Mini scule, Scherz & Hutter & Rakotoarison & Riemann & Rödel & Ndriantsoa & Glos & Roberts & Crottini & Vences & Glaw, 2019

Scherz, Mark D., Hutter, Carl R., Rakotoarison, Andolalao, Riemann, Jana C., Rödel, Mark-Oliver, Ndriantsoa, Serge H., Glos, Julian, Roberts, Sam Hyde, Crottini, Angelica, Vences, Miguel & Glaw, Frank, 2019, Morphological and ecological convergence at the lower size limit for vertebrates highlighted by five new miniaturised microhylid frog species from three different Madagascan genera, PLoS ONE 213314, pp. 1-45 : 18-24

Mini scule sp. nov.

urn:lsid:zoobank.org:act:AC570728-78AE-4FDB-B9BE-87DB553C5ABE

( Figs 1–3 , 7 and 8 , Tables 1 and 2 )

Remarks. This species was previously listed as Stumpffia sp. 9 [ 17]; Stumpffia sp. 16/Ca16 [ 15, 40, 46]; Stumpffia sp. aff. tetradactyla “Southeast” [ 41]; and Stumpffia sp. 16 MV-2009 ( KC351485 ) [ 20].

ZFMK 53775 ( Fig 7D and 7E ), a specimen from Nahampoana in southeastern Madagascar (ca. 24.975˚S, 46.980˚E, ca. 60 m a.s.l.), collected by F. Glaw and J. Müller on 4 January 1992, is similar to M. scule sp. nov. and we consider it possible that it is a member of this species. However, in the absence of genetic data from this specimen and population, we here do not consider it within the definition of M. scule sp. nov. and refer to it as M. cf. scule .

Holotype ( Figs 2 , 7 and 8 ). ZSM 5943/2005 ( FGZC 2662 , GenBank accession number KC351485 for 16S rRNA gene), an adult presumed male specimen collected in Sainte Luce Reserve forest at the QMM climate station (24.7798˚S, 47.1713˚E, 23 m a.s.l.), Anosy Region, former Toliara province, southeastern Madagascar on 4 February 2005 by F. Glaw and P. Bora.

Paratype ( Fig 7 ). ZSM 5942/2005 ( FGZC 2661 , GenBank accession number EU341081 for 12S rRNA gene, tRNA-Val, and 16S rRNA e genes), an adult presumed male specimen with the same collection data as the holotype. The hands and feet of this specimen were all removed as tissue samples. ZSM 265/2018 ( SHR 09112018 ), an adult male specimen collected while calling in Sainte Luce Reserve parcel S9 (24.7606˚S, 47.1732˚E, 28 m a.s.l.) on 8 November 2018 by S. Hyde Roberts. Additionally, the following five specimens collected by S. Hyde Roberts between 8 and 20 October 2016 in Sainte Luce Reserve: UADBA-A Uncatalogued ( ACZCV 0 386 , GenBank accession number MK459315 for 16S rRNA gene) an unsexed adult specimen, and UADBA-A Uncatalogued ( ACZCV 0 387 , GenBank accession number MK459316 for 16S rRNA gene), a juvenile female specimen (sexed by incision, small dark brown egg follicles present), both collected at 24.754– 24.755˚S, 47.173˚E, ca. 20 m a.s.l. ; ZSM 577 /2016 ( ACZCV 0 383, GenBank accession

number MK459312 for 16S rRNA gene), an adult unsexed specimen collected at 24.7600˚S, 47.1746˚E, ca. 20 m a.s.l.; UADBA-A Uncatalogued ( ACZCV 0 384 , GenBank accession number MK459313 for 16S rRNA gene), an adult male specimen collected at 24.7604˚S, 47.1737˚E, ca. 20 m a.s.l. ; ZSM 578/2016 ( ACZCV 0 385 , GenBank accession number MK459314 for 16S rRNA gene), an adult unsexed specimen collected at 24.7550˚S, 47.1735˚E, ca. 20 m a.s.l.

Diagnosis. An extremely miniaturised frog assigned to Mini gen. nov. on the basis of its small size, curved clavicles, laterally displaced and reduced nasals, and fusion or loss of carpal

2. This assignment is supported by its genetic affinities ( Fig 1 ; [ 15, 17, 20]). It is separated by uncorrected p-distances of 10.4–11.2% in the analysed 3’ fragment of the 16S rRNA gene from other members of the genus Mini gen. nov., and 9.7–13.3% from all members of the genus Plethodontohyla .

Mini scule sp. nov. is characterised by the unique combination of the following characters (n = 3 probable male and 3 adult unsexed specimens): (1) male SVL 9.9–10.5 mm (adult SVL up to 10.8 mm); (2) ED/HL 0.40–0.51; (3) HW/SVL 0.31–0.38; (4) FARL/SVL 0.34–0.39; (5) TIBL/SVL 0.39–0.47; (6) HIL/SVL 1.41–1.68; (7) fingers 1, 2, and 4 strongly reduced; (8) toe 1 absent, toes 2 and 5 quite reduced; (9) maxillary and premaxillary teeth present; (10) vomerine teeth absent; (11) lateral colour border occasionally present; (12) black inguinal spots generally absent; (13) postchoanal vomer present, spatulate, medially fused to parasphenoid; (14) nasal cultriform and laterally displaced; (15) quadratojugal-maxilla contact weak; (16) zygomatic ramus of squamosal short, thick, and horizontal; (17) clavicles present, curving with simple lateral articulations, medially not bulbous; (18) prepollex small or absent; (19) carpal 2 absent or fused to post-axial carpal 3–5 element; (20) finger phalangeal formula 0-2-3-2; (21) toe phalangeal formula 1-2-3-4-3; (22) single-note, unpulsed calls, not emitted in series; (23) non-frequency modulated calls; (24) call dominant frequency 6675 ± 64 Hz (n = 51); (25) call duration 121.9 ± 8.7 ms (n = 51); (26) inter-call interval 1905.1 ± 398.3 ms (n = 50).

Within the genus Mini gen. nov., the new species can be distinguished from M. mum sp. nov. by the presence of maxillary and premaxillary teeth (vs absence), and less distinct lateral colour border. For diagnosis against M. ature sp. nov., see the diagnosis of that species, below.

This species is particularly similar to some extremely miniaturised Stumpffia species, but it can be distinguished from all Stumpffia based on the condition of the carpals, from almost all Stumpffia by the possession of maxillary and premaxillary teeth (present only in S. spandei, S. miovaova, S. makira, S. diutissima; unpublished data), and from all Stumpffia except S. tridactyla, S. contumelia, and S. obscoena by the extremely reduced fingers and toes. It differs from these latter three species in lacking a strong lateral colour border (vs present), curved clavicles (vs absent in S. contumelia and S. obscoena and straight or absent in S. tridactyla; unpublished data), and presence of neopalatine and divided vomer (vs absence of neopalatine and nondivided vomer in S. obscoena, S. tridactyla, and S. contumelia; unpublished data).

Calls differ significantly from M. mum sp. nov. in frequency, duration, and inter-call intervals (see Table 2 ), but resemble those of numerous Stumpffia species. For distinction, compare the values given in Table 5 of Rakotoarison et al. [ 14]. In call duration, the calls are most similar to S. gimmeli, S. larinki, and S. tridactyla, but they are higher in dominant frequency than S. gimmeli and S. larinki (6675 ± 64 Hz vs 4823 ± 302 Hz in S. gimmeli and 2914 ± 124 Hz in S. larinki), and lower in dominant frequency with a longer inter-call interval than S. tridactyla (dominant frequency 6675 ± 64 Hz Hz vs 7244 ± 200 Hz; inter-call interval 1905.1 ± 398.3 ms vs 1012 ± 39 ms).

Holotype description. Specimen in a moderately good state of preservation, the left arm removed as a tissue sample. Body oblong; head wider than long, narrower than body width; snout rounded in dorsal view, squared in lateral view; nostrils directed laterally, not protuberant, equidistant between tip of snout and eye; canthus rostralis indistinct, straight; loreal region flat, vertical; tympanum indistinct, round, about 55% of eye diameter; supratympanic fold absent; tongue long and thin, attached anteriorly, not notched; maxillary teeth present; vomerine teeth absent; choanae small and round, located very far forward. Forelimbs slender; subarticular tubercles single, indistinct; outer/palmar metacarpal tubercle rounded; inner metacarpal tubercle small and indistinct; hand without webbing; first, second, and fourth fingers strongly reduced, third finger basally broadened; relative length of fingers 1 <4 <2 <3, fourth finger slightly more reduced than second; finger tips not expanded into discs. Hind limbs slender; TIBL 43% of SVL; lateral metatarsalia strongly connected; inner metatarsal tubercle indistinguishable from completely reduced first toe; outer metatarsal tubercle absent; no webbing between toes; first toe absent, second and fifth toes extremely reduced; relative length of toes 2 <5 <3 <4; fifth toe distinctly shorter than third. Skin on dorsum smooth, without distinct dorsolateral folds. Ventral skin smooth.

After 12 years in 70% ethanol, the dorsum is metallic silver over the whole body, excepting brown colour in the inguinal region and the posterior surface of the thigh ( Fig 2 ). There is a moderately distinct colour border between the dorsal and ventral colouration that runs the length of the flank. The side of the head is dark brown, but this becomes increasingly flecked with cream posteriorly. The ventral and lower lateral colouration is cream flecked with brown, most densely on the anterior abdomen, and most loosely at the posterior abdomen. This flecking becomes akin to ocelli on the ventral surfaces of the legs. Colour pattern in life was the same as in preservative, but dorsal colouration was bronze instead of silver (compare Fig 7A and 7B with Fig 2 ). Iris was rust red.

Variation. For measurements, see Table 1. The paratypes strongly resemble the holotype in morphology. Colouration among paratypes is highly variable. ZSM 5942/2005 resembles the holotype but is steelier in colour ( Fig 7C ). Dark markings in the inguinal region are present in ZSM 5942/2005 and ZSM 265/2018, and both specimens have a more distinctly black flank than the holotype ( Fig 7 C–7F). ZSM 265/2018 additionally has broad burnt umber crossband on its thighs and shanks.

Bioacoustics. Calls recorded from ZSM 265/2018 by S. Hyde Roberts ( Fig 4B , Table 2 ) on 8 November 2018 at 10h16, at an air temperature of 30.4˚C. The individual was found in forest habitat (parcel S9 at 24.7606˚S, 47.1732˚E, 28 m a.s.l.), ca. 3 m from a lentic stream, with an estimated canopy height of 11 m and canopy cover of ~70% after a night of heavy rain. Call details (n = 51 in all cases except inter-call intervals, where n = 30): Calls consisted of a single note and were emitted at regular intervals without defined call series. Calls were not or only very slightly frequency modulated. For detailed call parameters, see Table 2 .

Osteology ( Fig 8 ). Based on ZSM 5942/2005 (not figured) and ZSM 5943/2005 (figured).

Cranium ( Fig 8 C–8F). Shape and proportions. Skull narrow, longer than wide, widest at the level of the dorsal end of the squamosal and the anterior edge of the otic capsule. Braincase proportionally broad, with a short rostrum.

Neurocranium. Ossification varies: highly ossified in ZSM 5942/2005 with ossified otic capsules, less ossified in ZSM 5943/2005, without otic capsule ossification. Only the anterior cone of the sphenethmoid is ossified and contacts the frontoparietal dorsally but is not in contact with any other bones. Prootic in dorsal contact with lateral flange of frontoparietal, ventral contact with parasphenoid alae, not approaching contralateral ventrally. Septomaxilla miniscule, very tightly curled, with a long and thin posterior ramus. Columella (stapes) well ossified, pars media plectra (stylus) long and nearly straight, posteriorly and dorsally oriented toward the elongated pars interna plectra (baseplate). Nasal narrow and cultriform, laterally displaced, curved downward laterally, the acuminate maxillary process not closely approaching maxillary pars facialis, broadly separated from contralateral. Frontoparietal with rounded anterior edge, laterally rather straight-edged, with short lateral flange covering prootic, posteriorly strongly (ZSM 5942/2005) or weakly (ZSM 5943/2005) connected to exoccipital, anteroventrally contacting sphenethmoid, lacking any dorsal process, separated from contralateral by a narrow gap, with a clear, rhomboid facet at the level of the prootics, which may represent a pineal foramen.

Parasphenoid with narrow, rather straight-edged cultriform process and slightly broader posterior-curved alae, considerably shorter than frontoparietals, in contact with exoccipitals posterodorsally, prootics dorsally along the edges of the alae, anteroventrally in contact with postchoanal vomer and not in contact with neopalatine; posteromedial process not participating in foramen magnum. Vomer divided into pre- and postchoanal portions; prechoanal portion narrow, simple, curved, with a suggestion of a lateral ramus; postchoanal portion spatulate and edentate, narrowly separated from its contralateral on the midline, in dorsal contact with the parasphenoid proximally and the neopalatine distally, lacking an anterior projection. Neopalatine simple, straight, weakly distinguishable from lateral postchoanal portion of vomer, laterally broadly separated from the maxilla, not exceeding the lateral-most point of the postchoanal vomer.

Maxillary arcade gracile, maxilla and premaxilla bearing numerous small teeth, anterior extension of maxilla exceeding lateral extent of premaxilla but not in contact with it. Premaxilla with a narrow acuminate dorsal alary process rising laterally, pars palatina shallowly divided into a narrow palatine process and broad lateral process. Maxilla with a low triangular pars facialis and a narrow pars palatina, its posterior tip acuminate and barely contacting the quadratojugal, the lingual surface of the pars palatina in contact with the anterior ramus of the pterygoid. Pterygoid with an exceptionally short medial ramus, long anterior ramus, and broad posterior ramus, posteriorly calcified to the quadratojugal complex. Quadratojugal weakly bowed laterally, broadly connected to the ventral ramus of the squamosal, bearing a small posteroventral knob, weakly anteriorly connected to the maxilla; the articulation of the mandible is apparently fortified by the mineralisation of the posterior ramus of the pterygoid +squamosal+quadratojugal posteroventral knob. Squamosal with a slender, sigmoid ventral ramus, broadened otic ramus, and short, thick zygomatic ramus, the otic ramus oriented dorsally and posteriorly, the zygomatic ramus horizontal.

Mandible slim and edentate, largely unremarkable, with a moderately raised coronoid process on the angulosplenial. Mentomeckelians separated from the dentary, with slightly bulbous, almost hooked ventrolateral projections sometimes present (present in ZSM 5942/2005, absent in ZSM 5943/2005).

Posteromedial processes of hyoid proximally rounded with a broad medial crista.

Postcranial skeleton ( Fig 8A, 8B, 8G and 8H ). Eight procoelous presacrals, with some differentiation errors in ZSM 5942/2005 leading to a transverse process forming on the head of the urostyle; all presacrals much broader than long, lacking neural spines, with round posterior articular processes, presacral I with a more or less complete neural arch, presacrals II–IV with thicker and longer transverse processes than V–VIII. Sacrum with expanded diapophyses, the leading and trailing edges roughly equally angled, the articulation type IIB sensu Emerson [ 42]. Urostyle bicondylar, long, not broadening posteriorly, with a somewhat flared head in ZSM 5943/2005 and with a low dorsal ridge.

Pectoral girdle without ossified prezonal or postzonal elements, with ossified clavicles, badly fractured in ZSM 5942/2005. Clavicle thin and weakly curved, with a simple lateral junction, shorter than the coracoid. Coracoid fairly narrow, not flared laterally, strongly flared medially with a curved medial articular surface with the contralateral. Scapula slender, with a thin pars acromialis, the cleithral border straight. Cleithrum ossified for two thirds the width of the scapular border, thickened anteriorly. Suprascapula unossified.

Arms and legs described only from ZSM 5943/2005, as the limbs of ZSM 5942/2005 were removed for DNA sequencing. Humerus with a well-developed crista ventralis and no medial or lateral cristae. Radioulna slender with a distinct sulcus intermedius. Carpals poorly ossified, composed of radiale, ulnare, element Y, and large post-axial element formed by carpals 3–5. Carpal 2 has either been lost or fused to the latter element. Finger phalangeal formula is reduced (0-2-3-2), and the terminal phalanges of the second and fourth fingers are small, round elements. Prepollex absent.

Pubis unossified in ZSM 5943/2005 and fully ossified in ZSM 5942/2005; iliac shafts passing ventral to and beyond sacrum, oblong in cross-section, with a weak dorsal crest and without a dorsal prominence and with a shallow oblique groove. Femur weakly sigmoid, lacking a posterior crest. Tibiofibula equal to femur in length, with a sulcus intermedius. Tibiale and fibulare fused proximally and distally. T1 and T2+3 tarsals present, T1 considerably smaller than T2+3. Centrale present, slightly smaller than T2+3. Prehallux diminutive. Phalangeal formula reduced (1-2-3-4-3). Terminal phalanges of toes 3 and 4 with knobs, those of other toes small, round elements.

Distribution, natural history, and conservation status. This species is known only from Sainte Luce, southeast Madagascar ( Fig 6 ). Records of ‘ Stumpffia tridactyla ’ from Mandena [ 47], and Vohimena mountains and the southern Anosy mountain chain [ 48], and of ‘ Stumpffia sp. aff. tetradactyla “Southeast”‘ from Tsitongambarika [ 49] may refer to this species but require verification. A specimen from Nahampoana (ZFMK 53775) resembles this species, but due to the lack of genetic data, we cannot confirm its identity. Calls of Stumpffia -like frogs from Nahampoana were described in Glaw and Vences [ 50], but these were lower in dominant frequency (ca. 5 kHz), and longer in call duration (ca. 250 ms) than those recorded in Sainte Luce that are here assigned to M. scule sp. nov. Two separate ‘ Stumpffia ’ calls from Nahampoana were included in Vences et al. [ 51], one as Track 51, ‘ Stumpffia sp. (Nahampoana)’, and a second as Cut 2 of Track 37, ‘ Stumpffia tetradactyla ’.

This species appears restricted to areas of deep leaf litter concomitant with semi-permanent water bodies such as shallow and slow-moving forest streams. Individuals call from concealed positions on adjacent stream banks during the day. Sainte Luce consists of 17 forest fragments (numbered S1–S17), covering approximately 1600 Ha of littoral forest. At present we assume that this species is microendemic to these forest fragments, and we have directly observed it in fragments S7, S8, and S9, but it appears to be absent from S1 and S2. It may also occur in other parcels of lowland forest nearby. Based on its current estimated Extent of Occurrence (= Area of Occupancy) of <10 km 2 in forest that is threatened and declining in quality despite protection status, we recommend this species be listed as Critically Endangered according to the IUCN Red List Criterion CR B1ab(iii) [ 44]. So far, no other described amphibian species are known to be restricted to Sainte Luce.

Etymology. We use the specific epithet ‘scule’ as an arbitrary combination of letters, in order to form a pun on ‘miniscule’ from the name in apposition, in reference to the fact that it is among the smallest known frogs from Madagascar and in the world. It is to be regarded as an invariable noun.

Figures

Fig 1. Stylised representation of the latest phylogeny of the microhylid subfamily Cophylinae. Based on the phylogenetic tree of Tu et al. [20] (reconstructed from a concatenated multi-gene DNA sequence data set) with numerous nomenclatural updates. See that paper for support values and phylogenetic context. The sister species of Rhombophryne proportionalis sp. nov. is an undescribed candidate species (R. sp. Ca7) that is not included in this phylogeny; its taxonomy will be treated elsewhere. Taxa highlighted in red are those described herein. Inset photos show a selection of the new species described herein, and representatives of the other small-bodied genera, Stumpffia and Anilany. Inset photos are not to scale. https://doi.org/10.1371/journal.pone.0213314.g001

Fig 2. Holotypes of the new species described in this paper and their hands. Whole specimens in dorsal (left) and ventral (right) view. Hand images not to scale. https://doi.org/10.1371/journal.pone.0213314.g002

Fig 3. Currently known localities of the new taxa described in this paper. The base map is USGS SRTM 1-Arc second digital elevation model. https://doi.org/10.1371/journal.pone.0213314.g003

Fig 4. Mini mum gen. et sp. nov. in life and its habitat in Manombo Special Reserve. (a-c) ZSM 861/2014, holotype, in (a) anterolateral view on a thumbnail, (b) dorsolateral view on a leaf, (c) ventral view. (d, e) ZSM 862/2014, paratype, in (d) ventral view, and (e) lateral view on a thumbnail. (f, g) ZMB 83194, paratype, in (f) dorsolateral view, and (g) ventral view. (h) ZMB 81993, paratype, in dorsolateral view. (i) ZMA 20172 in posterodorsolateral view. (j) Habitat of the new species in Manombo Special Reserve. https://doi.org/10.1371/journal.pone.0213314.g004

Fig 6. Osteology of Mini mum gen. et sp. nov. holotype (ZSM 861/2014). (a-c) Whole skeleton in (a) dorsal, (b) lateral, and (c) ventral view. (d-g) Skull in (d) lateral, (e) ventral, (f) anterior, and (g) dorsal view. (h) Foot in ventral view. (i) Hand in ventral view. Abbreviations for all osteological figures: angspl, angulosplenial; col.pip, pars interna plectra of columella; col.pmp, pars media plectra of columella; cpl(s), carpal(s); den, dentary; exoc, exoccipital; exoc.occ, occipital condyle of exoccipital; fpar, frontoparietal; max, maxilla; max.fp, facial process of maxilla; mmk, mentomeckelian; n, nasal; n.mp, maxillary process of nasal; npl, neopalatine; oc, otic capsule; prsph. ap, parasphenoid alary process; prsph.cp, parasphenoid cultriform process; prsph.pmp, parasphenoid posteromedial process; pmx, premaxilla; pmx.ap, premaxilla ascending process; pmx.lp, premaxilla lateral process; pmx.pp, premaxilla palatine process; povom, postchoanal portion of vomer; pro, prootic; prvom, prechoanal portion of vomer; pt.ar, pterygoid anterior ramus; pt.mr, pterygoid medial ramus; pt.vr, pterygoid ventral ramus; qj, quadratojugal; smx, septomaxilla; spheth, sphenethmoid; sq. or, squamosal otic ramus; sq.vr, squamosal ventral ramus; sq.zr, squamosal zygomatic ramus; tsl(s), tarsals; vt, vomerine teeth. https://doi.org/10.1371/journal.pone.0213314.g006

Fig 7. Mini scule gen. et sp. nov. in life and its habitat in Sainte Luce Reserve. (a, b) ZSM 5943/2005, holotype, in (a) dorsolateral and (b) ventral view. Black lines in the two pictures are scanning artefacts from damaged analogue slides. (c) Probably ZSM 5942/2005, paratype, in dorsolateral view. (d-f) ZSM 265/2018 (SHR 09112018) in (d) dorsal, (e) dorsolateral, and (f) ventral view. Note the numerous pink cf. Endotrombicula mites on the abdomen and legs. (g) Habitat in Sainte Luce Special Reserve. (h, i) Mini cf. scule from Nahampoana, ZFMK 53775 in (h) lateral and (i) ventral view. https://doi.org/10.1371/journal.pone.0213314.g007

Fig 8. Osteology of Mini scule gen. et sp. nov. holotype (ZSM 5943/2005). (a, b) Whole skeleton in (a) dorsal and (b) ventral view. (c-f) Skull in (c) lateral, (d) ventral, (e) anterior, and (f) dorsal view. (g) Foot in ventral view. (h) Hand in ventral view. For abbreviations, see Fig 6. https://doi.org/10.1371/journal.pone.0213314.g008