Leptobrachella melanoleuca (Matsui 2006)

Rongchapho, Peerasit, Chuaynkern, Chantip, Duangjai, Sutee, Chalermwong, Pornthawat & Chuaynkern, Yodchaiy, 2024, Molecular Identification and Description of the Tadpole of Leptobrachella melanoleuca (Matsui, 2006) (Amphibia: Anura: Megophryidae), Ecologica Montenegrina 80, pp. 152-170 : 158-170

Leptobrachella melanoleuca (Matsui 2006)

Leptolalax melanoleucus Matsui, 2006: 822 .

Material examined: PR 03399.1–11 (11 tadpoles) and PR 03307–08 (2 adult males). The specimens were collected from a small stream in the Thum Chian Protected Unit, Khlong Saeng Wildlife Sanctuary, Surat Thani Province, southern Thailand (9°07'11"N, 98°43'06"E, 129 m a.s.l.). The external morphological description of the tadpole is based on a DNA-vouchered specimen at stage 35 (PR 03399.2, BL 15.5 mm), while the buccopharyngeal description is based on specimens at stage 38 (PR 03399.7, BL 15.3 mm). The tadpoles were found in small rivulets within the woods, 500 meters from the Ratchaprapha dam's riverbank. Tadpoles were collected from a stream with a slight current, where they were hidden among pebbles, dead leaves, and stones at the bottom ( Figure 9 ). Adult frogs were found along rocky rivulets, in these streams, other species such as Leptobrachium sp. , Megophry sp., Alcalus sp. and Limnonectes sp. were also observed.

Description of external morphology: In dorsal view ( Figure 3A , 6A ), body elliptical oblong, snout rounded, two sizable lateral lymphatic sacs from spiracle to end of body. In lateral view ( Figure 3C, D ), body oval, slightly depressed, BW 101.19% of BH, snout oval. Eyes moderately small, ED 8.96% of BL, bulging and not visible in ventral view, positioned dorsolaterally, directed anterolaterally. Pineal ocelli absent, nasolacrymal ducts are not visible. Nare slightly small, round with an oval opening, rimmed, positioned dorsolateral, directed anterolaterally, closer to snout than to pupils, RN 110.52% of NP; NN 155.78% of PP. Spiracle sinistral, square, small, positioned nearly haft of head and body and situated at ventrolaterally, SD 5.16% of BL; opening at closed against body wall, caudal myotomes and hind limb insertion, oriented posterodorsally. Tail musculature strong; TMH 97.36% of BH and 70.24% of MTH, TMW 92.65% of BW, gradually tapering, reaching tail tip. Tail fins moderately large; UF 36.68% of MTH, upper fin extending onto body, SU 93.17% of BL, slightly convex; lower fin extending onto body, convex; MTH 138.61% of BH, tail tip acute angle. Anal tube large, approximately conical, medial, entirely attached to lower fin, opening medial, posteriorly directed.

Oral disc ( Figure 4A, B ). Oral disc medium, positioned ventrally and directed anteroventrally, not emarginated laterally, moderately large, ODW 23.21% of BL and 85.31% of BW, semicircular without median notch on the lower labium, upper labium with two lobes, the median separated by narrow fold forming a median fold with marginal papillae, lower labium with median deeper from median fold without ventral marginal papillae. Row of papillae at lateral sides of upper labium, 3–4 submarginal papillae, No papilla row on lower labium. The KRF of 1:(2–2)/0, A2> A3> A1, A1 very short, A2 shortly interrupted in the middle, A3 separated by the upper beak, Keratodonts upper teeth obclavate shaped ( Figure 4D ), Jaw sheaths strong, dentate serrated, black with brown blurring slightly above the serrations ( Figure 4C ); upper sheath shaped as reverse U, upper sheath large arch with gap median lower sheath hidden, deep fitted under the upper jaw sheath.

Lateral line present ( Figure 5 ), 6 pairs, large, visible by eye. Canthoinfraorbital line pair, 11/14 openings, visible in dorsal and lateral views, running from anterior nostril upward and curved above the nostril to infraorbital. Supraorbital line pair, 8/8 openings, visible in dorsal and lateral views, running from anterior to posterior eye. Dorsal line pair, 42/39 openings, visible in dorsal and lateral views, running from posterior eye along the dorsum of body until half of the tail. Angular line single, 22/22 openings, visible in dorsal, lateral, and ventral views, running from the posterior eye downward across the lateral and ventral sides of the throat, then upward along the lateral side to the posterior eye. Medial line pair, 33/33 openings, visible in dorsal and lateral views, running from anterior and upward spiracle (left side) to posterior end of body. Ventrolateral line pair, 45/45 openings, visible in lateral and ventral views, running forward from the posterior end of the body along the belly to the chest, then turning back and extending along the belly to the base of the vent tube.

Coloration. In preservative ( Figure 3 ), dorsal and lateral of body dark brown with light brown spots, throat brown with light brown spots, chest dark gray. Tail musculature gray with light brown spots. Fins creamy transparent with less light brown spots. In life ( Figure 6 ), dorsal and lateral of body brown with creamy spots, throat brown with creamy spots, chest gray. Tail musculature gray with creamy spots. Fins creamy transparent with less creamy spots.

Description of buccal anatomy. Buccal roof ( Figure 7A ): Prenarial arena semicircle, concave on anteromedially, arched ridge bearing knobby projections (3/4 anterolaterally, 1 posteromedially) located ¾ from beak to choanae, without lateral papillae. Choanae narrow, with an angle of 50° to body axis, internarial distance approximately half of the length of choana, anterior wall pustular; no papillae on narial valve. Prenarial papillae present. Postnarial papillae present. Postnarial areas narrowly transverse and rectangular, with few papillae. Medial ridge transverse elliptical, wider than long, smooth with 6 notches on medial ridge edge, lateral ridge papilla absent. Buccal roof arena rectangular, with buccal roof arena papillae on anterior and lateral sides; densely numerous pustulate within the arena, more gradually dense to posterior; no papillae on anterior esophageal funnel. Posterolateral ridge with papillae. No glandular zone. Dorsal velum continuous, margin curved, medial portion curved toward esophagus.

Buccal floor ( Figure 8A ): Prelingual arena trapezoid: its floor smooth. Two pairs of infralabial papillae, equal size, long and large separate into three branches. Tongue anlage triangular, large, without a posterior protuberance, no bearing lingual papillae. Buccal floor arena rectangular, delimited by 14 large and long buccal floor arena papillae, flattened and densely pustular formed as triangular shape. Buccal pockets oval, anteroposteriorly oriented, with a wide opening, deep, closer to medial end of ventral velum than to tongue anlage; large and long buccal floor arena papillae in inner side of the buccal pockets, numerous pustules in posterior of the buccal pockets. Numerous pustules between posterior part of buccal floor arena and end of ventral velum. Ventral velum discontinuous, with specular support, not wavy, with 4–5 projections on margin of velum, medial notch medially concave, little developed and made up by two most medial projections; glottis large, visible below velum, branchial baskets large, oblique, longer than wide, with three filters cavities; filter rows dense with tertiary folds.

Variation with the series. A total of 11 tadpoles were used in this study. The variation among these tadpoles was assessed across 10 developmental stages, distributed as follows: 26 (9%), 29 (9%), 31 (9%), 32 (9%), 33 (9%), 34 (9%), 35 (18%), 36 (9%), 37 (9%), and 38 (9%). The variation in KRF ranged from 2+2/0 (18%, two specimens) at stage 34 and 36 to 1:2+2/0 (82%, nine specimens) at stage 26, 29 31–33, 35, and 37–38. Tadpole development did not influence the differentiation between lateral lines. The number of openings in each lateral line varied both among specimens and between the left and right sides of the same lateral line. The canthoinfraorbital line had 10–15 openings, the supraorbital line contained 5–8 openings, the angular line had 18–25 openings, the dorsal line had 35–45 openings, the medial line had 30-33 openings, and the ventrolateral line had 43–47 openings. Measurements, meristic counts, KRF, and lateral line values for all available stages are provided in Table 4.

Table 5

L. minima 25–37 14.4 1:4+4/3+3:1 Visible Visible Grey

L. oshanensis 25-36 17.1±1.49 (13.4–18.6) 1:3+3/2+2:1, 1:3+3/3+3:1 Visible Visible

L. pelodytoides – – 2:4+4/3+3:1 – Visible Dark black

Ecologica Montenegrina , 80, 2024, 152-170

ventripunctata (1:2+2/2+2:1) have keratodont rows on both the upper and lower lips, differing from L. melanoleuca . However, species with absent lower lip keratodont rows, like L. baluensis , L. itiokai , L. mangshanensis , and L. juliandringi (0/0), as well as L. arayai (1/0), L. bidoupensis (1+1/0), still present fewer or no upper lip keratodont rows compared to L. melanoleuca . Third, a distinct lateral line was documented in this study, with visible lateral line organs in L. melanoleuca tadpoles, unlike the invisible lateral lines observed in species such as L. dringi , L. gracilis , and L. petrops . This is the first detailed report of the lateral line in Leptobrachella tadpoles. Fourth, the gut tubes were not visible in the abdominal region when they were fixed in preserved solution, although they were clearly apparent when the tadpoles were alive. Only one other species, L. kalonensis , exhibited these characteristics. Lastly, the color patterns of L. melanoleuca tadpoles—brown with light brown blotches—further distinguishes them from several other species, including L. baluensis , L. bidoupensis , L. bidoupensis , L. botsfordi , L. dringi , L. itiokai , L. kajangensis , L. mangshanensis , L. mjorbergi , L. oshanensis , L. rowleyae , L. yeae , and L. juliandringi , all of which lack spots, dots, or blotches.

The lateral line system in L. melanoleuca tadpoles, observed from stages 26 to 38, shows significant variation both between individuals and between the left and right sides of the same line. Multiple lateral lines, including the canthoinfraorbital, supraorbital, angular, dorsal, medial, and ventrolateral lines, are present, each with varying numbers of openings. These structures likely play important sensory roles throughout development ( Quinzio & Fabrezi, 2006; Quinzio et al., 2014), with their distribution unaffected by growth stages. The exact timing of the first appearance and disappearance of lateral lines remains unknown due to missing data, highlighting the need for further research to better understand their developmental timeline and function.

The buccal anatomy of Leptobrachella (formerly Leptolalax ) tadpoles has been previously reported, notably in the work of Kaewtongkum et al. (2014), which focused on the tadpole of Le. pelodytoides . However, that study did not employ DNA analysis for the identification of the tadpoles. Le. pelodytoides is particularly interesting due to its ventrolateral abdominal wall, which contains distinct lymphatic spaces that likely enhance stability in rocky habitats ( Altig et al., 2009). Additionally, the external narial apertures of megophryid tadpoles are characterized by a range of ornamentations, from simple rims to lobate rims and distinct tubes ( Altig et al., 2009). Furthermore, buccal anatomy data have been documented for other genera within the family Megophryidae , such as Leptobrachium ailaonicum ( Grosjean, 2001) and Brachytarsophrys feae ( Chuaynkern et al., 2022) . Consequently, we believe that further exploration of the buccal anatomy of Leptobrachella tadpoles will be essential and advantageous moving forward.

Tadpoles play a crucial role in enhancing estimates of anuran richness, since many species either move significant distances from their breeding habitats or remain dormant during the non-breeding season, making them challenging to detect. Thus, tadpole data is particularly important for studying the natural history and ecological requirements of various anuran species ( Le et al., 2023). This study addresses some of the knowledge gaps regarding this particular tadpole, which is vital for taxonomic studies as well as natural history and ecology. However, research on pects such as development, reproduction, and high-level molecular investigations of this species remains limited. Further studies are necessary to bridge these gaps. It is anticipated that this work will stimulate additional research on lateral line characteristics and physiology, tadpole feeding behaviors, and overall tadpoles’ behavior.

Acknowledgements

We would like to extend our deepest gratitude to the Faculty of Science at Khon Kaen University, the Faculty of Forestry at Kasetsart University, and the Khlong Saeng Wildlife Sanctuary under the Department of National Parks, Wildlife and Plant Conservation for their invaluable support in facilitating this research. Our sincere appreciation goes to the dedicated team at Khlong Saeng Wildlife Sanctuary, with special thanks to Anantachai Isaraphakdee and Sitthichai Prikdaeng for their vital assistance during fieldwork. We also wish to thank Nuttapong Khajitmathee for his contributions to map creation, and Boonsong Kongsook and Saksit Suwan for their assistance with CPD and SEM preparation. This research was partially funded by the Khon Kaen University Research and Graduate Studies and Faculty of Science Research Grant (2019-2020), awarded to Chantip Chuaynkern and Yodchaiy Chuaynkern. We further acknowledge the financial support of the National Research Council of Thailand (NRCT) (N34E670115) and the Centre of Excellence on Biodiversity (MHESI) (BDC-PG1- 167003).

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Figures

Figure 3. The specimen of the tadpole of L. melanoleuca (PR 03399.2, stage 35). (A) dorsal view of specimens; (B) ventral view of specimens, (C) lateral view of left side and (D) lateral view of right side.

Figure 4. The oral disc of tadpole of L. melanoleuca (PR 03399.2, stage 35) showing (A) photography of oral disc, (B) diagram of oral disc, (C) lower jaw sheath and (D) labial tooth row.

Figure 5. The lateral line system of L. melanoleuca, (A, C, E) lateral lines on specimen; (B, D, F) diagram of lateral lines, (A–B) Dorsal, (C–D) ventral, (E–F) lateral view of body.

Figure 6. Photographs showing the living tadpole of L. melanoleuca (PR 03399.2) in dorsal view.

Figure 7. Buccal roof of L. melanoleuca tadpole. (A) Buccal roof, (B) prenarial arena protrusion, (C) prenarial arena, (D) narial and (E) medial ridge (F) buccal roof arena papilla, and (G) tubercles in lower area of buccal roof arena [BR1: upper labium, BR2: upper jaw sheath, BR3: prenarial arena, BR4: prenarial arena protrusion, BR5: prenarial papilla, BR6: choana, BR7: postnarial papilla, BR8: median ridge, BR9: postnarial arena, BF10: buccal roof arena papilla, BF11: buccal roof arena].

Figure 8. Buccal floor of L. melanoleuca tadpole (PR 03399.7). (A) Buccal floor, (B) infralabial projection, (C) lingual papilla, (D) buccal floor arena papilla, (E) infralabial papilla, (F) tubercles in central area of buccal floor arena, and (G) tubercles in lower area of buccal floor arena [BF1: lower jaw sheath, BF2: infralabial projection, BF3: infralabial papilla, BF4: lingual papilla, BF5: tongue anlage, BF6 buccal floor arena papilla, BF7: buccal floor arena, BF8: buccal pockets, BF9: medial notch, BF10: filter rows, BF11: glottis, BF12: ventral velum].

Figure 9. Photo showing of stream of collected, rocky streams, tadpoles collected.