International Journal of Stem Cells and Medicine

Abstract

Extensive and exhaustive multidisciplinary studies for several decades covering many parts of India on population biology of the genus and various species of Ophioglossum L and related genera of the family ophioglossaceae (Goswamia Zhang & Zhang) have revealed that the genus Ophioglossum in particular has been exhibiting certain teratological traits of great evolutionary significance. Elsewhere, it has been opined that many new traits or teratologies might be expressed in some segregates of natural hybridizations but appearance of some totally unknown remarkable features observed in different populations of species inhabiting distant far remote areas must be an epigenetic expression within the genome. Leafy sporophores (fertile spikes), presence of many abnormal forked or twisted ones are common ones, known for several years but find of spore mass developing on the margins of tropohylls (leaves)without the development of sporangium in some plants of many species is a unique feature.  Additionally, Goswamia costata Zhang & Zhang (=Ophioglossum costatum) shows large number of variables in shapes and full mid-vein variations of tropophores (leaves) resembling exactly like leaves of Glossopterisgenus Senotheca Banerjee never known or described for any genera of the group. Yet another new feature has been observed in some plants of Goswamia bispora Vadhiya et al., the rhizome possesses very fine unicellular and branched hairs. Since the stem cells are innately undifferentiated cells located in the meristems of plants and are also totipotent cells equipped with regenerative powers  facilitating plant growth and production of new organs throughout lifetime, we hypothesize that repeated over expression of certain totally unknown traits will have to be considered as expressions of some epigenetic mechanisms triggered within the genomes of Ophioglossum and sister genus Goswamia.  This triggered molecular mechanism might be imposing revitalized or mutated function of a few genes in stem cells in plants of a few selected species. Obviously, stem cells also serve as repositories of conserved DNA sequences related to ancestral lineages.

Introduction

Taxonomy is a branch of science which is based on classification of species and genera in a way which offers technical parameters for exact identification and proper placement of species into the evolutionary ladder suggesting common evolutionary lineages. Therefore, whenever some genera or species exhibit many similarities as well as gross and major differences in morphological and or anatomical characters, they are sub grouped into sister genera (Zhang, Zhang 2022; Lima et al., 2024) Lately, most International collaborative teams of taxonomists have been inferring results on the basis of chloroplast genes. A large group of botanists weaving PPG 16 have recognized two pteridophyte classes: Lycopodiopsida (lycophytes) and Polypodiopsida (ferns). These are distinct lineages within the tracheophyte tree of life, with ferns resolved as more closely related to seed plants than to lycophytes.Within Lycopodiopsida, PPG16 mentions three orders (Lycopodiales, Isoëtales, and Selaginellales). Order Lycopodiales includes one family and 16 genera, whereas orders Isoëtales and Selaginellales each contain a single monogeneric family. Within Polypodiopsida, the group members have recognized four subclasses: Equisetidae (horsetails); Ophioglossidae; Marattiidae; and Polypodiidae. The Extant Equisetidae (Hauke, 1978) includes a single order, a single small family, and a single genus (Equisetum L.). Subclass Ophioglossidae encompasses two orders, each with a single family, and a total of 12 genera. Marattiidae includes just one order, one family, and six genera.

Recent revised classification and taxonomic placements of genera have been based on chloroplast genes. Chloroplasts are a type of double membrane–bound cytoplasmic organelle found in plants, algae, and some protists. The ‘‘energy factories’’ of the cell, are the site where photosynthesis takes place, converting light energy into chemical energy to sustain life on Earth. Chloroplasts are likely derived from an ancient photosynthetic cyanobacterium that was engulfed by a eukaryotic host cell, giving rise to an ancestral eukaryotic plant cell more than 1.5 billion years ago (see Zhang et al., 2023). On average, the chloroplast genomes of land plants (Hauk et al., 2023; Zhang et al., 2020) have retained about 120 genes with conserved content. Most of these genes are particularly important for plant viability because they encode core subunits of the photosynthetic apparatus and the gene expression system of chloroplasts (i.e., RNA polymerase subunits, ribosomal proteins, ribosomal RNAs [rRNAs], and transfer RNAs [tRNAs]) Needless to mention, an extensive  comparative search for similarities and differences among chloroplast genes has become of vital importance, At the same time, detailed studies on morphological, anatomical and chromosomal are also absolutely essential and should not be ignored; these all traits are also controlled by genes (Goswami, 2008, 2019, 2023)

A recent development has been announced by Zhang, Zhang (2022); the publication has been based on Sanger sequences of eight plastid markers of 184 accessions, 22 plastomes, 29 morphological characters, and combined Sanger and morphological data. These authors have emphatically concluded that there are 07 genera within the family Ohioglossaceae. These are: Botrychium, Botrypus,Japanobotrychium, Sceptridium, Ophioglossum, Goswamia, Haukia, and Whittieria. Precisely, the genus Ophioglossum has three additional sister genera Goswamia, Haukia, and Whittieria which are all deeply diverged from the rest of Ophioglossum (Ophioglossums.s.) on the basis of comparative phylogenetic studies on a large number of plastomes. Accordingly, originally conventional Ophioglossum plants (Fig.1 A) with bulbous or cylindrical corm / rhizome with thick but if thin, then with a bunch of roots (like O. costatum) are being identified as Goswamia Zhang & Zhang. Similarly, plants with small, linear corm with thin and often not many roots are identified as species of Ophioglossum L. Hereunder, we have used revised nomenclature; older conventional names of species are also mentioned in parenthesis for convenience of identity. The genus Ophioglossum as well as recently erected genus Goswamia have been exhibiting series of common as well as extremely rare abnormal teratologies (Goswami, Khandelwal, 1973; Goswami, 1987, 2007) some of them have been being of evolutionary significance.

This paper presents a very rare observation of possessing spores developed on the margins of tropophylls (leaves) besides the fact that the plant also has a regular properly developed fertile organ the “sporophore” (the spike). On one hand this extremely rare trait supports foliar origin of the sporophore (Goswami, 1987, 2007), and on the other, it poses a question as to “what compels the tissue within the margins of some leaves of some plants to produce spores directly on the surface without developing a sporangium?.Since this highly abnormal feature is also recorded in many species with a gap of decades from different parts of India (Goswami et al., 2024) we presume this “triggering” of genes at an undesired place to be a rare molecular-genetic phenomenon related to stem cells within the leaf tissue. We also mention some altogether new and unusual features not known so far among any species of this group.

Materials and Methods

One of us (HKG) has been studying the members of Ophioglossales (Ophioglossum, Botrychium and Helminthostachys) for nearly six decades on the basis of his own collections from various parts of Central, Northern and Western parts of India. Since 1973 onwards series of publications on these plants by HKG and his associates have been published based on following standard methodologies and protocols depending upon specific approaches (eg. morphological, anatomical, biochemical, chromosomal and molecular phylogenetic etc). Publications have also been reviewed in exhaustive papers with relevant figures and additional details (Goswami, Khandelwal, 1973; Khandelwal, Goswami,1977; Goswami, 1987, 2007, 2023; Goswami, Patel, 2019; Goswami et al., 2024; Sharma et al., 2019).

Observations

Several other workers interested in these groups of plants (Ophioglossales) have also described various developmental teratological features mainly involving malformed, bifid, and partially sterile sporophores (spikes) but leafy spikes or lower half as leaf with venation and the other half being a fertile normal sporophore (Figs 1 A-D) region have been extremely rare (Goswami, 1987). Our earlier and present repeated observations of the highly unusual traitshave been very interesting discoveries because as all these features indicate “awakening of the genome and revitalizing” of some inherently conserved genes within the parental genome of the genus Ophioglossum L and now also referable to Goswamia Zhang & Zhang. These traits are mentioned below:

Leafy Sporophore (spike)

These abnormal sporophores have been observed in many collections among populations of several species (Fig 1 A- E); always arising from the base of the tropophyll. Sometimes one normal tropophyll gives rise to a normal spike and the other tropophyll of the same plant gives rise to an abnormal spike.

Periderm

Khandelwal & Goswami (1977) have described the rare development of periderm tissue in rhizomes of several species and genera of the order ophioglossales. This anatomical feature is a feature of the fossil group of pteridosperms and among gymnosperms and many angiosperms. As opined earlier, the development of periderm in some plants of many species of Ophioglossum is a selective polygenic expression which is now verified by the presence of such a tissue in the long petiole of Ophioglossum aletum, Patel et al 2019).

 Spores on the Margins of tropophyll

Earlier papers on this observation have revealed it categorically that the expression of this feature is evolutionary indication of the rare molecular genetic activity inherent within the genome to suggest the foliar nature of evolution of a sporophore by inward rolling of the leaf margin (Goswami, 1987, 2007; Goswami et al., 2024). But most intriguing fact is that spores on the margins of such leaves (Fig. 2 A –B) are in clusters and many such spores have triradiate mark suggesting that there might have been some diploid cells functioning as spore mother cells which in turn divided “meiotically” (?) to produce irregular tetrads. We have not seen any spore mother cell nor any trace of a sporangium, except small round clusters of spores and some clusters of triradiate spores.

Unicellular Hairs on Rhizome

As far as known none of the species of any Ophioglossaceous genera has shown or possessed rhizomes being covered by unicellular branched hairs. This trait (Fig 2 C-E) observed in six plants of Goswamia bispora Vadhiya et al., (2024) is altogether an uncommon trait which can be termed as a “Profern” feature.

Shape and mid-vein variations among tropophylls (leaves)

There are a large number of tropophyll (leaf) variations, specifically among Goswamia costata (= Ophioglossum costatum) populations which do not resemble typical wellknown figure for the species but offer direct comparable structures to the Glossopterid leaf genus Senotheca Baneerjee. Stephen McLoughlin (2012) has published an exhaustive review on the Glossopterid leaf genus Senotheca Banerjee and midvein, shape and nodulated structures are exactly similar as seen in Figure 3. These observations are not altogether new but earlier Kato (1988) had also pointed out a resemblance between ophioglossoid fertile leaves and glossopterid fructifications, which some workers have suggested as a possible archetype for the angiosperm carpel (Kato, 1990).

Goswamia costata (=O.costatum) : Note the large cylindrical rhizome/corm with large number of roots.(A).  This species always has a large stuffy cylindrical or round rhizome with bunch of roots., irrespective of collections emanating from any parts of the country, even from abroad; B & C show  upper side as the  fertile sided of the sporophore (spike)  while lower half shows incurving and veins like a leaf ( half leaf and half spike both in half circination) ; Lower side enlarged to show veins in mesophyll-like tissue  (After Goswami, 1987); D. Normal Tropohyll shows midvein,  two leafy Sporophores arise on bifurcated petiole; E. The Sporophore looking as a flat leaf shows sporangia on the margins; such a feature has been observed in several plants belonging to Goswamia and Ophioglossum species for the last 50 years.

Fig 1. Ill developed sporangia present on the margins of tropophyll and leaf-like sporophore (arrowed)

Fig 2.A commonly found with forked tropophyll a plant of Goswamia bispora may also show spore mass ; B, Cluster of  triradiate Spores with the mesophyll tissue first seen in Goswamia eliminata (= Ophioglossum eliminatum); C, Enlarged rhizome of Goswamia bispora, arrows indicate fine white hairs which are (D & E) unicellular branched hairs. This feature has not been known to be typical trait on rhizomes of ophioglossum ss.  Thick Roots emerge from the rhizome which as expected do not have hairs

Fig.3.Among Goswamia costata (=Ophioglossum costatum) plants (A) a few plants are being collected which sometimes possess several variations in shapes and sizes (A & B ) of tropophyll (leaf); most important variations are found  on the  lower surface of tropophyll which  exhibit highly thickened variations in the mid rib showing nodulated thickening of the tissue (B, C). Not all plants of this species nor all leaves of the variant plant show such peculiar modifications. This is an extremely rare expression, D. Extremely rare patches of elevated tissue on the margins and also on the midrib; on teasing these structures, mass of cells and sterile sporelike cells or “nurse cells” are observed (See Fig.2). Some fossil leaves of the Glossopterid genus Senotheca show this feature.

Discussion

During the Late Devonian and Early Carboniferous, at least five vascular plant lineages,eg. (lycophytes, ferns, progymnosperms, sphenopsids and seed plants, independently evolved laminate leaves and followed the same early sequence of morphological evolution. Practically, leaves are believed to have evolved by progressive vascularization and have evolved several times (Boyce, Knol 2002, Boyce, 2007, 2008) during evolutionary path; even liverworts and moss have leaf like forms but with divergent anatomy and unrelated to tracheophyte leaf. Development can be considered in terms of two related processes: (1) growth, including cell division and the differentiation of individual cells, and (2) the patterning of differentiated cells to form functional tissues (Wolpert,1971; Sachs,1991).. The parallel evolution of a marginal meristem by the modification of developmental mechanisms available in the common ancestor of all groups resulted in the pattern of leaf evolution.

Recent molecular genetic studies have however presented on the basis of expression of KNOX and APR gene complexes in the sporangia, a very interesting evolutionary phenomenon that even if leaves have different origins and independent evolutionary affiliation, the mechanism for the determinacy has always been present for the differentiation of sporangia and or axially borne organ among all tracheophytes. This genetic trait is inherent among all plants and has been faithfully passed on from ancestors for millions and millions of years.   Quite possibly this trait and its transmission might be a basic function of the stem cells present in the meristematic leaf tissue.  The evolution of a marginal meristem by the modification of developmental mechanisms available in the common ancestor of all groups resulted in the pattern of leaf evolution.

Possible Role of Stem Cells

As emphasized earlier, the repeated observations (Goswami, 2007; Goswami et al., 2024) of the development of spores in clusters and in irregular or proper tetrads on the margins of tropophylls (leaves) of Goswamia eliminata (Ophioglossum eliminatum), Goswamia costata, (Ophioglossum costatum) Goswamia bispora and Ophioglossum gramineum will have to be considered to be the expressions of some epigenetic mechanismstriggered within the genomes of Ophioglossum. Such an expression on margins of leaves may be a revitalized action of meristems of margins of certain leaves; as meristems sometimes can be over-activated (Boyce, 2008).

The stem cells are innately undifferentiated cells located in the meristems of plants are totipotent cells equipped with regenerative powers that facilitate plant growth and production of new organs throughout lifetime. Two distinct areas of stem cell, the apical meristem and the lateral meristem are recognized on the basis of prevalence. These cells (see: Weigle, Jurgens 2002; Singh, Bhalla, 2006)   serve as the origin of plant vitality by providing a steady supply of precursor cells to form differentiated tissues and organs in plants . These stem cells are characterized by unique ability to create all differentiated cell types and the ability to self-renewal (so that the number of stem cells is maintained). Plant stem cells never undergo aging process but immortally give rise to new specialized and unspecialized cells, and possess the molecular potential to grow into any organ, tissue, or cell in the plant body. 

Probable Molecular genetics of reformation of stem cells

Tissue specific stem cells are multipotent populations residing in localized niches and are responsible for maintaining all lineages of their resident tissue/system throughout life. Overall, evidence suggests tissue-specific stem cells accrue DNA damage during aging despite being considered as protected populations. This accumulation differs among the various stem cell compartments, and divergence could derive from stem cells’ inherent properties (e.g., DNA methylation profile, potency, etc. Epigenetic mechanisms are regulators of gene expression, and epigenetic changes can alter functioning of some genes. Recent investigations among cellular mechanisms have offered sufficient evidence that an important layer of gene regulation, epigenetics– which encompasses the mechanisms of DNA methylation, histone modification, and chromatin organization also contributes to unusual dysmorphological developments (Dexheimer, 2013; Guo et al., 2023). Molecular geneticists have also documented that hypomethylation-associated silencing of large chromatin domains can influence certain. transcriptionally silenced genes.

We do not know nor can imagine, whether any specific molecular or epigenetic mechanism is operative within the genome of the genera Ophioglossum (plants with linear rhizome and thin roots)and Goswamia (plants with bulbous / knot or cylindrical rhizome with proportionately thick roots) but we are certain that repeated occurrence of development of spores on the margins of leaves without the development of sporangia and spore mother cells has to be considered as a rare mechanism of triggering of functions of stem cells regulating such an expression. Exactly, similar situation is quite apparent on very few but regularly occurring of unusual traits of leaves of Goswamia costata resembling leaves of the Glossopteris genus Senotheca (see Banerjee, 1969; McLoughlin 2012, 2019) He has published an exhaustive review on the Glossopterid leaf genus Senotheca Banerjee and as suggested by Kato (1988,1990) most variations among the leaves of Glossopterids are exactly similar to variations encountered in the genus Ophioglossum. There are many features expressed similar to pteridosperms and our field experience supports the hypothesis that Goswamia costata (=O costatum) in particular exhibits many exactly similar traits to indicate that Ophioglossum ss are moreclose to Glossopterids.

Precisely, the re-appearance of certain highly unusual or altogether new traits must be taken seriously as most such genes responsible for such expressions convey the inherent and deeply conserved gene sequences within the genome. Hypothetically, these appear as evolutionary obligations on stem cells so as to produce variability and to test the survival fitness of these traits. Needless to mention, the stem cells also serve as store house for conserved genes because the appearance of such totally unknown traits in the extant population can become revived only due to unusual activation within the stem cells.

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