Bioacoustics of Limnonectes megastomias

Diagnosis. Limnonectes megastomias , a large species of Limnonectes allied to the kuhlii species group as characterized by the following combination of characters: (1) adult male SVL 40.0– 123.7 mm (mean = 80.2; SD ± 23.66; n = 26), adult female SVL 53.5–86.3 mm (mean = 74.0; SD ± 11.29; n = 10); (2) tympanum obscured by thickened skin; (3) vocal sac and vocal slits absent in males; (4) males have advertisement call; (5) males with nuptial pads on Fingers I and II; (6) males with thick, elongate odontoid processes; females with reduced odontoid processes; (7) males with enlarged heads (HL 41–56% of SVL; 39–45% in females); (8) prominent supratympanic fold from posterior superior corner of eye to angle of jaw; (9) throat, venter, and borders of thigh and leg heavily pigmented (mottled) in males, females with lightly pigmented throat, moderate pigmentation on venter and borders of thigh and leg; (10) small, low glandular warts tipped with translucent horny spinules on flanks of body, around vent, and on dorsum of shank and foot; dorsal skin moderately rugose except for regions covered with warts, ventral skin smooth; (11) Finger II longer than Finger I when adpressed; (12) no webbing between fingers, toes fully webbed ( I 0–0+ II 0–0+ III 0+–0 IV 0–0 V ); (13) larval labial tooth row formula: 2(2)/3(1); (14) narrow gap in ventral oral papillae of larvae; (15) eggs pigmented; eggs deposited in large numbers with no parental care. Comparisons. As noted by Emerson et al. (2000) , frogs of the genus Limnonectes are phenotypically similar. Whereas the most distinguishing feature of Limnonectes megastomias is its tremendous maximum size (SVL) when compared to other species allied to L. kuhlii , reliance on external morphological characters alone for species determination is inadequate for this group. Based on the results of the molecular phylogenetic analysis placing the new species within Limnonectes , and because of the genetic distinctiveness of Limnonectes megastomias , I have elected to limit morphological comparisons of the new species to those species with which it is (1) most closely related, (2) geographically proximate to, and (3) with those it would most easily be confused: Limnonectes kuhlii (from the type locality, Java, and specimens currently recognized as L. kuhlii from Thailand and Laos ), the phenotypically similar Limnonectes laticeps Boulenger (1882) , and the large-bodied Limnonectes namiyei Stejneger (1901) . Emerson and Berrigan’s (1993) clade consisting of L. kuhlii , L. namiyei , and L. laticeps is united by six synapomorphies, three of which are considered here (an obscured tympanum, short vomerine tooth row, and the presence of nuptial pads), features also shared by L. megastomias .

Distribution and ecology. Limnonectes megastomias is known from only three localities in eastern Thailand ( Fig. 6 ). This aquatic frog inhabits streams in dry evergreen forest with year-round water supply and is rarely found away from water. Pang Si Da National Park, located on the southwestern margin of the Khorat Basin, was sampled at elevations between 90 and 600 m along roads, around buildings, in disturbed bamboo and evergreen forests, and in hill evergreen forest. Phu Luang Wildlife Sanctuary, located on the northwestern margin of the Khorat Basin, was sampled at elevation between 850 and 1460 m in hill evergreen, bamboo mixed with evergreen, and rhododendron heath forests. The last forest type was encountered at 1460 m on the summit of Kok Nok Kraba, where weather conditions were cool and foggy. At SERS, located on the southwest margin of the Khorat Basin, this frog is known from only one closedcanopy stream. For most of the year, this spring-fed stream is reduced to standing pools connected by a continuous trickle of water. At the time of our visit, the pools were 10–15cm deep, except the largest pool at the head of the stream, was slightly less than 1m at the deepest point. Stream substrate is sand-silt overlaid with an accumulation of leaf detritus, under which these frogs are quick to seek cover when disturbed. Males were observed calling from 1400–2200 hrs. During two visits to the SERS site ( 2003 and 2005 ), only one other anuran ( Limnonectes gyldenstolpei ) was found to co-occur with L. megastomias . Clutches of newly deposited eggs were found in = 10 mm water at the SERS site on 15 March 2005 . There was no evidence of parental egg care. Individual jelly capsules of eggs were 12–15 mm in diameter, with brown-pigmented ova 2.3–2.8 mm in diameter. Embryos maintained in captivity began hatching within 2 days of collection and had completed hatching after 7 days. Larvae had reached Gosner (1960) Stage 39 by 12 July when all remaining specimens were preserved. Limnonectes megastomias seems to be a sit-and-wait predator and is known to consume insects, frogs ( L. gyldenstolpei ), and birds. Stage 28 30 30 31 32 34 34 36 40* 40 40 40 41 41 BL 10.2 10.3 11.1 11.0 11.2 12.1 11.0 13.8 14.4 13.7 13.1 13.6 13.7 12.9 TL 17.8 20.8 20.4 20.7 22.0 20.0 18.8 23.2 28.0 23.5 26.8 25.9 30.3 30.8 ToL 28.0 31.1 31.5 31.8 33.3 32.1 29.8 37.0 42.4 37.2 40.0 39.5 44.0 43.7 BW 4.7 5.2 5.3 5.3 5.4 5.9 5.2 7.6 8.5 8.1 7.4 7.4 7.5 7.0 BH 3.5 2.6 2.6 3.5 3.5 2.6 3.6 3.6 3.5 3.8 2.8 4.0 3.8 4.0 LED 1.3 1.3 1.4 1.5 1.5 1.5 1.5 1.8 1.8 1.8 1.5 1.8 1.8 1.7 LIO 2.5 2.3 2.7 2.7 2.7 2.8 2.6 3.0 3.5 3.5 3.5 3.5 3.5 3.6 LIN 1.8 1.8 1.9 1.9 1.9 1.9 1.9 2.3 2.2 2.1 2.0 2.0 1.8 1.7 LNS 1.5 1.0 1.8 1.5 1.7 1.8 1.3 1.8 1.8 2.0 1.4 2.0 1.5 1.5 LEN 1.1 1.1 1.2 1.1 1.2 1.3 1.1 1.3 1.4 1.5 1.5 1.2 1.3 1.3 SW 0.7 0.7 0.7 0.7 0.7 0.5 0.7 0.8 0.8 0.8 0.5 1.0 0.7 1.0 TMH1 2.8 2.8 2.8 2.8 3.0 3.0 3.0 3.5 3.5 3.5 3.3 3.5 3.3 3.3 TMH2 2.0 2.5 2.5 2.5 2.4 2.5 2.7 2.9 3.3 3.0 2.8 3.4 2.0 2.5 TH 4.9 8.0 5.3 5.2 5.5 5.8 5.5 7.3 7.0 8.0 6.0 6.8 8.0 5.6 BH/BL 0.34 0.25 0.24 0.31 0.31 0.21 0.32 0.26 0.24 0.27 0.21 0.30 0.28 0.31 BL/ToL 0.36 0.33 0.35 0.35 0.34 0.38 0.37 0.37 0.34 0.37 0.33 0.34 0.31 0.29 BW/BL 0.46 0.51 0.48 0.48 0.48 0.49 0.47 0.55 0.59 0.59 0.56 0.55 0.55 0.54 LED/BL 0.12 0.12 0.13 0.13 0.13 0.12 0.14 0.13 0.12 0.13 0.11 0.13 0.13 0.13 LED/BH 0.36 0.48 0.53 0.42 0.41 0.56 0.42 0.49 0.50 0.47 0.53 0.44 0.46 0.41 LIO/BW 0.53 0.43 0.50 0.50 0.49 0.47 0.50 0.39 0.41 0.43 0.47 0.47 0.46 0.51 LIN/LIO 0.70 0.78 0.72 0.70 0.72 0.69 0.73 0.76 0.61 0.60 0.57 0.57 0.51 0.46 TL/ToL 0.64 0.67 0.65 0.65 0.66 0.62 0.63 0.63 0.66 0.63 0.67 0.66 0.69 0.71 TL/BL 1.75 2.02 1.83 1.88 1.96 1.65 1.71 1.67 1.95 1.72 2.04 1.90 2.22 2.39 TH/ToL 0.17 0.26 0.17 0.16 0.17 0.18 0.18 0.20 0.17 0.22 0.15 0.17 0.18 0.13 TH2/TH 0.41 0.31 0.48 0.48 0.44 0.43 0.49 0.40 0.46 0.38 0.46 0.50 0.25 0.45 Discussion The genetic distinctiveness of Limnonectes megastomias and the morphological differences that separate it from its congeners are certainly sufficient to warrant the recognition of this frog as a new species. There is, however, a frustrating amount of external morphological similarity between the new species and L. kuhlii , enough to warrant a brief discussion of the “ kuhlii complex” and some of the characters shared between L. kuhlii and L. megastomias . Historically, the identification of Limnonectes kuhlii was credited to Dumeril and Bibron (1841) who provided a description of a single specimen from Java. Tschudi (1838) , however, had previously (albeit briefly) described the same taxon under the same name, also from Java. Several names have been synonomized with L. kuhlii , including Rana conspicillata Günther (1872) from Sarawak (Borneo), Nyctibatrachus sinensis Peters (1882) from Guangzhou Province, China , Rana khasiana Boulenger (1882) from the Khasi Hills, India , and Rana paradoxa Mocquard (1890) from Sabah (Borneo) ( Frost, 2007 ). Boulenger (1920) indicated that though he observed great intraspecific variation in Limnonectes kuhlii he was unable to find characters that defined “geographic races. ” Inger (1966) also noted variation among the populations of L. kuhii from Borneo, but found insufficient evidence using external morphology to warrant the recognition of separate species. While examining specimens of the “ kuhlii ” complex for this study, I have observed a great deal of similarity between L. megastomias and L. kuhlii , enough so that it would be difficult to identify individuals of the same approximate size to the level of species in the field. Population-level variation in morphological characters (in particular, the presence and location of nuptial pads, and the degree to which the tympanum is obscured) is potentially confounding when one is trying to diagnose Limnonectes kuhlii and separate it from Limnonectes megastomias . All males of L. megastomias examined in this study (26 individuals from three populations; SVL 40–123 mm ) all have nuptial pads on Fingers I and II. Inger (1966) noted that specimens of L. kuhlii from China and Thailand also possessed nuptial pads on Fingers I and II, whereas specimens from Sabah and Sarawak presented nuptial pads only on Finger I. These observations are corroborated by specimens I examined, with the exception of a single male from Thailand (Chiang Mai Province), which had nuptial pads only on Finger I. Additionally, specimens examined from Laos also have nuptial pads on Fingers II and III. Three adult males from Java examined in this study (sex determined by enlarged head and odontoids, and gonadal evidence): a poorly preserved and bleached lectotype (MNHN 4469), a paralectotype (RMNH 4297), and a recently preserved specimen. It is clear that the RMNH paralectotype and recent specimens lacks nuptial pads. It appears that the MNHN lectotype also lacks nuptial pads, but the poor state of preservation makes it difficult to say this with certainty. Examination of additional specimens hopefully will resolve this uncertainty. Emerson & Berrigan (1993) described L. kuhlii , L. laticeps , and L. namiyei as all having concealed, but present, tympana. In general, the same character also diagnoses Limnonectes megastomias , yet in 12 of 37 specimens examined, the tympanic annulus was partially obscured, but visible enough to permit a measurement of the tympanic diameter. Similarly, Boulenger (1920) , considered the tympanum of L. kuhlii hidden or slightly distinct, but provided measurements of the tympanic diameter. Inger (1966) noted that in Bornean populations of L. kuhlii , the tympanic annulus usually was not visible under the skin. Although Tschudi (1838) does not mention the tympanum of L. kuhlii , Duméril and Bibron (1841) discuss its size in relation to the upper eyelid, presumably visible and measurable without dissection of the single Javan specimen from which they based their description. In Javan specimens of L. kuhlii examined here, 12 of 13 have obscured tympana, but still visible enough to allow for measurement. The tympanic annulus is not visible in the poorly preserved lectotype . The specimens of L. kuhlii that I examined from Thailand and Laos also varied in the degree to which the tympanum was obscured (obscure, but visible in 7 of 14, and 4 of 5 specimens , respectively), sometimes even varying from side to side in the same animal. It is possible that the state of preservation and hydration of a specimen affects the degree to which the tympanic annulus is visible through the skin. Because most systematic works are based on fluid-preserved specimens, this may influence how this character appears to the investigator. It is reasonable to consider that as a specimen dries, the skin covering of the head covering the tympanic structures tightens and presents an “obscure, but visible” tympanic annulus. The variation in this character among populations of L. kuhlii and its allies warrants closer examination (both in preserved and live specimens). The presence of bifid subarticular tubercles (proximal or distal, or both) on Fingers II or III (or both) was noted only in some specimens of Limnonectes megastomias . This same character was also observed in four individuals of L. kuhlii from Chiang Mai ( Thailand ), one specimen of L. kuhlii from Laos , and in none of the specimens from Java. In the Chiang Mai population, only Finger III has bifid tubercles (proximal in all cases, distal in one). The specimen from Laos has a bifid distal subarticular tubercle on Finger III. It is interesting that in the SERS population of L. megastomias bifid subarticular tubercles are present in all individuals examined, but in other populations it seems to be highly variable. The apparent small size and isolation of the SERS population to only one stream may affect the frequency of inheritance of this trait. In Thailand , Limnonectes kuhlii is distributed along the mountainous western edge of the Kingdom from north to south, including the central portion of the Thai-Malay Peninsula ( Chan-ard, 2003 ). The type locality of L. megastomias and Pang Si Da National Park are more than 400 km distant from the western edge of the known distribution of L. kuhlii in Thailand . There are records, however, of L. kuhlii (a small-bodied form; SVL < 90 mm ) from Loei Province where specimens of L. megastomias also were collected (Phu Luang Wildlife Sanctuary). The amount of genetic divergence between samples of L. kuhlii from Chiang Mai Province ( Thailand ) and L. kuhlii from Java ( type locality) (14–16%) would suggest that what is currently recognized, as “ kuhlii ” in Thailand is a distinct species. Furthermore, based on the genetic distance between L. megastomias and Chiang Mai populations of L. kuhlii (±9%), it is plausible that there are multiple species of “ kuhlii ”-like frogs in Thailand and that some of those populations may occur sympatrically with L. megastomias . Because of the wide distribution of L. kuhlii and the broad range of morphological variation, it is also possible that populations currently recognized, as L. kuhlii will be subsumed by L. megastomias . A broad and robust sampling of specimens from across the known distribution of L. kuhlii is currently underway to investigate the molecular structure of this species at the population level (McLeod, in prep). There remains a great deal of work necessary to resolve fully the identity of the frogs in the kuhlii -complex. Phylogenetic resolution of this enigmatic group, and other groups like it, will not be possible without the detailed study of internal and external morphology coupled with a robust molecular analysis (McLeod, in prep).