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

MARIA E. MCNAMARA, PATRICK J. ORR, STUART L. KEARNS, LUIS ALCALA, PERE ANADON AND

ENRIQUE PENALVER-MOLLA

McNamara, M.E., Orr, P.J., Kearns, S.L., Alcala, L., Anadon, P. & Penalver-Molla, E.2010: Exceptionally preserved tadpoles from the Miocene of Libros, Spain: ecomorpho-logical 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 abundantfrog tadpoles (Rana pueyoi) preserved in finely laminated lacustrine mudstones. The tad-poles exhibit a depressed body, short tail, low tail fins, dorso-laterally directed eyes andjaw sheaths; these features identify the Libros tadpoles as members of the benthic lenticecomorphological guild. This, the first ecomorphological reconstruction of a fossil tad-pole, supports phylogenetic evidence that this ecology is a conserved ranid feature. Thesoft-tissue features of the Libros tadpoles are characterized by several modes of preserva-tion. The space occupied previously by the brain is defined by calcium carbonate, thenerve cord is defined by calcium phosphate, and jaw sheaths and bone marrow are pre-served as organic remains. Gut contents (and coprolites adjacent to specimens) compriseingested fine-grained sedimentary detritus and epiphyton. The body outline and the eye-spots, nares, abdominal cavity, notochord, caudal myotomes and fins are defined by acarbonaceous bacterial biofilm. A similar biofilm in adult specimens of R. pueyoi fromLibros defines only the body outline, not any internal anatomical features. In the adultfrogs, but not in the tadpoles, calcium phosphate and calcium sulphate precipitated inassociation with integumentary tissues. These differences in the mode of preservationbetween 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 ofBristol, Wills Memorial Building, Queen’s Rd, Bristol BS8 1RJ, UK; Luis Alcala [[email protected]], Fundacion Conjunto Paleontologico de Teruel-Dinopolis, Avda. Saguntos ⁄ n, 44002 Teruel, Aragon, Spain; Pere Anadon [[email protected]], Consejo Superior deInvestigaciones Cientıficas, Institut de Ciencies de la Terra ‘Jaume Almera’, Lluıs Sole i Sab-arıs s ⁄ n 08028, Barcelona, Spain; Enrique Penalver-Molla [[email protected]], MuseoGeominero, Instituto Geologico y Minero de Espana, C ⁄ Rıos Rosas 23, E-28003 Madrid,Spain; manuscript received on 30 ⁄ 12 ⁄ 2008; manuscript accepted on 24 ⁄ 06 ⁄ 2009.

Exceptionally preserved anurans (Rana pueyoi) fromthe lacustrine-hosted Miocene Libros biota (Spain)comprise both larvae (representing a range of develop-mental stages) (n = 72) (Figs 1, 2) and adults(n = 73). The taphonomy of the non-biomineralized(soft) tissues of the adult frogs has been studied com-prehensively (McNamara et al. 2006, 2009). Previousobservations of the larval frogs comprise only briefreferences to a brown carbonaceous bacterial biofilmthat defines the general body outline and, in a singlespecimen, the presence of organically preserved bonemarrow (McNamara et al. 2006). This paper thereforeconsiders in detail the taphonomy of the larval frogsfrom Libros.

Notably, the soft-tissue morphology of modernanuran larvae varies strongly with ecology and habi-tat (Table 1). Reconstruction of the soft-tissue

morphology of the Libros tadpoles could thereforeconstrain hypotheses of their palaeoecology. Theseresults would have significant wider implications.Soft tissues are preserved in larval anurans fromnumerous Late Cretaceous and Cenozoic localities(Young 1936; Nevo 1968; Spinar 1972; Estes et al.1978; Wassersug & Wake 1995; Maus & Wuttke2002; Toporski et al. 2002; Rocek & Van Dijk 2006).Collectively, this material has improved our under-standing of anuran phylogeny, most importantly viathe construction of ontogenetic series (e.g. Maus &Wuttke 2002; Rocek & Van Dijk 2006). Complemen-tary studies of larval anuran taphonomy and ecology,however, are rare (but see Maus & Wuttke 2002;Toporski et al. 2002).

This paper also considers how biological factorscontrol exceptional fossil preservation. Exceptional

DOI 10.1111/j.1502-3931.2009.00192.x � 2009 The Authors, Journal compilation � 2009 The Lethaia Foundation

faunas often include various developmental stages ofthe same taxon: the preservation potential and themode of preservation of each should not be assumedto be constant. Different developmental stages canvary in their ecology, and thus different biostratinom-ic processes may affect carcasses. Factors that influencedecay and mineralization, such as physiology and tis-sue chemistry, can also vary during ontogeny. Theoccurrence of both adult and larval stages of R. pueyoiin the Libros biota is therefore an opportunity to elu-cidate the extent to which differences in the mode,and fidelity, of soft-tissue preservation of the sametaxon have been influenced by factors relating to theirontogeny.

Geological background

The Libros lacustrine system developed within theEarly Miocene–Late Pliocene Teruel Basin in NESpain (Ortı et al. 2003). The Teruel basin-fill in theLibros region comprises up to 500 m of alluvialterrigenous facies, lacustrine carbonates andevaporites. The deepest water facies of the Librossequence is the 150-m-thick late Miocene (Vallesian)Libros Gypsum Unit. The lowest part of this unit,the bituminous–calcareous subunit, comprises inter-calated charophytic limestones and laminatedmudstones (including oil shales) with deposits ofnative sulphur and rare primary gypsum (Ortı et al.2003). The exceptional biota comprises salamanders,frogs, birds, snakes, insects, arachnids and leaves(Navas 1922a,b; Olson 1995; Penalver 1996) and ishosted within the laminated mudstones. This faciesrepresents deposition within the profundal regionsof a permanently stratified bench-type lake, in whichlaterally extensive shallow-water zones (<10 mdepth) were separated from deeper waters by steepslopes. The monimolimnion was anoxic and sulphi-dic, and there was intense bacterial sulphate reduc-tion in the uppermost sediment column (Anadonet al. 1992; de las Heras et al. 2003; Ortı et al.2003).

Material and methods

A total of 72 specimens were examined from thecollections of the following institutions: Forschungs-institut und Naturmuseum Senckenberg, Frankfurt,Germany (FNS), Museu de Geologia de Barcelona,Barcelona (MGB), Museo Nacional de CienciasNaturales, Madrid (MNCN) and the Natural HistoryMuseum, London (NHM). Specimens occur mainlyas individuals, on slabs with trimmed edges. All

specimens were recovered during commercial exploi-tation of the sulphur and oil shales of the LibrosGypsum Unit in the early 20th century; the originalfield context and way up of specimens is unknown.The precise fossiliferous horizons are unknown andit is therefore impossible to determine to whatextent the curated specimens are an unbiased sampleof the fossil assemblage. All developmental stagesthat were preserved may not be represented. Intui-tively, smaller specimens, broadly synonymous withearlier developmental stages, are more likely to beabsent. Similarly, all states of preservation may notbe represented; disarticulated specimens and thoselacking obvious soft-tissue outlines are more likelyto be under-represented.

Not all of the soft-tissue features discussed belowcould be identified in each specimen, e.g. if a speci-men was incomplete, truncated by the edge of theslab, or if soft tissues were obscured in part by sedi-ment. The number of specimens in which a particularfeature is present is therefore indicated in the text inparentheses.

Ontogeny

The general categories of larval anuran developmentsensu Gosner (1960) are: (1) embryo (stages 1–20); (2)hatchling (stages 21–25); (3) tadpole (stages 26–41)and (4) metamorph (stages 42–46). Twelve incom-plete R. pueyoi larvae could not be staged. The remain-ing 60 specimens fall within a narrow range ofdevelopmental stages (Gosner stages 30–41) andrepresent fossil tadpoles.

More detailed resolution of the data is difficult. TheGosner (1960) staging system (and also the Shumway1940; Taylor & Kollros 1946 systems) for modernranid larvae is based primarily upon the presence ofspecific soft-tissue morphological features and cannotbe applied easily to fossil anuran larvae. The skeletalossification sequence has been described for Ranapipiens (Kemp & Hoyt 1969); however, the relativetiming of ossification of skeletal elements, and theorder in which they ossify, varies considerably amongranids (Sheil 1999), making extrapolation to othertaxa difficult. For example, certain cranial elementsbegin to ossify 11 stages earlier in R. temporaria (deJongh 1968) than in R. pipiens (Kemp & Hoyt 1969).The Libros specimens present additional difficulties.The skeleton is often obscured in part by sediment,gut contents or diagenetic minerals; the ossified partsof elements diagnostic of certain stages (e.g. the max-illa, femur, humerus and ischium) are less than 1 mmlong initially and therefore difficult to identify.

It is not possible to assign any tadpole of R. pueyoito a single developmental stage. Each specimen can,

LETHAIA 43 (2010) Miocene fossil tadpoles 291

A F

G

H

B

C

D

E

292 McNamara et al. LETHAIA 43 (2010)

however, be assigned to one of three groups of consec-utive developmental stages, described below. These aredefined by the skeletal elements present and theirdegree of ossification.

1 Gosner stages 30–35 (six specimens). The parasphe-noid, exoccipitals and neural arches of the verte-brae are the only skeletal elements visible; each ispoorly ossified; total length (as defined by soft tis-sues) 35–70 mm (Fig. 1H).

2 Gosner stages 36–37 (ten specimens). Ossificationof the fronto-parietals has begun; total length60–100 mm (Fig. 1F).

3 Gosner stages 38–41. Ossification of the prooticshas initiated; ossification of the parasphenoid,fronto-parietals, exoccipitals and vertebrae isadvanced. The femur, tibiofibulare, humerus, pre-maxilla, maxilla and angular may be visible; totallength 110–145 mm (Fig. 1A–E, G).

A

B

Fig. 2. Detail of MNCN 63786 A, showing soft tissue features preserved and B, interpretative drawing.

Fig. 1. Photographs of exceptionally preserved tadpoles of Rana pueyoi from Libros. A, Gosner stages 38–41; dorso-ventral. Small arrow indi-cates coprolite (R4999). B, Gosner stages 38–41; lateral. Small arrow indicates coprolite, large arrow indicates the point of origin of the tailfin, white arrowhead indicates the margin of a hind limb, and black arrowhead indicates the ventral margin of the notochord (MNCN63786). C, Gosner stages 38–41; oblique. Arrowhead indicates the margin of a hind limb. Note definition of the lungs as a speckled texture inthe abdomen (MNCN 63799). D, Gosner stages 38–41; dorso-ventral. Small arrow indicates the gut contents (27147, MGB). E, Gosner stages38–41; 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 thecranium (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.


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Sedimentary context

In vertical section, each fossil-bearing slab exhibits adistinctive, ‘bar code’-like striped pattern, generatedby alternations of laminae of different composition,colour and thickness. To determine whether two ormore fossil-bearing slabs include the same horizon,digital images of the vertical edge of each slab wereprinted to the same scale and the position of the fos-sil-bearing lamina marked. The lamina succession inone image was compared with that in all others (simi-lar to the technique used by Trewin 1986). The imagewas then rotated by 180� and the process repeated (asthe way-up of each slab is unknown). The most parsi-monious interpretation is that two or more slabs that

exhibit an identical succession of laminae representthe same stratigraphic interval.

Analytical methods

The methods used to analyse the soft tissues have beendescribed previously (McNamara et al. 2006). In sum-mary, SEM analyses of carbon- or gold-coated sam-ples were performed with a Hitachi S-3500Nmicroscope at an accelerating voltage of 15 kV andacquisition times of 60 s for EDS spectra of carbon-coated samples. Unstained resin-embedded sampleswere sectioned with a diamond knife, and TEM analy-ses performed using a JEOL 2000TEMSCAN at 80 kVwith an objective aperture of 10 lm diameter.

Terminology

The terminology used to describe specimens followsthat of Altig & McDiarmid (1999). The body is theregion between the anterior tip of the head (the snout)and the junction between the posterior body wall andthe axis of the tail myotomes (the body terminus); thetail is posterior to the body terminus. The transverselyoriented buccopharyngeal wall in the plane of the firstvertebra divides the body into two sections: the cra-nium and buccopharynx are anterior, and the abdomenposterior, of the wall.

The following variables were measured (Fig. 3A, B):body length (the horizontal distance from the snout tothe body terminus), body height (the maximum heightof the body in lateral aspect), body width (the maxi-mum width of the body in dorso-ventral aspect) andtail length (the distance from the body terminus to thetip of the tail). The positions of the maximum heightand width of the body were recorded. As growth dur-ing tadpole stages is typically isometric (McDiarmid &Altig 1999), the ratio of any two components is con-sistent between developmental stages. The complete-ness and orientation of individual specimenscontrolled which of these variables could be recorded.

Body and tail morphology are defined sensuMcDiarmid & Altig (1999). The body is compressed ifheight exceeds length and depressed if the reverse. Anoval body is the widest near the centre of the abdomenand tapers anterior and posterior of this; spherical andglobular denote spherical and more irregular bodyshapes respectively. Tail fins are low if the periphery ofeach fin is close to the periphery of the myotomes andhigh if not. No standard definition exists for taillength; examination of published literature indicatesthat tails more than 1.5 times body length are longand short if less. Variables measured for the body andtail of the 16 most complete Libros tadpoles are sum-marized in Table 2.

A

B

C

D

Fig. 3. Tadpole body plan (A, B) and anatomy (C). A, B, sche-matic illustrations of the body plan of a tadpole in lateral (A) anddorsal (B) views. BH, body height; BL, body length; BW, bodywidth; TAL, tail length; TL, total length. C, simplified tadpole anat-omy. Left-hand diagram shows a tadpole in lateral view; for clarity,the notochord is shaded in grey and the caudal myotomes are notshown in the anterior part of the tail. Right-hand diagram is aschematic vertical section through the tadpole tail along line X–Yin the left-hand diagram. a, abdomen; br, brain; bu, buccopharynx;e, eye; f, fin; i, intestine; j, jaw sheath; l, lung; m, caudal myotomes,ma, manicotto; ms, myotome sheath; na, external nares; nc, nervecord; no, notochord; ns, notochord sheath.


The orientation of each specimen is described asfollows: dorso-ventral if the sagittal plane is perpendic-ular to bedding, oblique if the sagittal plane is inclinedto bedding and lateral if the sagittal plane is parallel tobedding.

Cause of death

In addition to predation, mechanisms that result inthe death of anuran larvae include asphyxiation dueto increases in epilimnetic temperature (Elder &Smith 1988), and the release of toxic metabolites intothe epilimnion during algal blooms (Boyer 1981;Buchheim & Surdam 1981). Such environmentalstresses often generate mass mortalities. In the fossilrecord, mass mortality events are most apparentwhere high numbers of individuals co-occur in eitherthe same bed or, in laminated sequences, on the samehorizon (see, e.g. Martill et al. 2008; Fig. 4). The phe-nomenon is difficult to identify in cases, such asherein, where the data set comprises individual speci-mens. Comparison of the lamina succession of eachfossil tadpole-bearing slab from Libros, however, dem-onstrates that most specimens (52; n = 70) are fromdifferent horizons; the remaining 18 are from sevenhorizons. This distribution implies that the supply ofspecimens to the site of deposition was on an on-going basis over an extended time interval. This isinconsistent with, but, on its own, does not eliminatecompletely, the death of the tadpoles having occurredduring mass mortality events. The cause of the tad-poles’ death therefore remains unknown.

Physical taphonomy

All specimens are highly articulated. This, combinedwith the preservation of soft tissues (see below), indi-cates that specimens were deposited in profundallake zones shortly after death. The absence of fish inthe lake would have eliminated scavenging of

Table 2. Various ratios calculated from measurements taken fromfossil specimens in which the body margin is defined by softtissues.

SpecimenBody length:tail length

Body length:body height

Body length:body width

1 1.1 2 –2 0.89 1.83 –3 1.22 – 1.584 – – 2.035 0.79 – 1.476 – – 1.957 1.08 – 1.978 0.74 – 2.149 0.85 – 210 – 1.65 –11 – 1.92 –12 – – 1.4113 – 2.04 –14 – – 1.9515 – – 1.7216 0.93 – –Mean 0.95 1.89 1.82SD 0.17 0.16 0.26

A

B

C

Fig. 4. Structure of the bacterial biofilm. A, light micrograph ofsurface of biofilm showing polygonal fracturing and, inset, frac-tured vertical section through biofilm (MNCN 63796). B, scanningelectron micrograph of fossilized bacteria (27143, MGB). C, trans-mission electron micrograph of bacteria showing homogenous,extremely low electron lucency (MNCN 63821). r, resin.


carcasses in the period between death and deposi-tion; after deposition, bottom water conditions pre-cluded disturbance of carcasses by bioturbators orscavengers. All specimens are orientated with theanterior–posterior axis of the body and tail parallelto bedding; this suggests the lake floor was firm andcohesive at the time of deposition. Specimens occurin dorso-ventral (19 specimens; n = 43), lateral (16specimens) or oblique (seven specimens) aspect. Dif-ferences in the number of specimens in each aspectare not statistically significant (5.57; d = 2;v2

2 = 13.82; P < 0.001), i.e. the larvae do not occurin any preferred orientation. This reflects the near-circular geometry of cross-sections of the tadpolebody in vivo.

Soft-tissue preservation

Bacterial biofilm

General features. – The general outline of the soft tis-sues is defined by a brown layer (Fig. 1) that envelopsthe bones and gut contents; where thick, it is fracturedinto a series of polygons (Fig. 4A). The layer is thethickest and most extensive in specimens at moreadvanced developmental stages. Its thickness also var-ies within an individual specimen: it is thick (up to1.3 mm; average �300 lm), and continuous, in theabdomen, thin (5–10 lm) and discontinuous in thetail and buccopharynx, and of intermediate thickness(80 lm) in the eyespots. The layer comprises denselypacked, predominantly ovoid, micro-structures, eachca. 1 lm long (Fig. 4B). The possibility that thesestructures represent fossilized melanosomes (see Vin-ther et al. 2008) is yet to be considered in detail.Herein, we follow previous authors who have studiedsimilarly preserved faunas (e.g. Wuttke 1983a; Topor-ski et al. 2002) and consider this layer to represent abiofilm of fossilized bacteria (a Hautschatten (skinshadow) sensu Wuttke 1983a). The structureless mate-rial associated with the bacteria is interpreted as extra-cellular polymeric substance (EPS), rather than thedegraded remains of the original tissues.

In TEM images the bacteria are uniformly electronlucent (Fig. 4C). There is no evidence for authigenicminerals in association with either the bacteria orEPS; both comprise primarily C and S, which is attrib-uted to a high abundance of organosulphur com-pounds (McNamara et al. 2006). Experimentaldegradation of anurans has demonstrated that micro-bial consumption of soft tissues initiates in the gutand mouth (Wuttke 1983b); the fossil bacteria aretherefore probably anaerobic heterotrophs derivedfrom the indigenous intestinal community.

In many specimens, the biofilm pseudomorphs thegeneral morphology of the body and tail (Figs 1A–E,H, 2A). The biofilm can also define the following ana-tomical features.

Abdominal cavity. – In 42 specimens (n = 53) (allGosner stages 36–37 or 38–41) the biofilm is signifi-cantly thicker in the posterior of the body than else-where (Figs 1A–E, G, 2A). This more likely defines theovoid abdominal cavity than the yolk sac: in modernranid larvae the latter is resorbed fully by Gosner stage24. In vivo, the abdominal cavity houses a tightlycoiled intestine, the liver, pancreas and lungs (Viertel& Richter 1999). The preferential development of athick biofilm in this region, but not in the anteriorpart of the body, reflects the greater volume of soft tis-sues available for bacterial consumption within theabdomen; in particular, the digestive tract comprises�50% of the biomass of a tadpole (Wassersug 1974).The anterior of the body, however, includes extensivevoid space (the buccopharangeal cavity).

Lungs. – In six specimens (n = 53), one, or a pair of,elongate structures occurs in the abdominal cavity. Indorso-ventral orientation, each originates either sideof, and extends posteriorly at an acute angle to, thevertebral column (at VI–VII) (small arrows in Fig. 1F,H). The position, size and geometry of these struc-tures match those of the primordial lungs in tadpolesof modern Rana, and resemble those of lungs identi-fied in other exceptionally preserved anuran tadpoles(see Spinar 1972). The structures are evident in speci-mens from Libros only at Gosner stages 30–35. This isattributed to variation in biofilm thickness, not differ-ences in the preservation potential of the lungs duringontogeny. The biofilm in the abdomen is notably thinin specimens at Gosner stages 30–35 (Fig. 1F, H),enhancing the likelihood of identifying anatomicalfeatures that are defined subtly. In larger specimens,the abdominal cavity can exhibit a distinctively speck-led texture if the biofilm is thin (Fig. 1C). Lungs inmodern anurans have a honeycomb-like lattice struc-ture (Maina 1989), degradation of which could poten-tially generate the speckled texture evident in thefossils.

Eyespots. – One or both eyespots occur(s) in 30 spec-imens (n = 49) (Figs 1A, B, D, F–H, 3A). Their out-line is circular in specimens in lateral aspect (Fig. 1B).In dorso-ventrally orientated specimens the eyespotsare elliptical (with the long axis of the ellipse parallelto the body axis (Fig. 1F)), and contained inside thebody margin (Fig. 1A). The eyes of the tadpoles there-fore faced laterally and were on the dorsal body sur-face (Altig & McDiarmid 1999). Preservation of the


eyespots reflects the high recalcitrance of the sclera rel-ative to the surrounding soft tissues. The sclera com-prises layers of collagen, a decay-resistant tissue(Briggs & Kear 1994) that would have retarded micro-bial infiltration of the eye cavity in the initial stages ofdecay; the surrounding tissues would have degradedmore rapidly, leaving the eyespots as isolated outliersof biofilm.

Nares. – The nares (the external extensions of thenostrils), approximately midway between the snoutand the parasphenoid, are defined in nine specimens(n = 49) (arrowheads in Fig. 1G). In vivo the marginsof the nares have distinct thick, fleshy protuberances;definition of the nares may therefore have beenenhanced by the greater thickness of soft tissues rela-tive to surrounding areas of the buccopharynx.

Notochord. – The former position of the notochordis defined in 14 specimens in dorso-ventral aspectand, more tentatively, in three in lateral aspect(n = 36). In dorso-ventral aspect, the notochord isdefined as a pair of thin, closely spaced (average 1 and1.5 mm apart in specimens at Gosner stages 30–35and 38–41 respectively), parallel, brown lines that runalong the tail axis (Fig. 5A, B). This spacing is similarto the breadth of the notochord in the extant R. cates-beiana at Gosner stage 30 (Bruns & Gross 1970); itsheight is greater as the notochord is highly com-pressed laterally (Wassersug 1980). These lines canextend from the posterior terminus of the vertebralcolumn along the visible length of the tail. Theyresemble structures noted in the tails of primitivechordate fossils, and those generated during experi-mental decay of the lancelet Branchiostoma (by col-lapse of the notochord sheath; Briggs & Kear 1994,

and references therein). The apparent absence of thenotochord inside the body of the fossil specimenscould result from its being obscured by the vertebraeand ⁄ or thick biofilm in the abdomen.

In laterally orientated specimens, the biofilmdefines a single brown line parallel to, and 2–4 mmventral of, the tail axis (black arrowhead, Figs 1B, 2A).This is presumably the position of the ventral marginof the notochord in vivo. The dorsal margin is difficultto identify but could be obscured by the dorsal nervecord that runs along the tail axis (see below) (Figs 1B,2A).

In vivo the notochord comprises a core of vacuo-lated cells and a tough collagenous outer sheath(Wassersug 1980). It is a recalcitrant structure: inBranchiostoma decayed under anoxic conditions thenotochord persisted in recognizable form for up to124 days (Briggs & Kear 1994). Preservation of thenotochord in two dimensions as a pair of parallellines, not a solid band, is notable. After preferentialdecay of the core of vacuolated cells, the notochordsheath would have been hollow and elliptical in cross-section. Its subsequent, decay-induced, collapse wouldproduce a structure in which the thickness of tissuewould have been greater at the two edges than medi-ally. Thus, the margins have greater preservationpotential simply as a function of their being thicker.This applies to specimens preserved in both lateraland, especially, dorso-ventral aspect (Fig. 6).

Caudal musculature. – In 14 specimens (n = 35) themargin of the caudal myotomes is defined by a thinbrown line (Figs 1A–C, 2A). This occurs irrespectiveof specimen orientation and thus defines the left andright sides of the myotomes (in specimens in dorso-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-ventralaspect, showing preservation of margins of notochord sheath as a pair of thin parallel brown lines (arrows) along the tail axis. Near-linearwhite feature in A is the nerve cord, defined by calcium phosphate. Scale: 5 mm.


(in those in lateral aspect). It was therefore continu-ous, surrounding the myotomes, and probably corre-sponds to the collagenous sheath that enclosed themyotomes in vivo.

Fins. – The outlines of the tail fins are preserved inonly four specimens (n = 35), primarily as a thinbrown line that marks their margin; their surfaces areindistinct. The latter reflects the limited volume of softtissue in the fins (epidermis enclosing a thin layer ofloose connective tissue; McDiarmid & Altig 1999) andtheir having degraded relatively rapidly. The dorsal finoriginates close to the junction between the body mar-gin and caudal myotomes (large arrow in Figs 1B, 2).The margin of each fin lies 5 mm outside, and runsapproximately parallel to, the margin of the caudalmyotomes.

Other caudal features defined by biofilm. – Althoughextant ranids vary in both the timing and pattern ofskeletal ossification, hind limb buds begin to developby, at the latest, Gosner stage 30; hind limbs are typi-cally large and distinct by Gosner stage 41. Assumingthe same applied to the Libros tadpoles, at least themajority specimens should exhibit hind limbs. Devel-oping hind limbs are, however, present in only twospecimens from Libros (both at Gosner stages 38–41):in each, the biofilm defines two or more parallel linesin the abdomen that are ventral of, and at an angle to,the vertebral column (white arrowheads in Figs 1B, C,2A). The absence of hind limbs in fossil specimensfrom earlier Gosner stages could reflect the small sizeof both the buds and the specimens. Their absence in

all but two specimens from later stages is more prob-lematic. In laterally orientated specimens, other softtissues might obscure the outline of the developinglimbs. In many fossil specimens at advanced develop-mental stages the relevant parts of the body are eitherconcealed by sediment or the specimen is truncatedanterior to the tail ⁄ body junction.

Other soft-tissue features

White masses in cranium. – In 64 specimens (n = 69)friable white material occurs in the cranium betweenthe parasphenoid and fronto-parietals, and within theprootics (Figs 1A–H, 2A). The material compriseseuhedral, 5- to 15-lm-long crystals of calcium car-bonate, probably aragonite (a strong strontium peakis present in EDS) (Fig. 7A). In modern anurans,deposits of calcium carbonate occur within theendolymphatic sac that is associated with the nervoustissues (Kawamata et al. 1987), i.e. the brain (locatedclose to the fronto-parietals and parasphenoidin vivo), nervous ganglia of the prootics and nervecord. In ranid tadpoles, the endolymphatic sac extendsalong the entire length of the nerve cord (Kawamataet al. 1987) and, in the cranium, diverges into twoirregularly shaped diverticula; one marginal to andeither side of the brain (Whiteside 1922; see plate 1in Pilkington & Simkiss 1966). This distribution,however, does not correspond to that of the calciumcarbonate in the Libros tadpoles. The latter exhibit noevidence of calcium carbonate in the postcranial bodyand tail; in the cranium, the calcium carbonate occursas a single, unpaired, white mass that is continuous

A B

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 itsmargins. The thickness of the notochord sheath (which is exaggerated for clarity) along each of sections A–B and C–D is greater than thecumulative thickness of the sheath along sections E–F and G–H.


across the sagittal axis. It is possible that some of thecalcium carbonate in the tadpoles could represent ori-ginal endolymphatic deposits. The distribution and

extent of the calcium carbonate, however, suggest thatit is better interpreted as a very early diagenetic precip-itate that defines the former position of the brain andminor nervous ganglia. The calcium carbonate doesnot replicate any tissue per se, but infills the space cor-responding to the latter. The process therefore reflectsthe generation of a micro-environment rich in calciumand bicarbonate ions, the latter sourced via rapid liq-uefaction of nervous tissue (McNamara et al. 2009).The deposits of the endolymphatic sac would haveconstituted a particularly rich source of such ions.

Dorsal nerve cord. – In 24 specimens (n = 67) whitematerial defines a continuous or fragmented, 0.5-mm-wide structure that runs along the axis of the bodyand ⁄ or tail (Figs 1A–E, 2A). This white material is fria-ble and comprises sub- to anhedral, 0.8- to 3-lm-longcrystals of calcium phosphate (Fig. 7B). The linear nat-ure of the structure is most obvious in the tail, whereit runs parallel to, and dorsal of, the notochord(Fig. 1B); in the body, the structure is often obscuredin part by the vertebral column. The geometry andposition of the white material correspond to that ofthe nerve cord; this is situated within a recess in thedorsal margin of the notochord in vivo (Wassersug1980). In one specimen from Libros, the linear struc-ture is cohesive and exhibits a fibrous texture(Fig. 7C), possibly representing mineralized bundlesof nerve fibres. In the remainder, the material definingthe nerve cord lacks structure. In these cases the cal-cium phosphate probably precipitated on the templateprovided by the decaying tissues of the nerve cord.

The nerve cord occurs as: (1) a near-continuousstraight or undulating line (Fig. 1A, B); (2) a series ofaligned short fragments of varying length, some ofwhich are displaced from life position but rarely occuroutside the body margin (Fig. 1C); (3) many short,variably orientated fragments, which are often reflexedand ⁄ or occur outside the body margin (Fig. 1D, E);and (4) short, isolated, highly dispersed fragments.This sequence represents progressive stages in the deg-radation of the nerve cord. Fragments of nerve cordare not contiguous even in decay stages 1 and 2, andalways have rounded termini; this suggests that con-traction and at least the initial separation of the cordoccurred prior to mineralization. Fragments in somespecimens are approximately equal in length (Fig. 1C,D). The pattern of fragmentation may have been con-trolled by the nerve cord being segmented along itslength; this feature is widespread among most verte-brate classes (at least during their embryonic and ⁄ orlarval stages) (Keynes & Stern 1984).

Gut contents. – These occur in the abdomen of 15specimens (n = 54), mainly as light brown-coloured

A

B

C

Fig. 7. Anatomical features defined by white material. A, scanningelectron micrograph of white material in the cranium (27143,MGB) showing euhedral crystals of calcium carbonate. B, scanningelectron micrograph of white material in the nerve cord (MNCN63786) showing sub- to anhedral crystals of calcium phosphate. C,light micrograph of part of part of the nerve cord (27146, MGB)that exhibits a fibrous texture.


curvilinear features 0.3–0.5 mm wide and up to80 mm long (arrow, Figs 1D, 8A); they can defineaccurately the geometry of part of the coiled intestine(Fig. 8A). The contents have a granular texture(Fig. 8B) and usually comprise silt-sized grains of cal-cium carbonate and fragmented diatom frustules(Fig. 8C); siliceous sponge spicules also occur.

Granular gut contents in other fossil tadpoles havebeen interpreted as ingested shallow-water, fine-grained sediment (Spinar 1972; Maus & Wuttke2002). The high abundance of diatoms in the gut con-tents of the Libros tadpoles suggests that rasping ofalgal communities associated with submerged vegeta-tion (i.e. epiphyton) was an important feeding strat-egy. Gut clearance in modern anuran larvae typicallyoccurs within 4–6 h of ingestion (Alford 1999). Thepresence of gut contents in the fossils therefore indi-cates that death occurred shortly after ingestion of ameal.

Coprolites. – These occur within 5–8 mm of thebody margin in six specimens (n = 53) (smallarrows in Fig. 1A, B, G) and are similar in composi-tion to the gut contents. In specimens in lateralaspect, coprolites consistently occur close to the ven-tral margin of the body. This association indicatesthat the coprolites represent evacuation of the bow-els during the very early stages of decay due topost-mortem relaxation of the bowel muscles, inparticular the sphincters. Given that gut clearance islikely to have been rapid in vivo (see above), thesetadpoles were deposited on the lake floor shortlyafter their death. Notably, these larvae are not char-acterized by an appreciably higher fidelity of preser-vation (i.e. higher articulation, more extensivebiofilm, greater definition of anatomical features)than the remainder. This suggests that the latterwere also deposited shortly after death and that, col-lectively, the specimens did not experience pro-nounced differences in either the extent or durationof transport in the interval between death and theirdeposition.

Jaw sheath. – This is preserved in ten specimens(n = 44), all at Gosner stages 38–41. It comprises a3-mm-wide, �300-lm-thick, black-coloured, arcuatestructure (arrow in Figs 1F, 9A). The sheath is usuallyanterior of the cranial bones but slightly posterior ofthe 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 moderntaxa the jaw sheath does not articulate with the skele-ton and separates from the carcass within a few daysof death (Altig & McDiarmid 1999). As in moderntadpoles (Altig & McDiarmid 1999, p. 39), the fossilsheaths have a fissile ultrastructure. Each is carbona-ceous with distinctive micro-serrations on its oral sur-face (Fig. 9B). Each micro-serration is ca. 32 lm long,25 lm wide and 4 lm thick, has an irregular oralmargin and is parallel-sided and concavo-convextransversely (Fig. 9C). There are ca. 30 micro-serra-tions per mm in the fossils; this is at the lower end of

A

B

C

Fig. 8. Gut contents and coprolites. A, detail of A322 (FNS) show-ing definition of part of the coiled intestine as a series of overlap-ping, light-coloured curved features (small arrow) in the abdomen.Gosner stages 38–41; lateral. B, light micrograph of gut contentsshowing granular texture (2712, MGB). b, biofilm. C, scanningelectron micrograph of gut contents showing highly fragmenteddiatom frustules and silt-sized grains of calcium carbonate (27148,MGB).


the range observed in modern anuran larvae (30–80micro-serrations per mm) (Altig & McDiarmid 1999).The preservation of the jaw sheaths, but not othercomponents of the complex oral apparatus, is attrib-uted to their being keratinous. The keratin molecule ischaracterized by a scaffold of inter- and intramolecu-lar hydrogen- and disulphide bridges (Takahashi et al.2004): these render keratin insoluble and resistant toproteolytic attack (Coulombe & Omary 2002).

Bone marrow. – Very small (10–50 lm long) frag-ments of bone marrow occur in the cranium andvertebrae of one specimen. The marrow is preservedas a sulphur-rich organic residue that retains the origi-nal red colour of haematopoietic bone marrow. Itspreservation reflects the protective nature of the ostealmicro-environment (which retarded microbial degra-dation of the marrow); sulphurization of organic mol-ecules within the tissue during early diagenesisenhanced its preservation potential (McNamara et al.2006).

Ecology

Ecomorphological reconstruction

The body of the R. pueyoi tadpole is the tallest andbroadest in the plane of the sixth vertebra (Fig. 1A–D)and is oval in lateral aspect. The ratios of body lengthto body height and body length to body width arenearly identical, and the body is longer than it is highor wide (Table 2); i.e. the body is depressed, with anear-circular transverse section. The terminus of thetail is not visible in most specimens; extrapolation ofthe lines that define the margins of the caudal myoto-mes indicates that the tail is 1.1–1.3 times body length,i.e. short. The fins are low. This anatomical configura-tion, in conjunction with: (1) the dorsal position ofthe eyes; (2) origin of the dorsal fin close to the dorsaltail ⁄ 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 inhabitniches close to the sediment–water interface innon-flowing (lentic) water systems; they feed by pas-sive filtration of phytoplankton and rasping peri- andepiplankton, bacteria and sedimentary detritus fromsubmerged substrates (Larson & Reilly 2003; Grosjeanet al. 2004). This interpretation is consistent with thepresence of silty and diatomaceous gut contents inmany Libros tadpoles.

This ecological reconstruction has significant impli-cations for our understanding of the evolution ofbehavioural patterns in ranids. Most modern Ranaspecies deposit their eggs in lentic habitats (Holman2003). Larval morphology in modern Ranidae ishighly conserved: larvae typically exhibit primitive fea-tures such as an oval depressed body, dorsal eyes andlow fins, and many species belong to the lentic benthicguild (Holman 2003). The similarity between the lar-val ecomorphologies in modern Rana, and R. pueyoiindicate that a lentic benthic ecological strategy hasbeen utilized for at least the last 10 Myr. This strategy

A

B

C

Fig. 9. Details of the jaw sheath. A, light micrograph of MNCN63783 with, inset, position of jaw sheath, which is disarticulatedand rotated by �70� from its position in vivo. Gosner stages 38–41,oblique. B, C, light (B (MNCN 63865)) and scanning electron (C,MNCN 63842) micrographs of micro-serrations on oral surface ofjaw sheath.


need not, however, be the primitive condition: Ranaappears first in the late Oligocene.

Cannibalism is common amongst modern anurantadpoles (Alford 1999). No Libros specimen, however,includes vertebrate skeletal remains in its gut contents.The anatomy of the Libros tadpoles is also inconsis-tent with this behavioural strategy. The widest pointof the body of cannibalistic tadpoles is near the planeof the eyes (due to hypertrophy of the jaw muscles);jaw sheaths are usually located terminally (Altig &McDiarmid 1999) and are highly modified, typicallybearing long sharp projections (Grosjean et al. 2004).Further, cannibalism is most common in taxa thatinhabit small ephemeral water bodies subject to dessi-cation and ⁄ or other environmental instability (Alford1999). The Libros larvae, however, inhabited a perma-nent water body.

Absence of specific developmental stages

Despite the high abundance of specimens, the Librostadpoles represent only certain developmental stages(Gosner stages 30–41). A bias towards specific develop-mental stages has been noted in other exceptionallypreserved larval anuran faunas (Chipman & Tchernov2002; Maus & Wuttke 2002; Rocek 2003); tadpolesfrom Enspel (Maus & Wuttke 2002) exhibit biastowards developmental stages similar to that of theLibros specimens. Both the earliest (embryos andhatchlings) and metamorphic developmental stages areabsent in the Libros material. This implies that progres-sive changes through ontogeny, e.g. increased ossifica-tion, are not the explanatory factor. Instead, selectivepreservation of certain developmental stages is mod-elled as having been controlled primarily by changes inecology during ontogeny. The anuran embryo is sur-rounded by a tough vitelline membrane and severalcapsules (Holman 2003) and should have a high pres-ervation potential (Raff et al. 2006). Despite this,embryonic larvae are highly unlikely to be fossilized:they are non-motile and typically develop in low-energy, usually densely vegetated, extremely shallow-water, environments (Holman 2003). Transport ofembryos offshore and deposition into environmentsmore favourable for soft-tissue preservation is there-fore extremely unlikely. Hatchlings, although motile,remain in littoral habitats and, for the same reasons,are unlikely to be fossilized. Tadpole stages, however,are characterized by a major behavioural shift towardsa more active lifestyle (Alford 1999); they frequentlyrange beyond highly vegetated littoral zones and arethus less likely to become entrained in marginal vegeta-tion after death. This ecological shift is reversed subse-quently: metamorphs retreat into littoral vegetationuntil metamorphosis is complete. The preservational

potential of metamorphs is enhanced by their near-complete ossification, but this is more than offset bythe shift in their ecology into an environmental nicheless conducive to preservation.

Additional factors would have enhanced the preser-vational potential of specific developmental stages.For instance, the vast majority of embryos are con-sumed by predators prior to hatching (Alford 1999).Different developmental stages vary in their duration.Tadpole stages comprise the bulk of development time(for example 56.7% in Bufo) (McDiarmid & Altig1999); even if all other variables are constant thesestages should provide the majority of fossil specimens.

Ontogenetic controls uponpreservation

The taphonomic history reconstructed for adult andlarval R. pueyoi from Libros is broadly similar. Speci-mens consistently exhibit high degrees of articulationand completeness and are always orientated parallel tobedding; carcasses came to rest on the sediment–waterinterface in an anoxic profundal environment andwere buried subsequently by fine-grained laminatedsediments. Shared features include definition of thebody outline and eyespots by a carbonaceous bacterialbiofilm, definition of the former position of the brainby calcium carbonate, and organic preservation ofbone marrow.

There are, however, several important taphonomicdifferences between the adult frogs and tadpoles thatcan (to varying extents) be attributed to ontogeneticfactors. No soft tissues are replicated in authigenicminerals in the tadpoles. In the adult frogs, however,collagen fibres of the mid-dermal Eberth–Katschenko(EK) layer (which also contains interstitial glycosami-noglycans (GAGs) and granules of calcium phosphatein vivo) are replicated extensively in calcium phos-phate (McNamara et al. 2009). The source ofphosphate has been interpreted as the carcass, in par-ticular, the granules of calcium phosphate in the EKlayer (McNamara et al. 2009). The absence of the EKlayer in the Libros tadpoles reflects the developmentalbiology of anuran larvae: the EK layer does notdevelop until metamorphosis (Elkan 1968).

In the adult frogs, the position of collagen fibres ofthe lower dermis is defined by calcium sulphatediscoids that precipitated as the collagen decayed(McNamara et al. 2009). This mineral phase does notoccur in the fossil larvae. In modern Rana, sulphatedGAGs, a potential source of sulphate, comprise 75%of the total integumentary GAGs in adults but lessthan 4% of that in tadpoles (Lipson & Silbert 1968).


The biofilm defines the outlines of upper dermalglands in only adult specimens (McNamara et al.2009). In modern anurans, many integumentary glandsdo not develop until metamorphosis (Rosenberg& Warburg 1978). Other anatomical structures (tailmusculature and lungs) are defined by the biofilm inonly tadpoles from Gosner stages 30–35. Such subtleinternal features may be obscured in tadpoles at laterdevelopmental stages and in the adult frogs by thethicker biofilm and, in the latter, by the preservedremains of the EK layer. Further, in the adult frogs, thebiofilm medial to the EK layer comprises bacteria ofdifferent morphotypes organized into discrete layers(McNamara et al. 2009). The stratified chemicalmicro-environments implied by this complex structuremay have developed only in the adult frogs due to thepersistence of their integument during decay (collagenfibres of the EK layer were replicated during the latestages of decay; McNamara et al. 2009).

Bone marrow occurs in 10% of adult frogs but onlyone tadpole; limited ossification, and thus high poros-ity, of bone in the tadpoles would have facilitated bac-terial infiltration of marrow cavities and enhanced therate of microbial degradation (McNamara et al.2006).

Calcium phosphate is a common infill of the stom-ach in adult specimens, but does not occur inside thestomach (the manicotto) of the tadpoles. The stomachof both adults and tadpoles would have had abundanthydrolytic enzymes and a low pH (less than 3); decay-ing ingested organic matter would have been a poten-tial source of phosphate ions. Critically, the manicottolacks the muscular sphincters that separate, and couldhave sealed, the stomach from the remainder of thedigestive tract (Viertel & Richter 1999). In theirabsence it may not have been possible to sustain amicro-environment conducive to precipitation of cal-cium phosphate within the manicotto during decay.

Lastly, the nerve cord is defined by calcium phos-phate in only the tadpoles. The reason for this isunclear, especially as the nerve cord in both adult andtadpoles has a broadly similar structure and composi-tion. Problematically, the timing of mineralization ofthe nerve cord is difficult to constrain. Mineralizationoccurred after fragmentation of the nerve cord, butthis does not imply that it preceded displacement ofthe fragments from life position. Mineralization afterdispersal of the fragments could imply that the sourceof calcium and phosphorous was not the carcass itself.In this case, replication of the nerve cord, and poten-tially other tissues, in at least some adult frogs wouldbe expected. There is no evidence for this in the Librosspecimens; the source of phosphate ions for replica-tion of the mid-dermal collagen fibres in the adultfrogs was the carcass itself (McNamara et al. 2009). It

is therefore more likely that the nerve cord in the tad-poles was mineralized before dispersal of the frag-ments. This implies that the carcass was the source ofthe relevant ions.

In summary, almost all the taphonomic differencesbetween the adult frogs and tadpoles of the Librosbiota, and, to a lesser extent, between different devel-opmental stages of the tadpoles, can be attributed toontogenetic factors. Foremost among these are differ-ences in the structure of certain organs and tissues,and the absence of certain tissues at specific develop-mental stages. These ontogenetic changes in biologycontrolled which early diagenetic authigenic mineralsprecipitated in association with the decaying carcasses,which soft tissues were replicated in authigenic miner-als, the extent to which bone marrow was preservedorganically, and the extent to which internal anatomi-cal features were defined by the bacterial biofilm. Inother exceptional biotas it is likely that the taphonomyof a taxon will vary depending on its developmentalstage. These subtle differences are superimposed onthe more generalized taphonomic history, i.e. themode of preservation, of the exceptional biota as awhole.

Wider implications

The larval anurans from Libros exhibit various soft-tis-sue anatomical features that would have also been pres-ent in other fossil chordates. Relevant anatomicalfeatures include the broadly fusiform body shape, alarge anterior feeding chamber, a hollow nerve corddorsal to a notochord and a terminally tapered tail withfins and segmented myotomes. Insights into thetaphonomy of larval anurans could therefore constraininterpretations of the anatomy, and affinities, of otherfossils. This includes examples that, with varyingdegrees of confidence, have been considered as chor-dates, e.g. Haikouella (Chen et al. 1999; Shu et al.2003), Myllokunmingia (Shu et al. 1999; Hou et al.2002), Pikaia (Conway Morris 1998) and Yunnanozoon(Chen et al. 1995), or that may have chordate affinities(e.g. vetulicolians; Aldridge et al. 2007). Such excep-tionally preserved fossils are crucial to attempts to deci-pher the origins of vertebrates, but interpretations ofpreserved anatomical features have been controversial(Donoghue & Purnell 2009). Further, aspects of thefunctional morphology and ecology are intrinsic tomodels of early vertebrate evolution (Purnell 2001).These aspects of the biology of primitive chordate fos-sils have received little attention. Herein, we have dem-onstrated that an understanding of the taphonomy,and ecological significance, of specific anatomical fea-tures allows the ecology of fossil anuran tadpoles to be


resolved. Similar studies could facilitate more detailedecological reconstructions of early chordates, putativeexamples thereof, and related taxa, thus enhancing ourunderstanding of vertebrate origins.

Acknowledgements. – We thank Drs Cormac O’Connell and DaveCottell for assistance using TEM facilities at University CollegeDublin, Eric Callaghan for assistance with preparation of histologi-cal sections, Laura Tormo for ESEM analyses of the nerve cord andjaw sheath, and the Aragon Government (exp. 103 ⁄ 2003 from theDireccion General de Patrimonio Cultural de Aragon and FO-CONTUR project) for access to the Libros site. Hermano MiguelPerez (Museo del Colegio de la Salle, Teruel, Spain), Ms SandraChapman (Natural History Museum, London), Dr EberhardSchindler (Forschungsinstitut Senckenberg, Frankfurt, Germany)and Dra Begona Sanchez (Museo Nacional de Ciencias Naturales,Madrid, Spain) provided access to specimens. The manuscript wasimproved greatly by the comments of Dr Mark Purnell and a sec-ond, anonymous, reviewer. Funded by Enterprise Ireland BasicResearch Grant SC ⁄ 2002 ⁄ 138 to PJO.

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