Limnonectes bannaensis, Ye, Fei, Xie & Jiang, 2007, Ye, Fei, Xie & Jiang, 2007

We   GoogleMaps present a description of the tadpole

L. bannaensis View in CoL

from the extreme south of their known range based on 12 specimens ( ITBCZ 3636–47 ) collected at night in evergreen forest in Loc Tien Commune , Phu Loc District, Thua Thien Hue Province, Vietnam (N16.23319, E107.99256, 134 m asl; Fig. 1A View FIGURE 1 ) on the 08 th April, 2019. GoogleMaps At the time of collection, the air temperature was 27.0 oC, water temperature 25.4 oC and ambient humidity was 89.8% (measured by a Kestrel 3500 weather meter). The tadpoles were collected from a small pool beside a large stream. The puddle had a 0.5 m radius, and a maximum depth of 0.2 m. At the time of collection, approximately 15 tadpoles were active and feeding. The substrate consisted of rocks and stone and a layer of submerged leaf litter ( Fig. 1B View FIGURE 1 ). Adult L. bannaensis were not seen at the collection site. A further 8 specimens ( ITBCZ 3628–35 ) collected at night in evergreen forest in Sao La A Luoi Nature Reserve , A Luoi District, Thua Thien Hue Province, Vietnam (N16.11176, E107.47611, 458 m asl; Figs. 1A and 1B View FIGURE 1 ) on the 15 th of January 2024 at 21:15 GoogleMaps . At the time of collection the air temperature was 20.1 oC, the water temperature was 19.2 oC and ambient humidity was 99.2% (measured by Kestrel 3500 weather meter). The tadpoles were collected from a puddle in a dry stream bed. The puddle had a 0.5 m radius, and a maximum depth of 0.2 m. At the time of collection, approximately 11 tadpoles were active and feeding. The substrate consisted of rocks and stone and a layer of submerged leaf litter ( Fig. 1B View FIGURE 1 ). Adult L. bannaensis were not seen at the collection site.

Specimens were photographed in life before being euthanised using a 20% solution of benzocaine which was dissolved in stream water held in food grade plastic bags of water containing live tadpoles. Tissue samples (tail clips) for molecular analyses were extracted from freshly euthanised specimens and stored in absolute ethanol prior to fixation of specimens with 10% formalin for 24 hours and subsequent storage in absolute ethanol. Specimens were subsequently deposited at the Institute of Tropical Biology Zoological Collection (ITBCZ), Ho Chi Minh City, Vietnam.

Total genomic DNA was extracted from ethanol-preserved tissues using a DNeasy® Blood and Tissue Kit (QIAGEN GmbH, Hilden, Germany), following the manufacturer’s protocols for purification of genomic DNA from animal tissues. We amplified a section (550 bp) of 16S (mtDNA) using the primers 16SL2021 (5’ CCTACCGAGCTTAGTAATAGCTGGTT-3’) modified from Hedges (1994) 16SH1 (5’-CTCCGGTCTGAACTC AGATCACGTAGG-3’) by Hedges & Maxson (1993). PCR amplification was carried out in 25-μL reactions volume, including 12.5-μL 2 x ES Master Mix (CWBIO, China), 0.75-μL of each primer (10 pmol/μL), 1-μL of cDNA template, and 10-μL H 2 O. Negative controls were included in each PCR batch. Thermocycling was performed on an Eppendorf Mastercycler EpS (Eppendorf, Hamburg, Germany) under the following conditions: initial denaturation 94°C (5 mins), followed by 35 cycles of 94°C (1 min) denaturation, 55°C (1 min) annealing and 72°C (1 min) extension, followed by a final extension step at 72°C (10 mins). All PCR products were purified using ExoSap-ITTM (USB Corporation, Ohio USA), and sequenced in both 5’ and 3’ directions at Macrogen (Seoul, South Korea). Sequence chromatograms were edited and checked by eye for quality using BioEdit V. 7.0.5.3 ( Hall 1999). The new sequences were then checked on BLAST (The National Center for Biotechnology Information) ( Altschul et al. 1990) to verify their approximate identity and sequences were deposited in GenBank.

Tadpoles at both sites were assigned to L. bannaensis based upon mitochondrial 16S RNA sequences taken from the tails of ITBCZ 3628 (GenBank accession number PQ757240 ) collected from Sao La A Luoi Nature Reserve and ITBCZ 3638 (GenBank accession number PQ757247 ) collected from Loc Tien Commune . Uncorrected p-distances of 16S mtDNA between these two specimens was 0.19%. Uncorrected p-distances of 16S mtDNA between the 2 tadpoles collected from Thua Thien Hue Province and from L. bannaensis collected from their approximate type locality 1350 km to the northwest, in Xishuangbanna, Yunnan Province, China ( KIZ011728 , GenBank accession number KU599852 ) differed by 1.7–1.9% . Uncorrected p-distance (with partial deletion of gaps and missing data) was calculated using MEGA 11 ( Tamura et al. 2021).

We used ImageJ 1.49 ( Schneider et al. 2012) to measure preserved tadpoles from photographs. Staging followed the Gosner (1960) table and we used Altig & McDiarmid (1999) for the terminology for tadpole description. Measurements followed McLeod (2008) and include: body length (BL), tail length (TL), total length (ToL), body width (BW), body height (BH), eye diameter (LED), interorbital distance (LIO), internarial distance (LIN), naris– snout distance (LNS), eye–naris distance (LEN), spiracle tube width (SW), tail muscle height at body junction (TMH1) and mid length (TMH2), and tail height (TH).

The description of tadpole morphology is based on 20 specimens collected in Sao La A Luoi Nature Reserve and Phu Loc District ( Figs. 1–4 View FIGURE 1 View FIGURE 2 View FIGURE 3 View FIGURE 4 , Table 1 View TABLE 1 ) at Stages 26–41. Body elongated (BW 31.0–62.0% BL), and depressed (BH 46.0–61.0% of BW); snout rounded ( Figs. 2–4 View FIGURE 2 View FIGURE 3 View FIGURE 4 ); nares anterodorsally positioned, typically closer to eyes than to tip of snout, rim not raised; eyes positioned dorsolaterally, oriented laterally, comparatively small (LED 7.0–11.0% of BL); conical tube shaped sinistral spiracle, positioned anteriorly to the widest part of the body, closer to eye than to end of body, fused to body with a free short distal portion; tail muscle long, tail length approximately twice body length (TL 157.0–236.0% of BL) pointed tail tip, tail musculature distinct, muscle height greatest at tail base and gradually tapers towards tail tip, tail fins prominent, both upper and lower tail fins reach maximum height approximately half way along tail length; cup-like, emarginated oral disc, anteroventrally positioned, fringed with single row of short, lobed papillae interrupted in a wide dorsal gap and a short ventral gap. Two upper rows of keratinised labial teeth with medial gap in 2 nd row; three lower rows of keratinised labial teeth with a medial gap in 1 st row [labial tooth row formula 2(2)/3(1)]; jaw sheaths black, robust; upper jaw sheath broadly curved, with distinctly serrated edges and a distinct medial convexity, lower jaw sheath developed with distinctly serrated edges ( Fig. 4 View FIGURE 4 ).

Colouration. In preservative ( Fig. 2 View FIGURE 2 ), body and tail cream with dark brown flecks which increase in density on the dorsal surface of body posterior to eyes, these flecks form a brown bar running from anterior edge of eye to snout; tail fin similar to colouration in life; pupil milky, iris black. In life ( Fig. 3 View FIGURE 3 ), light brown body with dark brown flecks which increase in density on dorsal surface of body posterior to eyes, these flecks form a brown bar running from anterior edge of eyes, joining at tip of snout; oral disc translucent; whitish neuromasts arranged in two mirroring lines running from snout between nares and eyes, running dorsolaterally along body before continuing along lateral surfaces of tail muscle, whitish neuromasts surround each eye; pupil black and round; iris orange, speckled with black dots; internal gills distinctly reddish; tail beige-brown, upper and lower tail fins cream, tail fins with two equally spaced, bold, broad, black bars, stripes and in some individuals, blotches; one saddle at body tail junction, other immediately after it. These saddles extend to stripes on latter half of tail; translucent skin on ventral surface of body, gut coil clearly visible.

Variation.Tail tips missing in ITBCZ 3639 and ITBCZ 3642. The colouration of L. bannaensis tadpoles collected from Thua Thien Hue Province in this study differs from those collected further north from their type locality and from Nghe An Province. The equally spaced, bold, broad, black bars and blotches are absent in these populations ( Ye et al. 2007; Cao et al. 2020). In the original species description, tadpoles we assigned to L. bannaensis without associated molecular data and only the second half of the tadpole tail is reported as being black ( Ye et al. 2007); however this is not apparent in the associated figure ( Fig. 2B View FIGURE 2 of Ye et al. 2007). Cao et al. (2020), who confirmed species identification with molecular data, report 4–5 black stripes on the tail fins but the associated figure (Fig. 6) shows a tadpole at Stage 25 with diffuse black vertical markings rather than bold, broad, black bars and blotches. Tadpoles we collected in this study have a wide dorsal gap and a short ventral gap in the papillae which fringe the oral disc; the short ventral gap is not present in the figure of the oral disc in Cao et al. (2020).

We compared the tadpole of L. bannaensis with other Limnonectes tadpoles reported from mainland southeast Asia where species identity has been confirmed with molecular data. The tadpole of L. bannaensis differs from the tadpole of L. dabanus (Smith) by the presence of bold black vertical stripes on the tail (absent in L. dabanus at Gosner Stages 29 and 31; Rowley et al. 2014); from L. isanensis by the presence of bold black vertical stripes on the tail (vs. absent in L. isanensis at Gosner Stage 38; Ampai et al. 2015); from L. megastomias McLeod by the presence of bold black vertical stripes on the tail (vs. absent in L. megastomias at Gosner Stage 40; McLeod 2008); from L. nguyenorum McLeod, Kurlbaum & Hoang by having a larval tooth row formula 2(2)/3(1) [vs. 1/3(1–2) in L. nguyenorum at Gosner Stage 28; Ziegler et al. 2015] and the presence of bold black vertical stripes on the tail (absent in L. nguyenorum at Gosner Stages 25–29); and from L. selatan Matsui, Belabut & Ahmed by a short ventral gap in marginal papillae (no ventral gap in L. selatan at Gosner Stage 40) and by the presence of bold black vertical stripes on the tail (vs. dark bands in L. selatan ).

This is the only Limnonectes species currently known to exhibit such bold black striped tail colouration, although dark bars are present in L. selatan and tadpoles putatively assigned to L. utara Matsui, Belabut & Ahmed. A similar pattern has been reported in other tadpoles, such as in the hylids Pseudis platensis ( Dixon et al. 1995) , Litoria micromembrana ( Altig & Channing 1993) and Xenohyla truncata ( Dias et al. 2023) . The bold colouration on the tails of tadpoles has been suggested to attract the attention of potential predators to less vital regions of the body (e.g., Thibaudeau & Altig 2012), to confuse predators, or to provide camouflage in linearly arranged habitats ( Altig & Channing 1993). Elucidating the function of the bold black vertical bars on the tail of L. bannaensis tadpoles would require field experiments.