Contributions to Zoology, 86 (1) – 2017R.G. Bina Perl; Sarig Gafny; Yoram Malka; Sharon Renan; Douglas C. Woodhams; Louise Rollins-Smith; James D. Pask; Molly C. Bletz; Eli Geffen; Miguel Vences: Natural history and conservation of the rediscovered Hula painted frog, Latonia nigriventer

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Appendix 1 - Morphological description of adult specimens of Latonia nigriventer

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Body width about two times head width. Head flattened and about as long as wide with a projecting snout that appears rounded in dorsal as well as lateral view. Nostrils positioned dorsally and closer to the tip of the snout than to the eyes. Eyes protruding, with heart-shaped pupil and golden iris. A distinct transversal fold present in the neck (absent from species of Discoglossus, the sister genus of Latonia), varying in depth depending on the posture. In a bent-forward posture with stretched dorsal skin the fold merely discernible as a slight groove. Tympanic membrane indistinct and comparatively small. Forearms robust and muscular with four fingers. Fingers free with rounded or pointed tips (order of length: 1 < 4 < 2 < 3). Hind limbs muscular with five toes (order of length: 1 < 2 < 5 < 3 < 4). Metatarsalia largely separated by webbing. Webbing formula difficult to assess as subarticular tubercles absent: 1(1), 2(1), 3(1), 4(1), 5(1), with webbing starting more or less inconspicuous as dermal ridges. Snout-vent length (SVL; mean value (range)): ♀♀ 94.8 mm (69.0–128.4 mm; N = 64), ♂♂ 98.0 mm (66.6–121.4 mm; N = 44). Skin smooth with numerous small round tubercles on dorsal and ventral surfaces as well as limbs, but no distinct rows of tubercles. Tubercles on limbs closer to each other and less rounded, resulting in a rougher surface.

Colouration in life: Dorsally with ochre, brownish or auburn background colour and dark brown or olive-grey colour patches on head and dorsum; rarely with rusty coloured flecks. Characteristic dark V-shaped pattern on head with broad end positioned between eyes and posteriorly narrowing up to level of arms present in all L. nigriventer individuals. Likewise characteristic lateral stripes at head mostly starting posterior (sometimes anterior) of eyes and extending up to level of arms. Dark patterns often to some degree rimmed by a light beige line. Almost all individuals with an incomplete middorsal band of the background colour in the posterior part of the dorsum. No distinctly ocellated pattern, nor prominent middorsal band extending over the whole dorsum as present in the two colour morphs displayed by both, Discoglossus galganoi and D. pictus (Vences, 2012a, b), in any L. nigriventer individual examined. Ventrally, greyish-black with numerous characteristic white dots corresponding to raised tubercles and distributed more or less evenly across all ventral surfaces including arms, legs and plantar surfaces. In small juveniles (SVL 20–30 mm), white spots not raised as in adults. Border between ventral and dorsal pattern mostly gradually and rather inconspicuous; in some individuals colour transition associated with marbling in different hues of ochre, brown and grey that might include arms and legs. Femur and tibia often with more or less conspicuous dark crossbands. Single individuals almost without dorsal pattern and rather uniform. Ventral side sometimes rather light grey with inconspicuous white tubercles. Individuals found within deep leaf litter during winter months often displayed such a rather pale colouration.

Appendix 2 - Morphological data on the holotype of Latonia nigriventer

The holotype specimen of L. nigriventer (described as Discoglossus nigriventer Mendelssohn & Steinitz, 1943) is preserved in the collection of the Hebrew University of Jerusalem under the collection number HUJ 236 (Fig. 13 A–B). It is a subadult specimen, possibly a female. We here provide some additional morphological data complementing those of Mendelssohn and Steinitz (1943). Upon examination in November 2011 (measurements by MV) it had a SVL of 39.9 mm. Webbing formula was 1(2), 2i(2), 2e(2), 3i(2.5), 3e(2.5), 4i(2.5), 4e (2.75), 5(2.25). In addition, we also examined a second historical specimen collected in 1955 (no type status; Fig. 13 C–D). This specimen, HUJ 544, has faint remains of keratinised excrescences on some fingers but no clear nuptial pads. It therefore is likely a female. SVL is 76.6 mm, webbing formula is 1(0.5), 2i(1.5), 2e(1.5), 3i(2), 3e(2), 4i(2.75), 4e(2.75), 5(2). Measurements of the two specimens (all in mm; type specimen measurements followed by those of the second specimen in parentheses) are as follows: head width 13.3 (24.4), head length 14.5 (ca. 26.5), tympanum diameter 2.3 (4.6), ED 4.0 (6.7), eye-nostril distance 3.7 (6.1), nostril-snout tip distance 3.0 (5.0), nostril-nostril distance 3.1 (4.7), HAL 9.4 (17.0), forelimb length 17.5 (not measured), hind limb length 52.5 (not measured), foot length including tarsus 25.5 (49.7), foot length 15.7 (not measured), tibia length 16.5 (34.5).


Fig. 13. Latonia nigriventer specimens preserved in the collection of the Hebrew University of Jerusalem. A) Subadult holotype (collection number HUJ 236) in dorsal view; B) HUJ 236 in ventral view; C) specimen collected in 1955 (collection number HUJ 544) in dorsal view; D) HUJ 544 in ventral view. Both individuals are assumed to be females.

Appendix 3 - Morphological description of tadpoles of Latonia nigriventer

The following description refers to a single, preserved tadpole in developmental stage 34, fixed in 70% ethanol and preserved in 5% formalin (field number ZCMV 12962, BL 8.9 mm, TL 23.8 mm). Abbreviations used: A1 (first upper biserial keratodont row), A2 (second upper biserial keratodont row), A1-2 den (density of the keratodonts in biserial row A1-2 ), A1-2 dist (distance between biserial rows A1-2 , between outer biserial rows at centre of oral disk), A1-2 num (number of keratodonts in A1-2 ), BH (maximum body height), BL (body length), BW (maximal body width), DF (dorsal fin height at mid-tail), DG (size of the dorsal gap of marginal papillae), DMTH (distance of maximal tail height from tail-body junction), ED (horizontal eye diameter), EH (eyes height – from the lower curve of the belly to the centre of the eye), HAB (body height where axis of tail myotomes contacts body – from the lower curve of belly), IND (inter-narial distance – from the centre), IOD (inter-orbital distance – from the centre), JL (maximum jaw sheath length), LR (number of lower rows of keratodonts), LTRF (labial tooth row formula), MP (marginal papillae), MTH (maximal tail height), ND (naris diameter), NH (naris height – from the lower curve of the belly to the centre of the naris), NP (naris-pupil distance), ODW (maximum oral disk width), P1 (first lower biserial keratodont row), P1gap (medial gap in P1 ), P2 (second lower biserial keratodont row), P3 (third lower biserial keratodont row), P1-3 den (density of keratodonts in biserial row P1-3 ), P1-3 dist (distance between biserial rows P1-3 , between outer biserial rows at centre of oral disk), P1-3 num (number of biserial keratodonts in row P1-3 ), RN (rostro-narial distance), SBH (distance between snout and point of maximal body height), SBW (distance between snout and point of maximal body width), SE (snout-eye distance), SL (spiracle length – from the visible edges), SS (snout-spiracle distance), TAL (tail length), TH (tail height at beginning of tail), THM (tail height at mid-tail), Thorn-pap (thorn-shaped papillae), TL (total length), TMH (tail muscle height at beginning of tail), TMHM (tail muscle height at mid-tail), TMW (tail muscle width at beginning of tail), UR (number of upper rows of keratodonts), VF (ventral fin height at mid-tail) and VL (vent tube length).

In dorsal view, body elliptical with small constrictions of the body wall at the plane of the spiracle, maximal body width attained at anterior 1/3 of mid-body length (SBW 28.4% of BL), and snout rounded. In lateral view, body depressed (BW of 5.17 corresponds to BH), maximal body height attained between 2/5 and 3/5 of body length (SBW 58% of BL), and snout sloping. Eyes medium-sized (ED 8% of BL), not visible from ventral view, positioned far dorsally (EH 64% of BH) and directed laterally, situated between the 2/10 and 3/10 of the body length (SE 22% of BL), medium distance between eyes (IOD 55% of BW). Small rounded nares (ND 3% of BL), positioned at medium height (NH 55% of BH) and oriented anteriorly, situated distinctly closer to snout than to eye (RN 35% of NP) and lower than eye (NH 85% of EH), rather close distance between nares (IND 45% of IOD), dark spot posterior to nare present, other ornamentation absent. Spiracle crescentic, opening situated at centre of ventral side of the body (SL 4% of BL), directed posteriorly with inner wall present as a slight ridge, visible from ventral view, invisible from dorsal and lateral view; opening situated between the 2/5 and 3/5 of the body length (SS 56% of BL), Long medial vent tube (VL 19% of BL), attached directly to ventral fin. Tail moderately long (TAL 169% of BL), maximal tail height lower than body height (MTH 89% of BH), tail height at mid-tail lower than both body height and maximal tail height (THM 83% of BH and THM 93% of MTH), tail height at the beginning of tail slightly lower than body height (TH 92% of BH). Caudal musculature moderately developed (TMW 39% of BW, TMH 50% of BH and 57% of MTH, TMHM 32% of THM and 30% of MTH). Tail muscle does not reach tail tip (distance of tail muscle to tail tip 6% of TAL). Moderately high fins (DF 105% of TMHM, VF 110% of TMHM), at mid tail ventral fin higher than dorsal fin (VF 104% of DF). Dorsal fin inserts at the tail muscle posterior to the dorsal body-tail junction, first remains almost parallel to dorsal border of tail muscle, then rises gradually until the posterior 1/3 of the tail where it increases to attain its maximal height, and then decreases gradually towards tail tip. Ventral fin originates at the ventral terminus of the body, rises minimally until middle of tail, and then remains almost parallel to the ventral border of the tail muscle until 1/3 of the tail where it likewise decreases gradually towards the tail tip. Maximal tail height located at anterior 1/3 of tail (DMTH 35% of TAL), lateral line vein imperceptible, myosepta faintly discernible in lateral view in the proximal 2/5 of the tail, point where the axis of the tail myotomes contacts the body located centred in body height (HAB 51% of BH). Tail tip rounded. Oral disk relatively large (ODW 29% of BL and 49% of BW), positioned and directed anteroventrally, not emarginated. Oral disk not visible from dorsal view, upper labium is a continuation of snout. Single row of marginal papillae interrupted by a narrow gap on the upper labium (DG 12% of ODW), gap on the lower labium absent. Total number of marginal papillae 64. Short and moderately sized conical papillae, longest marginal papillae measured 0.1 mm. LTRF 2/3(1) after Altig and McDiarmid (1999), with two rows of keratondonts per ridge that are positioned at a distance of 0.5 mm from each other. P3 row generally biserial, but occasionally additional keratodons in between the two main rows. Very long A1 rows (88% of ODW). Density of keratodonts varies from 64/mm to 76/mm, A1 73/mm (total 206). Narrow gap in the first posterior interrupted P1 rows (P1gap 6% of P1 ). Rows alignment regular. Short discernible keratodont (0.08 mm). Distal keratodont same length as those in the middle. Space between marginal papillae and keratodont rows only absent in P2 . Partially keratinised jaw sheath, with the half part close to the edge being black and the remainder whitish coloured; finely pointed serrations; moderately wide jaw sheath (JW 51% of ODW) without medial concavity on the upper sheath. Lower jaw sheath V-shaped, partially keratinised and partially hidden by the upper jaw sheath (Table S3).

Colouration in life: Medium brown, translucent and with a uniformly distinct reticulation, inner anatomy well discernible. Dorsally, and with exception of the eye region, body covered by homogenous dark brown melanophoric pigments and golden speckles probably corresponding to guanophores. Tail of a slightly lighter hue than rest of body. Fins translucent, with dark brown spots of variable sizes and irregularly distribution, spot density higher in dorsal fin. Ventrally, oral disk and gular region reticulated, branchial regions reddish (Fig. 7).

Colouration in preservative: Uniformly dark brown, body covered by homogenous dark melanophoric pigments giving it a granular appearance, colour slightly darker above brain and trunk region. Tail musculature overlaid by dark brown speckles. Fins translucent and, with exception of outer rim, covered with brown speckles. Venter including spiracle pale with irregularly distributed pale grey spots, spiracle hardly discernible, intestinal coils visible. Besides the regular pigmentation, whole body distinctly reticulated as was previously known to be characteristic for the tadpoles of only the genera Bombina, Discoglossus and Pelodytes (Boulenger, 1891). The fine pigmentary network is most apparent on fins and paler ground colour, e.g. venter.

Variation: in total, three tadpoles were investigated, two of which were in developmental stage 34 (ZCMV 12962 and ZCMV 12963) and one was at developmental stage 25 (ZCMV 12961). The observation of the other voucher specimens from the same locality shows the same general morphology and typical oral disk configuration of the described specimen, independent of their developmental stage.