Limnonectes larvaepartus, Iskandar, Djoko T., Evans, Ben J. & McGuire, Jimmy A., 2014

Iskandar, Djoko T., Evans, Ben J. & McGuire, Jimmy A., 2014, A Novel Reproductive Mode in Frogs: A New Species of Fanged Frog with Internal Fertilization and Birth of Tadpoles, PLoS ONE 9, pp. 1-14 : 3-10

Limnonectes larvaepartus new species ( Figs. 1, 2)

Urn:lsid:zoobank.org:act: 60AA7136-89A0-4DBB-9FBC-BD0FAF8A214C

This species has been referred to in the literature under the names Limnonectes larviparus [11] and Limnonectes ‘‘ovovivipar’’ [4], both of which created nomina nuda. This species also corresponds to Limnonectes sp. V in [2, 3].

Etymology

The species name larvaepartus (from ‘larvae’, plural of larva, the early form of an animal, and ‘partus’, to give birth to) reflects the unique reproductive mode of this tadpole-bearing species.

Holotype

MZB.Amph.23755 (Field Number: BSI 0 605, see Fig. 1), an adult male, collected from Dunu Village , (0.92353o N; 122.64386o E) at 189 m elevation, Kecamatan Anggrek, Kabupaten Gorontalo , Provinsi Gorontalo, Sulawesi , Indonesia by J.A. McGuire & team, 18 October 2004 .

Paratypes

Paratypes (n=30) are from Sulawesi Utara, Gorontalo, Sulawesi Tengah, and Sulawesi Barat Provinces: MZB 2834 , a gravid female with 33 translucent tadpoles (Gosner stage 23) from the left oviduct and 2 from outside the body, from Toraut (00'33.72o N, 123'54.23o E), Bogani Nani Wartabone National Park, Kabupaten Bolaang Mongondow , Sulawesi Utara at 370 m elevation, by D. T. Iskandar, 15 August 1991 ; FMNH 252453 , a female, from Toraut, Bogani Nani Wartabone National Park, Kabupaten Bolaang Mongondow , Sulawesi Utara by D. T. Iskandar, August 1991. MVZ 255545 , 256009-11, 256013 from Desa Lombongo (‾1.43346, 120.30800), Kecamatan Suwawa, Kabupaten Bone Bolango, Bogani Nani Warta Bone National Park , Provinsi Gorontalo at 75 m elevation by J. A McGuire and team, 20 October 2004. ZRC 1.3258 (left femur removed) from Toraut, Bogani Nani Wartabone National Park, Kabupaten Bolaang Mongondow , Sulawesi Utara at 370 m elevation, by D. T. Iskandar, 15 August 1991. MZB 2835– 2841 from Toraut, Bogani Nani Wartabone National Park, Kabupaten Bolaang Mongondow , Sulawesi Utara at 370 m elevation, by D. T. Iskandar, 15 August 1991 & 12 July 1992. MVZ 255548–49 from Desa Pontak (‾2.62910, 118.99300), Kecamatan Motoling, Kabupaten Minahasa Selatan , Provinsi Sulawesi Utara at 285 m elevation by J. A. McGuire and team, 13 October 2004. MZB 3117, near Potolok river, Lolak, Bogani Nani Wartabone National Park at 350 m elevation, Kabupaten Bolaang Mongondow, Sulawesi Utara ; MZB 3118 , male from seashore forest near Bungbungan River, Lolak, Bogani Nani Wartabone National Park, Kabupaten Bolaang Mongondow , Sulawesi Utara. MZB 3120 from Tangkorak River, Desa Pindol, Kecamatan Lolak, Bolaang Mongondow, Sulawesi Utara, by Mumpuni, 26 June 1995 ; MZB 3121 from Tangaga Forest, Dudepo, Bolaang Mongondow , Sulawesi Utara, by I. Maryanto, 20 October 1995 ; MZB 3122 from Potolok River, Bogani Nani National Park, Lolak , Sulawesi Utara by Mumpuni , 19 June 1995 ; MZB 3124 from Bungbungan River, Bogani Nani National Park, Lolak , Sulawesi Utara by Mumpuni , 19 June 1995 ; MZB Amph.8108 from Toraut, near Bogani Nani Wartabone National Park , Sulawesi Utara. LSUMZ 84209 , 84214 , 84221 , 84224 from Desa Torout, Bogani Nani Wartabone National Park, Kabupaten Bolaang Mongondow , Provinsi Sulawesi Utara at 267 m elevation by J. A. McGuire on 6 and 11 September 2001 .

doi:10.1371/journal.pone.0115884.g002

Other referred specimens

LSUMZ 84218 , 84219 from Desa Torout, Bogani Nani Wartabone National Park, Kabupaten Bolaang Mongondow , Provinsi Sulawesi Utara by J.A. McGuire. FMNH 130991 , 106930 from Buang-buang Island , Sulawesi Utara. MZB 3119 , juvenile from Seashore forest near Bungbungan River, Lolak, Bogani Nani Wartabone National Park, Kabupaten Bolaang Mongondow , Provinsi Sulawesi Utara. AMNH 167199 from Tangkoko National Park (1.570083o N, 125.156933o E), Kabupaten Minahasa , Provinsi Sulawesi Utara. MVZ 255546 from Desa Salumpaku (‾1.60757, 119.29900), Kecamatan Banawa, Kabupaten Donggala , Provinsi Sulawesi Tengah. MVZ 255547 from Desa Kelapa Dua (‾1.60757, 119.29900), Kecamatan Anreapi, Kabupaten Polewali Mandar , Provinsi Sulawesi Barat. MVZ 268426 , 268428–30 , 268432 from Polewali-Masawa Road (River 1) (‾2.65490, 118.93300), Kecamatan Polewali, Kabupaten Polewali Mandar , Provinsi Sulawesi Barat. MVZ 268309–10 , 268313 , 268317–19 , 268322 , 268325 , MZB.Amph. 20675 , 20677–80 from Desa Uaemate ( Tasiu-Tibo Road ; S02.61550, E119.14417), Kecamatan Kaluku, Kabupaten Mamuju , Provinsi Sulawesi Barat. MZB.Amph.20663 from Desa Kabiraan (‾2.62460, 119.14700), Kecamatan Ulumunda, Kabupaten Majene , Provinsi Sulawesi Barat .

Distribution

Limnonectes larvaepartus occurs across the Northern Peninsula, as well as on the western margin of Sulawesi’s Central Core ( Fig. 3). We do not know the full extent of the species’ range in the Central Core because the central highlands of Sulawesi remain poorly explored herpetologically. Several genera have species range boundaries in this same general region (e.g. the flying lizards Draco spilonotus and D. walker [12], the fanged frogs Limnonectes sp. I and L. sp. D [3], and the tarsiers Tarsius lariang and T. dentatus [13].

Diagnosis

Prior workers have recognized substantial species diversity in the genus Limnonectes on Sulawesi. However, diagnosing many of these lineages on the basis of morphology is challenging, and several authors have instead opted to apply names to Sulawesi specimens representing species from outside Sulawesi. Consequently, the following names have all been incorrectly applied to Sulawesi populations: the Lesser Sundas species (type locality: Flores Island) L. dammermani (Mertens, 1927) , the Bornean species L. finchii (Inger, 1966), the Mollucan species (type locality: Ambon) L. grunniens (Daudin, 1801) , and the Philippine taxa L. leytensis (Boettger, 1893) , L. magnus (Stejneger, 1909) , and L. palavanensis (Boulenger, 1894) . These names should not be applied to Sulawesi populations, as was verified phylogenetically for several of these taxa [3]. Only four Sulawesi species have been described: L. arathooni (Smith, 1927) , L. heinrichi (Ahl, 1933) , L. modestus (Boulenger, 1882) , and L. microtympanum (van Kampen, 1909). However, we will show elsewhere that L. heinrichi is a junior synonym of L. modestus and the species complex that we have referred to previously [2, 3] as L. modestus remains undescribed – thus, at present there are but three valid described species of Sulawesi Limnonectes .

Limnonectes larvaepartus can be distinguished from all other described species of Limnonectes by its reproductive mode ( Fig. 2). It can be further differentiated from all described Sulawesi species by its combination of body size (mean male SVL =37.4; female SVL = 40.2 mm), coloration, tympanum size, and degree of hind foot webbing, as well as on the basis of phylogenetic placement ( Fig. 3). Limnonectes arathooni is endemic to Sulawesi’s Southwestern ( SW) Peninsula south of the Tempe Depression, and thus does not occur within the range of L. larvaepartus . It is similar in size to the new species (male SVL = 36.6 mm, females= 39.6 mm), but differs in having substantially reduced webbing (extending to penultimate phalange of fourth toe vs. to toe disc), in lacking fine granular dorsal tubercles, and in having melanic spots above the forelimb insertion, a fine ridge extending posteriorly behind each eye, and an alternative derived reproductive mode in which males guard clutches of terrestrial eggs that hatch into tadpoles that then make their own way to an adjacent stream by sliding down steep stream-side embankments [14]. Limnonectes microtympanum , like L. arathooni , is restricted to the SW Peninsula south of the Tempe Depression, and thus does not overlap in geographical range with L. larvaepartus . Limnonectes microtympanum is moderately large (male SVL =78.4; female SVL = 72.4 mm), and thus much larger than the new species. Limnonectes microtympanum also differs from the new species in having proportionally smaller tympana ( TY / SVL =0.05 + 0.01 in males, 0.06 + 0.01 in females versus 0.08 + 0.01 in both sexes in L. larvaepartus ). The new species occurs in broad sympatry with L. modestus , which is a moderate sized (male SVL = 70.2 mm; female SVL =64.0 mm) inhabitant of fast-moving streams and substantially larger than L. larvaepartus . Like L. larvaepartus , L. modestus has nearly complete hindfoot webbing (slightly more extensive in L. modestus than in L. larvaepartus but reaching the toe disc in both species), a dusky throat with melanic pigments extending onto the pectoral region (in a clear wedge shape in L. modestus , more randomly distributed in L.

larvaepartus ), and skin with extensive fine granular tubercles. Limnonectes modestus also exhibits a derived reproductive mode involving production of a relatively small number of large (10 mm diameter) eggs that are deposited along the edge of fast moving streams.

Description of the holotype

An adult male ( Fig. 1) 48 mm SVL, body moderately robust, head not broader than body, head about 65% longer than wide, length 45% of snout-vent length, snout 17% of snout-vent length, moderately pointed, projecting above lower jaw, nostril lateral, closer to tip of snout than to eye, lore essentially straight, canthus rostralis distinct, eye about equal to snout length, pupil diamond-shaped, upper eyelid with tubercles; interorbital region smooth, width 69% of internarial distance, tympanum moderate, slightly wider than interorbital distance, supratympanic fold distinct, extending from posterior corner of eye to supraaxillary region, in contact with tympanic annulus, temporal muscle slightly enlarged; odontoid process 2.1 mm. Dentigerous process of vomer distinct, angled anterolaterally, approximately at 45o angle, posterior ends separated by distance approximately equal to one-third diameter of choanae. Limbs relatively slender, tibia width at thickest part 7.5 mm; femur length 52% of snout-vent length, heels moderately overlapping when placed perpendicular to body axis; tibia length 64.9% of foot length, 48.9% of snout-vent length; foot length 71% of snout-vent length; tarsal fold indistinct, only evident as a ridge; toe discs moderately expanded, circum-marginal groove horseshoe-shaped, pointed anteriorly. Plantar surface of foot smooth, subarticular tubercle rounded, relative length of toes 4.3.5.2.1. Inner metatarsal tubercle prominent, elongate, ovoid with a sharp spade like ventral edge; outer metatarsal tubercle absent; hind foot webbing full, extending to toe discs, slightly emarginated. Manus length 51.3% of foot length, fingers slender, terminal discs slightly expanded, length formula 3.1$4$2, with slight differences in length, subarticular tubercle rounded, convex; supernumerary tubercles absent; inner and outer metacarpal tubercles enlarged, nuptial pads and webbing absent, forearm muscle not enlarged. See Table 1 for measurements and variation.

Coloration

The dorsal coloration is highly variable, typically brownish-grey, but can be darker brown on the dorsolateral region, and some individuals are reddish-brown or golden-tan (see Fig. 2). ~23% of specimens have a bold mid-dorsal stripe. The venter is either yellowish or cream colored, with the upper end of the tibia usually bearing a prominent dark spot. A light bar is often present in the interorbital region, and the coloration of the snout to interorbital region may be lighter than the remainder of the dorsum. The tympanum is often masked in black leaving only the lower rim sharing the predominant body coloration. The gular region is usually darker in males and may have a finely mottled wedge-shaped melanic patch. The dorsal half of the iris is golden-orange in coloration in at least some individuals (we do not know of exceptions, but have not documented iris coloration for most specimens).

Limnonectes larvaepartus

doi:10.1371/journal.pone.0115884.t001

Eggs and tadpoles

Females produce ~100 non-pigmented eggs (see [15]), though the most we have observed is 55, which possibly represents the contents of just one oviduct. The eggs lack a jelly-coat and reach at least ~ 3 mm in diameter. These eggs develop within the oviducts into pigmented tadpoles that reach at least Gosner stage 35 prior to parturition (see [16] for staging). The gut is initially provisioned with substantial yolk ( Fig. 2 b), and developing tadpoles prematurely removed from the oviducts at approximately stage 21 progressed to approximately stage 25 over the course of two weeks in a water bottle without supplemental food, suggesting plasticity in terms of the timing of parturition. A detailed description of the tadpole is provided in the accompanying paper by Kusrini et al. [15].

Natural history

Limnonectes larvaepartus occurs in natural and disturbed forest habitats of Sulawesi, generally in sympatry with at least one, and sometimes as many as five other Limnonectes species. In the western Central Core of Sulawesi, we have always found L. larvaepartus living in sympatry with a much larger species that has been referred to in the literature as L. sp. D (2,3). Whereas L. sp. D is generally found on rocks in fast-moving streams or within a meter of water on the banks of fastflowing streams, L. larvaepartus is usually found further from the stream (2–10 meters from water) on rocky substrates, in leaf-litter, or secluded in grassy vegetation. Because we have observed that L. sp. D predates other frogs, including other Limonectes, it is possible that L. larvaepartus avoids large streams in response to predation pressure from larger Limnonectes species. Male L. larvaepartus typically call from the margins of seeps, puddles, or small pools away from the main stream. Notably, we have found many males calling from small pools that were already inhabited by L. larvaepartus tadpoles ( Fig. 2 c), with as many as three size-classes of tadpoles represented. It is unclear whether some or all of the observed tadpoles were sired by the accompanying male. We have furthermore collected at least one pregnant female from a small stream-side puddle already inhabited by tadpoles of two size classes, again suggesting the possibility that individual pools may be visited repeatedly by the same adult males and females during the reproductive season.

Tables

Table 1. Summary of univariate morphological variation among mensural characters in Limnonectes larvaepartus.

Males (31)	Females (30)	Holotype
Snout-Vent Length (SVL)	39.8 + 4.7	41.2 + 3.1	48.0
Range	31.3‾48.3	35.7‾47.5	-
Head Length (HL)	17.6 + 2.0	17.9 + 1.3	21.5
Head Width (HW)	15.4 + 2.1	15.1 + 1.5	18.9
Snout Length (SL)	7.0 + 0.9	7.2 + 0.5	8.2
Eye-Narial Distance (EN)	3.6 + 0.5	3.9 + 0.4	4.0
Nostril-Tip of Snout (NT)	3.6 + 0.4	3.8 + 0.4	4.3
Internarial Distance (IN)	4.5 + 0.6	4.4 + 0.4	4.9
Snout Width at Eye (SWE)	10.5 + 1.2	10.6 + 1.0	12.2
Snout Width at Nostril (SWN)	6.0 + 0.8	5.8 + 0.7	7.5
Eye-Tympanum Distance (ET)	1.8 + 0.5	1.6 + 0.4	1.7
Odontoid Process Length (OP)	2.0 + 0.3	1.6 + 0.2	2.1
Tympanum Diameter (TY)	3.1 + 0.5	3.3 + 0.4	3.6
Interorbital Distance (IO)	3.0 + 0.5	3.0 + 0.4	3.9
Eye Diameter (EY)	7.2 + 0.8	7.4 + 0.5	8.7
Femoral Length (FE)	21.1 + 2.6	22.0 + 2.4	23.7
Tibial Length (TI)	22.7 + 2.8	24.1 + 2.3	23.5
Foot Length (FL)	32.1 + 3.6	33.5 + 3.0	36.2
Humeral Length (UA)	11.5 + 1.0	11.4 + 1.1	13.5
Lower Arm Length (LA)	8.4 + 1.0	9.1 + 1.0	9.5
Hand Length (HA)	9.7 + 1.2	9.9 + 1.0	11.8
HL/SVL	0.44 + 0.02	0.44 + 0.03	0.45
HW/SVL	0.39 + 0.02	0.37 + 0.02	0.39
SL/SVL	0.18 + 0.01	0.18 + 0.01	0.17
EN/SVL	0.09 + 0.01	0.09 + 0.01	0.08
IN/SVL	0.11 + 0.01	0.11 + 0.01	0.10
SWE/SVL	0.26 + 0.02	0.26 + 0.01	0.25
SWN/SVL	0.15 + 0.01	0.14 + 0.01	0.16
ET/SVL	0.04 + 0.01	0.04 + 0.01	0.04
OP/SVL	0.05 + 0.01	0.04 + 0.01	0.04
TY/SVL	0.08 + 0.01	0.08 + 0.01	0.08
IO/SVL	0.08 + 0.01	0.07 + 0.01	0.08
EY/SVL	0.18 + 0.01	0.18 + 0.01	0.18
FE/SVL	0.53 + 0.03	0.53 + 0.03	0.49
TI/SVL	0.57 + 0.04	0.59 + 0.04	0.49
PL/SVL	0.81 + 0.03	0.81 + 0.04	0.75
SL/SWE	0.67 + 0.07	0.69 + 0.07	0.67
IN/SWN	0.74 + 0.08	0.76 + 0.08	0.65
HW/HL	0.87 + 0.07	0.84 + 0.08	0.88
SL/HW	0.46 + 0.05	0.48 + 0.03	0.43
IO/IN	0.68 + 0.07	0.67 + 0.08	0.80
TI/TD	3.6 + 0.4	3.8 + 0.2	3.6