Research Article |
Corresponding author: Bryan L. Stuart ( bryan.stuart@naturalsciences.org ) Academic editor: Angelica Crottini
© 2019 Somphouthone Phimmachak, Stephen J. Richards, Niane Sivongxay, Sengvilay Seateun, Yodchaiy Chuaynkern, Sunchai Makchai, Hannah E. Som, Bryan L. Stuart.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Phimmachak S, Richards SJ, Sivongxay N, Seateun S, Chuaynkern Y, Makchai S, Som HE, Stuart BL (2019) A new caruncle-bearing fanged frog (Limnonectes, Dicroglossidae) from Laos and Thailand. ZooKeys 846: 133-156. https://doi.org/10.3897/zookeys.846.33200
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A new species of the dicroglossid frog genus Limnonectes is described from recent and historical museum specimens collected in central and southern Laos and northeastern Thailand. Limnonectes savan sp. nov. has males that bear a caruncle on top of the head, and most closely resembles L. dabanus from adjacent southern Vietnam and eastern Cambodia. However, the new species is readily distinguished from L. dabanus, and all other caruncle-bearing species of Limnonectes in mainland Southeast Asia, by its adult and larval morphology, mitochondrial DNA, and advertisement call. Its description brings the total number of caruncle-bearing species of Limnonectes to six.
Amphibia, bioacoustics, larval morphology, Limnonectes dabanus, mitochondrial DNA, Southeast Asia
The dicroglossid frog genus Limnonectes Fitzinger, 1843 currently contains 73 species that are distributed from southern China and the Ryukyu Islands of Japan south and eastward to Papua New Guinea (
Our collective fieldwork during 1998–2016 at multiple localities in central and southern Laos and northeastern Thailand, and examination of historical museum specimens and the literature (
Specimens collected in the field were humanely euthanized by immersion in tricaine methanesulfonate (MS-222;
Measurements were taken to the nearest 0.1 mm using dial calipers under an ocular microscope. Adult measurements followed
EYE eye diameter;
FTL pes length from tip of fourth toe to base of inner metatarsal tubercle;
HDL head length from tip of snout to rear of jaws;
HDW maximum head width;
HND manus length from tip of third digit to base of palmar tubercle;
IML inner metatarsal tubercle length;
IMW inner metatarsal tubercle width;
IND internasal distance;
IOD interorbital distance, measured as minimum distance between eyes on top of head;
LAL forearm length, from elbow to base of palmar tubercle;
SHK shank length;
SNT snout length from tip of snout to anterior margin of eye;
SVL snout-vent length;
TGH thigh length, from knee to midline of vent;
TMP horizontal diameter of tympanum.
Terminology for the cap-like structure, “caruncle,” followed
Total genomic DNA was extracted from liver or muscle samples from 53 individuals of Limnonectes (Appendix 2) using the ArchivePure DNA Cell/Tissue Kit (5 Prime) or the DNeasy Blood and Tissue Kit (Qiagen). An 551–1,949 nucleotide base pair (bp) fragment of mitochondrial (mt) DNA that encodes parts of the 12S rRNA, tRNA valine, and 16S rRNA genes was amplified by the polymerase chain reaction (PCR; 94–95° C 45s, 53–60° C 30s, 72° C 1 min) for 35 cycles using at least one of four primer combinations: (i) 12L1 (
Newly generated sequences were aligned to the same homologous sequences used by
Advertisement calls were recorded from one male (
Comparisons of “Lao-Thai” specimens to all other Limnonectes species having males that bear a caruncle on top of the head revealed consistent differences in body size, shape and position of the head caruncle, shape and length of odontoid processes, dorsal skin texture, toe length, and larval tooth rows.
The aligned dataset contained 2,533 characters. The standard deviation of split frequencies was 0.005711 among the four Bayesian runs, and the Estimated Sample Sizes (ESS) of parameters were ≥ 1,092. The “Lao-Thai” specimens were recovered as a well-supported monophyletic group (Fig.
Fifty percent majority-rule consensus phylogram resulting from Bayesian analysis of 2,533 aligned characters of mitochondrial DNA from dicroglossid frogs in the genus Limnonectes, and the outgroup taxa Fejervarya limnocharis and Quasipaa spinosa (not shown). Black circles at nodes indicate Bayesian posterior probabilities ≥ 0.99, and open circles at nodes indicate Bayesian posterior probabilities ≥ 0.95. Numbers at terminal tips are GenBank accession numbers. Voucher and locality data for sequenced samples are provided in Appendix 2.
Nine advertisement calls from the “Lao-Thai” male specimen
On the basis of these corroborated lines of evidence from multiple, independent datasets (larval morphology, adult male morphology, mitochondrial DNA, and male advertisement calls), we hypothesize that the “Lao-Thai” specimens represent a distinct evolutionary lineage that should be recognized as a species, described herein as:
Limnonectes sp. Chan-ard, 2003: 120.
Laos, Savannakhet Province, Vilabouli District:
Laos, Khammouan Province, Boualapha District:
Laos, Champasak Province, Pakxong District:
Thailand, Ubon Ratchatani Province, Na Chaluai District:
Thailand, Ubon Ratchatani Province, Buntharik District:
Thailand, Ubon Ratchatani Province, Sirindhorn District:
The specific epithet savan means paradise in the Lao language, and is a commonly used, truncated form of the name for Savannakhet Province, Laos, that contains the holotype and most paratype localities of the new species. The specific epithet savan is a noun in apposition.
Savan Fanged Frog (English), Kop Hone Savan (Lao), Kop Panomdongrak (Thai).
Assigned to the genus Limnonectes on the basis of its inferred phylogenetic position (Fig.
Habitus moderately stocky; body broad anteriorly, tapering to narrow groin. Head broad and depressed, head width equal to head length. Snout obtusely pointed in dorsal view; round, projecting well beyond lower jaw in profile; nostril dorsolateral, much closer to tip of snout than to eye, below canthus; internarial distance 72% of interorbital distance; canthus rostralis indistinct, rounded, slightly constricted behind nostrils; lores concave, oblique; eye diameter 59% of snout length, upper eyelid width 50% of interorbital distance; pineal ocellus visible; tympanum imperfectly circular, not elevated from side of head, annulus visible, tympanum diameter equal to eye diameter and greater than distance between tympanum and eye; small, slit-like vocal sac openings on floor of mouth near lateral margin of tongue; vomerine teeth on two oblique ridges, equidistant to each other as to choanae; two large odontoid processes at front of mandible, triangular, tapered, length subequal to depth of mandible at base of process; median triangular symphysial knob at mandibular symphysis.
Forelimbs robust. Fingers relatively slender, without webbing, with fringe of skin on preaxial and postaxial sides of all fingers, fringes on Fingers II-III movable; tips of fingers rounded, expanded into discs; relative finger lengths II < I < IV < III; distinct, rounded subarticular tubercles, one on Fingers I–II, two on Fingers III–IV; distinct thenar tubercle; two palmar tubercles in contact at base of Fingers II–IV; nuptial pad absent.
Hindlimbs robust. Toes relatively slender; tips of toes rounded, expanded into small discs; relative toe lengths I<II<III=V<IV on right foot, I<II<V<III<IV on left foot; webbing on Toe I to base of disc, on preaxial side of Toe II to level midway between subarticular tubercle and disc and continuing as fringe to base of tip, on postaxial side of Toe II to base of disc, on preaxial side of Toe III to level of distal subarticular tubercle and continuing as fringe to base of disc, on postaxial side of Toe III to base of disc, on preaxial and postaxial sides of Toe IV to level of distal subarticular tubercle and continuing as fringe to base of disc, and on Toe V to base of disc; moveable fringe of skin on outer margins of Toes I and V; distinct fold on distal two-thirds of tarsus; distinct, elongate, oval, inner metatarsal tubercle, length approximately 59% distance between tip of toe I and tubercle; no outer metatarsal tubercle.
Skin on dorsum and flank shagreened with large, irregular, scattered tubercles; tubercles tipped with single, whitish spinule on loreal region, eyelid, lower back near groin, around vent, and ventral surfaces of tibiotarsus and foot; dense clusters of warts (enlarged tubercles), each tipped with numerous whitish spinules, on dorsal surfaces of shank; interorbital caruncle consisting of low-profile swelling without free posterior margin, extending from level of anterior margin of eye to level midway between posterior margin of eye and tympanum, with highest point between eyes; hypertrophied jaw musculature forming two low postorbital swellings on top of head at level of tympanum; distinct supratympanic fold from posterior corner of eye to axilla; rictal gland absent; dorsolateral fold absent; aberrant, triangular skin tag near midline of back; skin on throat with weak longitudinal wrinkles, that on remaining ventral surfaces smooth.
Dorsum tan. Loreal region, tympanic region, and dorsal surfaces of digits whitish gray. Back of head, dorsolateral region, under canthus and dorsoposterior region of tympanum with gray mottling. Dorsal and posterior surfaces of thigh, posterior surface of shank, and groin with yellowish wash. Lips with irregular, broad, gray bars, dorsal surfaces of limbs with cross-bands. Interorbital bar tannish yellow. Iris bronze with black vermiform mottling, black vertical and horizontal bars forming shape of a single plus sign (“+”) over eye (Fig.
Based on
Dorsal surfaces nearly uniformly brown with indistinct, scattered, dark brown mottling, lips with indistinct dark brown bars, and dorsal surfaces of limbs with indistinct dark brown cross-bands. Tympanum and forelimbs with lighter brown than remaining dorsum. Interorbital bar indistinct. Ventral surfaces light brown with dark brown mottling, becoming uniformly dark brown on ventral surfaces of hands and feet (Fig.
Based on subsample of eggs (
Limnonectes savan sp. nov. A oviposition site with dead palm frond in situ in Savannakhet Province, Laos B eggs (
Based on largest individual in series of 28 larvae (
Females lack caruncle and postorbital swellings (Fig.
Measurements (mm) of types of Limnonectes savan sp. nov. and L. dabanus. Abbreviations defined in the text. Values are presented as range; mean ± standard deviation (SD).
Measurement | L. savan | L. savan | L. savan | L. dabanus | L. dabanus |
Holotype male |
Paratype males | Paratype females | Males | Females | |
n = 22 | n = 14 | n = 24 | n = 14 | ||
SVL | 56.2 | 39.0–53.6; 45.9 ± 4.8 | 38.9–55.2; 47.2 ± 5.5 | 48.8–64.4; 56.9± 4.5 | 42.3–57.4; 48.2 ± 4.8 |
HDL | 26.8 | 16.6–26.0; 20.4 ± 2.9 | 15.7–22.2; 19.2 ± 2.2 | 21.2–38.0; 26.8 ± 3.5 | 17.3–24.1; 20.0 ± 1.9 |
HDW | 26.9 | 17.0–26.1; 20.8 ± 2.7 | 16.1–22.1; 19.8 ± 2.3 | 20.8–35.4; 26.5 ± 3.2 | 17.1–23.9; 19.8 ± 1.8 |
SNT | 9.9 | 6.1–9.9; 7.8 ± 1.1 | 6.1–9.2; 7.6 ± 0.9 | 8.9–14.3; 11.0 ± 1.1 | 7.0–9.8; 8.2 ± 0.8 |
EYE | 5.8 | 4.5–6.9; 5.4 ± 0.7 | 4.4–6.8; 5.5 ± 0.8 | 5.4–7.3; 6.2 ± 0.5 | 5.1–7.1; 5.9 ± 0.6 |
IOD | 7.6 | 3.0–7.5; 5.1 ± 1.2 | 3.1–4.9; 4.2 ± 0.4 | 5.2–7.9; 6.4 ± 0.7 | 3.8–5.3; 4.3 ± 0.3 |
TMP | 5.8 | 3.4–6.3; 4.7 ± 0.8 | 3.4–5.5; 4.3 ± 0.7 | 5.0–9.5; 6.5 ± 1.3 | 3.7–5.3; 4.4 ± 0.6 |
IND | 5.5 | 3.6–6.0; 4.6 ± 0.6 | 3.9–5.6; 4.6 ± 0.5 | 4.6–7.7; 5.7 ± 0.6 | 4.1–5.9; 4.7 ± 0.5 |
SHK | 26.9 | 18.6–25.8; 22.2 ± 2.1 | 18.9–25.4; 22.5 ± 2.4 | 23.0–31.1; 27.8 ± 2.7 | 21.9–29.3; 24.5 ± 2.1 |
TGH | 28.9 | 18.4–29.1; 23.9 ± 2.8 | 20.0–28.0; 24.3 ± 3.2 | 22.0–33.1; 29.1± 3.1 | 21.9–31.1; 24.8 ± 2.3 |
LAL | 11.0 | 7.1–10.4; 8.8 ± 0.9 | 7.7–10.8; 9.2 ± 1.2 | 8.5–14.1; 11.6 ± 1.3 | 8.2–11.6; 9.8 ± 0.9 |
HND | 13.8 | 9.7–14.6; 12.1± 1.3 | 9.3–14.0; 12.2 ± 1.4 | 12.4–16.6; 15.1 ± 1.3 | 11.1–14.1; 12.5 ± 1.0 |
FTL | 26.6 | 18.8–25.7; 22.3 ± 2.1 | 18.0–25.4; 22.5± 2.4 | 22.4–31.4; 27.3 ± 2.7 | 21.8–27.0; 23.8 ± 1.6 |
IML | 4.1 | 2.3–4.0; 3.2 ± 0.4 | 2.9–4.0; 3.4± 0.4 | 3.0–4.6; 3.8 ± 0.4 | 2.8–4.2; 3.3 ± 0.4 |
IMW | 1.3 | 1.0–2.1; 1.3 ± 0.3 | 0.8–1.6; 1.3 ± 0.2 | 1.1–2.0; 1.5 ± 0.2 | 0.9–1.6; 1.1 ± 0.2 |
TMP:EYE | 1.0 | 0.7–1.1; 0.9 ± 0.1 | 0.7–0.9; 0.8 ± 0.1 | 0.8–1.5; 1.0 ± 0.2 | 0.7–0.8; 0.8 ± 0.1 |
TMP:SVL | 0.1 | 0.1–0.1; 0.1 ± 0.0 | 0.1–0.1; 0.1 ± 0.0 | 0.1–0.2; 0.1 ± 0.0 | 0.1–0.1; 0.1 ± 0.0 |
The holotype is the largest male in the type series, with the next largest male (
Dorsal surfaces are lighter brown, or have more gray mottling, in some specimens than in the holotype. Lip bars on lips and crossbands on dorsal surfaces of limbs more distinct in some specimens than in the holotype. Six paratypes (
Limnonectes savan is known to occur in central and southern Laos (Khammouan, Savannakhet, and Champasak Provinces), and northeastern Thailand (Ubon Ratchatani; Fig.
Localities of studied specimens of Limnonectes savan sp. nov. at the holotype locality at Savannakhet Province, Vilabouli District, Laos (star) and the paratype localities at Khammouan Province, Boualapha District, Laos (square), Champasak Province, Pakxong District, Laos (asterisk), Ubon Ratchathani Province, Sirindhorn District, Thailand (circle), and Ubon Ratchathani Province, Na Chaluai and Buntharik Districts, Thailand (triangle).
Limnonectes savan breeds in puddles on the forest floor during the rainy season. A chorus of calling males, including paratype male
Limnonectes savan occurs in sympatry with L. lauhachindai in Ubon Ratchathani Province in northeastern Thailand (Appendix 1), but its geographic distribution appears to be parapatric to that of L. dabanus in southern Laos, and to that of L. gyldenstolpei in central and southern Laos and northeastern Thailand (Appendix 1).
Limnonectes savan differs from all other species of mainland Southeast Asian Limnonectes, except L. gyldenstolpei, L. lauhachindai, L. dabanus, L. macrognathus, and L. plicatellus, by having mature males with an interorbital caruncle (sensu
Comparisons of males of four similar, caruncle-bearing species of Limnonectes in preservative: Dorsal (above) and ventral (below) views of A L. savan sp. nov. holotype male (
Comparisons of Limnonectes dabanus and L. savan sp. nov. in preservative A lateral view of head of males A1 L. macrognathus (
Limnonectes savan further differs from L. gyldenstolpei, L. lauhachindai, L. dabanus, and L. macrognathus by having relative toe lengths I<II<III=V<IV, with the tips of Toes III and V reaching the base of the distal subarticular tubercle on Toe IV (vs. relative toe lengths I<II<V<III<IV, with the tip of Toe III shorter not reaching the distal subarticular tubercle on Toe IV in L. gyldenstolpei, L. lauhachindai, L. dabanus, and L. macrognathus). Limnonectes savan further differs from L. plicatellus by having males with much larger body size (SVL ≤ 43.0 in L. plicatellus;
Limnonectes savan is phenotypically most similar and phylogenetically most closely related (Fig.
Male secondary sexual characters are unknown in L. khammonensis (
New species of Limnonectes continue to be discovered and described in mainland Southeast Asia (e.g.
This study found that L. savan is phylogenetically related to the caruncle-bearing species L. dabanus, L. gyldenstolpei, and L. lauhachindai (Fig.
Finally, the taxonomic identity of the Lao-endemic L. khammonensis, known only from the female holotype specimen taken near Ban Na Pe (“Napé;
Fieldwork in Laos was conducted with permission of the Ministry of Agriculture and Forestry, Vientiane, Laos; Bounnhot Namsena, Deputy Head of the Forestry Section, Savannakhet Province; and Phonekham Sayasombath, District Governor of Vilabouli District. Fieldwork in Thailand was conducted under the auspices of the Thailand Natural History Museum, the National Research Council of Thailand, and the head of Phu Jong-Na Yoi National Park. The Wildlife Conservation Society Laos Program, Lane Xang Minerals Ltd. (Sepon), and MMG Ltd. (Sepon) provided invaluable logistical support. Chatchay Chuechat, Derin Henderson, and Singthong Sanvixay assisted with fieldwork. David Kizirian and Lauren Vonnahme (
Appendix 1. Comparative specimens examined
Data type: species data
Appendix 2. Fejervarya, Quasipaa, and Limnonectes 16S sequences used in the phylogenetic analysis
Data type: species data