Limnonectes coffeatus, Phimmachak & Sivongxay & Seateun & Yodthong & Rujirawan & Neang & Aowphol & Stuart, 2018

Phimmachak, Somphouthone, Sivongxay, Niane, Seateun, Sengvilay, Yodthong, Siriporn, Rujirawan, Attapol, Neang, Thy, Aowphol, Anchalee & Stuart, Bryan L., 2018, A new Limnonectes (Anura: Dicroglossidae) from southern Laos, Zootaxa 4375 (3), pp. 325-340 : 329-336

Limnonectes coffeatus sp. nov.

Holotype: NCSM 77788 (field tag BLS 13957 ), adult male ( Fig. 2 ), Laos, Champasak Province, Pakxong District, Bolaven Plateau , Dong Hua Sao National Protected Area , Houay Tad Seua Stream , 15.06237°N 106.21075°E, 1,235 m elev., coll. 3 August 2010 by Bryan L. Stuart, Niane Sivongxay, and Sengvilay Seateun.

Paratypes: NCSM 77786, adult female, same data as holotype except coll. 30 July 2010. NCSM 77785, NCSM 77787 ( Fig. 3–4 ), two adult females; NUOL 0 0 0 60, one immature female; same data as holotype except coll. 15.06515°N 106.21394°E, 1,245 m elev., 28 July–01 August 2010. FMNH 258440, immature female; FMNH 258441, FMNH 258530, two immature males, same data as holotype except coll. near 15.06528°N 106.21750°E, 1,200 m elev., coll. 22–23 September 1999 by Bryan L. Stuart and Harold F. Heatwole.

Referred Specimens: NCSM 77944, larvae (n =13; Fig. 5 ), same data as holotype except coll. 30 July 2010.

Etymology: The specific epithet refers to the coffee plant genus Coffea , in reference to the extensive coffee plantations that are encroaching into the natural habitat of the new species at upper elevations on the Bolaven Plateau of southern Laos.

Diagnosis: Assigned to the genus Limnonectes on the basis of its inferred phylogenetic position ( Fig. 6 ), the presence of fang-like odontoid processes on the lower jaw ( Emerson et al. 2000; Lambertz et al. 2014), and males with hypertrophied heads ( Lambertz et al. 2014). A small-sized Limnonectes having the combination of the single available adult male with SVL 37.9, adult females with SVL 38.1–42.1; male lacking postorbital caruncle; male with hypertrophied head; odontoid processes on anterior margin of lower jaw, larger in male than in females; male with horizontal diameter of tympanum greater than that of eye, females with horizontal diameter of tympanum less than or equal to that of eye; tubercles on dorsum enlarged and rounded, but not arranged in rows; interdigital webbing of foot present; throat uniformly gray in male; tympanum uniformly colored, without distinct dark marking on upper half; flank without distinct spots; and ova with pigmented poles.

Description of holotype: Habitus moderately stocky; body tapering to groin. Head broad and depressed; head width greater than head length. Snout obtusely pointed in dorsal view, round in profile, projecting well beyond lower jaw in profile; nostril dorsolateral, much closer to tip of snout than to eye, below canthus; internarial distance greater than interorbital distance; canthus rostralis indistinct, rounded, slightly constricted behind nostrils; lores concave, oblique; eye diameter 55% of snout length, interorbital distance greater than upper eyelid width; pineal ocellus visible; tympanum round, not elevated from side of head, annulus visible, tympanum diameter approximately 117% 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, closer to choanae than to each other; two large odontoid processes at front of mandible; median triangular protuberance at mandibular symphysis.

Forelimbs moderately robust. Fingers relatively slender, without webbing or skin flaps; tip of fingers rounded, weakly expanded into discs; relative finger lengths II = IV <I <III; distinct 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 moderately robust. Toes relatively slender; tips of toes rounded, expanded into small discs; relative toe lengths I <II <V <III <IV; webbing on Toe I to base of disc, on preaxial side of Toe II to distal margin of subarticular tubercle and continuing as a fringe to base of tip, on postaxial side of Toe II to base of tip, on preaxial side of Toe III to level of distal subarticular tubercle continuing as a fringe to base of tip, on postaxial side of Toe III to base of tip, on preaxial and postaxial sides of Toe IV to level of penultimate subarticular tubercle and continuing as a fringe to base of tip, and on Toe V to base of tip; moveable flap 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 shagreened with large round tubercles, not elongated or arranged in rows; tubercular ridges from posterior margin of eyelid to occiput forming “W”-shape; caruncle absent from interorbital region; distinct supratympanic ridge from posterior corner of eye to axilla; rictal gland absent; dorsolateral fold absent; skin on throat wrinkled, that on remaining ventral surfaces smooth.

Color of holotype in life: Dorsum brown with ochre wash; larger tubercles on dorsal surface of head and back orange-red with black margins, those on occiput forming “W”-shape; dark brown triangular marking between eyes with yellow anterior margin; dark brown alternating white-gray bands on upper and lower lips; dorsal surfaces of limbs with indistinct gray-brown bands; dorsal surfaces of thighs with yellowish-wash. Chin and throat gray; ventral surfaces of chest, belly and limbs yellow; ventral surfaces of hands and feet brown; iris bronze.

Description of larvae NCSM 77944: Based on Gosner stage 28 individual in series of thirteen larvae ( Fig. 5 ). Body oval in dorsal view, slightly compressed dorsoventrally, maximum body width slightly anterior to level of spiracle. Nares dorsal, without raised rim. Eyes dorsolateral, not visible from below. Spiracular tube single, sinistral on left side, angled slightly dorsally, aperture near midline of side and projecting posteriorly, approximately midway between eye and end of body. Tail slender, tapering gradually in distal one-third to rounded tip, origins of dorsal and ventral fins at end of body, dorsal fin only slightly deeper than ventral fin. Oral disk ventral, subterminal, width about 36% maximum width of body. Anterior labium with single row of papillae on lateral margins; posterior labium with two staggered rows of papillae on lateral and posterior margins; papillae homogenous in length. Labial tooth row formula 2(2)/3(1). All posterior tooth rows subequal in length. Upper and lower jaw sheaths black with serrated margins, upper sheath without median convexity. No distinct cluster of glandules posterior to eye on dorsal surface of body, subequal to interorbital distance. Color in life brown with gold flecking on belly and distinct black spots on dorsal surface of body, caudal muscle, and dorsal and ventral tail fins.

In preservative, brown faded to light brown, black faded to dark brown, gold flecking absent. Measurements of larvae are summarized in Table 3.

Variation: Females have narrower heads in dorsal view than holotype male ( Fig. 4 ). Females have relatively smaller tympana than holotype male, with the horizontal diameter of the tympanum less than or equal to that of the eye. Females have smaller odontoid processes at front of mandible than holotype male. Female NCSM 77787 has wider black margins on larger tubercles on dorsal surface of head and back than holotype, and with black rather than brown or gray-brown bands on interorbital region, lips, and dorsal surfaces of limbs. Females lack wrinkled skin and have paler coloration on throat than holotype male. Females contain ova with pigmented poles. Measurements of adults are summarized in Table 2.

SVL 37.9 38.1–42.1; 40.5 ± 2.1 41.1 37.6–46.4; 40.9 ± 2.9 36.0–41.2; 38.1 ± 2.2 HDL 17.9 15.8–17.7; 16.9 ± 1.0 19.8 16.0–21.4; 18.9 ± 1.6 14.4–16.8; 15.7 ± 0.9 HDW 18.6 15.2–17.3; 16.6 ± 1.2 18.0 17.6–23.2; 20.0 ± 1.8 13.1–17.7; 15.9 ± 1.7 SNT 7.6 5.9–7.3; 6.8 ± 0.8 6.7 6.1–9.0; 7.4 ± 0.8 5.6–6.9; 6.5 ± 0.5 EYE 4.2 3.6–4.4; 4.1 ± 0.4 5.5 4.3–5.7; 5.0 ± 0.4 4.2–5.1; 4.7 ± 0.3 IOD 3.8 3.2–3.3; 3.3 ± 0.1 4.4 4.0–6.2; 5.1 ± 0.7 2.6–4.6; 3.7 ± 0.7 TMP 4.9 3.5–4.3; 3.9 ± 0.4 5.3 4.4–7.3; 5.6 ± 0.9 2.8–3.8; 3.4 ± 0.4 IND 4.6 4.3–4.6; 4.5 ± 0.2 4.8 4.3–6.2; 5.3 ± 0.6 3.6–5.2; 4.6 ± 0.6 SHK 20.2 19.4–20.0; 19.7 ± 0.3 20.5 17.6–22.1; 20.4 ± 1.3 17.6–20.5; 19.5 ± 1.2 TGH 20.3 19.4–21.0; 20.5 ± 0.9 18.0 18.9–22.9; 20.9 ± 1.4 18.5–20.7; 20.0 ± 0.9 LAL 7.2 7.9–8.4; 8.2 ± 0.3 n/a 7.5–8.7; 8.0 ± 0.4 6.7–8.0; 7.6 ± 0.6 HND 10.5 10.0–11.0; 10.4 ± 0.5 n/a 9.1–12.0; 10.6 ± 0.8 8.5–10.9; 10.0 ± 0.9 FTL 19.3 18.7–20.7; 19.9 ± 1.0 n/a 17.5–22.4; 20.3 ± 1.5 16.7–20.1; 19.3 ± 1.4 IML 3.0 2.5–2.6; 2.5 ± 0.0 n/a 1.8–2.7; 2.3 ± 0.3 1.8–3.1; 2.5 ± 0.4 IMW 1.2 0.9–1.2; 1.1 ± 0.1 n/a 0.8–1.1; 1.0 ± 0.1 0.7–1.2; 0.9 ± 0.2 TMP:EYE 1.2 0.9–1.0; 1.0 ± 0.0 1.0 1.0–1.4; 1.1 ± 0.2 0.5–0.8; 0.7 ± 0.1 TMP:SVL 0.1 0.1–0.1; 0.1 ± 0.0 0.1 0.1–0.2; 0.1 ± 0.0 0.1–0.1; 0.1 ± 0.0

Molecules: The aligned dataset contained 1,633 characters. The standard deviation of split frequencies was 0.003805 among the four Bayesian runs, and all parameters had an Estimated Sample Size (ESS) ≥267. The new species was recovered as a member of a clade containing L. doriae , L. plicatellus , L. hascheanus , L. limborgi , and L. macrognathus , and this clade was sister to L. kohchangae ( Fig. 6 ). The exact sister taxon relationship of the new species was unresolved, but the new species was not recovered to be the sister species to L. kohchangae ( Fig. 6 ). The holotype, six paratypes, and referred larvae of L. coffeatus sp. nov. are identical in the 16S gene fragment (differing only by a single insertion-deletion event), but have an uncorrected pairwise divergence of 10.13–11.72% from L. kohchangae (n =11), the species to which it is phenotypically most similar (but not phylogenetically related; Fig. 6 ).

Distribution, natural history, and conservation: Limnonectes coffeatus sp. nov. is only known from wet evergreen forest ( Fig. 7 ) near to the top (≥ 1,200 m elev.) of the Bolaven Plateau in Dong Hua Sao National Protected Area, Pakxong District, Champasak Province, southern Laos ( Fig. 1 ). Most of the known specimens were collected at night on the forest floor within 3 m of the bank, or in shallow water, of small, rocky streams. The immature female (NUOL 00060) was found at night (2215 h) during heavy rain on a forest trail, and one adult female (NCSM 77787) was found during the morning (1030 h) on leaf litter on the forest floor 20 m from a stream. Larvae (NCSM 77944) were collected at night (2130 h) in a 1.5 m wide slow-moving stream, approximately 20 cm deep, with a substrate of sand and algae-covered rocks ( Fig. 7 ). An adult female (NCSM 77786) was collected in shallow water at the same time and place as the larvae. Calls and oviposition sites are unknown.

Coffee ( Coffea spp.) is the major agricultural export from Laos (for example, it was the largest agricultural export from the country in 2010), and 95% of Lao coffee is grown on the Bolaven Plateau (85% from Pakxong District alone) ( Schönweger & Messerli 2015). Rainfall, temperature and physical properties of the soil render the upper elevations of the Bolaven Plateau highly favorable for growing coffee ( Trelo-Ges et al. 2010; Schönweger & Messerli 2015), and a system of small landholder coffee production has been in place on the Bolaven Plateau since French colonial times ( Delang et al. 2012). Today, approximately 15,000 households (69% of farms) on the Bolaven Plateau depend on coffee production as their primary source of income ( Delang et al. 2012; Schönweger & Messerli 2015). Large-scale coffee plantations have also been established in recent years ( Delang et al. 2012; Schönweger & Messerli 2015). As such, forest at upper elevations on the Bolaven Plateau has been extensively converted into coffee plantations ( Thewlis et al. 1998), with human settlements (and accompanying plantations) encroaching even inside the boundaries of Dong Hua Sao National Protected Area ( Delang et al. 2012). Bauxite mining, rubber plantations, and hydroelectric dam construction place additional pressures on forested areas of the Bolaven Plateau ( Delang et al. 2012). All known records of the new species were obtained in closed-canopy forest (and not in coffee plantations), and so we expect that the continued conversion of forest into coffee plantations at upper elevations of the Bolaven Plateau likely presents a significant threat to the persistence of the new species.

Comparisons: Four other species of Limnonectes in the region ( Laos, Vietnam, Cambodia and Thailand) have the combination of males lacking a cephalic caruncle (swollen or cap-like structure; sensu Lambertz et al. 2014); adult SVL <60 mm; and reduced interdigital foot webbing: L. limborgi ( Sclater, 1892) , L. hascheanus ( Stoliczka, 1870) , L. doriae ( Boulenger, 1887) and L. kohchangae ( Smith, 1922) .

Limnonectes coffeatus sp. nov. differs from L. limborgi by having webbing on Toe V reaching base of tip (webbing more reduced in L. limborgi ); dorsum with rounded tubercles, not arranged in rows (if present, tubercles elongated and arranged in rows in L. limborgi ), and having a uniformly gray throat in males (absent in L. limborgi ).

Limnonectes coffeatus sp. nov. differs from L. hascheanus by having adults with SVL> 26 (adults with SVL <26 in L. hascheanus ); having males with two large odontoid processes at front of mandible (absent in L. hascheanus ); and having ova with pigmented poles (enlarged, non-pigmented ova in L. hascheanus ).

Limnonectes coffeatus sp. nov. differs from L. doriae by having males with two large odontoid processes at front of mandible (absent in L. doriae ); having adults with SVL <43 (SVL 56 in L. doriae ); and males with TMP> EYE (TMP <EYE in L. doriae ).

Limnonectes coffeatus sp. nov. is phenotypically most similar (but not phylogenetically related; Fig. 6 ) to L. kohchangae ( Fig. 8 ). Limnonectes coffeatus sp. nov. differs from L. kohchangae by having dorsum with rounded tubercles, not arranged in rows (distinctly elongated and arranged in rows in L. kohchangae ; Fig. 9 ); by lacking a distinctly bi-colored tympanum, with the upper half heavily pigmented (present in L. kohchangae ; Fig. 8 ); by lacking dark spots on flank (present in L. kohchangae ; Fig. 8 ); and having narrower limb bands, with the maximum shank band width <50% horizontal diameter of eye (maximum shank band width>50% horizontal diameter of eye in L. kohchangae ; Fig. 9 ).

Figures

FIGURE 1. Map illustrating the type locality (white star) of Limnonectes coffeatus sp. nov. in Champasak Province, Laos; the type locality (black star) of L. kohchangae in Trat Province, Koh Chang Island, Thailand; and localities of studied specimens (black circles) of L.kohchangae in Pursat and Kampong Speu Provinces,Cambodia.

FIGURE 2. Holotype male of Limnonectes coffeatus sp. nov. (NCSM 77788, field tag BLS 13957) in dorsal (A), ventral (B), profile (C), and posterior (D)views immediately prior to preservation.

FIGURE 3. Paratype female of Limnonectes coffeatus sp. nov. (NCSM 77787, field tag BLS 13934) in lateral (A) and dorsal (B) views in life, and in ventral (C) and lateral (D) views immediately prior to preservation.

FIGURE4. Paratypefemale (NCSM 77787, left)and holotype male (NCSM77788,right)of Limnonectes coffeatussp.nov. in dorsal (A) and ventral (B) views in preservative.

FIGURE 5. Larvae of Limnonectes coffeatus sp. nov. (NCSM 77944, field tag BLS 13928) immediately prior to preservation (A), and inlateral (B), ventral (C) and oral (D) views in preservative.

FIGURE 6. Fifty percent majority-rule consensus phylogram resulting from Bayesian analysis of 1,633 aligned characters of the mitochondrial 16S gene from frogs of the dicroglossid genera Fejervarya, Quasipaa, and Limnonectes. Black circles at nodes indicate Bayesian posterior probabilities ≥ 0.99, open circles at nodes indicate Bayesian posterior probabilities ≥ 0.95, and no circle at nodes indicate Bayesian posterior probabilities <0.95. Numbers at terminal tips are GenBank accession numbers.

FIGURE 7. Limnonectes coffeatus sp. nov. habitat of adults (A) and larvae (B) at the type locality on the Bolaven Plateau, Pakxong District, Champasak Province, Laos.

FIGURE 8. Adult males of Limnonectes kohchangae in life at the type locality of Ko Chang Island, Ko Chang District, Trat Province, Thailand (A; ZMKU AM 01158) and at Phnom Aural, Kampong Speu Province, Aural District, Cambodia (B; CBC 01907).Lectotype male (BMNH 1947.2.3.60) of L. kohchangae in dorsal (C) and ventral (D) views.

FIGURE 9. Dorsal views in preservative of males (A) of L. kohchangae (left, NCSM 79541) and L. coffeatus sp. nov. (right, holotype NCSM 77788) and females (B) of L. kohchangae (left, NCSM 79542) and L. coffeatus sp. nov. (right, paratype NCSM 77787).