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. Ganesan
1,a, J. A. West
2,*, G. C. Zuccarello
3, S. Loiseaux de Goër
4and J. Rout
51Instituto 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 fila- ment 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, nar- row 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. ma- nipurensis 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 commonly in terrestrial and freshwater habitats as unicel- lular, colonial, simple filamentous, flat-bladed and more complex pseudoparenchymatous forms with quite varied reproductive patterns. The order Batrachospermales is an exclusively freshwater group comprised of three distinct evolutionary lineages, all with uniaxial shoots, pseudo-
parenchymatous morphologies. The molecular phylog- eny and classification are somewhat controversial. The order includes two families, Batrachospermaceae with 6 genera (Batrachospermum, Nothocladus, Petrohua, Psilosiphon, Sirodotia and Tuomeya) and Lemaneaceae with two genera [Lemanea Bory (1808) and Paralema- nea (Silva) Vis and Sheath (Vis and Sheath 1992)]. Lema-
Received January 13, 2015, Accepted March 12, 2015
*
Corresponding Author E-mail: [email protected]Tel: +61-3-8344-8080, Fax: +61-3-9347-5460
aPresent address: 3-A Srinivas Terrace, 52, II Main Road, Gandhi This is an Open Access article distributed under the
terms of the Creative Commons Attribution Non-Com- mercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
algae recorded in India, Lemanea has been reported for more than 4 decades (1973-2015) under 5 specific epi- thets (Ganesan and West 2013a, 2013b). Judging from the descriptions and figures for Lemanea plants in India and comparing them with current literature, the generic dis- position and specific identities of these Indian records are problematic.
The precise taxonomic identity of the Indian Lema- nea specimens is relevant in view of their various uses:
1) as an edible alga (called Nungsham-“hair of stones”) (Fig. 1A) collected in rivers of Manipur and sold fresh in local markets (Fig. 1B) and in dried packets (Fig. 1C) also sold on the internet, 2) medical uses (Bhosale et al. 2012) by numerous tribal populations of the remote northeast states of India, and 3) potential for biofuels (Rout et al.
2011). Based on freshly collected material (Fig. 1A) from Manipur state, we presented preliminary morphological nea and Paralemanea occur in boreal to warm temper-
ate regions of the northern hemisphere (Vis and Sheath 1992, Eloranta and Kwandrans 2007) with a single record of Paralemanea in the southern hemisphere from Brazil (Necchi and Zucchi 1995). Petrohua Saunders (in Vis et al. 2007), a Lemanea-like monotypic genus from Chile, has structural and reproductive features similar to Lema- neaceae but differs molecularly. Psilosiphon, a monotypic genus from Australia (Entwisle 1989), is related to these other genera and was placed previously in its own fam- ily, Psilosiphonaceae (Sheath et al. 1996, Vis et al. 1998), but currently is placed in the family Batrachospermaceae (Entwisle et al. 2009).
Khan (1973) was the first to report a member of the Le- maneaceae (Lemanea mamillosa Kützing) in the Indian subcontinent. Among the approximately 70 taxonomic entities distributed in 19-20 genera of non-marine red
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.A
C D
B
Nemalionopsis of the sister order Thoreales (Müller et al.
2002, Johnston et al. 2014).
Sequences were edited, assembled and aligned us- ing the Geneious software package (Biomatters, avail- able from http://www.geneious.com/). Alignment was straight forward as no gaps were found in the data set.
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 (Stamatakis 2006). RAxML was performed with all threes codons partitioned and the GTR + gamma model and 500 non-parametric bootstrap replicates.
Bayesian inference was performed with MrBayes v3.1.2 (Ronquist and Huelsenbeck 2003). Analyses con- sisted of two independent simultaneous runs of one cold and three incrementally heated chains, and 3 × 106 gen- erations with sampling every 1,000 generations. Codons were partitioned. The log files of the runs were checked with Tracer v1.5 (Rambaut and Drummond 2007) and a burn-in sample of 100 trees was removed from each run before calculating the majority rule consensus tree.
RESULTS
Our results indicate that the species collected in the Chakpi River is a new species of Lemanea that we de- scribe here.
Lemanea manipurensis E. K. Ganesan, J. A. West, Zuccarello et J. Rout sp. nov.
Diagnosis. Plants to 10 cm long, growing in clusters on stones, green to dark purple, with or without a basal constriction to form a stalk, shoots to 650 µm diam., with sparse irregular branching in the lowermost region of the erect shoots (Figs 1A & 2A), ultimate branches with at- tenuated apices (Fig. 2B); basal Chantransia phase some- times evident; hair cells absent, four ray cells of a single type formed at right angles on each cell of the single central axial filament (Fig. 2C & D), internal corticating filaments lacking (Fig. 2C); outer cortex cells cylindrical, compact (7-9 µm diam. × 11-12 µm long) in side view, ir- regular to quadrate shape in surface view (Fig. 1B & C);
monoecious, spermatangia (3-5 µm diam. × 10-12 µm long) in irregular sub-prominent patches or incomplete irregularly shaped rings (to 130 µm wide) around the axial nodes (Fig. 3A-C); carposporophytes projecting into the thallus cavity, gonimoblast filaments short and simple to repeatedly branched (Fig. 3D-G), each terminal cell observations on this entity (Ganesan and West 2013b).
Manipur (Fig. 1D) is in the Indo-Burma Biodiversity Hotspot region and requires more extensive sampling.
The previous morphological observations are supported here with molecular rbcL data to show that these Manipur specimens belong to the genus Lemanea and represent a new species.
MATERIALS AND METHODS
Lemanea specimens were collected by J. Rout and M. Thajamandhi in the Chakpi River, Thoubal district, Imphal, Manipur, India on December 23, 2013. For mor- phological studies, the materials were preserved in 4%
formalin, voucher specimens were mounted on herbar- ium paper and specimens for molecular analyses were blotted dry with tissue and placed in small snap lock plas- tic bags containing silica gel beads.
For microscopic observations a mounting medium in- cluding stain was prepared as 0.02% aniline blue WS in 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- ing water carefully with blotting paper, adding a drop of mounting medium and arranging the specimens with a fine needle and forcep tip. The medium was stirred gently to allow good penetration of the stain without disrupting the sections. The material was allowed to absorb the stain for about thirty minutes. A cover slip was then gently placed on the slide. After 1-2 days each slide was sealed with a clear fingernail polish to prevent the medium from drying and receding under the cover slip margins.
Photographs were taken with a Zeiss binocular research compound microscope (Carl Zeiss, Jena, Germany) and Canon G3 camera (Canon, Tokyo, Japan). The holotype photo (Fig. 2A) was taken with a Canon G10 camera. Pho- tographic plates were made using Adobe Photoshop CS4.
Total DNA was isolated from silica gel-dried material using a modified CTAB procedure (Zuccarello and Lok- horst 2005). Amplification and sequencing of the plas- tid-encoded large subunit of the ribulose bisphosphate carboxylase / oxygenase gene (rbcL) used amplification primers presented in Freshwater and Rueness (1994) (F8- R753, F577-rbcL-st). Amplified products were checked for correct length (approximate 1,000 bp length), purity and yield on 1% agarose gels. Polymerase chain reaction products were cleaned using ExoSAP-IT (USB, Cleveland, OH, USA) and commercially sequenced (Macrogen Inc., Daejeon, Korea). The outgroups used were Thorea and
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.A
C
D B
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.A C
D B
E
G F
toria, Australia (MEL 2381649). Isotype specimens are also deposited in the University of Michigan Herbarium (MICH 1210723) and University / Jepson Herbaria, Uni- versity of California, Berkeley (UC / JEPS). Specimens (Figs 1A & 2A) were collected December 23, 2013, near Chakpi village on the Chakpi River (24°16′ N, 93°52′ E) growing profusely on stones at 755 m above sea level (wa- ter temperature was 14°C at the time of collection). The forming a single, large, elongate cylindrical carpospore
with round ends, 38-54 µm diam. and 85-115 µm long, filled with abundant, colourless spheres (3-5 µm diam.) and a prominent central nucleus (Fig. 3F). There is no os- tiole opening (Fig. 3D) for carpospore release so presum- ably release occurs when the gametophyte disintegrates.
The holotype specimen is deposited in the Royal Bo- tanic Gardens Melbourne, National Herbarium of Vic-
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.
and online stores (Fig. 1A-C) as food and for ethnobotani- cal / medical uses (Bhosale et al. 2012). Manipur state is located at elevations of 50-3,300 m above sea level) and has various rivers, of which the Chakpi, Imphal, Iril, Ma- nipur and Thoubal are well known (24°16′ N, 93°52′ E) (Singh and Gupta 2014, Fig. 1). Lemanea specimens have been collected and reported from all five rivers (Singh and Gupta 2010a, 2010b, 2011, Gupta and Singh 2012).
Critical observations on the anatomical and reproduc- tive details, supplemented by molecular sequences data are lacking for specimens collected here. Five different specific names, i.e., L. australis Atkinson [now Paralema- nea grandis (Wolle) Kumano], L. catenata Kützing [now Paralemanea catenata (Kützing) M. Vis et R. Sheath], L.
fluviatilis (Linneaus) C. Agardh, L. mamillosa Kützing, and L. torulosa Sirodot have been assigned during more than four decades (1973-2015) for the Indian Lemanea / Paralemanea populations (Table 2).
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 were formed.” More importantly, Khan (1973, p. 173, Fig.
B) misjudged large carpospores with numerous, small, colourless spheres and a large central nucleus as sper- matangia.
Deb et al. (1986) questioned Khan’s identification of L.
mamillosa and referred their specimens to L. australis.
A careful analysis of the description and figures by Deb et al. (1986) also raised serious doubts about the taxo- nomic identity of specimens they examined. The central axial filament was covered by slender descending corti- river flows through the villages of Serou and Sugnu in the
Thoubal district, 75 km from the capital city Imphal of Manipur state. L. manipurensis is probably more wide- spread in Manipur river systems, but more sampling is needed to confirm this. The species is named for the state of Manipur, the only location in India where Lemanea is currently known.
Phylogenetic analysis. The phylogenetic tree (Fig. 4) of the Batrachospermales and Thoreales using rbcL gene se- quences clearly shows a monophyletic Lemaneaceae, in- cluding both Lemanea and Paralemanea. The phylogeny reveals that L. manipurensis is molecularly distinct and in a sister lineage to other Lemanea samples accessed from Genbank, with weak support (ML BP 68). The rbcL phy- logeny shows great genetic variation in the genus Lema- nea and the most commonly sequenced species (L. flu- viatilis) is not monophyletic based on that species name assigned to nucleotides in Genbank.
GenBank accession number. KP407869.
DISCUSSION
Morphologically the specimens we examined clearly belong to the genus Lemanea (Table 1).
Among the approximately 19-20 non-marine red algal genera known hitherto from the Indian sub-continent (Ganesan and West 2013a, 2013b), Lemanea is reported only from Manipur state, and occurs mostly during the colder months (December to February) when harvesting, drying and packaging are done for sale in local markets
Table 1.
Diagnostic differences between Lemanea and ParalemaneaFeature Lemanea Paralemanea
Basal part Stalked or unstalked Unstalked
Hair cells Generally present Absent
Internal cortical filaments Not present Present
Ray cells One type
T or L shaped, closely applied to the outer cortex
Two types
Proximal cells not touching the outer cortex Distal cells Y shaped and attached to the
centre cortex Spermatangia In isolated sori or patches and not in
complete bands around the axis
In continuous rings or 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).
River, (1) L. australis, (2) L. torulosa from Manipur and Imphal Rivers, (3) L. mamillosa from Iril River, and (4) L. catenata from Iril River in the Manipur River systems.
However, critical details on thallus anatomy and repro- ductive features were not provided. Gupta and Singh (2012, p. 23, Table 2) and Singh and Gupta (2010a, p. 52, Table 2) further confused this issue showing in a com- parative table that some populations had axial cortical filaments (L. mamillosa and L. catenata), while in others (L. australis, L. torulosa, and L. fluviatilis) these filaments were not shown. Also, excepting L. fluviatilis, all the other species had spermatangia in rings. The bionomial L. cat- enata was used throughout their studies but the pres- ent binomial is Paralemanea catenata (Kützing) Vis et Sheath. Most of their illustrations were habit views with- out anatomical details in illustrations i.e., with or without axial filaments in the 5 species they identified. Singh and Gupta (2010a, p. 54, Pl. 5b) illustrated “spermatangial sori in rings between the internodes” in L. catenata. These ap- pear to be the large carpospores characteristic of L. ma- nipurensis. Finally, Deb et al. (1986), Bhosale et al. (2012), Gupta and Singh (2012), and Singh and Gupta (2010a) did not provide appropriate magnification scales in their photographic and line-drawing illustrations. Many of the above identifications referred to species unknown in In- dia and lacked photographic evidence of critical details.
Among the records of the Lemanea / Paralemanea species complex in countries neighbouring India, Faridi (1971) mentioned Lemanea and four other freshwater red algae, Chroodactylon (as Asterocytis), Audouinella, Batrachospermum, and Compsopogon, in a diagnostic key to the freshwater red algae of Pakistan. Later (Faridi 1975) mentioned L. mamillosa from Northwest frontier (Parachinar), but without description or illustration of the specimens he examined. Nizamuddin (1988) remarked that “Faridi’s collections are not available to make any comments on their occurrence in Pakistan.”
Xie et al. (2004) studied 4 species of Lemanea and 2 spe- cies of Paralemanea from China. Lemanea simplex C. -C.
Jao and L. sinica C. -C. Jao were described by Jao (1941) and L. crassa S. L. Xie et Z. X. Shi and L. ramosa S. L. Xie et Z. X. Shi were described by Xie et al. (2004). Since there are no other reports of these species they may be endemic to different regions of China. The carposporophyte struc- ture and carpospore shape and size of L. simplex and L.
sinica are similar to those of L. manipurensis specimens.
The specific differences between L. manipurensis and L.
sinica are shown in Table 3. Except for thallus height and branching mode, L. manipurensis is remarkably similar to L. sinica although carpospore size is slightly larger. No cating filaments, two types of ray cells were seen and the
outermost cortex with spermatangial nodal rings that are features of Paralemanea (Deb et al. 1986, Pl. II, Fig.
2). They pointed out that the carpospores of the Indian material were comparatively larger (45-63 × 72-126 μm) than those of L. australis given by Atkinson (1890) for the North American specimens. Voucher specimens of Deb and others were stated to have been deposited at the Bo- tanical Survey of India: “Sugnu, Feb 1971 & Dec 1973, Sh.
Singh. S. n. (BSIS).” However, these were not traceable during our (EKG and JW) visit to the BSIS Herbarium, Kol- kata, W. Bengal in November 2013. Sugnu is near where our specimens for the present study were collected.
Desikachary et al. (1990) concluded, based on observa- tions of Deb et al. (1986), that they were L. mamillosa. The issue was further clouded by Desikachary et al. (1990, Fig.
20A-P) stating that several figures of North American L.
mamillosa populations (Mullahy 1952, Figs 17-23 & 25- 33) were similar to the Indian populations. Both sets of figures clearly show that axial filaments with many down- growing cortical filaments characteristic of Paralemanea.
Bhosale et al. (2012) studied the morphology of abun- dant fresh material from the same general area as our present collection site (Chakpi River, Thoubal District) and concluded that the Indian specimens were mis- identified earlier and that they should be referred to the widely distributed Lemanea fluviatilis. However, these authors failed to observe some critical anatomical and reproductive details, e.g., there is no mention or illustra- tion to show if the axial filament is naked or surrounded by cortical filaments. More importantly, the nature of the mature carpospores in their studies (Bhosale et al. 2012, Fig. 2G) clearly showed a single, large and terminal carpo- spore on each gonimoblast branch which is in agreement with our specimens. In other words, the nature, shape and size of carpospores shown by Bhosale et al. (2012) are in sharp contrast to L. fluviatilis, where carpospores are generally spherical and in simple or branched chains (see Sheath and Sherwood 2011, Pl. 42E & F, Guiry and Guiry 2015). Identification as L. fluviatilis by Bhosale et al. (2012) was followed subsequently in other studies on biofuel production (Rout et al. 2011), nutrient content (Singh and Gupta 2010a, 2010b), treatment of diabetes (Devi 2011), trace elements (Bino-Devi et al. 2011), bio- synthesis of gold nanoparticles (Sharma et al. 2014) and anti-urolithiatic agent (Mikawlrawng et al. 2014).
Singh and Gupta (2010a, 2010b, 2011, 2014) and Gupta and Singh (2012) extensively studied the ecology of Le- manea populations and identified five different species.
These are, in addition to L. fluviatilis from the Thoubal
Table 2.
Synopsis of references (1973-2014) for the five specific epithets applied to Lemanea plants from Manipur StateReferences 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 and Chakpi Rivers
Taxonomy
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), Gupta and Singh (2012)
L. australis Imphal and Manipur Rivers Ecology and nutrient contents 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
(2014)
L. fluviatilis Imphal Rivers Hipatoprotective and antioxi-
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 from L. 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 between Lemanea manipurensis sp. nov. and L. sinica JaoExternal features L. manipurensis (present study) L. sinica (Jao 1941) Morphology Plants up to 10 cm long, sparsely and irregularly
branched with or without a stipe and
constriction, growing on stones in river beds of Manipur, India
Up to 16 cm long, with a long stipe, branched more at the basal part, branching opposite, alternate above, apical branches capillary, China
Anatomy No internal corticating filaments No internal corticating filaments
Spermatangia In isolated irregular patches, sometimes
completely covering the axis Mostly in complete, distinct rings, rarely in isolated patches
Carposporophyte structure Gonimoblast filaments few that are sparsely branched, apical cell bearing a single, terminal, elongate-obovoid carpospore
Gonimoblast filaments with a few and sparsely branched cells, apical cell 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.
lacks cortical filaments and commented that it is a “pecu- liar combination of the characteristics of two genera” in L. sinica. Kučera and Marvan (2004), in a taxonomic study of the Lemaneaceae in the Czech Republic, characterized Lemanea with a central axis with or without cortical fila- ments. According to them, “the regular formation of sper- matangia in rings varies with age and the magnification used.” These variable features in spermatangial forma- tion in their material of L. fluviatilis were also shown in illustrations (Kučera and Marvan 2004, p. 168, Fig. 3A-D, Simić and Djordjević 2011, p. 515). Xie et al. (2004) in their study of Chinese Lemaneaceae, mentioned that in Para- lemanea parvula (Sirodot) S. L. Xie & Z. X. Shi, spermatan- gia are found in patches (Xie et al. 2004, p. 886, Fig. 13).
Eloranta et al. (2011, p. 96) preferred to keep P. parvula in Lemanea as an uncertain species, but notably remarked that “spermatangial papillae form separate or sometimes coalescent tubercles.” Anatomical details, i.e., whether cortical filaments are present or not in P. parvula is not known in Chinese and European specimens. Eloranta et al. (2011) did not include the reference of Xie et al. (2004).
The above mentioned instances clearly show the ambigu- ity of using some anatomical and reproductive features to differentiate Lemanea and Paralemanea.
It is significant that the five species of Lemanea (L.
crassa, L. manipurensis, L. ramosa, L. simplex, and L. si- nica) from the Southeast Asian continent are remarkably similar, producing just a single and elongate terminal car- pospore on each short simple to branched carposporo- phyte filament with narrow cylindrical cells. Most other species of Lemanea and Paralemanea produce spherical carpospores serially in simple or branched chains (cate- nate). Detailed and comparative morphological and mo- lecular data would clarify whether this group of Lemanea species warrants segregation into a new genus, different from closely related genera like Lemanea, Paralemanea, and Petrohua. A case in point is that extensive molecu- lar and morphological data on Batrachospermum spe- cies sensu lato resulted in the segregation of Kumanoa from Batrachospermum with spirally twisted carpogo- nial branches and axial carposporophytes (Entwisle et al.
2009) and Sheathia with hetero-corticated thalli, carpo- gonia on undifferentiated cells and exserted carposporo- phytes (Salomaki et al. 2014).
It is noteworthy that Necchi et al. (2013) found a very low genetic diversity between species of the two genera Composopogon and Compsopogonopsis (Compsopogo- nales, Rhodophyta) based on global sampling and mo- lecular data. Species of these 2 genera were distinguished hitherto by caeruleus morphology (without cortication) other reports of L. sinica are known in China or elsewhere.
Future molecular studies on L. sinica would undoubtedly reveal whether or not it differs from L. manipurensis. The type locality of L. sinica is Chun-tien, Yunnan (32°03′01″
N, 110°36′50″ E), while that of the Indian species is Chak- pi, Thoubal district, Manipur (24°16′ N, 93°52′ E), perhaps not a very significant geographical distance but the many diverse river systems and mountain ranges between these localities may be formidable barriers for dispersal.
In the five Manipur river systems, Lemanea popula- tions may be abundantly distributed in the different riv- er segments. Hence the possibility of cryptic speciation among the different populations cannot be ruled out.
Only critical molecular studies would clarify this point.
Lemanea and Paralemanea are known to grow together in the same ecological habitat and there are instances where the same herbarium packet contained more than one species (Palmer 1941). Harvested material of Lema- nea from different places and the processed dried packets sold in the local markets may be helpful on morphologi- cal and molecular bases in resolving whether Paralema- nea species also occur in India.
Lemanea, Paralemanea, Petrohua, and Psilosiphon species offer few reliable morphological characteristics to separate them at the generic level. As stated by Sheath (2003, pp. 208, 216 & 217) the presence or absence of axial filament cortication and the configuration of spermatan- gial rings or patches should be the most useful features in defining Lemanea and Paralemanea, but these 2 ge- neric attributes apparently are not consistent, thus rais- ing doubts about the generic status of Paralemanea as different from Lemanea. In any case molecular analyses in this and other published work suggest Paralemanea is paraphyletic leaving the issue unresolved.
The landmark publication by Jao (1941) on L. sinica is overlooked in many later publications on the Lema- neaceae. Jao (1941, p. 271, Pl. VII, Figs 50-52) described L.
sinica specimens with “the antheridial bands are mostly in complete rings around the antheridial zones. In older portions of the thallus they are occasionally slightly in- terrupted but also kept a band-shape. Probably the inter- rupting is due to the hypertrophy of the tissue beneath the antheridial bands. In addition to the ordinary anther- idial bands, small antheridial sori sometimes appear on the old carpogonial zones.” Xie et al. (2004) said in their Lemanea species key that L. sinica has “Spermatangia in perfect rings” but they did not obtain any new L. sinica specimens. Hu and Wei (2006) stated “the antheridia are always in well-defined bands” without further evidence.
Jao (1941) clearly mentioned the “naked central axis”
Chowdhury, A. N., Das, H. B. & Singh, S. T. 1974. An ed- ible alga of Manipur (Lemanea australis): presence of silver. Curr. Sci. 43:629.
Deb, D. B., Krishna, B. & Singh, S. T. 1986. Life history of a Lemanea species from Manipur. In Desikachary, T. V. &
Raja Rao, V. N. (Eds.) Taxonomy of Algae. University of Madras, Madras, pp. 213-221.
Desikachary, T. V., Krishnamurthy, V. & Balakrishnan, M.
S. 1990. Rhodophyta. Vol. 1. Part II A. Madras Science Foundation, Madras, 279 pp.
Devi, A. P. 2011. Plants used by Meitei community of Ma- nipur for the treatment of diabetes. Assam Univ. J. Sci.
Technol. Biol. Environ. Sci. 7:63-66.
Devi, W. I., Devi, G. S. & Singh, C. B. 2011. Traditional herbal medicine used for the treatment of diabetes in Manipur, India. Res. J. Pharm. Biol. Chem. Sci. 2:709-715.
Eloranta, P. & Kwandrans, J. 2007. Freshwater red algae. Rho- dophyta. Identification guide to European taxa, particu- larly to those found in Finland. Norrilinia 15:1-103.
Eloranta, P., Kwandrans, J. & Kusel-Fetzmann, E. 2011. Rho- dophyceae and Phaeophyceae. In Schagerl, M. (Ed.) Süßwasserflora von Mitteleuropa Band 7. Freshwater Flora of Central Europe. Vol. 7. Spectrum Akademischer Verlag, Heidelberg, pp. 1-155.
Entwisle, T. J. 1989. Psilosiphon scoparium gen. et sp. nov.
(Lemaneaceae), a new red alga from south-eastern Aus- tralian streams. Phycologia 28:469-475.
Entwisle, T. J., Vis, M. L., Chiasson, W. B., Necchi, O. Jr. & Sher- wood, A. R. 2009. Systematics of the Batrachospermales (Rhodophyta): a synthesis. J. Phycol. 45:704-715.
Faridi, M. A. F. 1971. The genera of fresh-water algae of Paki- stan and Kashmir. Biologia 12:123-142.
Faridi, M. A. F. 1975. Batrachospermum in Pakistan. Biologia 21:107-109.
Freshwater, D. W. & Rueness, J. 1994. Phylogenetic relation- ships of some European Gelidium (Gelidiales, Rho- dophyta) species, based on rbcL nucleotide sequence analysis. Phycologia 33:187-194.
Ganesan, E. K. & West, J. A. 2013a. Nomenclatural changes for some freshwater red algae from India. Algae 28:45- 51.
Ganesan, E. K. & West, J. A. 2013b. On the identity of an edible macroscopic freshwater (riverine) red alga from Mani- pur, north-east India. In National Conference on ”Fron- tiers in Algology and Algal Biotechnology” [NCFAAB], Visva-Bharati University, Santiniketan, p. 6.
Guiry, M. D. & Guiry, G. M. 2015. AlgaeBase. World-wide electronic publication, National University of Ireland, Galway. Available from: http://www.algaebase.org. Ac- cessed Mar 8, 2015.
for Compsopogon species and leptoclados morphology (with cortication) for Compsopogonopsis species. Nec- chi et al. (2013) concluded that only one genus, Compso- pogon, can be recognized.
It is still too early to provide a complete molecular and morphological story concerning generic and species de- limitations for Lemanea and Paralemanea.
ACKNOWLEDGEMENTS
Morgan Vis kindly provided a copy of Atkinson (1890).
Interlibrary loan facilities of the University of Melbourne, Michael Wynne and S. Tripathi obtained some early and recent literature not easily accessible to us. P. S. Yadava (Manipur University, Imphal) provided liquid preserved material and some information about the edible Nung- sham sold in the local markets. Ms. M. Thajamandhi as- sisted J. Rout in field collections and processing of liquid preserved, herbarium and silica gel material for DNA ex- traction. Tim Entwisle provided very helpful advice on the manuscript before submission. Funding was provided mostly from the personal pensions of Ganesan and West.
REFERENCES
Atkinson, G. F. 1890. Monograph of the Lemaneaceae of the United States. Ann. Bot. 4:177-229.
Bhosale, R. A. & Chaugule, B. B. 2010. Freshwater algae as po- tential source of polyunsaturated fatty acids: review. Int.
J. Algae 12:335-356.
Bhosale, R., Rout, J. & Chaugule, B. 2012. The ethnobotanical study of an edible freshwater red alga, Lemanea fluviati- lis (L.) C. Ag. from Manipur, India. Ethnobot. Res. Appl.
10:69-76.
Bino-Devi, Ch., Sharat Singh, N. K., Singh, N. R., Rajendro Singh, N., Sudarshan, M., Chakraborty, A. & Ram, S. S.
2011. Trace elements in Nungsham, the red edible algae of Manipur. Int. J. Appl. Biol. Pharm. Technol. 2:198-203.
Bory de Saint-Vincent, J. B. 1808. Mémoire sur le genre Le- manea de la famille des Conferves. Ann. Mus. Hist. Nat.
12:177-190.
Carmona Jimenez, J. & Necchi, O. Jr. 2002. Taxonomy and distribution of Paralemanea (Lemaneaceae, Rhodophy- ta) in central Mexico. Cryptogam. Algol. 23:39-49.
Darriba, D., Taboada, G. L., Doallo, R. & Posada, D. 2012.
jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9:772.
Deb, D. B., Krishna, B., Mukherjee, M. K., Bhattacharya, S.,
Necchi, O. Jr., Fo, A. S. G., Salomaki, E. D., West, J. A., Aboal, M. & Vis, M. L. 2013. Global sampling reveals low genet- ic diversity within Compsopogon (Compsopogonales, Rhodophyta). Eur. J. Phycol. 48:152-162.
Necchi, O. Jr. & Zucchi, M. R. 1995. Record of Paralemanea (Lemaneaceae, Rhodophyta) in South America. Algol.
Stud. 78:33-38.
Nizamuddin, M. 1988. Occurrence of the genus Bangia Lyn- gbye (Bangiales, Rhodophyta) from Chitral, North West of Pakistan. Pak. J. Bot. 20:45-48.
Palmer, C. M. 1941. Lemanea herbarium packets containing more than one species. Butler Univ. Bot. Stud. 5:222-223.
Rambaut, A. & Drummond, A. J. 2007. Tracer. Available from:
http://beast.bio.ed.ac.uk/Tracer. Accessed Aug 30, 2012.
Ronquist, F. & Huelsenbeck, J. P. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinfor- matics 19:1572-1574.
Rout, J., Chaugule, B. & Bhosale, R. 2011. Potential of a red alga, Lemanea for use as a source of biofuel. In Bhat- nagar, S. K., Saxena, A. & Krann, S. (Eds.) Algae Biofuel.
Stadium Press, New Delhi, pp. 111-115.
Salomaki, E. D., Kwandrans, J., Eloranta, P. & Vis, M. L. 2014.
Molecular and morphological evidence for Sheathia gen. nov. (Batrachospermales, Rhodophyta) and three new species. J. Phycol. 50:526-542.
Sharma, B., Purkayastha, D. D., Hazra, S., Thajamanbi, M., Bhattacharjee, C. R., Ghosh, N. N. & Rout, J. 2014. Bio- synthesis of fluorescent gold nanoparticles using an ed- ible freshwater red alga, Lemanea fluviatilis (L.) C. Ag., and antioxidant activity of biomatrix loaded nanopar- ticles. Bioprocess Biosyst. Eng. 37:2559-2565.
Sheath, R. G. 2003. Red Algae. In Wehr, J. D. & Sheath, R. G.
(Eds.) Freshwater Algae of North America: Ecology and Classification. Academic Press. San Diego, CA, pp. 197- 224.
Sheath, R. G., Müller, K. M., Vis, M. L. & Entwisle, T. J. 1996.
A re-examination of the morphology, ultrastructure and classification of genera in the Lemaneaceae (Batracho- spermales, Rhodophyta). Phycol. Res. 44:233-246.
Sheath, R. G., Müller, K., Sherwood, A. & Shea, T. 2006. Phylo- genetic relationships of members of the freshwater red algal family Lemaneaceae from North America, Europe and Africa. In Stekoll, M. S. (Ed.) Phycol. Soc. Am. 60th Annu. Meet. Northwest Algal Symp., Phycological Soci- ety of America, Juneau, AK, p. 57.
Sheath, R. G. & Sherwood, A. R. 2011. Rhodophyta. In John, D. M., Whitton, B. A. & Brook, A. J. (Eds.) The Freshwa- ter Algal Flora of the British Isles. An Identification Guide to Freshwater and Terrestrial Algae. 2nd ed. Cambridge University Press, Cambridge, pp. 159-180.
Gupta, A. & Singh, R. M. 2012. Lemanea in lotic environment of Manipur, North-East India: distribution, ecology and need for conservation of Lemanea. Lamhat Academic Publishing, Saarbrücken, 80 pp.
Hu, H. & Wei, Y. 2006. The freshwater algae of China: system- atics, taxonomy and ecology. Science Press, Beijing, 1023 pp.
Jain, A., Roshnibala, S., Kanjilal, P. B., Singh, R. S. & Singh, H. B. 2007. Aquatic/semi-aquatic plants used in herbal remedies in the wetlands of Manipur, Northeastern In- dia. Ind. J. Tradit. Knowl. 6:346-351.
Jain, A., Sundriyal, M., Roshnibala, S., Kotoky, R., Kanjilal, P.
B., Singh, H. B. & Sundriyal, R. C. 2011. Dietary use and conservation concern of edible wetland plants at Indo- Burma Hotspot: a case study from northeastern India. J.
Ethnobiol. Ethnomed. 7:29.
Jao, C. C. 1941. Studies on the freshwater algae of China. VIII.
A preliminary account of the Chinese freshwater Rhodo- phyceae. Sinensia 12:245-290.
Johnston, E. T., Lim, P. -E., Buhari, N., Keil, E. J., Djawad, M. I.
& Vis, M. L. 2014. Diversity of freshwater red algae (Rho- dophyta) in Malaysia and Indonesia from morphologi- cal and molecular data. Phycologia 53:329-341.
Khan, M. 1973. On edible Lemanea Bory de St. Vincent, a fresh water red alga from India. Hydrobiologia 43:171- 175.
Kučera, P., Grulich, V., Fránková, M. & Bureš, P. 2008. Dis- tribution of freshwater red algal family Lemaneaceae (Rhodophyta) in the Czech Republic: an update. Fottea 8:125-128.
Kučera, P. & Marvan, P. 2004. Taxonomy and distribution of Lemanea and Paralemanea (Lemaneaceae, Rhodophy- ta) in the Czech Republic. Presilia (Praha) 76:163-174.
Kumano, S. 2002. Freshwater red algae of the world. Biopress Ltd., Bristol, 375 pp.
Mannino, A. M., Barone, R. & Raimondo, F. M. 2003. First re- cord of Paralemanea catenata (Rhodophyta) from Ital- ian Peninsula. Bocconea 16:1053-1058.
Mao, A. A., Hynniewta, T. M. & Sanjappa, M. 2009. Plant wealth of northeast India with reference to ethnobotany.
Indian J. Tradit. Knowl. 8:96-103.
Mikawlrawng, K., Kumar, S. & Vandana. 2014. Current sce- nario of urolithiasis and the use of medicinal plants as antiurolithiatic agents in Manipur (North East India): a review. Int. J. Herb. Med. 2:1-12.
Mullahy, J. H. 1952. The morphology and cytology of Lema- nea australis Atk. Bull. Torrey Bot. Club 79:471-484.
Müller, K. M., Sherwood, A. R., Pueschel, C. M., Gutell, R. R. &
Sheath, R. G. 2002. A proposal for a new red algal order, the Thoreales. J. Phycol. 38:807-820.
Climate Change. Pointer Publishers, Jaipur, pp. 118-134.
Stamatakis, A. 2006. RAxML-VI-HPC: maximum likelihood- based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688-2690.
Vis, M. L., Harper, J. T. & Saunders, G. W. 2007. Large subunit rDNA and rbcL gene sequence data place Petrohua ber- nabei gen. et sp. nov. in the Batrachospermales (Rho- dophyta), but do not provide further resolution among taxa in this order. Phycol. Res. 55:103-112.
Vis, M. L., Saunders, G. W., Sheath, R. G., Dunse, K. &
Entwisle, T. J. 1998. Phylogeny of the Batrachospermales (Rhodophyta) as inferred from rbcL and 18S ribosomal RNA gene sequences. J. Phycol. 34:341-350.
Vis, M. L. & Sheath, R. G. 1992. Systematics of the freshwater red algal family Lemaneaceae in North America. Phyco- logia 31:164-179.
Xie, S. -L., Shi, Z. -X. & Wang, R. -N. 2004. Taxonomy of the Le- maneaceae (Batrachospermales, Rhodophyta) in China.
Acta Bot. Sin. 46:883-888.
Zuccarello, G. C. & Lokhorst, G. M. 2005. Molecular phylog- eny of the genus Tribonema (Xanthophyceae) using rbcL gene sequence data: monophyly of morphologically simple algal species. Phycologia 44:384-392.
Simić, S. & Djordjević, N. 2011. Lemanea fucina Bory, 1808 (Lemaneaceae, Rhodophyta), a rare species with a vari- able morphology: first record in the Republic of Monte- negro. Arch. Biol. Sci. Belgrade 63:511-515.
Singh, I. L. & Muralidharan, P. 2014. Evaluation of hepatopro- tective and anti-oxidant activity of Lemanea fluviatilis on CCI4 induced hepatotoxicity in rats. Res. J. Pharm.
Biol. Chem. Sci. 5:976-982.
Singh, M. R. & Gupta, A. 2010a. Morphological characteris- tics of rare and endangered species of Lemanea (Rho- dophyta) from the rivers of Manipur, North-east India.
Botanique 14:47-54.
Singh, M. R. & Gupta, A. 2010b. Nutritional composition of edible Lemanea australis Atkinson (Lemaneaceae, Rho- dophyta) from Manipur river systems, India. PGTD of Botany, R. T. M. University, Nagpur, pp. 100-107.
Singh, M. R. & Gupta, A. 2011. Factors affecting the occur- rence of Lemanea, Rhodophyta from the Rivers of Ma- nipur, North-east India. In Tiwari, D. (Ed.) Advances in Environmental Chemistry. Excel India Publishers, New Delhi, pp. 233-236.
Singh, M. R. & Gupta, A. 2014. Biomass and productivity study of red algae from rivers of Manipur, North East India. In Gupta, A. (Ed.) Ecoplanning, Biodiversity and