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Progress in Natural Science 19 (2009) 1403–1408 www.elsevier.com/locate/pnsc Short communication Morphological evolution from aquatic to terrestrial in the genus Oreolalax (Amphibia, Anura, )

Gang Wei a,b,c, Bin Wang c, Ning Xu b, Zizhong Li a, Jianping Jiang c,*

a Institute of Entomology, Guizhou University, Guiyang 550025, China b Centre of Ecology, Guiyang College, Guiyang 550005, China c Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China

Received 18 January 2009; received in revised form 23 February 2009; accepted 23 February 2009

Abstract

A phylogeny of 17 species in the genus Oreolalax is reconstructed based on 21 morphological characters from adult specimens, skel- eton specimens, tadpoles and eggs. Four species groups are recognized, of which the O. rugosus species group is the most primitive, the O. weigoldi species group is the second, the O. omeimontis species group is the third and the O. pingii species group is the most recently diversified. Based on the evolutional tendency of the morphological characters on the phylogenetic tree, it is proposed that the evolution of tympanum, tympanic annulus, columella, spoon-like cartilage and the web between toes reflect the habit changes from aquatic to ter- restrial. Thus, Oreolalax is regarded as one important representative genus to study further the evolution of morphological characters from aquatic to terrestrial. Ó 2009 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

Keywords: Aquatic; Terrestrial; Morphology; Evolution; Oreolalax

1. Introduction evolution, geographic distribution and differentiation of high-altitude pelobatid toads (=Megophryidae) in Asia [5]. It is well known that are transitional verte- Megophryid genus Oreolalax Myers and Leviton, 1962, just brates from aquatic to terrestrial. There are more than occurred in this area and mainly distributed in the middle- 6000 species in the class Amphibia in the world [1]. However, southern part of the Hengduan Mountains and adjacent in this group with so high a biodiversity, the evolutionary areas, restrained by the Qinghai-Tibet Plateau in the west tendency of morphological characters from aquatic [6]. Therefore, genus Oreolalax could be used as an example to terrestrial animals based on phylogeny remains unclear. evolving from aquatic to terrestrial in vertebrates around the The southern part of the Qinghai-Tibet Plateau was ocean Hengduan Mountains. The validity of Oreolalax was reeval- during the Paleocene Epoch [2]. The Indian block colliding uated by Fei et al. [7] and has widely been accepted [1,6,8– with Eurasia led to a large-scale Qinghai-Tibet Plateau as 10], which has only 17 species and could be classified into well as adjacent areas (including the Hengduan Mountains) four species groups: the O. rugosus species group, the O. wei- [3]. The historically stable environment of the Hengduan goldi species group, the O. omeimontis species group and the Mountains was in high favor for maintaining biodiversity O. pingii species group [6]. This study aims to reconstruct [4]. The Hengduan Mountains might be the centre of origin, phylogeny within the genus based on morphological charac- ters, study relationships between morphological transfor- * Corresponding author. Tel.: +86 13668280192. mation and environment change and further investigate E-mail address: [email protected] (J. Jiang). the origin and differentiation centre of the genus.

1002-0071/$ - see front matter Ó 2009 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved. doi:10.1016/j.pnsc.2009.02.010 1404 G. Wei et al. / Progress in Natural Science 19 (2009) 1403–1408

2. Materials and methods Table 2 Morphological characters of the genus Oreolalax. 2.1. Specimens Character number Character state 01 23 Our analyses included 1307 morphological specimens of 1. Spoon-like cartilage Absent Present 17 species and 50 skeleton specimens of 16 species within 2. Columella Short Long Oreolalax (Table 1), of which information of O. weigoldi 3. Post-otic projection of Absent Present was obtained from the description of Ohler and Dubois squamosal [11]. In addition, 44 morphological specimens and 5 skele- 4. Linea musculinae Absent Present 5. Spinal patches on the Not Apparent ton specimens of two species of genus were lower arm apparent selected as the outgroup in our analyses, as Scutiger is a sis- 6. Ratio of spinal patches Large Small (<0.3) ter to Oreolalax based on the results of Delorme and width on the chest to the (>0.4) Dubois [12] and our work (unpublished) on the phylogeny chest width of Megophryidae. All specimens were deposited in the 7. Density of spines on the Small Large (<5 chest (>10 granules/ Chengdu Institute of Biology (CIB), the Chinese Academy granules/ mm2) of Science (CAS). mm2) 8. Spines on both sides of Absent Present 2.2. Cladistic analysis the belly 9. Tympanum Absent Concealment Apparent 10. Ratio of tympanic Tympanic Small (<0.2) large According to the criteria of Ye et al. [13], plesiomorphy annulus to eye diameter annulus (>0.3) (0) or apomorphy (1), or autopomorphic character (2 or 3) absent was determined for the 21 morphological characters used 11. Opening of eustachian Small Large in this study (Table 2). tube The morphological data matrix of the 17 species with 12. Color pattern on belly Present Absent 13. Web between toes Present Absent their character state (Table 3) was used in this phylogenetic 14. Spines in lip margins Present Absent analysis using PAUP version 4.10b [14]. All characters 15. Ratio of the length of Short Long (>0.6) were ordered and equally weighted. The MP tree was fore arm and hand to (<0.6) reconstructed by a heuristic search with 1000 randomly- body length added-sequence replicates. Tree-bisection-reconnection 16. Ratio of tibia length to Short Long (>0.45) body length (<0.45) branches were swapped, and only one parsimonious tree 17. Spines on the back Present Absent was held each time. The clade robustness in the MP tree 18. Color pattern on back Absent Present was assessed by the bootstrap analysis: 1000 replicates, 19. Spines on the metatarsal Absent Present 100 randomly added samples and one tree held at each 20. Labial tooth formula of <4 row 4 row 5 or 6 >6 step. tadpole row row 21. Egg pigment Present Absent

Table 1 Specimen information. 3. Results Species Location Specimens Skeleton Others (China number number We obtained one maximum parsimonious tree for the Province) genus Oreolalax from the parsimony analysis based on 21 Oreolalax popei #25$2 #5$1 Eggs, tadpoles morphological characters. The parameters of this tree were O. omeimontis Sichuan #3$3 #1 Eggs, tadpoles O. major Sichuan #4$2 #1 Eggs, tadpoles as follows: tree length = 60, consistency index (CI) = O. liangbeiensis Sichuan #50$88 #4 Eggs, tadpoles 0.4167 and retention index (RI) = 0.6569. O. pingii Sichuan #168$137 #4 Eggs, tadpoles The maximum parsimonious tree showed that O. shmidti O. schmidti Sichuan #68$4 #4 Eggs, tadpoles was a sister to O. puxiongensis supported by synapomor- O. puxiongensis Sichuan #236$18 #4 Eggs, tadpoles phies 12 (color pattern on belly absent), 13 (web between O. lichuanensis Hubei #71$3 #4 Eggs, tadpoles O. rhodostigmatus Guizhou, #3$2 #1 Eggs, tadpoles toes absent) and 20 (labial tooth formula of tadpole >4 Hubei row). Oreolalax omeimontis was a sister to O. lichuanensis O. chuanbeiensis Sichuan #25$2 #4 Eggs, tadpoles supported by synapomorphies 16 (ratio of tibia length to O. rugosus Sichuan #43$46 #3$1 Eggs, tadpoles body length >0.45) and 20 (labial tooth formula of tadpole O. jingdongensis Yunan #32$2 #4 Eggs, tadpoles >4 row). Oreolalax pingii was a sister to node 15 supported O. granulosus Yunan #13$2 #4 Eggs, tadpoles O. weigoldi Sichuan #1 Eggs by synapomorphies 5 (spinal patches on the lower arm O. multipunctatus Sichuan #2 #1 Eggs, tadpoles apparent), 17 (spines on the back absent) and 21 (egg pig- O. nanjiangensis Sichuan #11$8 #2 Tadpoles ment absent). Node 13 was supported by synapomorphies Scutiger sikimmensis Tibet #5$1 #3 Eggs, tadpoles 2 (columella long), 3 (post-otic projection of squamosal S. nyingchiensis Tibet #35$3 #2 Eggs, tadpole present), 4 (linea musculinae present) and 9 (tympanum G. Wei et al. / Progress in Natural Science 19 (2009) 1403–1408 1405

Table 3 Morphological data matrix for phylogenetic analysis of the genus Oreolalax. Species Character code 123456789101112131415161718192021 O. omeimontis 11111000121010011102 1 O. popei 11100110120010111102 0 O. liangbeiensis 11100000110100011101 0 O. chuanbeiensis 11100000110000010101 0 O. major 11100001121000010101 1 O. lichuanensis 11111110121000011112 1 O. rugosus 00000100000000010101 0 O. xiangchengensis 00100000000100011001 0 O. pingii 11111000121110001101 1 O. schmidti 11110000121111000102 1 O. puxiongensis 11110000121110000112 0 O. granulosus 00000000000000010101 0 O. jingdongensis 01000010000000011101 0 O. nanjiangensis 111000101211110101029a O. multipunctatus 10990000121111011102 1 O. weigoldi 99990001199101110009 1 O. rhodostigmatas 11100000221011001103 1 S. nyingchiensis 00000000000100000001 0 S. sikkimensis 01000000000010000000 0 a “9” means the character state unknown. concealment). Oreolalax multipunctatus was a sister to O. The node consisting of O. xiangchengensis, O. jingdongensis, rhodostigmatas supported by synapomorphies 16 (ratio of O. rugosus and O. granulosus was a sister to the clade tibia length to body length long >0.45), 17 (spines on the consisting of O. chuanbeiensis, O. liangbeiensis, O. major back absent) and 21 (egg pigment absent). Oreolalax nanji- and O. weigoldi supported by synapomorphy 16 (ratio of angensis was a sister to node 12 supported by synapomor- tibia length to body length long). phies 13 (web between toes absent) and 14 (spines in lip margins absent). Node 10 was supported by synapomorphy 4. Discussion 11 (opening of the eustachian tube is large). Oreolalax popei was a sister to node 10 supported by synapomorphies 4.1. Morphological evolution from aquatic to terrestrial 1 (spoon-like cartilage present), 10 [ratio of tympanic annu- lus to eye diameter small (<0.2)] and 18 (color pattern on Morphological evolution might be related to the envi- back present). Oreolalax major was a sister to O. weigoldi ronment [15]. This rule also holds true for the genus Oreo- supported by synapomorphies 8 (spines on both sides of lalax, and some characters of it appeared to adapt to the the belly present), 9 (tympanum concealment), 16 (ratio environmental changes (Table 4). The evolution of charac- of tibia length to body length long >0.45) and 21 (egg pig- ter spoon-like cartilage (absent ? present) may be due to ment absent). Oreolalax rugosus was a sister to O. granulo- the change of the respiration environment, since the emer- sus supported by synapomorphies 16 (ratio of tibia length gence of spoon-like cartilage was accompanied by the tran- to body length long >0.45), 18 (color pattern on back pres- sition from gill respiration in aquatic animals to lung ent) and 20 (labial tooth formula of tadpole >4 row). respiration in terrestrial animals. Meanwhile, the evolution There was no synapomorphy on nodes 1, 3, 4, 5, 6 and 7 of columella (short ? long), tympanum (absent ? present for supporting the monophyly of related clades (Fig. 1). but concealed ? apparent) and tympanic annulus (absent Then, we collapsed the branches without support by ? present) reflected the transition from the sidetrack organ synapomorphy and manually searched for synapomorphies of aquatic animals sensing water pressure to the ears of again and revised the clades. We found that O. xiangcheng- terrestrial animals sensing air pressure and sound waves. ensis was a sister to O. jingdongensis supported by synapo- Furthermore, the disappearance of the web between toes morphy 17 (spines on the back absent). Synapomorphy 20 reflected the transition from fins of aquatic animals to (labial tooth formula of tadpole >4 row) supported the sister limbs of terrestrial animals. relationship of node 2 and the node including O. xiangcheng- The phylogenetic relationships and synapomorphies of ensis and O. jingdongensis. Oreolalax chuanbeiensis was a sis- clades within Oreolalax on our MP tree (Fig. 1) indicated ter to O. liangbeiensis supported by synapomorphies 1 the tendency of morphological evolution from aquatic to (spoon-like cartilage present), 2 (columella long), 3 (post- terrestrial (Fig. 1). The Oreolalax rugosus species group otic projection of squamosal present), 10 (ratio of tympanic as the basic clade within Oreolalax on the tree was more annulus to eye diameter <0.2), 18 (color pattern on back primitive with more aquatic characters, and the O. pingii present) and 20 (labial tooth formula of tadpole >4 row). species group on the top of the tree was more evolved with 1406 G. Wei et al. / Progress in Natural Science 19 (2009) 1403–1408

Fig. 1. The most maximum parsimonious tree of genus Oreolalax from MP analysis based on 21 morphological characters (left), and the revised cladogram of four species groups within it (right) based on our suggested phylogenetic classification and taxonomical classification of Fei et al. [6]. Node numbers were shown in bold circles. Bold histograms means the presence of synapomorphies supporting the branches, and the number of synapomorphies is above the histogram. The “2” below the histograms means one state of character. Broken lines separated four species groups in Fei et al. [6]. more terrestrial characters (Fig. 1; Table 4). Thus, the In addition, some other characters of a Oreolalax were genus Oreolalax contains the crucial characters of transi- in favor of organism survival on land; for example, the tion from aquatic to terrestrial, and it could be regarded ratio of tibia length to body length became larger to confer as the representative genus of the vertebrate from aquatic advantages in prey and escape, linea musculinae, spinal to terrestrial. patches on the lower arm, spines on the chest, the ratio G. Wei et al. / Progress in Natural Science 19 (2009) 1403–1408 1407

Table 4 The state of characters reflecting the evolutionary tendency from aquatic to terrestrial of species groups, and the distribution area of species groups. Position on O. rugosus species group O. weigoldi species group O. omeimontis species group O. pingii species group tree Basic Middle Top Top Character Spoon-like cartilage Spoon-like cartilage present, Spoon-like cartilage present, Spoon-like cartilage present, states absent, columella short, columella long, tympanum columella long, tympanum columella long, tympanum related with tympanum absent, concealed, tympanic annulus a concealed, tympanic annulus big concealed (O. rhodostigmatus hydrophily tympanic annulus absent little (O. major big) and web and web absent (O. lichuanensis apparent), tympanic annulus big or and web present present present) and web absent triphibian Distribution Southern part of the Middle part of the Hengduan From the eastern side of the Eastern side of the middle part of Hengduan Mountains Mountains middle part of the Hengduan the Hengduan Mountains Mountains to the Daba, Wuling and Dalou Mountains of the spinal-patch width in the chest to the chest width and the basal clade to the top clade on the MP tree and the evo- spines at both sides of belly were related with the mating, lutional tendency of them from aquatic to terrestrial. The and color patterns were related with different environ- parallel relationships indicated that the tendency of expan- ments. However, the significance of post-otic projection sion of Oreolalax was probably from southwest (south of squamosal, large eustachian-tube opening, spines in lip Hengduan Mountains) to northeast (north Hengduan margins, spines on the back, spines on the metatarsal, Mountains and Mountains around the Sichuan Basin). egg pigment and labial tooth rows of tadpoles for Oreola- However, specific divergence is mainly related to the vicari- lax species are still unclear. ance in their evolutional history [17,18]. The formation and differentiation of the present Oreolalax might be related 4.2. Geographic diversification of Oreolalax with the Himalayas orogeny. As the southern part of the Qinghai-Tibet Plateau was ocean [2] and the Indian block The genus Oreolalax distributed in the middle-southern colliding with Eurasia led to a large-scale Qinghai-Tibet Pla- part of the Hengduan Mountains, mountains around the teau as well as adjacent areas [3], the geological ecological Sichuan Basin (about 99°–110°E and 24°–33°N) at altitude environment of ancestral Oreolalax experienced dramatic 700–3300 m, but most species were at altitude 1500–3300 m. changes. These changes supplied obvious obstacles for However, 12 species of this genus (71% species in the genus) vicariance of ancestral populations of Oreolalax and after distributed in a narrow and long zone in the western moun- a long time these populations probably evolved to different tains of the Sichuan Province and the middle-southern part species. So, we suggested that the diversification of Oreola- of the Hengduan Mountains (100°–105°E, 24°–32°N) [5]. lax might be attributed to their earlier geographic expansion The distribution area of basal clade on the phylogenetic tree and latter long-time vicariance among different geological was often suggested as the divergence centre or origin area ancestral populations. of the entire group on the tree [15,16]. The O. rugosus species group was the most primitive group suggested by its basic Acknowledgments position on our phylogenetic tree, which hold more charac- ters adapted for the aquatic environment and distributed in This work was supported by the National Natural Sci- the southern parts of the Hengduan Mountains (Fig. 1; ences Foundation of China (Grant Nos. 30670245, Table 4). This result indicated that the distribution area of 30730029) to Jiang, the grant from the Guizhou Education the O. rugosus species group (southern parts of the Hengduan Department (No. 2007057), the Guizhou Science and Tech- Mountains) might be the diversified centre of Oreolalax. nology Department (Nos. 20072031, 20082083) and the The O. pingii species group and O. omeimontis species group Guiyang Science and Technology Department (No. diverged more recently and at the top of the tree. These two 200838) to Wei. The authors thank L. Fei and C.Y. Ye groups are distributed far away from the Qinghai-Tibet Pla- for their tremendous field and anatomy work. teau and evolve more characters of survival on land. O. rho- dostigmatus distributed in Mount Wuling and Mount References Dalou, far away from the distribution centre, which keeps the standard middle ear with clear outside tympanum. In [1] Frost DR. Species of the World: an Online Reference. addition, the O. weigoldi species group distributed at the Available from: http://research.amnh.org/herpetology/amphibia/ middle of the Hengduan Mountains connected the distribu- [2008-10-12]. tion of the O. rugosus species group and the O. omeimontis [2] Qiu ZD, Li CK. Evolution of mammal fauna in China and the uplift of the Tibetan Plateau. Sci China (Ser D) 2004;34(9):845–54. species group. The continuity from the southwest to the [3] Wang EQ, Burchfiel BC, Ji JQ. Calculation of the Cenozoic crustal northeast of the distribution area of these species groups shortening in eastern Himalayan syntaxis and its geological evi- corresponded to the continuous positions of them from dences. Sci China (Ser D) 2001;31(1):1–9. 1408 G. Wei et al. / Progress in Natural Science 19 (2009) 1403–1408

[4] Zhang RZ. Geological events and mammalian distribution in China. [12] Delorme M, Dubois A. Une nouvelleespe´ce de Scutiger du Bhutan, et Acta Zoologica Sinica 2002;48(2):141–53. quelques remarques sur la classification subge´ne´rique du genre [5] Fei L, Ye CY. Geographical distribution, origin, evolution and centre Scutiger (Megophryidae, Leptobrachiinae). Alytes 2001;19: of differentiation of high-altitude pelobatid toads in Asia (Amphibia: 141–53. Pelobatidae). Zool Res Kunming 1989;10(4):295–302, [in Chinese]. [13] Ye CY, Fei L, Wei G, et al. Study on the phylogenetic relationship [6] Fei L, Ye CY, Jiang JP, et al. An illustrated key to Chinese between species of Scutiger genus in Qinghai-Xizang Plateau amphibians. 1st ed. Chengdu: Sichuan Publishing House of Science (Amphibian: Pelbatidae). Acta Herpetologica Sinica, Guiyang and Technology; 2005, p. 1–340 [in Chinese]. 1992;1:27–39, [in Chinese]. [7] Fei L, Ye CY, Li SS. On the generic classification of Asian high [14] Swofford DL. PAUP*. Phylogenetic Analysis Using Parsimony (* altitude pelobatid toads (Amphibia: pelobatidae). Acta Zoologica and Other Methods). Sunderland MA: Sinauer Associates; 2002. Sinica 1989;35(4):381–9, [in Chinese]. [15] Roelants K, Jiang JP, Bossuyt F. Endemic ranid (Amphibia: Anura) [8] Huang YZ, Fei L, Ye CY. Studies on internal oral structures of genera in southern mountain ranges of the Indian subcontinent tadpoles of Chinese Pelobatidae. Acta Biol Plateau Sinica Xining represent ancient lineages: evidence from molecular data. Mol 1991;10:71–99, [in Chinese]. Phylogenet Evol 2004;31:730–40. [9] Xu N, Wei G, Li DJ, et al. Study on the phylogenetic relationship [16] Wilkinson JA, Drewes RC, Tatum OL. A molecular phylogenetic between species of genus Oreolalax (Amphibia: Pelobatidae). Acta analysis of the family Rhacophoridae with an emphasis on the Asian Herpetologica Sinica 1992;1:40–9, [in Chinese]. and African genera. Mol Phylogenet Evol 2002;24:265–73. [10] Delorme M, Dubois A, Grosjean S, et al. Une nouvelle ergotaxinomie [17] Zhang Y. Evolution of biology. Beijing: Beijing University Press; des Megophryidae (Amphibia, Anura). Alytes 2006;24(1–4):6–21. 1998, p. 1–250 [in Chinese]. [11] Ohler A, Dubois A. The holotype of Megophrys weigoldi Vogt, 1924 [18] Chen YY, Liu HZ. New progress of biogeography. Bull Biol (Amphibia: Anura, Pelobatiddae). Herpetology 1992;26(3):245–68. 1995;30(6):1–4.