Digestive structures in Ordovician trilobites Colpocoryphe and Flexicalymene from the Barrandian area of Czech Republic Old ř

Two recently discovered specimens of the abundant calymenoid trilobite Colpocoryphe Novák in Perner, 1918 from the Middle Ordovician Šárka Formation and one specimen of Flexicalymene (Flexicalymene) pragensis Vaněk & Vokáč, 1997 from the Upper Ordovician Bohdalec Formation, all from the Prague Basin, display remains of the digestive system. In Colpocoryphe, an internal mould of an articulated exoskeleton contains a post-stomach part of the alimentary canal preserved through the narrow axial region of the occipital ring, all thoracic segments as well as in the axial part of the pygidial shield. The anterior part of the digestive system is poorly known as the specimen shows the hypostome preserved in situ and the space between the glabella and the hypostome is represented by an empty cavity associated with probable remains of gut diverticulae on both sides of the cephalon. The second, incomplete specimen consists of five posterior thoracic segments articulated with the pygidium. The axial region of this specimen is preserved with a clearly discernible segmented intestine which terminates at the axial tip and then bends ventrally. In an enrolled specimen of Flexicalymene pragensis, supposed remains of the alimentary tract are comparatively poorly preserved but discernible in the middle and posterior parts of the thoracic axis and in the anterior part of the pygidial axis. Digestive structures within the family Calymenidae have not been described previously. Earlier discoveries of the digestive system in Ordovician trilobites of the Barrandian area are briefly reviewed.


INTRODUCTION
Skeletal remains of invertebrates, including trilobite exoskeletons, are abundant in the Barrandian area (e.g.Barrande 1852Barrande , 1872;;Chlupáč et al. 1998;Bruthansová et al. 2007 and references therein).More than 900 trilobite species have been described from Cambrian to Devonian rocks of this area (Valíček & Vaněk 2001;Vaněk & Valíček 2002) but remains of their soft parts are extremely rare (e.g.Šnajdr 1990).Exceptionally preserved trilobites with remains of the digestive system were discovered in the Cambrian Buchava and Jince formations of the Skryje-Týřovice and Příbram-Jince basins as well as from the Ordovician Mílina and Letná formations of the Prague Basin (Fatka et al. 2014;Fig. 1C herein).
Here, we describe the remains of an undoubted digestive system in three specimens of calymenoid trilobites; Colpocoryphe bohemica (Vaněk, 1965), C. cf.bohemica and Flexicalymene (F.) pragensis Vaněk & Vokáč, 1997.Both specimens of the genus Colpocoryphe were recently collected at two localities situated in the lower levels of the Middle Ordovician Šárka Formation (Darriwilian = Oretanian Regional Stage; see Fig. 1C) in the western part of the Prague Basin (Fig. 1B); the specimen of Flexicalymene (F.) pragensis was collected by the junior author at the Brumlovka locality in shales of the Upper Ordovician Bohdalec Formation (Katian = Late Berounian Regional Stage; see Fig. 1C) in the eastern part of the Prague Basin (Fig. 1B).Two specimens are deposited in the palaeontological collections of the Czech Geological Survey Prague (ČGS MD 001, 007).The third specimen is stored in the West-Bohemian Museum in Plzeň (specimen number WBM S 06 160).
With the exception of several probably heterochronous, stratigraphically and geographically restricted horizons, the dark shales of the Šárka Formation are usually quite sparsely fossiliferous.Our knowledge of the contained fauna comes mainly from loose siliceous nodules.These nodules have long attracted the attention of private collectors by an abundant occurrence of wellto excellently preserved fossils.Unfortunately, such loose nodules do not provide information about their stratigraphic position and/or about the depositional setting (see Budil et al. 2007;Mergl et al. 2008).
black shale was assigned to the 'pelagic fauna dominated by graptolites' of Havlíček & Vaněk (1990) and Havlíček (1998).Mergl (2002) proposed that the generally poorly fossiliferous shales with phyllocarids (Caryocaris), graptolites, conulariids, nautiloids and trilobites should be classified as the Rafanoglossa Community within his Paterula Community Group.Vavrdová (1982) assigned samples from the Šárka Formation to the MPM (assemblage with dominant Micrhystridium) as well as to the SPH (assemblage with a dominance of small sphaeromorphs) phytoplankton communities.Mikuláš (1991Mikuláš ( , 1998) ) ranged the ichnofossil assemblages, though with some reservations, to a transition from the Cruziana to the Zoophycos ichnofacies.Lefebvre (2007, p. 164) distinguished two biofacies based on stylophoran echinoderms.The western part of the Prague Basin was assigned to the comparatively shallower Mitrocystitid Biofacies representing the Echinoderm Taphofacies D, the eastern part of the basin was classified as an example of the deeper Lagynocystitid Biofacies of the Echinoderm Taphofacies E (Fig. 2).
Mitrocystites mitra and other echinoderm taxa occur in the fields at Mýto and Rokycany, e.g. in the area from which both exceptionally preserved trilobite specimens originate, and the faunal association fits well with the Mitrocystitid Biofacies.However, M. mitra and Lagynocystis pyramidalis, i.e. typical forms of different echinoderm biofacies, are known to occur together in fields north of Rokycany as well as in the eastern part of the basin.Both taxa occur together with typical representatives of the Euorthisina-Placoparia Association.

Trilobite associations of the Šárka Formation
More than 50 species of trilobites occur as dominant elements of these fossil associations (Havlíček & Vaněk 1966;Budil et al. 2007;Mergl et al. 2007).Mergl et al. (2008) prefer the name Placoparia Association instead of the Euorthisina-Placoparia Community of Havlíček & Vaněk (1990); associations with common cyclopygids, Bohemilla and Girvanopyge were interpreted as an indication of the proximity of the Cyclopygid Biofacies of Fortey (1985).Bruthansová (2003) briefly discussed associations of Ordovician illaenid trilobites and preferred the designation 'dalmanitid-illaenid-calymenacean Fig. 2. Sketch showing the distribution of major biofacies associated with the late Darriwilian Šárka Formation.The shallowest part of the basin was inhabited by a sparse orthid brachiopod association.In the offshore direction it was replaced by the Placoparia Association which is characterized by a rich skeletal fauna with trilobites and brachiopods.In the offshore slope settings it grades into a low-diversity atheloptic trilobite association that also includes 'gardens' of benthic dendroids.The water column was inhabited by planktonic graptolites and taxa of the poorly diverse Caryocarid and Cyclopygid biofacies.Poorly oxygenated black shales in the central part of the basin were dominated by the Paterula Association.Modified after Fatka & Mergl (2009, fig. 11d).
assemblage'; she also noted that illaenids do occur together with cyclopygids.Fatka & Mergl (2009) compared the Euorthisina Community of Havlíček with the atheloptic trilobite association of Fortey & Owens (1987) and considered the Middle Ordovician trilobite fauna for an example of the Dalmanitid-Calymenacean Fauna of Cocks & Fortey (1988).
The studied specimens of Colpocoryphe were collected from loose siliceous nodules at two localities in the western part of the Prague Basin.The diverse skeletal fauna found in nodules occurring at both localities belongs to the Placoparia Association of Mergl et al. (2008).The presence of the ichnogenus Arachnostega in the left part of the cephalon of the Colpocoryphe cf.bohemica specimen (Fig. 3) excludes deposition in an anoxic environment.

Trilobite associations of the Bohdalec Formation
More than 20 species of trilobites are abundant in fossil associations in the Bohdalec Formation (Havlíček & Vaněk 1966, 1990;Vaněk & Vokáč 1997).Two associations have been distinguished on the basis of trilobites: the Onnia abducta Community, defined originally by Havlíček & Vaněk (1990), and the interval with Declivolithus alfredi established in the lower part of the formation (see Röhlich 2006).This comparatively deeper environment provided rare pelagic elements such as Cyclopyge, Bohemilla and Sculptaspis, associated with scarce shallow-water elements.
The enrolled specimen of Flexicalymene (F.) pragensis, described here, is preserved as internal and external moulds.The dark shale in which it occurs represents a typical lithology of the lower part of the Polyteichus Facies (Onniella michlensis Community, Bohdalec Formation).The absence of ichnofossils in the sample with this trilobite argues for poorly oxygenated depositional environment.

METHODS
The methods used to analyse all trilobite specimens include standard light microscopy of external surfaces (Microscope NIKON SMZ 1500, Leica S8APO).Photographs were taken using digital cameras NIKON D 300 and Olympus SZX-ILLB200 after coating the samples with ammonium chloride.Drawings were made from photographs using Corel Draw X3 and Adobe Photoshop CS5.The terminology follows that proposed by Whittington & Kelly (1997), including the following abbreviations: sag.(sagittal), tr.(transverse).The chemical analyses was performed using Scan TESCAN Vega, EDS X-MAX 50 (Oxford Instruments).
Discussion.Seven species and subspecies of the genus have been recorded in the Barrandian area (Fig. 1C; Table 1).The two specimens described herein are preserved as internal moulds in hard siliceous nodules, which represents a typical lithology of the Middle Ordovician Šárka Formation (Kukal 1962;Chvátal 2003;Drost et al. 2003).External moulds of both specimens were destroyed during the splitting.
Specimen ČGS MD 001, Colpocoryphe cf.bohemica (Vaněk, 1965) Figure 3 Description.It represents a late meraspid (M-10), preserved as an internal mould of an articulated thorax associated with the cephalon and pygidium.The right pleural part of the thorax and pygidium are partly covered by rock matrix; the original exoskeletal material is dissolved.The left side of the cephalon bears an about 1 mm long cavity, representing a tiny ichnofossil of the genus Arachnostega (see Fatka et al. 2011; Ar in Fig. 3B).
Alimentary canal.A comparatively narrow, threedimensionally preserved tube-like relic is visible inside the thoracic and pygidial axes.This centrally placed and nearly parallel-sided tube extends from the posteriormost part of the glabella, through the occipital ring to the sixth thoracic segment.In the seventh and eighth segments, the alimentary canal bends to the left and seems to be interrupted in the ninth and tenth segments (G in Fig. 3B).Remains of the alimentary canal reappear in the left anterolateral part of the pygidial axis.The alimentary canal is visible on the occipital ring, but its anterior continuation on the glabella is not known, as the axial part of the cephalon shows only the lower ventral surface of the hypostome (Hy in Fig. 3B).The major part of the glabella is broken off and consequently the anteriormost parts of the alimentary canal are preserved only in narrow postero-lateral borders of the glabella, where imprints of two cephalic gut diverticulae are seen on the left side (Dc2 and Dc3 in Fig. 3B); one cephalic gut diverticula is developed in the right side (Dc3 in Fig. 3B).Small cavities developed left and right from the gut in the occipital ring most probably belong to thoracic gut diverticulae (Dt1 in Fig. 3B).
In dorsal view, the transversal width of the alimentary canal ranges from 0.47 to 0.52 mm in the thorax.The canal is narrower in the pygidium, where it measures only about 0.4 mm in tr.width.In dorsal and lateral views, the alimentary canal shows a bead-shaped contour.
Remarks.Because of relatively poor preservation, this specimen is affiliated to Colpocoryphe bohemica only tentatively.Assignment to this species is, however, the most parsimonious because the two other species identified from the Šárka Formation, C. inopinata (Novák in Perner, 1918) and the poorly known C. zmudai Vaněk, 1995, have been found only from the eastern part of the Prague Basin.In addition, these species are very rare, whereas C. bohemica is known from several hundred specimens (see Table 1).
Specimen WBM S 06 160, Colpocoryphe bohemica (Vaněk, 1965) Figure 4 Description.The posterior five thoracic segments are articulated with the pygidium.The preserved part of the exoskeleton is 9.4 mm long (sag.) and 8.8 mm wide (tr.); the pygidium measures 4.8 mm in sag.length and 6.4 mm in tr.width.The axis of the thoracic segments as well as the pygidial axis display a distinct, axially placed, three-dimensionally preserved tube-like relic (Fig. 4), which we interpret as a remnant of the alimentary canal.
Alimentary canal.This structure extends through the thorax and pygidium to the axial tip.In dorsal view, the transversal width of the alimentary canal ranges around 1.4 mm in the thorax, being narrower in the pygidium, where it measures only about 0.85 mm in tr.width.
The alimentary canal shows a bead-shaped contour in dorsal as well as in lateral view (Fig. 4).Fine and short transversal constrictions producing the bead-shaped contour of the alimentary canal seem to be related to the segmentation of the exoskeleton.
Although only incompletely preserved, this specimen shows morphological features indicating that it was partly or fully enrolled when entombed.A comparable preservation of the gut in an enrolled Cambrian trilobite Jiumenia anhuiensis was recently described and discussed by Zhu et al. (2014), who stressed the importance of enrollment for the preservation of trilobite soft parts.
Locality.This specimen was collected by the late V. Kordule (amateur collector of fossils in Příbram).After his sudden death the sample was purchased by the West-Bohemian Museum in Plzeň in 2010.The outcrop  (Vaněk, 1965) from the Šárka Formation (Middle Ordovician, Darriwilian = Oretanian Regional Stage); West-Bohemian Museum in Plzeň, WBM S 06 160; fields near Mýto (locality 1 in Fig. 1B).A, dorsal view.B, lateral view.from which the specimen was collected was not registered.However, the lithological characters of the nodule and the composition of the associated fossils agree with concretions occurring in fields north of Rokycany (P.Kraft, pers. comm. 2013; locality 2 in Fig. 1B).
Remarks.The affiliation of this specimen to Colpocoryphe bohemica is, in comparison with the previous sample, more reliable because the pygidium has 7-8 rings and a distinct postaxial elevation reaching the pygidial margin.
Based on a comparison with complete specimens of Colpocoryphe bohemica, the length (sag.) of the studied specimen is estimated to have been approximately 18 mm.The missing part of the body was likely destroyed during diagenetic processes, because it was situated outside the boundary of the nodule.Such a type of preservation suggests that the nodules were formed during early diagenesis (for a detailed discussion see Dabbard & Loi 2012).
Remarks.Only three Upper Ordovician species of the genus Flexicalymene have been recorded from the Barrandian area (Fig. 1C, Table 2).The studied specimen is assigned to Flexicalymene (F.) pragensis Vaněk & Vokáč, 1997, the only species of this genus known from the Bohdalec Formation.The systematic status of F. (F.) pragensis is somewhat problematic, as the species was established on more or less flattened specimens preserved in fine silty shale.
Specimen ČGS MD 007, Flexicalymene (F.) pragensis Vaněk & Vokáč, 1997 Figure 5 Description.The exoskeleton is enrolled and strongly flattened; the cephalon and pygidium are visible on one surface, whereas the articulated thorax is visible on the opposite side of the mould (Fig. 5).Enrolment combined with flattening preclude direct measurement of some dimensions and allows of only approximate assessment of the original size.The specimen reaches 47 mm in maximum width (tr.).The length of the exoskeleton in a prone attitude is estimated to be about 105 mm (sag.).
Alimentary canal.A comparatively narrow but quite prominent swelling is visible along the inner axial surface of the external mould in the middle and posterior parts of the thorax (G in Fig. 5A).A furrow  of comparable width and length is developed on the surface of the internal mould of the counterpart (G in Fig. 5B).A shallow, poorly cut furrow is seen also in the axial part of the internal mould of the pygidium (?G in Fig. 5C).
We interpret the axially placed and nearly parallelsided structures that have developed as swellings and furrows as a poorly preserved remnant of the alimentary tract.This tube-like structure occurs in the medial part of the thorax, whereas its anterior continuation in the cephalon is not preserved.In dorsal view, the alimentary canal seems to have a bead-shaped contour, as in the two specimens of Colpocoryphe described above (cf.Fig. 4).
Locality.This specimen was recently collected by the junior author (M.D.) at the Brumlovka locality in Prague (locality 3 in Fig. 1B, C).

Decay of soft parts
Experiments on modern Limulus show that decay of the internal soft parts begins only a few hours after death and lasts for no more than one month (Babcock & Chang 1997;Babcock et al. 2000).The preservation of the gut in both specimens of Colpocoryphe and in Flexicalymene means that the processes of mineral precipitation leading to the preservation of delicate remains of internal soft parts must have started very early after the entombment of carcasses.
Dabbard & Loi (2012, pp.100-101) suggested a model, in which the early diagenetic phosphogenesis linked to carbonate fluor-apatite (CFA) precipitation was more intensive in the upper few centimetres (5-10 cm) of sediment under oxic to suboxic conditions.The following silicification invokes a decrease in pH linked to sulphate reduction and pyrite precipitation in the anoxic zone.

Morphology of the alimentary tract
Recently, Lerosey-Aubril et al. (2011) distinguished two major morphological types of the alimentary tract: (1) a simple tube with a crop and (2) a simple tube flanked laterally with metamerically paired caeca (= gut diverticulae).Both morphologies have been found in Ordovician trilobites of the Prague Basin (Fatka et al. 2014).
As in the recently described Cambrian specimen of Jiumenia anhuiensis (see Zhu et al. 2014), a bell-shaped, anteriorly narrow glabella is present in Colpocoryphe.Metamerically paired caeca could be expected in such a glabella.We also agree with Zhu et al. (2014), who noted that the preservation of the gut as a sediment-like infilling is more common (see also Šnajdr 1991) than the phosphatization of metamerically paired digestive caeca.

Preservation by phosphatization
Two of the studied specimens (ČGS MD 001 and ČGS MD 007) were collected from strongly weathered rocks which preclude chemical analyses.The third specimen (WBM S 06 160) is preserved in a dark grey, comparatively lightly weathered siliceous nodule.We have examined this specimen using scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analyses with the intention of testing the suspected preservation of the digestive system by phosphatization (see Lerosey-Aubril et al. 2012;Zhu et al. 2014).
The SEM examination did not show any apparent differences between the enclosing rock matrix and the internal mould of the trilobite specimen.
The EDX analyses were undertaken at four different areas of the rock sample.Two spots were placed inside the supposed gut (guts 1 and 2 in Table 3), and two spots were located outside the trilobite body (matrices 1 and 2 in Table 3).This investigation did not show statistically important differences in the percentages of Table 3. Results of the energy dispersive X-ray (EDX) analyses performed on the gut and the surrounding sediment.The composition is expressed in weight percentage (wt%).Some elements were not detectable and/or the estimated proportions was below the equipment's lower limit of reliability (referred as n) most of the analysed elements.The chemical composition of the material in the supposed gut and the surrounding rock matrix are very similar (Table 3).The only exception is the distinctive enrichment by C and the relative depletion of Al inside the supposed gut.The interpretation of these data is quite difficult as the content of all other elements varies only very slightly.We do not have any explanation for the depletion of Al.It could not be excluded, however, that the enrichment by C reflects the differences in the initial composition of the bottom mud (= surrounding matrix) and the gut filled by partly digested food particles.

CONCLUSIONS
Two specimens belonging to Colpocoryphe show a simple tube in the occipital ring as well as in all thoracic segments and in the pygidium.In a complete specimen of Colpocoryphe, possible relics of two cephalic diverticulae and one thoracic diverticula are visible.
The glabella expands posteriorly and thus a simple gut associated with gut diverticulae is the preferred morphology of the intestine in Colpocoryphe (Fig. 6).The anterior part of the digestive tract is not preserved in Flexicalymene, but the posterior part shows a morphology comparable with that of Colpocoryphe.

Fig. 1 .
Fig. 1.Location of discovery sites, and the Ordovician stratigraphy in the Barrandian area.A, map of the Czech Republic and the Bohemian Massif showing the distribution of Ordovician rocks in the Barrandian area.B, Ordovician of the Prague Basin with the location of three outcrops that yielded the studied specimens: 1, field near Mýto; 2, fields north of Rokycany; 3, Brumlovka locality.C, chart showing the correlation between global series, stages, stage slices, time slices (TS), time units (TU) and the regional chronostratigraphic and lithostratigraphic units recognized in the Ordovician of the Prague Basin, and ranges of species of Colpocoryphe and Flexicalymene (stratigraphy modified fromBergström et al. 2008, Fatka et al. 2013 and Vodička et al. in press).

Fig. 6 .
Fig. 6.Reconstruction of the morphology of the preserved parts of the digestive system in the cephalic, thoracic and pygidial regions of Colpocoryphe in dorsal view.

Table 1 .
Species of the genus Colpocoryphe Novák in Perner, 1918 in the Ordovician of the Prague Basin

Table 2 .
Species of the genus Flexicalymene Shirley, 1936 in the Ordovician of the Prague Basin