Exceptionally Preserved Tadpoles from the Miocene of Libros, Spain: Ecomorphological Reconstruction and the Impact of Ontogeny Upon Taphonomy

Exceptionally preserved tadpoles from the Miocene of Libros, Spain: ecomorphological reconstruction and the impact of ontogeny upon taphonomy

MARIA E. MCNAMARA, PATRICK J. ORR, STUART L. KEARNS, LUIS ALCALA´ , PERE ANADO´ N AND ENRIQUE PEN˜ ALVER-MOLLA´

McNamara, M.E., Orr, P.J., Kearns, S.L., Alcala´, L., Anado´n, P. & Pen˜alver-Molla´,E. 2010: Exceptionally preserved tadpoles from the Miocene of Libros, Spain: ecomorphological reconstruction and the impact of ontogeny upon taphonomy. Lethaia,Vol.43, pp. 290–306.

The Libros exceptional biota from the Upper Miocene of NE Spain includes abundant frog tadpoles (Rana pueyoi) preserved in finely laminated lacustrine mudstones. The tadpoles exhibit a depressed body, short tail, low tail fins, dorso-laterally directed eyes and jaw sheaths; these features identify the Libros tadpoles as members of the benthic lentic ecomorphological guild. This, the first ecomorphological reconstruction of a fossil tadpole, supports phylogenetic evidence that this ecology is a conserved ranid feature. The soft-tissue features of the Libros tadpoles are characterized by several modes of preservation. The space occupied previously by the brain is defined by calcium carbonate, the nerve cord is defined by calcium phosphate, and jaw sheaths and bone marrow are preserved as organic remains. Gut contents (and coprolites adjacent to specimens) comprise ingested fine-grained sedimentary detritus and epiphyton. The body outline and the eyespots, nares, abdominal cavity, notochord, caudal myotomes and fins are defined by a carbonaceous bacterial biofilm. A similar biofilm in adult specimens of R. pueyoi from Libros defines only the body outline, not any internal anatomical features. In the adult frogs, but not in the tadpoles, calcium phosphate and calcium sulphate precipitated in association with integumentary tissues. These differences in the mode of preservation between the adult frogs and tadpoles reflect ontogenetic factors. h Anuran, ecology, soft- tissue, tadpoles, taphonomy.

Maria E. McNamara [[email protected]], Patrick J. Orr [[email protected]], UCD School of Geological Sciences, University College Dublin, Belfield, Dublin 4, Ireland; Stuart L. Kearns [[email protected]], Department of Earth Sciences, University of Bristol, Wills Memorial Building, Queen’s Rd, Bristol BS8 1RJ, UK; Luis Alcala´ [alcala@ dinopolis.com], FundaciońConjuntoPaleontolo´gico de Teruel-Dino´polis, Avda. Sagunto s ⁄ n, 44002 Teruel, Aragoń, Spain; Pere Anadoń [[email protected]], Consejo Superior de Investigaciones Cientı´ficas, Institut de Cie`ncies de la Terra ‘Jaume Almera’, Lluı´s Sole´ iSab- arı´ss⁄ n 08028, Barcelona, Spain; Enrique Penãlver-Molla´ [[email protected]], Museo Geominero, Instituto Geolo´gico y Minero de Espanã, C ⁄ Rıós Rosas 23, E-28003 Madrid, Spain; manuscript received on 30 ⁄ 12 ⁄ 2008; manuscript accepted on 24 ⁄ 06 ⁄ 2009.

Exceptionally preserved anurans (Rana pueyoi)from morphologyoftheLibrostadpolescouldtherefore the lacustrine-hosted Miocene Libros biota (Spain) constrain hypotheses of their palaeoecology. These comprise both larvae (representing a range of develop- results would have significant wider implications. mental stages) (n = 72) (Figs 1, 2) and adults Soft tissues are preserved in larval anurans from (n = 73). The taphonomy of the non-biomineralized numerous Late Cretaceous and Cenozoic localities (soft) tissues of the adult frogs has been studied com- (Young 1936; Nevo 1968; Sˇpinar 1972; Estes et al. prehensively (McNamara et al. 2006, 2009). Previous 1978; Wassersug & Wake 1995; Maus & Wuttke observations of the larval frogs comprise only brief 2002; Toporski et al. 2002; Rocˇek & Van Dijk 2006). references to a brown carbonaceous bacterial biofilm Collectively, this material has improved our under- that defines the general body outline and, in a single standing of anuran phylogeny, most importantly via specimen, the presence of organically preserved bone the construction of ontogenetic series (e.g. Maus & marrow (McNamara et al. 2006). This paper therefore Wuttke 2002; Rocˇek & Van Dijk 2006). Complemen- considers in detail the taphonomy of the larval frogs tary studies of larval anuran taphonomy and ecology, from Libros. however, are rare (but see Maus & Wuttke 2002; Notably, the soft-tissue morphology of modern Toporski et al. 2002). anuran larvae varies strongly with ecology and habi- This paper also considers how biological factors tat (Table 1). Reconstruction of the soft-tissue control exceptional fossil preservation. Exceptional

DOI 10.1111/j.1502-3931.2009.00192.x 2009 The Authors, Journal compilation 2009 The Lethaia Foundation LETHAIA 43 (2010) Miocene fossil tadpoles 291 faunas often include various developmental stages of specimens were recovered during commercial exploi- the same taxon: the preservation potential and the tation of the sulphur and oil shales of the Libros mode of preservation of each should not be assumed Gypsum Unit in the early 20th century; the original to be constant. Different developmental stages can field context and way up of specimens is unknown. vary in their ecology, and thus different biostratinom- The precise fossiliferous horizons are unknown and ic processes may affect carcasses. Factors that influence it is therefore impossible to determine to what decay and mineralization, such as physiology and tis- extent the curated specimens are an unbiased sample sue chemistry, can also vary during ontogeny. The of the fossil assemblage. All developmental stages occurrence of both adult and larval stages of R. pueyoi that were preserved may not be represented. Intui- in the Libros biota is therefore an opportunity to elu- tively, smaller specimens, broadly synonymous with cidate the extent to which differences in the mode, earlier developmental stages, are more likely to be and fidelity, of soft-tissue preservation of the same absent. Similarly, all states of preservation may not taxon have been influenced by factors relating to their be represented; disarticulated specimens and those ontogeny. lacking obvious soft-tissue outlines are more likely to be under-represented. Not all of the soft-tissue features discussed below Geological background could be identified in each specimen, e.g. if a specimen was incomplete, truncated by the edge of the The Libros lacustrine system developed within the slab, or if soft tissues were obscured in part by sedi- Early Miocene–Late Pliocene Teruel Basin in NE ment. The number of specimens in which a particular Spain (Ortı´ et al. 2003). The Teruel basin-fill in the feature is present is therefore indicated in the text in Libros region comprises up to 500 m of alluvial parentheses. terrigenous facies, lacustrine carbonates and evaporites. The deepest water facies of the Libros Ontogeny sequence is the 150-m-thick late Miocene (Vallesian) Libros Gypsum Unit. The lowest part of this unit, The general categories of larval anuran development the bituminous–calcareous subunit, comprises inter- sensu Gosner (1960) are: (1) embryo (stages 1–20); (2) calated charophytic limestones and laminated hatchling (stages 21–25); (3) tadpole (stages 26–41) mudstones (including oil shales) with deposits of and (4) metamorph (stages 42–46). Twelve incom- native sulphur and rare primary gypsum (Ortı´ et al. plete R. pueyoi larvae could not be staged. The remain- 2003). The exceptional biota comprises salamanders, ing 60 specimens fall within a narrow range of frogs, birds, snakes, insects, arachnids and leaves developmental stages (Gosner stages 30–41) and (Nava´s 1922a,b; Olson 1995; Penãlver 1996) and is represent fossil tadpoles. hosted within the laminated mudstones. This facies More detailed resolution of the data is difficult. The represents deposition within the profundal regions Gosner (1960) staging system (and also the Shumway of a permanently stratified bench-type lake, in which 1940; Taylor & Kollros 1946 systems) for modern laterally extensive shallow-water zones (<10 m ranid larvae is based primarily upon the presence of depth) were separated from deeper waters by steep specific soft-tissue morphological features and cannot slopes. The monimolimnion was anoxic and sulphi- be applied easily to fossil anuran larvae. The skeletal dic, and there was intense bacterial sulphate reduc- ossification sequence has been described for Rana tion in the uppermost sediment column (Anadoń pipiens (Kemp & Hoyt 1969); however, the relative et al. 1992; de las Heras et al. 2003; Ortı´ et al. timing of ossification of skeletal elements, and the 2003). order in which they ossify, varies considerably among ranids (Sheil 1999), making extrapolation to other taxa difficult. For example, certain cranial elements Material and methods begin to ossify 11 stages earlier in R. temporaria (de Jongh 1968) than in R. pipiens (Kemp & Hoyt 1969). A total of 72 specimens were examined from the The Libros specimens present additional difficulties. collections of the following institutions: Forschungs- The skeleton is often obscured in part by sediment, institut und Naturmuseum Senckenberg, Frankfurt, gut contents or diagenetic minerals; the ossified parts Germany (FNS), Museu de Geologia de Barcelona, of elements diagnostic of certain stages (e.g. the max- Barcelona (MGB), Museo Nacional de Ciencias illa, femur, humerus and ischium) are less than 1 mm Naturales, Madrid (MNCN) and the Natural History long initially and therefore difficult to identify. Museum, London (NHM). Specimens occur mainly It is not possible to assign any tadpole of R. pueyoi as individuals, on slabs with trimmed edges. All to a single developmental stage. Each specimen can, 292 McNamara et al. LETHAIA 43 (2010)

A F

E LETHAIA 43 (2010) Miocene fossil tadpoles 293

Fig. 2. Detail of MNCN 63786 A, showing soft tissue features preserved and B, interpretative drawing. however, be assigned to one of three groups of consec- 2 Gosner stages 36–37 (ten specimens). Ossification utive developmental stages, described below. These are of the fronto-parietals has begun; total length defined by the skeletal elements present and their 60–100 mm (Fig. 1F). degree of ossification. 3 Gosner stages 38–41. Ossification of the prootics has initiated; ossification of the parasphenoid, 1 Gosner stages 30–35 (six specimens). The parasphe- fronto-parietals, exoccipitals and vertebrae is noid, exoccipitals and neural arches of the verte- advanced. The femur, tibiofibulare, humerus, pre- brae are the only skeletal elements visible; each is maxilla, maxilla and angular may be visible; total poorly ossified; total length (as defined by soft tis- length 110–145 mm (Fig. 1A–E, G). sues) 35–70 mm (Fig. 1H).

Fig. 1. Photographs of exceptionally preserved tadpoles of Rana pueyoi from Libros. A, Gosner stages 38–41; dorso-ventral. Small arrow indicates coprolite (R4999). B, Gosner stages 38–41; lateral. Small arrow indicates coprolite, large arrow indicates the point of origin of the tail ﬁn, white arrowhead indicates the margin of a hind limb, and black arrowhead indicates the ventral margin of the notochord (MNCN 63786). C, Gosner stages 38–41; oblique. Arrowhead indicates the margin of a hind limb. Note deﬁnition of the lungs as a speckled texture in the abdomen (MNCN 63799). D, Gosner stages 38–41; dorso-ventral. Small arrow indicates the gut contents (27147, MGB). E, Gosner stages 38–41; lateral (27143, MGB). F, Gosner stages 36–37; dorso-ventral. Small arrows indicate the lungs; arrowhead indicates the jaw sheath (MNCN 389). G, Gosner stages 38–41; dorso-ventral. Small arrow indicates a coprolite; arrowheads indicate the nares (MNCN 63821). H, Gosner stages 30–35; dorso-ventral. Small arrows indicate the lungs (MNCN 63771). Note also the concentration of white material in the cranium (A–H) and the thin light-coloured line in varying degrees of fragmentation along the length of the tail (A–E). Scale bar: 20 mm. 294 McNamara

Table 1. Primary ecomorphologies of modern anuran larvae; data compiled from Altig & Johnston (1989) and McDiarmid & Altig (1999).

Anatomical ⁄ morphological

features (2010) 43 LETHAIA al. et

Fin Keratinous jaw Position of Distinctive EcomorphologyHabitat Body shape Eye position morphology sheath present? oral disc features

Lentic ⁄ lotic Nektonic Within water column Compressed Lateral High with pointed tip; Yes – Upturned oral disc PO anterior to TBJ Neustonic Near water surface Depressed Lateral Low Not always Upturned Benthic Near sediment–water Depressed Dorsal Low; PO at ⁄ near TBJ Yes; high density Ventral interface of micro-serrations Lentic Arboreal Terrestrial; isolated Variable – Variable Usually – Several (non-flowing pockets of water systems) esp. in trees Carnivorous Variable Widest closest Lateral – Yes Terminal Jaw sheaths highly to eyespots modified Macrophagous Variable Variable Variable – Yes Terminal Gut contents contain Suspension feeder Within water column Circular in Lateral – No Terminal large particles dorsal aspect or absent Suspension rasper Within water column Variable Lateral Ventral fin higher Yes – Tail flagellum than dorsal Benthic Near sediment–water Depressed, Dorsal Low; PO at ⁄ near TBJ Yes; low density Ventral interface short tail of micro-serrations Lotic Clasper Fast flowing water Globular ⁄ Variable Variable – Ventral Modified oral disc (flowing depressed systems) Adherent Fast flowing water Depressed Variable Variable – Ventral Modified oral disc Suctorial Fast flowing water Depressed Variable Variable – Ventral Highly modified oral disc with large sucker Fossorial Leaf mats in backwaters Vermiform Variable Variable – Ventral Modified jaw sheaths Gastromyzophorous Fast and turbulent water – Variable Low; PO well –Ventralwith large oral sucker posterior to TBJ Psammonic Bury in sand – Variable Variable No Ventral Vermiform body shape Semiterrestrial Terrestrial rock faces Vermiform Variable Variable – Ventral and forest floor

PO, point of origin (of tail ﬁn); TBJ, tail–body junction. LETHAIA 43 (2010) Miocene fossil tadpoles 295

A exhibit an identical succession of laminae represent the same stratigraphic interval.

Analytical methods The methods used to analyse the soft tissues have been described previously (McNamara et al. 2006). In sum- B mary, SEM analyses of carbon- or gold-coated samples were performed with a Hitachi S-3500N microscope at an accelerating voltage of 15 kV and acquisition times of 60 s for EDS spectra of carbon- coated samples. Unstained resin-embedded samples C were sectioned with a diamond knife, and TEM analyses performed using a JEOL 2000TEMSCAN at 80 kV withanobjectiveapertureof10lmdiameter.

Terminology The terminology used to describe specimens follows D that of Altig & McDiarmid (1999). The body is the region between the anterior tip of the head (the snout) and the junction between the posterior body wall and the axis of the tail myotomes (the body terminus); the tail is posterior to the body terminus. The transversely oriented buccopharyngeal wall in the plane of the first vertebra divides the body into two sections: the cranium and buccopharynx are anterior, and the abdomen posterior, of the wall. The following variables were measured (Fig. 3A, B): Fig. 3. Tadpole body plan (A, B) and anatomy (C). A, B, schematic illustrations of the body plan of a tadpole in lateral (A) and body length (the horizontal distance from the snout to dorsal (B) views. BH, body height; BL, body length; BW, body the body terminus), body height (the maximum height width; TAL, tail length; TL, total length. C, simplified tadpole anat- of the body in lateral aspect), body width (the maxi- omy. Left-hand diagram shows a tadpole in lateral view; for clarity, the notochord is shaded in grey and the caudal myotomes are not mum width of the body in dorso-ventral aspect) and shown in the anterior part of the tail. Right-hand diagram is a tail length (the distance from the body terminus to the schematic vertical section through the tadpole tail along line X–Y tip of the tail). The positions of the maximum height in the left-hand diagram. a, abdomen; br, brain; bu, buccopharynx; e, eye; f, fin; i, intestine; j, jaw sheath; l, lung; m, caudal myotomes, and width of the body were recorded. As growth dur- ma, manicotto; ms, myotome sheath; na, external nares; nc, nerve ing tadpole stages is typically isometric (McDiarmid & cord; no, notochord; ns, notochord sheath. Altig 1999), the ratio of any two components is consistent between developmental stages. The completeness and orientation of individual specimens Sedimentary context controlled which of these variables could be recorded. Body and tail morphology are defined sensu In vertical section, each fossil-bearing slab exhibits a McDiarmid & Altig (1999). The body is compressed if distinctive, ‘bar code’-like striped pattern, generated height exceeds length and depressed if the reverse. An by alternations of laminae of different composition, oval body is the widest near the centre of the abdomen colour and thickness. To determine whether two or and tapers anterior and posterior of this; spherical and more fossil-bearing slabs include the same horizon, globular denote spherical and more irregular body digital images of the vertical edge of each slab were shapes respectively. Tail fins are low if the periphery of printed to the same scale and the position of the fos- each fin is close to the periphery of the myotomes and sil-bearing lamina marked. The lamina succession in high if not. No standard definition exists for tail oneimagewascomparedwiththatinallothers(simi- length; examination of published literature indicates lar to the technique used by Trewin 1986). The image that tails more than 1.5 times body length are long was then rotated by 180 and the process repeated (as and short if less. Variables measured for the body and theway-upofeachslabisunknown).Themostparsi- tail of the 16 most complete Libros tadpoles are sum- monious interpretation is that two or more slabs that marized in Table 2. 296 McNamara et al. LETHAIA 43 (2010)

Table 2. Various ratios calculated from measurements taken from A fossil specimens in which the body margin is deﬁned by soft tissues.

Body length: Body length: Body length: Specimen tail length body height body width

11.12 – 20.891.83– 3 1.22 – 1.58 4 – – 2.03 5 0.79 – 1.47 6 – – 1.95 7 1.08 – 1.97 8 0.74 – 2.14 90.85– 2 10 – 1.65 – 11 – 1.92 – 12 – – 1.41 B 13 – 2.04 – 14 – – 1.95 15 – – 1.72 16 0.93 – – Mean 0.95 1.89 1.82 SD 0.17 0.16 0.26

The orientation of each specimen is described as follows: dorso-ventral if the sagittal plane is perpendic- ular to bedding, oblique if the sagittal plane is inclined to bedding and lateral if the sagittal plane is parallel to bedding. C Cause of death

In addition to predation, mechanisms that result in the death of anuran larvae include asphyxiation due to increases in epilimnetic temperature (Elder & Smith 1988), and the release of toxic metabolites into the epilimnion during algal blooms (Boyer 1981; Buchheim & Surdam 1981). Such environmental stresses often generate mass mortalities. In the fossil record, mass mortality events are most apparent where high numbers of individuals co-occur in either the same bed or, in laminated sequences, on the same horizon (see, e.g. Martill et al. 2008; Fig. 4). The phe- Fig. 4. Structure of the bacterial biofilm. A, light micrograph of surface of biofilm showing polygonal fracturing and, inset, frac- nomenon is difficult to identify in cases, such as tured vertical section through biofilm (MNCN 63796). B, scanning herein, where the data set comprises individual speci- electron micrograph of fossilized bacteria (27143, MGB). C, trans- mens. Comparison of the lamina succession of each mission electron micrograph of bacteria showing homogenous, extremely low electron lucency (MNCN 63821). r, resin. fossil tadpole-bearing slab from Libros, however, dem- onstrates that most specimens (52; n = 70) are from different horizons; the remaining 18 are from seven horizons. This distribution implies that the supply of Physical taphonomy specimens to the site of deposition was on an on- going basis over an extended time interval. This is All specimens are highly articulated. This, combined inconsistent with, but, on its own, does not eliminate with the preservation of soft tissues (see below), indi- completely, the death of the tadpoles having occurred cates that specimens were deposited in profundal during mass mortality events. The cause of the tad- lake zones shortly after death. The absence of fish in poles’ death therefore remains unknown. the lake would have eliminated scavenging of LETHAIA 43 (2010) Miocene fossil tadpoles 297 carcasses in the period between death and deposi- In many specimens, the biofilm pseudomorphs the tion; after deposition, bottom water conditions pre- general morphology of the body and tail (Figs 1A–E, cluded disturbance of carcasses by bioturbators or H, 2A). The biofilm can also define the following ana- scavengers. All specimens are orientated with the tomical features. anterior–posterior axis of the body and tail parallel to bedding; this suggests the lake floor was firm and Abdominal cavity. – In 42 specimens (n =53)(all cohesive at the time of deposition. Specimens occur Gosner stages 36–37 or 38–41) the biofilm is signifi- in dorso-ventral (19 specimens; n = 43), lateral (16 cantly thicker in the posterior of the body than else- specimens) or oblique (seven specimens) aspect. Dif- where(Figs1A–E,G,2A).Thismorelikelydefinesthe ferences in the number of specimens in each aspect ovoid abdominal cavity than the yolk sac: in modern are not statistically significant (5.57; d =2; ranid larvae the latter is resorbed fully by Gosner stage 2 v2 = 13.82; P < 0.001), i.e. the larvae do not occur 24. In vivo, the abdominal cavity houses a tightly in any preferred orientation. This reflects the near- coiled intestine, the liver, pancreas and lungs (Viertel circular geometry of cross-sections of the tadpole & Richter 1999). The preferential development of a body in vivo. thick biofilm in this region, but not in the anterior part of the body, reflects the greater volume of soft tissues available for bacterial consumption within the Soft-tissue preservation abdomen; in particular, the digestive tract comprises 50% of the biomass of a tadpole (Wassersug 1974). Bacterial biofilm The anterior of the body, however, includes extensive void space (the buccopharangeal cavity). General features. – The general outline of the soft tissues is defined by a brown layer (Fig. 1) that envelops Lungs. – In six specimens (n = 53), one, or a pair of, the bones and gut contents; where thick, it is fractured elongate structures occurs in the abdominal cavity. In into a series of polygons (Fig. 4A). The layer is the dorso-ventral orientation, each originates either side thickest and most extensive in specimens at more of, and extends posteriorly at an acute angle to, the advanced developmental stages. Its thickness also var- vertebral column (at VI–VII) (small arrows in Fig. 1F, ies within an individual specimen: it is thick (up to H). The position, size and geometry of these struc- 1.3 mm; average 300 lm), and continuous, in the tures match those of the primordial lungs in tadpoles abdomen, thin (5–10 lm) and discontinuous in the of modern Rana, and resemble those of lungs identi- tail and buccopharynx, and of intermediate thickness fied in other exceptionally preserved anuran tadpoles (80 lm) in the eyespots. The layer comprises densely (see Sˇpinar 1972). The structures are evident in speci- packed, predominantly ovoid, micro-structures, each mens from Libros only at Gosner stages 30–35. This is ca. 1 lm long (Fig. 4B). The possibility that these attributed to variation in biofilm thickness, not differ- structures represent fossilized melanosomes (see Vin- ences in the preservation potential of the lungs during ther et al. 2008) is yet to be considered in detail. ontogeny. The biofilm in the abdomen is notably thin Herein, we follow previous authors who have studied in specimens at Gosner stages 30–35 (Fig. 1F, H), similarly preserved faunas (e.g. Wuttke 1983a; Topor- enhancing the likelihood of identifying anatomical ski et al. 2002) and consider this layer to represent a features that are defined subtly. In larger specimens, biofilm of fossilized bacteria (a Hautschatten (skin the abdominal cavity can exhibit a distinctively speck- shadow) sensu Wuttke 1983a). The structureless mate- led texture if the biofilm is thin (Fig. 1C). Lungs in rial associated with the bacteria is interpreted as extra- modern anurans have a honeycomb-like lattice struc- cellular polymeric substance (EPS), rather than the ture (Maina 1989), degradation of which could poten- degraded remains of the original tissues. tially generate the speckled texture evident in the In TEM images the bacteria are uniformly electron fossils. lucent (Fig. 4C). There is no evidence for authigenic minerals in association with either the bacteria or Eyespots. – One or both eyespots occur(s) in 30 spec- EPS; both comprise primarily C and S, which is attrib- imens (n = 49) (Figs 1A, B, D, F–H, 3A). Their out- uted to a high abundance of organosulphur com- line is circular in specimens in lateral aspect (Fig. 1B). pounds (McNamara et al. 2006). Experimental In dorso-ventrally orientated specimens the eyespots degradation of anurans has demonstrated that micro- areelliptical(withthelongaxisoftheellipseparallel bial consumption of soft tissues initiates in the gut to the body axis (Fig. 1F)), and contained inside the and mouth (Wuttke 1983b); the fossil bacteria are body margin (Fig. 1A). The eyes of the tadpoles there- therefore probably anaerobic heterotrophs derived fore faced laterally and were on the dorsal body sur- from the indigenous intestinal community. face (Altig & McDiarmid 1999). Preservation of the 298 McNamara et al. LETHAIA 43 (2010) eyespots reflects the high recalcitrance of the sclera rel- and references therein). The apparent absence of the ative to the surrounding soft tissues. The sclera com- notochord inside the body of the fossil specimens prises layers of collagen, a decay-resistant tissue could result from its being obscured by the vertebrae (Briggs & Kear 1994) that would have retarded micro- and ⁄or thick biofilm in the abdomen. bial infiltration of the eye cavity in the initial stages of In laterally orientated specimens, the biofilm decay; the surrounding tissues would have degraded defines a single brown line parallel to, and 2–4 mm more rapidly, leaving the eyespots as isolated outliers ventralof,thetailaxis(blackarrowhead,Figs1B,2A). of biofilm. This is presumably the position of the ventral margin of the notochord in vivo. The dorsal margin is difficult Nares. – The nares (the external extensions of the to identify but could be obscured by the dorsal nerve nostrils), approximately midway between the snout cord that runs along the tail axis (see below) (Figs 1B, and the parasphenoid, are defined in nine specimens 2A). (n =49)(arrowheadsinFig.1G).In vivo the margins In vivo the notochord comprises a core of vacuo- of the nares have distinct thick, fleshy protuberances; lated cells and a tough collagenous outer sheath definition of the nares may therefore have been (Wassersug 1980). It is a recalcitrant structure: in enhanced by the greater thickness of soft tissues rela- Branchiostoma decayed under anoxic conditions the tive to surrounding areas of the buccopharynx. notochord persisted in recognizable form for up to 124 days (Briggs & Kear 1994). Preservation of the Notochord. – The former position of the notochord notochord in two dimensions as a pair of parallel is defined in 14 specimens in dorso-ventral aspect lines, not a solid band, is notable. After preferential and, more tentatively, in three in lateral aspect decay of the core of vacuolated cells, the notochord (n = 36). In dorso-ventral aspect, the notochord is sheath would have been hollow and elliptical in cross- defined as a pair of thin, closely spaced (average 1 and section. Its subsequent, decay-induced, collapse would 1.5 mm apart in specimens at Gosner stages 30–35 produceastructureinwhichthethicknessoftissue and 38–41 respectively), parallel, brown lines that run would have been greater at the two edges than medi- along the tail axis (Fig. 5A, B). This spacing is similar ally. Thus, the margins have greater preservation to the breadth of the notochord in the extant R. cates- potential simply as a function of their being thicker. beiana at Gosner stage 30 (Bruns & Gross 1970); its This applies to specimens preserved in both lateral height is greater as the notochord is highly com- and, especially, dorso-ventral aspect (Fig. 6). pressed laterally (Wassersug 1980). These lines can extend from the posterior terminus of the vertebral Caudal musculature. – In 14 specimens (n =35)the column along the visible length of the tail. They margin of the caudal myotomes is defined by a thin resemble structures noted in the tails of primitive brown line (Figs 1A–C, 2A). This occurs irrespective chordate fossils, and those generated during experi- of specimen orientation and thus defines the left and mental decay of the lancelet Branchiostoma (by col- right sides of the myotomes (in specimens in dorso- lapse of the notochord sheath; Briggs & Kear 1994, ventral aspect) and their ventral and dorsal margins

A B

Fig. 5. Subtle internal features defined by the biofilm. A, B, detail of tail of R4999 (NHM) (A) and MNCN 63848 (B), each in dorso-ventral aspect, showing preservation of margins of notochord sheath as a pair of thin parallel brown lines (arrows) along the tail axis. Near-linear white feature in A is the nerve cord, defined by calcium phosphate. Scale: 5 mm. LETHAIA 43 (2010) Miocene fossil tadpoles 299

Fig. 6. Notochord modelled as a hollow cylinder with an elliptical cross-section preserved in dorso-ventral aspect (A) and lateral aspect (B). In each case (especially A) its vertical collapse downwards as a result of decay generates a structure in which the tissue is the thickest at its margins. The thickness of the notochord sheath (which is exaggerated for clarity) along each of sections A–B and C–D is greater than the cumulative thickness of the sheath along sections E–F and G–H.

(in those in lateral aspect). It was therefore continu- all but two specimens from later stages is more prob- ous, surrounding the myotomes, and probably corre- lematic. In laterally orientated specimens, other soft sponds to the collagenous sheath that enclosed the tissues might obscure the outline of the developing myotomes in vivo. limbs. In many fossil specimens at advanced developmental stages the relevant parts of the body are either Fins. – The outlines of the tail fins are preserved in concealed by sediment or the specimen is truncated only four specimens (n = 35), primarily as a thin anterior to the tail ⁄body junction. brown line that marks their margin; their surfaces are indistinct. The latter reflects the limited volume of soft Other soft-tissue features tissue in the fins (epidermis enclosing a thin layer of loose connective tissue; McDiarmid & Altig 1999) and White masses in cranium. – In 64 specimens (n = 69) their having degraded relatively rapidly. The dorsal fin friable white material occurs in the cranium between originates close to the junction between the body mar- the parasphenoid and fronto-parietals, and within the gin and caudal myotomes (large arrow in Figs 1B, 2). prootics (Figs 1A–H, 2A). The material comprises The margin of each fin lies 5 mm outside, and runs euhedral, 5- to 15-lm-long crystals of calcium car- approximately parallel to, the margin of the caudal bonate, probably aragonite (a strong strontium peak myotomes. is present in EDS) (Fig. 7A). In modern anurans, deposits of calcium carbonate occur within the Other caudal features defined by biofilm. – Although endolymphatic sac that is associated with the nervous extant ranids vary in both the timing and pattern of tissues (Kawamata et al. 1987), i.e. the brain (located skeletal ossification, hind limb buds begin to develop close to the fronto-parietals and parasphenoid by,atthelatest,Gosnerstage30;hindlimbsaretypi- in vivo), nervous ganglia of the prootics and nerve cally large and distinct by Gosner stage 41. Assuming cord. In ranid tadpoles, the endolymphatic sac extends the same applied to the Libros tadpoles, at least the along the entire length of the nerve cord (Kawamata majority specimens should exhibit hind limbs. Devel- et al. 1987) and, in the cranium, diverges into two oping hind limbs are, however, present in only two irregularly shaped diverticula; one marginal to and specimens from Libros (both at Gosner stages 38–41): either side of the brain (Whiteside 1922; see plate 1 in each, the biofilm defines two or more parallel lines in Pilkington & Simkiss 1966). This distribution, in the abdomen that are ventral of, and at an angle to, however, does not correspond to that of the calcium the vertebral column (white arrowheads in Figs 1B, C, carbonate in the Libros tadpoles. The latter exhibit no 2A). The absence of hind limbs in fossil specimens evidence of calcium carbonate in the postcranial body from earlier Gosner stages could reflect the small size and tail; in the cranium, the calcium carbonate occurs of both the buds and the specimens. Their absence in as a single, unpaired, white mass that is continuous 300 McNamara et al. LETHAIA 43 (2010)

A extent of the calcium carbonate, however, suggest that it is better interpreted as a very early diagenetic precip- itate that defines the former position of the brain and minor nervous ganglia. The calcium carbonate does not replicate any tissue per se, but infills the space cor- responding to the latter. The process therefore reflects the generation of a micro-environment rich in calcium and bicarbonate ions, the latter sourced via rapid liq- uefaction of nervous tissue (McNamara et al. 2009). The deposits of the endolymphatic sac would have constituted a particularly rich source of such ions.

Dorsal nerve cord. – In 24 specimens (n = 67) white material defines a continuous or fragmented, 0.5-mm- wide structure that runs along the axis of the body B and ⁄or tail (Figs 1A–E, 2A). This white material is friable and comprises sub- to anhedral, 0.8- to 3-lm-long crystals of calcium phosphate (Fig. 7B). The linear nature of the structure is most obvious in the tail, where it runs parallel to, and dorsal of, the notochord (Fig. 1B); in the body, the structure is often obscured in part by the vertebral column. The geometry and position of the white material correspond to that of the nerve cord; this is situated within a recess in the dorsal margin of the notochord in vivo (Wassersug 1980). In one specimen from Libros, the linear structure is cohesive and exhibits a fibrous texture (Fig. 7C), possibly representing mineralized bundles of nerve fibres. In the remainder, the material defining the nerve cord lacks structure. In these cases the calcium phosphate probably precipitated on the template C provided by the decaying tissues of the nerve cord. The nerve cord occurs as: (1) a near-continuous straight or undulating line (Fig. 1A, B); (2) a series of aligned short fragments of varying length, some of which are displaced from life position but rarely occur outside the body margin (Fig. 1C); (3) many short, variably orientated fragments, which are often reflexed and ⁄or occur outside the body margin (Fig. 1D, E); and (4) short, isolated, highly dispersed fragments. This sequence represents progressive stages in the degradation of the nerve cord. Fragments of nerve cord are not contiguous even in decay stages 1 and 2, and always have rounded termini; this suggests that con- traction and at least the initial separation of the cord occurred prior to mineralization. Fragments in some Fig. 7. Anatomical features defined by white material. A, scanning electron micrograph of white material in the cranium (27143, specimens are approximately equal in length (Fig. 1C, MGB) showing euhedral crystals of calcium carbonate. B, scanning D). The pattern of fragmentation may have been con- electron micrograph of white material in the nerve cord (MNCN trolled by the nerve cord being segmented along its 63786) showing sub- to anhedral crystals of calcium phosphate. C, light micrograph of part of part of the nerve cord (27146, MGB) length; this feature is widespread among most verte- that exhibits a fibrous texture. brate classes (at least during their embryonic and ⁄or larval stages) (Keynes & Stern 1984). across the sagittal axis. It is possible that some of the calcium carbonate in the tadpoles could represent ori- Gut contents. – These occur in the abdomen of 15 ginal endolymphatic deposits. The distribution and specimens (n = 54), mainly as light brown-coloured LETHAIA 43 (2010) Miocene fossil tadpoles 301 curvilinear features 0.3–0.5 mm wide and up to Granular gut contents in other fossil tadpoles have 80 mm long (arrow, Figs 1D, 8A); they can define been interpreted as ingested shallow-water, fine- accurately the geometry of part of the coiled intestine grained sediment (Sˇpinar 1972; Maus & Wuttke (Fig. 8A). The contents have a granular texture 2002). The high abundance of diatoms in the gut con- (Fig. 8B) and usually comprise silt-sized grains of cal- tents of the Libros tadpoles suggests that rasping of cium carbonate and fragmented diatom frustules algal communities associated with submerged vegeta- (Fig. 8C); siliceous sponge spicules also occur. tion (i.e. epiphyton) was an important feeding strategy. Gut clearance in modern anuran larvae typically occurs within 4–6 h of ingestion (Alford 1999). The A presence of gut contents in the fossils therefore indicates that death occurred shortly after ingestion of a meal.

Coprolites. – These occur within 5–8 mm of the body margin in six specimens (n =53) (small arrows in Fig. 1A, B, G) and are similar in composition to the gut contents. In specimens in lateral aspect, coprolites consistently occur close to the ventral margin of the body. This association indicates that the coprolites represent evacuation of the bow- els during the very early stages of decay due to post-mortem relaxation of the bowel muscles, in B particular the sphincters. Given that gut clearance is likely to have been rapid in vivo (see above), these tadpoles were deposited on the lake floor shortly after their death. Notably, these larvae are not characterized by an appreciably higher fidelity of preservation (i.e. higher articulation, more extensive biofilm, greater definition of anatomical features) than the remainder. This suggests that the latter were also deposited shortly after death and that, collectively, the specimens did not experience pro- nounced differences in either the extent or duration of transport in the interval between death and their deposition. C Jaw sheath. – This is preserved in ten specimens (n = 44), all at Gosner stages 38–41. It comprises a 3-mm-wide, 300-lm-thick, black-coloured, arcuate structure (arrow in Figs 1F, 9A). The sheath is usually anterior of the cranial bones but slightly posterior of the snout; i.e. in approximately life position (Fig. 1F). It can, however, be displaced by up to 15 mm and re- orientated by up to 70 from this (Fig. 9A); in modern taxa the jaw sheath does not articulate with the skeleton and separates from the carcass within a few days of death (Altig & McDiarmid 1999). As in modern tadpoles (Altig & McDiarmid 1999, p. 39), the fossil sheaths have a fissile ultrastructure. Each is carbona- Fig. 8. Gut contents and coprolites. A, detail of A322 (FNS) show- ceous with distinctive micro-serrations on its oral sur- ing definition of part of the coiled intestine as a series of overlap- ping, light-coloured curved features (small arrow) in the abdomen. face (Fig. 9B). Each micro-serration is ca. 32 lmlong, Gosner stages 38–41; lateral. B, light micrograph of gut contents 25 lm wide and 4 lm thick, has an irregular oral showing granular texture (2712, MGB). b, biofilm. C, scanning margin and is parallel-sided and concavo-convex electron micrograph of gut contents showing highly fragmented diatom frustules and silt-sized grains of calcium carbonate (27148, transversely (Fig. 9C). There are ca. 30 micro-serra- MGB). tions per mm in the fossils; this is at the lower end of 302 McNamara et al. LETHAIA 43 (2010)

A Bone marrow. – Very small (10–50 lmlong)fragments of bone marrow occur in the cranium and vertebrae of one specimen. The marrow is preserved as a sulphur-rich organic residue that retains the original red colour of haematopoietic bone marrow. Its preservation reﬂects the protective nature of the osteal micro-environment (which retarded microbial degradation of the marrow); sulphurization of organic mol- ecules within the tissue during early diagenesis enhanced its preservation potential (McNamara et al. 2006).

Ecology B Ecomorphological reconstruction The body of the R. pueyoi tadpole is the tallest and broadest in the plane of the sixth vertebra (Fig. 1A–D) and is oval in lateral aspect. The ratios of body length to body height and body length to body width are nearly identical, and the body is longer than it is high or wide (Table 2); i.e. the body is depressed, with a near-circular transverse section. The terminus of the tail is not visible in most specimens; extrapolation of the lines that define the margins of the caudal myotomes indicates that the tail is 1.1–1.3 times body length, C i.e. short. The fins are low. This anatomical configura- tion, in conjunction with: (1) the dorsal position of the eyes; (2) origin of the dorsal fin close to the dorsal tail ⁄body junction; (3) presence of keratinized mouth- parts; and (4) low density of jaw sheath micro- serrations, indicate that the Libros tadpoles are repre- sentatives of the lentic benthic ecomorphological guild (Table 1). Tadpoles of this guild typically inhabit niches close to the sediment–water interface in non-flowing (lentic) water systems; they feed by pas- sive filtration of phytoplankton and rasping peri- and epiplankton, bacteria and sedimentary detritus from submerged substrates (Larson & Reilly 2003; Grosjean Fig. 9. Details of the jaw sheath. A, light micrograph of MNCN et al. 2004). This interpretation is consistent with the 63783 with, inset, position of jaw sheath, which is disarticulated presence of silty and diatomaceous gut contents in and rotated by 70 from its position in vivo. Gosner stages 38–41, oblique. B, C, light (B (MNCN 63865)) and scanning electron (C, many Libros tadpoles. MNCN 63842) micrographs of micro-serrations on oral surface of This ecological reconstruction has significant impli- jaw sheath. cations for our understanding of the evolution of behavioural patterns in ranids. Most modern Rana the range observed in modern anuran larvae (30–80 species deposit their eggs in lentic habitats (Holman micro-serrations per mm) (Altig & McDiarmid 1999). 2003). Larval morphology in modern Ranidae is The preservation of the jaw sheaths, but not other highly conserved: larvae typically exhibit primitive fea- components of the complex oral apparatus, is attrib- turessuchasanovaldepressedbody,dorsaleyesand uted to their being keratinous. The keratin molecule is low fins, and many species belong to the lentic benthic characterized by a scaffold of inter- and intramolecu- guild (Holman 2003). The similarity between the lar- lar hydrogen- and disulphide bridges (Takahashi et al. val ecomorphologies in modern Rana,andR. pueyoi 2004): these render keratin insoluble and resistant to indicate that a lentic benthic ecological strategy has proteolytic attack (Coulombe & Omary 2002). been utilized for at least the last 10 Myr. This strategy LETHAIA 43 (2010) Miocene fossil tadpoles 303 need not, however, be the primitive condition: Rana potential of metamorphs is enhanced by their near- appears first in the late Oligocene. complete ossification, but this is more than offset by Cannibalism is common amongst modern anuran the shift in their ecology into an environmental niche tadpoles (Alford 1999). No Libros specimen, however, less conducive to preservation. includes vertebrate skeletal remains in its gut contents. Additional factors would have enhanced the preser- The anatomy of the Libros tadpoles is also inconsis- vational potential of specific developmental stages. tent with this behavioural strategy. The widest point For instance, the vast majority of embryos are con- of the body of cannibalistic tadpoles is near the plane sumed by predators prior to hatching (Alford 1999). of the eyes (due to hypertrophy of the jaw muscles); Different developmental stages vary in their duration. jaw sheaths are usually located terminally (Altig & Tadpole stages comprise the bulk of development time McDiarmid 1999) and are highly modified, typically (for example 56.7% in Bufo)(McDiarmid&Altig bearing long sharp projections (Grosjean et al. 2004). 1999); even if all other variables are constant these Further, cannibalism is most common in taxa that stages should provide the majority of fossil specimens. inhabit small ephemeral water bodies subject to dessication and ⁄or other environmental instability (Alford 1999). The Libros larvae, however, inhabited a perma- Ontogenetic controls upon nent water body. preservation

Absence of specific developmental stages The taphonomic history reconstructed for adult and larval R. pueyoi from Libros is broadly similar. Speci- Despite the high abundance of specimens, the Libros mens consistently exhibit high degrees of articulation tadpoles represent only certain developmental stages and completeness and are always orientated parallel to (Gosner stages 30–41). A bias towards specific develop- bedding; carcasses came to rest on the sediment–water mental stages has been noted in other exceptionally interface in an anoxic profundal environment and preserved larval anuran faunas (Chipman & Tchernov were buried subsequently by fine-grained laminated 2002; Maus & Wuttke 2002; Rocˇek 2003); tadpoles sediments. Shared features include definition of the from Enspel (Maus & Wuttke 2002) exhibit bias body outline and eyespots by a carbonaceous bacterial towards developmental stages similar to that of the biofilm, definition of the former position of the brain Libros specimens. Both the earliest (embryos and by calcium carbonate, and organic preservation of hatchlings) and metamorphic developmental stages are bone marrow. absent in the Libros material. This implies that progres- There are, however, several important taphonomic sive changes through ontogeny, e.g. increased ossifica- differences between the adult frogs and tadpoles that tion, are not the explanatory factor. Instead, selective can (to varying extents) be attributed to ontogenetic preservation of certain developmental stages is mod- factors. No soft tissues are replicated in authigenic elled as having been controlled primarily by changes in minerals in the tadpoles. In the adult frogs, however, ecology during ontogeny. The anuran embryo is sur- collagen fibres of the mid-dermal Eberth–Katschenko rounded by a tough vitelline membrane and several (EK) layer (which also contains interstitial glycosami- capsules (Holman 2003) and should have a high pres- noglycans (GAGs) and granules of calcium phosphate ervation potential (Raff et al. 2006). Despite this, in vivo) are replicated extensively in calcium phos- embryonic larvae are highly unlikely to be fossilized: phate (McNamara et al. 2009). The source of they are non-motile and typically develop in low- phosphate has been interpreted as the carcass, in par- energy, usually densely vegetated, extremely shallow- ticular, the granules of calcium phosphate in the EK water, environments (Holman 2003). Transport of layer (McNamara et al. 2009). The absence of the EK embryos offshore and deposition into environments layer in the Libros tadpoles reflects the developmental more favourable for soft-tissue preservation is there- biology of anuran larvae: the EK layer does not fore extremely unlikely. Hatchlings, although motile, develop until metamorphosis (Elkan 1968). remain in littoral habitats and, for the same reasons, In the adult frogs, the position of collagen fibres of areunlikelytobefossilized.Tadpolestages,however, the lower dermis is defined by calcium sulphate are characterized by a major behavioural shift towards discoids that precipitated as the collagen decayed a more active lifestyle (Alford 1999); they frequently (McNamara et al. 2009). This mineral phase does not range beyond highly vegetated littoral zones and are occur in the fossil larvae. In modern Rana,sulphated thus less likely to become entrained in marginal vegeta- GAGs, a potential source of sulphate, comprise 75% tion after death. This ecological shift is reversed subse- of the total integumentary GAGs in adults but less quently: metamorphs retreat into littoral vegetation than 4% of that in tadpoles (Lipson & Silbert 1968). until metamorphosis is complete. The preservational 304 McNamara et al. LETHAIA 43 (2010)

The biofilm defines the outlines of upper dermal is therefore more likely that the nerve cord in the tad- glands in only adult specimens (McNamara et al. poles was mineralized before dispersal of the frag- 2009). In modern anurans, many integumentary glands ments. This implies that the carcass was the source of do not develop until metamorphosis (Rosenberg the relevant ions. & Warburg 1978). Other anatomical structures (tail In summary, almost all the taphonomic differences musculature and lungs) are defined by the biofilm in between the adult frogs and tadpoles of the Libros only tadpoles from Gosner stages 30–35. Such subtle biota, and, to a lesser extent, between different devel- internal features may be obscured in tadpoles at later opmental stages of the tadpoles, can be attributed to developmental stages and in the adult frogs by the ontogenetic factors. Foremost among these are differ- thicker biofilm and, in the latter, by the preserved ences in the structure of certain organs and tissues, remains of the EK layer. Further, in the adult frogs, the and the absence of certain tissues at specific develop- biofilm medial to the EK layer comprises bacteria of mental stages. These ontogenetic changes in biology different morphotypes organized into discrete layers controlled which early diagenetic authigenic minerals (McNamara et al. 2009). The stratified chemical precipitated in association with the decaying carcasses, micro-environments implied by this complex structure which soft tissues were replicated in authigenic miner- may have developed only in the adult frogs due to the als, the extent to which bone marrow was preserved persistence of their integument during decay (collagen organically, and the extent to which internal anatomi- fibres of the EK layer were replicated during the late cal features were defined by the bacterial biofilm. In stages of decay; McNamara et al. 2009). other exceptional biotas it is likely that the taphonomy Bone marrow occurs in 10% of adult frogs but only of a taxon will vary depending on its developmental one tadpole; limited ossification, and thus high poros- stage. These subtle differences are superimposed on ity, of bone in the tadpoles would have facilitated bac- the more generalized taphonomic history, i.e. the terial infiltration of marrow cavities and enhanced the mode of preservation, of the exceptional biota as a rate of microbial degradation (McNamara et al. whole. 2006). Calcium phosphate is a common infill of the stomach in adult specimens, but does not occur inside the Wider implications stomach (the manicotto) of the tadpoles. The stomach of both adults and tadpoles would have had abundant The larval anurans from Libros exhibit various soft-tis- hydrolytic enzymes and a low pH (less than 3); decay- sue anatomical features that would have also been pres- ing ingested organic matter would have been a poten- ent in other fossil chordates. Relevant anatomical tial source of phosphate ions. Critically, the manicotto features include the broadly fusiform body shape, a lacks the muscular sphincters that separate, and could large anterior feeding chamber, a hollow nerve cord have sealed, the stomach from the remainder of the dorsal to a notochord and a terminally tapered tail with digestive tract (Viertel & Richter 1999). In their fins and segmented myotomes. Insights into the absence it may not have been possible to sustain a taphonomy of larval anurans could therefore constrain micro-environment conducive to precipitation of cal- interpretations of the anatomy, and affinities, of other cium phosphate within the manicotto during decay. fossils. This includes examples that, with varying Lastly, the nerve cord is defined by calcium phos- degrees of confidence, have been considered as chor- phate in only the tadpoles. The reason for this is dates, e.g. Haikouella (Chen et al. 1999; Shu et al. unclear, especially as the nerve cord in both adult and 2003), Myllokunmingia (Shu et al. 1999; Hou et al. tadpoles has a broadly similar structure and composi- 2002), Pikaia (Conway Morris 1998) and Yunnanozoon tion. Problematically, the timing of mineralization of (Chen et al. 1995), or that may have chordate affinities the nerve cord is difficult to constrain. Mineralization (e.g. vetulicolians; Aldridge et al. 2007). Such excep- occurred after fragmentation of the nerve cord, but tionally preserved fossils are crucial to attempts to deci- this does not imply that it preceded displacement of pher the origins of vertebrates, but interpretations of the fragments from life position. Mineralization after preserved anatomical features have been controversial dispersal of the fragments could imply that the source (Donoghue & Purnell 2009). Further, aspects of the of calcium and phosphorous was not the carcass itself. functional morphology and ecology are intrinsic to In this case, replication of the nerve cord, and poten- models of early vertebrate evolution (Purnell 2001). tially other tissues, in at least some adult frogs would These aspects of the biology of primitive chordate fos- be expected. There is no evidence for this in the Libros sils have received little attention. Herein, we have dem- specimens; the source of phosphate ions for replica- onstrated that an understanding of the taphonomy, tion of the mid-dermal collagen fibres in the adult and ecological significance, of specific anatomical fea- frogs was the carcass itself (McNamara et al. 2009). It tures allows the ecology of fossil anuran tadpoles to be LETHAIA 43 (2010) Miocene fossil tadpoles 305 resolved. Similar studies could facilitate more detailed Donoghue, P.C.J. & Purnell, M.A. 2009: Distinguishing heat from light in debate over controversial fossils. BioEssays 31, 178–189. ecological reconstructions of early chordates, putative Elder, R.L. & Smith, G.R. 1988: Fish taphonomy and environmen- examples thereof, and related taxa, thus enhancing our tal inference in palaeolimnology. Palaeogeography, Palaeoclima- understanding of vertebrate origins. tology, Palaeoecology 62, 577–592. Elkan, E. 1968: Mucopolysaccharides in the anuran defense against Acknowledgements. – We thank Drs Cormac O’Connell and Dave dessication. Journal of Zoology 155, 19–53. Cottell for assistance using TEM facilities at University College Estes, R., Sˇpinar, Z.V. & Nevo, E. 1978: Early Cretaceous pipid tad- Dublin, Eric Callaghan for assistance with preparation of histologi- poles from Israel (Amphibia: Anura). Herpetologica 34, 374–393. cal sections, Laura Tormo for ESEM analyses of the nerve cord and Gosner, K.L. 1960: A simplified table for staging anuran embryos jaw sheath, and the Aragoń Government (exp. 103 ⁄ 2003 from the and larvae with notes on identification. Herpetologica 16, Direccioń General de Patrimonio Cultural de Aragoń and FO- 183–190. CONTUR project) for access to the Libros site. Hermano Miguel Grosjean, S., Vences, M. & Dubois, A. 2004: Evolutionary signifi- Pe´rez (Museo del Colegio de la Salle, Teruel, Spain), Ms Sandra cance of oral morphology in the carnivorous tadpoles of tiger Chapman (Natural History Museum, London), Dr Eberhard frogs, genus Hoplobatrachus (Ranidae). Biological Journal of the Schindler (Forschungsinstitut Senckenberg, Frankfurt, Germany) Linnean Society 81, 171–181. and Dra BegonãSańchez (Museo Nacional de Ciencias Naturales, de las Heras, F.X.C., Anadoń, P. & Cabrera, L. 2003: Biomarker Madrid, Spain) provided access to specimens. The manuscript was record variations in lacustrine coals and oil shales: contribution improved greatly by the comments of Dr Mark Purnell and a sec- from Tertiary basins in NE Spain. In Valero Garce´s, B.L. (ed): ond, anonymous, reviewer. Funded by Enterprise Ireland Basic Limnogeology in Spain: A Tribute to Kerry Kelts, 187–228. Span- Research Grant SC ⁄ 2002 ⁄ 138 to PJO. ish Research Council (Consejo Superior de Investigaciones Cientı´ficas (CSIC), Madrid. Holman, J.A. 2003: Fossil Frogs and Toads of North America,246 pp. Indiana University Press, Bloomington. Hou, X.-G., Aldridge, R.J., Siveter, D.J., Siveter, D.J. & Feng, X.-H. References 2002: New evidence on the anatomy and phylogeny of the earli- Aldridge, R.J., Hou, X.-G., Siveter, D.J., Siveter, D.J. & Gabbott, est vertebrates. Proceedings of the Royal Society of London B 269, S.E. 2007: The systematics and phylogenetic relationships of 1865–1869. vetulicolians. Palaeontology 50, 131–168. de Jongh, H.J. 1968: Functional morphology of the jaw apparatus Alford, R.A. 1999: Ecology: resource use, competition, and preda- of larval and metamorphosing Rana temporaria. Journal of Zool- tion. In McDiarmid, R.W. & Altig, R. (eds): Tadpoles: The Biol- ogy 18,1–103. ogy of Anuran Larvae, 240–278. University of Chicago Press, Kawamata, S., Takaya, K. & Yoshida, T. 1987: Light- and electron Chicago. microscopic study of the endolymphatic sac of the tree frog, Altig, R. & Johnston, G.F. 1989: Guilds of anuran larvae: relation- Hyla arborea japonica. Cell and Tissue Research 249, 57–62. ships among developmental modes, morphologies, and habitats. Kemp, N.E. & Hoyt, J.A. 1969: Sequence of ossification in the skel- Herpetological Monographs 3, 81–109. eton of growing and metamorphosing tadpoles of Rana pipiens. Altig, R. & McDiarmid, R.W. 1999: Body plan: development and Journal of Morphology 129, 415–444. morphology. In McDiarmid, R.W. & Altig, R. (eds): Tadpoles: Keynes, R.J. & Stern, C.D. 1984: Segmentation in the vertebrate The Biology of Anuran Larvae,24–51.UniversityofChicago nervous system. Nature 310, 786–789. Press, Chicago. Larson, P.M. & Reilly, S.M. 2003: Functional morphology of feed- Anadoń, P., Rosell, L. & Talbot, M.R. 1992: Carbonate replacement ing and gill irrigation in the anuran tadpole: electromyography of lacustrine gypsum deposits in two Neogene continental and muscle function in larval Rana catesbeiana. Journal of Mor- basins. Sedimentary Geology 78, 201–216. phology 255, 202–214. Boyer, B.W. 1981: Tertiary lacustrine sediments from Sentinel Lipson, M.J. & Silbert, J. 1968: Glycosaminoglycans of adult frog Butte, North Dakota, and the sedimentary record of ectogenic back skin. Biochimica et Biophysica Acta 158, 344–350. meromixis. Journal of Sedimentary Petrology 51, 429–440. Maina, J.N. 1989: The morphology of the lung of the East African Briggs, D.E.G. & Kear, A.J. 1994: Decay of the lancelet Branchios- tree frog Chiromantis petersi with observations of the skin and toma lanceolatum (Cephalochordata): implications for the inte- buccal cavity as secondary gas exchange organs. A TEM and pretation of soft-tissue preservation in conodonts and other SEM study. Journal of Anatomy 165,29–43. primitive chordates. Lethaia 26, 275–287. Martill, D.M., Brito, P.M. & Washington-Evans, J. 2008: Mass Bruns, R.R. & Gross, J. 1970: Studies on the tadpole tail I. Structure mortality of fishes in the Santana Formation (Lower Cretaceous, and organization of the notochord and its covering layers in ?Albian) of northeast Brazil. Cretaceous Research 29, 649–658. Rana catesbiana. American Journal of Anatomy 128, 193–224. Maus, M. & Wuttke, M. 2002: Comparative anatomical and tapho- Buchheim, H.P. & Surdam, R.C. 1981: Palaeoenvironments and nomical examination of the larvae of Pelobates decheni Troschel fossil fishes of the Laney Member, Green River Formation, Wyo- 1861 and Eopelobates anthracinus Parker 1929 (Anura: Pelobati- ming. In Gray, J., Boucot, A.J. & Berry, W.B.N. (eds): Communi- dae) found at the Upper Oligocene sites at Enspel (Wester- ties of the Past, 415–452. Dowden, Hutchinson and Ross, wald ⁄ Germany) and Rott (Siebengebirge ⁄ Germany). Courier Stroudsburg. Forschungsinstitut Senckenberg 237, 129–138. Chen, J.-Y., Dzik, J., Edgecombe, G.D., Ramskold, L. & Zhou, G.Q. McDiarmid, R.W. & Altig, R. 1999: Research: materials and tech- 1995: A possible early Cambrian chordate. Nature 377, 720–722. niques. In McDiarmid, R.W. & Altig, R. (eds): Tadpoles: The Chen, J.-Y., Huang, D.-Y. & Li, C.-W. 1999: An early Cambrian Biology of Anuran Larvae, 7–23. University of Chicago Press, craniate-like chordate. Nature 402, 518–522. Chicago. Chipman, A.D. & Tchernov, E. 2002: Ancient ontogenies: larval McNamara, M., Orr, P.J., Kearns, S.L., Anadoń, P., Alcala´,L.& development of the Lower Cretaceous anuran Shomronella Penãlver-Molla´, E. 2006: High-fidelity organic preservation of jordanica (Amphibia: Pipoidea). Evolution and Development 3, bone marrow in c. 10 million year old amphibians. Geology 34, 86–95. 641–644. Conway Morris, S. 1998: The Crucible of Creation: the Burgess Shale McNamara, M., Orr, P.J., Kearns, S.L., Anadoń, P., Alcala´,L.& and the Rise of Animals, 276 pp. Oxford University Press, New Penãlver-Molla´, E. 2009: Soft tissue preservation in Miocene York. frogs from Libros (Spain): insights into the genesis of decay mic- Coulombe, P.A. & Omary, M.B. 2002: ‘Hard’ and ‘soft’ principles roenvironments. Palaios 24, 104–117. defining the structure, function and regulation of keratin inter- Nava´s, L. 1922a: Algunos fo´siles de Libros (Teruel). Boletıńdela mediate filaments. Current Opinions in Cell Biology 14, 110–122. Sociedad Ibe´rica de Ciencias Naturales 21,52–61. 306 McNamara et al. LETHAIA 43 (2010)

Nava´s, L. 1922b: Algunos fo´siles de Libros (Teruel). Adiciones y Shumway, W. 1940: Stages in the normal development of Rana correcciones. Boletıń de la Sociedad Ibe´rica de Ciencias Naturales pipiens. I. External form. Anatomical Record 78, 139–147. 21, 172–175. Sˇpinar, Z.V. 1972: Tertiary Frogs from Central Europe, 286 pp. Aca- Nevo, E. 1968: Pipid frogs from the Early Cretaceous of Israel and demia, Prague. pipid evolution. Bulletin of the Museum of Comparative Zoology Takahashi, K., Yamamoto, H., Yokote, Y. & Hattori, M. 2004: 136, 255–318. Thermal behaviour of fowl feather keratin. Bioscience Biotechnol- Olson, S.L. 1995: Thiornis sociata Nava´s, a nearly complete Mio- ogy Biochemistry 68, 1875–1881. cene Grebe (Aves: podicipedidae). Courier Forschungsinstitut Taylor, A.C. & Kollros, J.J. 1946: Stages in the normal development Senckenberg 181, 131–140. of Rana pipiens larvae. Anatomical Record 94,7–24. Ortı´, F., Rosell, L. & Anadoń, P. 2003: Deep to shallow lacustrine Toporski, J.K.W., Steele, A., Westall, F., Avci, R., Martill, D.M. & evaporites in the Libros gypsum (southern Teruel Basin, Mio- McKay, D.S. 2002: Morphologic and spectral investigation of cene, NE Spain): an occurrence of pelletal gypsum rhythmites. exceptionally well-preserved bacterial biofilms from the Oligo- Sedimentology 50, 361–386. cene Enspel formation, Germany. Geochimica et Cosmochimica Penãlver, E. 1996: Los yacimientos con insectos fo´siles de Aragoń Acta 66, 1773–1791. (Espanã). Boletıń de la Sociedad Entomolo´gica Aragonesa Paleo- Trewin, N.H. 1986: Palaeoecology and sedimentology of the Entomologıá16, 139–146. Achanarras fish bed of the Middle Old Red Sandstone, Scotland. Pilkington, J.B. & Simkiss, K. 1966: The mobilization of the cal- Transactions of the Royal Society of Edinburgh: Earth Sciences 77, cium carbonate deposits in the endolymphatic sacs of metamor- 21–46. phosing frogs. Journal of Experimental Biology 45, 329–341. Viertel, B. & Richter, S. 1999: Anatomy: viscera and endocrines. In Purnell, M.A. 2001: Feeding in extinct jawless heterostracan fishes McDiarmid, R.W. & Altig, R. (eds): Tadpoles: The Biology of and testing scenarios of early vertebrate evolution. Proceedings of Anuran Larvae, 92–148. University of Chicago Press, Chicago. the Royal Society of London B 269,83–88. Vinther, J., Briggs, D.E.G., Prum, R.O. & Saranathan, V. 2008: The Raff, E.C., Villinsk, J.T., Turner, F.R., Donoghue, P.C.J. & Raff, colour of fossil feathers. Biology Letters 4, 522–525. R.A. 2006: Experimental taphonomy shows the feasibility of fos- Wassersug, R.J. 1974: The evolution of anuran life cycles. Science sil embryos. Proceedings of the National Academy of Sciences USA 185, 377–378. 103, 5846–5851. Wassersug, R.J. 1980: Internal oral features of tadpoles from eight Rocˇek, Z. 2003: Larval development in Oligocene palaeobatrachid anuran families: functional, systematic, evolutionary and ecolog- frogs. Acta Palaeontologica Polonica 48, 595–607. ical considerations. Miscellaneous Publications of the Museum of Rocˇek, Z. & Van Dijk, E. 2006: Patterns of larval development in Natural History of the University of Kansas 68, 1–146. Mesozoic pipid frogs. Acta Palaeontologica Polonica 51, 111–126. Wassersug, R.J. & Wake, D.B. 1995: Fossil tadpoles from the Mio- Rosenberg, M. & Warburg, M.R. 1978: Changes in the structure of cene of Turkey. Alytes 12, 145–157. ventral epidermis of Rana ridibunda during metamorphosis. Cell Whiteside, B. 1922: The development of the saccus endolymphati- and Tissue Research 195, 111–122. cus in Rana temporaria L. American Journal of Anatomy 30, Sheil, C.A. 1999: Osteology and skeletal development of Pyxicepha- 231–266. lus adspersus (Anura: Ranidae: Raninae). Journal of Morphology Wuttke, M. 1983a: ‘Weichteil-Erhaltung’ durch lithifizierte Mikro- 240,49–75. organismen bei mittel-eoza¨nen Vetebraten aus den O¨ lschiefern Shu, D.G., Luo, H.L., Conway Morris, S., Zhang, X.L., Hu, S.X., der ‘Grube Messel’ bei Darmstadt. Senckenbergiana lethaea 64, Chen, L., Han, J., Zhu, M., Li, Y. & Chen, L.Z. 1999: Lower 509–527. Cambrian vertebrates from south China. Nature 402, 42–46. Wuttke, M. 1983b: Aktuopalaöntologische studien u¨ber den Zerfall Shu, D.G., Luo, H.L., Conway Morris, S., Zhang, X.L., Hu, S.X., von Wirbeltieren. Teil 1: Anura. Senckenbergiana lethaea 64, Chen, L., Han, J., Zhu, M., Li, Y. & Chen, L.Z. 2003: A new 529–560. species of yunnanozoan with implications for deuterostome Young, C.C. 1936: A Miocene fossil frog from Shantung. Bulletin of evolution. Science 299, 1380–1384. the Geological Society of China 15, 189–197.