Discovery of a new species of Adder’s tongue fern from India with comparative analysis of morphological and molecular attributes

Eusporangiate fern genus Ophioglossum L. is commonly known as Adder’s tongue fern as its fertile frond gives the appearance of snake tongue. A new species in this fern genus, O. trilokinathii belonging to Ophioglossaceae family has been discovered from the plateau region of Rajasthan State of northwestern India. The new species can be distinguished from other taxa of this genus by its smaller habit, subglobose-tuberous rhizome, basipetal emergence of young roots, aggregation of old decaying roots on rhizome apex, fertile stalk as well as spike short and thick, trophophylls in rosette, ovate or orbicular and a unique sporoderm sculpture pattern under SEM having broad reticulations with thick and raised muri enclosing large hexagonal or irregular areas on the distal and proximal faces of the spores hitherto unreported in any of the presently known taxa of Ophioglossum. In addition, comparative study of stomatal structure, foliar anatomy and nucleotide sequence data of its three chloroplast DNA markers (trnL-F, rbcL and psbA-trnH) was carried out. In view of all the attributes including habitat, ecology, morphology, foliar anatomy, stomatal features, palynology and molecular phylogenetic data, the present study suggests that the Ophioglossum specimen collected from plateau region of Rajasthan represents a hitherto undescribed species thereby warranting its establishment as O. trilokinathii sp. nov. A detailed comparative account of the new taxon with its allied species has also been provided.

Etymology. The specific epithet has been chosen in honour of Professor Triloki Nath Bhardwaja (Professor T. N. Bhardwaja), Former Vice-Chancellor, V. M. Open University, Kota, Rajasthan, India for his commendable work in the field of pteridology.
Diagnosis. O. trilokinathii sp. nov. is unique among the species of this genus in having basipetal emergence of roots, aggregation of old decaying roots at rhizome apex, trophophylls in rosette touching the substratum, peculiar broad reticulate spore wall ornamentation with thick and raised muri enclosing large hexagonal or irregular areas, granulose perine (perispore), differentiation of mesophyll cells into palisade and spongy tissues and elliptic stomata with thin and smooth inner margin of outer stomatal ledge.
Description. Plants terrestrial, small, 1.1-2.4 cm in height (Fig. 1A); rhizome subglobose-tuberous, 0.3-0.5 cm long, 0.1-0.2 cm broad (Fig. 1B); roots thick, fleshy, brownish white, stoloniferous, young roots on basal part of rhizome, aggregation of old decaying roots on rhizome apex (Fig. 1B,C); common stalk 0.2-0.4 cm, subterranean, white (Fig. 1B); trophophylls1-4 sometimes 5, arranged in a rosette, horizontal touching the substratum, broadly ovate or ovate orbicular, thick, margin entire, apex acute or apiculate, base cuneate, lamina surface sometimes with 1-3 longitudinal shallow furrows, midrib absent, 0.6-0.9 cm long, 0.4-0.7 cm broad (Fig. 1D);   1D); fertile segment arises from the junction of face of lamina and common stalk, 0.7-1.5 cm long, thick, spike short, thick, 0.4-0.9 cm long, with two rows of lateral sporangia and a sterile tip, sometimes sterile tip absent; sporangia 5-10 pairs; spores trilete, globose, perinnate ( Fig. 2A), 18-30 µm in size, laesural arms short, straight not reaching to the margins of central cavity of proximal face, sporoderm reticulate under Light Microscope ( Fig. 2A,B). SEM imaging revealed that spores were globose and perinate. Interestingly, spores have a unique exine ornamentation pattern of broad reticulations with thick and raised muri, enclosing large hexagonal or irregular areas on distal and proximal faces, proximal face with distinct short tri-radiate mark and reticulations, laesural arms smooth and straight, not reaching to the margins of cavity of proximal face. To the best of our knowledge, the exine ornamentation pattern as noted in this case has not been reported so far in any other presently known species of Ophioglossum (Fig. 2C,D).
Distribution and ecology. India-Rajasthan state, Chittorgarh district, Mainal locality. The species grow in dense populations on moist soil in open terrestrial habitat at an elevation of ~ 507 m. Plants of this species sprout from the underground rhizome after a fortnight period of first showers in the month of June-July and dry up by the mid of September every year.
Conservation status. The species has been recorded from Mainal area which is known for its famous waterfall and nearby temple of Jogniyamata. It is a plateau region supporting the occurrence of O. costatum, O. gramineum, O. parvifolium, O. indicum, O. petiolatum, and O. gujaratense., Therefore it is one of the richest localities of Adder's tongue fern where contiguous occurrence of two or more species is frequent. The species population occurs in 1.0 × 1.0 m 2 area and is represented by 300-350 individuals. Future explorations are needed to get its entire range of distribution and therefore, at present the species is treated under the category "Data Deficient" (DD) of IUCN 33 .

Species recognition.
Comparison of the new taxon with its allied species provided in Table 1  nov. by its marshy habitat, larger size, roots non stoloniferous, acropetal emergence of young roots, trophophylls upward from the ground, spore exine shows beaded strings of exine grains forming polygonal or round spaces on the distal pole and granulate proximal face 23 .
Basipetal emergence of young roots and 1-4 or 5 trophophylls are also found in O. costatum but in this species bottom part of subglobose rhizome is devoid of roots, roots non stoloniferous, trophophylls are larger with distinct midrib, not arranged in rosette form and reticulate exine with vermiculate muri, muri thin and raised enclosing deep polygonal lumina on distal face and almost pitted ornamentation on proximal face. Leasural arms of triradiate ridge straight reaching to the margins of the central cavity of spore (Fig. 3C,D) which has also been described by earlier workers 34  www.nature.com/scientificreports/ different in having thick and raised muri enclosing large hexagonal or irregular areas on distal and proximal faces, proximal face with distinct short tri-radiate mark and reticulations, laesural arms smooth and straight, not reaching to the margins of cavity of proximal face (Fig. 3A,B). Comparison of the new species (O. trilokinathii sp. nov.) with O. gujaratense indicates that these two taxa resemble in having stoloniferous roots, subglobose tuberous rhizome, number of trophophylls which are 1-4 sometimes 5, appressed to the ground and common stalk subterranean but O. gujaratense differs from O. trilokinathii by its larger size, acropetal emergence of roots, trophophylls lanceolate, fertile stalk thin and long 20,32 and exine with muri of uneven heights along with some flat and wider areas, enclosing shallow depressions on distal face, proximal face reticulated bearing prominent triradiate ridge, laesural arms straight sometimes wavy, reaching to the margins of the central cavity of the spore (Fig. 3E,F). www.nature.com/scientificreports/ O. trilokinathii. also has some similarities with O. parvifolium, as in these two species roots are stoloniferous, common stalk subterranean, trophophylls horizontal touching the soil surface, trophophyll apex sometimes apiculate but the later species differs from the new taxon in, size (upto 10 cm vs 1.1-2.4 cm), emergence of roots (acropetal vs basipetal) number of trophophylls (1-2 vs 1-4), trophophyll base (cordate vs cuneate), fertile stalk (thin and long vs thick and short). Sporoderm structure of O. parvifolium is entirely different from that of O. trilokinathii. In former species exine is reticulate, muri thin enclosing funnel shaped lumina on the distal face and on proximal face exine is pitted, laseural arms of triradiate ridge straight reaching to the margins of central cavity of the spore (Fig. 3G,H).
Another noteworthy feature of the new taxon is the mesophyll tissue of its trophophyll which shows differentiation into palisade and spongy parenchyma (Fig. S1A). Mesophyll cells towards upper epidermis are elongate and closely packed while those towards lower epidermis are rounded or ovoid and are loosely arranged with intercellular spaces. Such differentiation is not found in other species (Fig.S1B,C) The new taxon is quite different from its allied species in its stomatal shape and inner margin of outer stomatal ledge. Trophophylls are amphistomatic. Stomata are elliptic in the new taxon (    Table S6).

Discussion
Ophioglossum is a fern well known for confused state of affairs prevailing in respect of inter-specific differences 5 Table 1. Besides the shape of rhizome, acropetal and basipetal pattern of root emergence on the rhizome of Ophioglossum species has been recognized as feature of taxonomic importance and this character was used to distinguish different species of this fern 31,35 . O. trilokinathii sp. nov. shares the character of basipetal pattern of emergence of young roots with O. costatum but later species is larger in size with subglobose rhizome whose bottom part being devoid of roots in contrast to the former one which is smaller in size with subglobose-tuberous rhizome.
In majority of taxa, the mesophyll tissue is homogenous made up of isodiametric spongy cells. The trophophylls in the present taxon are in rosette and lie flat on the substratum resulting into the unequal illumination of the two surfaces of the blades which lead the differentiation of mesophyll tissue into palisade and spongy parenchyma. Such differentiation of mesophyll is unknown in species of Ophioglossum except O. nudicaule 36 . The structure and design of mesophyll tissue in the plant leaf is one of the key traits playing important role in the regulation of photosynthesis. Elongated chlorenchyma cells of palisade layer with large amount of chloroplast which tend to stay very close to the walls of the cells to harvest maximum amount of illumination enable the plants to make optimal advantage of available light. Palisade parenchyma also helps in distribution of light more uniformly to chloroplast within the cell 37 .
Owing to significant variation among species in stomatal features particularly the stomatal shape, shape and ornamentation of inner margin of outer stomatal ledge, they have been used taxonomically by the earlier workers 38 in separating the complex taxa of Dryopteridaceae in pteridophyta. Epidermal features and mesophyll characteristics have also been suggested by various researchers as supporting evidences in differentiating some    17,22 . Outer stomatal ledges prevent wide opening of the stomatal pore and its lifting above leaf epidermis 39 . This lip like structure around the stomatal pore also helps in preventing water loss by sealing the pore during physiological stress 40,41 . Thus, the differentiation of mesophyll tissue and stomatal ledges are the features which provide survival potential to the new taxon as it is a heliophyte occurring in open terrestrial habitat. Sporoderm features were treated as of taxonomic importance in distinguishing the European species of Ophioglossum 42,43 . Spore coat structure could be valid mark for identification of some Ophioglossum species 43 . Spore characters are not easily prone to environmental influences compared to vegetative features and are thus more dependable in systematic consideration 44 . Some workers 45 have concluded for Japanese species of Ophioglossum that the ornamentation pattern of a particular species collected from different localities and with distinct morphological variability possesses a specific pattern of spore exine.
Spore morphology has now assumed a significant role in pteridophytic taxonomy. Several workers 9,23,34,46-51 have treated the exine ornamentation pattern as the most reliable character for systematic purposes in Ophioglossum. Thus, spore studies fully justify the relevance and validity of SEM investigation in the species delimitation of Ophioglossum. Three types of exine ornamentation patterns namely reticulate, verrucate and scabrate are known in species of Ophioglossum 52 . The exine ornamentation pattern recorded in O. trilokinathii sp. nov. is entirely different consisting of thick and raised muri enclosing large hexagonal or irregular areas on the proximal and distal poles of the spore. Such an exine ornamentation is not known so far in any of the species of the genus Ophioglossum.
DNA barcoding has been used increasingly for shedding light on species delineation in many fern species including Ophioglossum. For a valid plant barcode, it is emphasized to include conservative coding region like rbcL together with rapidly evolving non-coding region like trnL-F 53 . Similarly, The psbA-trnH intergenic region is among the most variable regions in the angiosperm chloroplast genome and is a popular tool for plant population genetics and species level phylogenetics. This region has also been suggested to be suitable for DNA barcoding studies 54 . Various researchers have successfully used these DNA barcodes for molecular identification of Ophioglossum 20-23 .
In the present study, the comparative phylogenetic analysis of seemingly allied species of Ophioglossum suggests considerable degree of differences between O. trilokinathii and O. hitkishorei with respect to rbcL and psbA-trnH regions as confirmed by determination of evolutionary divergence (p-distance) and percent identity matrix. The sequence difference between O. trilokinathii and O. hitkishorei in terms of percent identity matrix was as high as 6.9% for rbcL region. The sequence data of O. sp. SAD 2020a were available in GenBank only for psbA-trnH region and not for the remaining two regions (rbcL and trnL-F). Although, the data on p-distance could not provide evolutionary divergence between O. trilokinathii and O. sp. SAD-2020a respectively, percent identity matrix suggested considerable differences in the nucleotide sequences of these specimens with respect to psbA-trnH region. Furthermore, the morphological description of O. sp. SAD-2020a was not available in the published literature. Based on the information available in NCBI database with respect to the sequence data of psbA-trnH marker, O. SAD2020a specimen belongs to Kerala (Southern India) while O. trilokinathii is reported from northwestern part of India (Rajasthan). Both the regions have altogether different bio-geoclimatic conditions.
Taking all the attributes including habitat, ecology, morphology, foliar anatomy, stomatal features, palynology and molecular phylogenetic data into consideration we conclude that the Ophioglossum specimen collected from plateau region of Rajasthan represents a hitherto undescribed species thereby warranting its establishment as O. trilokinathii sp. nov.  Alignment and phylogenetic analysis. Newly generated sequence data were aligned using Gene Tool v1.0 and their contigs were prepared. NCBI-BLAST search was conducted to reveal the sequence identity. The Sequence data were then deposited to NCBI database (Accession numbers: trnL-F, MW081148; rbcL, MW081146; psbA-trnH, MW081147). Further, nucleotide sequences of Ophioglossum species (28 sequences of rbcL, 18 sequences of trnL-F and 22 sequences of psbA-trnH) were retrieved from the GenBank and used for comparative analysis with nucleotide sequences of O. trilokinathii sp. nov. Botrychium lunaria of the same family was included in the analysis as an outgroup taxon. Nucleotide sequences of all three chloroplast markers as mentioned above were subjected to pairwise alignment and multiple sequence alignment (MSA) using Clustal-W embedded in MEGA X with default settings. Also, MEGA X was used to find out the best fit model for phylogenetic analyses 60 . The evolutionary history was inferred by using the Maximum Likelihood method wherein the models with the lowest BIC scores (Bayesian Information Criterion) were considered to describe the substitution pattern the best and selected as best-fit model 61 . Accordingly, K2 (Kimura-2 parameter) model was found to be the best-ft model in case of the rbcL dataset whereas T92 (Tamura-3 parameter) was the best-fit model for of psbA-trnH and trnL-F datasets. The bootstrap consensus tree inferred from 1000 replicates 62 was taken to represent the evolutionary history of the taxa analyzed 62 . Initial tree (s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the corresponding best-fit model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+ G, parameter = 10.3173 for psbA-trnH, 1.8565 for rbcL and 3.7709 for trnL-F datasets). All positions with less than 95% site coverage were eliminated, i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). The trees with the highest log likelihood (−2272.44 for psbA-trnH, −2007.65 for rbcL and −1758.23 for trnL-F datasets) are shown as Figs. 5, 6 and 7. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches in the trees 61 . The evolutionary divergence (p-distance) between sequences was calculated using MEGA X 60 . The rate variation among sites was modeled with a gamma distribution (shape parameter = 1). Codon positions taken into consideration were 1st + 2nd + 3rd + Noncoding. All positions with less than 95% site coverage were removed with partial deletion option. In total, there were 453, 226 and 297 positions in the final datasets corresponding to rbcL, trnL-F and psbA-trnH regions respectively. The percent identity matrix was calculated using Clustal Omega (https:// www. ebi. ac. uk).