Zeylanidiummanasiae, a new species of Podostemaceae based on molecular and morphological data from Kerala, India

Abstract We present the description of Zeylanidiummanasiae (Podostemaceae), a new species from Kerala, India, which is proposed based on molecular, macro- and micromorphological data. This species is characterised by its ribbon-like dichotomous thallus, floriferous shoots produced along the margins and dichotomy of the thallus, inflorescence with two bracts, unequal stigmatic lobes, ellipsoid fruits and large seeds.


Introduction
Podostemaceae represents a very distinct family of fresh water aquatic angiosperms, with unique evolutionary, ecological, morphological, developmental and embryological attributes (Cook and Rutishauser 2007, Katayama et al. 2016, Khanduri et al. 2014. It is the most diverse family of fresh water aquatic flowering plants, comprising ca. 54 genera and ca. 300 species (Koi et al. 2012, Cheek et al. 2017) distributed worldwide, but with most species presenting restricted distribution and a high degree of endemism (Philbrick et al. 2010). Podostemaceae is subdivided into three monophyletic subfamilies: Podostemoideae, Tristichoideae and Weddellinoideae (monogeneric and monospecific) (Koi et al. 2012). Southern Asia is one of the main centres of diversity for the podostemads, ac-counting for 17 genera and 80 species from the region (Kato 2016). India harbours 28 species of Podostemaceae, in which 23 are endemics (Khanduri et al. 2014).
The genus, Zeylanidium (Tul.) Engl. (subfamily Podostemoideae), is characterised by plants with crustose or ribbon-shaped thalli and caducous leaves. The flowering shoots in these species may be located either in the sinuses of the thallus lobes or borne randomly on the dorsal surface of the thallus (Mathew and Satheesh 1997). Each shoot bears a solitary, terminal and bracteate flower. The other key features include persistent spathella, anisolobous ovarian locules and many seeded capsules (Kato and Koi 2018).
Zeylanidium is currently represented by seven species: Z. olivaceum Engl., Z. johnsonii Engl., Z. lichenoides Engl., Z. maheshwarii C.J.Mathew & V.K.Satheesh, Z. sessile (Willis) C.D.K.Cook & Rutish, Z. crustaceum M.Kato and the recently described Z. tailichenoide M. Kato & Koi (Mathew and Satheesh 1996, Cook and Rutishauser 2001, Kato et al. 2015, Kato and Koi 2018. Out of these seven species, the status of Z. johnsonii Engl. is doubtful as it has never been reported by any study after Engler's (1930) description. All species are confined to peninsular India, Sri Lanka, Myanmar and Thailand (Mathew and Satheesh 1997, Kato et al. 2015, Kato and Koi 2018. The taxonomic delimitation of the genus is still under dispute. Two species, originally described under the New World genus Podostemum, were transferred to Zeylanidium by Cusset (1992) as Z. barberi (Willis) C. Cusset and Z. subulatum (Gardn.) C. Cusset. However, a recent combined morphological and molecular phylogenetic analysis does not support these new combinations (Khanduri et al. 2014). Therefore, these species are here excluded from Zeylanidium and it is our opinion that their generic placement requires further studies (pers. observ.).
During field studies in the riverine areas of Kerala, India, between 2014 and 2016, a new morphological variant of Zeylanidium was found. The ribbon-shaped specimens with solitary flowering shoots appeared morphologically similar to Z. lichenoides, but the fruits were remarkably distinct in size and shape. Detailed morphological and molecular studies revealed that the specimens were different from the remaining species on several other characters. Hence, the specimens are documented and described here as a new species, Zeylanidium manasiae. A detailed description with photographic documentation, illustrations, phylogenetic placement within Podostemaceae and an identification key are provided.

Morphology
Plant specimens were collected from Thommenkuthu waterfalls, Thodupuzha, Idukki, Kerala, India (9°57'21.59"N 76°50'01.87"E, Fig. 1). Collections were made from various spots at the rapids for three consecutive years (2014)(2015)(2016). Voucher specimens have been deposited at the Delhi University Herbarium (DUH), Department of Botany, University of Delhi and Calicut University Herbarium (CALI). The morphological details of the plants were recorded in the field and documented photographically. Morphometric details of randomly collected plants (N=30) were measured by using a digital Vernier calliper and calibrated ocular micrometre. The specimens were compared with other members of Zeylanidium either by using preserved materials from our spirit collections [i.e. Z. olivaceum, Z. lichenoides, Z. sessile and Z. maheshwarii (Suppl. material 1)] or information available in literature (i.e. Z. johnsonii, Z. crustaceum and Z. tailichenoides; Mathew and Satheesh 1997, Suzuki et al. 2002, Kato et al. 2015, Kato and Koi 2018. The differences between the taxa were compiled and are presented below (Table 1, Suppl. material 2). The terminology for vegetative and floral characters follows Mathew and Satheesh (1997), Marinho et al. (2014) and Jäger-Zürn (2003). The distribution map was constructed using ArcGIS 9.2 version (Zhan and Huang 2004).

Anatomy
For anatomical details, flower buds of desired specimens were fixed in Karnowsky's fixative (Karnowsky 1965) and then processed to prepare resin blocks for sectioning (Feder and O'Brien 1968). Semi-thin sections (4 and 5 μm) were obtained with the help of a rotary microtome, stained with 0.1% toluidine blue O' (pH 4.4) and mounted in DPX (O'Brien and McCully 1981). The observations were recorded with the help of a photo microscope (Carl Zeiss, Axio scope A1) with an attached digital camera (Axiocam).

Scanning electron microscopy
For palynological and seed micromorphological studies, anthers and seeds, respectively, were fixed in Karnowsky's fixative, dehydrated in a graded series of cold acetone (10-100%, 30 min interval each), critical point dried (CPD), mounted on aluminium stubs and coated with gold-palladium alloy before making observations. The samples were examined by using a scanning electron microscope (SEM, JEOL, JSM-6610LV) at the Department of Botany, University of Delhi, India.

DNA extraction, amplification and sequencing
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 ITS 1 and ITS 2 (White et al. 1990). The polymerase chain reaction (PCR) was executed using standard protocol with one unit of itaq (Taq Intron, Intron Biotechnology Inc.), 2.5 μl of 10 X buffer, 2.5 μl dNTPs, 1 μl of 10 pM solution of each primer, 1 μl of genomic DNA and 16.7 μl distilled water. PCR products were purified using QIAquick Gel Extraction Kit (QIAGEN) and the purified product was ligated into a pGEM-T vector (Promega, USA). The ligated mix was transformed using competent E. coli DH5α strain. The blue-white selection method was employed for transformation (Sambrook and Russell 2001). Three clones per PCR product were sequenced at SciGenome Labs Pvt. Ltd. (Cochin, India). Contigs were assembled using DNA star Laser gene version 5.07 software (Burland 2000). Nucleotide BLAST was performed to estimate sequence similarity by using the acquired nucleotide sequence as the query. The sequences have been submitted in the GenBank (Suppl. material 3).

Taxon sampling
ITS sequences of Z. manasiae and Z. maheshwarii were added to a dataset consisting of 39 species of Podostemaceae, produced by Khanduri et al. (2014). Hypericum perforatum L. and Hypericum kouytchense H.Lév. were included as outgroups based on the results of Ruhfel et al. (2011). The final data matrix comprised of a total of 43 accessions, representing 41 species of Podostemaceae and the two outgroups. Out of the seven species of Zeylanidium, five were included in the present phylogenetic analysis.

Phylogenetic analysis
ITS sequences of all the taxa were aligned using ClustalX ver. 2.0.11 (Thompson et al. 1997) and checked manually using ClustalW (Thompson et al. 1994). Phylogenetic reconstruction was carried out using MrBayes 3.1 (Ronquist and Huelsenbeck 2003) with the best sequence evolution model i.e. JC model under Model Test version 0.1.1 (Guindon and Gascuel 2003). Analyses were run for 1,300,000 generations until stationarity (standard deviation < 0.01). In each run, trees were sampled after every 100 generations with a sample frequency of 10. All the parameters were summarised after excluding 25% of the samples (burn-in fraction), based on the inspection of log-likelihoods of sampled trees. The summary table provides mean and mode with 95% credibility interval. The potential scale reduction factor approached 1.0 for all the parameters. Branch length information was recorded and averaged across all the retained trees and a majority rule consensus tree was computed to obtain the posterior probabilities (PP). Trees were summarised by the sump burn-in command yielding a cladogram showing PP, clade credibility for each split and a phylogram with mean branch lengths. The values between 0.95 and 1.0 were only taken into consideration for Bayesian analysis.
Palynology. The dyads of Z. manasiae are of the acalymmate type and measure 30.25 ± 2.41 μm in length and 19.62 ± 1.99 μm in width. Individual pollen grains   are sub-prolate in shape with tricolpate aperture. The exine wall has microechinate ornamentation. The echinations on the apertural surface are larger than those on the non-apertural surface (Fig. 3B).
Additional specimens seen ( Etymology. The specific epithet 'manasiae' honours the late Dr. Manasi Ram née Ghosh for her contributions to the study of embryology and systematics of Santalaceae (Ghosh 1956) and Trapa L. (Lythraceae; Ghosh 1954).
Distribution and ecology. Zeylanidium manasiae is highly endemic and is known from only one location so far, i.e. Thommenkuthu waterfalls (Figs 1 and 3A). According to a previous report, rocks of this waterfall are hornblende biotite gneiss type (Girija 2008). According to Mathew and Satheesh (1996), water in Kaliyar River is well-oxygenated hard water with low chloride content. Zeylanidium manasiae grows along with Z. lichenoides and Z. sessile. Diatoms (Cymbella C.Agardh species) were also observed forming colonies on the spathella of some of the plants.
Conservation status. This species is currently known to occur from a single location in Kerala and, hence, we suggest its placement in the Data Deficient category of IUCN (2017).
Phenology. Flowering and fruiting occurs from December to January when the water level recedes to partly expose the rocks.
Zeylanidium tailichenoides and Z. sessile, the other two ribbon-shaped species in the genus, can be easily distinguished from Z. manasiae on the basis of (i) unilocular ovary in Z. tailichenoides vs. bilocular ovary in Z. manasiae; and (ii) sessile flowers and smooth capsules in Z. sessile vs. pedunculate flowers and ribbed capsules in Z. manasiae. The remaining congenerics (i.e. Z. olivaceum, Z. maheshwarii, Z. johnsonii and Z. crustaceum) have crustose thallus with leaves and flowering shoots scattered on the dorsal surface and, hence, are distinct from Z. manasiae.
Palynological studies also revealed the presence of tricolpate apertures with microechinate exine ornamentation. These characters are similar to the other Zeylanidium species, which confirms its generic placement. The structure of the pistil further supports generic identity of the species, since the anisolobous ovary is a characteristic feature of Zeylanidium. This characteristic feature separates the genus from Polypleurum.

Conclusion
Based on the evidence drawn from the present work, it is clear that Z. manasiae should be recognised as a new species of Zeylanidium. The recognition of Z. manasiae brings the total number of Zeylanidium species to eight. The finding of new species of Zeylanidium indicates that the region is splendidly diverse but remains poorly explored.