The earliest direct evidence of frogs in wet tropical forests from Cretaceous Burmese amber

Frogs are a familiar and diverse component of tropical forests around the world. Yet there is little direct evidence from the fossil record for the antiquity of this association. We describe four fossil frog specimens from mid-Cretaceous (~99 mya) amber deposits from Kachin State, Myanmar for which the associated fauna provides rich paleoenvironmental context. Microcomputed tomographic analysis provides detailed three-dimensional anatomy for these small frogs, which is generally unavailable for articulated anurans in the Mesozoic. These crown-group anuran specimens provide the earliest direct evidence for anurans in a wet tropical forest. Based on a distinct combination of skeletal characters, at least one specimen has clear similarities to living alytoid frogs as well as several Mesozoic taxa known from the Jehol Biota in China. Whereas many Mesozoic frogs are from seasonal and mesic paleoenvironments, these fossils provide the earliest direct evidence of anurans in wet tropical forests.

We report the first specimens of frogs preserved in amber from northern Myanmar. These are the oldest records of frogs preserved in amber, with the only two previous reports from Cenozoic amber deposits of the Dominican Republic 23,24 . These Burmese fossils provide the earliest direct evidence of anurans in a wet tropical forest ecosystem. Etymology. Electrorana is feminine and derives from the Latin electrum (amber) and rana (frog). The specific epithet, limoae, is a matronym in the genitive singular for Mrs. Mo Li, who purchased and provided these specimens for study.  (Figs 1 and 2) that also contains an unidentified beetle (Coleoptera; D. Grimaldi, pers. comm.). Three other specimens preserve either an incomplete anuran forelimb (DIP-V-16121, 16127) or the general body shape of a frog that x-ray computed tomography reveals contains no skeletal material within (DIP-V-16119). Diagnosis. Electrorana differs from extant and extinct anurans by the combination of a premaxilla with a prominent and bifurcated alary process, a V-shaped parahyoid bone, and free ribs (preserved on Presacral vertebra II), as well as the absence of a palatine.

Material.
Description. The holotype is a partial skeleton of a small frog (~22 mm snout-vent length) preserving a skull, a nearly complete left forelimb, pectoral girdle, partial right forelimb, a partial preaxial vertebral column, and a partial left hindlimb. The neurocranium has been flattened such that the frontoparietals and sphenethmoid rest alongside the parasphenoid. The sacrum, urostyle, and pelvis are not preserved, nor are the dorsal components of the vertebrae (Supplemental Materials). Ossified sternal elements and posteromedial processes of the hyoid might have been displaced and not preserved.
The skull is approximately as wide as long (~9 mm in length and width) and relatively flat in lateral view. In dorsal view, the jaw joint is approximately even with the occiput. The dermal elements of the skull are not sculptured. The triangular nasals are widely spaced, form the anterior border of the orbit, have a weakly concave anterolateral margin, and taper posteriorly; the nasal does not bear a rostral process or articulate with the maxilla. The frontoparietals are paired and unfused, bordering a large fontanelle. The frontoparietal does not have a supraorbital flange or posterolateral process. The squamosal is present but difficult to discern due to pyritization of overlying soft tissues. The premaxilla is dentigerous (~10-12 teeth) and bears a stout, forked alary process extending dorsally. The palatine process of the premaxilla is weakly developed. A small triradiate septomaxilla is posterolateral to the alary process of the premaxilla. The stout maxilla articulates with the premaxilla, but is relatively shorter, and bears a deep facial process that tapers posteriorly along the orbit. The maxilla bears teeth along three-fourths of its length (~25 teeth). Posteriorly, the maxilla has an overlapping articulation with a slender quadratojugal. The vomer bears pre-and post-choanal processes with a dentigerous process (5-6 teeth) medial to the prechoanal process. There is no palatine. The sphenethmoid is present as a thin posteriorly directed C-shaped mineralization extending across the midline at the anterior margin of the parasphenoid; an ossified nasal septum is not present. The pterygoid is thin and tri-radiate, abutting the maxilla, lateral margin the parasphenoid ala, and the pyritized mass containing the squamosal and quadrate. The parasphenoid is triradiate, tapering anteriorly and nearly reaching the vomers, with alae extending across the ventral surface of the prootics and exoccipitals, which are not co-ossified. The occipital condyles are widely space.
The pectoral girdle was likely arciferal, as suggested by the anteriorly curved clavicles. The clavicle tapers medially and is approximately twice the length of the stout scapula. The coracoid is approximately the same length as the clavicle, and is weakly expanded medially. Both the coracoid and clavicle articulate with the scapula. The clavicles are widely spaced, and there are no ossified sternal elements. There may be a cleithrum present but it is not clearly demarcated from the suprascapula. The humerus and fused radiulna are approximately equal in length. The metacarpals are relatively straight and lack processes along their lateral margins; metacarpal III is the longest. The carpals are not fully ossified and thus difficult to discern. The phalangeal formula of the manus is 2-2-3-3, and a prepollex is absent. The terminal phalanges of the manus are subtriangular and weakly expanded at their distal tip.
The vertebral column is incomplete, but portions of presacral vertebrae I-VII are preserved. Neural arches are not preserved and a transverse process is only preserved on Presacral II. The atlas does not bear transverse processes, and the atlantal cotyles are widely spaced and not contiguous. A free rib is preserved in articulation with the right transverse process of Presacral II, possibly with a posteriorly directed uncinate process. The centra of the presacral vertebrae are difficult to discern.
The hindlimb is incompletely preserved, but longer than the forelimb. Neither the femur nor the fused tibiofibula is preserved in its entirety. The tarsals are not fully ossified. An enlarged prehallux is absent. The metatarsals are all similar in length. The phalangeal formula of the pes is 2-2-3-4-3.
Remarks. The holotype of Electrorana is likely not an adult. This is based on the absence of the columella and incomplete ossification of the carpals, prootic-exocciptal, and sphenethmoid. These often form or fully ossify after metamorphosis 26 30 resulted in four equally parsimonious trees (score = 243); the matrix contains 20% missing data, including 36 of 66 characters for Electrorana.
The topology based on the matrix from Gao & Chen 29 clearly indicates that Electrorana is a crown-group anuran and supports an affinity with the extinct taxon Aerugoamnis from the Early Eocene Green River Formation of Wyoming (Fig. 4). However, analyses based on the matrices of Henrici et al. 30 and Báez 28 suggest that Electrorana may be an earlier diverging lineage of crown-group anurans. Notably, analysis of the matrix of Henrici et al. 30 which was used in the recognition and description of Aerugoamnis, does not suggest a close relationship between Electrorana and Aerugoamnis. Because of both the large amount of missing data in these matrices, as well as the relatively small number of characters (ranging from 66 to 97 characters), the phylogenetic affinities of Electrorana remain uncertain. However, based on the phylogenetic analysis, we are confident that Electrorana is not within crown-group Acosmanura, which includes Anomocoela and Neobatrachia. Further, among extant taxa, Electrorana has strong anatomical similarities to extant alytoids (formerly referred to as Discoglossoidea 31 ).

Discussion
Evolutionary relationships. Electrorana bears strong similarities to extant taxa that form the clade Alytoidea, including Bombinatoridae and Alytidae. These are the only extant taxa exhibiting the unique combination of a V-shaped parahyoid bone, free ribs, and lacking a palatine. Among extinct Mesozoic anurans, Electrorana is similar to anurans from the older Jehol Biota. These are recognized as either the single genus Liaobatrachus 32 or a collection of crown-group anurans with affinities to extant Alytoidea 33 . Other taxa with similarities to Electrorana include Eodiscoglossus from the Jurassic and Lower Cretaceous of Europe 1,34,35 and two taxa from the Lower Cretaceous of Japan 36 . However, phylogenetic analyses conducted by different authors result in conflicting patterns of relationships for these Mesozoic taxa, including whether these are all within crowngroup Anura 28,[32][33][34][35][36] . The incompleteness of the holotype of Electrorana, especially the lack of a sacrum, urostyle, and pelvis, complicates determining its affinities among these Mesozoic anurans. It is diagnosable as a member of crown-group Anura by the presence of a V-shaped parahyoid which is not known in stem anurans.

Comparisons. Among extant anurans, Electrorana is most similar to taxa in the families Alytidae and
Bombinatoridae, which together comprise the Alytoidea. While today representing only five genera, many extinct taxa extending into the late Jurassic have been referred to this clade 34,37 . In comparison to Ascaphus (Ascaphidae) and Leiopelma (Leiopelmatidae), which comprise the earliest diverging lineage of extant anurans, Electrorana has more robust premaxillae that are approximately as tall as wide in anterior view. In both Ascaphus and Leiopelma, the premaxillae are wide with a narrow alary process. The alary process of the premaxilla in Electrorana is robust, comprising approximately half the length of the bone, which is most similar to the condition of Alytes (Alytidae) and Bombina (Bombinatoridae). The alary process is also forked dorsally, a condition similar to that recently reported for Genibatrachus by Gao & Chen 29 . The nasals of Electrorana are widely spaced and slender, similar to Ascaphus and Bombina (Bombinatoridae) and believed to be the ancestral condition for anurans 38 . The frontoparietals of Electrorana are widely spaced as in Ascaphus, Leiopelma, and Alytes, though unlike Bombina and Barbourula (Bombinatoridae) in which the fontanelle is partially or fully covered. The dentigerous vomer of Electrorana is more complicated in shape than the simple vomer found in Ascaphus (which does not have a postchoanal ramus of the vomer 38 ), but similar in structure to the vomer of other anurans including Leiopelma, Alytes, Bombina, or Pelodytes (Pelodytidae). Unlike these other taxa, the vomer of Electrorana is relatively small and displaced anteriorly, which would suggest a relatively anterior location for the choana; however, this may be a result of either the holotype not being an adult specimen or being displaced postmortem. While thin and small, the sphenethmoid of Electrorana does appear to be contiguous across the midline, unlike the condition found in Ascaphus and Leiopelma in which it is generally poorly ossified 38 . The sphenethmoid is clearly not as robust as seen in Alytes, Discoglossus, Bombina, and many other anurans, but this is likely due to the holotype of Electorana not being an adult specimen. The pterygoids lack the enlarged ventrolaterally projecting flange found in Barbourula.
There is a thin V-shaped parahyoid bone, as found in Alytes, Discoglossus, and Pelodytes 38 , but there are no plate-like parahyoid bones as found in Bombina, Barbourula, or Rhinophrynus (Rhinophrynidae). A similar V-shaped parahyoid is found in the various anuran taxa referred to as Liaobatrachus 32,34 , including Callobatrachus, Mesophryne, Dalianbatrachus, and Yizhoubatrachus 33 . Unlike the condition in Pelodytes, the parahyoid bone does not appear 'x-shaped' with posterolateral processes that approach the bony posteromedial processes of the hyoid.
The one preserved rib of Electorana is relatively longer than seen in Alytes and Bombina. In Bombina, the ribs of Presacral II and III can bear a similar posteriorly directed uncinate process to what appears to be present in Electrorana. Unlike taxa in the Megophryidae, Pelobatidae, Pelodytidae, and many neobatrachian frogs, Electrorana does not exhibit ossified sternal elements along the midline.
Paleoenvironment. The paleoecological context for most Mesozoic anurans is either unknown or not described as being a tropical forest. The paleoecology of the Jehol Biota in China (~130-122 mya 39 ) is probably the best documented for a Cretaceous faunal assemblage that contains anurans. It is characterized as having seasonal and mesic environments 40 and possibly open forested habitats 41 . The Lower Cretaceous Crato Formation of Brazil is another example with anuran fossils coming from lacustrine deposits that were also likely formed in a semiarid environment 42  Triassic stem-anuran Triadobatrachus likely lived in semi-arid, near-shore riparian forests near floodplains 45,46 . Transitions to more arid paleoenvironments have even been cited as a possible driver of morphological evolution in the earliest anurans 47 . Electrorana from the mid-late Cretaceous amber deposits in Myanmar provides the oldest definitive association of anurans with tropical forests and suggests that lineages today associated with temperate regions may have occupied a larger diversity of habitats in the past.

Methods
We performed high-resolution x-ray computed tomography (CT-scanning) at the University of Florida's Nanoscale Research Facility. We used a Phoenix v|tome|x M (GE Measurement & Control, Boston, USA) scanner with a 180 kv x-ray tube with a diamond-tungsten target and with the following settings: 75 kV, 150 mA, a 0.5 second detector time, averaging of three images per rotation and a voxel resolution of 8.6-10.7 µm. Raw 2D x-ray data were processed using the datos|x software v. 2.3 with post-processing, analyses (including segmentation), and visualization conducted using VG StudioMax v. 3.1 (Volume Graphics, Heidelberg, Germany).
A replica of DIP-L-0826 (enlarged by 300%) was created by 3D-printing.stl files that were extracted from the CT volumetric data. The replica was printed using rigid polypropylene materials using an Objet260 Connex2 3D printer (Stratasys, Eden Prairie, USA) based at the University of Florida's Nanoscale Research Facility.
Morphological comparisons to other anurans were based on CT-scans of extant species also scanned at the University of Florida and fossil specimens housed at the Institute for Vertebrate Paleontology and Paleoanthropology in Beijing, China (see Supplementary Table 1), as well as published descriptions of extinct anurans.
To evaluate potential evolutionary relationships of Electrorana, we coded this new taxon into three different recent matrices used in phylogenetic analyses of extinct and extant anurans [28][29][30] . We conducted phylogenetic analyses using parsimony as an optimality criterion in PAUP* v4.0a (build 158) 48 . For the analysis of each matrix, we conducted 10 replicate heuristic searches with starting trees obtained through random stepwise addition. Missing (?) or inapplicable (−) character states were both treated as missing data in the analyses; taxa with multistate characters were treated as uncertainties (rather than polymorphisms). We summarized the results using majority rule consensus and these are presented in Fig. 4.  Numbers adjacent to nodes represent the percentage of equally parsimonious trees containing that node. The phylogeny based on Henrici et al. 30 is rooted using the extant alytoid clade, that based on Báez 28 is rooted with Ascaphus, and that based on Gao & Chen 29 is rooted with Triadobatrachus. Extinct taxa are indicated with a dagger, and Electrorana is indicated in boldface.