Persistent and Widespread Occurrence of Bioactive Quinone Pigments During Post-Paleozoic Crinoid Diversification

Persistent and widespread occurrence of bioactive quinone pigments during post-Paleozoic crinoid diversification

Klaus Wolkenstein1

Department of Geobiology, Geoscience Centre, University of Göttingen, 37077 Göttingen, Germany

Edited by Tomasz K. Baumiller, University of Michigan, Ann Arbor, MI, and accepted by the Editorial Board January 20, 2015 (received for review September 9, 2014) Secondary metabolites often play an important role in the adapta- (14, 15), closely related to the gymnochromes, suggesting that the tion of organisms to their environment. However, little is known fossil pigments may be only slightly changed during diagenesis. about the secondary metabolites of ancient organisms and their Although violet coloration has been occasionally observed in fossil evolutionary history. Chemical analysis of exceptionally well-pre- crinoids (e.g., refs. 16 and 17), until now, only very few chemical served colored fossil crinoids and modern crinoids from the deep sea proofs of quinone pigments have been found for crinoids other than suggests that bioactive polycyclic quinones related to hypericin Liliocrinus (14, 15, 18, 19). Recent measurements suggested the were, and still are, globally widespread in post-Paleozoic crinoids. presence of quinone-like compounds in Paleozoic (Mississippian) The discovery of hypericinoid pigments both in fossil and in present- crinoids (20), but the identity of these compounds has been ques- day representatives of the order Isocrinida indicates that the pig- tioned on methodological grounds (21). ments remained almost unchanged since the Mesozoic, also suggest- To investigate the general occurrence of polycyclic quinone ing that the original color of hypericinoid-containing ancient crinoids pigments in the Crinoidea, the coloration of numerous fossil may have been analogous to that of their modern relatives. The crinoids from collection material was evaluated, and diverse persistent and widespread occurrence, spatially as well as taxo- samples with colorations ranging from distinct violet to almost nomically, of hypericinoid pigments in various orders during the gray were chemically analyzed by HPLC–diode array detection– adaptive radiation of post-Paleozoic crinoids suggests a general mass spectrometry (HPLC-DAD-MS). For comparison, a set of functional importance of the pigments, contributing to the evolu- distinctly colored extant stalked crinoids was analyzed for their tionary success of the Crinoidea. pigment contents.

crinoids | molecular preservation | marine natural products | polyketides | Results and Discussion liquid chromatography–mass spectrometry Evaluation of fossil crinoids revealed that specimens with a distinct violet coloration occur not only at several localities in the rinoids are the most ancient group of extant echinoderms Swiss Jura in the vicinity of the original “Fringeli” locality, but Cwith a fossil record extending to the Ordovician (1). Fol- also at many other localities in Europe and even in East Africa lowing the end-Permian mass extinction, which almost led to the (Table S1). Analysis using HPLC-DAD-MS unambiguously disappearance of the Crinoidea, the post-Paleozoic crinoids, all showed that the spatial distribution of hypericinoid pigments grouped in the subclass Articulata (2), underwent a major evolu- among fossil crinoids is almost worldwide, the stratigraphic tionary radiation and diversification that led to the development distribution ranges at least back to the Middle Triassic, and the of free-living crinoids and to the offshore displacement of stalked taxonomic distribution comprises representatives from at least crinoids (2, 3). Quinone and pyrone pigments are well-known secondary Significance metabolites from present-day free-living comatulids (order Coma- tulida) (4), the dominant group of modern crinoids. By contrast, the Marine organisms produce a wide diversity of natural products constituents of extant stalked crinoids are still almost unknown due with specific bioactivities, representing a valuable source for to their occurrence in the deep sea. Although early attempts to drug discovery. These secondary metabolites are the result of characterize the pigments of stalked crinoids were performed in the adaptation and natural selection during the evolution of the 19th century (5), until now, only those of the isocrinid Proisocri- producing organisms. However, little is known about the evo- nus ruberrimus and of the cyrtocrinids Neogymnocrinus richeri lutionary persistence and distribution of these secondary and Holopus rangii have been elucidated. A group of bromi- metabolites through deep time, because original secondary nated anthraquinones (proisocrinins) was isolated from the metabolites of ancient organisms usually are not preserved. former (6), whereas a group of brominated phenanthroper- Here the unique example of a group of bioactive quinone pig- ylene quinones (gymnochromes) was found in the latter (7, 8). ments is shown to have persisted almost unchanged and with From the fossil record only a group of polycyclic quinone a wide distribution from the Early Mesozoic (about 240 MyBP) pigments (fringelites) is known, which was discovered in violet- until today within the echinoderm class Crinoidea, whereas the colored specimens of the Jurassic stalked crinoid Liliocrinus class underwent a major adaptive radiation and diversification (extinct order Millericrinida) from northern Switzerland (9). after the end-Permian mass extinction. These fossil pigments have been described as a series of phenanthroperylene quinones differing in the number of their hydroxy Author contributions: K.W. designed research, performed research, analyzed data, and groups (10–12) and were originally interpreted as diagenetic con- wrote the paper. densation products of primary naphthoquinones (9, 10). Based on The author declares no conflict of interest. comparison with present-day comatulid pigments, the fossil pig- This article is a PNAS Direct Submission. T.K.B. is a guest editor invited by the Editorial ments were later thought to be geochemical transformation prod- Board. ucts of primary anthraquinones or bianthrones (4, 13). However, 1Email: [email protected]. using more advanced spectroscopic techniques, the pigments have This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. been characterized as a homologous series of hypericinoid pigments 1073/pnas.1417262112/-/DCSupplemental.

2794–2799 | PNAS | March 3, 2015 | vol. 112 | no. 9 www.pnas.org/cgi/doi/10.1073/pnas.1417262112 Downloaded by guest on October 1, 2021 EVOLUTION

Fig. 1. Examples of fossil post-Paleozoic crinoids with distinct purple to violet coloration due to preservation of polycyclic quinone pigments (hypericinoids). (A) CHEMISTRY Encrinid Encrinus aculeatus (GPIT/CR/373), Middle Triassic, Anisian, Górazd_ ze,_ Poland. (B) Isocrinid Pentacrinites dargniesi (PIMUZ 24616), Middle Jurassic, Bajocian, Develier, Switzerland. (C) Comatulid Solanocrinites beltremieuxi (SMNS 67702), Upper Jurassic, Kimmeridgian, Île de Ré, France. (D) Millericrinid Apiocrinites roissyanus (longitudinal section of cup, MJSN QLP005-884), Upper Jurassic, Oxfordian, Boncourt, Switzerland. (Scale bar, 1 cm.)

four (Encrinida, Isocrinida, Comatulida, and Millericrinida) of foresti (Fig. 2B and Table S2). Considering the rarity of fossil crinoids the eight post-Paleozoic crinoid orders (Figs. 1, 2A, and 3 and with preservation of organic pigments and the poor fossil record of Table 1). intact Cenozoic crinoids (2, 3), the analytical data of fossil and Chemical proofs of fossil hypericinoid pigments were obtained extant crinoids strongly suggest that hypericinoids were common from a large variety of representatives of the order Millericrinida crinoidal pigments throughout the Mesozoic and Cenozoic, pre- from the Jurassic of Europe, the Middle East, and East Africa sumably with a monophyletic origin within the ancestral Articulata (Fig. 2A and Table 1). Moreover, the example of the diverse and (Fig. 3). exceptionally well-preserved Upper Jurassic millericrinid fauna Fossil as well as extant crinoid pigments show the typical UV- of the La Rochelle area in France shows that, in addition to the visible spectra of phenanthroperylene quinones (Fig. S2) (24), hypericinoid-containing Liliocrinus polydactylus (Table 1), almost differing mainly in the position of the long wavelength maxima. all species from this assemblage exhibit a distinct violet colora- Among the extant crinoids, several series of brominated hyper- tion (Fig. S1). The combined data suggest that coloration by icinoid pigments and their isomers were identified by comparison hypericinoid pigments was ubiquitous in Jurassic millericrinids with authentic standards of gymnochrome B and A from the and, regarding the stratigraphic occurrence of the order (Middle cyrtocrinid Neogymnocrinus (Fig. 2B). Accurate mass data and Triassic to Lower Cretaceous) (Fig. 3), likely beyond. Fossil isotopic patterns indicate that the individual pigments differ in hypericinoids were also detected in representatives of the Tri- the number of their bromine atoms, the length of the side chains assic order Encrinida, including the oldest polycyclic quinones (with characteristic C2H4 intervals due to the biosynthesis of the known (Fig. 2A and Table 1). However, most interesting are polyketide compounds via the acetate–malonate pathway), and chemical proofs of these pigments in Jurassic representatives of the presence or lack of sulfate moieties (Fig. S3 and Table S4). two orders that still exist today, Isocrinida and Comatulida (Figs. Apart from obvious differences in relative amounts, the crinoids 2A and 3 and Table 1), allowing direct comparison of ancient show only minor variations in the composition of pigments (Fig. and modern crinoid pigments within the same clade. 2B). Based on these results and the observations made by Indeed, chemical analysis of ethanolic extracts shows that hyper- Moseley in 1877 (5), it is very likely that the “purple pentacrinin” icinoid pigments are still widespread in extant isocrinids (Fig. 2B and pigments extracted from different stalked crinoids were also Table S2)withHypalocrinus belonging to the same subfamily (Iso- brominated hypericinoids. crininae) as the fossil Hispidocrinus (Table1).Inadditiontothe Among the fossil crinoids, several series of nonbrominated known occurrences in extant cyrtocrinids (7, 8) and comatulids (4), hypericinoid pigments and their isomers were identified by com- hypericinoid pigments were found in the cyrtocrinid Cyathidium parison with the standard compounds fringelite F and hypericin

Wolkenstein PNAS | March 3, 2015 | vol. 112 | no. 9 | 2795 Downloaded by guest on October 1, 2021 A B

Fig. 2. HPLC chromatograms of crinoid pigment analysis. (A) Extracts of fossil crinoids in comparison with reference compounds. From Top to Bottom,the chromatograms correspond to fringelite F and hypericin; millericrinid, Upper Jurassic, Tanzania; comatulid Solanocrinites beltremieuxi, Upper Jurassic, France; isocrinid Pentacrinites dargniesi, Middle Jurassic, United Kingdom; and encrinid Encrinus cf. brahli, Middle Triassic, Germany. (B) Extracts of extant crinoids in comparison with reference compounds. From Top to Bottom, the chromatograms correspond to gymnochrome B and gymnochrome A isolated from Neo- gymnocrinus richeri, New Caledonia (note that according to ref. 7 the relative positions of the two side chains in gymnochrome B remain to be established); cyrtocrinid Cyathidium foresti, Azores; isocrinid Saracrinus nobilis, western Pacific; isocrinid Endoxocrinus (Diplocrinus) wyvillethomsoni, Bay of Biscay; and isocrinid Hypalocrinus naresianus, western Pacific. Peaks were identified by comparison of the retention times, UV-visible spectra, accurate mass data, and isotopic patterns with those of the standard compounds (Figs. S2 and S3). Molecular formulas of indicated compounds are listed in Tables S3 and S4.

(Fig. 2A, Fig. S3,andTable S3). Almost the same pigments and in isocrinids, with a supposed last common ancestor in the were found in all samples independent of occurrence, stra- Early Mesozoic (Fig. 3). However, although the fossil hypericinoids tigraphy, or taxon of the crinoids (Table 1). Despite minor represent almost unchanged colorful secondary metabolites, it diagenetic changes, as indicated by the presence of homologs should be noted that the purple to violet color currently visible in [due to a stepwise demethylation of the side chains during the Mesozoic fossils does not necessarily represent the original diagenesis (15)] and the predominance of the demethylated color of the crinoids. Because deprotonation and protonation lead fringelite F (Table S1), the pigments preserved in the fossil to dramatic changes in the absorption behavior of hypericinoid crinoids show astonishing similarities to those of their extant pigments (24) (Fig. S4), and hypericinoid-containing living crinoids relatives. In particular, the common occurrence of hypericinoids (e.g., Cyathidium, Saracrinus,andHypalocrinus) commonly exhibit in fossil and living representatives of the order Isocrinida sug- a green color (Table S2), hypericinoid-containing ancient crinoids gests that the fossil pigments are almost unchanged natural may have exhibited an analogous greenish color. products and not diagenetic condensation products as pre- Structural conservatism in natural products over geological viously supposed. time indicates that the compounds had functions that were im- Moreover, because halogenated organic compounds generally portant for the organisms that produced them. In the case of the are not documented in the fossil record (25) (probably as a result crinoid pigments, these compounds were widespread over a pe- of the bromine–carbon bonds being less stable than the car- riod of about 240 million years while the crinoids adapted to bon–carbon bonds), there is no reasonable explanation why diverse habitats after the end-Permian mass extinction (26). hypericinoid pigments of extant isocrinids are brominated but those Despite significant morphological changes among crinoids dur- of Mesozoic isocrinids were not. Therefore, it is likely that the fossil ing this diversification, their pigments remained almost un- pigments originally were also brominated and lost the bromine changed. Hypericinoid pigments occurred in benthic encrinids, during diagenesis. This is also supported by the corresponding millericrinids, and isocrinids as well as in free-living comatulids. presence of brominated hypericinoids both in extant cyrtocrinids Moreover, the pigments can be found both in shallow-water

2796 | www.pnas.org/cgi/doi/10.1073/pnas.1417262112 Wolkenstein Downloaded by guest on October 1, 2021 adinircotryC

a from fouling organisms, and antifouling properties have been

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lu di reported for ethanolic extracts of echinoderms (32).

nirco amoC It is striking that fossil crinoids with preserved hypericinoid

s pigments generally are found associated with hardgrounds, oolitic

Quaternary dinircaev limestones, or reefal carbonates (Table1),whichweredepositedin Ma ? Neogene 23 well-oxygenated environments where exceptional preservation of organic substances usually would be least expected. In the case of

Paleogene o the Tendaguru locality, which is mainly known for the discovery 66 R of dinosaur remains (33), the pigmentsarepreservedeveninfossils from a sandstone-dominated coastal lithofacies. The embedding

a din sediments are predominantly light colored and show low contents

Cretaceous cirelliM of organic matter. By contrast, no colored crinoids are known from bituminous sediments such as the Posidonia Black Shale, which is 145 famous for the occurrence of well-preserved isocrinids and fossils with preserved organic tissue material (34). Therefore, it may be

a supposed that a shallow-water carbonate depositional environment

dini

Jurassic d i is crucial for the preservation of hypericinoid compounds. Because

nirc

col 201 the pigments occur within the carbonate cement that fills the for-

o ? n

H E mer pores of the crinoid endoskeleton (15), and early precipitation of carbonate cements is typical for the high-energy depositional Triassic environments where the crinoids were buried (35), it is likely that 252 early marine cementation is a key factor for the preservation of Permian hypericinoid pigments in fossil crinoids. Furthermore, the carbonate depositional environment may be critical for the Fig. 3. Diversification of the post-Paleozoic crinoids (subclass Articulata) preservation of the pigments, because of the strong acidity of with occurrences of hypericinoid pigments (solid circles). Phylogenetic rela- the pigment bay region hydroxy groups that favors the for- + tionships are based on morphological data from ref. 2. Recent investigations mation of highly insoluble salts with bivalent ions such as Ca2 based on molecular data support the hypothesis that all living crinoids ra- (36). It is also striking that the pigments generally are pre- EVOLUTION diated from a small clade that passed through the end-Permian mass ex- served in articulated crinoid remains, suggesting rapid burial tinction, but suggest an earlier divergence of the order Comatulida from other clades (22, 23). In present-day comatulids the most abundant pigments of the animals, and in more massive structures such as the roots are anthraquinones, pyrones, and bianthrones, whereas hypericinoid pig- of millericrinids. The observed occurrence of hypericinoid pigments have been detected only as minor pigments (4). ments in fossil crinoids thus very likely is the result of differences among depositional environments of crinoid habitats, preservation potential, and diagenesis and does not reflect the original

Mesozoic crinoids and in present-day stalked crinoids from the occurrence of the pigments, which was certainly much more CHEMISTRY deep sea, suggesting a general functional importance of the widespread. pigments. In contrast to the Mesozoic crinoids, no traces of hypericinoid Although a potential role of the pigments in visual predator– pigments could be detected in Paleozoic crinoids (Table S1). prey interactions cannot be excluded, this would be mainly rel- This also applies for specimens with a slight violet hue such as evant for shallow-water crinoids. Considering the greenish color Strimplecrinus from the exceptionally well-preserved Mississippian of present-day hypericinoid-containing crinoids, a role in cam- crinoid fauna of LeGrand (Iowa), which is known for species- ouflage may be suggested rather than a role in aposematism. specific coloration (37, 38). One explanation for the apparent lack However, secondary metabolites such as quinones may also play of polycyclic quinones in Paleozoic crinoids would be that those a role in chemical defense. Hypericin and gymnochromes are pigments have not survived diagenesis. However, although the well known for their biological activity (8, 24), and antifeedant occurrence of hypericinoids and related pigments in Paleozoic activities against fish have been reported for structurally related crinoids cannot be excluded, it is also possible that these pigments anthraquinone pigments from comatulids (27, 28). Previous are an invention of the Articulata, the only crinoid group that observations on two extant isocrinids, Endoxocrinus parrae and survived the end-Permian mass extinction. Neocrinus decorus, suggested that stalked crinoids lack chemical Materials and Methods defense against fish predation, supporting the hypothesis that Samples and Reference Compounds. Fossil crinoid specimens showing a col- restriction of stalked crinoids to deep-water habitats may have oration similar to the characteristic purple to violet of the Liliocrinus speci- resulted from the Mesozoic radiation of durophagous fishes in mens from the original Fringeli locality near Bärschwil in Switzerland (9, 15) shallow seas (29). Chemical analyses also indicated that these and additional specimens with a more gray to black coloration were selected crinoid species are virtually devoid of quinone pigments and from collection material (Table S1). Ethanolic solutions containing dissolved aromatic polyketides (30). However, the present study shows that pigments of colored extant crinoids were obtained either by sampling of the many other species of stalked crinoids do contain quinone pig- ethanolic solutions used for preservation of the specimens or by extraction ments, indicating that potentially deterrent quinones are com- of dried specimens (C. foresti, Metacrinus levii, and Porphyrocrinus cf. ver- rucosus) with ethanol (Table S2). For reference, authentic samples of gym- mon both in comatulids and in stalked crinoids. Therefore, nochrome B (containing small amounts of gymnochrome A) from N. richeri currently there is no evidence for fundamental differences in the (7) and hypericin from St. John’s wort (Hypericum perforatum) as well as chemical defense of comatulids and stalked crinoids that might a synthetic sample of fringelite F prepared according to ref. 39 were used. explain the offshore displacement of stalked crinoids. In contrast Specimens cited in this study are housed in the following institutions: to a possible deterrent function of the pigments against fishes, Bayerische Staatssammlung für Paläontologie & Geologie (BSPG), Field Mu- such a function against benthic predation by echinoids is not seum of Natural History (FMNH), Institut für Geowissenschaften der Uni- versität Tübingen (GPIT), Geowissenschaftliches Zentrum der Universität supported, because indications of predation by echinoids have Göttingen (GZG), National Natural History Collections at The Hebrew Uni- been observed for (likely hypericinoid-containing) crinoids of the versity of Jerusalem (HUJ), Ludwig Maximilians Universität München (LMU), order Encrinida (31). A further defensive function of the pig- Museum für Naturkunde Berlin (MB), Musée Jurassien des Sciences Naturelles ments might be antifouling, because most echinoderms are free Porrentruy (MJSN), Muséum National d’Histoire Naturelle Paris (MNHN),

Wolkenstein PNAS | March 3, 2015 | vol. 112 | no. 9 | 2797 Downloaded by guest on October 1, 2021 Table 1. Occurrences of hypericinoid pigments in fossil crinoids Species Stratigraphy Locality Sediment Hypericinoids* (ref.)

Millericrinida Millericrinid U. Jurassic, Ti Solnhofen, DE Hardground 8, 10 Millericrinid U. Jurassic, Ki/Ti Tendaguru, TZ Sandstone 1–10 Millericrinus lusitanicus U. Jurassic, Ki Vestiaria, PT Marl 5, 8–10 Liliocrinus polydactylus U. Jurassic, Ki Angoulins, FR Reef flank 5, 7–10 † ?Pomatocrinus mespiliformis U. Jurassic, Ki Małogoszcz, PL Oolite 5, 7 , 8 (18) Apiocrinites roissyanus U. Jurassic, Ki Tonnerre, FR Chalky limestone 1, 3–10 Millericrinid U. Jurassic, Ox/Ki Hannover, DE Bioclastic limestone 1, 3–10 Liliocrinus munsterianus U. Jurassic, Ox L’Isle-sur-le-Doubs, FR Oolite 1, 3–10 † L. munsterianus U. Jurassic, Ox Bärschwil, CH Marl 5, 7 , 8 (15) A. roissyanus U. Jurassic, Ox Boncourt, CH Reef flank 1, 3–10 Angulocrinus echinatus U. Jurassic, Ox Malton, GB Oolitic limestone 1, 3–10 Millericrinid U. Jurassic, Ox Villers-sur-Mer, FR Limestone 3, 5, 6, 8, 10 Apiocrinites negevensis M. Jurassic, Ca Hamakhtesh Hagadol, IL Marl 5, 8–10 Apiocrinites parkinsoni M. Jurassic, Bt Bradford-on-Avon, GB Hardground 1, 3–10 Ailsacrinus abbreviatus M. Jurassic, Bt Eastington, GB Sandy bio-oosparite 5, 7–10 Comatulida Solanocrinites sp. U. Jurassic, Ki/Ti Tendaguru, TZ Sandstone 5, 7–10 Solanocrinites beltremieuxi U. Jurassic, Ki Île de Ré, FR Limestone 5, 8–10 Isocrinida Pentacrinites dargniesi M. Jurassic, Bt Malmesbury, GB Not documented 5, 7–10 Hispidocrinus leuthardti M. Jurassic, Bt Liestal, CH Marl 5, 7–10 P. dargniesi M. Jurassic, Bj Develier, CH Oolite 5, 7–9 Encrinida Chelocrinus schlotheimi M. Triassic, An Willebadessen, DE Limestone 5, 8–10 Carnallicrinus carnalli M. Triassic, An Freyburg/Unstrut, DE Oolite 5, 7†, 8 (15) Encrinus aculeatus M. Triassic, An Raciborowice, PL Limestone 5, 7†, 8 (19) † Encrinus cf. aculeatus M. Triassic, An Górazd_ ze,_ PL Limestone 5, 7 , 8 (14) Encrinus cf. brahli M. Triassic, An Weißenborn, DE Hardground 5, 7

A more comprehensive dataset is given in Table S1. Geological stages indicated: An, Anisian; Bj, Bajocian; Bt, Bathonian; Ca, Callovian; Ki, Kimmeridgian; Ox, Oxfordian; Ti, Tithonian. M., Middle; U., Upper. Countries indicated: CH, Switzerland; DE, Germany; FR, France; GB, United Kingdom; IL, Israel; PL, Poland; PT, Portugal; TZ, Tanzania. *Molecular formulas of indicated compounds are listed in Table S3. † Indicated compound and/or isomer.

Natural History Museum London (NHMUK), Naturhistorisches Museum Wien Schleicher & Schuell). Mass spectra were acquired in the negative-ion mode – (NHMW), Naturhistorisches Museum Basel (NMB), Nagoya University Museum (nebulizer gas pressure 60 psi, drying gas flow 12 L·min 1, drying gas tem- (NUM), Paläontologisches Institut und Museum der Universität Zürich perature 350 °C, capillary voltage 4.0 kV) over the 100- to 1,300-m/z range. (PIMUZ), Staatliches Museum für Naturkunde Stuttgart (SMNS), University Mass calibration and continuous reference correction were obtained using

Museum of the University of Tokyo (UMUT), and Systematische Zoologie am purine and the HP-0921 acetate adduct (C20H21O8N3P3F24) introduced via an Museum für Naturkunde Berlin (ZMB). internal reference mass kit. Extracts from two additional fossil samples (Apiocrinites negevensis and millericrinid from Solnhofen) were analyzed Sample Preparation and Extraction of Fossil Crinoids. Samples of fossil crinoids only by HPLC-DAD, using an Agilent 1200 Series HPLC system, by applying were cleaned with acetone. After dissolution of the carbonate with 10 M the same chromatographic conditions as described above. HCl, the residues were separated by centrifugation, washed thoroughly with distilled water, and dried overnight at room temperature under Absorption Spectroscopy of Extant Crinoid Pigments. UV-visible spectra were vacuum (about 10 Torr). Residues were then sequentially extracted by recorded on a Jasco V-630 spectrophotometer, using 1-cm quartz cuvettes. The sonication (10 min at 40 °C) and centrifugation in toluene (3×) and di- same amounts of crude Saracrinus pigments were dissolved in ethanol and methyl sulfoxide (DMSO) (1×). The pigment-containing DMSO extracts ethanol containing 1% 1 M HCl. Solutions were filtered before measurements, were cleaned up by solid-phase extraction. The sorbent (Bondesil C18, using 0.2-μm polytetrafluoroethylene filters (ReZist; Schleicher & Schuell). 40 μm) was conditioned by washing with acetonitrile. The extracts then were loaded onto the column, and the pigments were eluted ACKNOWLEDGMENTS. I thank J. Ayer (Musée Jurassien des Sciences Nature- with acetonitrile. lles Porrentruy), U. Bielert, S. Charbonnier [Muséum National d’Histoire Naturelle Paris (MNHN)], T. Ewin (Natural History Museum London), HPLC-DAD-MS Analysis. Aliquots of extracts obtained from fossil crinoids and L. Kaecke, A. Kroh (Naturhistorisches Museum Wien), S. Lidgard (Field Mu- seum of Natural History), J. Mainguy, C. Neumann (Museum für Naturkunde extracts of extant crinoids were analyzed using the same method. HPLC- Berlin), S. Schneider (Bayerische Staatssammlung für Paläontologie & DAD-MS measurements were carried out on an Agilent 1100 Series HPLC Geologie), G. Schweigert (Staatliches Museum für Naturkunde Stutt- system with a diode array detector coupled to an Agilent 6520 Q-TOF LC/MS gart), S. Seppelt, O. Schmidt [Naturhistorisches Museum Basel (NMB)], mass spectrometer equipped with an electrospray ionization (ESI) source. A. Weissmüller, and M. A. Wilson (College of Wooster) for fossil crinoid Separation was performed at 50 °C on a Phenomenex Gemini C18 column material; N. Améziane and M. Eléaume (MNHN), T. Oji (Nagoya University (250 × 4.6 mm i.d., 5 μm). The HPLC program consisted of a linear gradient Museum), M. Wisshak (Senckenberg am Meer), and G. Wörheide [Ludwig of acetonitrile/20 mM aqueous ammonium acetate [60:40 (vol/vol)] to 100% Maximilians Universität München (LMU)] for recent crinoid material; M. Iorizzi (University of Molise) and W. Steglich (LMU) for references acetonitrile in 20 min, followed by isocratic elution at 100% acetonitrile · –1 of gymnochrome B and fringelite F, respectively; H. Furrer (Paläontolo- at a flow rate of 1 mL min . The diode array detector wavelengths were gisches Institut und Museum der Universität Zürich) for providing the 570 nm, 590 nm, and 600 nm and UV-visible spectra of each peak were image for Fig. 1B; W. Buchberger (Johannes Kepler Universität Linz) for recorded in the 200- to 800-nm wavelength range. Extracts were filtered access to laboratory facilities; J. H. Gross (Universität Heidelberg) for ini- before injection, using 0.2 μm polytetrafluoroethylene filters (ReZist; tial ESI-MS measurements of the pigments of Hypalocrinus;T.Ojiand

2798 | www.pnas.org/cgi/doi/10.1073/pnas.1417262112 Wolkenstein Downloaded by guest on October 1, 2021 M. Roux for information on the natural color of extant crinoid species; and discussions. This work was supported by the Deutsche Forschungsgemein- H. Hess (NMB) and H. Falk (Johannes Kepler Universität Linz) for helpful schaft (WO 1491/1-1 and WO 1491/4-1).

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