In Trial 1, the specimens were placed in an aquarium filled with sediment composed of grain sizes ranging from 0.1–1.0 mm. When the specimen was placed between the glass plates, it remained on the surface in a rigid posture for 5–10 minutes (Fig. 5A). The specimen retained sand grains from its holding tank due to contraction of the body wall and podia. Once relaxed, the specimen moved along the surface and investigated the area for approximately 20 minutes before burrowing began (Fig. 5B). Once burrowing was initiated, the specimen pushed itself easily into the sand taking approximately 3 hours for the specimen to completely move below the sediment-water interface.

Fig. (5) Complete burrowing sequence of T. gemmata in fine-grained carbonate sand. A) The specimen remains rigid when placed on the sediment surface. B) Once relaxed, the specimen begins muscular contractions and starts to penetrate the sediment. C) Podia aid in burrowing by carrying sand grains away from the growing depression beneath the center of the animal. D) The specimen continues to contract its body into a U shape. E) Sand grains fill the depression forming above the center of the animal. F) Podia attach to grains of sand and form a cover around the animals body. G) Once burrowing is complete, the anterior and posterior ends of the animal are left in contact with the surface. The dashed line outlines the base of the U-shaped burrow. H) Over time the holothurians body may relax and form a broader U shape.

To begin burrowing, T. gemmata used the middle portion of its body to penetrate the sand creating a small depression (Fig. 5B). Muscular contractions within the body wall played a large role in this phase of downward movement. The podia were then used to carry sand grains away from the depression (Fig. 5C-D). The specimen continued to penetrate the sediment while acquiring a more pronounced U-shape by contracting the muscles in the body wall and using its podia to transport sediment grains (Fig. 5D-5E). As the specimen burrowed deeper into the substrate, the surrounding sand collapsed into the depression formed in the middle of the “U” (Fig. 5E-F). While burrowing, the specimen also attached grains of sand to its body surface using the podia (Fig. 5F). As a result, the specimen within its burrow was not completely visible in the side of the glass plates. During the final stages of burrowing, the posterior end of the specimen remained in contact with the water column and the anterior end was slightly exposed at the sediment-water interface (Fig. 5G-H). The specimen later extended its tentacles to feed. The final depth of this burrow was approximately 4 cm. When the specimen was disturbed, it quickly burrowed deeper into the substrate to a depth of approximately 8–10 cm, often reaching the bottom of the aquarium. At the end of the experiment, the specimen was still buried deeply so it was removed from the substrate. When excavated, the holothurian retained the body shape it had maintained in its deep burrow (Fig. 3C).

In Trial 2, the specimens were placed on a layer of 0.5– 1.5 mm carbonate sand. The specimens burrowed easily and in similar fashion and time frame to the previous trial performed using the fine-grained sand (Fig. 6). Once the specimens had fully burrowed, they were disturbed using a glass rod. Each of the specimens was able to quickly burrow up to 10 cm into the sediment for protection.

Fig. (6) Complete burrow sequence in medium-grained carbonate sand. A) Initial penetration of the sediment in a U shape. B) Passive fill of the depression formed above the center of the animal. C) Final position beneath the sediment surface.

In Trial 3, the specimens were placed on a layer of 1.0– 2.0 mm carbonate sand. The specimens experienced difficulty burrowing compared to the efficient burrowing that took place within the fine- and medium-grained sand. Complete burrowing of the animal took approximately two hours longer than in finer-grained sand. Once burrowed in the coarse sand, the holothurian’s body was in the shape of an asymmetrical “U” (Fig. 7A). Later the specimen was able to further adjust its body in the substrate and form a more uniform U-shape (Fig. 7B). When disturbed at the surface, the specimen moved its entire body below the sediment- water interface, but did not burrow any deeper into the sand as the specimens did within the fine- and medium-grained sand.

Fig. (7) Specimen burrowed into coarse-grained (A-B) or layered (C-D) carbonate sand. A) The initial shape of the burrow formed in coarse-grained sand is a lop-sided U. B) Later, the specimen’s body contracted into a more symmetrical U shape. C) When encountering interbedded medium- and coarse-grained sand, the holothurian penetrated the medium-grained surface layer but did not fully penetrate the underlying coarse-grained layer. Medium-grained sand is transported downward by the burrowing animal. D) When disturbed at the surface, the specimen pushes downward through the coarse-grained layer. The dashed line outlines the burrow.

Trial 5 involved the use of the alternating layers of medium- and coarse-grained carbonate sand (Fig. 4). When placed upon these substrates, the specimens efficiently burrowed into the top layer of medium-grained sand and transported some of the medium-grained sand into the coarse-grained layer below (Fig. 7C-D). The specimens did not burrow through the coarse-grained sand layer. When disturbed at the surface, the specimens only contracted into their burrows but did not increase their burrow depth.

When placed in a 2.5-gallon aquarium filled with water with a salinity of 40‰ for Trial 1, the specimen explored the substrate with its tentacles for approximately 20 minutes. The specimen then moved to the side wall of the tank and began to burrow in a similar fashion to the burrowing that took place in the fine- and medium-grained sand. The specimen burrowed with efficiency and did not appear to be affected by the increased salinity.

When exposed to water with a salinity of 20‰ in Trial 2, the specimen of T. gemmata appeared to be detrimentally affected by the lowered salinity. The specimen did not move from the place in which it was initially set within the aquarium. It instead investigated the sand in its immediate area with its tentacles for approximately 15 minutes before withdrawing. The posterior portion of the specimen’s body then began to curl. It was at that point that the specimen was withdrawn from the small tank, acclimated to water of a higher salinity in a small container, and then returned to its holding tank with normal marine salinity.

Raising the temperature to 32º C did not have any observed effect on the burrowing behavior T. gemmata. The specimens burrowed in the same manner and time as they did in 21ºC water.

The holothurian Thyonella gemmata produced distinct biogenic structures in each of the experimental trials with the exception of the reduced salinity experiment. In each of the trials, as the specimens burrowed into the sediment, the overlying sand filled a U-shaped void between the shafts of the burrow creating a disrupted fill (Fig. 5D-F). An open burrow consisting of two irregular, subvertical shafts connected by a short tunnel at the base of the structure was maintained by the intrusion of the animal’s body into the sediment. The resulting structure was an irregular U-shaped burrow with a zone of layered to homogenized sediment in between the paired shafts.

Arenicolites and Diplocraterion are two common U- shaped burrows typically found in shallow marine deposits. Arenicolites and Diplocraterion are both characterized by a vertically oriented pair of shafts connected by a tunnel at the base of the structure. Arenicolites is traditionally described as an open U-shaped burrow with no spreite between the burrow shafts [11]. In the modern this type of burrow is often produced in low energy environments, such as the lower shoreface or tidal flats, and serves as the dwelling burrow for suspension-feeding organisms [12]. Diplocraterion is described as a U-shaped burrow with spreite between or below the burrow shafts [11]. The spreite are produced as the organism adjusts its position within the sediment in response to sediment deposition or erosion [9, 12]. In the modern this type of burrow is typically produced by suspension-feeding organisms inhabiting higher energy environments, such as the middle shoreface or high energy tidal flats [12].

The T. gemmata burrows do not possess distinct spreite as defined in Diplocraterion, and are therefore more similar to Arenicolites. From the observations of T. gemmata behavior, however, it is possible that spreite could be produced during long-term occupation of a burrow. These spreite would likely extend from the base of the burrow resulting from the upward movement of the holothurian within the sediment as a result of sediment accumulation. These would be similar to the spreite produced by the holothurian Thyone briareus as it moves its U-shaped body upward in the substrate in response to gradual sediment deposition [5, 13]. The U-shaped shafts of both Arenicolites and Diplocraterion typically possess sharp wall boundaries and passive fill which indicates that the burrows were kept open and free of sediment by the inhabiting organism [12]. Thyonella gemmata, however, did not leave open shafts when the animals evacuated their burrows. The observations in the experiments also indicate that T. gemmata does not reinforce its burrow walls to maintain an open burrow; the presence of the holothurian itself maintains the sediment-free space in the subsurface. While it is possible that the sediment of the burrow walls may become indurated and self-supporting after long-term use, this result was not observed in the laboratory. For these reasons, the U-shaped burrow produced by T. gemmata appears to be unique and cannot be assigned to either ichnogenus.

Since the burrows produced by T. gemmata cannot be assigned to an established ichnogenus, it is necessary to examine the burrow structures produced by these modern organisms in order to determine which architectural properties of the burrow may be recognized in the fossil record. The specimens used in this study produced the same general burrow morphology every time they burrowed. The burrow morphology consists of a wide, U-shaped concentration of disrupted sediment and spreite may occur below the burrow if the organism moved vertically in the substrate. While T. gemmata is in its burrow, the open burrow takes the shape of the body of the holothurian. A potential open burrow left behind when the holothurian leaves the sediment would have a total length of approximately 8 cm. The diameter of the burrow would be wider at the base of the “U” and would decrease upward along the limbs. The total width and depth of the burrow would vary depending on the individual organism’s size, position within the substrate, and substrate consistency.

Despite the common occurrence of burrowing holothurians in modern shallow to deep marine environments, there has been little documentation on the subsurface biogenic structures produced by these animals in either the modern or the ancient. The extant holothurians Thyone briareus [5, 13-17], Holothuria scabra [4], and Protankyra bidentata [18] are three exceptions. Pearse [5] conducted various experiments with T. briareus including some designed to observe the animals burrowing methods. Thyone briareus was found to burrow in a similar manner to T. gemmata, wedging its way down into the sediment through muscular contractions in a rough U-configuration with the two ends of the body turned upward. Despite the description of the burrowing methods, however, Pearse [5] did not describe the structures produced by T. briareus in the sediment. Howard [13] conducted experiments with several marine animals including Thyone sp for the purpose of investigating the potential application of X-radiography to the study of bioturbation. The experiments were designed to test the reaction of the organisms to changes in environment; for the holothurnians sediment layers were gradually added to determine the types of escape patterns produced by Thyone. The X-radiographs revealed that cup-in-cup structures were produced as the holothurians gradually moved upward in response to sediment aggradation [13]. Field observations of Thyone have shown that they produce irregular U-shaped burrows similar to those of T. gemmata in intertidal settings although these have not been described in detail [16, 17]. Laboratory experiments with H. scabra indicated that this species burrowed in response to daylight and decreasing salinity, however, neither the burrowing method nor the burrows were described in this study [4]. Field observations on tidal flats have shown that P. bidentata produces L- to J- shaped burrows with a single surface opening and some broadly U-shaped burrows with a single opening [18]. The burrows are typically 5–10 cm deep and occur in muddy sediment. These holothurians are deposit feeders and appear to obtain most of their sediment for ingestion in the course of burrow excavation [18]. Further feeding takes place at the sediment surface near the burrow opening [18]. This difference in behavior accounts for the difference in burrow morphology between P. bidentata and T. gemmata.

The ichnospecies Artichnus pholeoides from the Middle to Late Eocene Hieroglyphic Beds of the Polish Carpathians was interpreted as the dwelling burrow of holothurians [19]. This ichnofossil is described as a wide, J-shaped passively filled burrow surrounded by thickly laminated, mostly retrusive spreite best developed in the lower part of the structure [19]. The structure is interpreted as having one opening for the tentacle crown to pass through during feeding. With the absence of a second opening, fecal material would be deposited around and beneath the holothurian producing a laminated halo, or spreite, around the burrow [19]. The single opening would suggest a different style of burrowing than T. gemmata. The holothurian Synapta has been found to burrow ‘head first’ using its tentacles to move sediment particles and intrude its body into the substrate [20]. This process of burrow construction would likely produce an elongate, cylindrical structure similar to A. pholeoides. The burrows produced by P. bidentata are also morphologically similar to A. pholeoides although they do not possess a wide basal chamber [18].

Ichnofossils with morphological characteristics similar to the burrows produced by T. gemmata in the laboratory have been found in fossiliferous wackestone and packstone of the Ames Member of the Glenshaw Formation (Kasimovian). The ichnofossil is a U-shaped structure with a fill of poorly sorted bioclasts and spar (Fig. 8). The diameter of the structure is greatest along the base and tapers upward along the two upturned shafts. The upper margin of the ichnofossil is characterized by a zone of disruption that is distinct from the surrounding sedimentary texture. An animal, likely a holothurian, burrowing using a method similar to T. gemmata could have created the disrupted fill. When the animal reached its required position within the sediment its body occupied open burrow. When the organism evacuated the burrow or possibly died within the burrow, the resulting void was passively filled with coarse fossil debris and later calcite spar crystallized in the remaining pore spaces.

Fig. (8) A U-shaped ichnofossil from the Pennsylvanian Ames Member of the Glenshaw Formation (Kasimovian), southeast Ohio. The ichnofossil resembles the burrow morphology of Thyonella gemmata.

The experiments described in this paper demonstrate that holothurians can produce biogenic structures that can be distinguished from the surrounding primary sedimentary fabric. Since holothurians have a long-ranging, but extremely incomplete fossil record, the identification of their burrows provides an additional means of interpreting their true evolutionary and biogeographic history.

Once an ichnofossil resembling that produced by T. gemmata is recognized in the fossil record, interpretations regarding the depositional environment can also be made. The burrowing activities and resulting morphologies of T. gemmata reflect habitation in a subtropical, shallow, nearshore environment. The specimens did not react to an increase in water temperature to 32º C. This is consistent with dwelling in intertidal environments in equatorial regions where intense heat from the sun can increase water temperatures to over 32º C during low tides [6]. The specimens also did not react to an increase in salinity to 40‰, which can be produced by increased ambient temperature resulting in the excess evaporation of sea water in intertidal environments [6]. There was an effect on the specimen placed in water with a salinity of 20‰ indicating that in the natural environment, T. gemmata would not inhabit an area with freshwater input. Altering substrate consistency had the greatest effect on burrowing activities and the burrow morphology. The test specimens were able to easily burrow in fine- and medium-grained carbonate sand, but had difficulty burrowing in coarse-grained sand. This suggests that the burrowing method of T. gemmata restricts them to finer grained sediments. In marine settings finer-grained sediments are typically deposited in the nearshore, low energy environments, such as in bays or on tidal flats [18, 21, 22].

Given the know behaviors and environmental preferences of extant holothurians, the recognition of a burrow that resembles that of T. gemmata in the fossil record allows for the interpretation of: 1) the presence of a holothurian or holothurian-like organism; 2) the presence of suspended nutrients in the water column to support at least limited suspension-feeding; 3) a salinity level that does not decrease greatly from normal, but may increase from normal; 4) a low energy, shallow water paleoenvironment where the substrate is composed mainly of fine- to medium-grained sediment; and 5) the sediment-water interface at or near the top of the burrow. Additionally, the preservation of a delicate burrow produced near the sediment-water interface indicates that there was not an abundance of other burrowing organisms in the paleoenvironment. Generally, shallow tier burrows are obliterated by deeper tier burrows as sediment accumulates on the sea floor [9]. The presence of a burrow like that of T. gemmata may indicate that the environmental conditions, such as oxygen content or nutrient availability within the sediment, were not adequate to support shallow-tier, deposit- feeding organisms.