Phycologia Volume 55 (1), 45-54 Published 8 January 2016

Morphological and molecular characterization of Ulva chaugulii sp. nov., U. lactuca and U. ohnoi (, ) from India

M udassar A nisoddin K a z i', M onica G. K a vale'* a n d V eeren d r a V. Sinqh^ 'Department o f Botcmy, I.C.S. College, Khed, Rcilnagiri, Maharashtra 41S 709, huUa ^ICAR-Mumbai Research Centre of Central Marine Fisheries Research Inslitule, Mumbai 400061, Indki

A b s t r a c t : Detailed morphological, aiialomical and molecular characteristics of Uha on the west coast of India revealed the presence of U. chaugulii sp. nov. Vim climigiiiii is characterized by tubular, compressed and fragile tliaili with infundibuliform shape, a dilated apex and the presence of two pyrenoids per cell. The molecular phylogeny based on the rbcL gene and internal transcribed spacer rDNA sequences support the recognition of a new lineage within Uha. The present study also verifies the presence of U. ohnoi in western India. Ulva lactuca and U. olmoifrom India weie resolved as separate lineages based on rbcL phylogeny. The genetic divergence between rbcL sequences of these two species was 0.4%.

K e y W o r d s ; Clilorophyta, India, Molecular phylogeny, rbcL, Ulva, Ulva cluiiigiilii

INTRODUCTION The molecular data also helped in understanding the biogeographic history, cryptic diversity and introduction of U/va Linnaeus, commonly referred to as '’, with species of U ha in different regions (Heesch et al. 2009; 128 taxonomically accepted species, is the most species-rich Hofmann et al. 2010; Kraft et al. 2010; Wolf et al. 2012; genus in the family (Guiry & Guiry 2015). Several Kirkendale et al. 2013). As a consequence, recent studies species of Ulva are associated with notorious blooms called recommended an integrated approach to the taxonomy of ‘green tides’ (Fletcher 1996; Blonister ef al. 2002; Zhao et a!. Ulva that used both morphological and molecular charac­ 2010). The uncertainty in taxonomic status of most (axa in terization (Loughnane el al. 2008; Hofmann et al. 2010; this genus is again a major concern for taxonomists. This Matsumoto & Shimada 2015). uncertainty is directly attributed to the morphological In this study, we used a polyphasic approach to Uha that plasticity resulting in recognition of large numbers of included morphological, ecological and molecular analyses varieties, forms and ecotypes. The plasticity was also jjroved to resolve the taxonomy from the west coast of India. The at the higher taxonomic level, wherein the artificial nature of study is a part of broader goal of biodiversity assessment of a generic character distinguishing Uha and Enteromorpha economically important marine macroalgae from India. Link (i.e. distromatic thalll in Uha and tubular-monostro- Preliininary data suggested that most species of Ulva matic thalli in Enteromorpha) was established (Hayden et al. identified previously (e.g. Silva et al. 1996) needed extensive 2003; Shimada et al. 2003). Species of Ulva exhibit a range of revision. For example, several niorphotypes that we identi­ morphology in thallus architecture, that is, foliose, lanceo­ fied as well-established species turned out to be U. ohnoi late, linear, ovate, cuneate or tubulose. Other important Hiraoka & Shimada, a new report from India. In India, 17 taxonomlcal cliaracters include cell size, shape and arrange­ species of Ulva were reported (Silva et al. 1996), including the ment; thallus thickness; number of pyrenoids per cell; and recently identified new species U. pascliima F. Bast (Bast et morphology of holdfast and basal region (Bliditig 1968; al. 2014) from the west coast. Among these, U. lactuca was Koeman & van den Hoek 1981). However, intraspecific abundant. During surveys of the west coast of India, we variations were observed for these characters in different encountered a new species of Uha. This study focuses mainly environments and growth phases (Blomster et al. 1998). on the detailed morphological, ecological and molecular Over the last two decades, remarkable progress has been characterization of the new species U. chaugulii M.G. Kavale made in molecular genetic techniques. The advent of & M.A. Kazi and of U. lactuca and U. ohnoi from western molecular markers has provided new insights Into organis- India. mal systematics. DNA sequence-based taxonomy has helped to resolve many challenging and difficult taxonomic issues. Recent inolecuiar studies in Uha have contributed signifi­ MATERIAL AND METHODS cantly to its taxonomy, such as the merging of Ulva and Enteromorpha (Hayden et al. 2003), synonymization of U. lactuca Linnaeus and U.fasciala Delile (O’Kelly et al. 2010) The samples were collected from the west coast of India. and identification of green tide-forming taxa (Liu et al. 2010; Sixteen sites were visited to survey the occurrence of Uha Zhao et al. 2010; Guidone el al. 2013; Gtioying et al. 2014). (Fig. I, Table SI), A total of 26 samples were handpicked at low tide and analysed. The specimens were washed with sterile filtered seawater to remove epiphytes and adhered * Corresponding author ([email protected]). DOlr 10.2216/15-11.1 debris. Specimens were examined for morphological and © 2016 Iiiterjiational Phycological Society anatomical characters by using compound and phase 46 Phycologia, Vol. 55 (1)

68“ 72° 76° / 80 ° 84° 88 ° 4 - .. y - ; 24°

INDIA / ....1 y

■y,

16° 1.AdrI 2. Circuit House, Veraval 3. 8hldbhanjan 4. Harihareshwar 5. Ladghar 6. Kolthsre 7. Panchanadi 12° 8. Palshet 9. Vayangani 10. Purnagad 11. Malwan 12. Redi 13. Vagalor 14. Ullal 15. Viihinjam 16. Kovalam

Fig, I, Map of sampling sites for species of Uha from India.

contrast microscopes (Molic BA310 Phase, Hong Kong, al. 1998). The PCR amplification cycle consisted of a cycle of China). Morphometric data were based on a minimum of 30 5 mill at 94°C, 35 cycles of 1 niin at 94°C, 1 niin at 52°C, 2 min measurements for each character. The photographic docu­ at 72°C and one cycle of 7 mJn at 72°C. The PCR products mentation and measurements were made with tlie software were purified with a GenElufe Gel Extraction Kit (Sigma Motic Images Plus 2.0 ML (Motic, Hong Kong, China). Aldrich) and sent for connnercial sequencing (Xcelris Labs Voucher herbarium specimens were made and submitted to Ltd, Ahmedabad, riidia). The sequences were deposited in the Central National Herbarium, Botanical Survey of India, GenBank (see Tables S2 and S3 for accession numbers). Kolkata (CAL), A portion of each sample was frozen at Sequences amplified in this study were aligned with those -20°C for DNA extraction. The biomass was determined by published sequences retrieved from GenBank. The rhch measuring average wet weight of an identifying accompany­ sequences were aligned using the CiustalW algorithm in ing species from at least 10 quadrats (I m^). MEGA v6 (Taniura el al. 2013), and the ITS rDNA Total genomic DNA was extracted with the Gene Elute sequences were aligned using M AFFT v7 (E-lNS-i iterative Plant Genomic DNA miniprep kit (Sigma Aldrich, St Louis, refinement method) (Katoh el al. 2005), The aligned rbch IVIissouri USA) following the manufacturer’s protocol. data set consisted of 1361 characters that included 132 Amplification by polymerase chain reaction (PCR) was parsimony informative, 1152 conserved and 209 variable performed In a master mix of volume 25 nl containing 5 sites. The aligned ITS rDNA data set consisted of 1159 pmol of each primer, 200 ).iM of each dNTP, IX assay buffer characters that included 297 parsimony informative, 509 and 1.25 units of Taq DNA polymerase. The partial rbch conserved and 482 variable sites. Gaps in the alignments (large subunit of ribulose-l,S-bisphosphate carboxylase/oxy­ were treated as missing data. The alignments were subjected genase) gene was amplified using primer pairs RHI to phylogenetic analysis by the maximum likelihood (ML) (5'ATGTCACCACAAACAGAAACTAAAGC-3') and approach. The analyses were performed using the general I385r (5'-AATTCAAATTTAATTTCTTTCC-3') (Manhart time reversible model with gamma distributed site rates and 1994). PCR amplification was carried out for rbcl. following considering certain fraction of invariable sites (I), as Hayden et al, (2003). For the nuclear ribosomal internal obtained in jModelTest v2.l.4 (Darriba et al. 2012) based transcribed spacer (ITS rDNA) region, the primer pair used on Akaike information criterion scores, ML analysis was was 18S1505 (Hayden el al. 2003) and ENT26SA (Blomster el carried out using raxmlGUI vl.l (Silvestro & Michalak 2012) with 1000 bootstrap replicates. Umbrauha olivcisceiis up to 0.5 cm (0.37 ± 0,11) long. There were frequent (P.J.L. Daiigeard) G. Furnari, U.japonica (Holmes) Bae & proliferations on the stipe. Proliferations were elongated into I.K. Lee, Gemina tetterslecltokka V.J. Chapman, Ulvaria horn-like projections. Blades expanded above the stalk and obsciiya var. Z)/J’^^/7(Areschoug) Sliding, Percursaria perciiisa became flat and infundibuliform; margins stnooth, apex (C. Agardh) Rosenvinge and Ulvaria fusca (Wittrock) dilated, sometimes constricted at intervals. Plants were Vinogradova were included as out-groups in the analyses. attached by basal discs, occasionally several arising from a The pairwise distance between sequences was calculated common disc. Cells in surface view were polygonal, regularly using MEGA v6 (Tainura et al. 2013). arranged in longitudinal rows in greater part of the thallus. Cell size was 14.9-18.7 nm (16.7 ± 1.4) at their greatest dimensions. In transverse section, cells were longer than wide RESULTS with blunt corners and rectangular, 16.5-24.0 fim (19,5 ± 2.8) high and 9.6-20.3 )un (14.7 ± 4.3) wide. Chloroplasts Morphological characterization were parietal with two pyrenoids. In transverse section, stipes were cylindrical, 308-432 juii (357 ± 56) in diameter, Species identifications based on morphological characters with the upper portion traversed by trabeculae. At the end of (attachment, shape and size of blade, nature of margin, cell stipe and at the edge of blade expansion, thalli were shape and size in surface view and cross section, type of monostromatic with a hollow ring-like structure. The chloroplast and number of pyrenoids per cell) were made inonostromatic ring gradually became distromatic except at using descriptions given by Krishnamurthy (2000). Uha marginal regions in the middle to upper parts of the thallus. fasciatci and V. laduca were initially identified. However, The two layers rarely adhered all the way to the margin. The after molecular analysis, most of the specimens were thickness of the monostromatic layer was 37.1-63.3 |.ini (50.7 recognized as U. ohiioi (previously identified as U. kictuca). ± 9.2); whereas, the combined thickness of the two layers In the present study, specimens of U.fasciata were treated as was 51.0-107,1 nm (80.0 ± 23,0). Cell mucilage was 1.1-2.7 U. laduca following O ’Kelly et al. (2010). The detailed )ini (2.0 ± 0.6); whereas, thickness of mucilage ranged from morphological study was conducted to analyze the intra- and 2,2-4,4 nm (3,3 ± 0,9), Rhizoids were 36.7-112,9 ^tm (76.0 interspecific variations in U. lacliiai and U. olmoi (Tables S2 ±31.3) long, and S3). Ulva iactuca Linnaeus Ulva cliaiigiilii M,G. Kavale & M.A. Kazi sp. not'. Plants were 4-100 cm high (26.29 ± 13.21), 1^0 cm (9.48 ± Figs 2-13 7.36) wide, dark green to pale green and attached by a circular I3ESCR1PTI0N: Thalli tubular, light green, fragile and compressed, up disc, with or without a small stipe (Figs 14-19). The thalli were to 4,0 cm (2.93 ± 0.93, mean ± s) long, 0.6 cm (0.48 ± 0.11) broad. deeply divided with lanceolate lobes, broadly expanded or Thalli simple or branched at base, with a cylindrical hollow silpe up to with less divided, lanceolate to irregular lobes (Fig. SI). The 0.5 cm (0.37 ± 0.11) long. Apex of thalli obtuse and dilated. Frond blade margin was smooth, undulate to ruffled and inore or less margins smoolh or irregularly constricted. Cells in surface view spinulous. Spines were 2,0-69,3 ntu (20,4 ± 12,4) in height. polygonal 14.9-18.7 jim (^16.7 ± 1.4) in greatest dimension. Cliloroplasts parietal with two pyrenoids. In transverse section, cells Broadly expanded thalli had numerous perforations; whereas, longer than wide with blunt corjiers and rectangular, 16.5-24.0 ^im deeply lobed, lanceolate thalli had few perforations, Thalli (19.5 ± 2,8) high, 9.6-20.3 ^mi (14.7 ± 4.3) wide. Cells aligned in were 43,2-105,5 pm (72,4 ± 10.6), 62.3-192,9 )tm (106,0 ± longitudinal series through the thallus. Frond consisted of two layers 30,0) and 85.0-375.5 nm (173,7 ± 50,9) thick in apical, tniddle adhering with each other except in the margin, Monostromatic layer 37.1-63.3 |.im (50,7 ± 9,2) thick. and basal regions, respectively. Vegetative cells in surface view were round to polygonal and ranged from 8,8 to 30,7 nm (17,3 HOLOTYPE: CAL/ALG./029, collected 6 September 2014, deposited in ± 2,3) in its greatest dimension. The chloroplasts almost filled Central National Herbarium, Botanical Survey of India, Kolkata. the cells and had one to three pyrenoids.

(SOTYPE: CAL/ALG.030 and CAL/ALG./031, collected 6 September Ulva o/iiioi I-Ilraoka & Shimada 2014, deposited in Central National Herbarium, Botanical Survey of India. Kolkata. Plants were orbicular, lanceolate or irregularly expanded,

ETYMOLOGY: This species is named In honour of Prof. B.B. Chaugule wider than long, 1-12 ctn (4.20 ± 2.80) in height and 2-35 crn in appreciation of his immense contribution to phycology in India. (6.71 ± 5.19) in width and attached by circular disc with small stipe (Figs 20-25). These plants were pale green to dark green GENBANK ACCESSrON: KP710829-K.P710833 represent the rbcL in colour, easily separated in two layers, larger thalli with sequences, and KT429218-KT4292I9 are the ITS rDNA seqtiences. numerous perforations. Tufts of thalli developed from a common disc. The thallus margin was ruffled, with micro­ TYPE LOCALITY: Vayangani (16°55.52'N, 73“17.orE), Maharashtra, India. scopic spines ranging from 4.5 to 44.3 ).tm (15.9 ± 7.5). Thalli were 33.2^9.7 \xm (48.7 ± 7.6), 41.3-82.2 nm (58.0 ± 7.6) Thalli of U. cli(iiigulii were light green, tubular, com­ and 45.0-182.8 |.tm (83.2 ± 32,1) in apical, central and basal pressed and fragile, up to 4.0 cni (2.93 ± 0.93) long and 0.6 regions, respectively. Cells in surface view were polygonal and cm (0.48 ± 0.11) broad, simple or branched at base and completely filled by a chloroplast with one to three pyrenoids. conspicuously septate. The stipe was cylindrical and hollow. In transverse section, vegetative cells were rectangular with ,s . " - v /■; ';V,.'T V '/iflij--, ■ ' ^

': ^ 1 ■ ■ '. '■

Figs 2-13. t//w e/)«»g<(/(r. Fig. 2, scale bar = I cm; Figs 3,4, scale bar = 1 mm; Figs 5,7-9, 12, 13, scale bar = 100 urn; Figs 6, 10, II,scale bar = 10 [im. Fig. 2. Holotype (CAL/ALG./029). Fig. 3. Stipitate thalliis. Fig. 4. Dilated apex of lhallus (arrow). Fig. 5. Transverse section of basal portion of stipe. Fig. 6. Transverse section of thallus showing two layers adhering to each other. Fig. 7. Transverse section of middle portion of stipe, arrow indicates trabeculae. Fig. 8. Transverse section of upper portion of stipe, arrow indicates trabeculae. Fig. 9. Transverse section of lamina showing middle (arrow) and miirginal portion. Fig. 10. Monostomatic layer of (halhis. Fig. II. Surface view of thallus. Fig. 12. Thallus showing branching and cell arrangement. Fig. 13, Proliferations on branches (arrow). blunt cornet's and ranged from 14.3 to 45.2 [.iin (23.4 ± 6.9) in Uh’o lacluca was distributed along the entire west coast of height and from 8.8 to 19.9 jim (13.9 ± 1.2) in width. India. The plants grew luxuriantly on the entire intertidal region of rocky shore attached to the substratum. The rocky Distribution and ecology substratum consisted mainly of basaltic wave-cut platforms with overhanging clilTs, numerous huge boulders and water Uha cimugiilii was found in tlie upper littoral zone in patches pools. The growth of plants started early in the monsoon during the monsoon season from July to September. The highest biomass recorded was 20 g nr^ witli a percent cover of season, and plants were found until the middle of winter (July- 1.9 ± 0.5%. Ulva lacluca, Pyropia acanthophora (E.C. Oliveira December), The associated genera with U. lacluca at collection & Coli) M.C. Oliveira, D. Milstein & E.C. Oliveira and sites in the states of Maharashtra, Goa, Karnataka and Kerala Pyropia vieinamensis (Tak. Tanaka & P.H. Ho) J.E. Suther­ were Pyropia, Chaetomorpha and Graleloupia', whereas, in land & Monotilla were also observed associated with U. Gujarat state, Gracilaria, Sargassiiiu, Caiikrpa, Sfoechosper- chaugulii. The air temperature was 25-29°C, and the seawater /wtin, Padina, Ceiilroceros, Jania, Dictyow, Spoiigonwrpha, had pH 8.5 and salinity of 10 practical salinity units. Haliitieda, Spaloglossum, Boodka and so on were observed. > *

Figs 14-19. Representalive specimen of Ulva hciuca. Fig. 14, scale bar = 2 cm; Fig. 15, 16, 18, scale b a r= 10 nm; Fig. 17, scale bar = 2 mm; Fig, 19, scale bar = 20 i-im Fig. 14. Habit. Fig, IS. Surface view showing pyienoids. Fig. 16. Surface view showing marginal spines. Fig. 17. Basal portion showing rhizoidal disc. Fig. 18. Transverse section of thallus. Fig. 19. Transverse section of thallus showing rhizoids.

The biomass of U. lactiica ranged from 0.5 to 4.0 kg with were clearly monophyletic and clustered with U. fasciata average percent cover of 64.0 ± 24.1%. (AY255872) and U. laciuca (GU138294), The specimens of U. Ulvci olmoi was encountered mostly on northwestern coasts olmoi showed a monophyletic association with U. olmoi (states of Maharashtra and Gujarat). Growth initiated at the (ABl 16040 and GU138284). The genetic divergence between end of the monsoon and continued until the middle of summer sequences of these two clades was 0.4%. The ITS rDNA (Septeniber-April), In Maharashtra, the associated flora phylogenetic data set consisted of 49 gene sequences. In the found with U. ohiioi were species of Porphyra, Sargassum, ML tree (Fig. 27), U. chaitguiii formed a sister lineage to U. Paclina, Didyola, Gnici/ana, Cauterpa, Chaeloniorpha, Spoii- paschima F. Bast. The interspecific sequence divergence gomorpha and Jama\ whereas, in Gujarat, all above-men- between U. cbaugulii and other species of Ulva ranged from tioned species except Pyropia were observed. The biomass 11.4% to 35,1%, estimated was comparatively lower than U. lactuca and varied from 0.5 to 1.5 kg with average percent cover of 41.2 ±19,9%. DISCUSSION

Molecular characterization Species of Ulva Linnaeus [including Enleromorpha (Hayden The rbcl^ phylogenetic data set consisted of 69 gene sequences, el al. 2003)] have been regularly reported and studied from including 25 generated in the present study. In ML India due to their widespread occurrence. The most recent phylogenetic analysis {Fig. 26), U. chaiigiilii did not cluster detailed taxonomic account of the genus was by Krishna- with any published rbcL sequences of Ulva. The interspecific murthy (2000), based on the morphological characters. Here sequence divergence between U. chaugidii and other species of we describe a new species of Uha in addition to character­ Ulva ranged from 2.3% to 4.0%. All specimens of U. laciucti izing U. ktciuca and U. oluwi froni western India. a / ’/ •M

■' / I,

^ : - i , ' - ' i- . \ \ ■' -qj < ■ ;

° ^'^?«*■yt^^21 ^ - U :

24

Figs 20-25, Representative specimen of U. olmoi. Fig, 20, scale bar = 2 cm; Fig, 21, scale bar = 20 (ini; Figs 22, 24, 25, scale b ar= 10 |.im; Fig, 23, scale bar = 2 nnn. Fig, 20. Habit. Fig. 21. Surfacc view showing marginal spines. Fig. 22. Surface view showing pyrenoids. Fig. 23. Basal portion showing rhizoidal disc. Fig. 24. Transverse section of thallus. Fig. 25. Transverse section of thailus showing rhizoids.

Ulva c/wugiilii can be distinguished from the other species Molecular data also confirmed the distinction of U. chaugulii of Ulva based mainly on the smaller size and shape of the from the above-mentioned species. The estimated sequence plant (infundibuliform), the long stipe and dilated apex and divergence for rhch and ITS rDNA sequence was well within the presence of two pyrenoids per cell. Ulva chaiiguUi showed interspecific range reported in earlier studies (Hayden & strong resemblance to U. lima Linnaeus with respect to Waaland 2002; Shimada et al. 2003; Ichihara el al. 2009). dimensions of cells, thickness of thalli and the loosely This warrants its recognition as a new species in the genus adherent two layers of cells, except at the thalhis margin. Ulva. U ha Utiza is simple, uiibranched and up to 45 cm in height Ulva fasciata is now considered as junior synonym of U. and 6 cm in width, with a single pyrenoid per cell. kictuca (O’Kelly 2010). However, in recent studies, they It also showed similarities with other species of Ulva, were still treated as distinct species (Guidone et al. 2013; including U. lingulaia J.G. Agardh, U. chihraia (Roth) Kirkendale et al. 2013) on the grounds that no formal Greville, U. flexuosa Wulfen, U. flexuosa (Wulfen) subsp. revision has been made. In the present study, we identified paradoxa Sliding, U. gujarateiisis Kale, U. dathratioides samples of U. laciiica and U. fasciata following the L.G. Kraft, Kraft & R.F. Waller and U. prolifewides L.G. morphological keys of Krishnamurthy (2000). In molec­ Kraft, Kraft & R.F. Waller, but all these species are ular analysis of these specimens, sequences of U. fasciata branched throughout the thallus. Ulva proHfera O. Muller clustered with the specimen designated as U. lactiica by and U. compressa Linnaeus have single pyrenoids per cell. O'Kelly el al. (2010). Therefore, in the present study, The cells of U. iiifesliiialis Linnaeus and U. ovata Thivy specimens of U. fasciata from India were also treated as &Visalakshmi ex Joshi & Krishnamurthy are irregularly U. lactiwa. Most of the morphological characters of arranged throughout the thallus. Uha brisbanensis L.G. collected specimens were relatively constant and consis­ Kraft, Kraft & R.F. Waller and U. nierklioiialis Horinioto & tent with the description of U. fasciata by Krishnamurthy Shimada lack a stipe. A newly identified species, U. sapora (2000), except for the presence of marginal teeth and J.A. Phillips, R.J. Lawton & C. Carl from Australia, has 2- greater thallus thickness. In addition, at some sites (e.g. 10 pyrenoids and filiform thalli (Phillips et al. 2016). Redi and Kovalam), lamina were palmately lobed. The 60 Ulva ohnoi (KP279696-KP279702.ABU6OiO.Gui3a2a4)

34 - Ulva spjnulosa AB097S36 aXva reticulata AB097S35

Ulva lactuca (KP279685-KP279695.ayz55872,gui38294)

50 - ulva gigantea SU4S440S - Viva scandlnavica AY255S70 90 - Ulva Jaetevirens SU9339S1

_95_ Ulva aroiorJcana AB097S32 Diva clatiratfl AY2558S2 Ulva gigantea Bu4Si414 Wva prollfera AF499S70 Ulva flexuosa AB097S19 i Ulva sEipitata var. linzoides BU93391B I— Ulva califojrnica Ai’lSSSSS 54 [flva tannneri ASfJSSeSS aiva clathratioides El/933940 100 [)lva procara Ay2S5eS3 SO Ulva linza AB097S20 61 ------I/Ji'a flexMsa subsp. p J J ife ra HH447573 9^ IfJva lin z a AY255S61 — Ulva linsa E?ll03Si 57 - Wva p to lile ra AY255B6i

100 Ulva taeniata Ar2SS875 Wva atenophylla AY255374 100 Ulva Chaugulii (KP710829-KP710833) Ulva flexuoea suisp. paradoxa EU933963 ------Ulva Umnetica AB42S$6e 100 I Ulva pertusa AY255873 ! Wva australis EU933953 ■ Ulva ita sa kii ABD97S21 63 74 ------Ulva feasstrata AF499S(8 71 I ------Ulva rotundata BU484396 ------Ulva lobata AY255d6S 83 100 yiva aonpreasa AY25S859 98 Viva pseudocurvaCa AY255869 80 ~ Ulva stenophylloides SU933955 ------Ulva in te s tin a lis Ar255S60 57 Ulva stenophylloides BU9339SS - Ulva brisbanensis EU93394S 89 ----- Umbraulva ollvascene BF110112 100 ------Unibraulva japonica AB097612 88 ------Semina le tte ra te d tio id e a EFJ100S9 Vivaria oijscura var. blytC li AF4SSS73

0.02

Fig. 26. ML phylogenetic tiee based on rbcL sequence data. Bootstrap values foi' ML analysis aie shown at nodes. Samples sequenced in this study are shown in bold. Ulva armoricana AB097661 27 Ulva Bcandinavica AJ234317 100 Ulva laetevirens EU933970 Viva rigida AY422522 Ulva stenophylloidea EU933977 Ulva clathrata AF127170 Ulva inu8Coide8AJ234307 p Ulva epinulosa AB097666 93 Ulva reticulata AB097665 Ulva obnoi AB116034 Ulva fasciata AB097663 Ulva fasciata AY260561 Ulva taeniata AY422525 100 -| Ulva chaugulii (KT429218, KT429219)

100 Ulva paechima KF385505 L Ulva paschima KF385504 97 Ulva lima AB097649 Ulva procera AY260558 — Ulva prolifera AJ234305 Ulva prolifera AY422510 Ulva flexuosa AB097644 Ulva Btipitata var. linzoides EU933988 75J Ulva californica AY260560 Ulva tanneri AB894331

72 — Ulva clathratioides EU933967 — Ulva Btenophylla AY2605S9 ^ Ulva linza AY260557 85j— l/2va flexuosa aubsp, paradoxa AJ234306 - Ulva flexuosa Bubep,flexuosa HM447564 Ulva aylindracea AJ234308 — Ulva flexuoBa aubap. pilifera HM447579 99 Ulva feneatrata AY260562 79 ■ Ulva lactuca AJ234309 L- U lv a araaakii AB097S50 84r ------Ulva adhaerena AB894334 I— Wva Jobata AY2605$3 100i— l^Iva au stralis KC411881 Ulva pertuaa AB894330 100 100 f Ulva cowpresBa AB097641

64 ■ Ulva paeudocurvata AJ234314 I— Ulva inteatinaloidea AJ234303 - Ulva intestinalis EU933966 Ulva proliferoidea BU933975 ------Ulva limnetica AB830513 Ulva briabaneiisia EU933972 ------W mbraulva japonica AB097638 100 — Percaraaria percursa AY260570 Ulvaria fuaca AB097637

0.1

Fig. 27. ML phylogcncUc tree based on ITS rDNA scquencc dala. Bootstrap values for ML analysis are shown at nodes. Samples scqucnccd from this studv are shown in bold. presence of spines was not reported for this species by REFERENCES earlier workers. All of our specimens had a more or less dentate margin. Detailed field and culture studies are Bast F., John A .A. & Bhushan S. 2014. Strong endemism of required to find the factors governing these morphological bloom-forming tubular Ulva in Indian West Coast, with description of Ulra pascliima sp. nov. (Ulvntes, Clilorophyta). variations. PLoS One 9: e l09295. The morphological and anatomical observations for the B l id in g C. 1968. A critical survey of European taxa in . II. specimens collected as U. lactuca corroborated the descrip­ Uha, 'Ul\'aria, Momstroma, Koiimimiia. Botcmiska NoHser 121: tion given by Krishnanuirthy (2000). However, a short stipe 535-629. B l o m s t e r J., M a g g s C.A. & St a n h o p e M.J. 1998. Molecular and and microscopic marginal teeth were observed. These morphological analysis of Eiileiomorplm imestinalis and E. plants were fragile and easily torn to separate into two coiiipressa (Clilorophyta) in the British Isles. Journal of Pliycology distinct layers. The molecular analysis using the rbcL gene 34: 319-340. showed that thalli, identified morphologically as U. lactuca, Blomster J., Back S., F ewer D.P., K urekki M ., L ehvo A., M acgs instead belonged to the U. olmoi clade rather than the U. C.A. & Stanhope M.J. 2002. Novel morphology in Etuerontorpba (Ulvophyccae) forming green tides. American Journal o f Bolony lactuca clade. The position of taxa in the rbch phylogenetic 89: 1756-1763. tree was congruent with data presented by Hiraoka et al. D arriba D ., T aboada G.L., D oallo R. & Posada D. 2012. (2004). The ibcL sequences were also identical to the JModelTest 2: more models, new heuristics and parallel comput­ sequences of Hawaiian specimens reported by O’Kelly et al. ing. Nature Methods 9; 772. (2010). Later, comparison of morphological characters with F letcher R.L. 1996. The occurrence o f ‘green tides’ - a review. In; Marine benthic vegetation in Europe: recent changes ami the effect U. olmoi reported by Hiraoka el al. (2004) also supported of eutrophication (Ed. by W. Schramm & P.H. Nienhuis), pp.7- the identity of Indian specimens of U. lactuca as U. olmoi 43. Springer, Heidelberg, Germany. The only difference was plant size; Indian U. obnoi ranged G uidone M., T hornber C., W vsor B. & O'K ellv C.J. 2013. from 1 to 12 cm; whereas, Hiraoka et al. (2004) reported Molecular and morphological diversity of Narragansett Bay (RI, USA) Viva (Ulvales, Chlorophyta) populations. Journal of thalli from Japan to be 20-30 cm. No intraspecific Phycoiogy 49; 979-995. variations were observed in rbch sequences. The intraspe­ G uiry M.D. & G u ir v G.M. 2015. AlgaeBase. World-wide cific variation in morphological characters was observed electronic publication. National University of Ireland, Galway. only in plant size. http://www.algaebase.org; searched on 2 September 2015. Our results also showed some morphological characters G uoyinq D., F eikei W., Y unxiang M., Shenohua G ., H oncpKan X. & Guto B. 2014. DNA barcoding assessment of green macroalga that can be utilized for preliminary routine identification of in coastal zone around Qingdao, China, Journnt o f Ocean specimens. For example, U. olmoi had mostly orbicular University o f Chinn 13: 97-103. shape, and the two layers of lamina separated easily. In U. H ayden H.S. & W aaland J.R. 2002. Phylogenetic systematics of lactuca, the thallus was broadly expanded, lanceolate, the Ulvaceae (Ulvales, Ulvophyceae) using chloroplast and nuclear DNA sequences. Journal of Phycoiogy 38: 1200-1212. ribbon-like and more or less deeply divided. These two H ayden H.S., Blomster J., M aggs C.A., Stlva P.C., Stanuope species also differed in thallus thickness. The thallus of U. M.J. & W aaland J.R. 2003. Linnaeus was right all along: Ulva ohnoi was thinner compared to U. lactuca. However, to and Enteromorpha arc iiot distinct genera. European Join'nal of confirm species identity, it is essential to subject the Phycoiogy 38: 277-294. specimens to molecular characterization. H eesch s., B room J.E.S., N eill K .F., F arr T.J., D alen J.L. & N elson W.A. 2009. Ulva, Umhraulva and Gemina'. genetic survey In conclusion, the present study substantiates the occur­ of New Zealand taxa reveals diversity and introduced species. rence of three species of Ulva from the west coast oflndia: U. European Journal o f Phycoiogy 44: 143-154, chaugulii sp. nov., U. lactuca and U. olmoi. The present work H iraoka M ., S himada S., U enosono M , & M asuda M . 2004. A new has implications for future studies related to life cycles, green-tide-forming alga, Ulva obnoi Hiraoka et Shimada sp. nov. (Ulvales, Ulvophyceae) from Japan. Phycological Research 52: morphogenesis and cultivation and further biochemical and 17-29. molecular characterization of these taxa. H o f m a n n L.C., N e t t l e t o n J.C., N e efu s C.D. & M a t h ie s o n A.C. 2010. Cryptic diversity of Ulva (Ulvales, Chlorophyta) in the Great Bay estuarine system (Atlantic USA): introduced and indigenous distromatic species. European Journal of Phycoiogy 45: SUPPLEMENTARY DATA 230-239. Ichihara K., A rai s., U chimura M,, F ay E.J., E bata H., H iraoka Supplementary data associated with this article can be found M. & Shimada S. 2009. New species of freshwater Ulva, Ulva online at http://dx.doi.org/10.2216/15-l 1.1.si. linmetica (Ulvales, Ulvophyceae) from the Ryukyu Islands, Japan. Phycohgicai Research 57: 94-103. K a to h K., K u m a K., Toh H. & M iy a ta T. 2005. M AFFT version 5: improvement in accuracy of multiple sequence alignment. ACKNOWLEDGEiVIENTS Nucleic Acids Research 33: 511-518. K irkendalf, L., Saunders G.W. & W inbero P. 2013. A molecular survey of Ulva (Chlorophyta) in temperate Australia reveals The financial support received from National Agriculture enhanced levels of cosmopolitanism. Journal of Phycoiogy 49: 69- Science Fund (NASF), ICAR, under project no. 81. NFBSFARA/CA-3002/2012-13, is thankfully acknowl­ KohMAN R.P.T. & V a n Den H o e k C. 1981. The taxonomy of Ulva edged. The authors are thankful to the editor and reviewers (Chlorophyceae) in (he Netherlands. British Phycohgicai Journal for their constructive suggestions to improve the manuscript. 16: 9-53. K r a it L.G.K., K r a it G.T. & W aller R.F. 2010. Investigations The authors also thank Kiran More and Nitesh Marchande into southern Australia Ulva (Ulvophyceae, Chlorophyta) taxon­ for their assistance in the field collections and laboratory omy and molecular phytogeny indicate both cosmopolitanism experiments. and endemic cryptic species. Journal o f Phycoiogy 46: 1257-1277. K rishnamurthy V. 2000. Algcie o f India and iteigliboiiriitg coimieies Enieromorpha (Ulvales, Ulvophyceae), with special lefcreiice to I. Cblorophycota. Oxford & IBH Publishing Co. Pvt. Ltd, New the free-floating Uha. Pliycological Research 51: 99-108. Delhi. 209 pp. SiLVA P.C., Basson P.W. & M oe R.L. 1996. Catalogue o f the benihic Liu F., P ang S.J., Xu N., Shan T.F., S un S., Hu X. & Y anq J.Q. marine algae of the Indian Ocean. University of California Press, 2010.[//vfl diversity in the Yellow Sea during the large-scale green Berkeley. 1259 pp. algal blooms in 2008-2009. Phycohgica! Research 58: 270-279. Silvestro D. & M ichalak I. 20l2.raxnilGUI; a graphical front-end L ouohnane C.J., M cIvor L.M., R indi F., Stengel D.B. & G uiry for RAxML. Organis/iis Diversity & Evolution 12: 335-337. M.D. 2008. Morphology, rbcL phytogeny and distribution of T amura K., Stecher G., P eterson D., F ilipski A. & K umar S. distroniatic U/vo (Ulvophyceae, Chloro]3liyta) in Ireland and 2013. MEGA6: molecular evolutionary genetics analysis version southern Britain. Pkycologia 47; 416-429. 6.0. Molecular Phylogenetics and Evolution 30: 2725-2729. M anhart J. R. 1994. Phylogenetic analysis of green plant rbcL W olf M .A., Sciurc K., A nd reo h C. & M oro I. 2012. Uha sequences. Molecular Pliylogenelics and Ewhaion 3: 114-127. (Chlorophyta, Ulvales) biodiversity in the North Adriatic Sea M atsumoto K. & S himada S. 2015. Syslematics of green algae (Mediterranean, Italy): cryptic species and new introductions. resembling Viva coiiglobala, with a description of Uha adhaerens Journal of Phycohgy 48: 1510-1521. sp. nov, (Ulvales, Ulvophyceae). European Journal of Phycology Z hao J., J iang P., Liu Z., W ang J., Cui Y. & Q(n S. 2010. Genetic 50: 100-111. variation of Uha (Enieromorpha) prolifera (Ulvales, Chlorophy­ O ’K elly C.J., K urihara A., Shiplkv T.C. & Sherwood A.R, 2010. ta) - the causative spccies of the green tides in the Yellow Sea, Moleculai assessment of Uha spp. (Ulvophyceae, Chlorophyla) China. Journal o f Applied Phycohgy 23: 227-233. in the Hawaiian Islands. Jouninl o f Phycohgy 46: 728-735, pH[LLii>s J.A., L awton R.J., D enys R., P aul N.A. & C arl C. 2016. A new abimdaiit tubular species of Uha (Ulvaks) from the tropical Pacific Ocean. Phycohgia 55: 55-64. Shimada S., H iraoka M „ N adata S„ L ima M . & M asuda M . 2003. Received 17 Ocloher 2015: accepted 10 November 2015 Molecular phylogenetic analyses of the Japanese Uha and Associate Editor: Charles Yarish