Phytotaxa 428 (2): 104–112 https://www.mapress.com/j/pt/ Copyright © 2020 Magnolia Press ISSN 1179-3155 (print edition) Article PHYTOTAXA ISSN 1179-3163 (online edition) https://doi.org/10.11646/phytotaxa.428.2.4 Sida sivarajanii (Malvaceae): a new species from India GAJANAN TAMBDE1, MILIND SARDESAI2,* & ARUN K. PANDEY3 1 Department of Botany, Shri. Vyankatesh Arts, Commerce and Science College, Deulgaon Raja, 443204, MS, India. Department of Botany, Savitribai Phule Pune University, Pune, 416004, MS, India. 3 Department of Botany, University of Delhi, Delhi-110007, India. *Corresponding author: sardesaimm@gmail.com 2 Abstract A new species, Sida sivarajanii (Malvaceae) from Aurangabad, Maharashtra, India is described. It is morphologically most similar to S. alnifolia and S. rhomboidea but differs in its conical ovary covered with stellate pubescence, 5 styles, 5 dehiscent mericarps and multireticulate spermoderm pattern. The ITS sequence data also confirm the distinctness of the new species. In this study, a comprehensive description, phenology, distribution, habitat and identification key of S. sivarajanii are provided. Keywords: Aurangabad, Maharashtra, New taxon, Novelty Introduction The genus Sida Linnaeus (1753: 684) includes approximately 250 species distributed in the pantropical and subtropical regions (Mabberley 2017). In India, the genus is represented by 20 species (Tambde et al. 2016), grouped under 6 sections: Sidae Linnaeus (1753: 684) (10 species), Nelavagae Borssum Waalkes (1966: 180) (5 species), Cordifoliae (de Candolle 1824: 463) Fryxell (1985: 77) (2 species), Malachroideae G. Don (1831: 498) (1 species), Spinosae Small (1933: 849) (1 species) and Stenindae Grisebach (1859: 76) (1 species) (Sivarajan & Pradeep 1996, Tambde et al. 2016). The taxonomy of the genus Sida is controversial due to morphological variability including the shape, size, and surface of mericarps (Ugborogho 1980, Fryxell 1985, Sivarajan et al. 1992, Sivarajan & Pradeep 1996). During field explorations in different parts of India, one of us (GMT), collected specimens of Sida that could not be assigned to any of the recognized species of the genus. Hence, the specimen is described and illustrated here as a new species, Sida sivarajanii sp. nov.. Material and Methods Morphology:—The morphological analysis and description of the new species are based on the examination of specimens from AHMA, BAMU, BSD, BSI, CAL, DUH, MH and SUK herbaria. The specimens were compared with existing collections of S. alnifolia Linnaeus (1753: 684) and S. rhomboidea Roxburgh ex Fleming (1810: 178) (all the species are morphologically similar to the unidentified specimens) and noted the differences among the three taxa. Taxon sampling:—Field surveys and collection trips were undertaken to different parts of India. The voucher specimens have been deposited in the Herbarium of the Department of Botany, Savitribai Phule Pune University, Pune (SPPU). Molecular Methods:—Genomic DNA was extracted using DNeasy Plant Mini Kit (Qiagen, Amsterdam, Netherlands). DNA amplification and sequencing of the entire ITS region (ITS1, 5.8S and ITS2) were performed using the primers ITS1 and ITS4 (White et al. 1990). The polymerase chain reaction (PCR) was performed with standard methods using Promega PCR mastermix (Promega Corporation, Madison, USA) in 25 µl volumes containing 50 units/ ml Taq DNA polymerase (supplied in a proprietary reaction buffer (pH 8.5), 400µM dNTPs and 3mM MgCl2). To this 2 µl of a 10 pM solution of each primer, 2 µl of genomic DNA and miliq water added to make a total volume of 25 µl. 104 Accepted by Prabhukumar K M: 7 Dec. 2019; published: 9 Jan. 2020 PCR amplification was performed with 35 cycles involving denaturation for 1 minute at 94ºC, annealing for 1 minute at 49º C, and 1 minute of extension at 72ºC followed by a last cycle of final extension for 5 minute at 72ºC. PCR products were checked for the presence of appropriate bands on a 0.8 % agarose gel, purified, and sequenced at SciGenom, Kochi, Kerala. Sequences comprised of ITS1, 5.8S, and ITS2 regions. Forward and reverse sequences were edited and assembled using the computer program Geneious® 10.2.2 (Drummond et al. 2010). The sequences have been deposited in GenBank (MK829605 & MK829606). FIGURE 1. Best ML tree inferred from analyses of nrDNA (ITS). 13 accessions including 2 accessions of Sida sivarajanii and one outgroup analyzed under TIM3e+G4 model of substitution. (RAxML Bootstrap values are indicated above branches) The ITS sequences of the genus Sida, Abutilon Miller (1754: 4) and Gossypium hirsutum Linnaeus (1763: 975) were retrieved from GenBank and added them to a dataset consisting of ITS sequences of two accessions of newly collected and sequenced material. The final data matrix comprised a total of 13 accessions. The sequence alignment is available from the corresponding author (MMS) on request. Sequences of the ITS region were aligned using ClustalX vers. 2.0.11 (Thompson et al. 1997) followed by manual adjustments in ClustalW (Thompson et al. 1994). Phylogenetic analyses were done using Maximum Likelihood approach using RAxML-HPC2 on XSDE v. 8.2.10 (Stamatakis 2014) on CIPRESS Science Gateway v.3.3 (Miller et al. 2010). TIM3e+G4 was found to be best-fit model (Kalyaanamoorthy et al. 2017). The following criteria were used to assess bootstrap support percentages (BP): 50–70 %, low; 71–84 %, moderate; 85–100 %, strong. The final tree was drawn using FigTree 1.4.0. A NEW SPECIES oF SIDA SIVARAJANII (MALvACEAE) Phytotaxa 428 (2) © 2020 Magnolia Press • 105 TABLE 1. Comparison between Sida sivarajanii sp. nov., S. alnifolia and S. rhomboidea. S. sivarajanii sp. nov. S. alnifolia S. rhomboidea Shape obovate or suborbicular obovate, rarely ellipticlanceolate obovate or suborbicular, rhomboid to lanceolate Apex sub-obtuse or acute retuse or emarginated or obtuse or acute subobtuse or acute 2–12 mm 2–4 mm 2–6 mm in flowers 0.4–0.6 cm 0.3–0.5 cm 1–1.5 cm in fruits 1–1.5 cm 1.5–2.5 cm 2–3 cm Calyx 3–6 mm across, tomentose with minute stellate hairs, margins brownish 5–7 mm across, stellate and simplehairy, margins brownish 5–6 across, tomentose with minute stellate hairs, margins purplish Corolla 25–40 mm diameter, minutely stellate hairy at base 8–10 mm diameter, stellate hairy at base 15–20 mm diameter, glabrous at base Staminal column 2–2.5 mm long 2–3 mm long 2.5–3 mm long ovary conical, stellate pubescent globose, glabrous globose, glabrous Styles 5 8–10 8–10 Number 5 8–10 8–10 Length 3–4 mm long 2.5–3 mm long 6–7 mm long Awns 1–1.5 mm long with densely stellate hairs 0.5–1 mm long with minute stellate hairs 3–4 mm long with simple, retrorse, bristly, hairs Surface dense long stellate hairs mixed with simple hairs minute stellate hairs minute simple hairs Mericarp dehiscent indehiscent indehiscent Hilum of seed glabrous pubescent glabrous Spermoderm pattern multireticulate reticulate reticulate Characters Leaf Petiole Pedicel Mericarps SEM study:—Mericarps and seeds of all the accessions were mounted on aluminium stubs with double adhesive tape and sputter-coated with platinum in Quorum Q150TES sputter coating unit (Fischer et al. 2012). Samples were examined using a Scanning electron microscope FEI Nova NanoSEM 450, at the Central Facility Centre, Savitribai Phule Pune University, Pune (Maharashtra). Results Molecular analyses:—The analyses of ITS sequences reveals that the aligned ITS region is 712 base pair long with 417 (=58.57 %) constant sites, 149 parsimonious sites and 339 distinct pattern sites. The result supports (BS=100) the inclusion of newly described species in the section Sidae recognized by Fryxell (1985) (Fig. 1). The new species described as S. sivarajanii is sister to S. alnifolia and S. rhomboidea, another species found in Peninsular India. The new species discovered varies at 9 positions in the alignment with its closely allied species: G/A at position 120; T/G at position 202; T/C at position 214; C/T at position 458; T/C at position 464; G/T at position 468; T/G at position 483; C/A at position 626; T/C at position 695 of 697 aligned characters. 106 • Phytotaxa 428 (2) © 2020 Magnolia Press TAMBDE eT Al. FIGURE 2. Sida sivarajanii sp. nov. (A) habit, (B) young twig showing leaves, (C) twig showing flowers and fruits, (D) flower, (E) young fruit, (F) mature fruit, (G) mericarp front view, (H) mericarp dorsal view. Photos: G. M. Tambde from G.M. Tambde 231. SEM study:—Mature seeds of S. sivarajanii show multi-reticulate spermoderm pattern whereas S. alnifolia and S. rhomboidea show reticulate pattern (Fig. 4). A NEW SPECIES oF SIDA SIVARAJANII (MALvACEAE) Phytotaxa 428 (2) © 2020 Magnolia Press • 107 FIGURE 3. Illustrations of Sida sivarajanii sp. nov.: (A)–(G) S. sivarajanii sp. nov. (A) flowering and fruiting twig, (B–D) leaves, (E) flower, (F) mature fruit with fruiting pedicel, (G) mature fruit (H) mericarp front view, (I) mericarp dorsal view, (J) seed. Illustrated by R. D. Gore from G.M. Tambde 231. 108 • Phytotaxa 428 (2) © 2020 Magnolia Press TAMBDE eT Al. FIGURE 4. Comparative Scanning Electron Micrographs (SEM) images of mericarps of Sida sivarajanii sp. nov., S. alnifolia and S. rhomboidea: (A–D) S. sivarajanii sp. nov. (A) mericarp dorsal view, (B) seed, (C) hilum, (D) seed spermoderm pattern from GM Tambde 231; (E–H) S. alnifolia (E) mericarp dorsal view, (F) seed, (G) hilum, (H) seed spermoderm pattern from GM Tambde 041; (I–L) S. rhomboidea (I) mericarp dorsal view, (J) seed, (K) hilum, (L) seed spermoderm pattern from GM Tambde 228. A NEW SPECIES oF SIDA SIVARAJANII (MALvACEAE) Phytotaxa 428 (2) © 2020 Magnolia Press • 109 Taxonomic treatment Sida sivarajanii Tambde, Sardesai & A.K. Pandey sp. nov. (Figs. 2 & 3) Sida sivarajanii sp. nov. morphologically most similar to S. alnifolia and S. rhomboidea but differs in its conical ovary covered with stellate pubescence, 5 styles, 5 dehiscent mericarps and multireticulate spermoderm pattern. (Figs. 2–4; Table 1) Type:—INDIA. Maharashtra: Aurangabad district, Kailas Nagar, N6-CIDCo, 19°52’50.0”N 75°20’43.9”E, 569 m alt., 17 october 2015, G.M. Tambde 231 (holotype: CAL!, isotypes: BAMU!, BSI!, BSD!, MH!, DUH!). Erect, branched, undershrub, upto 1 m tall. Stem terete, green, minutely pubescent with small stellate hairs. Leaf blades on younger shoot much larger (6–8 × 4–6 cm), concolorous, obovate or suborbicular, truncate or rounded at base, margins serrate to crenate, entire towards base, sub-obtuse or acute at apex; those on flowering shoots smaller, 1–4 × 0.5–3 cm, rhomboid to lanceolate, 3-nerved from base, lateral nerves 3–6 pairs, nerves raised on the lower surface, densely stellate-tomentose beneath, sparsely pubescent above. Petioles 2–12 mm long, stellate pubescent; stipules 5–7 mm long, stellate hairy, equal, linear, slightly purplish, caducous. Flowers axillary, solitary; pedicels 0.4–0.6 cm in flowers, to 1–1.5 cm in fruits, filiform, pubescent, articulated at about middle. Calyx 3–6 mm across, campanulate, 10–ribed at base, 5–lobed; the lobes 1.7–2 mm long, ovate, acuminate at apex, tomentose with minute stellate hairs. Corolla 2.5–4 cm in diameter, orange-yellow; petals 6–8 × 4–5 mm, obovate, retuse or emarginated at apex, minutely stellate hairy at base. Staminal column 2–2.5 mm long, stellate pubescent; antheriferous at apex, filaments 1–2 mm long, anthers reniform, pale-yellow. ovary 1–1.5 mm diameter, conical, stellate hairy; styles 5, covered by minute stellate hairs, stigma capitate, yellow. Schizocarps 2–3 mm long, ovoid-ellipsoid, raised at centre, green when immature, light brown at maturity, grooved between mericarps; mericarps 5, 3–4 × 1.5–2 mm, completely included in the calyx, completely covered densely with long stellate and sparsely with simple hairs; awns 1–1.5 mm long, covered with stellate hairs. Seed one per mericarp, 2 × 2 mm, brownish to black, laterally compressed, glabrous at the hilum. Phenology:—Flowering from September to February; fruiting from october to March. Distribution and habitat:—Sida sivarajanii is currently known to occur from different localities in Andhra Pradesh, Chattisgarh, Gujarat, Maharashtra, odisha, Uttarakhand and Tamil Nadu states. It is common, along road sides and waste lands, at about 569 m alt. found in association with Sida acuta Burman f. (1768: 147), Cynodon dactylon (Linnaeus 1753: 58) Persoon (1805: 85), Parthenium hysterophorus Linnaeus (1753: 988), Tridax procumbens Linnaeus(1753: 900), Senna obtusifolia (Linnaeus 1753: 377) H.S. Irwin & Barneby (1982: 252) and Malvastrum coromandelianum (Linnaeus 1753: 687) Garcke (1857: 295). Etymology:—The new species is named after late Prof. v.v. Sivarajan for his great contributions in the field of angiosperm taxonomy in India in general and family Malvaceae in particular. Additional specimens examined (Paratypes):—INDIA: Andhra Pradesh: Chittoor District, Nagari, 2 December 1987, D. Ranga Charyula 1879. (MH!). Chhattisgarh: Bilaspur, 22°03’35.5”N 82°08’42.4”E, 262 m alt., 26 September 2015, G.M. Tambde 210 (BAMU!). Gujarat: Dahod district, Piplod, 112053 (BSI!). Maharashtra: Kolhapur district, HSC Board Campus, Kolhapur, 16°40’44.10”N 74°14’35.30”E, 615 m alt., 4 December 2014, G.M. Tambde 75 (BAMU!); Pune district, Katraj, 9 August 1956, V.D. Vartak 5463. (AHMA!). Odisha: Padmayan, 18 November, 1950, 20575. (CNH!). Tamil Nadu: Kanchipuram district, vandalur, 12°53’44.40”N 80°5’14.80”E, 39.5 m alt., 10 February 2015, G.M. Tambde 125 (BAMU!). Uttarakhand: Dehra Dun district, Patanjali, 29°54’21.90”N 78°0’14.10”E, 253 m alt., 2 october 2015, G.M. Tambde 221 (BAMU!); Pithoragarh district, Kumaon, Askot, 1 September 1971, C.M. Arora 45494 (BSD!); Pithoragarh district, Nirayan, 7 November 1963, N.P. Singh 31626 (BSD!). Key to the species 1. 2. - Styles and mericarps 5; mericarps not prominently reticulate, thin-walled ............................................................... Sida sivarajanii Styles and mericarps 8–10; mericarps prominently reticulate laterally, thick-walled........................................................................2 Mericarp indehiscent .................................................................................................................................................... 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