Downloaded from http://rspb.royalsocietypublishing.org/ on May 13, 2015

Experimental taphonomy of Artemia rspb.royalsocietypublishing.org reveals the role of endogenous microbes in mediating decay and fossilization

Aodha´n D. Butler1,2, John A. Cunningham1,3, Graham E. Budd2 Research and Philip C. J. Donoghue1

Cite this article: Butler AD, Cunningham JA, 1School of Earth Sciences, University of Bristol, Life Sciences Building, 24 Tyndall Avenue, Bristol BS8 1TQ, UK Budd GE, Donoghue PCJ. 2015 Experimental 2Department of Earth Sciences, Palaeobiology Programme, Uppsala University, Villava¨gen 16, 75236 Uppsala, taphonomy of Artemia reveals the role of Sweden 3Department of Palaeobiology and Nordic Centre for Earth Evolution, Swedish Museum of Natural History, endogenous microbes in mediating decay 10405 Stockholm, Sweden and fossilization. Proc. R. Soc. B 282: ADB, 0000-0002-1906-0009 20150476. http://dx.doi.org/10.1098/rspb.2015.0476 Exceptionally preserved fossils provide major insights into the evolutionary history of life. Microbial activity is thought to play a pivotal role in both the decay of organisms and the preservation of soft tissue in the fossil record, though this has been the subject of very little experimental investigation. Received: 28 February 2015 To remedy this, we undertook an experimental study of the decay of the Accepted: 15 April 2015 brine shrimp Artemia, examining the roles of autolysis, microbial activity, oxygen diffusion and reducing conditions. Our findings indicate that endogenous gut bacteria are the main factor controlling decay. Following gut wall rupture, but prior to cuticle failure, gut-derived microbes spread into the body cavity, consuming tissues and forming biofilms capable of Subject Areas: mediating authigenic mineralization, that pseudomorph tissues and structu- palaeontology, evolution res such as limbs and the haemocoel. These observations explain patterns observed in exceptionally preserved fossil arthropods. For example, guts Keywords: are preserved relatively frequently, while preservation of other internal anat- explosion, palaeobiology, omy is rare. They also suggest that gut-derived microbes play a key role in the taphonomy, bilateria, metazoa preservation of internal anatomy and that differential preservation between exceptional deposits might be because of factors that control autolysis and microbial activity. The findings also suggest that the evolution of a through gut and its bacterial microflora increased the potential for exceptional fossil Authors for correspondence: preservation in bilaterians, providing one explanation for the extreme rarity Aodha´n D. Butler of internal preservation in those that lack a through gut. e-mail: [email protected] Philip C. J. Donoghue e-mail: [email protected] 1. Introduction The preservation of the soft-bodied anatomy of organisms in the fossil record occurs only rarely in Konservat-Lagersta¨tten. While such sites of exceptional preservation may be uncommon, their contributions to our knowledge of the history of life are disproportionate to their frequency [1]. It is ironic, therefore, that fossils exhibiting soft tissue preservation can be among the most difficult to interpret [2]. This is because fossils preserving soft tissues are invariably a melange of pristine to decayed structures preserved through mineral replication, while other aspects of anatomy are not preserved at all. Hence, the peculiarities of fossilization processes can lead to the preservation of different suites of anatomical structures, systematically distorting phylogenetic interpretations [3] but, by the same token, knowledge of such processes provides a framework for the correct interpretation of fossil remains. It is generally accepted that the principal vectors of decay are autolysis and microbial activity, and that microbial activity is a key mediator of authigenic min- eralization of soft tissue in the processes of exceptional fossilization [4]. However, the role of microbes has been treated largely as a black box that is not known or understood in detail, and the role of autolysis is essentially unknown in adult

& 2015 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. Downloaded from http://rspb.royalsocietypublishing.org/ on May 13, 2015

animals. Inspired by the discovery that embryos are buffer. A Leica binocular dissecting microscope, a Zeiss Photomi- 2 rapidly pseudomorphed and stabilized by external bacterial croscope III and a Leica M205C were used for light microscopy. rspb.royalsocietypublishing.org biofilms that probably become the substrate for fossilization Some specimens were stained with the fluorescent dye acridine [5], and as bacteria are major mediators of exceptional soft orange. In fixed specimens, the emission spectrum of tissues tissue preservation [4,6], we set out to determine whether simi- shifts to red/orange, while bacteria appear green, allowing the two to be distinguished. Scanning electron microscope (SEM) lar mechanisms might account for soft tissue preservation of analyses were carried out using a Hitachi S-3500 SEM at complex and macroscopic bilaterian animals. Using the brine 15 keV, and decay experiment specimens were cryo-sectioned. shrimp Artemia salina as our experimental organism, we Elemental data were collected using an EDAX x-ray dispersive analysed the pattern of autolytic and microbial decay, and bio- (EDX) analysis system with GENESIS software. film development, under different environmental conditions that controlled for oxygen diffusion, exogenous microbial activity and/or fungal activity. Our results show that the carcass (d) Semi-quantitative analyses B Soc. R. Proc. of the brine shrimp is consumed rapidly by gut-derived We captured the pattern of decay in a series of semi-quantitative indices [9] that delimit arbitrary points along the continuum of microbes after rupture of the gut wall and consequent release decay from complete organism to amorphous organic matter of endogenous bacteria to the rest of the body cavity, followed and allow us to describe the patterns of microbial and autolytic by microbially mediated tissue replacement through biofilm

processes observed in gross morphology, internal tissue struc- 282 development, irrespective of external controls. These biofilms ture, internal microbial infiltration and surface microbial cover. 20150476 : then mediate authigenic mineralization of soft tissues and of Six specimens were removed from each of the five experimental the biofilms themselves, within both the gut and body cavity. systems and coded at each sampling interval. The use of these These findings suggest that internally derived microbes are indices depends on description and assignment of a numerical the major control on internal preservation—a result that has value to each stage of decay (table 1). important implications for our understanding of the fossil record of exceptionally preserved animals. 3. Results 2. Material and methods (a) Gross morphological decay The overall pattern of morphological decay was not altered by the (a) Justification of experimental organism experimental treatments; only the rate at which decay progressed We selected the brine shrimp Artemia salina as the experimental differed. The sequence of gross morphological decay conformed organism, because its taphonomy has been well studied [7]; its to the pattern described previously [7]. Initial signs of decay were translucent body allows microbial activity to be observed readily observed in the distal portions of the thoracopods, which became via optical microscopy; it is available cheaply and in large num- shrivelled and matted (figure 1b). Next, the body became opaque bers, allowing statistically meaningful datasets to be obtained; because of microbial growth inside the cuticle (figure 1c). and because arthropods dominate many Konservat-Lagersta¨tten, The cuticle began to shrink and the distal parts of some appen- especially in Cambrian examples [8]. dages disarticulated (figure 1d). Eventually, the thoracic and abdominal cuticle failed and contents leaked from the carcass (b) Experimental conditions (figure 1e). Decay proceeded until only cuticular fragments and Animals were euthanized in standardized artificial seawater an unsupported gut remained (figure 1f–g). Samples in open (ASW) taken from a marine aquarium with sediment and organ- conditions decayed fastest, followed by closed, then reducing isms which was first supersaturated with carbon dioxide and environments (figure 4a). The combination of reducing and then sealed in an airtight Duran flask purged of remaining antimicrobial treatments progressed at the slowest rate. oxygen with a CO2/N2 gas mixture. Individual specimens were then transferred to 10 ml Wheaton serial crimp vials along with an aliquot of the ASW. Time-series replicates of five experimental (b) Tissue decay systems were established in order to isolate the effects of autolysis Tissue decay occurred very rapidly in open conditions. The rate and microbial activity on the decay and preservation of Artemia. of decay decreased in a series from closed conditions, through The five experimental systems were set up as follows: (i) open— reducing conditions, to reducing conditions with antimicrobial vials open to atmospheric diffusion, (ii) closed—sealed vials control. Anoxia alone was not sufficient to retard the decay of containing anoxic ASW, (iii) reducing—reducing environmental con- tissue by autolysis. It was only under reducing conditions, ditions consistent with an anoxic depositional environment with where autolytic tissue decay was greatly retarded, that tissues decay of organic matter by sulfate-reducing bacteria, simulated remained intact sufficiently long to become pseudomorphed with 10 Mmol beta-mercaptoethanol, (iv) antimicrobial— by biofilm bacteria (figure 2e–f). 10 mgml21 of rifampicin was introduced to anoxic ASW to control bacterial growth and (v) reducing and antimicrobial—inhibition of both exogenous microbial and autolytic processes by a combination (c) Microbial decay of the conditions in (iii) and (iv). The antifungal reagent methylene Initially, and in all the systems, the bulk of microbial activity blue was also used in the antimicrobial systems to prevent the oppor- was restricted to the gut (figure 2a). Bacterial populations tunistic growth of fungi. The experiments were run for 35 days with replaced the void left by decayed gut epithelial tissue in a dis- samples taken at 1, 2, 3, 5, 7, 9, 12, 15, 25, 30 and 35 days. The systems tinct biofilm (figure 2d). Bacteria were prevented from further were maintained at 158C for the duration of the experiments. infiltration of the body cavity by the basement membrane underlying the gut. In all instances, failure of the epithelium (c) Specimen preparation and analysis occurred first at the interface of mid and hindgut regions For imaging, specimens were fixed in a solution of 2.5% gluter- (a possible weak point), where microbes were observed aldehyde and 2% formaldehyde in a 0.1 M sodium acetate escaping into the body cavity (figure 2b). Here, they Downloaded from http://rspb.royalsocietypublishing.org/ on May 13, 2015

Table 1. Description of semi-quantitative indices of decay. 3 rspb.royalsocietypublishing.org internal microbial infiltration surface microbial cover gross morphology internal anatomy index index

1 fresh (figure 1a) fresh no microbes in body cavity, none/isolated microbes restricted to gut (figure 2a) 2 distal thoracopods shrivel and shrinkage of cells away from distal microbes escape from hindgut, isolated clusters of microbes mat (figure 1b) thoracopod setae, cells/nuclei in microbes visible in haemocoel visible integument still visible under and telson (figure 2b) fluorescent microscopy, some B Soc. R. Proc. evidence of degradation by autolysis 3 body appears opaque due to cellular details and outline microbes visible in distal patchy growth of biofilms 282 microbial growth inside degrade in integument, Z- appendages. Increase in (figure 2g) 20150476 : the cuticle (figure 1c) banding no longer visible in density in haemocoel muscle when viewed under cross-polarized light. Small droplets of what appear to be lipid form in association with tissues 4 shrinkage of cuticle and no cellular details visible, gross extensive infiltration by microbes enclosure—surface detail internal structure, loss of outline of muscle tissue still into body cavity and through obscured by biofilm some distal appendages visible. Lipid droplets are tissues. Individual microbes (figure 1d) present in large numbers in difficult to image (figure 2c) some cases outlining where tissue previously occurred 5 thoracic and abdominal no tissue structure visible except cuticle packed with biofilm, thick biofilm of diverse cuticle failure, internal unsupported basement individual microbes impossible morphotypes overgrows contents leaks from carcass membrane of gut, large to image by light microscopy initial community, when disturbed (figure 1e) spherulitic masses of lipid in appendages and body degradation of underlying proteinacous fragments visible segments due to high cuticle surface evident population density (figure 5a) (figure 2h) 6 cuticular fragments structural failure, unsupported gut cuticle fails releasing microbes structural failure of and unsupported gut all pulls away from cuticle, underlying cuticle that remains (figure 1f–g) amorphous decay fabric released into surrounding medium

proliferated until the haemocoel was filled with a dense bio- plateau that was possibly representative of a growth hiatus film. Eventually, the entire body was filled by biofilm as between seven and 12 days (figure 3). However, we failed internal tissues were replaced by microbes (figure 2c,e,f). to observe a difference between the systems with antimicro- Externally, surface biofilms formed rapidly (within 1–2 bial controls and the reducing and closed environments by days; figure 3), consisting initially of a relatively low diversity the end of the experiment for this index. of morphotypes (figure 2g) that gradually engulfed the car- Surface biofilm communities (figure 4d) were associated cass with a diverse community of filamentous, rod and with a visible degradation of the cuticle in the later stages coccoid microbial morphotypes (figure 2h). Despite engulfing of decay (figure 2h). While their development was retarded in the carcass, the bacteria did not penetrate the interior until closed and antimicrobial conditions, the most effective inhibi- the cuticle failed, which invariably occurred after the body tion of these surface microbial communities occurred in cavity had already been filled with endogenous gut-derived the reducing environmental conditions (figure 3), although microbes (figure 3). Neither antimicrobial nor reducing con- they were still present (figure 2e). Inhibition in the activity of sur- ditions on their own resulted in a significant reduction in face microbial communities in reducing conditions led to a the rate of internal microbial infiltration of the gut bacteria reduced rate of gross morphological decay (figure 4a)ofspeci- (figure 4b). When reducing conditions were applied in mens compared with those that displayed extensive surface tandem with antimicrobial controls the systems show a microbe colonization in the open (figure 4d) and closed systems. Downloaded from http://rspb.royalsocietypublishing.org/ on May 13, 2015

(a) (b) 4 rspb.royalsocietypublishing.org

(c) (d) B Soc. R. Proc. 282 20150476 :

(e)

( f ) (g)

(h) (i)

g

gf

bc

Figure 1. Generalized decay sequence in Artemia (a–g) and comparison to Burgess Shale-type fossils (h–i). (a) Undecayed specimen. (b) Thoracopods become matted. (c) Body and limbs (arrow) becomes opaque due to microbial activity. (d) Cuticle shrinks; some distal podomeres are disarticulated, cloudy appearance of limbs indicates internal biofilm. (e) Cuticle fails, internal biofilm is lost. (f) Cuticle disintegrates into fragments. (g) Only the unsupported gut remains. (h) Opabinia specimen (Burgess Shale, BC, Smithsonian National Museum of Natural History Washington DC, USNM 155600) showing the gut and associated microbial fabrics. (i) Enlargement of Opabinia gut area (Burgess Shale, BC, Geological Survey of Canada GSC 40251). Dark area, g gut; gf grey features corresponding to structures interpreted as gut diverticulae or limbs, bc lighter areas, body cavity. Scale bar: (a–c,i) ¼ 2 mm, (d–g) ¼ 1 mm, (h) ¼ 10 mm. (Online version in colour.)

(d) Microbial biofilms and co-associated mineralization 4. Discussion A crystalline fabric was observed under cross-polarized light and SEM in association with the gut-derived biofilms in (a) The role of autolysis, internal and external microbial both the body cavity and gut itself (figures 2d and 5a,b). activity in decay SEM-EDAX revealed enrichment of Ca, Mg and P in crystalline Our experimental conditions were designed in order to c masses (figure 5 ) suggesting earlyauthigenic mineral formation explore the various influences of autolysis, endogenous and of apatite or whitlockite in these biofilms. Downloaded from http://rspb.royalsocietypublishing.org/ on May 13, 2015

(a) (b) 5 rspb.royalsocietypublishing.org rc .Sc B Soc. R. Proc.

(c) (d) bm bf 282 20150476 :

gc m

(e) ( f )

(g) (h)

Figure 2. Microbial activity during Artemia decay. (a) Hindgut of live Artemia.(b) Microbes (arrow) escape at midgut/hindgut boundary and migrate into haemocoel. (c) Cuticle filled with microbes forming a dense biofilm; (d) replacement of gut epithelium by microbial pseudomorph. bm, basement membrane; bf, biofilm; gc, gut contents; m, musculature surrounding gut. (e) Section of appendage filled with biofilm (reducing conditions). (f) Enlargement of (e). (g) Surface microbes at early stage decay. (h) Diverse microbial community after 28 days in closed conditions. Scale bar (a) 500 mm, (b) 250 mm, (c)50mm, (d)20mm, (e) 200 mm, (f)10mm, (g)20mm, (h) 300 mm. (Online version in colour.) (a) open(b) reducing 6 day: 12 3 5 7 12 15 25day: 1 2 3 5 7 12 15 25 100 rspb.royalsocietypublishing.org

75

(%) 50

25 I I I I I I I I S S S S S S S S T T T T T T T T G G G G G G G 0 G I I I I I I I I S S S S S S S S T T T T T T T T G G G G G G G G (c)(closed d) antimicrobial and reducing

day: 12 3 5 7 12 15 25 day: 12 3 5 7 12 15 25 B Soc. R. Proc. 100 75 50 (%) 282 25 20150476 : 0 I I I I I I I I I I I I I I I I S S S S S S S S T T T T T T T T S S S S S S S S T T T T T T T T G G G G G G G G G G G G G G G G (e) antimicrobial day: 12 3 5 7 12 15 25 stage 6 G: gross anatomy index 100 stage 5 T: internal tissue index stage 4 I: internal microbes index 75 stage 3 S: surface microbes index stage 2 50 stage 1 (%) 25

0 I I I I I I I I S S S S S S S S T T T T T T T T G G G G G G G G Figure 3. Count data of decay experiments, six samples coded per interval. (Online version in colour.)

(a) gross morphology(b) internal tissues time (days) time (days) 0 5 10 15 20 25 30 0 5 10 15 20 25 30

1 1 2 2 3 3 4 4 decay stage 5 5 6 6 (c)(internal microbesd) surface microbes time (days) time (days) 0 5 10 15 20 25 30 0 5 10 15 20 25 30

1 1 2 2 3 3

decay stage 4

5 4

6 5 open closed antimicrobial reducing antimicrobial and reducing Figure 4. Graphs of median decay stage at each recorded time interval, error bars indicate median absolute deviation per interval where value is greater than zero. (Online version in colour.) exogenous bacteria in the post-mortem degradation and for exceptional preservation. In the open conditions, both biofilm replication of adult animal tissue level anatomy, pro- microbial and autolytic processes should proceed in an unin- viding important insights into the processes responsible hibited way, and, indeed, this is reflected in our results. The (a) (b) (c) 7 rspb.royalsocietypublishing.org c

Au o P Mg Ca 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60 4.00 Figure 5. Biofilm associated with gut and internal body cavity with phosphatic mineral spheroids growing in situ. (a) Internal body cavity biofilm. (b) Gut-associated biofilm. Arrows indicate phosphatic spheroids. (c) Point EDX spectrum from mineralized spheroid, note distinct Ca, Mg and P peaks (not present in surrounding tissue) Au peak results from gold coating used for SEM imaging. Scale bar: (a)20mm and (b)30mm. (Online version in colour.) rc .Sc B Soc. R. Proc. lowest rate of tissue decay was observed under the reducing decay, stabilization and, ultimately, the preservation of soft conditions, which have been shown previously to inhibit auto- tissues. Autolysis of tissues prior to stabilization by pseudo- lysis [10]. The stability of tissue structure for an extended morphing or microbially mediated mineralization may period post-mortem was also associated with the replication explain why the gut microenvironment is often preserved 282 of tissue structure by microbial pseudomorphs (figure 2e–f ). in three dimensions in deposits of exceptional preservation, In environmental conditions where autolysis was not inhib- whereas tissues associated with the body cavity are often 20150476 : ited, only homogeneous microbial fabrics were produced in much more poorly preserved, if at all [15]. Although the the body cavity. This corroborates the previous suggestion gut, especially if it is infilled at the time of death, may be that blocking autolysis is probably a necessary first step in readily stabilized by the presence of bacteria from the start the preservation of labile tissues [5,10]. of decay, the ability of the gut-derived bacteria to capture In all experimental conditions, the wall of the gut failed at other features of internal anatomy may depend on the the mid- to hindgut junction, allowing gut-derived microbes extent of autolysis of non-gut tissues at the time of gut rupture. to spread into the body where they proliferated. Under redu- If the rupture of the gut is delayed then, clearly, there may be cing conditions, where internal soft tissues remained intact little remaining of the original biological structure to serve as because autolytic degradation was blocked, the gut-derived a substrate for mineral replication, save amorphous remains microbes replicated these tissues. This implies a key role (figure 6). For example, in some Sirius Passet and Burgess for gut bacteria in the stabilization of tissue and organ level Shale arthropods (figure 1h–i) the gut is preserved and the mor- anatomy, as part of the process of exceptional preservation phology of internal structures is lost, with only amorphous through mineral replication. Exogenous microbes always features remaining. These may have been interpreted pre- played a subordinate role, regardless of the experimental con- viously as limb extensions [16,17] or gut diverticulae [15,18] ditions. Microbes derived from the gut had already filled the but based on our experimental observations and the resem- body by the time the cuticle failed allowing exogenous blance to decay derived fabrics (figure 1), these structures microbes to enter. We propose, therefore, that gut-derived may represent remnants of highly decayed tissues and their microbes are key to the replication of internal anatomy. How- associated microbial biofilm-derived fabrics. ever, because of the importance of the cuticle in retaining Fossilized internal biofilms have been observed directly in structural integrity of the decaying organism, limiting the animal remains from throughout the Phanerozoic, including activity of surface microbial activity that degrades cuticle also hyolith guts [19] and also Mesolimulus, an exceptionally pre- improves the preservation potential of a cuticular organism. served arthropod from the Upper of Germany [20], Size differences and variations in cuticle thickness probably where biofilms fill the haemocoel enveloping authigenically also affect preservation potential between separate taxa, but mineralized muscles, as in our experimental results. Fossi- since a similar pattern of gut rupture and similar progression lized guts are relatively common in Konservat-Lagersta¨tten of decay sequence is also observed in decay experiments of with observations from Ottoia, for example, showing that velvet worms [11], the primacy of gut-derived microbial the gut is retained in 75% of individuals [21]. Indeed, guts decay is probably universal, at least among ecdysozoans. are regularly the only portions of the internal anatomy that are preserved in fossils from these localities [21–24]. We recog- nize that the gut itself is readily fossilized as a product of both (b) The role of microbes in internal preservation its unique microenvironmental conditions, including high The fact that there is evidence for mineralization associated phosphate content and endogenous microbial flora. Our with the biofilms leads us to suggest that internal biofilms results provide experimental support that this is the fossi- are the precursors to exceptional preservation through the min- lization mechanism for bilaterian guts. The preservation of eral replication of soft tissue anatomy. This notion is supported detailed internal anatomy is much rarer, although records of by previous studies showing that microbial fabrics of the kind such preservation do exist, for example, musculature from observed in our experiments can produce high-fidelity pseudo- annelids [25] and arthropods [26] as well as nervous tissue [27]. morphs of soft tissues that are robust (at least on a small scale) The patterns observed in the fossil record are, therefore, [5,12,13] and are able to mediate mineralization [5,6]. compatible with those identified in our experiments. Bacteria Microbially mediated mineralization can probably occur in colonize the gut in living organisms and rapidly form biofilms the absence of pseudomorphing, resulting in the ‘substrate in the gut post-mortem, which is preserved comparatively fre- microfabrics’ that lack direct evidence of bacteria [14]. quently. On the other hand, biofilms can only pseudomorph Unlike experiments on animal embryos [5,13], where other features if the conditions allow them to do so before the biofilm-forming bacteria must be externally derived, in the structures decay. Hence, apart from the gut, internal anat- Artemia the endogenous gut bacteria are sufficient to control omy is rarely preserved [7]. Given this role for endogenous internal anatomy rapidly before stabilizing biofilms can form, 8 death leaving an unsupported gut in a sack of amorphous decayed rspb.royalsocietypublishing.org tissue (figure 6). If decay progresses unchecked, eventually autolysis unchecked: decay of labile soft tissue components external biofilms and decay mediating organisms digest and degrade the cuticle, that then ruptures and fragments microbial attack and replacement of gut epithelium (figure 7). Thus, the preservational state of a given fossil reflects its trajectory exiting the more general decay pathway, from exceptionally preserved Burgess Shale arthropods e.g. Bran- microbes escape from gut chiocaris and Opabinia (figure 1h–i), through branchiopods microbial digestion and infiltration of tissues and notostracans from the Upper Strud locality of inside haemocoel Belgium [29] to the small carbonaceous fossil record of recalci-

trant cuticular structures such as mandibles [30]. The finest B Soc. R. Proc. amorphous microbial infill of body cavity: unsupported gut level, and likely limit of fossilization, that of organic preser- vation of labile soft tissues, is restricted to highly specialized no pseudomorphing settings and confined to specific tissues, e.g. red bone marrow in frogs from Libros, Spain [31], reflecting stage 2 of 282 internal tissue preservation index in our model. More external microbial biofilm formation: digestion of cuticle common classic examples including Mesozoic Plattenkalk 20150476 : deposits, such as those from Solnhofen, Nu¨ splingen, Cerin, failure of cuticle: contents escape Lebanon or the Santana Formation, preserve internal muscle tissue relatively frequently [20,32–35]. Cambrian Konservat- large fragments of cuticle and isolated gut remain Lagersta¨tten preserve muscle tissue only rarely in Sirius Passet arthropods and annelids [24,25]. These exhibit high-fidelity internal preservation through inhibition of autolysis and fragmentary cuticular elements remain exogenous microbial decay, allowing endogenous biofilms to pseudomorph structures and catalyse authigenic mineralization, Figure 6. General model of decay in Artemia in open conditions. equivalent to stages 3–4 (figure 6). However, the effects of decay are readily apparent and information loss may preferentially act gut bacteria in mediating exceptional preservation, we propose on characters that are important for taxonomic classification [3,4]. that an endogenous source of bacteria may be essential Further along the decay pathway, Orsten phosphacopids for preservation of internal anatomy, as seen in Konservat- retain a cuticle, but internally preserve an unsupported gut Lagersta¨tten. The distribution of ‘substrate’ and ‘microbial’ and a spheroidal decay fabric [36], corresponding to stage 4, preservation fabrics [14] shows a degree of taxonomic control, with muscles preserved rarely in low resolution (figure 6). The stemming from differential rates of microbial penetration of effects of autolysis and microbial decay may have progressed carcasses. We hypothesize that this taxonomic control is the much further in both the Strud and Burgess Shale preservational result of the intrinsic arrangements of a body plan altering settings, eliminating structural information such as musculature the trajectory of microbial infiltration, e.g. open versus closed and most internal organs before fossilization occurred, leaving circulatory systems, an open haemocoel providing an ideal only recalcitrant gut and cuticle structures relatively intact with environment for microbes to proliferate, for example. amorphous decay derived fabrics replacing the majority of soft tissues (internal decay stages 4–5). The extensive record of (c) Reconciling differences between Konservat- small carbonaceous fossils [30], i.e. that of disarticulated cuticle fragments, corresponds to the end stage in our model. Lagersta¨tten Konservat-Lagersta¨tten characterized by exceptional preser- (d) The role of a through gut in modifying preservation vation are not generated equally. Instead, each fauna, indeed, each individual organism, can be viewed as a separate exit potential point from the normal progression of decay prior to fossiliza- The key role of gut-derived microbes in decay and, by infer- tion. It is the extent of autolysis versus microbial activity and ence, preservation means that the evolution of a through biofilm stabilization of anatomical structure that may account gut is likely to have important implications for preservation for the disparity of anatomical structures present in individual potential. Organisms that have blind guts, such as cnidarians, exceptionally preserved fossils. While the relative volatility and evert their guts such that they cannot maintain a gut flora. As a differential susceptibility of soft tissues to decay have been result, one might expect that such organisms would have little studied previously [4,28], the mechanisms underlying this chance of preserving internal anatomy. Preservation must are less apparent. We propose that our taphonomic model depend on the formation of favourable external biofilms that provides a deductive framework in which to understand invade inwards, similar to the process observed in embryos, the nature of taphonomic processes on soft tissue anatomy to stabilize their internal anatomical structure post-mortem, post-mortem and, consequently, inform the interpretation of allowing a much longer window for internal autolytic pro- exceptionally preserved fossils more generally. For example, cesses to take effect and thus resulting in a much lower in cases where highly labile structures are preserved by preservation potential for internal anatomy. This prediction is authigenic mineralization, where autolysis is inhibited and largely borne out by the fossil record. The overall quality of pseudomorphs that stabilize internal anatomy have the oppor- preservation is also often of a lower fidelity in described soft- tunity to form (figure 7). Conversely, in cases where autolysis bodied diploblast grade and blind-gut bearing organisms continues unchecked in open environments, decay obliterates relative to groups possessing through-guts. For example, 9 autolysis internal microbial activity rspb.royalsocietypublishing.org

death death

autolysis inhibited endogenous microbes autolysis unchecked (reducing conditions) restricted to gut rc .Sc B Soc. R. Proc.

internal anatomy microbial proliferation and labile soft-tissues persist destroyed by autolysis replacement of gut epithelium 282

pseudomorph of 20150476 : gut ruptures: microbes escape no pseudomorphing surviving original tissue into body cavity structure

no internal preservation cuticle remains intact microbially mediated exogenous decay mineralization microbes inhibited

pseudomorph mineralization (microbial microfabric)

surviving tissues autolithified (substrate microfabric)

internal structures preserved Figure 7. Hypothesized model of decay and pathways leading to exceptional preservation. arthropods, annelids, priapulids and hyoliths can in many the amount of anatomical information, that is preserved in cases preserve aspects of gut, musculature and, in rare cases, Konservat-Lagersta¨tten. Hence, the evolution of a through gut neural tissues. On the other hand, diploblastic organisms, is an important factor in both the ecology of metazoan diversi- such as cnidarians, are typically found as impressions or fication and its fossil record. This finding also suggests the outlines only (with the notable exception of very rare speci- bauplanofananimalmayactasastrongcontrolonthe mens of the probable cnidarian Olivooides [37,38]). This processes of subsequent taphonomic transformation into an may go some way to explain the mismatch between phyloge- exceptionally preserved fossil, when the basic conditions netic and molecular clock expectations that diploblasts required for the genesis of Konservat-Lagersta¨tten are met. existed long before diploblast bilaterians, yet the fossil records of diploblast and triploblast eumetazoans is approximately coincident [39,40]. Data accessibility. The datasets supporting this article are available at Under almost all circumstances, pseudomorphing of bio- doi:10.5061/dryad.f0tt2. logical anatomy by biofilm-forming microbes [5,13] may be Acknowledgements. Stuart Kearns, Jenny Greenwood and Simon Cobb limited to small structures. This process can provide a good are thanked for laboratory and SEM assistance and insightful explanation for the preservation of such as fossi- discussion. Rudy Lerosey-Aubril and anonymous others provided constructive reviews that helped to improve the manuscript. This lized embryos as well as internal microenvironments, such study represents the outcome of a research project completed by as guts, within larger fossils. However, it is only in the most A.D.B. in partial fulfilment of an MSc Palaeobiology at the University exceptional examples of exceptional fossil preservation that of Bristol. microbes replicate and preserve internal anatomy more gener- Funding statement. This project was funded by the Natural Environment ally. Bacterial biofilm pseudomorphing of anatomical Research Council (standard grant no. NE/F00348X/1 to P.C.J.D.; structure may not be an important mechanism in preserving Postdoctoral Fellowship NE/J018325/1 to J.A.C.), the Leverhulme Trust (Research Fellowship to P.C.J.D.) and the Royal Society (Wolf- macroscale animal remains, even though endogenous microbes son Merit Award to P.C.J.D.). are important vectors of the decay of visceral tissues that leaves Authors’ contributions. A.D.B., J.A.C. and P.C.J.D. designed the research cuticle articulated and intact in Burgess Shale-type preser- which was conducted by A.D.B. All authors contributed to the vation. Thus, endogenous microbes exert a fundamental interpretation of the results and the drafting of the manuscript. control on the amount of soft tissue morphology, and therefore Conflict of interests. We have no competing financial interests. References 10 rspb.royalsocietypublishing.org 1. Gould SJ. 1989 Wonderful life: the Burgess Shale 15. Butterfield NJ. 2002 Leanchoilia guts and the 28. Sansom RS, Gabbott SE, Purnell MA. 2011 Decay and the nature of history. New York, NY: WW interpretation of three-dimensional structures in of vertebrate characters in hagfish and lamprey Norton. Burgess Shale-type fossils. Paleobiology 28, (Cyclostomata) and the implications for the 2. Donoghue PCJ, Purnell MA. 2009 Distinguishing 155–171. (doi:10.1666/0094-8373(2002) vertebrate fossil record. Proc. R. Soc. B 278, heat from light in debate over controversial fossils. 028,0155:LGATIO.2.0.CO;2) 1150–1157. (doi:10.1098/rspb.2010.1641) BioEssays 31, 178–189. (doi:10.1002/bies. 16. Budd GE. 1996 The morphology of Opabinia regalis 29. Lagebro L, Gueriau P, Hegna TA, Rabet N, Butler A, 200800128) and the reconstruction of the arthropod stem group. Budd GE. 2015 The oldest notostracan (Upper 3. Sansom RS, Gabbott SE, Purnell MA. 2010 Non- Lethaia 29, 1–14. (doi:10.1111/j.1502-3931.1996. Devonian Strud Locality, Belgium). Palaeontology random decay of chordate characters causes bias in tb01831.x) 58, 497–509. (doi:10.1111/pala.12155) rc .Sc B Soc. R. Proc. fossil interpretation. Nature 463, 797–800. (doi:10. 17. Budd GE, Daley AC. 2012 The lobes and lobopods of 30. Harvey THP, Ve´lez MI, Butterfield NJ. 2012 1038/nature08745) Opabinia regalis from the middle Cambrian Burgess Exceptionally preserved crustaceans from western 4. Briggs DEG. 2003 The role of decay and Shale. Lethaia 45, 83–95. (doi:10.1111/j.1502- Canada reveal a cryptic Cambrian radiation. Proc. mineralization in the preservation of soft-bodied 3931.2011.00264.x) Natl Acad. Sci. USA 109, 1589–1594. (doi:10.1073/

fossils. Annu. Rev. Earth Pl. Sci. 31, 275–301. 18. Zhang X-G, Briggs DEG. 2007 The nature and pnas.1115244109) 282

(doi:10.1146/annurev.earth.31.100901.144746) significance of the appendages of Opabinia from 31. McNamara ME, Orr PJ, Kearns SL, Alcala L, Anadon 20150476 : 5. Raff EC et al. 2008 Embryo fossilization is a the Middle Cambrian Burgess Shale. Lethaia 40, P, Penalver-Molla E. 2006 High-fidelity organic biological process mediated by microbial biofilms. 161–173. (doi:10.1111/j.1502-3931.2007.00013.x) preservation of bone marrow in ca. 10 Ma Proc. Natl Acad. Sci. USA 105, 19 360–19 365. 19. Devaere L, Clausen S, Javier A´lvero J, Peel JS, amphibians. Geology 34, 641–644. (doi:10.1130/ (doi:10.1073/pnas.0810106105) Vachard D. 2014 Terreneuvian Orthothecid G22526.1) 6. Sagemann J, Bale SJ, Briggs DEG, Parkes RJ. 1999 (Hyolitha) digestive tracts from Northern Montagne 32. Briggs DEG, Wilby PR. 1996 The role of the calcium Controls on the formation of authigenic minerals in Noire, France; taphonomic, ontogenetic and carbonate/calcium phosphate switch in the association with decaying organic matter: an phylogenetic implications. PLoS ONE 9, e88583. mineralization of soft-bodied fossils. J. Geol. Soc. experimental approach. Geochim. Cosmochim. Acta (doi:10.1371/journal.pone.0088583) Lond. 153, 665–668. (doi:10.1144/gsjgs. 63, 1083–1095. (doi:10.1016/S0016-7037(99) 20. Briggs DEG, Moore RA, Shultz JW, Schweigert G. 153.5.0665) 00087-3) 2005 Mineralization of soft-part anatomy and 33. Wilby PR, Briggs DEG, Bernier P, Gaillard C. 1996 7. Gostling NJ, Dong XP, Donoghue PCJ. 2009 invading microbes in the horseshoe crab Role of microbial mats in the fossilization of soft Ontogeny and taphonomy: an experimental Mesolimulus from the Upper Jurassic Lagersta¨tte tissues. Geology 24, 787–790. (doi:10.1130/0091- taphonomy study of the development of the brine of Nusplingen, Germany. Proc. R. Soc. B 272, 7613(1996)024,0787:ROMMIT.2.3.CO;2) shrimp Artemia salina. Palaeontology 52, 169–186. 627–632. (doi:10.1098/rspb.2004.3006) 34. Fuchs D, Bracchi G, Weis R. 2009 New octopods (doi:10.1111/j.1475-4983.2008.00834.x) 21. Vannier J. 2012 Gut contents as direct indicators for (Cephalopoda: Coleoidea) from the Late 8. Butterfield NJ. 2003 Exceptional fossil preservation trophic relationships in the Cambrian marine (Upper Cenomanian) of Haˆkel and Haˆdjoula, and the Cambrian explosion. Integr. Comp. Biol. 43, ecosystem. PLoS ONE 7, e52200. (doi:10.1371/ Lebanon. Palaeontology 52, 65–81. (doi:10.1111/j. 166–177. (doi:10.1093/icb/43.1.166) journal.pone.0052200) 1475-4983.2008.00828.x) 9. Briggs DEG. 1995 Experimental taphonomy. Palaios 22. Vannier J, Liu J, Lerosey-Aubril R, Vinther J, Daley 35. Martill DM. 1990 Macromolecular resolution 10, 539–550. (doi:10.2307/3515093) AC. 2014 Sophisticated digestive systems in early of fossilized muscle tissue from an elopomorph 10. Raff EC, Villinski JT, Turner FR, Donoghue PCJ, arthropods. Nat. Commun. 5, 3641. (doi:10.1038/ fish. Nature 346, 171–172. (doi:10.1038/ Raff RA. 2006 Experimental taphonomy shows ncomms4641) 346171a0) the feasibility of fossil embryos. Proc. Natl Acad. 23. Lerosey-Aubril R, Hegna TA, Kier C, Bonino E, 36. Eriksson ME, Terfelt F, Elofsson R, Marone F. 2012 Sci. USA 103, 5846–5851. (doi:10.1073/pnas. Habersetzer J, Carre´ M. 2012 Controls on gut Internal soft-tissue anatomy of Cambrian ‘Orsten’ 0601536103) phosphatisation: the trilobites from the Weeks arthropods as revealed by synchrotron X-ray 11. Murdock DJ, Gabbott SE, Mayer G, Purnell MA. 2014 Formation Lagersta¨tte (Cambrian; Utah). PLoS ONE tomographic microscopy. PLoS ONE 7, e42582. Decay of velvet worms (Onychophora), and bias in 7, e32934. (doi:10.1371/journal.pone.0032934) (doi:10.1371/journal.pone.0042582) the fossil record of lobopodians. BMC Evol. Biol. 14, 24. Chatterton BDE, Johanson Z, Sutherland G. 1994 Form 37. Dong XP, Cunningham JA, Bengtson S, 222. (doi:10.1186/s12862-014-0222-z) of the trilobite digestive-system—alimentary Thomas CW, Liu JB, Stampanoni M, Donoghue PCJ. 12. Cunningham JA et al. 2012 Experimental structures in Pterocephalia. J. Paleontol. 68, 294–305. 2013 Embryos, polyps and medusae of the taphonomy of giant sulphur bacteria: implications 25. Conway Morris S, Peel JS. 2008 The earliest Early Cambrian scyphozoan Olivooides. Proc. R. Soc. B for the interpretation of the embryo-like Ediacaran annelids: Lower Cambrian polychaetes from the 280, 20130071. (doi:10.1098/rspb.2013.0071) Doushantuo fossils. Proc. R. Soc. B 1734, Sirius Passet Lagersta¨tte, Peary Land, North 38. Han J et al. 2013 Early Cambrian pentamerous 1857–1864. (doi:10.1098/rspb.2011.2064) Greenland. Acta Palaeontol. Pol. 53, 137–148. cubozoan embryos from South China. PLoS ONE 8, 13. Raff EC, Andrews ME, Turner FR, Toh E, Nelson DE, (doi:10.4202/app.2008.0110) e70741. (doi:10.1371/journal.pone.0070741) Raff RA. 2013 Contingent interactions among 26. Budd GE. 2011 Campanamuta mantonae gen. et sp. 39. Erwin DH, Laflamme M, Tweedt SM, Sperling EA, biofilm-forming bacteria determine preservation or nov., an exceptionally preserved arthropod from Pisani D, Peterson KJ. 2011 The Cambrian conundrum: decay in the first steps toward fossilization of the Sirius Passet Fauna (Buen Formation, lower early divergence and later ecological success in the marine embryos. Evol. Dev. 15, 243–256. (doi:10. Cambrian, North Greenland). J. Syst. Palaeontol. 9, early history of animals. Science 334, 1091–1097. 1111/ede.12028) 217–260. (doi:10.1080/14772019.2010.492644) (doi:10.1126/science.1206375) 14. Wilby PR, Briggs DEG. 1997 Taxonomic trends in the 27. Ma XY, Hou XG, Edgecombe GD, Strausfeld NJ. 2012 40. Benton MJ, Donoghue PCJ, Asher RJ, Friedman M, resolution of detail preserved in fossil phosphatized Complex brain and optic lobes in an early Cambrian Near TJ, Vinther J. 2015 Constraints on the soft tissues. Geobios Memoire Spe´cial 20, 493–502. arthropod. Nature 490, 258–261. (doi:10.1038/ timescale of animal evolutionary history. Paleontol. (doi:10.1016/S0016-6995(97)80056-3) nature11495) Electron. 18.1.1FC.