (frequency 88.1 vs. 11.9%, respectively) and (SVL 39.4–36.6 vs. 35.7–34.6 mm, respectively). There is a higher proportion of individuals with a middorsal stripe (59.7%) while absence of the occipital rusty spot is more common in the population (82.3%). We expect additional studies in different habitat types would help build our understanding of the relationship between color polymorphism and habitat, geography, and climate. Keywords dorsal ground color, middorsal stripe, occipital rusty spot, frequency, body size

1. Introduction sampled F. multistriata from a rice paddy near Lingnan Nature Reserve (Huangshan, Anhui), classified each frog Intraspecific color polymorphism (hereafter CP) is found based on several morphological characters related to CP widely in nature. In anurans, at least 225 different species and sequenced the DNA barcoding CO1 gene for each are known to be polymorphic (Hoffman and Blouin, described morph. 2000), showing discrete variation in adult coloration, such as dorsal ground color and state (presence or absence) of 2. Materials and Methods stripes and spots. Researchers have treated these color or pattern variants as true polymorphisms (Ford, 1975). For All sampling and procedures involving live frogs example, three color morphs, reddish-brown, gray, and were conducted in accordance with the Wild green occur in populations of Pseudacris ornata (Hylidae) Protection Law of the People’s Republic of China and in north Florida (Travis and Trexler, 1984), whereas Hyla approved by the Ethics Committee at Huangshan regilla shows variation in terms of presence/absence of University. dorsal stripes as well as an array of body colors (Resnick The sampling site is a rice paddy surrounded by and Jameson, 1963). mountains, about 9000 m2, located in the periphery The rice frog Fejervarya multistriata is one of the most of the Lingnan Nature Reserve (Huangshan, Anhui) widely distributed anuran species in China and South (Figure 1). A total of 599 samples of adult F. multistriata Asia. Xiong et al. (2011) investigated color spot diversity were collected on 27 April 2013 in about 3 hours within F. multistriata in a rice paddy environment and transported back to the laboratory at Huangshan in Maolan, Guizhou, China. To further understand University. intraspecific color polymorphism in this species, we The color morphs of the F. multistriata were classified by three relatively stable and distinctive morphological # Both authors contributed equally to this paper. * Corresponding author: Prof. Song HUANG, from Huangshan Univer- characters: dorsal ground color (green or gray), middorsal sity, with his research focusing on ophiology, molecular ecology and stripe (absence, wide, or narrow), and occipital rusty biogeography. E-mail: [email protected] spot (absence or presence). The individual number of Received: 16 May 2016 Accepted: 12 November 2016 every morph was counted, and the frequencies (%) were No. 1 Diancheng YANG et al. Intraspecific Polymorphism of Rice Frog 23

Figure 1 Sampling site of Fejervarya multistriata used in this study. calculated. In order to assess morphometric differences 3. Results among morphs, 11 characters were measured with dial calipers to the nearest 0.1 mm, after Fei et al. (2009). According to the three relatively stable and distinctive These characters are: (1) snout-vent length (SVL); (2) morphological characters, 12 phenotypes of F. head length (HL); (3) head width (HW); (4) snout length multistriata could possibly be observed. However, only (SL); (5) internasal space (INS); (6) diameter of eye 10 morphs were observed (Figure 2). The classification of (ED); (7) diameter of tympanum (TD); (8) length of these 10 morphs is listed in Table 1. lower arm and hand (LAHL); (9) hindlimb length or leg Significant differences were found in the frequency of length (HLL); (10) tibia length (TL); (11) foot length occurrence among the 10 morphs (Table 1). The morphs (FL). To standardize different over-all body sizes among of GyAbAb (dorsal ground color grey, middorsal stripe specimens, we converted each value to a percentage ratio absent and occipital rusty spot absent) and GyNrAb to SVL for comparisons of the different morphs (Djong (dorsal ground color grey, middorsal stripe narrow and et al., 2007). Independent Sample Test was used to occipital rusty spot absent) are common (26.4%, 36.5%), establish the significance of SVL between dorsal ground whereas the patterns of GnAbPr (dorsal ground color colors (green or gray) at a confidence level of 95%. green, middorsal stripe absence and occipital rusty spot Ten morphs were defined (see below) and a male and absence) and GnWdAb (dorsal ground color green, a female individual of each morph were euthanized. middorsal stripe wide and occipital rusty spot absence) Their fresh liver tissues were removed and immediately are rare (1.2%, 0.7%). preserved in 95% ethanol for molecular studies. These 20 The frequency of dorsal ground color grey (88.1%) specimens (Collection number: HS12001- HS12010) were is more common than green (11.9%); presence of the preserved and deposited in the Museum of Huangshan middorsal stripe (59.7%) is higher than absence (40.3%); University. Non euthanized frogs were released at point and absence of the occipital rusty spot (82.3%) is more of capture three days post capture. The mitochondrial frequent than presence (17.7%). DNA COI sequence was obtained by polymerase chain In relation to body size, the largest frogs typically reaction (PCR) and direct sequencing using the primers has the GyAbAb morph (SVL average 39.4±0.43 mm), and methods described in Che et al. (2011). while the morph with the smallest frogs is GnNrAb (SVL 24 Asian Herpetological Research Vol. 8

Figure 2 General view of ten morphs of Fejervarya multistriata of rice paddy in Lingnan, Huangshan, China. A: GyAbAb; C: GyNrAb; E: GyWdAb; G: GnAbAb; I: GnNrAb; B: GyAbPr; D: GyNrPr; F: GyWdPr; H: GnAbPr; J: GnWdAb. Note: First two letters, Gy: grey, Gn: green; middle two letters represent middorsal strip, Ab: absence, Nr: narrow, Wd: wide; last two represent occipital rusty spot, Ab: absence, Pr: presence. average 34.6 ± 0.30 mm). Generally, grey individuals the 20 specimens shared a single haplotype (GenBank (SVL 39.4–36.6 mm) are larger than green individuals accession number: KP901087). This fact confirms (SVL 35.7–34.6 mm) (p < 0.05, F = 1.145, t = 2.373, df that the differences in CP are the result of intraspecific = 89). All the SVL and other characters in ratios (/SVL) polymorphism, and the 10 morphs are conspecific. among all morphs (Table 2) agree with the description of this species in Fei et al. (2009). 4. Discussion We obtained a 579 bp segment of the CO1 gene for 20 specimens (10 morphs from both sexes). All In the present investigation, some other characters of

Table 1 Ten morphs and their frequency of Fejervarya multistriata collected from a rice paddy in Lingnan, Huangshan, China based on three characters.

Type of Ground color Middorsal stripe Occipital rusty spot Individual polymorphism Gray Green Absence Wide Narrow Absence Presence Number (abbreviation) (Gy) (Gn) (Ab) (Wd) Nr) (Ab) (Pr) (n, %) GyAbAb √ √ √ 158, 26.4%

GyAbPr √ √ √ 42, 7.0%

GyNrAb √ √ √ 219, 36.5%

GyNrPr √ √ √ 37, 6.2%

GyWdAb √ √ √ 52, 8.7%

GyWdPr √ √ √ 20, 3.3%

GnAbAb √ √ √ 34, 5.7%

GnAbPr √ √ √ 7, 1.2%

GnNrAb √ √ √ 26, 4.3%

GnWdAb √ √ √ 4, 0.7%

Note: n: number of specimens; %: frequency of each morph. No. 1 Diancheng YANG et al. Intraspecific Polymorphism of Rice Frog 25

Table 2 Comparison of 10 morphs of adult of snout-vent length (SVL) (mean) and ratios (medians) of measurements.

Morphometry (n) SVL (mm) HL/SVL HW/SVL SL/SVL INS/SVL ED/SVL TD/SVL LAHL/SVL HLL/SVL TL/SVL FL/SVL

GyAbAb (10) 39.4 ± 0.43 0.32 0.34 0.13 0.08 0.10 0.07 0.38 1.42 0.40 0.50 GyAbPr (10) 37.0 ± 0.51 0.37 0.34 0.14 0.08 0.11 0.07 0.37 1.39 0.46 0.48 GyNrAb (10) 36.6 ± 0.32 0.37 0.35 0.14 0.08 0.12 0.07 0.38 1.45 0.48 0.47

GyNrPr (10) 36.8 ± 0.33 0.34 0.36 0.14 0.08 0.10 0.07 0.38 1.41 0.42 0.50

GyWdAb (10) 38.6 ± 0.07 0.38 0.34 0.14 0.08 0.12 0.08 0.37 1.41 0.46 0.49

GyWdPr (10) 36.6 ± 0.32 0.38 0.36 0.15 0.09 0.12 0.07 0.38 1.45 0.46 0.48

GnAbAb (10) 35.7 ± 0.46 0.42 0.35 0.15 0.08 0.11 0.08 0.38 1.46 0.43 0.48

GnAbPr (7) 35.2 ± 0.37 0.37 0.34 0.15 0.08 0.12 0.07 0.38 1.51 0.50 0.48

GnNrAb (10) 34.6 ± 0.30 0.37 0.34 0.16 0.08 0.11 0.07 0.39 1.48 0.46 0.48

GnWdAb (4) 34.8 ± 0.37 0.36 0.32 0.16 0.09 0.11 0.07 0.36 1.45 0.43 0.49

(n): number of individual for measurement; SVL: snout-vent length; HL: head length; HW: head width; SL: snout length; NS: internasal space; ED: diameter of eye; TD: diameter of tympanum; LAHL: length of lower arm and hand; HLL: hindlimb length or leg length; TL: tibia length; FL: foot length

CP were not used in the study, e.g. number and situation the level of polymorphism, characters of morph(s) and the of spot, slight difference of color of spots, etc. These habitat, geography and climate. characters are considered random individual differences that are too unreliable to be used to classify the morphs. Acknowledgements This project was funded by the The three characters adopted for this study are relatively National Natural Science Foundation of China (NSFC stable, regular and clear, and easily distinguished among 31071891, 31471968), Natural Science Foundation of morphs (Stewart, 1967; Schiotz, 1975; Glaw and Vinces, the Education Department of Anhui Province of China 1992; O’Neill and Beard, 2010). (No. KJ2014A244), and the Zoology and Conservation Many animals possess protective coloration to reduce Biology Research Innovation Group of Huangshan the risk of predator detection by camouflage (Stevens, University. We thank Professor Christopher L. Parkinson 2007; Endler, 1978, 1984). Grey and green dominate the (University of Central Florida, USA) for helpful primary hue of the rice paddy used in this study. Grey, the comments and correction of English on a revision of this soil color, is the main color during four seasons. Green, manuscript. We thank Jinmin CHEN, Baolin ZHANG, the color of the crops, is the second major color in late Xiaoyu SUN, Tengfeng NI, Tianqi ZHOU, Xin ZHENG spring and whole summer to early autumn. The dorsal and Junsheng CUI (Huangshan University) for their help ground color of F. multistriata captured in the present in field work and morphometry. study matches with the two main colors of the rice paddy. Individuals with protective coloration are more likely to Reference escape predator attacks. Che J., Chen H. M., Yang J. X., Jin J. Q., Jiang K., Yuan Z. Y., The rice frog is one of the dominant in Murphy R. W., Zhang Y. P. 2011. Universal COI primers for its distribution range (Fei et al., 2009), and they are DNA barcoding amphibians. Mol Ecol Resour, 12(2): 247–258 a common prey for many animals. The tadpoles are Djong T. H., Matsui M., Kuramoto M., Belabut D. M., Sen Y. eaten by fish and birds. Neonates and adults are eaten H., Nishioka M., Sumida M. 2007. Morphological divergence, by many predators including some birds, rodents and reproductive isolating mechanism, and molecular phylogenetic snakes. The predator pressure is thus considered as relationships among , Malaysia, and Japan populations evolutionary dynamics which promote and maintain the of the Fejervarya limnocharis complex (Anura, Ranidae). Zool Sci, 24: 1197–1212 CP polymorphism of F. multistriata. Endler J. A. 1978. A predator’s view of animal color patterns. Evol More studies of polymorphism of F. multistriata at Biol, 11: 319–364 different types of habitat in its whole distribution range Endler J. A. 1984. Progressive background matching in moths, and are needed to further understand the relationship between a quantitative measure of crypsis. Biol J Linn Soc, 22: 187–231 26 Asian Herpetological Research Vol. 8

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