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 Macroneuropterisscheuchzeri (Pennsylvanian-age seed-fern)

Erwin L. ZodrowPalaeobiology Laboratory, Cape Breton University, Postal Box 303, Sydney, B1P 6L2 Nova Scotia, Canada

E-mail address: erwin_zodrow@cbu.ca.

0166-5162/$ – see front matter © 2013 Elsevier B.V. All rihttp://dx.doi.org/10.1016/j.coal.2013.11.002

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 September 2013Received in revised form 6 November 2013Accepted 7 November 2013Available online 13 November 2013

Keywords:Extracuticular depositEx “hair”FTIRCarboniferousM. scheuchzeri

Hoffmann (1827) erected the Carboniferous pteridophyll species Neuropteris Scheuchzeri without mentioning,nor illustrating, “hair” in the species' diagnosis. However, one to five millimeter-long hair-like structures onthe abaxial pinnule of the species, called hair or trichome in the literature, have been routinely used since1847 as one of the main taxonomic character states for determining the identity of this species. Results frompreparatory and microscopic observations, together with infrared spectrochemistry, have clarified that thesestructures are not the same as trichomes for the following reasons. The hair-like structures of M. scheuchzeri(1) are not organically attached to the abaxial surface; (2) differ spectrochemically from the organic materialof the lamina; (3) are composed, in contrast with the trichomes, of relatively long, unbranched aliphatic(polymythelinic) hydrocarbon chains [CH2)]n, and (4) are acellular andblack, unlike true trichomes of the speciesthat are multicellular. Overall, the sum-total of these experimental results supports the postulate for dynamicmolecular self-assembly. For this reason the term “extracuticular deposit” is proposed, reflecting the origin andemergent nature of such hair-like structures in the abaxial pinnule.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

The current concept of Macroneuropteris scheuchzeri (Cleal et al.,1990; Hoffmann, 1827) includes four character states on the abaxialpinnule, i.e., extracuticular deposits (this study), hair (trichome inbotanical Greek), files (unicellular transparent structures), and papillaewhich are comparatively small and curved. Of these features, thetrichomes very densely populate the abaxial lamina (see Barthel,1961; Cleal and Zodrow, 1989, and others). In contrast to these featuresof which extracuticular deposits are clearly observable under a loupe oreven by the naked eye as they are up to 5 mm long, Hoffmann did notmention the presence of any abaxial features in his diagnosis of thespecies. However, the presence of extracuticular deposits, using thename hair, has been taken as an important taxonomic character statesince 1847 to distinguish the identity ofM. scheuchzeri from among sim-ilar larger-leaved Pennsylvanian foliage (literature survey: Bunbury,1847 to Stull et al., 2012). A notable exception is Leo Lesquereux whoassigned American hairy, long-leaved pinnules to neuropteroid taxa,other than Neuropteris scheuchzeri (summarized by Darrah, 1969).However, Gothan (1916), made the pointed observation that the visiblehair ofN. scheuchzeri did not survive Schulze's chemical-oxidative treat-ment (cf. Cleal and Zodrow, 1989). Barthel (1961) described attached,pointed hairs of N. scheuchzeri, which instead I regard as extracuticulardeposits.

ghts reserved.

This paper focuses on the hypothesis that the long, hair-like struc-tures of M. scheuchzeri are really extracuticular deposits resulting fromthe process of dynamic molecular self-assembly (summary: Koch andEnsikat, 2008). A review of the taxonomy/systematics ofM. scheuchzeriis accordingly recommended.

2. Materials and methods

The Carboniferous seed-fern M. scheuchzeri bears polymorphicpinnate foliage that is seen above and below a basal frond dichotomy;individual pinnule lengths range from 3 mm to 120 mm (summaries:Cleal and Zodrow, 1989; Laveine, 1997; Laveine and Belhis, 2007;Zodrow, 2003, and many others).

The compression specimens for this study were collected by theauthor from the roof shale of the Lloyd Cove Seam, which is knownin the literature for its rich content in plant fossils that are well-preserved (Fig. 1). For the experimental work, only compressionsfreed from the rock matrix were used from which extracuticular de-positswere collected in a Petri dish. Trichomeswere obtained froma cu-ticle of a macerated compression (cf. Cleal and Zodrow, 1989).

Spectrochemical analyses of extracuticular deposits and trichomeswere performed using FTIR (Fourier transform infrared spectrometry)and the KBr-pellet technique. Interpretive details, particularly of IR(infrared) spectra ofM. scheuchzeri, supported by carbon13nuclearmag-netic resonance studies, are found in Lyons et al. (1995), or D'Angelo et al.(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 Cantabrianstrata.

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3. Results

3.1. Physical aspects of extracuticular deposits

The confusion in the literature between hair [trichomes] andextracuticular deposits stemsmainly from lack of observing the abaxialsurface of M. scheuchzeri compressions after being freed fromthe entombing rock matrix, though previous exceptions are noted(Barthel, 1961; Cleal and Zodrow, 1989; Gothan, 1916). To untanglethe confusion necessitates (1) examining freshly unearthed compres-sions immediately after collecting, (2) examining the HF solution usedfor freeing the compressions from the rock matrix, (3) real-time study-ing of the compressions during Schulze's (1855) maceration process,and (4) examining the ammonium hydroxide solution that producesthe cuticle. Summarizing my experimental results, extracuticulardeposits (1) tend to drop-off in storage because of dehydration of theexposed compression (not of the extracuticular deposits), (2) werefound loose in the plastic dishes, (3) had slowly solubilized on the com-pression in ca 3–5 h, and (4) intact trichomes were found in Petridishes, correlating with structural holes found subsequently in thecorresponding abaxial cuticles.

Microscopic observations include that trichomes (ca. 300 μm long)are not ordinarily visible on compressions, freed or still attached tothe rock matrix, but molds of extracuticular deposits are marked(Fig. 2A), and extracuticular deposits flatly overlie the abaxial venationat an acute angle in a more or less consistent parallel arrangement with

the midvein (Fig. 2B). The distribution pattern, as correctly drawn byBunbury in 1847, follows a trend, i.e., it is biogenetically non-random.Extracuticular deposits are straight in shape, doubly-pointed, black,solid, and fracture easily (Fig. 2C to E). Microscopic examination of theabaxial surfaces of compressions at ×250magnification, and critical ob-servation and photography of the compressions during maceration,show no evidence for organic attachment of the extracuticular deposits.Most importantly, they are acellular (Fig. 3).

Fig. 4A, B shows IR spectra of individual extracuticular deposit andtrichome, respectively. In particular, the former is relatively aliphatic-rich, as indicated by the larger CH2/CH3 ratio of 3.0, which at the sametime implies comparatively longer and straight hydrocarbon chainswith relatively few side branchings. This ratio is computed afterdeconvolution in the 3000–2800 cm−1 aliphatic stretching region (seeZodrow and Mastalerz, 2001, Figs. 6 or 7). The Al/Ox ratio of aliphaticsto oxygen-containing compounds [(3000–2800/1800–1500) cm−1

band] is comparatively very small at 0.32, which suggests a significantjoint contribution of oxygen-containing groups and aromatic carbon.The peak at ~3400 cm−1 is due to hydroxyl absorbance, at 1727 cm−1

(C_O) ester, at 1634 cm−1(C_O) ketones, at 1385 cm−1 (symmetricCOO\), and at 1029 cm−1 (C\O\C) ether, or Si\O stretch in silicates(see Chen et al., 2012). The inescapable conclusion is that extracuticulardeposits are the last physiological event in the development of theM. scheuchzeri pinnule, representing an excreted biochemical deposit.

In contrast, the trichomatous IR spectrum is relatively poor in termsof functional groups, confirmed by a second spectrum. In particular,

Fig. 2.Macroneuropteris scheuchzeri. (A) Impressions (molds) of extracuticular deposits. (B) Extracuticular deposits overlying lateral veins of the abaxial pinnule. (A) and (B) represent thesame 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.

Fig. 3.Macroneuropteris scheuchzeri. A partially solubilized extracuticular deposit after 2 3/4 h of maceration showing relict cross fractures, pointed tips not shown. Curved, orange–brown lateral veins are the background. Scale: the extracuticular deposit is ca. 3.5 mmlong. Slide 3-234/1. Nomarski phase-contrast micrsocopy. (For interpretation of the refer-ences to color in this figure, the reader is referred to the web version of this article.)

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absent are the aliphatic stretching bands (C\H) that are necessary forcalculating the two ratios mentioned (D'Angelo et al., 2010; D'Angeloet 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 fit theusual IR signature for medullosalean tree-fern compressions (comparewith Lyons et al., 1995; D'Angelo et al., 2010, 2013, and others). Inmedullosalean compressions, the Al/Ox ratio is generally much largerthan 0.32, but the CH2/CH3 ratio may be comparable. In comparisonwith vitrain from the Lloyd Cove Seam (sample location of theM. scheuchzeri specimens in this study), the Al/Ox ratio is similar, butthe CH2/CH3 ratio is not, being unity or less for the vitrain (D'Angeloet al., 2010, Table 5, analyses #17 and #19). In the original chemicalstudy ofM. scheuchzeri, Lyons et al. (1995) concluded that the compres-sion (understood without the cuticle) underwent chemical changessimilar to those of the vitrain in the Lloyd Cove Seam. However, the IRspectrum of the extracuticular deposit is different from that of the com-pression (understood without the cuticle) by having more aliphaticC\H groups, relative to carboxyl/carbonyl groups, and much reducedaromatic bands in the out-of-plane region. I mention, however, that

500 1000 1500 2000 2500 3000 3500 4000

Wavenumbers (cm-1)

29272851

1634

1385

1029

1634830

3398

A

B

1727

CH2 /CH

3 = 3

Ox1 = 0.32

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

Fig. 5.Alethopteris pseudograndinioides, cuticle. SEM image. The small elongate features areinterpreted as platelets, wax crystals (Stoyko et al., 2013).

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the CH2/CH3 and Al/Ox ratios for compressed trichomes of Alethopterispseudograndinioides correlate with the compressions to which theywere attached (unpublished research notes, 2013). This IR resultsuggests that the chemical signature of the mother tissue is carriedinto the epicuticular feature which is not the case for M. scheuchzeri,but supports the argument for a biochemical deposit. Moreover, thebiological reason and the significance why the spectrochemistry of thetrichome-mother tissue of M. scheuchzeri is non-convergent remainunknown for lack of experimental data.

4.2. Molecular self-assembly and plant surfaces

Self-assembly, more specifically molecular self-assembly, is defined“as a process by whichmolecules adopt a defined arrangement withoutguidance or management from an outside source” (Wikipedia, the freeencyclopedia). As a field of study, the concept originated with organicchemistry, and is applicable at all scales to a host of natural phenomena,including polymericmorphologies (Barrett et al., 2011). As such, it is notjust confined to nano or biomacromolecular scales. Self-assembly is notsynonymous with formation because the process involves “… pre-existing components… controlled by proper design of the components”(summary, Whitesides and Grzybowski, 2002, p. 2418). Moreover,Mandelbrot's fractal geometry (Mandelbrot, 1983) is an examplewhere, instead of molecules, geometric entities (fractal dimensions)adopt a defined arrangement by self-similarity, with application to frac-tal taxonomyof 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 themake-up of extant cuticles has been knownfor 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 microscopicwax crystals that impart to extant [and fossil] cuticles alike the hydro-phobic nature with well-known physiological properties that includeregulatory watermanagement (Szafranek et al., 2008). Wax crystalliza-tion is influenced by temperature, solvent and cuticular substrate, andself-assembly is carried out in solution at or near an interface, wherenon-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”,and they stressed that all true aliphatic waxes had X-ray diffraction

patterns, i.e., are crystalline (see Merk et al., 1997). But unansweredquestions remain about the influence of the cuticle as substrate onspatial distribution and pattern building of the epicuticular waxes. Theexistence of fossil-wax crystals is inferred from the micron-sizedplatelet-like particles on the cuticle of the Carboniferous seed-fernA. pseudograndinioides (Fig. 5), and thus the possibility exists that thehair-like structures may be of similar origin, but on a macroscale.Significant in this respect was the discovery by Gao (1992), usingtransmission-electron microscopy, that cuticles of M. scheuchzeri fromthe Sydney Coalfield showed microchannels that I now compare withthe lipoidic pathways. Consequently, I hypothesize a process of molec-ular self-assembly and propose the name extracuticular deposit, basedon the experimental results of this study. The name “extracuticulardeposit” reflects the origin and at the same time the emergent natureas cuticular sensu Koch and Ensikat (2008).

4.3. Systematic review

The reported experimental results raise three arguments forreviewing the systematics of the current concept of M. scheuchzeri,

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which are outside the scope of this study, but summarized briefly. Thefirst is if the position of Macroneuropteris Cleal et al., 1990 is tenablewithin the neuropterid taxa, given the presence of the character stateof extracuticular deposits that are not known from any other memberin the medullosalean seed-fern group (Zodrow, 2013; D'Angelo andZodrow, manuscript in progress)? The consequence of an untenableposition is a destabilized nomenclature for a widely-known Carbonifer-ous species. The second argument involves Hoffmann's types if they stillcan be the basis for M. scheuchzeri, i.e. is evolving science-technology afactor in nomenclatural change? The last argument is if a reexaminationof the neuropteroid type specimens known to show these long hair-likestructures shouldn't be the first step in a systematic review?

5. Conclusions

• The theory of dynamic molecular self-assembly is introduced intopalaeobotany.

• “Hair”, the long-time recognized 1–5 mm long diagnostic feature onthe abaxial surface of M. scheuchzeri, is a misnomer. Instead,extracuticular deposit is proposed as being an appropriate name.

• Extracuticular deposits, built-up of long-chain aliphatics, do notcompare morphologically or spectrochemically with trichomes norwith organic matter taken from the pinnule lamina of compressedM. scheuchzeri itself.

• The existence of extracuticular deposits paves theway to an answer ofthe question of the influence of the cutin matrix “on the spatial distri-bution of and pattern building by epicuticular waxes” posed by Kochand Ensikat (2008, p 768).

• A review of the neuropteroid systematics is recommended.

Acknowledgment

The author is very much indebted to Drs. Maria Mastalerz, IndianaGeological Survey, Indiana University, for the IR data and suggestionsfor improving the technical content, William A. DiMichele, SmithsonianInstitution, Department of Palaeobiology, for critical comments, andsuggesting the name “extracuticular deposit” of theMS, and toManfredBarthel, Humboldt University, for a copy of Hoffmann's, 1827 paper. Theauthor would like to thank Drs. M. Katherine Jones (Biology Depart-ment, Cape Breton University for the use of microscopes, Arthur Mar,Chemistry, University of Alberta, for the SEM micrograph, and JoséA. D'Angelo, IANIGLA–CCT–CONICET, for the production of Fig. 2. Thejournal reviewers, William A. DiMichele and anonymous, are cordiallythanked for editorial points that improved the presentation of the MS.

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