Discovery of the World’s Smallest Terrestrial Pteridophyte

Ophioglossum L. commonly known as “adder’s tongue fern”, has been of great interest due to the highest number of chromosomes in any organism so far known in biological world. Here, a new species of adder’s tongue fern has been discovered and reported from Western Ghats of India. It is prominently distinct from the other known taxa in Ophioglossaceae family. Phylogenetic analysis of three chloroplast DNA (cpDNA) regions (trnL-F, rbcL and psbA-trnH) unambiguously designate this adder’s tongue fern as the distinct lineage and is sister to the clade containing O. parvifolium and O. nudicaule. Azolla caroliniana – an aquatic fern (average size, 0.5–1.5 cm), is the smallest fern on the earth. Our discovery discloses a new species of adder’s tongue fern and ranking it among the smallest terrestrial fern in the world, attaining an average size of only 1–1.2 cm.

Description. Whole plant is minuscule, 1-1.2 cm in height; rhizome small, subglobose, tuberous, brown in colour, 0.4-0.6 cm in length, 0.2-0.3 cm in diameter; few 1-3 cm long brownish white (7-8 in numbers) roots emerges from the mid part of the rhizome; common stalk subterranean, 0.3-0.6 cm long, white in colour (Fig. 1A); rhizome bears trophophyll in set at its apex, parallel or marginally upward from the ground; trophophyll minute, 0.6-0.8 cm long, 0.4-0.6 cm wide, ovate to obovate in shape, obtuse at apex, cuneate at base, margin entire, thick and fleshy in texture, without midrib, yellowish green in shading (Fig. 1C); fertile spike inserted on adaxial side of trophophyll under trophophyll lamina, slightly yellowish green when young but brown on dry, 0.4-0.6 cm long; fertile apex very short, 0.2-0.4 cm long, 0.1 cm wide, ending with minute sterile tip; sporangia small, yellow, oppositely arranged, 4-8 in number on either side. Spores under light microscope (LM) are trilete tetrahedral type, globose, small in size (32-40 µm); presence of triradiate mark (laesurae) in the middle which is not extending up to the margins ( Fig. 2A,B). In scanning electron microscope (SEM), laesural arms are crassimarginate, less wavy and jointed up to the middle of the proximal cavity (Fig. 2F). Spores are covered with four layers; outer spore wall layer forms a distinct separated layer that may appear as a loose sack is known as perispore layer (perine) (Fig. 2D); below the perispore layer, thick, reticulate ectexine and endexine layers are present; below exospore layer, inner most endospore layer is present (intine) (Fig. 2C). The distal face is granulose to verrucate in LM but in SEM the depressed aeroles are united in to negative reticulation. In the distal face, sculpture is coarsely microrugulate-microreticulate.
Distribution and ecology. India -Gujarat state, Dang district, Jakhana village. The species grows in grassy area along with mosses on the grasslands of Jakhana village at an altitude of ~471 m, located in Ahwa forest division.
Conservation status. Currently, about 12 plants of O. malviae sp. nov. were found in the type locality. However, this area is poorly explored for the Pteridophytes diversity. Therefore, an assumption that the other population might be distributed around this area and more further explorations are needed to determine its full range distribution, the species should be considered as data deficient for now.  (Fig. 3C,F). As of now, such type of stomata were observed only in one species of family Ophioglossaceae, which is Helminthostachys zeylanica 1,[29][30][31][32] . In this regard, the new species O. malviae sp. nov. is unique among its congeners having such dome shape stomata.
However, our molecular results of trnL-F, rbcL and psbA-trnH sequence data support the status of O. malviae sp. nov. as a distinct species. It clearly and precisely indicates that it forms a sister clade (92% BS, 0.97 BPP) with a clade containing O. parvifolium and O. nudicaule (Fig. 4).
Phylogenetic relationships and genetic divergence. Significant sequence variances between O. malviae sp. nov. and other congeneric species were seen, ranging from 1.5% to 36.0% for psbA-trnH, 13.3% to 64.5% for trnL-F, and 56.7% to 59.6% for rbcL. Based on the molecular phylogenetics analysis, it is established that O. malviae sp. nov. is falls within the genus Ophioglossum. The concatenated alignment of three chloroplast genes (trnL-F, rbcL and psbA-trnH) contained 49 terminal dataset with 3325 characters. 21 likelihood trees of 2468 steps (consistency index, CI = 0.56, RI = 0.68) were generated by ML heuristic analysis. Topological comparison of ML and Bayseian are almost similar but they differ in ML bootstrap value and posterior probability number. O. malviae sp. nov. represent a distinct lineage and is sister to the clade containing O. parvifolium and O. nudicaule (Fig. 4).

Discussion
Members of Ophioglossum are well known not only to possess large amount of DNA, more than of most evolved angiosperms but some of its species display the extremes 33 misunderstood historically and has posed numerous complications for taxonomists due to the lack of morphometric characters upon which species are defined. Since, high likelihood of homoplasy is tricky and difficult in the genus Ophioglossum, morphology based species reports are known to be problematic 36 . Moreover, simple morphology and small size of Ophioglossum species poses challenges in identifying them from their related species. In the present study, a new species in the genus Ophioglossum has been identified and differentiated from all other known species using morphological, palynological and molecular methods. High degree of phenotypic distinct lineage and genetic divergence from the clade containing O. parvifolium and O. nudicaule, we described a new species (O. malviae) to which we have designated it as the smallest terrestrial pteridophyte of the world.
In ecology and evolutionary biology research, the most imperative element in the advancement and improvement of biodiversity management strategies is species delimitation. However, in many plant groups, studies on  species diversity is still poorly acknowledged, especially for those areas having exceptionally huge biodiversity and aboriginal such as Western Ghats of India.
The new species, O. malviae sp. nov. is reported from the Western Ghats of India which is home of more than 320 species of pteridophytes apart from Himalayas in the country 37 . The diversity of Pteridophytes of India appears to be well documented. However, many new species of Pteridophytes have been described from the country in recent years [38][39][40] , which highlight the need for more dedicated surveys across the country, specially in the less explored regions. Systematics & taxonomy of Indian Pteridophytes remains largely unattended and is in need of revision after incorporating detailed morphological, palynological as well as molecular data which will inevitably result in many more discoveries like this.

Methods
Morphological observations. New plant specimens were collected from the Jakhana village (20°37′36.79′′N, 73°44′31.47′′E) of Dang District, Gujarat, India during August-September, 2016. Photographs of the plant specimens were taken from the field showing their habitat. Plant specimens were collected and transferred to the laboratory for morphological as well as microscopic analysis and for herbarium preparation at Bapalal Vaidhya Botanical Research Centre (BVBRC), Gujarat, India. The study was based on examination and comparisons of specimens collected mainly from the Western Ghats regions and Central Gujarat regions of India, herbarium specimens including type collections from BLAT as well as high resolution images from SRGH (Image No: 14642, 14307, 11416, 14934, 14942, 14932, 15210, 16494, 15235, 15332) Isolation, amplification and sequencing of DNA. Sterilized collected plant material was used to extract the genomic DNA by modified method of Doyle and Doyle 41 . Quantification of isolated genomic DNA was done by the method of Sambrook et al. 42 . Purity of isolated genomic DNA was further assessed by electrophoresis in agarose gel (0.8%). Amplification was carried out by using extracted genomic DNA as template with three chloroplast DNA (cpDNA) regions (trnL-F, rbcL and psbA-trnH) [43][44][45] . All three chloroplast regions (rbcL, trnL-F, and psbA-trnH) were amplified via using a single protocol. 20 μL reaction mixture contained isolated genomic DNA template (2 μL) (1:10 dilution of the extracted DNA), forward primer (1 μL), reverse primer (1 μL), 1× final concentration of ReadyMix ™ Taq PCR reaction mix (Sigma) (10 μL) and nuclease free water (6 μL). The reaction was carried out in Thermal cycler (Applied BiosystemsVeriti ® ). PCR program was adjusted as: 94 °C for 4 minutes, 30 cycles of 94 °C for 30 seconds, 50 °C for 30 seconds, and 72 °C for 1.30 minutes, and a final elongation step at 72 °C for 10 minutes and stored at −4 °C for ∞ time. Amplified chloroplast regions (trnL-F, rbcL and psbA-trnH) were detected via agarose gel electrophoresis (1% agarose gel) under UV light by staining with ethidium bromide.
Amplified PCR products were purified using GenElute ™ PCR Clean-up kit and sequenced at Eurrofins Genomics India Pvt Ltd., Bangalore.
Alignment and phylogenetic analysis. Sequences of all three chloroplast genes were analyzed in BioEdit 7.2.5. To find out the common regions among all retrieved Ophioglossum species sequences, pairwise alignment and multiple sequence alignment (MSA) was carried out by using Clustal-W embedded in MEGA 7.0. with default settings. To evaluate congruence between different DNA regions, we analyzed each dataset (rbcL, psbA-trnH and trnL-F) separately to see if there is a similer topology. Incongruence Length Difference (ILD) test was also performed in PAUP* ver.4.0.a159 with 1000 replication of heuristic search 46 . Lower ILD value suggesting us to analysed three dataset in combination. Separate and combined molecular phylogenetic analyses were performed using Maximum likelihood (ML) and Bayesian inference (BI) methods.
To assess best fit model of phylogenetics analyses, jModeltest ver. 2.0 was used with the implemention of Akaike Information Criterion 47 . GTR + G model was set up as the best fit for psbA-trnH and trnL-F regions whereas GTR + I + G for rbcL regions. ML analyses were conducted in PAUP* ver.4.0.a159 with GTR + I + G model of nucleotide substitution using heuristic analysis of 1,000 random taxon addition using TBR (tree bisection-reconnection) branch swapping 48 . To evaluate internal node support, Bootstrap analyses (BS) were calculated by 1000 stepwise addition replicates with TBR branch swapping 49 . Bayesian inference of the phylogenetic analyses was performed in MrBayes v.3.1.2 50 and was run for independent MCMC analyses for two parallel searches from random starting trees for 5 million generations. At every thousand generations, trees were sampled. The analysis reached at a standard split frequency <0.005, and the analysis was not continued further. Twenty five percent of trees generated were discarded as burn-in and the post-burn-in samples were used as 50% majority rule consensus tree. Sequence divergence uncorrected "p-distance" was calculated by using MEGA 7.0 51 .