Molecular Self-Assembly: Hypothesized for “Hair” of Macroneuropteris Scheuchzeri (Pennsylvanian-Age Seed-Fern)

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International Journal of Coal Geology 121 (2014) 14–18

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International Journal of Coal Geology

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Molecular self-assembly: Hypothesized for “hair” of Macroneuropteris scheuchzeri (Pennsylvanian-age seed-fern)

Erwin L. Zodrow

Palaeobiology Laboratory, Cape Breton University, Postal Box 303, Sydney, B1P 6L2 Nova Scotia, Canada article info abstract

Article history: Hoffmann (1827) erected the Carboniferous pteridophyll species Neuropteris Scheuchzeri without mentioning, Received 5 September 2013 nor illustrating, “hair” in the species' diagnosis. However, one to five millimeter-long hair-like structures on Received in revised form 6 November 2013 the abaxial pinnule of the species, called hair or trichome in the literature, have been routinely used since Accepted 7 November 2013 1847 as one of the main taxonomic character states for determining the identity of this species. Results from Available online 13 November 2013 preparatory and microscopic observations, together with infrared spectrochemistry, have clarified that these structures are not the same as trichomes for the following reasons. The hair-like structures of M. scheuchzeri Keywords: Extracuticular deposit (1) are not organically attached to the abaxial surface; (2) differ spectrochemically from the organic material Ex “hair” of the lamina; (3) are composed, in contrast with the trichomes, of relatively long, unbranched aliphatic FTIR (polymythelinic) hydrocarbon chains [CH2)]n, and (4) are acellular and black, unlike true trichomes of the species Carboniferous that are multicellular. Overall, the sum-total of these experimental results supports the postulate for dynamic M. scheuchzeri molecular self-assembly. For this reason the term “extracuticular deposit” is proposed, reflecting the origin and emergent nature of such hair-like structures in the abaxial pinnule. © 2013 Elsevier B.V. All rights reserved.

1. Introduction This paper focuses on the hypothesis that the long, hair-like structures of M. scheuchzeri are really extracuticular deposits resulting from The current concept of Macroneuropteris scheuchzeri (Cleal et al., the process of dynamic molecular self-assembly (summary: Koch and 1990; Hoffmann, 1827) includes four character states on the abaxial Ensikat, 2008). A review of the taxonomy/systematics of M. scheuchzeri pinnule, i.e., extracuticular deposits (this study), hair (trichome in is accordingly recommended. botanical Greek), ﬁles (unicellular transparent structures), and papillae which are comparatively small and curved. Of these features, the trichomes very densely populate the abaxial lamina (see Barthel, 2. Materials and methods 1961; Cleal and Zodrow, 1989, and others). In contrast to these features of which extracuticular deposits are clearly observable under a loupe or The Carboniferous seed-fern M. scheuchzeri bears polymorphic even by the naked eye as they are up to 5 mm long, Hoffmann did not pinnate foliage that is seen above and below a basal frond dichotomy; mention the presence of any abaxial features in his diagnosis of the individual pinnule lengths range from 3 mm to 120 mm (summaries: species. However, the presence of extracuticular deposits, using the Cleal and Zodrow, 1989; Laveine, 1997; Laveine and Belhis, 2007; name hair, has been taken as an important taxonomic character state Zodrow, 2003, and many others). since 1847 to distinguish the identity of M. scheuchzeri from among sim- The compression specimens for this study were collected by the ilar larger-leaved Pennsylvanian foliage (literature survey: Bunbury, author from the roof shale of the Lloyd Cove Seam, which is known 1847 to Stull et al., 2012). A notable exception is Leo Lesquereux who in the literature for its rich content in plant fossils that are well- assigned American hairy, long-leaved pinnules to neuropteroid taxa, preserved (Fig. 1). For the experimental work, only compressions other than Neuropteris scheuchzeri (summarized by Darrah, 1969). freed from the rock matrix were used from which extracuticular de- However, Gothan (1916), made the pointed observation that the visible posits were collected in a Petri dish. Trichomes were obtained from a cu- hair of N. scheuchzeri did not survive Schulze's chemical-oxidative treat- ticle of a macerated compression (cf. Cleal and Zodrow, 1989). ment (cf. Cleal and Zodrow, 1989). Barthel (1961) described attached, Spectrochemical analyses of extracuticular deposits and trichomes pointed hairs of N. scheuchzeri, which instead I regard as extracuticular were performed using FTIR (Fourier transform infrared spectrometry) deposits. and the KBr-pellet technique. Interpretive details, particularly of IR (infrared) spectra of M. scheuchzeri, supported by carbon 13 nuclear mag- netic resonance studies, are found in Lyons et al. (1995),orD'Angelo et al. E-mail address: [email protected]. (2010, 2013).

Fig. 1. Location map. (A) Canada. (B) Sydney Sub-Basin as integral geological structure of the Maritimes Basin. (C) Coal lithostratigraphy and sample seam (S). CANT lower Cantabrian strata.

3. Results the midvein (Fig. 2B). The distribution pattern, as correctly drawn by Bunbury in 1847, follows a trend, i.e., it is biogenetically non-random. 3.1. Physical aspects of extracuticular deposits Extracuticular deposits are straight in shape, doubly-pointed, black, solid, and fracture easily (Fig. 2C to E). Microscopic examination of the The confusion in the literature between hair [trichomes] and abaxial surfaces of compressions at ×250 magnification, and critical ob- extracuticular deposits stems mainly from lack of observing the abaxial servation and photography of the compressions during maceration, surface of M. scheuchzeri compressions after being freed from show no evidence for organic attachment of the extracuticular deposits. the entombing rock matrix, though previous exceptions are noted Most importantly, they are acellular (Fig. 3). (Barthel, 1961; Cleal and Zodrow, 1989; Gothan, 1916). To untangle Fig. 4A, B shows IR spectra of individual extracuticular deposit and the confusion necessitates (1) examining freshly unearthed compres- trichome, respectively. In particular, the former is relatively aliphatic- sions immediately after collecting, (2) examining the HF solution used rich, as indicated by the larger CH2/CH3 ratio of 3.0, which at the same for freeing the compressions from the rock matrix, (3) real-time study- time implies comparatively longer and straight hydrocarbon chains ing of the compressions during Schulze's (1855) maceration process, with relatively few side branchings. This ratio is computed after and (4) examining the ammonium hydroxide solution that produces deconvolution in the 3000–2800 cm−1 aliphatic stretching region (see the cuticle. Summarizing my experimental results, extracuticular Zodrow and Mastalerz, 2001, Figs. 6 or 7). The Al/Ox ratio of aliphatics deposits (1) tend to drop-off in storage because of dehydration of the to oxygen-containing compounds [(3000–2800/1800–1500) cm−1 exposed compression (not of the extracuticular deposits), (2) were band] is comparatively very small at 0.32, which suggests a significant found loose in the plastic dishes, (3) had slowly solubilized on the com- joint contribution of oxygen-containing groups and aromatic carbon. pression in ca 3–5 h, and (4) intact trichomes were found in Petri The peak at ~3400 cm−1 is due to hydroxyl absorbance, at 1727 cm−1 dishes, correlating with structural holes found subsequently in the (C_O) ester, at 1634 cm−1(C_O) ketones, at 1385 cm−1 (symmetric corresponding abaxial cuticles. COO\), and at 1029 cm−1 (C\O\C) ether, or Si\O stretch in silicates Microscopic observations include that trichomes (ca. 300 μmlong) (see Chen et al., 2012). The inescapable conclusion is that extracuticular are not ordinarily visible on compressions, freed or still attached to deposits are the last physiological event in the development of the the rock matrix, but molds of extracuticular deposits are marked M. scheuchzeri pinnule, representing an excreted biochemical deposit. (Fig. 2A), and extracuticular deposits flatly overlie the abaxial venation In contrast, the trichomatous IR spectrum is relatively poor in terms at an acute angle in a more or less consistent parallel arrangement with of functional groups, confirmed by a second spectrum. In particular, 16 E.L. Zodrow / International Journal of Coal Geology 121 (2014) 14–18

Fig. 2. Macroneuropteris scheuchzeri. (A) Impressions (molds) of extracuticular deposits. (B) Extracuticular deposits overlying lateral veins of the abaxial pinnule. (A) and (B) represent the same compression. (C) Extracuticular deposit, round, opaque, solid. (D) Diagenetically altered extracuticular deposits in situ showing pointed terminals. No chemical treatment was ap- plied. (E) Extracuticular deposit, detail of a pointed terminus. (C) and (E) Nomarski phase-contrast micrsocopy.

absent are the aliphatic stretching bands (C\H) that are necessary for calculating the two ratios mentioned (D'Angelo et al., 2010; D'Angelo et al., 2013).

4. Discussion

4.1. Comparison of IR-spectra: extracuticular deposit vs compression

The combination of long and straight aliphatic chains with increas- ing contents of oxygenated/aromatic carbon groups does not ﬁtthe usual IR signature for medullosalean tree-fern compressions (compare with Lyons et al., 1995; D'Angelo et al., 2010, 2013, and others). In medullosalean compressions, the Al/Ox ratio is generally much larger

than 0.32, but the CH2/CH3 ratio may be comparable. In comparison with vitrain from the Lloyd Cove Seam (sample location of the M. scheuchzeri specimens in this study), the Al/Ox ratio is similar, but

the CH2/CH3 ratio is not, being unity or less for the vitrain (D'Angelo et al., 2010, Table 5, analyses #17 and #19). In the original chemical study of M. scheuchzeri, Lyons et al. (1995) concluded that the compression (understood without the cuticle) underwent chemical changes similar to those of the vitrain in the Lloyd Cove Seam. However, the IR Fig. 3. Macroneuropteris scheuchzeri. A partially solubilized extracuticular deposit after 2 3/ spectrum of the extracuticular deposit is different from that of the com- 4 h of maceration showing relict cross fractures, pointed tips not shown. Curved, orange– pression (understood without the cuticle) by having more aliphatic brown lateral veins are the background. Scale: the extracuticular deposit is ca. 3.5 mm \ long. Slide 3-234/1. Nomarski phase-contrast micrsocopy. (For interpretation of the refer- C H groups, relative to carboxyl/carbonyl groups, and much reduced ences to color in this ﬁgure, the reader is referred to the web version of this article.) aromatic bands in the out-of-plane region. I mention, however, that E.L. Zodrow / International Journal of Coal Geology 121 (2014) 14–18 17

1029 A

1385

2927 1634 2851 CH2 /CH3 = 3 Ox 1 = 0.32 1727

3398 B

1634 830

4000 3500 3000 2500 2000 1500 1000 500 Wavenumbers (cm-1)

Fig. 4. Macroneuropteris scheuchzeri. IR spectra. (A) Extracuticular deposit. (B) Trichome from a cuticle.

the CH2/CH3 and Al/Ox ratios for compressed trichomes of Alethopteris patterns, i.e., are crystalline (see Merk et al., 1997). But unanswered pseudograndinioides correlate with the compressions to which they questions remain about the influence of the cuticle as substrate on were attached (unpublished research notes, 2013). This IR result spatial distribution and pattern building of the epicuticular waxes. The suggests that the chemical signature of the mother tissue is carried existence of fossil-wax crystals is inferred from the micron-sized into the epicuticular feature which is not the case for M. scheuchzeri, platelet-like particles on the cuticle of the Carboniferous seed-fern but supports the argument for a biochemical deposit. Moreover, the A. pseudograndinioides (Fig. 5), and thus the possibility exists that the biological reason and the significance why the spectrochemistry of the hair-like structures may be of similar origin, but on a macroscale. trichome-mother tissue of M. scheuchzeri is non-convergent remain Significant in this respect was the discovery by Gao (1992), using unknown for lack of experimental data. transmission-electron microscopy, that cuticles of M. scheuchzeri from the Sydney Coalfield showed microchannels that I now compare with 4.2. Molecular self-assembly and plant surfaces the lipoidic pathways. Consequently, I hypothesize a process of molecular self-assembly and propose the name extracuticular deposit, based Self-assembly, more specifically molecular self-assembly, is defined on the experimental results of this study. The name “extracuticular “as a process by which molecules adopt a defined arrangement without deposit” reflects the origin and at the same time the emergent nature guidance or management from an outside source” (Wikipedia, the free as cuticular sensu Koch and Ensikat (2008). encyclopedia). As a field of study, the concept originated with organic chemistry, and is applicable at all scales to a host of natural phenomena, 4.3. Systematic review including polymeric morphologies (Barrett et al., 2011). As such, it is not just confined to nano or biomacromolecular scales. Self-assembly is not The reported experimental results raise three arguments for synonymous with formation because the process involves “… pre- reviewing the systematics of the current concept of M. scheuchzeri, existing components … controlled by proper design of the components” (summary, Whitesides and Grzybowski, 2002, p. 2418). Moreover, Mandelbrot's fractal geometry (Mandelbrot, 1983)isanexample where, instead of molecules, geometric entities (fractal dimensions) adopt a defined arrangement by self-similarity, with application to fractal taxonomy of Carboniferous spore-bearing ferns (Heggie and Zodrow, 1994). It is outside the scope of this paper to argue for synthesis of self- assembly and self-similarity. The role of waxes in the make-up of extant cuticles has been known for a long time (summary: Holloway, 1994), and Koch and Ensikat (2008) review the process of molecular self-assembly for epicuticular- wax crystallization on the cuticular substrate. It is these microscopic wax crystals that impart to extant [and fossil] cuticles alike the hydrophobic nature with well-known physiological properties that include regulatory water management (Szafranek et al., 2008). Wax crystallization is influenced by temperature, solvent and cuticular substrate, and self-assembly is carried out in solution at or near an interface, where non-covalent interactions among molecules are the driving forces, among other things. As Koch and Ensikat (2008) explained it, the pro- “ ” cess takes place by diffusion through the cuticle via lipoidic pathways , Fig. 5. Alethopteris pseudograndinioides, cuticle. SEM image. The small elongate features are and they stressed that all true aliphatic waxes had X-ray diffraction interpreted as platelets, wax crystals (Stoyko et al., 2013). 18 E.L. Zodrow / International Journal of Coal Geology 121 (2014) 14–18 which are outside the scope of this study, but summarized briefly. The Chen, Y., Mastalerz, M., Schimmelmann, A., 2012. Characterization of chemical functional fi groups in macerals across different coal ranks via micro-FTIR spectroscopy. Int. J. Coal rst is if the position of Macroneuropteris Cleal et al., 1990 is tenable Geol. 104, 22–33. within the neuropterid taxa, given the presence of the character state Cleal, C.J., Zodrow, E.L., 1989. Epidermal structure of some medullosan Neuropteris foliage of extracuticular deposits that are not known from any other member from the Middle and Upper Carboniferous of Canada and Germany. Palaeontology 32, 837–882 (pls. 97–106). in the medullosalean seed-fern group (Zodrow, 2013; D'Angelo and Cleal, C.J., Shute, C., Zodrow, E.L., 1990. A revised taxonomy for Palaeozoic neuropteroid Zodrow, manuscript in progress)? The consequence of an untenable foliage. Taxon 39, 486–492. position is a destabilized nomenclature for a widely-known Carbonifer- D'Angelo, J.A., Zodrow, E.L., Camargo, A., 2010. Chemometric study of functional groups in fi ous species. The second argument involves Hoffmann's types if they still Pennsylvanian gymnosperm plants organs (Sydney Coal eld, Canada): implications for chemotaxonomy and assessment of kerogen formation. Org. Geochem. 41, can be the basis for M. scheuchzeri, i.e. is evolving science-technology a 1312–1325. factor in nomenclatural change? The last argument is if a reexamination D'Angelo, J.A., Lyons, P.C., Mastalerz, M., Zodrow, E.L., 2013. Fossil cutin of Macroneuropteris scheuchzeri (Late Pennsylvanian seed fern, Canada). Int. J. Coal Geol. 105, 137–140. of the neuropteroid type specimens known to show these long hair-like fl fi Darrah, W.C., 1969. A critical review of the Upper Pennsylvanian oras of Eastern United structures shouldn't be the rst step in a systematic review? States with notes on the Mazon Creek Flora of Illinois. Self-published, Gettysburg 220 (plus 80 pls). Gao, Z., 1992. Ultrastructures of fossil plant cuticles from the Upper Carboniferous of 5. Conclusions Sydney Coalfield, Nova Scotia, Canada. Abstracts, Organisation Internationale de Paléobotanique, Muséum National d'Histoire Naturelle (Paris), IVéme Conference, 30 Août-3 Septembre 1992., 63. • The theory of dynamic molecular self-assembly is introduced into Gothan, W., 1916. Über die Epidermen einiger Neuropteriden des Carbons., Band XXXV – palaeobotany. (Teil II). Jahrbuch, Königlichen Geologischen Landesanstalt, Berlin, pp. 373 381. •“ ” – Heggie, M., Zodrow, E.L., 1994. Fractal lobatopterid frond (Upper Carboniferous Hair ,thelong-timerecognized1 5 mm long diagnostic feature on marattialean tree fern). Palaeontogr. Abt. B 232, 35–57. the abaxial surface of M. scheuchzeri, is a misnomer. Instead, Hoffmann, F., 1827. Über die Pflanzenreste des Kohlengebirges von Ibbenbühren und von extracuticular deposit is proposed as being an appropriate name. Piesberg bei Osnabrück. Keferstein, Ch. Teutschland, geognostisch-geologisch • dargestellt und mit Charten und Zeichnungen erläutert., Band 4 (II. Heft). Landes- Extracuticular deposits, built-up of long-chain aliphatics, do not Industrie-Comptoirs Weimar, pp. 156–166. compare morphologically or spectrochemically with trichomes nor Holloway, P.J., 1994. Plant cuticles: physicochemical characteristics and biosynthesis. In: with organic matter taken from the pinnule lamina of compressed Percy, K.E., et al. (Eds.), Air Pollutants and the Leaf Cuticle. NATO A S1 Series, Section 1 — Reviews, G 36. Springer-Verlag, pp. 1–13. M. scheuchzeri itself. Koch, K., Ensikat, H.-J., 2008. The hydrophobic coatings of plant surfaces: epicuticular wax • The existence of extracuticular deposits paves the way to an answer of crystals and their morphologies, crystallinity and molecular self-assembly. 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