The genus Isoetes from India : An overview

The genus Isoetes L., is an interesting and unique pteridophyte, popularly known as “quillworts or Merlin’s grass”. The Latin meaning is a small houseleek, for an annual plant (Plinius) while it is derived from two Greek words viz., isos “equal” and etos “a year” referring to the evergreen species or to the submerged species. It is considered as the most primitive lycopods and their presence on the planet is known since Triassic period (1). It is perennial, herbaceous, heterosporous pteridophyte, found growing in a variety of aquatic habitats. About 300– 350 species are distributed in world, of which 18 species, 1 subspecies and 4 varieties have been reported from India (2). All Indian species or infraspecies are identified on the basis of velum characters, megaspore ornamentation and recent few species are on the basis of chromosome counts (3,4). Total 23 species viz. Isoetes bilaspurensis Panigrahi, I. coromandeliana L. f., I. coromandeliana subsp. thanensis Shukla, S.K. Singh, P.K. Shukla, N.K. Dubey, H. Khanam & G.K. Srivast., I. debii S. C. Sinha, I. divyadarshanii P. K. Shukla, G. K. Srivast. & S. K. Shukla & P. K. Rajagopal, I. dixitii Shende, I. indica D. D. Pant & Srivast., I. indica var. harotiensis Bhardwaj & Gena, I. fuchsii Goswami & U. S. Sharma, I. mahadevensis G. K. Srivast., D. D. Pant & P. K. Shukla, I. mirzapurensis Panigrahi and Dixit, I. panchganiensis K. Srivast., D. D. Pant & P. K. Shukla, I. panchganiensis var. kemangundiensis G. K. Srivast., D. D. Pant & P. K. Shukla, I. panchananii D. D. Pant & G. K. Srivast., I. panchananii var. pachmarhiensis D. D. Pant & G. K. Srivast., I. pantii Goswami & Arya, I. pantii var. hybrida Goswami, I. rajasthanensis Gena & Bhardwaj, I. reticulata Gena & Bhardwaj, I. sampathkumaranii L. N. Rao, I. sahyadrii Mahable and I. tuberculata Gena & Bhardwaj, I. udupiensis P. K. Shukla, G. K. Srivast., S. K. Shukla & P. K. Rajagopal have been reported from India by earlier researchers. Amongst these, only four species viz., I. coromandeliana L.f., I. dixitii, I. sahyadrii and I. udupiensis, is accepted in annotated checklist of


Introduction
The genus Isoetes L., is an interesting and unique pteridophyte, popularly known as "quillworts or Merlin's grass".The Latin meaning is a small houseleek, for an annual plant (Plinius) while it is derived from two Greek words viz., isos "equal" and etos "a year" referring to the evergreen species or to the submerged species.It is considered as the most primitive lycopods and their presence on the planet is known since Triassic period (1).It is perennial, herbaceous, heterosporous pteridophyte, found growing in a variety of aquatic habitats.About 300-350 species are distributed in world, of which 18 species, 1 subspecies and 4 varieties have been reported from India (2).All Indian species or infraspecies are identified on the basis of velum characters, megaspore ornamentation and recent few species are on the basis of chromosome counts (3,4).
Total 23 species viz.Isoetes bilaspurensis pteridophytes of India -I (5).There are several species published by various authors that are yet to be recognised as independent species.Therefore, there is an urgent need to revise the Isoetes from India.The revision should be coupled with data on their ecology, phenology, spore morphology, cytology and molecular studies.

Taxonomic history of Isoetes
The name Isoetes was first introduced by Linnaeus (6) in his book "Species Plantarum".However, earlier botanist had described similar plants under different generic name viz., Marsilea, Calamaria and Subularia (7).Linnaeus (6) had described the genus Isoetes as a seed bearing plant produces unisexual flower without calyx and corolla.He also mentioned that capsule was hidden in the basal portion of the leaf and having many seeds per capsule.However, his son Linnaeus filius (8) described a new species, i.e.Isoetes coromandeliana, from Coromandel Coast, Tamil Nadu.Thereafter, Reichenback (9) introduced the family Isoetaceae to include the genus Isoetes.Subsequently, Braun (10,11) published a series of paper on Isoetes from Germany, Australia and Sardinia.Motelay and Vendryes (12) described nearly 47 species of Isoetes in their publication "Monographic der Isoeteae".Pfeiffer ( 13) also published a monograph on "Isoetaceae", while "Index Isoetales" was published by Reed (7) and suggested nomenclatural changes in the genus Isoetes.Taxonomy and spore morphology of Isoetes from North Eastern America was investigated by Kott and Britton (14).In last few decades modern tools like rbcL, nuclear and chloroplast DNA sequences have been used to study the cytology, and Karyology of Isoetes (15,16,17).
As far as Indian Isoetes history is concerned, Linnaeus filius (8)

Distribution of the Isoetes in India
The genus Isoetes is a semi-aquatic species, onetwo or more than two species are recorded from almost all the states of India (excluding few north Indian states).There are few reports on the distributional studies (32,35).Analysis of species distribution indicates that Isoetes coromandeliana is the most common species and it is recorded from almost all the states of India (except some Northern States).After I. coromandeliana, Isoetes indica is another common species that has been more frequently reported from different states viz., Madhya Pradesh, Maharashtra, Karnataka, Gujarat, West Bengal and Tamil Nadu (3) while others species have their restricted distribution.
A single species Isoetes debii is reported from North-East India and none of other species are reported from this region.Similarly, I. mirzapurensis is reported from Mirzapur (Utter Pradesh).
Regarding endemism, species like Isoetes rajasthanensis, I. reticulata and I. tuberculata are endemic to Rajasthan (India).Similarly, few species viz., I. panchananii, I. pantii, I. bilaspurensis, I. fuchsii, I. mahadevensis and few varieties viz., I. indica var.harotiensis, I. pantii var.hybrida and I. panchananii var.pachmarhiensis has been reported from Madhya Pradesh and are endemic to this region except I. panchananii that is reported from Maharashtra.Species like Isoetes sahyadrii, I. dixitii and I. panchganiensis are described from Table-Land Plateau Panchgani (Maharashtra).These species have been collected from different regions of Maharashtra (Mahabaleshwar, Kas plateau and Recently, Isoetes divyadarshanii is described from Maharashtra by Shukla et al. (34).The species I. sampathkumaranii, I. udupiensis and a variety I. panchganiensis var.kemangundiensis are endemic to Karnataka.
A single subspecies of Isoetes coromandeliana subsp.thanensis has been reported from Than, Gujarat state (2).
Madhya Pradesh shelters seven species and three varieties of Isoetes, hence it appears to be most suitable habitat for Isoetes.Followed by Karnataka, which has 5 species and 1 variety, Maharashtra possesses 5 species and Rajasthan that has 4 species.In contrast, only one species is reported from North East India i.e. from Manipur.

Mount
Abu, Panchmari, Panchgani and Bababuddhangiri are the ideal hills that favor growth, development and speciation of the quillworts in India.The state wise distribution of each species is given in Fig. 1.

Phytogeography distribution of Isoetes in India
Phytogeographically, India is situated at the trijunction of three realms-Afro-tropical, Indo-Malayan and Paleo-Arctic realms.India is one of the 12 mega biodiversity countries of the world and has a very rich and diverse heritage of biodiversity, including a wide range of habitats from tropical rainforests to alpine vegetation and from temperate forests to coastal wetlands including typical desert like great Rann of Kutchh.It provides shelter for many rare, threatened and endemic species that have massive ecological and commercial value.This assemblage of three different realms makes the country rich and unique in biological diversity.The subcontinent is bounded by efficient barriers like the Indian Ocean, the Himalayan ranges, the Western Ghats and the deserts of Sindh.
Whole country is divided into 10 biogeographic regions, including the Trans-Himalayan, the Himalayan, the Indian desert, the semi-arid zone(s), the Western Ghats, the Deccan Peninsula, the Gangetic Plain, North-East India, and the islands and coasts (36).The phytogeographic distribution of 12 species of Isoetes is studied by Shukla et al. (33), whereas remaining 11 species of Isoetes is given here in Fig. 2.
No records on Isoetes species are found from the Trans-Himalayan, the Himalayan region and The India desert.The maximum species were reported from the Deccan Peninsula ( 8 the Semi-arid zone possess 5 species and 1 subspecies (viz., Isoetes coromandeliana, I. indica, I. rajasthanensis, I. reticulata, Isoetes tuberculata and I. coromandeliana subsp.thanensis) whereas remaining phytogeographic zones having one, two or maximum three species.Among all these species I. coromandeliana is common and found in all phytogeographic regions.

Morphology and Palynology
Main characters like rhizomorph lobes, plant length, number of sporophylls, ligules, velum and megaspore ornamentation are used in the delimitation of different species of Isoetes.
Rhizomorph or corm is the underground, reduced stem and are bi-lobed or tri-lobed, rarely tetra-lobed or penta-lobed.This character is usually constant and reliable hence used to identify the species (3).Based on the lobe of rhizomorph all species of Indian Isoetes are grouped into two main categories i.e. bi-lobed and tri-lobed.
Bi-lobed category includes the species like Isoetes mahadevensis, I. Plant length and number of sporophylls are concerned with an expression of different degrees of ploidy levels and genomic constitution of the plants.Every species is having minimum and maximum number of sporophylls and length of sporophylls.On the basis of plant length and number of sporophylls different species can be identified.However, it may be influenced by soil type and climatic factors hence may be questionable and not much reliable one.
On the basis of plant length all Indian Isoetes are grouped into three categories i.e. small sized plant (height <20cm), medium sized plants (height >20 <40) and large sized plants (height >40 cm).Megaspores reticulate, at least on basal face e) Lavigate: Megaspores having fine powder Majority of Isoetes species from India were described on the basis of megaspore ornamentation.All Indian species are belonging to section tuberculatae and reticulate (Table 1, 2 and 3; Fig. 4) whereas no Indian taxa which could be assigned to the sections echinatae, cristatae and lavigate.In the section tuberculatae exine of megaspores having tubercles, they are either round apices or pointed apices (e.g.I. indica) whereas in reticulate section exine of megaspores having reticulated striations.
After a critical review of literature and observation of morphological as well as palynological characters, it was observed that "the species that are having velum possess reticulate spores except (Isoetes dixitii and I. sahyadrii) while species lacking velum show presence of tuberculate megaspore.On these observations, it may be concluded that I. dixitii and I. sahyadrii are the intermediate forms of tuberculate and reticulate spore groups.Similarly, species having reticulate megaspores possess bi-lobed rhizomorph while species having tuberculate megaspores show presence of tri-lobed rhizomorph.

Anatomy
Anatomy has always found to plays an important role as a supplementary discipline in identification or delimitation of taxa.However, in pteridophytes anatomical characters are rarely used for the identification.Anatomically Isoetes is characterized by presence of peripheral strands, stomata and air chambers.Amongst these, presence of peripheral strands is an important one while character like stomata and air chamber are used as supporting characters.Peripheral strand is an anatomical feature, which associated with "conductive tissue" present at the corners of leaves" and additionally it also provides mechanical strength to sporophylls in order to keep them stand erect.Taxonomically it is important feature that is used for the identification of closely related species (2,3,20,23,29,34,40).
The peripheral strands are present in the species viz., I.oromandeliana, I. coromandeliana subsp.thanensis, I. divyadarshanii, I. dixitii, I. indica, I. pantii, I. tuberculata and I. udupiensis whereas they are absent in Isoetes mahadevensis, I. panchganiensis, I. panchganiensis var.kemangundiensis, I. panchananii, I. panchananii var.pachmarhiensis, I. reticulata, I. sahyadrii and I. sampatkumarnii.However, it's presence or absence is unknown due to lack of studies on species viz., I. bilaspurensis, I. debii, I. fuchsii, I. indica var.harotiensis, I. mirzapurensis, I. pantii var.hybrida and I. rajasthanensis (2,3,20,23,29,34,40).It is interesting to note that all those species lacking peripheral strands are having reticulate spores while those species having peripheral strands show presence of tuberculate spores (expect I. sahyadrii).Therefore, above mentioned observation also support our previous statement that I. sahyadrii is the intermediate forms of tuberculate and reticulate spore groups.udupiensis spore germination starts after first shower of rains i.e. during June-July, become mature and produces spores during the month of August-September and undergoes dominant condition after October-November.However, in Isoetes panchganiensis germination starts at the end of rainy season i.e. in the month of September, saplings developed from spores become mature and produces sporangia in the month of November-December and undergoes dormant condition after January (2,34,40).Similar information is warranted on the remaining species and they are yet to be studied for the phenological events occurring in their life cycle.

Cytology
Cytological studies on Isoetes were initiated by Ekambaram and Venkatanathan (41) and described the process of sporogenesis in I. coromandeliana and reported that formation of two sterile and two fertile spores from a tetrad.Abraham and Ninan (42) studied cytology of I. coromandeliana and documented 2n = 22 (Table 1) while cytological and phylogenic studies of family Isoetaceae were carried out by Ninan (43).Verma (44) studied the cytology of I. coromandeliana explored the account of enucleate and nucleate spores.Subsequently, several cytological studies were also carried out by earlier researchers (45,46,47,48,49,50).Tripathi and Yadav (51) also described the cytology of Isoetes from Rajasthan and reported diploid, tetraploid and pentaploid conditions.
Cytological study conducted by Goswami and Bhu (52) led to the conclusion that I. pantii is a natural hybrid of I. sampathkumaranii and I. coromandeliana.Recently, Shukla et al. (2,34) described two new species and one subspecies on the basis of chromosome number.Nevertheless, several species of Isoetes remains to be explored for their cytology and further studies are warranted on this line (Table 2, 3 & 5).

Numerical Taxonomy
The morphometric study was also performed to delineate the taxa of Isoetes at the species and infra-species level.For numerical taxonomic study the 20-state characters are used the species as well as infra-species level with coded in a data matrix as '1' and '0' respectively (Table 1).They are viz., habitat characters (amphibious or submerged or terrestrial), plant size (< 20 cm, > 20 cm but <40 cm and > 40 cm), rhizomorph lobbing (bilobed or trilobed), ligule (cordate or deltoid), peripheral strands (present or absent), velum (present or absent), velum coverage (rudimentary, half, 3/4 or complete), microspore ornamentation (reticulate or tuberculate), megaspores nature (dimorphic, monomorpic or trimorphic) and megaspore ornamentation (reticulate or tuberculate).The dendrogram was generated by Bary-Curtis cluster analysis.In this dendrogram all the Isoetes L.f., species are fall under two major clades i.e. tuberculate megaspores with or without peripheral strands and reticulate spores without peripheral strands (Fig. 5).His conclusion appears to be based on study carried out on dry specimens from various herbaria and dry specimen may lose some fine details (like ligule, velum, etc.) during drying and these structures are important in delimitation of taxa.However, Isoetes is delicate fern with poorly developed vasculature; therefore, thorough revision is required to resolve the issue related with number of species in India.

Conclusion
After reviewing the genus Isoetes from India, it is observed that all 23 species are grouped into two groups i.e. reticulate and tuberculate on the basis of their spore morphology.Further it is also observed that reticulate species having bi-lobed rhizomorph, sporangia covered with velum and lacking peripheral strands.In contrast, tuberculate species show tri-lobed rhizomorph, sporangia without velum and show presence of peripheral strands (except Isoetes dixitii, I. sahyadrii and I. bilaspurensis).Another thing is that, due to high endemism and lack of data on cytology, is not quantifiable the taxonomic problems with reference to delimitation of species and excess naming of spore-variants as species.Moreover, morphology of certain species like Isoetes fuchsii, I. pantii var.hybrida and I. mirzapurensis is not known.Therefore, present study emphasizes the necessity of the inventorization by continuous searching and re-examination of different species of Isoetes distributed in India.Present study also suggest need of further studies to explore the taxonomy of the Indian Isoetes, coupled with the data on their ecology, phenology, spore morphology, cytology and molecular studies.

Fig. 5 :
Fig. 5: cluster analysis of the genus Isoetes L. from India Isoetes fuchsii is described by Bhu et al. (32) from Madhya Pradesh and stated as an intermediate form of I. coromandeliana and I. sampathkumaranii.Shukla et al. (33) studied distribution, endemism and species radiation of quillworts in India.Subsequently Shukla et al. (34) described two new species viz., I. udupiensis and I. divyadarshanii from Western Ghats of India.
was the first to describe a first species i.e.Isoetes coromandeliana from Coromandel Coast, Tamil Nadu.Later on Engelmann and Butler (18) reported the occurrence of Isoetes from Indian Territory.Thereafter, McCann (19) published a note on Isoetes of Bombay Presidency.Subsequently, Mahabale (20) described a new species I. sahyadrii (misspelled I. sahyadriensis) from Table-Land Plateau of Panchgani, Maharashtra, I. sampathkumaranii from Baba Budangiri hills, Karnataka by Rao (21), I. dixitii (misspelled I. dixitei) from Table-Land Plateau of Panchgani by Shende (22).Pant and Srivastava (23) reviewed the genus Isoetes in India and described two more new species i.e.I. panchananii and I. indica on the basis of velum character and morphology of plants and spores.Goswami and Arya (24) described another species as I. pantii from Madhya Pradesh.Panigrahi (25) second time reviewed taxonomy of Indian Isoetes and described two more new species, viz.I. bilaspurensis and I. mirzapurensis.Subsequently, additional species,

Table 1 :
Two-state characters used for Numerical Taxonomy I. divyadarshanii, I. indica, I. sahyadrii and I.

Table 2 :
Important characteristics of Indian Isoetes species along with their distribution and sporophytic count

Table 3 :
Important characteristics of Indian Isoetes species along with their distribution and sporophytic count

Table 4 :
Important characteristics of Indian Isoetes species along with their distribution and sporophytic countChandra et al. (53)studied status of threatened pteridophytes of India and commented on the doubtful taxonomic status of the Indian species.Fraser-Jenkins (54) considered the existence of at least two species of Isoetes in India, from which I. coromandeliana is a common one, while name of the second species remains undecided.Fraser-Jenkins (54) did the lectotypification and epitypification of I. sahyadriensis and decided a second species of Isoetes from India.In recent study, Fraser-Jenkins (5) accepted four species of Isoetes (viz., I. coromandeliana, I. dixitii, I. sahyadrii (i.e.I. sahyadriensis) and I. udupiensis from India in his publication "An Annotated Checklist of Indian Pteridophytes Part -1.He also mentioned about 25 species (19 species, 4 varieties and one subspecies) have been described by earlier researchers cataloguing variation in their micromorphology of megaspore, its types, their ornamentation, peripheral strands, velum, ligules, presence of different spores and fused spores in the sporangium.However, his critical observations on above mentioned features led him to conclude occurrence of only four species in India.