Bioacoustics of Nanorana aenea

Other specimens referred to the species Chaparana aenea ( Smith, 1922 ) To our knowledge, beside the two specimens described in detail above, only 11 other specimens referable to this species are known, which are briefly discussed below. Table II gives the measurements of the 13 specimens here discussed, and Figure 2 shows the known localities of this species in China, Thailand and Vietnam , as well as areas of known occurrence of the closely related species Chaparana unculuanus (see below). Specimens from Thailand BMNH 1974.1113 (ex MAS 5822). Juvenile (SVL 38.6 mm ), paratopotype of Rana aenea from Doi Chang. This specimen, slightly larger than the holotype , has similar characters. MNHN 1989.0712 ( Figure 3a–c ). Non-breeding adult male (SVL 77.4 mm ) from Doi Inthanon (18 ° 35'N, 98 ° 28'E), Chiangmai Province , Thailand . Except for its absence of secondary sex characters, this specimen is very similar to the three adult males from Fan Si Pan mentioned above and below. The major differences between them are: the complete absence of nuptial spines on fingers, breast and throat and the absence of enlargement of the forearms, as in the Vietnamese specimen MNHN 1999.5818. The ventral colour pattern is similar in all specimens, but the specimen from Doi Inthanon has a rather clear fawn dorsal colour, whereas the males from Fan Si Pan are dark brown on the back: this difference may be related to nycthemeral colour variation, as specimens collected at night are darker than specimens collected during the day. This specimen is clearly adult, and its absence of spines on the fingers and chest indicates that it was not collected during the breeding season. However, its first finger is notably enlarged, and this finger and the prepollex show structures which correspond to basements of spines. Metacarpal First Second Third Maximum Specimen number tubercle finger finger finger Breast total MNHN 1999.5818 20/19 98/109 32/36 3/7 35/31 207 MNHN 1999.5820 12/15 76/89 27/23 10/8 31/32 173 MNHN 1999.5821 21/21 93/91 34/29 19/20 42/44 212 For each item, the table gives the number of spines on the left side of the body followed by that on the right side: e.g. 20/19. The maximum total given in the last column is the total of the maximum value for each item on either the left or the right side of the body (see Dubois 1976 , p 59; Dubois and Matsui 1983 , p 900–901). , ) Pu Hoat, Luchun, Doi Chang, Thailand Doi Inthanon, Thailand Fan Si Pan, Vietnam Vietnam China Juv. female Juv. Adult male Juv. Juv. male Juv. male Adult male Adult male Adult male Juv. male Juv. male Juv. female Juv. male BMNH BMNH MNHN MNHN MNHN MNHN MNHN MNHN MNHN MNHN MNHN MNHN MNHN 1922.7.4.4 1974.1113 1989.0712 1989.0713 1989.0714 1948.0139 1999.5821 1999.5820 1999.5818 1999.5998 1999.5819 2000.9018 2001.0277 SVL 35.2 38.6 77.4 23.7 40.5 54.5 74.6 64.7 69.1 49.8 28.4 38.8 39.2 HW 14.2 15.0 31.2 9.2 16.6 22.0 30.1 25.7 25.8 19.5 11.9 15.6 15.2 HL 14.4 15.7 28.8 9.6 16.2 21.0 27.3 25.9 25.5 18.8 11.5 15.0 15.4 MN 11.5 13.3 23.2 8.2 13.1 17.2 23.1 19.7 20.4 14.9 10.0 12.1 12.6 MFE 8.9 10.4 19.2 6.5 11.0 14.2 18.0 16.2 16.3 12.0 8.0 9.5 10.8 MBE 5.3 6.7 12.0 3.4 6.3 8.7 11.4 9.9 10.3 7.2 4.9 5.4 6.4 IFE 6.3 7.2 12.5 5.1 7.6 8.6 11.2 10.2 11.6 8.8 5.1 7.2 7.9 IBE 10.5 11.2 18.5 7.1 12.3 14.8 17.9 15.6 17.4 12.9 8.2 11.1 11.1 IN 4.50 4.50 8.68 3.44 5.19 5.7 8.4 7.24 7.63 5.83 3.95 4.93 5.25 NS 3.25 6.49 7.90 2.72 4.74 3.7 7.1 6.05 6.84 4.90 3.29 3.82 4.34 EN 2.38 2.56 4.21 1.81 2.59 4.0 4.5 4.08 3.68 2.66 2.07 2.46 2.79 EL 4.51 5.19 8.16 3.37 5.12 5.7 7.7 6.97 7.76 6.42 3.63 4.80 5.19 TYD 2.63 2.38 3.82 0.84 2.07 2.9 nm 1.84 2.76 1.43 1.10 2.07 2.07 TYE 1.25 1.69 4.74 0.97 1.94 3.7 nm 3.55 4.21 3.24 1.62 1.94 1.62 IUE 2.63 2.75 6.58 2.14 3.24 4.4 4.7 3.16 5.40 3.89 2.98 3.11 2.98 UEW 3.19 3.63 5.79 2.40 3.89 4.2 5.4 5.92 5.79 4.41 2.59 3.95 3.56 SL 5.53 5.54 12.11 4.54 4.80 8.4 11.3 9.74 10.92 8.10 5.39 6.97 7.63 FLL 7.5 8.2 19.0 4.8 8.8 13.0 20.2 17.0 18.8 10.9 6.2 8.2 8.9 HAL 8.2 8.3 18.9 5.8 9.6 12.7 17.0 14.5 15.5 11.4 6.3 8.8 9.3 TFL 3.78 4.56 9.1 3.05 5.0 6.4 7.6 8.0 7.4 5.5 3.24 4.87 4.7 TL 21.1 23.7 49.6 14.1 25.9 34.8 48.4 40.9 41.9 28.3 16.5 22.9 24.0 TW 4.8 nm 17.3 2.8 6.7 11.0 12.8 10.6 11.1 6.9 3.7 6.2 4.9 FL 18.9 22.0 47.3 13.3 24.7 34.5 42.0 36.9 40.7 26.6 15.3 20.3 22.6 TFOL 29.4 nm 66.5 19.8 36.5 49.0 42.2 55.3 57.9 42.2 22.5 32.8 32.9 FOL 18.9 22.3 46.5 12.9 21.2 32.0 42.8 37.5 38.3 28.7 15.2 21.2 21.9 FTL 14.4 11.6 25.7 7.4 12.0 18.5 23.7 20.8 21.5 16.3 8.2 12.0 12.5 IMT 2.19 2.44 6.32 1.62 2.27 3.7 5.40 4.74 5.26 2.92 1.69 2.27 2.53 ITL 4.25 4.69 9.74 2.98 5.12 7.3 10.40 8.55 9.74 7.00 3.44 5.12 4.80 WTF 3.38 3.75 8.55 2.07 4.80 5.79 9.74 7.24 7.90 4.99 2.92 3.69 4.21 WFF 3.81 4.00 7.63 2.01 4.99 6.05 9.21 6.58 8.82 5.70 2.72 3.82 4.34 Pu Hoat, Luchun, Doi Chang, Thailand Doi Inthanon, Thailand Fan Si Pan, Vietnam Vietnam China WI 3.00 3.19 8.03 2.01 3.89 4.74 8.68 6.32 7.37 3.95 2.33 3.18 3.82 WII 2.88 3.19 7.37 1.75 4.34 5.00 8.42 5.79 7.24 4.34 2.07 3.11 3.68 MTTF 17.0 18.7 21.2 6.55 12.9 15.0 20.9 16.4 18.7 14.6 8.29 11.3 11.32 MTFF 18.4 19.5 24.4 7.13 14.6 18.6 21.4 16.0 20.3 16.2 8.55 11.6 12.11 TFTF 13.0 16.5 20.4 5.90 11.1 14.1 15.4 14.3 15.5 13.2 6.84 9.9 9.34 FFTF 14.1 16.8 20.2 6.16 10.5 14.0 17.7 15.4 14.0 12.5 7.11 9.5 10.26 MNHN 1989.0713. Juvenile female (SVL 23.7 mm ) from same locality as previous frog. This specimen is very similar in general aspect to both holotypes of Rana aenea and Rana fansipani . MNHN 1989.0714. Juvenile male (SVL 40.5 mm ) from same locality as both previous specimens, and very similar to them. Specimens from Vietnam MNHN 1948.0139 ( Figure 3d–f ). Juvenile male (SVL 54.5 mm ), holotype of Rana fansipani , collected in August 1938 by René Bourret on Fan Si Pan mountain (22 ° 19'N, 103 ° 47'E), Sa Pa District , Lao Cai Province , Vietnam . This specimen was described in detail by Dubois (1977) , who wondered whether this frog, clearly a male, was juvenile, subadult or adult. It is very similar in all its characters to the three adult males collected in 1997–1998 on the same mountain, except for its absence of male secondary sex characters: given its much smaller size (SVL 54.5 mm versus 64.7–74.6 mm in the three adult males), it is now clear that it is not adult, nor even subadult, but juvenile, hence its absence of any secondary sex character. This specimen is also very similar in all its characters to the lectotype and paralectotype of Rana aenea mentioned above. MNHN 1999.5820. Adult male (SVL 64.7 mm ), topotype of Rana fansipani Bourret, 1939 , collected on 17 November 1997 at Cat Cat ( 1280 m ; 22 ° 19'N, 103 ° 49'E), on Fan Si Pan mountain, Vietnam . This second specimen is very similar to the specimen MNHN 1999.5821 described in detail above. The major differences between them are the secondary sex characters that are less developed in MNHN 1999.5820: the spines on the breast are large and blackish, but isolated from each other and less numerous ( Table I ); on the throat, blackish spines are distinct, but small; the forearms are distinctly enlarged. MNHN 1999.5818 ( Figure 4a–c ). Adult male (SVL 69.1 mm ), topotype of Rana fansipani Bourret, 1939 , collected on 1 August 1998 at Ban Khoang ( 1500 m ; 22 ° 23'N, 103 ° 48'E), on Fan Si Pan mountain , Vietnam . This specimen is similar to the other two adult males mentioned above, but in a better condition of preservation. The secondary sex characters are similar to those of MNHN 1999.5820, but the blackish horny covering of the nuptial spines is lost on the breast and on the fingers, and the spines are less numerous ( Table I ). No spines are present on the throat, and the forearms are not enlarged. MNHN 1999.5819. Juvenile male (SVL 28.4 mm ) from O Qui Ho ( 1625 m ; 22 ° 23'N, 103 ° 47'E), on Fan Si Pan mountain, Vietnam . Its size is smaller but its morphology and colour pattern are similar to those of the adults from the same mountain. MNHN 1999.5998. Juvenile male (SVL 49.8 mm ) from Fan Si Pan mountain ( 1700 m ; 22 ° 23'N, 103 ° 47'E), Vietnam . This specimen is similar in colour pattern to the other specimens. MNHN 2000.9018. Juvenile female (SVL 38.8 mm ) from Pu Hoat ( 1300 m ; 19 ° 25'N, 104 ° 37'E), Nghe An Province , Vietnam . This specimen was collected much further south, in the Annamite mountain range . In general morphology and coloration it resembles the topotypes from Fan Si Pan . Specimen from China MNHN 2001.0277. Juvenile male (SVL 39.2 mm ) from Luchun Xian (altitude about 2050 m ; 23 ° 01'N , 104 ° 21'E), Huang Lian Shan Nature Reserve , Yunnan , China . This specimen, the only one of this species known from China , is very similar in aspect and coloration to the Vietnamese specimens. It has a very conspicuous chevron on the anterior part of the back. Morphometric comparisons Intraspecific comparisons within Chaparana aenea ( Smith, 1922 ) Despite the low number of specimens referable to the species Chaparana aenea available, we tried to carry out comparisons between them. As mentioned above, in their overall aspects and colours these 13 specimens are very similar. Part of the variation observed is related to age (size) and sex (especially in breeding adult males). However, the shapes and proportions of these specimens, as studied through 32 measurements ( Table II ), do not show any clear geographical trend, as shown in Figure 5 which presents the distribution of the 13 specimens according to the first two regression factor scores provided by the PCA. Before and after application of Bonferroni correction, Mann– Whitney U tests based on the first three regression factor scores of PCA (Table III) do not show significant differences between subgroups of these 13 specimens , based either on sex (males versus females) or on main geographic origin (mountains of northern Vietnam and southern Yunnan versus mountains of northern Thailand ). However , prior to Bonferroni correction, when ontogenetic stages (juveniles versus adults) are considered, the first regression factor scores of juveniles are statistically different from those of adults. After Bonferroni correction the value is slightly too high for significance. The existence of significant differences between different ontogenetic stages is meaningful as it points to allometric growth in this species, as is usual in frogs, and especially in this group of torrent frogs ( Dubois 1976 ). From these limited available data, we conclude that, for the time being, no significant difference is documented between populations from Thailand, Vietnam and China referred to this species, and we consider the name Rana fansipani Bourret, 1939 as a junior subjective synonym of Rana aenea Smith, 1922 . Further data, based on more specimens and using additional information (molecular data, bioacoustics, tadpole morphology) will be necessary to confirm or refute this conclusion. Comparisons between Chaparana aenea ( Smith, 1922 ) and Chaparana unculuanus (Liu, Hu and Yang, 1960) Besides Chaparana aenea ( Smith, 1922 ) and its synonym Chaparana fansipani ( Bourret, 1939 ) , Dubois (1992) included a third nominotypical species in his subgenus Chaparana (Chaparana) , namely Chaparana unculuanus (Liu, Hu and Yang, 1960) . The latter ( Figure 4d–f ) is indeed very similar to Chaparana aenea in general morphology, and both species occur in southern Yunnan , although in different areas ( Figure 2 ). However , these two species differ markedly in their male secondary sex characters: as discussed above, breeding males of Chaparana aenea have enlarged forelimbs and black cornified spines on the fingers and chest, but no spines around the vent. In contrast, breeding males of Chaparana unculuanus are devoid of spines on fingers and chest and their forelimbs are not enlarged, but the skin around their vent is distended and bears spines. We examined and measured two adult males and four adult females of Chaparana unculuanus from Jingdong Xian ( Yunnan , China ) ( Table IV ). Univariate comparisons by Mann– Whitney U tests of measurements (expressed as ratios of SVL, except for SVL itself) between the four adult specimens of Chaparana aenea listed in Table II and the six adult specimens of Chaparana unculuanus listed in Table IV show that the two samples differ statistically in seven measurements out of 27 (HL, FLL, TFL, TL, IN, TYE, IMT). If these 10 specimens are included in a PCA, the two samples appear as two well-separated groups ( Figure 6 ) and the second regression factor scores for the two samples are significantly different ( Table V ). These data confirm the morphological distinctness of the two samples, independently from their male secondary sex characters, and they support the interpretation that the two samples represent different biological species. This is consistent with the result of the analysis of partial sequences of mitochondrial 12S and 16S rRNA genes provided elsewhere (Jiang et al. forthcoming), which confirms that the two species have distinct, although closely similar, sequences. Mann– Subsamples Regression Whitney BBC ABC compared factor score First subsample Second subsample U test a 50.05 a '5 0.0167 Geographic RFS 1 G1 ( n 55) G2 ( n 58) U 520.0 ns ns origin 21.852–0.818 21.473–1.191 P 51.00 Geographic RFS 2 G1 ( n 55) G2 ( n 58) U 515.0 ns ns origin 20.482–1.727 21.497–1.378 P 50.52 Geographic RFS 3 G1 ( n 55) G2 ( n 58) U 513.0 ns ns origin 21.033–2.182 21.281–0.727 P 50.354 Sex RFS 1 S1 ( n 59) S2 ( n 52) U 52.0 ns ns 21.852–1.049 0.700–1.191 P 50.15 Sex RFS 2 S1 ( n 59) S2 ( n 52) U 56.0 ns ns 21.497–1.378 21.029–20.248 P 50.58 Sex RFS 3 S1 ( n 59) S2 ( n 52) U 56.0 ns ns 21.281–0.968 0.208–0.083 P 50.58 Ontogenetic RFS 1 D1 ( n 59) D2 ( n 54) U 53.0 * ns stage 21.473–1.191 21.852–20.125 P 50.02 Ontogenetic RFS 2 D1 ( n 59) D2 ( n 54) U 510.0 ns ns stage 21.497–1.727 21.129–20.159 P 50.26 Ontogenetic RFS 3 D1 ( n 59) D2 ( n 54) U 511.0 ns ns stage 21.033–2.182 21.281–0.968 P 50.33 Criteria of construction of subsamples: (G) geographic origin: Thailand (G1) versus Vietnam + China (G2); (S) sex: males (S1) versus females (S2); (D) ontogenetic stage: juveniles (D1) versus adults (D2). The sex of two juveniles is unknown, hence the total sample of 11 instead of 13 for the criterion ‘‘sex’’. For each criterion of comparison, minimum and maximum values of RFS given for subsamples are those obtained by PCA for the first (RFS 1), second (RFS 2) and third (RFS 3) regression factor scores. Significance levels before Bonferroni correction (BBC): ns, not significant, P .0.05; * P ǩ0.05; significance levels after Bonferroni correction (ABC): ns, not significant, P .0.0167; * P ǩ0.0167. Taxonomic consequences of these observations at the generic level Beside the synonymization of Rana aenea and Rana fansipani , the observations presented above have taxonomic consequences at the generic level. The nominotypical species Rana fansipani being the type-species of Chaparana Bourret, 1939 , the status of the latter name needs to be re-evaluated. Dubois (1992) recognized two genera in the tribe Paini Dubois, 1992 of the subfamily Raninae Rafinesque-Schmaltz, 1814 : Chaparana Bourret, 1939 (with four subgenera) and Paa Dubois, 1975 (with four subgenera). Based on unpublished molecular data, this tribe was transferred to the subfamily Dicroglossinae of the Ranidae Anderson, 1871 by Dubois et al. (2001), a taxonomy followed by Dubois (2003) and Roelants et al. (2004) . Furthermore, following the results of the work of Jiang and Zhou (2003), the genus Nanorana Günther, 1896 , placed by Dubois (1992) in the Ranini, must be included in the tribe Paini . In Dubois’s (1992) taxonomic arrangement, breeding males of all species of the genus Chaparana were stated to be devoid of breeding spines both on the forelimbs and on the breast, whereas most (but not all) species of Paa had such spines, at least on the first finger. Besides, breeding males of several species referred to Chaparana were known to have a specialized zone of skin, either swollen or bearing spines, around the vent. It is now known that the type-species of Chaparana is devoid of this latter character but has spines on the fingers and chest, thus fitting with the genus Paa as understood by Dubois (1992) . This species, but this species alone, must be removed from the group called Chaparana by Dubois (1992) , and placed in the group called Paa by this author. The name Chaparana has priority to designate this genus. Ad. female Ad. female Ad. male Ad. male Ad. female Ad. female MNHN CIB 581270 CIB 581267 CIB 581597 CIB 581664 CIB 581666 2001.0280 SVL 77.6 78.4 77.0 69.0 80.0 72.9 HW 29.6 30.2 27.5 25.9 29.3 27.9 HL 27.5 28.0 25.5 24.5 27.6 25.9 MN 22.8 23.3 21.0 20.0 22.3 22.1 MFE 18.5 19.0 16.4 16.1 18.1 19.1 MBE 11.7 11.7 9.5 9.8 11.4 12.5 IFE 11.7 12.0 10.5 11.3 12.2 11.5 IBE 19.5 19.5 17.8 17.3 20.0 19.3 IN 8.4 7.2 7.0 6.5 7.8 6.8 NS nm nm nm nm nm 7.5 EN 4.7 4.4 3.9 4.2 4.5 4.3 EL 6.8 7.6 7.3 7.1 7.6 9.0 TYD 2.9 2.7 2.7 2.5 3.0 3.3 TYE 3.8 4.5 4.1 2.7 3.4 4.0 IUE 4.5 4.6 4.3 3.9 4.0 5.0 UEW 5.6 5.7 6.1 5.4 5.8 6.1 SL nm nm nm nm nm 10.7 FLL 16.3 17.1 17.3 15.4 16.9 15.4 HAL 19.0 19.1 18.2 17.7 19.1 17.8 TFL 9.9 9.7 9.5 9.3 10.4 10.3 TL 44.9 45.5 43.1 40.0 45.5 42.6 TW nm nm nm nm nm 15.7 FL nm nm nm nm nm 39.6 TFOL nm nm nm nm nm 52.2 FOL 43.6 41.4 41.8 38.3 42.0 40.5 FTL 24.2 23.1 22.1 21.6 24.0 23.6 IMT 4.1 4.5 4.1 3.9 5.0 4.7 ITL 9.6 9.5 9.2 8.5 9.3 10.4 WTF 9.9 9.0 9.2 9.0 10.4 11.8 WFF 8.8 8.6 8.0 7.8 7.8 9.3 WI 9.0 8.7 7.5 7.7 9.6 9.9 WII 7.5 7.1 6.0 6.1 7.7 6.6 MTTF nm nm nm nm nm 22.1 MTFF nm nm nm nm nm 22.1 TFTF nm nm nm nm nm 14.2 FFTF nm nm nm nm nm 17.1 Ad., adult (as defined in Dubois 1976 , p 31–33); nm, measurement not taken on this specimen. Of the 36 measurements, only 27 are available for all six specimens and were used for the principal component analysis of Figure 6 and Table V. However, two significant differences remain between Chaparana aenea and all species referred by Dubois (1992) to the subgenus Paa (Paa) : (1) whereas, in breeding males of the former, both sides of the chest are covered by a well-delimited patch of densely packed spines, whose general shape recalls that of a bean or kidney ( Figure 7 ), in breeding males of those species of Paa that have breast spines (i.e. only a part of the species of this group), the two patches are composed of unequally spaced spines and have variable limits, so that these patches do not have a common general shape (see e.g. Dubois 1976 , p 52, 89, 105, 121); (2) in Chaparana , the first finger is usually longer than the second (both fingers may be subequal in some juveniles), whereas in Paa the first finger is usually shorter than the second (in some large adults, both fingers may be subequal, or the first one may be exceptionally longer in the species Paa liebigii (Günther, 1860) : see Dubois 1976 , p 65): in this character, Chaparana aenea is similar to the frogs placed by Dubois (1992) in the subgenus Paa (Quasipaa) . This suggests that the relationships between Chaparana and Paa should be explored further. As for the other groups referred by Dubois (1992) to several other subgenera of Chaparana and Paa , their taxonomy and nomenclature will also have to be modified as a result of a re-evaluation of the significance of some characters following the results presented above. To start clarifying this matter, we recently proposed a preliminary cladistic analysis of the Paini based on partial sequences of mitochondrial 12S and 16S rRNA genes of 17 species of the clade (Jiang et al. forthcoming), but this analysis only provides partial information. Therefore we explored the phylogenetic relationships in this clade on the basis of external morphological characters of adults and of a few other characters as available. These results and their implications will be presented elsewhere.