Abstract We describe a new species of fanged frog (Limnonectes larvaepartus) that is unique among anurans in having both internal fertilization and birth of tadpoles. The new species is endemic to Sulawesi Island, Indonesia. This is the fourth valid species of Limnonectes described from Sulawesi despite that the radiation includes at least 15 species and possibly many more. Fewer than a dozen of the 6455 species of frogs in the world are known to have internal fertilization, and of these, all but the new species either deposit fertilized eggs or give birth to froglets.

Citation: Iskandar DT, Evans BJ, McGuire JA (2014) A Novel Reproductive Mode in Frogs: A New Species of Fanged Frog with Internal Fertilization and Birth of Tadpoles. PLoS ONE 9(12): e115884. https://doi.org/10.1371/journal.pone.0115884 Editor: Matthias Stöck, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Germany Received: May 17, 2014; Accepted: November 23, 2014; Published: December 31, 2014 Copyright: © 2014 Iskandar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This research was funded by the United States National Science Foundation Division of Environmental Biology (DEB 0328700, DEB 0640967) to JAM, the Natural Science and Engineering Research Council of Canada (RGPIN/283102-2012) to BJE, and the Indonesian Directorate General of Higher Education, Ministry of Education and Culture (Basic Science Grant No. 60/P4M/DPPM/L-3311/MS/1993) to DTI. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction The Indonesian island of Sulawesi in central Indonesia is home to a largely unstudied radiation of fanged frogs in the genus Limnonectes Fitzinger, 1843 (Anura: Dicroglossidae Anderson, 1871). To date, only three species are recognized from Sulawesi, but at least 15 species occur on the island [1], [2], [3]. It was recently proposed that Sulawesi fanged frogs represent an adaptive radiation based on patterns of morphological diversification among co-distributed species, and correspondence of morphological features (body size, webbing) with habitat utilization [3]. Adult body size ranges from ∼2 g to more than 900 g in members of the Sulawesi assemblage, with large-bodied species found in close association with fast moving rivers, and small-bodied species occupying more terrestrial niches in forest leaf-litter and on the banks of slow moving streams. Iskandar and Tjan [4] first documented that there are multiple undescribed species of Limnonectes on Sulawesi, and indicated that one of the constituent species utilizes an unusual ovoviviparous reproductive strategy. Here we describe that species, and provide additional details on its reproductive mode. Anurans exhibit tremendous diversity in mode of reproduction. Not only have there been many independent origins of direct development, the condition in which a free-living tadpole stage is bypassed and froglets emerge from the egg capsules [5], but a bewildering array of mechanisms has evolved wherein parents care for their developing offspring. Examples of parental care include guarding of terrestrial eggs, transport of tadpoles on the back following hatching, carrying developing eggs in pouches or depressions on the back and flanks, males carrying developing tadpoles in the vocal sac, and even the now-extinct species of gastric brooding frogs in which the female carried her developing tadpoles in her stomach (see [6] for a summary). Despite this extreme reproductive diversity, internal fertilization has evolved only a few times among the 6455 known species of anurans [7], [8]. Only the African bufonid genera Nectophrynoides and Nimbaphrynoides and the now extinct Puerto Rican species, Eleutherodactylus jasperi, are documented to combine internal fertilization with egg retention and subsequent birth of froglets [9]. To this group, we can now add a species of Sulawesi fanged frog in the genus Limnonectes with internal fertilization and live birth of tadpoles, a reproductive mode that is unique among anurans.

Materials and Methods We analyzed more than 100 specimens representing the new species from 23 localities on the island of Sulawesi. This study was conducted under an approved Institutional Animal Care and Use Committee protocol issued to JAM by the University of California at Berkeley (Protocol #R279). Fieldwork conducted in Indonesia was undertaken under research permits issued by LIPI and RISTEK, with specimen exportation authorized under permits or loans issued by LIPI, the Museum Zoologicum Bogoriense, and Pusat Studi Biologi. Specimens were captured by hand in the field, sacrificed via emersion in an aqueous solution of MS-222 (tricaine methane sulfonate) buffered to neutral pH, and prepared as formalin-fixed specimens deposited primarily in the Museum Zoologicum Bogoriense (the national museum of Indonesia) or Museum of Vertebrate Zoology at UC Berkeley. As part of a comprehensive study of Sulawesi Limnonectes diversification, we obtained morphometric data for the full Sulawesi assemblage. Sex was determined by inspection of gonads. The following measurements were made to the nearest 0.1 mm using digital calipers: snout-vent length (SVL) – from cloaca to tip of snout; head width (HW) – widest distance between posterior end of lower jaw; head length (HL) – from posterior end of lower jaw to tip of snout; femur length (FE) – from cloaca to distal end of femur; tibia length (TI) – from knee to distal end of tibia; foot length (FL) – from distal end of tibia-fibula to tip of 4th toe; inner metatarsal tubercle (IM); humerus length (UA) – from proximal end of upper arm to elbow; lower arm length (LA) – from elbow joint to base of middle palmar tubercle; hand length (HA) – from lower border of middle tubercle to distal tip of third finger; snout length (SL) – from bony border of eye socket to tip of snout; snout width at eye level (SWE); snout width at nostril level (SWN); eye diameter (EY) – outer diameter, measured from bones bordering eye; interorbital distance (IO) – distance between orbits ( = width of frontoparietal bones); eye-tympanum distance (ET); eye-narial distance (EN) – eye socket border to posterior border of nostril; Internarial distance (IN) – shortest distance between nostrils; nostril to tip of snout (NT); tympanum diameter (TY); odontoid process length (OP) –length of fang-like odontoid process of lower jaw; tibia diameter measured at its widest point (TD). Digital webbing formulae from [10]. Nomenclatural acts The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSID (Life Science Identifier) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix “http://zoobank.org/”. The LSID for this publication is: urn:lsid:zoobank.org:pub: A758E03A–C054–4193–9768–D0C4AD588AEA. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

Discussion We first became aware of the unusual reproductive mode of Limnonectes larvaepartus while conducting fieldwork on Sulawesi. In several instances, we discovered tadpoles in the oviducts while preparing specimens. In each case, having sacrificed frogs for preparation, the abdominal wall was observed to quiver, and incision resulted in living tadpoles emerging from the opening (see S1 Movie). On one occasion, a gravid female gave birth to tadpoles in-hand at the moment of capture. On four other occasions, field-collected L. larvaepartus in our possession gave birth to tadpoles while being held individually in collecting bags. In total, we have either observed tadpoles in the oviducts or direct-birth of tadpoles on 19 occasions. Because we have not witnessed natural birth of tadpoles in free-living frogs, two possible alternative reproductive modes are possible for this species. Limnonectes larvaepartus reproduction may simply reflect what we have observed in the hand – direct birth of tadpoles. Alternatively, this species may be capable of retaining developing young in the oviducts through metamorphosis with subsequent birth of froglets, as is the case for Eleutherodactylus jasperi and members of the African bufonid genera Nectophrynoides and Nimbaphrynoides [9], [17], [18]. The latter mode seems unlikely for several reasons. First, we have collected 19 pregnant individuals carrying tadpoles in the oviducts but none carrying froglets. These 19 females were collected across different months, years, and localities. Second, we have observed and collected at least four clutches of free-living L. larvaepartus tadpoles in small pools of water on the margins of streams, with each of these samples exhibiting two or three size classes and thus possibly representing multiple clutches. Three of these clutches were accompanied by males, some of which were calling, and one was found in association with the gravid female that gave birth to 55 tadpoles (some in the hand of JAM, others subsequently deposited in a collecting bag). Finally, although adaptive plasticity has been demonstrated for many frog species, whereby tadpoles or froglets are capable of hatching from their eggs prematurely when attacked by predators [19], this has only been documented for a single direct-developer, Eleutherodactylus coqui [20]. In the case of E. coqui, the capacity for early hatching commenced at stages 13 or 14, whereas normal hatching occurred at stage 15 (see [21] for staging). Thus, E. coqui were capable of premature hatching only as froglets, with toe pads and eyelids present but before the tail was completely resorbed, a stage much later than would be required by L. larvaepartus if it were simultaneously capable of hatching as either a tadpole or full-term froglet. Most direct-developers, as well as the few frog species that give birth to froglets, pass through the tadpole stage in a form poorly suited for free-living. In many such species, the tadpole has a broad, highly vascularized ‘respiratory tail’ specialized for intra-egg or intra-oviductal gas exchange rather than for swimming, as is the case for Eleutherodactylus jasperi [17], and may lack mouthparts and functional gills [22], [23], [24]. In others, the tail remains rudimentary and limb buds appear early in development such that a typical tadpole phenotype never occurs [22], [23]. Given these observations, it appears unlikely that any one species would be characterized by the combination of (1) internal fertilization, (2) complete metamorphosis within the oviducts and live-birth in the form of froglets, as well as (3) an oviductal tadpole stage that is capable of premature birth and free-living. Although we think that L. larvaepartus is much more likely to give birth to tadpoles as its sole mode of reproduction as opposed to exhibiting adaptive plasticity allowing for birth of either tadpoles or froglets depending on the circumstances confronting the frog, we note that either condition would be unique among Anura. In either case, L. larvaepartus requires internal fertilization, which is, itself, extremely rare among anurans [8], [25]. The mechanism by which internal fertilization takes place is unknown, and there is no obvious intromittent organ present to facilitate sperm transfer. If L. larvaepartus reproduction always involves birth of tadpoles, it is likely that they are ovoviparous given that the tadpoles are well provisioned with yolk and appear fully capable of developing without nutrient transfer from the mother. Prior to parturition, the tadpoles have well-developed tails and pigmentation, and well developed mouthparts. Given this morphology, it is unlikely that these tadpoles are endotrophic (never feed before metamorphosing into froglets). It is more likely that the tadpoles are born after exhausting their yolk supply, and are subsequently self-feeding prior to metamorphosis. Nevertheless, it is clear that much remains to be learned about this unusual frog, the discovery of which brings to light yet another axis of diversity characterizing the remarkable Sulawesi fanged frog adaptive radiation.

Supporting Information S1 Movie. Video showing the characteristic quivering abdomen caused by movement of tadpoles within a pregnant female Limnonectes larvaepartus. https://doi.org/10.1371/journal.pone.0115884.s001 (MOV)

Acknowledgments We thank K. N. Tjan, U. Arifin, K. Laras, Suwatio, A. Rachmansah, R. M. Brown, and C. J. Hayden for assistance in the field. Boeadi and Mumpuni for MZB specimens; R. F. Inger for discussion and access to comparative materials from FMNH, USNM and MCZ; D. N. Cannatella, M. H. Wake, and members of the McGuire Lab for helpful discussion.

Author Contributions Conceived and designed the experiments: DTI BJE JAM. Performed the experiments: DTI BJE JAM. Analyzed the data: DTI BJE JAM. Contributed reagents/materials/analysis tools: DTI BJE JAM. Wrote the paper: DTI BJE JAM.