Abstract

Citation:Rittmeyer EN, Allison A, Gründler MC, Thompson DK, Austin CC (2012) Ecological Guild Evolution and the Discovery of the World's Smallest Vertebrate. PLoS ONE 7(1): e29797. https://doi.org/10.1371/journal.pone.0029797

Editor:William J. Etges, University of Arkanas, United States of America

Received:September 28, 2011;Accepted:December 3, 2011;Published: January 11, 2012

Copyright: © 2012 Rittmeyer 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.

Funding:This research was funded by National Science Foundation grants DEB 0103794 and DEB 0743890 to AA and DEB 0445213 and DBI 0400797 to CCA. 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

Living vertebrates range in size over 3,000 fold. The breadth and limits on vertebrate size have been of great interest to biologists due to the functional and physiological constraints associated with extreme body size. The largest extant vertebrate is the blue whale (Balaenoptera musculus, average adult size 25.8 m)[1] while the smallest is a fish (Paedocypris progenetica, adult size 7.9–10.3 mm)[2]. Both species are aquatic and biologists have speculated that the buoyancy of water may play a role in facilitating the evolution of both large and small size[3]–[5]. Extreme miniaturization, however, has evolved independently at least eleven times in terrestrial frogs. Here we describe two new species of diminutive terrestrial frogs from the island of New Guinea, one of which represents the smallest known vertebrate species, attaining an average body size of only 7.7 mm (range 7.0–8.0 mm). We identify ecological similarities among the most diminutive frog species suggesting that the independent origins of minute frogs are not merely evolutionary outliers, but represent a previously undocumented ecological guild found in moist leaf litter of tropical wet-forests.

Results

Taxonomic treatment

Amphibia, Linnaeus, 1758

Anura, Rafinesque, 1815

Microhylidae, Günther, 1858

Asterophryinae, Günther, 1858

Paedophryne, Kraus 2010

Paedophryne amauensis, sp. nov. (urn:lsid:zoobank.org:act:496F26AB-CD82-4A9C-944C-070EC86ADAA4)

Etymology.

The species epithet refers to the type locality, near Amau Village, Central Province, Papua New Guinea.

Holotype.

LSUMZ 95000 (field tag CCA 5739), adult male, collected by C.C. Austin and E.N. Rittmeyer near Amau Village, Central Province, Papua New Guinea, 09.9824°S, 148.5785°E, 177 m, 7 August 2009.

Paratypes.

LSUMZ 95001, same data as holotype, except collected 6 August 2009; LSUMZ 95002, same data as holotype, except collected 10 August 2009; LSUMZ 95003-4, same data as holotype, except collected 12 August 2009; LSUMZ 95005-6, same data as holotype, except collected 14 August 2009.

Diagnosis.

A minute microhylid (male SVL = 7.0–8.0 mm) of the genusPaedophryne based on the following combination of characters: eleutherognathine jaw, 7 presacral vertebrae, first digits of hand and foot reduced to single elements, prepollex and prehallux reduced to single elements (Fig. 1). Legs moderately long (TL/SVL = 0.478–0.507), snout broad and short (EN/SV = 0.075–0.084, EN/IN = 0.667–0.765), and eye relatively large (EY/SVL = 0.127–0.150). Digits un-webbed with slightly enlarged discs (3F/SVL = 0.025–0.033; 4T/SVL = 0.036–0.050). First finger and first toe reduced to vestigial nubs, second and fourth fingers and second and fifth toes also markedly reduced. Dorsal coloration dark brown with irregular tan to rusty-brown blotches; lateral and ventral surfaces dark brown to slate grey with irregular bluish-white speckling. Detailed mensural characters and proportions provided inTable 1 andTable 2.

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Figure 1. Osteological characters ofPaedophryne amauensis,P. swiftorum.

A. X-ray of paratype ofPaedophryne amauensis (LSUMZ 95002).B. X-ray of paratype ofP. swiftorum (BPBM 31886).C,E,G,I. Photos of cleared and double-stained paratype ofP. amauensis (LSUMZ 95002).C. Whole body.E. Head.G. Hand.I. Foot.D,F,H,J. Photos of cleared and double-stained paratype ofP. swiftorum (BPBM 31886).D. Whole body.F. Head.H. Hand.J. Foot. Skeletal elements labeled as follows: Fp, frontoparietal; Il, illium; Mc1-4, metacarpals 1-4; Mt1-5, metatarsals 1-5; Mx, maxilla; N, nasal; S, Sacrum; Sp, sphenethemoid; Sq, squamosal; U, urostyle; V1, first presacral vertebra; V7, seventh presacral vertebra.

https://doi.org/10.1371/journal.pone.0029797.g001

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Table 1. Mensural characters ofPaedophryne amauensis andP. swiftorum.

https://doi.org/10.1371/journal.pone.0029797.t001

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Table 2. Relevant proportions ofPaedophryne species.

https://doi.org/10.1371/journal.pone.0029797.t002

Paedophryne amanuensis is distinguished from all congeners by its smaller size (SVL = 10.1–10.9 mm inP. kathismaphlox, 11.3 mm inP. oyatabu, 8.3–8.9 mm inP. swiftorum) and longer legs (TL/SVL = 0.35–0.39 inP. kathismaphlox, 0.40 inP. oyatabu, 0.427–0.471 inP. swiftorum).Paedophryne amauensis is further distinguished fromP. oyatabu andP. swiftorum by its longer, narrower head (EN/SV = 0.062, EN/IN = 0.64 inP. oyatabu; EN/SV = 0.064–0.071, EN/IN = 0.579–0.632 inP. swiftorum), and fromP. kathismaphlox by its shorter, broader head (EN/SV = 0.067–0.079; EN/IN = 0.78–0.80 inP. kathismaphlox). The call ofP. amauensis differs from that ofP. swiftorum by its higher dominant frequency (7300 Hz inP. swiftorum) and by consisting of single notes, rather than eight paired notes as inP. swiftorum. The calls ofP. kathismaphlox andP. oyatabu are unknown.

Call.

This species is crepuscular and calls from within leaf litter in primary forest at dawn and dusk. Its call consists of a continuous series of high-pitched notes with a dominant frequency of ∼8400–9400 Hz. Individual notes range in duration from 2–14 ms and are produced at a rate of 1.5 notes/s (Fig. 2;Table 3). The overall acoustic impression is that of a stridulating insect. Individuals generally call from one to three minutes and then rest briefly before resuming. In a 5.5 minute recorded sequence, one individual (NS2,Table 3) produced a total of 355 calls in four groups, with the interval between groups ranging from 3.3 to 40.8 s.

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Figure 2. Type localities, call sonograms, and photographs ofPaedophryne species.

A. Photograph of paratype ofPaedophryne swiftorum in life (BPBM 31880).B. Waveform (upper right), power spectrum (lower left) and spectrogram (lower right) of a single call series consisting of four double notes of the holotype ofP. swiftorum (BPBM 31883).C. Type localities of the four species ofPaedophryne. Blue:P. swiftorum; red:P. amauensis; yellow:P. kathismaphlox; purple:P. oyatabu.D. Photograph of paratype ofP. amanuensis (LSUMZ 95004) on U.S. dime (diameter 17.91 mm).E. Waveform (upper right), power spectrum (lower left) and spectrogram (lower right) of the first four notes of the call of the holotype ofP. amauensis (LSUMZ 95000).

https://doi.org/10.1371/journal.pone.0029797.g002

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Table 3. Call characters ofPaedophryne amauensis.

https://doi.org/10.1371/journal.pone.0029797.t003

Paedophryne swiftorum, sp. nov. (urn:lsid:zoobank.org:act:6F724864-05A5-4729-AB27-7093A64F90F2)

Etymology.

The species epithet honors the Swift family, in recognition of their generous contributions that enabled the establishment of the Kamiali Biological Station, where the type series was collected.

Holotype.

BPBM 31883 (field tag AA 19195), adult male, collected by A. Allison, M.C. Gründler, E.N. Rittmeyer, and D.K. Thompson at Kamiali Wildlife Management Area, 1.3 km N, 6.2 km W of Cape Dinga, Cliffside Camp, Morobe Province, Papua New Guinea, 07.255997°S, 147.092879°E, 500 m elevation, 14 July 2008.

Paratypes.

BPBM 31879, same data as holotype, except collected 8 July 2008; BPBM 31880, same data as holotype, except collected 10 July 2008; BPBM 31881-82, same data as holotype, except collected 11 July 2008; BPBM 31884 collected by M. Gründler at Kamiali Wildlife Management Area, Pinetree Camp, Morobe Province, Papua New Guinea, 07.257906°S, 147.06335°E, 950 m elevation, 12 July 2008; BPBM 31885, same data as BPBM 31884, except an unsexed juvenile collected on 13 July 2008; BPBM 31886, same data as holotype, except collected 13 July 2008.

Diagnosis.

A minute microhylid (SVL = 8.25–8.90 mm) of the genusPaedophryne based on the following combination of characters: eleutherognathine jaw, 7 presacral vertebrae, first digits of hand and foot reduced to single elements, prepollex and prehallux reduced to single elements (Fig. 1). Legs moderately long (TL/SVL = 0.427–0.471), snout short and broad (EN/SV = 0.064–0.071; EN/IN = 0.579–0.623), and eyes relatively large (EY/SVL = 0.139–0.149). Fingers lacking enlarged discs (3F/SVL = 0.018–0.024), toes with slightly enlarged discs (4T/SVL = 0.041–0.047). Digits un-webbed; first finger and first toe reduced to vestigial nubs, second and fourth fingers and second and fifth toes also markedly reduced. Dorsum dark brown with irregular tan to rusty brown blotches or a broad tan mid-dorsal stripe; chin and throat dark brown, abdomen lighter brown, occasionally mottled with tan. Detailed mensural characters and proportions provided inTable 1 andTable 2.

Paedophryne swiftorum is distinguished fromP. oyatabu andP. kathismaphlox by its smaller size (SVL = 10.1–10.9 mm inP. kathismaphlox, 11.3 mm inP. oyatabu), longer legs (TL/SVL = 0.35–0.39 inP. kathismaphlox, 0.40 inP. oyatabu,), and larger eyes (EY/SV = 0.12 inP. kathismaphlox, 0.13 inP. oyatabu).Paedophryne swiftorum is further distinguished fromP. kathismaphlox by its broader head (EN/IN = 0.78–0.80 inP. kathismaphlox). It is distinguished fromP. amauensis by its larger size (SVL = 7.0–8.0 mm inP. amauensis), shorter legs (TL/SVL = 0.478–0.507 inP. amauensis), and shorter, broader head (EN/SV = 0.075–0.084; EN/IN = 0.667–0.765 inP. amauensis). The call ofP. swiftorum differs from that ofP. amauensis by its lower dominant frequency (8400–9400 Hz inP. amauensis), and by consisting of a series of four double notes, rather than repeated single notes as inP. amauensis. The individual notes are otherwise similar toP. amauensis. The calls of otherPaedophryne species are unknown.

Call.

The calling ecology ofPaedophryne swiftorum is similar to that ofP. amauensis – it is generally crepuscular; however, it calls diurnally during particularly wet conditions. It does not call nocturnally regardless of rainfall. The call generally consists of four double notes (Fig. 2;Table 4) delivered in a continuous series at the rate of 0.66 calls/s. Each note is around 7 ms in duration and the entire call lasts approximately 0.5 seconds. The interval between notes is 40–50 ms within a double note series and 85–100 ms between each double note series. The dominant frequency averages 7300 Hz. Some individuals occasionally produce calls of only six notes, invariably consisting of double notes, and otherwise similar to eight-note calls. The acoustic characteristics of the call and the tendency of males to call continuously within a chorus produces an uncanny resemblance to stridulating orthopteran insects.

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Table 4. Call characters ofPaedophryne swiftorum.

https://doi.org/10.1371/journal.pone.0029797.t004

Discussion

Miniaturization, the reduction in body size necessitating drastic alterations to an organism's physiology, ecology, and behavior, is known from every major vertebrate lineage and nearly all major groups of animals[6]. Yet among vertebrates only teleost fishes approach the extreme size ofPaedophryne amauensis; the smallest known actinopterygian fish isPaedocypris progenetica, maturing at 7.9 mm[2], whereas the smallest known vertebrate excluding teleosts and anurans is a gecko (Spherodactylus ariasae, mean SVL = 16.3 mm)[14] or a salamander (Thorius arboreus, mean SVL = 17.0 mm)[15]. Miniaturization has occurred repeatedly in anurans: the 29 smallest species (maximum male SVL<13 mm) include representatives from 5 families and 11 genera (Table 5)[7],[13],[16]–[24]. Several large frog families (e.g. Bufonidae, Hylidae, Ranidae) lack extremely miniaturized species, whereas other families include numerous minute taxa: 15 of these species are microhylids, including representatives of 7 genera. This distribution of miniaturization among frog families suggests that the evolution of miniaturization has been nonrandom with respect to phylogeny.

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Table 5. Sizes and mode of reproduction in extremely miniaturized frogs.

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Miniaturized animals typically show reduced overall fecundity and increased egg size relative to larger congeners[6]. Of the 29 smallest frogs, 24 (83%) lack a larval tadpole stage and develop directly[7],[13],[16]–[20],[22]–[24], and only two congeners (Microhyla supracilius,M. perparva) have a typical anuran tadpole stage[21]. These direct developing species belong to clades that include much larger direct developing species, thus direct development may facilitate the evolution of extreme miniaturization in frogs[7]. Miniaturized species also typically express a generally reduced and simplified morphology[6],[8],[9]. These changes are also apparent inPaedophryne, which exhibit a reduced number of presacral vertebrae, reduced ossification of several cranial elements, and phalangeal and digital reduction on both the hand and foot (Fig. 1).

All but two species of extremely miniaturized frogs inhabit tropical wet-forest leaf litter; the two exceptions (Choerophryne burtoni,Oreophryne minuta) inhabit dense moist moss. Frogs are sensitive to water loss[25]–[27] and small species, which have a high surface to volume ratio, are particularly susceptible to desiccation[28]. Indeed, one of smallest known amniote species (Sphaerodactylus parthenopion) loses water at much higher relative rates than larger congeners, and is known to select moist microhabitats to compensate[28]. A disproportionate number of tropical wet-forest frogs occur on or near the ground and have life histories dependent on the near constant high moisture content of leaf litter[29]. This may explain the absence of diminutive frogs from temperate forests and tropical dry-forests, where the leaf litter is seasonally dry. Alternatively, the absence of minute frogs from temperate forests may be explained by the evolution of clades including miniaturized species in the wet tropics (i.e. tropical niche conservatism)[30]–[32]; however, this would not explain the apparent absence of these species from tropical dry-forests. Thus, the wet-forest leaf litter may represent an adaptive zone for diminutive frogs. Their small size likely increases their susceptibility to predation by invertebrates[33]–[35], which may account for the absence of diminutive anurans from aquatic habitats, where invertebrate predation is particularly high[33]. This may also explain a tendency for these frogs to inhabit upland regions where invertebrate diversity is less than in the lowlands.

Phylogenetic analyses corroborate the monophyly ofPaedophryne (albeit with moderate support) and suggest a relationship withBarygenys andCophixalus balbus (Fig. 3,Fig. S1). Divergences among species withinPaedophryne are surprisingly deep (mean uncorrected p-distance ≥0.102) and on par with, or greater than, divergences observed among distinct genera of asterophryine frogs (e.g. mean uncorrected p-distance betweenAlbericus andChoerophryne = 0.11, betweenHylophorbus andMantophryne+Pherohapsis = 0.113). These deep divergences withinPaedophryne suggest that the extremely diminutive size exhibited by the genus arose early in the radiation of microhylid frogs in New Guinea, thus indicating that these minute anurans have long been a component of the leaf litter community where they occur. Indeed,Paedophryne amanuensis andP. swiftorum appear to be relatively common inhabitants of leaf litter, judging by the level of calling, and we estimate that calling maleP. swiftorum are spaced only approximately 50 cm from one another within the leaf litter. Thus, these minute species are likely an important component of the tropical wet-forest ecosystem, both as a predator of small invertebrates such as acarians and collembolans, and as a prey item for larger invertebrates and vertebrates.

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Figure 3. Phylogenetic position ofPaedophryne and evolution of body size in Asterophryinae.

Maximum likelihood phylogeny ofPaedophryne and asterophryine frogs. Colors of branches correspond to maximum male SVL (Paedophryne) or average SVL within each clade on a logarithmic scale (Table 6). Circles above branches correspond to posterior probabilities: black: >0.95; grey: 0.85–0.95; white: 0.5–0.85. Circles below branches correspond to maximum likelihood bootstrap support: black: >95%; grey: 75–95%; white: 50–75%.

https://doi.org/10.1371/journal.pone.0029797.g003

The discovery ofPaedophryne amauensis andP. swiftorum also greatly expands the distribution of the genus westward, both north and south of the central mountains. The genus remains restricted to the East Papuan Aggregate Terrain that composes the Papuan Peninsula in eastern New Guinea[36]–[38], supporting Kraus's[13] conclusion on the importance of this geologic entity for the evolution ofPaedophryne. However, the poorly explored nature of New Guinea and the extremely minute size and atypical, insect-like call ofPaedophryne species leaves the possibility of a much broader distribution.

These discoveries further reveal intriguing patterns of amphibian diversity in a megadiverse hotspot region and highlight ecological similarities among the most diminutive anurans, suggesting that these species are not merely curiosities, but represent a previously unrecognized ecological guild. Phylogenetic analysis also show genetic divergences amongPaedophryne species are deep, equal to or greater than among genera of asterophryine frogs, suggesting that the evolution of this miniaturized vertebrate guild arose early in the radiation of New Guinea microhylid frogs. Such discoveries are increasingly critical in this time of global amphibian declines and extinctions.

Materials and Methods

Ancestral State Reconstructions

To examine the evolution of body size in asterophryine frogs, we used weighted squared-change parsimony[51], which is computationally equivalent to maximum likelihood based ancestral state reconstructions[52],[53], as implemented in Mesquite v.2.72[54]. The maximum likelihood phylogeny of asterophryine frogs (Fig. S1) was trimmed to a single representative per generic-level clade for use in ancestral state reconstructions. We tested several different measures of body size for each clade, including mean size, maximum size of the smallest species, and maximum size of the largest species. Results did not differ substantially among analyses (data not shown), thus the results of ancestral state reconstructions with mean size for each clade are shown (Fig. 3). Mean size for each clade used in the analysis are provided inTable 6.

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Table 6. Average sizes of asterophryine genera.

https://doi.org/10.1371/journal.pone.0029797.t006

Supporting Information

Acknowledgments

We thank the local people from Papua New Guinea for the privilege to conduct fieldwork on their land. We thank B. Roy, V. Kula, and B. Wilmot from the Papua New Guinea Department of Environment and Conservation, J. Robins from the Papua New Guinea National Research Institute. J. Animiato, I. Bigilale, and B. Iova from the Papua New Guinea National Museum provided research assistance in Papua New Guinea. P. Chakrabarty and S. Gotte took x-rays; S. Gotte and F. Kraus performed clearing and staining. We also thank C. McMahan for his assistance with examining cranial osteology. Photos of some asterophryine frogs were provided by R. Günther, F. Kraus, and S. Richards. This manuscript was improved from comments from the Austin lab group. All research was carried out under LSU IACUC protocol 06-071.

Author Contributions

Conceived and designed the experiments: CCA ENR AA. Performed the experiments: CCA ENR AA. Analyzed the data: CCA ENR AA. Contributed reagents/materials/analysis tools: CCA AA. Wrote the paper: CCA ENR AA. Fieldwork: CCA ENR AA MCG DKT.

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