Lemanea Manipurensis Sp. Nov. (Batrachospermales), a Freshwater Red Algal Species from North-East India

Home , Batrachospermales, Chitral (princely state), Compsopogon, Lemanea fluviatilis, Sirodotia

Research Article

Algae 2015, 30(1): 1-13 http://dx.doi.org/10.4490/algae.2015.30.1.001 Open Access

Lemanea manipurensis sp. nov. (Batrachospermales), a freshwater red algal species from North-East India

E. K. Ganesan1,a, J. A. West2,*, G. C. Zuccarello3, S. Loiseaux de Goër4 and J. Rout5 1Instituto Oceanográfico, Universidad de Oriente, Cumaná, 6101, Venezuela 2School of Botany, University of Melbourne, Parkville, VIC 3010, Australia 3School of Biological Sciences, Victoria University of Wellington, Wellington, 6140, New Zealand 411 Rue des Moguerou, 29680 Roscoff, France 5Department of Ecology and Environmental Science, Assam University, Silchar, 788011, Assam, India

A new macroscopic riverine red algal species, Lemanea manipurensis sp. nov. (Batrachospermales) is described from Manipur in northeast India. It has a sparsely branched, pseudoparenchymatous thallus with a single, central axial filament that lacks cortical filaments. Spermatangia occur generally in isolated, low and indistinct patches or form an almost continuous ring around the axis. Carposporophytes project into the hollow thallus cavity without an ostiole. The most striking morphological feature is the carposporophyte with very short gonimoblast filaments having cylindrical, narrow and sparsely branched sterile filaments, the terminal cell of each branch with a single, large, elongate carpospore. The widely distributed L. fluviatilis has spherical carpospores in long branched chains. Phylogenetic analysis of rbcL sequence data and comparison with other Batrachospermales clearly show that our specimens do not align with other species of Lemanea and Paralemanea investigated thus far. Five specific names attributed in previous literature (1973- 2014) to Lemanea from Manipur, L. australis, L. catenata, L. fluviatilis, L. mamillosa, and L. torulosa are rejected until critical anatomical and molecular evidence is available for specimens from the Manipur river systems. Taxa referable to Paralemanea were not confirmed for India in this study. In view of the high demand for food and medical uses of L. manipurensis in northeast India, conservation measures are needed for its long term survival. The present paper constitutes the first combined morphological / molecular study on a freshwater red alga from India.

Key Words: Batrachospermales; carpospores; India; Lemanea manipurensis sp. nov.; Manipur; molecular phylogeny; rbcL; spermatangia

INTRODUCTION

The red algae occur widely in marine habitats and less parenchymatous morphologies. The molecular phylog- commonly in terrestrial and freshwater habitats as unicel- eny and classification are somewhat controversial. The lular, colonial, simple filamentous, flat-bladed and more order includes two families, Batrachospermaceae with complex pseudoparenchymatous forms with quite varied 6 genera (Batrachospermum, Nothocladus, Petrohua, reproductive patterns. The order Batrachospermales is an Psilosiphon, Sirodotia and Tuomeya) and Lemaneaceae exclusively freshwater group comprised of three distinct with two genera [Lemanea Bory (1808) and Paralema- evolutionary lineages, all with uniaxial shoots, pseudo- nea (Silva) Vis and Sheath (Vis and Sheath 1992)]. Lema-

This is an Open Access article distributed under the Received January 13, 2015, Accepted March 12, 2015 terms of the Creative Commons Attribution Non-Com- Corresponding Author mercial License (http://creativecommons.org/licenses/by-nc/3.0/) which * permits unrestricted non-commercial use, distribution, and reproduction E-mail: [email protected] in any medium, provided the original work is properly cited. Tel: +61-3-8344-8080, Fax: +61-3-9347-5460 aPresent address: 3-A Srinivas Terrace, 52, II Main Road, Gandhi Nagar, Adyar, Chennai, 600020, Tamil Nadu, India

A B

C D

Fig. 1. (A) Lemanea manipurensis growing on stones from Chakpi River. (B) Woman selling freshly collected Lemanea (Nungsham) at a market in Manipur. (C) Packet (100 g) of dried Nungsham for sale on the internet (20 rupees). (D) Map of NE India showing Manipur and other states in the Indo-Burma Biodiversity Hotspot region. Scale bar represents: A, 5 mm.

nea and Paralemanea occur in boreal to warm temper- algae recorded in India, Lemanea has been reported for ate regions of the northern hemisphere (Vis and Sheath more than 4 decades (1973-2015) under 5 specific epi- 1992, Eloranta and Kwandrans 2007) with a single record thets (Ganesan and West 2013a, 2013b). Judging from the of Paralemanea in the southern hemisphere from Brazil descriptions and figures for Lemanea plants in India and (Necchi and Zucchi 1995). Petrohua Saunders (in Vis et comparing them with current literature, the generic dis- al. 2007), a Lemanea-like monotypic genus from Chile, position and specific identities of these Indian records are has structural and reproductive features similar to Lema- problematic. neaceae but differs molecularly. Psilosiphon, a monotypic The precise taxonomic identity of the Indian Lema- genus from Australia (Entwisle 1989), is related to these nea specimens is relevant in view of their various uses: other genera and was placed previously in its own fam- 1) as an edible alga (called Nungsham-“hair of stones”) ily, Psilosiphonaceae (Sheath et al. 1996, Vis et al. 1998), (Fig. 1A) collected in rivers of Manipur and sold fresh in but currently is placed in the family Batrachospermaceae local markets (Fig. 1B) and in dried packets (Fig. 1C) also (Entwisle et al. 2009). sold on the internet, 2) medical uses (Bhosale et al. 2012) Khan (1973) was the first to report a member of the Le- by numerous tribal populations of the remote northeast maneaceae (Lemanea mamillosa Kützing) in the Indian states of India, and 3) potential for biofuels (Rout et al. subcontinent. Among the approximately 70 taxonomic 2011). Based on freshly collected material (Fig. 1A) from entities distributed in 19-20 genera of non-marine red Manipur state, we presented preliminary morphological

http://dx.doi.org/10.4490/algae.2015.30.1.001 2 Ganesan et al. Lemanea manipurensis sp. nov.

observations on this entity (Ganesan and West 2013b). Nemalionopsis of the sister order Thoreales (Müller et al. Manipur (Fig. 1D) is in the Indo-Burma Biodiversity 2002, Johnston et al. 2014). Hotspot region and requires more extensive sampling. Sequences were edited, assembled and aligned us- The previous morphological observations are supported ing the Geneious software package (Biomatters, avail- here with molecular rbcL data to show that these Manipur able from http://www.geneious.com/). Alignment was specimens belong to the genus Lemanea and represent a straight forward as no gaps were found in the data set. new species. The program jModeltest version 2 (Darriba et al. 2012) was used to find the model of sequence evolution. Maxi- mum likelihood (ML) was performed with RAxML 7.2.8 MATERIALS AND METHODS (Stamatakis 2006). RAxML was performed with all threes codons partitioned and the GTR + gamma model and 500 Lemanea specimens were collected by J. Rout and non-parametric bootstrap replicates. M. Thajamandhi in the Chakpi River, Thoubal district, Bayesian inference was performed with MrBayes Imphal, Manipur, India on December 23, 2013. For mor- v3.1.2 (Ronquist and Huelsenbeck 2003). Analyses con- phological studies, the materials were preserved in 4% sisted of two independent simultaneous runs of one cold formalin, voucher specimens were mounted on herbar- and three incrementally heated chains, and 3 × 106 gen- ium paper and specimens for molecular analyses were erations with sampling every 1,000 generations. Codons blotted dry with tissue and placed in small snap lock plas- were partitioned. The log files of the runs were checked tic bags containing silica gel beads. with Tracer v1.5 (Rambaut and Drummond 2007) and a For microscopic observations a mounting medium in- burn-in sample of 100 trees was removed from each run cluding stain was prepared as 0.02% aniline blue WS in before calculating the majority rule consensus tree. 50% corn syrup and 0.5% phenol (to prevent contami- nation by bacteria and fungi). Surface views and cross sections were prepared on a microscope slide, remov- RESULTS ing water carefully with blotting paper, adding a drop of mounting medium and arranging the specimens with a Our results indicate that the species collected in the fine needle and forcep tip. The medium was stirred gently Chakpi River is a new species of Lemanea that we de- to allow good penetration of the stain without disrupting scribe here. the sections. The material was allowed to absorb the stain for about thirty minutes. A cover slip was then gently Lemanea manipurensis E. K. Ganesan, J. A. West, placed on the slide. After 1-2 days each slide was sealed Zuccarello et J. Rout sp. nov. with a clear fingernail polish to prevent the medium from drying and receding under the cover slip margins. Diagnosis. Plants to 10 cm long, growing in clusters Photographs were taken with a Zeiss binocular research on stones, green to dark purple, with or without a basal compound microscope (Carl Zeiss, Jena, Germany) and constriction to form a stalk, shoots to 650 µm diam., with Canon G3 camera (Canon, Tokyo, Japan). The holotype sparse irregular branching in the lowermost region of the photo (Fig. 2A) was taken with a Canon G10 camera. Pho- erect shoots (Figs 1A & 2A), ultimate branches with at- tographic plates were made using Adobe Photoshop CS4. tenuated apices (Fig. 2B); basal Chantransia phase some- Total DNA was isolated from silica gel-dried material times evident; hair cells absent, four ray cells of a single using a modified CTAB procedure (Zuccarello and Lok- type formed at right angles on each cell of the single horst 2005). Amplification and sequencing of the plas- central axial filament (Fig. 2C & D), internal corticating tid-encoded large subunit of the ribulose bisphosphate filaments lacking (Fig. 2C); outer cortex cells cylindrical, carboxylase / oxygenase gene (rbcL) used amplification compact (7-9 µm diam. × 11-12 µm long) in side view, ir- primers presented in Freshwater and Rueness (1994) (F8- regular to quadrate shape in surface view (Fig. 1B & C); R753, F577-rbcL-st). Amplified products were checked monoecious, spermatangia (3-5 µm diam. × 10-12 µm for correct length (approximate 1,000 bp length), purity long) in irregular sub-prominent patches or incomplete and yield on 1% agarose gels. Polymerase chain reaction irregularly shaped rings (to 130 µm wide) around the axial products were cleaned using ExoSAP-IT (USB, Cleveland, nodes (Fig. 3A-C); carposporophytes projecting into the OH, USA) and commercially sequenced (Macrogen Inc., thallus cavity, gonimoblast filaments short and simple Daejeon, Korea). The outgroups used were Thorea and to repeatedly branched (Fig. 3D-G), each terminal cell

3 http://e-algae.kr Algae 2015, 30(1): 1-13

A B

Fig. 2. Lemanea manipurensis E. K. Ganesan, J. A. West, Zuccarello et J. Rout sp. nov. Specimens for B-D lightly stained with aniline blue. (A) Holotype. Dried thallus showing natural colour, minimal branching and elongate basal stolon. (B) Slightly squashed apical sector of branch showing the outer cell layers and central axial filament. (C) Slightly squashed midsection of branch showing the axial filament devoid of cortical filaments. (D) Cross section of branch showing the four basal cells of the outer whorl branchlets at a node. Scale bars represent: A, 5 mm; B & C, 30 μm; D, 20 μm.

http://dx.doi.org/10.4490/algae.2015.30.1.001 4 Ganesan et al. Lemanea manipurensis sp. nov.

A C

B D

F G

Fig. 3. Lemanea manipurensis E. K. Ganesan, J. A. West, Zuccarello et J. Rout sp. nov. All specimens lightly stained with aniline blue. (A) Small spermatangial patches around an old branch perimeter. (B) Spermatangial patches extend almost continuously around a young branch perimeter. (C) Branch cross section with small spermatangial patch erumpent on outer cortex. (D) Cross section with carposporophytes projecting into the hollow branch center. (E) Surface view of several carposporophytes in a whorl projecting inwardly. (F) Single carpospores terminal on carposporophyte branches. Numerous small colourless spherical bodies (arrowheads) and central nucleus (arrows) visible in each carpospore. (G) Individual carposporophyte removed from thallus to show repeated branching and terminal carpospores. Scale bars represent: A, 65 μm; B, 25 μm; C, 100 μm; D-G, 40 μm.

5 http://e-algae.kr Algae 2015, 30(1): 1-13

Fig. 4. Maximum-likelihood topology, created in RAxML, of rbcL sequence data of the Batrachospermales. Support values at each node are bootstrap values from RAxML (left) and Bayesian posterior probability (right). Asterisk (*) indicates strong support (≥95%, ≥0.95) in all analyses. Outgroups were Thorea and Nemalionopsis (Thoreales). B., Batrachospermum.

forming a single, large, elongate cylindrical carpospore toria, Australia (MEL 2381649). Isotype specimens are with round ends, 38-54 µm diam. and 85-115 µm long, also deposited in the University of Michigan Herbarium filled with abundant, colourless spheres (3-5 µm diam.) (MICH 1210723) and University / Jepson Herbaria, Uni- and a prominent central nucleus (Fig. 3F). There is no os- versity of California, Berkeley (UC / JEPS). Specimens tiole opening (Fig. 3D) for carpospore release so presum- (Figs 1A & 2A) were collected December 23, 2013, near ably release occurs when the gametophyte disintegrates. Chakpi village on the Chakpi River (24°16′ N, 93°52′ E) The holotype specimen is deposited in the Royal Bo- growing profusely on stones at 755 m above sea level (wa- tanic Gardens Melbourne, National Herbarium of Vic- ter temperature was 14°C at the time of collection). The

http://dx.doi.org/10.4490/algae.2015.30.1.001 6 Ganesan et al. Lemanea manipurensis sp. nov.

river flows through the villages of Serou and Sugnu in the and online stores (Fig. 1A-C) as food and for ethnobotani- Thoubal district, 75 km from the capital city Imphal of cal / medical uses (Bhosale et al. 2012). Manipur state is Manipur state. L. manipurensis is probably more wide- located at elevations of 50-3,300 m above sea level) and spread in Manipur river systems, but more sampling is has various rivers, of which the Chakpi, Imphal, Iril, Ma- needed to confirm this. The species is named for the state nipur and Thoubal are well known (24°16′ N, 93°52′ E) of Manipur, the only location in India where Lemanea is (Singh and Gupta 2014, Fig. 1). Lemanea specimens have currently known. been collected and reported from all five rivers (Singh Phylogenetic analysis. The phylogenetic tree (Fig. 4) of and Gupta 2010a, 2010b, 2011, Gupta and Singh 2012). the Batrachospermales and Thoreales using rbcL gene se- Critical observations on the anatomical and reproduc- quences clearly shows a monophyletic Lemaneaceae, in- tive details, supplemented by molecular sequences data cluding both Lemanea and Paralemanea. The phylogeny are lacking for specimens collected here. Five different reveals that L. manipurensis is molecularly distinct and in specific names, i.e., L. australis Atkinson [now Paralema- a sister lineage to other Lemanea samples accessed from nea grandis (Wolle) Kumano], L. catenata Kützing [now Genbank, with weak support (ML BP 68). The rbcL phy- Paralemanea catenata (Kützing) M. Vis et R. Sheath], L. logeny shows great genetic variation in the genus Lema- fluviatilis (Linneaus) C. Agardh, L. mamillosa Kützing, nea and the most commonly sequenced species (L. flu- and L. torulosa Sirodot have been assigned during more viatilis) is not monophyletic based on that species name than four decades (1973-2015) for the Indian Lemanea / assigned to nucleotides in Genbank. Paralemanea populations (Table 2). GenBank accession number. KP407869. We believe the binomial L. mamillosa was misapplied by Khan (1973). He said “from the outer ends of the T shaped cells, ascending and descending lateral filaments DISCUSSION were formed.” More importantly, Khan (1973, p. 173, Fig. B) misjudged large carpospores with numerous, small, Morphologically the specimens we examined clearly colourless spheres and a large central nucleus as sper- belong to the genus Lemanea (Table 1). matangia. Among the approximately 19-20 non-marine red algal Deb et al. (1986) questioned Khan’s identification ofL. genera known hitherto from the Indian sub-continent mamillosa and referred their specimens to L. australis. (Ganesan and West 2013a, 2013b), Lemanea is reported A careful analysis of the description and figures by Deb only from Manipur state, and occurs mostly during the et al. (1986) also raised serious doubts about the taxo- colder months (December to February) when harvesting, nomic identity of specimens they examined. The central drying and packaging are done for sale in local markets axial filament was covered by slender descending corti-

Table 1. Diagnostic differences betweenLemanea and Paralemanea Feature Lemanea Paralemanea Basal part Stalked or unstalked Unstalked Hair cells Generally present Absent Internal cortical filaments Not present Present Ray cells One type Two types T or L shaped, closely applied to the Proximal cells not touching the outer cortex outer cortex Distal cells Y shaped and attached to the centre cortex Spermatangia In isolated sori or patches and not in In continuous rings or bands around the axis complete bands around the axis “Chantransia” stage Transitory Permanent Holotype species L. fluviatilis (Linn.) C. Agardh P. catenata (Kutz.) Vis et Sheath Phylogenetic status Monophyletic Paraphyletic? Based on Vis and Sheath (1992), Sheath et al. (1996, 2006), Carmona Jimenez and Necchi (2002), Kumano (2002), Mannino et al. (2003), Sheath (2003), Kučera and Marvan (2004), Xie et al. (2004), Eloranta and Kwandrans (2007), Kučera et al. (2008), Eloranta et al. (2011) (compare however with Kučera and Marvan 2004, p. 168, Fig. 3A-D), Sheath and Sherwood (2011).

7 http://e-algae.kr Algae 2015, 30(1): 1-13

cating filaments, two types of ray cells were seen and the River, (1) L. australis, (2) L. torulosa from Manipur and outermost cortex with spermatangial nodal rings that Imphal Rivers, (3) L. mamillosa from Iril River, and (4) are features of Paralemanea (Deb et al. 1986, Pl. II, Fig. L. catenata from Iril River in the Manipur River systems. 2). They pointed out that the carpospores of the Indian However, critical details on thallus anatomy and repro- material were comparatively larger (45-63 × 72-126 μm) ductive features were not provided. Gupta and Singh than those of L. australis given by Atkinson (1890) for the (2012, p. 23, Table 2) and Singh and Gupta (2010a, p. 52, North American specimens. Voucher specimens of Deb Table 2) further confused this issue showing in a com- and others were stated to have been deposited at the Bo- parative table that some populations had axial cortical tanical Survey of India: “Sugnu, Feb 1971 & Dec 1973, Sh. filaments L.( mamillosa and L. catenata), while in others Singh. S. n. (BSIS).” However, these were not traceable (L. australis, L. torulosa, and L. fluviatilis) these filaments during our (EKG and JW) visit to the BSIS Herbarium, Kol- were not shown. Also, excepting L. fluviatilis, all the other kata, W. Bengal in November 2013. Sugnu is near where species had spermatangia in rings. The bionomial L. cat- our specimens for the present study were collected. enata was used throughout their studies but the pres- Desikachary et al. (1990) concluded, based on observa- ent binomial is Paralemanea catenata (Kützing) Vis et tions of Deb et al. (1986), that they were L. mamillosa. The Sheath. Most of their illustrations were habit views with- issue was further clouded by Desikachary et al. (1990, Fig. out anatomical details in illustrations i.e., with or without 20A-P) stating that several figures of North American L. axial filaments in the 5 species they identified. Singh and mamillosa populations (Mullahy 1952, Figs 17-23 & 25- Gupta (2010a, p. 54, Pl. 5b) illustrated “spermatangial sori 33) were similar to the Indian populations. Both sets of in rings between the internodes” in L. catenata. These ap- figures clearly show that axial filaments with many down- pear to be the large carpospores characteristic of L. ma- growing cortical filaments characteristic ofParalemanea . nipurensis. Finally, Deb et al. (1986), Bhosale et al. (2012), Bhosale et al. (2012) studied the morphology of abun- Gupta and Singh (2012), and Singh and Gupta (2010a) dant fresh material from the same general area as our did not provide appropriate magnification scales in their present collection site (Chakpi River, Thoubal District) photographic and line-drawing illustrations. Many of the and concluded that the Indian specimens were mis- above identifications referred to species unknown in In- identified earlier and that they should be referred to the dia and lacked photographic evidence of critical details. widely distributed Lemanea fluviatilis. However, these Among the records of the Lemanea / Paralemanea authors failed to observe some critical anatomical and species complex in countries neighbouring India, Faridi reproductive details, e.g., there is no mention or illustra- (1971) mentioned Lemanea and four other freshwater tion to show if the axial filament is naked or surrounded red algae, Chroodactylon (as Asterocytis), Audouinella, by cortical filaments. More importantly, the nature of the Batrachospermum, and Compsopogon, in a diagnostic mature carpospores in their studies (Bhosale et al. 2012, key to the freshwater red algae of Pakistan. Later (Faridi Fig. 2G) clearly showed a single, large and terminal carpo- 1975) mentioned L. mamillosa from Northwest frontier spore on each gonimoblast branch which is in agreement (Parachinar), but without description or illustration of the with our specimens. In other words, the nature, shape specimens he examined. Nizamuddin (1988) remarked and size of carpospores shown by Bhosale et al. (2012) that “Faridi’s collections are not available to make any are in sharp contrast to L. fluviatilis, where carpospores comments on their occurrence in Pakistan.” are generally spherical and in simple or branched chains Xie et al. (2004) studied 4 species of Lemanea and 2 spe- (see Sheath and Sherwood 2011, Pl. 42E & F, Guiry and cies of Paralemanea from China. Lemanea simplex C. -C. Guiry 2015). Identification asL. fluviatilis by Bhosale et Jao and L. sinica C. -C. Jao were described by Jao (1941) al. (2012) was followed subsequently in other studies on and L. crassa S. L. Xie et Z. X. Shi and L. ramosa S. L. Xie biofuel production (Rout et al. 2011), nutrient content et Z. X. Shi were described by Xie et al. (2004). Since there (Singh and Gupta 2010a, 2010b), treatment of diabetes are no other reports of these species they may be endemic (Devi 2011), trace elements (Bino-Devi et al. 2011), bio- to different regions of China. The carposporophyte struc- synthesis of gold nanoparticles (Sharma et al. 2014) and ture and carpospore shape and size of L. simplex and L. anti-urolithiatic agent (Mikawlrawng et al. 2014). sinica are similar to those of L. manipurensis specimens. Singh and Gupta (2010a, 2010b, 2011, 2014) and Gupta The specific differences between L. manipurensis and L. and Singh (2012) extensively studied the ecology of Le- sinica are shown in Table 3. Except for thallus height and manea populations and identified five different species. branching mode, L. manipurensis is remarkably similar These are, in addition to L. fluviatilis from the Thoubal to L. sinica although carpospore size is slightly larger. No

http://dx.doi.org/10.4490/algae.2015.30.1.001 8 Ganesan et al. Lemanea manipurensis sp. nov.

Table 2. Synopsis of references (1973-2014) for the five specific epithets applied toLemanea plants from Manipur State References Specific name(s) River names of collection sites Type of study Khan (1973) Lemanea mamillosa Imphal and Chakpi Rivers Taxonomy Deb et al. (1974) L. australisa Confluence of Manipur and Chakpi Rivers Analysis of ash content Deb et al. (1986) L. australis Confluence of Manipur and Chakpi Rivers Taxonomy Desikachary et al. (1990) L. australis Liwanglee stream, confluence of Manipur Taxonomy and Chakpi Rivers Jain et al. (2007) L. australis NA Herbal remedy Mao et al. (2009) L. australis NA Ethnobotany Bhosale et al. (2012) L. fluviatilis Chakpi and Manipur Rivers Taxonomy and indigenous information Jain et al. (2011) L. australis NA Dietary use Devi et al. (2011) L. australis NA Diabetes treatment Devi (2011) L. australis NA Diabetes treatment for Metei community of Manipur Bino-Devi et al. (2011) L. australis Chakpi and Imphal Rivers Trace elements analysis Rout et al. (2011) L. fluviatilis Rivers of Manipur Biofuel production Singh and Gupta (2011), L. australis Imphal and Manipur Rivers Ecology and nutrient contents Gupta and Singh (2012) L. catenatab Iril L. fluviatilis Thoubal L. mamillosac Iril L. torulosad Manipur Rivers Sharma et al. (2014) L. fluviatilis Chakpi Rivers Biosynthesis of gold nano par- ticles Singh and Muralidharan L. fluviatilis Imphal Rivers Hipatoprotective and antioxi- (2014) dant activity Mikawlrawng et al. (2014) L. fluviatilis NA Antiurolithiatic uses aLemanea australis: A taxonomic synonym of Paralemanea grandis (Wolle) Kumano. bL. catenata: A taxonomic synonym of Paralemanea catenata (Kutzing) Vis et Sheath. cL. mamillosa: A species of Lemanea with entirely different set of features morphologically quite distinct fromL. fluviatilis. dL. torulosa: A taxonomic synonym of Paralemanea torulosa (Roth) Sheath and Sherwood (see for more details, Vis and Sheath 1992, Kumano 2002, Eloranta et al. 2011, Guiry and Guiry 2015). NA, not available.

Table 3. Synopsis of salient differences betweenLemanea manipurensis sp. nov. and L. sinica Jao External features L. manipurensis (present study) L. sinica (Jao 1941) Morphology Plants up to 10 cm long, sparsely and irregularly Up to 16 cm long, with a long stipe, branched with or without a stipe and branched more at the basal part, constriction, growing on stones in river beds of branching opposite, alternate above, Manipur, India apical branches capillary, China Anatomy No internal corticating filaments No internal corticating filaments Spermatangia In isolated irregular patches, sometimes Mostly in complete, distinct rings, rarely completely covering the axis in isolated patches Carposporophyte structure Gonimoblast filaments few that are sparsely Gonimoblast filaments with a few and branched, apical cell bearing a single, terminal, sparsely branched cells, apical cell elongate-obovoid carpospore bearing a single, terminal, elongate- oboviod carpospore Carposporangial size 30-50 µm broad, 80-120 µm long 30-50 µm broad, 75-90 µm long Based on Jao (1941), Xie et al. (2004), Hu and Wei (2006), and the present study.

9 http://e-algae.kr Algae 2015, 30(1): 1-13

other reports of L. sinica are known in China or elsewhere. lacks cortical filaments and commented that it is a “pecu- Future molecular studies on L. sinica would undoubtedly liar combination of the characteristics of two genera” in reveal whether or not it differs from L. manipurensis. The L. sinica. Kučera and Marvan (2004), in a taxonomic study type locality of L. sinica is Chun-tien, Yunnan (32°03′01″ of the Lemaneaceae in the Czech Republic, characterized N, 110°36′50″ E), while that of the Indian species is Chak- Lemanea with a central axis with or without cortical fila- pi, Thoubal district, Manipur (24°16′ N, 93°52′ E), perhaps ments. According to them, “the regular formation of sper- not a very significant geographical distance but the many matangia in rings varies with age and the magnification diverse river systems and mountain ranges between these used.” These variable features in spermatangial forma- localities may be formidable barriers for dispersal. tion in their material of L. fluviatiliswere also shown in In the five Manipur river systems, Lemanea popula- illustrations (Kučera and Marvan 2004, p. 168, Fig. 3A-D, tions may be abundantly distributed in the different riv- Simić and Djordjević 2011, p. 515). Xie et al. (2004) in their er segments. Hence the possibility of cryptic speciation study of Chinese Lemaneaceae, mentioned that in Para- among the different populations cannot be ruled out. lemanea parvula (Sirodot) S. L. Xie & Z. X. Shi, spermatan- Only critical molecular studies would clarify this point. gia are found in patches (Xie et al. 2004, p. 886, Fig. 13). Lemanea and Paralemanea are known to grow together Eloranta et al. (2011, p. 96) preferred to keep P. parvula in in the same ecological habitat and there are instances Lemanea as an uncertain species, but notably remarked where the same herbarium packet contained more than that “spermatangial papillae form separate or sometimes one species (Palmer 1941). Harvested material of Lema- coalescent tubercles.” Anatomical details, i.e., whether nea from different places and the processed dried packets cortical filaments are present or not in P. parvula is not sold in the local markets may be helpful on morphologi- known in Chinese and European specimens. Eloranta et cal and molecular bases in resolving whether Paralema- al. (2011) did not include the reference of Xie et al. (2004). nea species also occur in India. The above mentioned instances clearly show the ambigu- Lemanea, Paralemanea, Petrohua, and Psilosiphon ity of using some anatomical and reproductive features to species offer few reliable morphological characteristics differentiate Lemanea and Paralemanea. to separate them at the generic level. As stated by Sheath It is significant that the five species ofLemanea (L. (2003, pp. 208, 216 & 217) the presence or absence of axial crassa, L. manipurensis, L. ramosa, L. simplex, and L. si- filament cortication and the configuration of spermatan- nica) from the Southeast Asian continent are remarkably gial rings or patches should be the most useful features similar, producing just a single and elongate terminal car- in definingLemanea and Paralemanea, but these 2 ge- pospore on each short simple to branched carposporo- neric attributes apparently are not consistent, thus rais- phyte filament with narrow cylindrical cells. Most other ing doubts about the generic status of Paralemanea as species of Lemanea and Paralemanea produce spherical different from Lemanea. In any case molecular analyses carpospores serially in simple or branched chains (cate- in this and other published work suggest Paralemanea is nate). Detailed and comparative morphological and mo- paraphyletic leaving the issue unresolved. lecular data would clarify whether this group of Lemanea The landmark publication by Jao (1941) on L. sinica species warrants segregation into a new genus, different is overlooked in many later publications on the Lema- from closely related genera like Lemanea, Paralemanea, neaceae. Jao (1941, p. 271, Pl. VII, Figs 50-52) described L. and Petrohua. A case in point is that extensive molecu- sinica specimens with “the antheridial bands are mostly lar and morphological data on Batrachospermum spe- in complete rings around the antheridial zones. In older cies sensu lato resulted in the segregation of Kumanoa portions of the thallus they are occasionally slightly in- from Batrachospermum with spirally twisted carpogo- terrupted but also kept a band-shape. Probably the inter- nial branches and axial carposporophytes (Entwisle et al. rupting is due to the hypertrophy of the tissue beneath 2009) and Sheathia with hetero-corticated thalli, carpo- the antheridial bands. In addition to the ordinary anther- gonia on undifferentiated cells and exserted carposporo- idial bands, small antheridial sori sometimes appear on phytes (Salomaki et al. 2014). the old carpogonial zones.” Xie et al. (2004) said in their It is noteworthy that Necchi et al. (2013) found a very Lemanea species key that L. sinica has “Spermatangia in low genetic diversity between species of the two genera perfect rings” but they did not obtain any new L. sinica Composopogon and Compsopogonopsis (Compsopogo- specimens. Hu and Wei (2006) stated “the antheridia are nales, Rhodophyta) based on global sampling and mo- always in well-defined bands” without further evidence. lecular data. Species of these 2 genera were distinguished Jao (1941) clearly mentioned the “naked central axis” hitherto by caeruleus morphology (without cortication)

http://dx.doi.org/10.4490/algae.2015.30.1.001 10 Ganesan et al. Lemanea manipurensis sp. nov.

for Compsopogon species and leptoclados morphology Chowdhury, A. N., Das, H. B. & Singh, S. T. 1974. An ed- (with cortication) for Compsopogonopsis species. Nec- ible alga of Manipur (Lemanea australis): presence of chi et al. (2013) concluded that only one genus, Compso- silver. Curr. Sci. 43:629. pogon, can be recognized. Deb, D. B., Krishna, B. & Singh, S. T. 1986. Life history of a It is still too early to provide a complete molecular and Lemanea species from Manipur. In Desikachary, T. V. & morphological story concerning generic and species de- Raja Rao, V. N. (Eds.) Taxonomy of Algae. University of limitations for Lemanea and Paralemanea. Madras, Madras, pp. 213-221. Desikachary, T. V., Krishnamurthy, V. & Balakrishnan, M. S. 1990. Rhodophyta. Vol. 1. Part II A. Madras Science ACKNOWLEDGEMENTS Foundation, Madras, 279 pp. Devi, A. P. 2011. Plants used by Meitei community of Ma- Morgan Vis kindly provided a copy of Atkinson (1890). nipur for the treatment of diabetes. Assam Univ. J. Sci. Interlibrary loan facilities of the University of Melbourne, Technol. Biol. Environ. Sci. 7:63-66. Michael Wynne and S. Tripathi obtained some early and Devi, W. I., Devi, G. S. & Singh, C. B. 2011. Traditional herbal recent literature not easily accessible to us. P. S. Yadava medicine used for the treatment of diabetes in Manipur, (Manipur University, Imphal) provided liquid preserved India. Res. J. Pharm. Biol. Chem. Sci. 2:709-715. material and some information about the edible Nung- Eloranta, P. & Kwandrans, J. 2007. Freshwater red algae. Rho- sham sold in the local markets. Ms. M. Thajamandhi as- dophyta. Identification guide to European taxa, particu- sisted J. Rout in field collections and processing of liquid larly to those found in Finland. Norrilinia 15:1-103. preserved, herbarium and silica gel material for DNA ex- Eloranta, P., Kwandrans, J. & Kusel-Fetzmann, E. 2011. Rho- traction. Tim Entwisle provided very helpful advice on the dophyceae and Phaeophyceae. In Schagerl, M. (Ed.) manuscript before submission. Funding was provided Süßwasserflora von Mitteleuropa Band 7. Freshwater mostly from the personal pensions of Ganesan and West. Flora of Central Europe. Vol. 7. Spectrum Akademischer Verlag, Heidelberg, pp. 1-155. Entwisle, T. J. 1989. Psilosiphon scoparium gen. et sp. nov. REFERENCES (Lemaneaceae), a new red alga from south-eastern Aus- tralian streams. Phycologia 28:469-475. Atkinson, G. F. 1890. Monograph of the Lemaneaceae of the Entwisle, T. J., Vis, M. L., Chiasson, W. B., Necchi, O. Jr. & Sher- United States. Ann. Bot. 4:177-229. wood, A. R. 2009. Systematics of the Batrachospermales Bhosale, R. A. & Chaugule, B. B. 2010. Freshwater algae as po- (Rhodophyta): a synthesis. J. Phycol. 45:704-715. tential source of polyunsaturated fatty acids: review. Int. Faridi, M. A. F. 1971. The genera of fresh-water algae of Paki- J. Algae 12:335-356. stan and Kashmir. Biologia 12:123-142. Bhosale, R., Rout, J. & Chaugule, B. 2012. The ethnobotanical Faridi, M. A. F. 1975. Batrachospermum in Pakistan. Biologia study of an edible freshwater red alga, Lemanea fluviati- 21:107-109. lis (L.) C. Ag. from Manipur, India. Ethnobot. Res. Appl. Freshwater, D. W. & Rueness, J. 1994. Phylogenetic relation- 10:69-76. ships of some European Gelidium (Gelidiales, Rho- Bino-Devi, Ch., Sharat Singh, N. K., Singh, N. R., Rajendro dophyta) species, based on rbcL nucleotide sequence Singh, N., Sudarshan, M., Chakraborty, A. & Ram, S. S. analysis. Phycologia 33:187-194. 2011. Trace elements in Nungsham, the red edible algae Ganesan, E. K. & West, J. A. 2013a. Nomenclatural changes of Manipur. Int. J. Appl. Biol. Pharm. Technol. 2:198-203. for some freshwater red algae from India. Algae 28:45- Bory de Saint-Vincent, J. B. 1808. Mémoire sur le genre Le- 51. manea de la famille des Conferves. Ann. Mus. Hist. Nat. Ganesan, E. K. & West, J. A. 2013b. On the identity of an edible 12:177-190. macroscopic freshwater (riverine) red alga from Mani- Carmona Jimenez, J. & Necchi, O. Jr. 2002. Taxonomy and pur, north-east India. In National Conference on ”Fron- distribution of Paralemanea (Lemaneaceae, Rhodophy- tiers in Algology and Algal Biotechnology” [NCFAAB], ta) in central Mexico. Cryptogam. Algol. 23:39-49. Visva-Bharati University, Santiniketan, p. 6. Darriba, D., Taboada, G. L., Doallo, R. & Posada, D. 2012. Guiry, M. D. & Guiry, G. M. 2015. AlgaeBase. World-wide jModelTest 2: more models, new heuristics and parallel electronic publication, National University of Ireland, computing. Nature Methods 9:772. Galway. Available from: http://www.algaebase.org. Ac- Deb, D. B., Krishna, B., Mukherjee, M. K., Bhattacharya, S., cessed Mar 8, 2015.

11 http://e-algae.kr Algae 2015, 30(1): 1-13

Gupta, A. & Singh, R. M. 2012. Lemanea in lotic environment Necchi, O. Jr., Fo, A. S. G., Salomaki, E. D., West, J. A., Aboal, of Manipur, North-East India: distribution, ecology and M. & Vis, M. L. 2013. Global sampling reveals low genet- need for conservation of Lemanea. Lamhat Academic ic diversity within Compsopogon (Compsopogonales, Publishing, Saarbrücken, 80 pp. Rhodophyta). Eur. J. Phycol. 48:152-162. Hu, H. & Wei, Y. 2006. The freshwater algae of China: system- Necchi, O. Jr. & Zucchi, M. R. 1995. Record of Paralemanea atics, taxonomy and ecology. Science Press, Beijing, 1023 (Lemaneaceae, Rhodophyta) in South America. Algol. pp. Stud. 78:33-38. Jain, A., Roshnibala, S., Kanjilal, P. B., Singh, R. S. & Singh, Nizamuddin, M. 1988. Occurrence of the genus Bangia Lyn- H. B. 2007. Aquatic/semi-aquatic plants used in herbal gbye (Bangiales, Rhodophyta) from Chitral, North West remedies in the wetlands of Manipur, Northeastern In- of Pakistan. Pak. J. Bot. 20:45-48. dia. Ind. J. Tradit. Knowl. 6:346-351. Palmer, C. M. 1941. Lemanea herbarium packets containing Jain, A., Sundriyal, M., Roshnibala, S., Kotoky, R., Kanjilal, P. more than one species. Butler Univ. Bot. Stud. 5:222-223. B., Singh, H. B. & Sundriyal, R. C. 2011. Dietary use and Rambaut, A. & Drummond, A. J. 2007. Tracer. Available from: conservation concern of edible wetland plants at Indo- http://beast.bio.ed.ac.uk/Tracer. Accessed Aug 30, 2012. Burma Hotspot: a case study from northeastern India. J. Ronquist, F. & Huelsenbeck, J. P. 2003. MrBayes 3: Bayesian Ethnobiol. Ethnomed. 7:29. phylogenetic inference under mixed models. Bioinfor- Jao, C. C. 1941. Studies on the freshwater algae of China. VIII. matics 19:1572-1574. A preliminary account of the Chinese freshwater Rhodo- Rout, J., Chaugule, B. & Bhosale, R. 2011. Potential of a red phyceae. Sinensia 12:245-290. alga, Lemanea for use as a source of biofuel. In Bhat- Johnston, E. T., Lim, P. -E., Buhari, N., Keil, E. J., Djawad, M. I. nagar, S. K., Saxena, A. & Krann, S. (Eds.) Algae Biofuel. & Vis, M. L. 2014. Diversity of freshwater red algae (Rho- Stadium Press, New Delhi, pp. 111-115. dophyta) in Malaysia and Indonesia from morphologi- Salomaki, E. D., Kwandrans, J., Eloranta, P. & Vis, M. L. 2014. cal and molecular data. Phycologia 53:329-341. Molecular and morphological evidence for Sheathia Khan, M. 1973. On edible Lemanea Bory de St. Vincent, a gen. nov. (Batrachospermales, Rhodophyta) and three fresh water red alga from India. Hydrobiologia 43:171- new species. J. Phycol. 50:526-542. 175. Sharma, B., Purkayastha, D. D., Hazra, S., Thajamanbi, M., Kučera, P., Grulich, V., Fránková, M. & Bureš, P. 2008. Dis- Bhattacharjee, C. R., Ghosh, N. N. & Rout, J. 2014. Bio- tribution of freshwater red algal family Lemaneaceae synthesis of fluorescent gold nanoparticles using an ed- (Rhodophyta) in the Czech Republic: an update. Fottea ible freshwater red alga, Lemanea fluviatilis (L.) C. Ag., 8:125-128. and antioxidant activity of biomatrix loaded nanopar- Kučera, P. & Marvan, P. 2004. Taxonomy and distribution of ticles. Bioprocess Biosyst. Eng. 37:2559-2565. Lemanea and Paralemanea (Lemaneaceae, Rhodophy- Sheath, R. G. 2003. Red Algae. In Wehr, J. D. & Sheath, R. G. ta) in the Czech Republic. Presilia (Praha) 76:163-174. (Eds.) Freshwater Algae of North America: Ecology and Kumano, S. 2002. Freshwater red algae of the world. Biopress Classification. Academic Press. San Diego, CA, pp. 197- Ltd., Bristol, 375 pp. 224. Mannino, A. M., Barone, R. & Raimondo, F. M. 2003. First re- Sheath, R. G., Müller, K. M., Vis, M. L. & Entwisle, T. J. 1996. cord of Paralemanea catenata (Rhodophyta) from Ital- A re-examination of the morphology, ultrastructure and ian Peninsula. Bocconea 16:1053-1058. classification of genera in the Lemaneaceae (Batracho- Mao, A. A., Hynniewta, T. M. & Sanjappa, M. 2009. Plant spermales, Rhodophyta). Phycol. Res. 44:233-246. wealth of northeast India with reference to ethnobotany. Sheath, R. G., Müller, K., Sherwood, A. & Shea, T. 2006. Phylo- Indian J. Tradit. Knowl. 8:96-103. genetic relationships of members of the freshwater red Mikawlrawng, K., Kumar, S. & Vandana. 2014. Current sce- algal family Lemaneaceae from North America, Europe nario of urolithiasis and the use of medicinal plants as and Africa. In Stekoll, M. S. (Ed.) Phycol. Soc. Am. 60th antiurolithiatic agents in Manipur (North East India): a Annu. Meet. Northwest Algal Symp., Phycological Soci- review. Int. J. Herb. Med. 2:1-12. ety of America, Juneau, AK, p. 57. Mullahy, J. H. 1952. The morphology and cytology of Lema- Sheath, R. G. & Sherwood, A. R. 2011. Rhodophyta. In John, nea australis Atk. Bull. Torrey Bot. Club 79:471-484. D. M., Whitton, B. A. & Brook, A. J. (Eds.) The Freshwa- Müller, K. M., Sherwood, A. R., Pueschel, C. M., Gutell, R. R. & ter Algal Flora of the British Isles. An Identification Guide Sheath, R. G. 2002. A proposal for a new red algal order, to Freshwater and Terrestrial Algae. 2nd ed. Cambridge the Thoreales. J. Phycol. 38:807-820. University Press, Cambridge, pp. 159-180.

http://dx.doi.org/10.4490/algae.2015.30.1.001 12 Ganesan et al. Lemanea manipurensis sp. nov.

Simić, S. & Djordjević, N. 2011. Lemanea fucina Bory, 1808 Climate Change. Pointer Publishers, Jaipur, pp. 118-134. (Lemaneaceae, Rhodophyta), a rare species with a vari- Stamatakis, A. 2006. RAxML-VI-HPC: maximum likelihood- able morphology: first record in the Republic of Monte- based phylogenetic analyses with thousands of taxa and negro. Arch. Biol. Sci. Belgrade 63:511-515. mixed models. Bioinformatics 22:2688-2690. Singh, I. L. & Muralidharan, P. 2014. Evaluation of hepatopro- Vis, M. L., Harper, J. T. & Saunders, G. W. 2007. Large subunit tective and anti-oxidant activity of Lemanea fluviatilis rDNA and rbcL gene sequence data place Petrohua ber-

on CCI4 induced hepatotoxicity in rats. Res. J. Pharm. nabei gen. et sp. nov. in the Batrachospermales (Rho- Biol. Chem. Sci. 5:976-982. dophyta), but do not provide further resolution among Singh, M. R. & Gupta, A. 2010a. Morphological characteris- taxa in this order. Phycol. Res. 55:103-112. tics of rare and endangered species of Lemanea (Rho- Vis, M. L., Saunders, G. W., Sheath, R. G., Dunse, K. & dophyta) from the rivers of Manipur, North-east India. Entwisle, T. J. 1998. Phylogeny of the Batrachospermales Botanique 14:47-54. (Rhodophyta) as inferred from rbcL and 18S ribosomal Singh, M. R. & Gupta, A. 2010b. Nutritional composition of RNA gene sequences. J. Phycol. 34:341-350. edible Lemanea australis Atkinson (Lemaneaceae, Rho- Vis, M. L. & Sheath, R. G. 1992. Systematics of the freshwater dophyta) from Manipur river systems, India. PGTD of red algal family Lemaneaceae in North America. Phyco- Botany, R. T. M. University, Nagpur, pp. 100-107. logia 31:164-179. Singh, M. R. & Gupta, A. 2011. Factors affecting the occur- Xie, S. -L., Shi, Z. -X. & Wang, R. -N. 2004. Taxonomy of the Le- rence of Lemanea, Rhodophyta from the Rivers of Ma- maneaceae (Batrachospermales, Rhodophyta) in China. nipur, North-east India. In Tiwari, D. (Ed.) Advances in Acta Bot. Sin. 46:883-888. Environmental Chemistry. Excel India Publishers, New Zuccarello, G. C. & Lokhorst, G. M. 2005. Molecular phylog- Delhi, pp. 233-236. eny of the genus Tribonema (Xanthophyceae) using rbcL Singh, M. R. & Gupta, A. 2014. Biomass and productivity gene sequence data: monophyly of morphologically study of red algae from rivers of Manipur, North East simple algal species. Phycologia 44:384-392. India. In Gupta, A. (Ed.) Ecoplanning, Biodiversity and

13 http://e-algae.kr