Pleione 11(2): 241 - 248. 2017. ISSN: 0973-9467 © East Himalayan Society for Spermatophyte Taxonomy doi:10.26679/Pleione.11.2.2017.241-248

Observations on indusia in Adiantum L. (Pteridaceae : Vittarioideae) of North-East India

S. D. Yumkham1, P. K. Singh1 and S. D. Khomdram2 1Ethnobotany & Plant Physiology Laboratory, Centre of Advance Studies in Life Sciences, Manipur University, Canchipur - 795 003, Manipur, India 2Corresponding author: Department of Botany, Mizoram University, Aizawl-796004, Mizoram, India E-mail: [email protected] [Received 09.10.2017; Revised 29.11.2017; Accepted 07.12.2017; Published 31.12.2017]

Abstract The present paper highlights the indusial character in eight (8) species of Adiantum L. (Pteridaceae- Vittarioideae) found in North East India. These include A. capillus-veneris L., A. caudatum L., A. edgeworthii Hook., A. flabellulatum L., A. incisum Forssk., A. peruvianum Klotzsch, A. philippense L. and A. raddianum C. Presl. Data on the morphology of indusia, spore size and exine ornamentation are studied in order to assess their systematic significance. A key to species based on indusial characters is also incorporated. Key words: Adiantum, North East India, Morphology, Indusia, Exine ornamentation

INTRODUCTION The Maiden-hair ferns, Adiantum L. (Pteridaceae: Vittarioideae) are well known and popular as ornamentals for their beauty with graceful and attractive evergreen fronds. The genus is represented by 200 species distributed in tropical and sub-tropical to temperate regions (Prado et al. 2007). They usually grow in moisture rich areas with low intensity of sunlight. Sometimes, they are seen growing as base epiphyte on moss-humus laden trees like Ficus benghalensis L., Mimusops elengi L., Phoenix sylvestris (L.) Roxb., Kigelia pinnata (Jacq.) DC. etc. This group of ferns is regarded as a wonder herb and found using in various traditional therapies including the Chinese Herbal Medicine, Pharmacopoeia of United States, Indian Medicine of Ayurveda and Homeopathy. They possess various medicinal, pharmacological properties and used in traditional healing system of various communities. Many alkaloids and bioactive components responsible for their therapeutic properties have been isolated and identified (Brahmachari et al. 2003). It is used in curing various skin diseases, diabetes, fever, bronchial disease, bone fracture, respiratory problems, snake bite, asthma etc. (Ansari & Ekhlasi-Kazaj 2012; Saha et al. 2016). Some of the important bioactive compounds responsible for their medicinal properties are isoadiantone, hydroxyadiantone, â- Sitosterol, daucosterol, di-tert-butyl p-cresol, 16-hentriacontanone, hentriacontane, isoquercetin, neohop-13 (18)-ene, nonanoic acid, n-decanoic acid etc. (Gupta et al. 1990; Shiojima & Ageta 1994; Tsuzuki et al. 2001). Reproduction in Adiantum mainly takes place asexually through sporulation in which thousands of spores are produced inside a sporangium. A group of sporangium form sporangial clusters called sori which are again protected by a membranous covering called indusium. This extra protective covering is a unique feature encompassing the pteridophytes. In ferns, indusia differ in shape, size and structure according to different 242 Indusia structure in Adiantum L. species. Some of the unique indusia in Polypodiales are cup-shaped in Cyathea Sm., two lipped in Dennstaedtia Bernhard, kidney-shaped (reniform) in Dryopteris Adans., linear in Asplenium L. etc. In fact, indusia played a key role in the systematics of many ferns. Indusia may be either true or false based on their origin. True indusium originates from lower side of pinnae as an epidermal outgrowth (Dryopteris). In Adiantum, the indusium is formed by the curving of pinnae margin and regarded as false indusia. The shape and placement of indusia, mode of sporangia dehiscence, nature of spore, ornamentation of exine layer etc. plays a significant role in their systematics. It serves as a reliable source of unit to circumscribe specificity or to establish hypothetical relationships between or among species within a genus (Devi 1980; Das & Dixit 2001). In the present paper, the morphology of indusia in eight (8) species of maiden ferns (A. capillus-veneris L., A. caudatum L., A. edgeworthii Hook., A. flabellulatum L., A. incisum Forssk., A. peruvianum Klotzsch, A. philippense L., A. raddianum C. Presl) found in North-East India is critically studied. Details on spore structure, shape, size, exine ornamentation and key to species based on indusial characters are also incorporated.

MATERIALS AND METHODS The present study was conducted from June 2015 to June 2017 in the seven North-Eastern (NE) states of India (except Sikkim) viz Arunachal Pradesh (AP), Assam, Manipur, Meghalaya, Mizoram, Nagaland and Tripura. Identification was based on data recorded from mature sporophytes, indusia, spores and exine ornamentation. An updated nomenclature is provided based on Smith et al. (2006), Brummit and Powell (1992), and the Plant List (www.theplantlist.org). Photomicrographs of indusia were taken from live specimen using Motic BA 210 and Cooling Tech USB-LED Microscope. For Scanning Electron Microscopy (SEM, Quanta 250, Manipur University), dried undamaged and unacetolysed spores were fixed on brass stubs by electron conductive point and kept on the iron sputter after coating with gold vapour (300 AU). Size of spores is expressed as polar diameter × equatorial diameter and determined from the average of 10 readings. Spore terminology by Devi (1977) and Punt et al. (2007) is followed to describe the characters of spores. Voucher specimens were prepared according to Jain and Rao (1977) and deposited in Manipur University Museum of Plants (MUMP). A comparison on morphology of indusium, spore size and ornamentation of the eight (8) species is highlighted in Table 1. A key to species and illustration on fertile fronds, indusia and spores are also incorporated (PLATES I & II).

RESULTS AND DISCUSSION In maiden-hair ferns, indusia are formed by folded over ends of pinnae/ pinnules and therefore called as false indusium. It protects the sporangial cluster until it matures from external environments like extreme dryness, rain, pest, wind etc. During the present investigation, eight (8) species of Adiantum (A. capillus-veneris, A. caudatum, A. edgeworthii, A. flabellulatum, A. incisum, A. peruvianum, A. philippense, A. raddianum) were collected from different places of NE India. A. Indusia structure: We observed that the indusia among the eight species differ a lot from each other. The shape, persistent or deciduous nature of indusia, presence or absence of indusial hairs and number of sori per pinnae are very unique for each species. In A. capillus-veneris, indusia are orbicular-reniform in shape and present at the edges of wedge- shaped pinna. They are membranous, pale coloured, turn black with maturity, persistent and devoid of hairs. A single pinna bears around 4 – 9 indusia (PLATE I: A – C). Indusium in A. caudatum is orbicular-oblong in shaped with straight unicellular hairs and single pinna bears S.D. Yumkham et al. 243

PLATE – I: Fronds of Maidenhair Ferns showing indusia and stages of maturation. A – C. A. capillus-veneris; D – F. A. caudatum; G – I. A. edgeworthii; J – L. A. incisum 244 Indusia structure in Adiantum L. 8 – 17 clusters of sori. The hairs are translucent, stiff and remain attached to the indusia even when the colour of indusia turns brown from green. On maturity, the indusia begins to tear off (deciduous nature) from inner side of pinnae (PLATE I: D – F). Indusia in A. edgeworthii are either orbicular-oblong or reniform in shaped, membranous, bear no hairs and presence of faint lines resembling veinlets. In extreme environmental condition like limited rainfall and excessive exposure to sunlight, the indusium shrinks in size and assumes oval to round shaped. Basal and middle pinnae usually bears around 4 – 6 sori, however the number of sori in upper pinnae tend to decrease. Indusia are deciduous in nature (PLATE I: G – I). In case of A. flabellulatum, indusia are semi-orbicular to elliptical in shaped, devoid of hairs and remain persistent. About 4 – 7 sori are present on dimidiate semi-orbicular pinnae (PLATE II: A & B). The indusia in A. incisum resembles more or less to that of A. caudatum, except that here the indusia are slightly oblong-lunate in shaped and the number of sori per pinnae ranges from 4 – 8 in number. Indusia are deciduous and long stiff translucent unicellular hairs are also present. The indusial flap begins to tear off first from the inner side and completely dispatched off at the margin of the pinnae thus exposing bunches of sporangia (PLATE I: J – L). The Silver Dollar Maiden Fern, A. peruvianum also possess unique indusia at the abaxial surface of the pinnae very different from the rest of the maidens. The indusia are reniform in shaped and thick in texture rather than the usual membranous nature as in case of other maiden ferns. Around 18 – 23 sori are present on the large curvy pinnae and arranged very close to one another. Indusia are glabrous and deciduous on maturity (PLATE II: C & D). In A. philippense, the indusia are large and linear-oblong in shaped. The upper margin remain slightly depressed compared to the rest of the species and is membranous without any hairs. The inner margin of the indusia is wavy and remains attached to the surface of the pinnae mainly at the area where the veinlets are present. Number of sori per pinna is 3 – 5 (PLATE II: E & F). The indusia in A. raddianum are reniform, small in size, translucent with 2 – 8 sori per pinna and arranged beautifully in U-shaped pattern at the edge of veins of pinnae. It was observed that the indusia shrivel and shades off only when the sporangia are ready enough for dehiscence (PLATE II: G). B. Dehiscence of sporangia: In Adiantum, the sporangia remain fixed to the surface of pinnae by slender stalks. It may range from 110 ìm to 140 ìm in length. Each sporangium consists of a rounded sac which is composed of a single layer of cells towards the outer rim called annulus. This annulus which numbers around 11 – 13 in Adiantum extends upto two- third of the entire length of the sporangium. These are dead water filled cells with thinner outer walls as compared to the inner wall. During extreme environmental condition, dehydration takes place from the annulus and leads in distortion of outer wall. This causes annulus to peel backward and transverse dehiscence takes place in a specialized region called stomium. The tensile strength of water is gradually decrease with loss of water and leads to snapping back of annulus. In this process, the spores are catapulted effectively and dispersal takes place in Adiantum. After release of spores, the leaves slowly start to degenerate. C. Spores and ornamentation: With maturation, indusial flap opens up exposing a bunch of sporangia (PLATE I: F, I, L). The spores are produced inside the sporangium and get exposed in the air through the stomium. In most of the Adiantum species, overproduction of spores takes place which is a mechanism for ensuring the perpetuation of the species (Gifford & Foster 1989). According to Poppinga et al. (2015), a mature A. peruvianum plant with five small and three large fronds, possessing ~300 pinnules with ~6000 false indusia produces ~1, 600, 000 sporangia each producing 64 spores. This generally means that the entire genus of Adiantum produces large number of spores. These enable effective dissemination and check the natural gene flow, maintenance of population and colonization of new habitats. S.D. Yumkham et al. 245

PLATE – II: Indusia and exine ornamentation in spores of Maidenhair Ferns. A – B. Indusia in A. flabellulatum; C – D. Reniform indusia in A. peruvianum; E – F. Linear- oblong indusia in A. philippense; G. Sori embedded below indusia in A. raddianum; H. Granulose exine in A. capillus-veneris; I. Verrucate exine in A. caudatum; J. Faintly granulose exine in A. edgeworthii; K. Densely verrucate exine in A. incisum; L. Verrucate exine in A. flabellatum; M. Granulose-smooth exine of A. peruvianum; N. Close-up view of verrucate exine in A. philippense; O. Granulose exine of A. raddianum 246 Indusia structure in Adiantum L. Table 1. List of species of Adiantum found in NE India along with their occurrence, uses, indusial and sporal characters.

Species Occurrence in NE Uses Indusium shape & Spore size & India character ornamentation A. capillus-veneris Entire NE region, very Dried powder Orbicular-reniform, (42×57) µm, exine L.; Yumkham - common, 700 – 1300 m taken as herbal tea persistent, hairs absent, 4–9 layer 2.4 µm thick, 003492 sori per pinna granulose (Fig. 2H) A. caudatum L.; Entire NE region, Sold in market as Orbicular-oblong, (39×44) µm, exine Yumkham - 003493 common, 800 – 1300 m basket ferns deciduous, hairs present, 8– layer 2.7 µm thick, 17 sori per pinna verrucate (Fig. 2I) A. edgeworthii Meghalaya, AP, Taken as herbal Orbicular-oblong or (32×35) µm, exine 2.5 Hook.; Yumkham - Manipur, Not common, tea in Mao area of reniform, deciduous, hairs µm thick, exine faintly 003494 found in hills above Manipur absent, 4–6 sori per pinna granulose (Fig. 2J) 1000 m A. flabellulatum L.; Assam, AP, Manipur, Semi-orbicular or elliptical, (32×43) µm, exine 3 Yumkham - 003495 Nagaland; not persistent, hairs absent, 4– µm thick, exine common, grows in - 7 sori per pinna verrucate (Fig. 2L) acidic red soil and strives well in pine forest floor, below 1000 m A. incisum Forssk.; Manipur; not common, Whole plant Oblong-lunate, deciduous, (35×40) µm, exine 3.5 Yumkham - 003491 grows in rock crevices extract as fish hairs present, 4–8 sori per µm thick, densely above 1100 m altitude poison by pinna verrucate (Fig. 2K) Monsang tribe of Manipur A. peruvianum Introduced species in Highly Reniform, deciduous, hairs (30×38) µm, exine 3 Klotzsch; Yumkham NE India; grows in ornamental, sold absent, 18–23 sori per µm thick, granulose to - 003490 rainforest, above 1500 in market pinna smooth (Fig. 2M) m A. philippense L.; Entire NE region; Dried powder Linear-oblong, deciduous, (32×45) µm, exine 3.3 Yumkham - 003489 common in wet humid taken as herbal hairs absent, 3–5 sori per µm thick, finely places, along water drink in diabetes pinna verrucate (Fig. 2N) courses, indicator of by locals in Assam acidic soil A. raddianum C. Tropical rainforest; Highly Reniform, deciduous, hairs (42×48) µm, exine 2 Presl; Yumkham - exotic, naturalized in ornamental, sold absent, 2–8 sori per pinna µm thick, faintly 003488 many parts of NE India in markets granulose (Fig. 2O) like Meghalaya and Assam Spores exhibit specific characteristics and served as an important criterion in taxonomic and phyletic studies. In Adiantum, spores are trilete (characterized with three radiating arms from the proximal pole) and tetrahedral in shape. Usually spores are dark-brown in colour and remain enveloped by a perine which is an extra-exinous sporoderm layer enveloping the exine. Exine ornamentation is granulose (granules like protuberances which are less than 1µm) in case of A. capillus-veneris, A. edgeworthii, and A. raddianum. Spores of A. caudatum, A. flabellulatum, A. incisum and A. philippense exhibit verrucate ornamentation. These are warty protuberances which are hemispherical in shape. In A. peruvianum, inconspicuous granules are distributed irregularly and interrupted by smooth exine surfaces in between them. In Table 1, list of maidens found in NE India along with details on their occurrence, indusium and spore ornamentation in exine layer are highlighted. CONCLUSION The present study is an attempt to include indusial characters as one of the reliable taxonomic parameters in the identification of Adiantum. Most of the fern researchers tend to overlook S.D. Yumkham et al. 247

An artificial Key for distinguishing the seven species of Adiantum L. found in NE India based on indusial characters: 1a. Indusia persistent, hairs absent ..………………..….………..… 2 1b. Indusia non-persistent, hairs present or absent .....…….….…… 3 2a. Orbicular-reniform, 4–9 sori per pinna …………….….... A. capillus-veneris 2b. Semi-orbicular or elliptical, 4–7 sori per pinna ..…..…..… A. flabellulatum 3a. Hairs present ...……….………………………………..……… 4 3b. Hairs absent …………………………………………..…..…… 5 4a. Orbicular-oblong, 8–17 sori per pinna ……………….….. A. caudatum 4b. Oblong-lunulate, 4–8 sori per pinna ………….….……… A. incisum 5a. Reniform shape ...……………………………………………… 6 5b. Non-reniform shape ……………………………………...…… 7 6a. 18–23 sori per pinna ……………………….………..….. A. peruvianum 6b. 2–8 sori per pinna ………………………………………...A. raddianum 7a. Orbicular-oblong, 4–6 sori per pinna ...……………….….. A. edgeworthii 7b. Linear-oblong, 3–5 sori per pinna .……...…………….…. A. philippense the indusial characters, and emphasizes on other domain characters of the sporophytic body. It is thus concluded that studies on indusia can be used as a reliable source not only in the Maidenhair ferns, but also in other Pteridophytes.

Acknowledgements The present work was supported by Science & Engineering Board (SERB), New Delhi, India (Grant No. SB/YS/LS-138/2014). Authors are thankful to the Department of Physics (Manipur University) for the SEM photographs and other relevant data. Thanks are due to all locals, florists, forest dwellers and folks who extended their kind cooperation during the survey.

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