Alliumnegianum (Amaryllidaceae): a new species under subg. Rhizirideum from Uttarakhand Himalaya, India

Abstract A new species, Alliumnegianum (Amaryllidaceae), belongs to the genus Alliumsubg.Rhizirideum, sect. Eduardia is described here from the Uttarakhand Himalayan region of India. This taxon grows in Malari region of Niti valley in Chamoli district and Dharma valley of Pithoragarh district, Uttarakhand, India. It is a narrowly distributed species and morphologically more closer to A.przewalskianum Regel but differentiated by its tunic color of bulb, umbel with lax flowers, peduncle length, perigone colour, size and shape and leaf anatomy. Taxonomic delineation and relationship analysis based on nuclear ribosomal Internal Transcribed Spacers (ITS) region indicated that A.negianum is distinct and related to A.przewalskianum. This study provided a comprehensive description and comparison with A.przewalskianum, an identification key and notes on the distribution of the species.


Introduction
Allium L., one of the largest genera in the family Amaryllidaceae, has about 1,100 species distributed world-wide (Li et al. 2010;Govaerts et al. 2021). The genus Allium naturally occurs in dry seasons in the northern hemisphere and South Africa (Friesen et al. 2006;Nguyen et al. 2008;Neshati and Fritsch 2009). The primary centre of evolution for the ge-nus extends across the Irano-Turanian bio-geographical region, and the Mediterranean basin and western North America are considered as the secondary centres of diversity (Friesen et al. 2006). The genus is characterized by bulbs that are enclosed within the membranous or fibrous tunics, free tepals, often a subgynobasic style and well-known characteristic plant odour and taste due to the presence of cysteine sulphoxides (Friesen et al. 2006). The classification of global species in the genus Allium is based on molecular phylogenetic analyses, which includes 15 subgenera and 56 sections (Friesen et al. 2006). The Indian Allium includes over 10 subgenera, 22 sections and 35-40 taxa excluding cultivated species distributed in different eco-geographical areas of the temperate and alpine regions of Himalayas sharing many taxa of Chinese origin (Pandey et al. 2008(Pandey et al. , 2017Li et al. 2010). Indian Himalayan region has two distinct centres of diversity, the western Himalaya (over 85 per cent of total diversity) and the eastern Himalaya (6 per cent), covering the alpinesub temperate region (2500-4500 m a.s.l.) (Gohil 1992;Pandey et al. 2008).
Globally Allium subg. Rhizirideum (G.Don ex Koch) Wendelbo s.str. has ca. 37 taxa that are included in four sections distributed mainly in Europe-East Asia, in China (Friesen et al. 2006;Choi et al. 2012;Jang et al. 2021) and also in Russia, Mongolia and Kazakstan (Sinitsyna et al. 2016;Friesen et al. 2020). Allium senescens L. of sect. Rhizirideum a species native to northern Europe and Asia from Siberia-Korea and also naturalized in parts of Europe, is an exception (Xu and Kamelin 2000;Li et al. 2010).
Taxa of the subg. Rhizirideum belong to the third and the most advanced evolutionary line, which is phylogenetically sister to taxa of the subg. Allium L., Cepa L., Reticulatobulbosa (Kamelin) N.Friesen and Polyprason Radic. (Friesen et al. 2006;Memariani et al. 2007;Li et al. 2010;Choi et al. 2012). The sect. Eduardia N.Friesen of the subg. Rhizirideum is mainly distributed in the western Himalaya with Pakistan on the west and Nepal and Tibet in the centre, and southwest China on the eastern side. Its habitat mainly comprises of mountainous, snow peak grassland, dry or rocky places in forests, subalpine meadows, steppes, sunny, saline areas, sandy deserts, stony and gravelly slopes, rocky crevices along the stream banks and damp places (Fritsch and Friesen 2002;Choi and Oh 2011;Choi et al. 2012).
Despite the importance of the genus Allium for the Indian region, meagre comprehensive studies have been attempted pertaining to molecular and taxonomic evaluation that led to gaps in the status of interspecific and infraspecific relationships among the taxa. Meagre taxonomic studies on the native taxa, unavailability of material for research, sporadic collections from under-explored/unexplored areas and lack of the published literature have led to the possibility of finding new taxonomic records from the Indian region (Pandey et al. 2008(Pandey et al. , 2017(Pandey et al. , 2021. The subg. Rhizirideum is the smallest subgenus of Allium as per the flora of India, and it is represented only by the sect. Eduardia containing only one species, A. przewalskianum Regel. This taxon occurs in the scrub, drier slopes, ravines and rocky crevices (2000-4500 m a.s.l.) in Leh, Jammu and Kashmir and Spiti in Himachal Pradesh. The taxa under subg. Rhizirideum are characterized by the presence of several narrowly ovoid-cylindric bulbs, which borne on creeping rhizome usually covered with a common reticulate membrane, leaves shorter than scape, adaxially channeled and stamens slightly longer than perigone segments, spathe with a long beak, nearly 2 to 3 times longer than the base and hemispherical umbel. Most species share a basic chromosome number of x = 8 and 2n = 16 or 32. Occurrence of a polyploid complex in different sections of the subgenus Rhizirideum indicated recent origin of taxa as supported by phylogenetic and biogeographical evidences (Li et al. 2010). Areas with geographical isolation are the driving force of underestimated speciation (Seregin et al. 2015).
A new taxon, Allium negianum, was collected from the Indo-Tibetan border area of Malari village, Niti valley of Chamoli district in Uttarakhand (India) in 2019 and identity was confirmed by the authors. It is distinct from its closest relative, A. przewalskianum Regel (Table 2), the only taxon of subg. Rhizirideum, sect. Eduardia in India. It is characterized by finely reticulated red-brown outer tunics, hemispherical umbel having lax flowers, spathe with a very long beak, deep purple tepals, asynchronous flowering and inner stamen filaments having longer and sharp teeth. In the present work, A. negianum, is described and illustrated here. Authors have examined the evidences from morphology, eco-geography, leaf anatomy, molecular study, and taxonomic delineation from other related species.

Taxon sampling and morphological descriptor
A total of 110 plants representing 7 accessions of the new species were collected from the type locality and farmers' fields in the Niti region of Uttarakhand, India. For delimitation of the taxon with other related species, plants were grown in the Field Gene Bank (FGB) at the ICAR-National Bureau of Plant Genetic Resources (ICAR-NBPGR), Regional Station Bhowali (Nainital), Uttarakhand for comparative study of morphological characters. Data were recorded using the Allium descriptor with modifications from the published literature. The floral characters were measured with separate parts to the nearest ten points of the decimal. The seeds having uniform size and maturity were recorded for ultra-features of the characters using the Stereozoom Microscope (LMI, England, model no. SZM167), and the images were captured as JPEG. Ten replicate voucher herbarium specimens of the new species were prepared as per standard procedure and deposited in the National Herbarium of Cultivated Plants (code-NHCP) (Holotype) and CAL (Isotype).
The new species was compared with its closest relative using data derived from the study of specimens preserved in the herbaria of CAL, DD, E, K and NHCP and available literature. Due to its closer affinity with A. przewalskianum, all the specimens from diverse sources were critically examined. Taxonomic description and identification key were provided for Allium negianum and affined species.

Leaf anatomy
For leaf anatomy live plants were grown in the FGB at Regional Station Bhowali (Nainital), Uttarakhand. Leaf-blades were taken from a point 3-4 cm above the sheaths and fixed in 70% alcohol. Cross-sections were made at three different lengths of leaf and stained with Sartur solution (a mix of sudan III, aniline, chloral hydrate, lactic acid, iodine), the structure was studied, and analyzed with the help of a light microscope (Olympus BH-2) and line diagrammes drawn. The outlines of cells were diagrammatically depicted (Fig. 1F).

DNA extraction, amplification and sequencing
Genomic DNA of nine known species and one new taxon (Table 1) which was collected from western Himalayan region and maintained as live material at Field Gene Bank (FGB), ICAR-National Bureau of Plant Genetic Resources, Regional Station, Bhowali, was isolated from fresh leaves using spin column-based Qiamp DNA kit according to the suppliers' protocol. Selection of taxa for this study was mainly based on the fact that all taxa belong the third evolutionary line representing same eco-geographical areas, and were known by similar local names. This has resulted in confusion of their identity in Indian literature. The quantity and purity of the isolated genomic DNA was tested using the spectrophotometric method. The universal primers ITS1 and ITS4 (White et al. 1990) were used to amplify the ITS regions. The PCR protocol was run at 94 ° C for 5 minutes; 30 cycles of 94 °C for 45 seconds, 55 °C for 45 seconds and 74 °C for 45 seconds and 74 °C for 5 minutes. PCR products were purified using Zymo DNA concentrator kit following the supplier's protocol. The purified PCR product was used in ABI 3730 DNA sequencer (Applied Biosystems) for generating sequences using PCR primers as sequencing primers. For remaining species from subgenus Rhizirideum and other related sub-genera, the ITS sequence were used from NCBI database.
Etymology. The specific epithet, "negianum", is named in honour of Late Dr. Kuldeep Singh Negi, an eminent explorer who has dedicated his life in collection of indigenous Allium species germplasm along with associated indigenous knowledge across the country. He was also instrumental in establishing the Allium Field Gene Bank (FGB) at the Regional Station, Bhowali, Uttarakhand. The entire germplasm of indigenous Allium species collected by him from remote areas of the country are characterized and successfully conserved at Allium FGB, Bhowali, Uttarakhand.
Leaf anatomy. The transverse section of the leaf of A. negianum showed an elliptical outline. The epidermis has small cells covered with a thin cuticle layer, and stomata are narrowly distributed along the surface area. Single layered compactly arranged palisade tissue comprised of long cylindrical cells. The mesophyll cells are spongy tissue and compact in young leaf as well in the proximal ends of mature leaf while in the centre part of mature leaf, broken mesophyll cells are confused with fistulous leaf ap-   pearance; 10-12 vascular bundles are arranged along with the palisade tissue across the entire circumference (Fig. 1F). Seed morphology. Seed characters and testa sculptures represents a good taxonomic character in Allium (Neshati and Fritsch 2009;Celep et al. 2012;Lin and Tan 2017). Apparently, the seeds of the newly described species were marginally bigger than the related taxon, A. przewalskianum. Baasanmunkh et al. (2020) have discussed on the seed testa structure and its taxonomic implication for taxa of the subg. Rhizirideum. The seed size in A. negianum (Fig. 1I) measured 3.2-4.0 × 1.9-1.9 mm in contrast to 2.7-2.9 × 1.5-1.5 mm in the later taxon (Fig. 1I). The seeds of A. negianum are obovate in shape with a prominent notch on one side, gradually concave from edge to centre, with deep black and wrinkled testa.
The testa cell shape was irregularly hexagonal-pentagonal, loose with clear meshes of reticulated tissue. The anticlinal walls are usually raised, prominently small to intermediate granulose verrucae. The periclinal cells wall has several verrucae with irregular depressions. Study indicated that in subg. Rhizirideum testa cell shape varied from oval to irregular or oval to hemispherical; and seed length 1.30-2.35 mm, anticlinal wall were distinguished by nearly S type to straight and periclinal wall was flat to nearly convex with densely granulated verrucae (Baasanmunkh et al. 2020). A. przewalskianum was distinguished by irregular testa cells in a loose arrangement with reticulated tissue, straight to arched anticlinal walls, and concave periclinal walls with small to intermediate verrucae and granules (Lin and Tan 2017).
Distribution and ecology.The sect. Eduardia of the subg. Rhizirideum is distributed in the southern most range of the Himalayan region of India extending to China which is the centre of diversification. Allium negianum is a species recorded from the southernmost transitional zone between India and China. The distribution of A. negianum is restricted to the phytogeographical region of western Himalaya from Sumna valley, Malari, Chamoli district of Uttarakhand, in western Himalaya, India where it commonly occurs along the open grassy meadows, sandy soils along rivers and streams occurring in the snow pasture lands along the alpine meadows (locally known as 'bugyal' or 'bugial') between 3000-4800 m a.s.l. (Fig. 1A; Fig. 3) in synanthropic habitats. It was reported growing as wild population in Darma valley of Pithoragarh, along Gori Ganga (also Gori Gad) river in the Munsiyari, Pithoragarh district, in Milam Glacier, in northeast of Nanda Devi, Uttarakhand, India. The seeds flowing with the melting snow led to its broader spread in the areas with good regeneration reported by the authors (Fig. 1A). Hence the taxon may be considered endemic in the area of study. Indiscriminate harvest of leaves and bulbs used for 'seasoning' purposes has threatened its wild population.
The first report on large scale cultivation of this taxon in Niti valley, Uttarakhand, as 'seasoning allium spice' called 'jambu' and 'phran' has been published (Pandey et al. 2021). Though the taxon was reported commonly under cultivation, the authors have observed the wild populations primarily from the above 'type' locality. The authors could not trace large scale cultivation of another taxon, A. stracheyi (used for same purpose and known by same local name) in the described locality in Uttarakhand (Pandey et al. 2021). Considering that A. stracheyi was a rare species reported from wild habitats in Uttarakhand Himalaya, the authors assume that the reports by Kuniyal and Negi (2018) on large scale cultivation may be referring to this newly described taxon which is also known by the same local name. Unfortunately, earlier studies on A. stracheyi did not provide any locality details, nor were the voucher specimens deposited in any herbaria of the material used in their study. Therefore, validation of the taxonomic identity could not be ascertained. Also, there is no occurrence record of the taxa belonging to subg. Rhizirideum from Uttarakhand, India. There are no records on the availability of this new taxon from Uttarakhand (Dasgupta 2006). Shah (2014) has raised doubts on reported cultivation of A. przewalskianum in Uttarakhand by Negi (2006). Also recorded data on the occurrence of allied taxon under A. przewalskianum from Gori, Kumaon, Uttarakhand (dated 16 June 2005) and Gori, Martoli, Uttarakhand (7 Oct. 2004) during the study of a total of 413 specimens in the GBIF database need critical study.
Note. Allium negianum was previously mistaken for identity as A. stracheyi as noted in the published records from India. Despite no morphological similarity with the latter taxon, Kuniyal and Negi (2018) referred 'phran' as A. stracheyi. In literature, it was also referred as A. auriculatum and A. przewalskianum due to morphologically similarity of the outer tunics (Pandey et al. 2021). However, the present study demonstrated that A. negianum is clearly distinguished from A. przewalskianum and A. stracheyi, particularly characters of the bulb tunic color when fresh, umbel, teeth in filament and perigone size and color (Fig. 1B-H; Table 1). Allium negianum is diploid (2n = 2x = 16) (data not produced), whereas A. przewalskianum is reported to be tetraploid (2n = 4x = 32) as well as diploid with no stated morphological variation except the stout habit. Authors noted that A. negianum has robust plant habit, stronger plant aroma in wild habitat as compared to plants growing under cultivation. In contrast, the related taxon of the subg. Rhizirideum is currently distributed in Jammu and Kashmir, Himachal Pradesh and adjoining parts in Nepal. A. negianum is reported from areas of Uttarakhand and only known from the type locality (altitude 3200-4800 m a.s.l.) and has never been collected from elsewhere in India and other parts of the world. Therefore A. negianum is said to be localized in distribution.
Upon critical examination of specimen of A. auriculatum deposited in the NHCP, all plant characters were found to be closer to A. negianum. Four specimens of this taxon were noted in label data as frequently growing on flat rocks in Brahmmathya, district Chamoli (3800 m asl.), Uttarakhand, used as leaves cooked as a vegetable.
Allium negianum is morphologically allied to a Chinese species A. eduardi Stearn that occurs on the dry slopes and plains in the adjoining regions of Mongolia and Russia and shares characters of spathe beak size, hemispherical umbel and perigone shape, but differs in having yellowish-brown bulb tunic color, tepal apex with a reflexed point and shorter stamen teeth length.

Taxnomic treatment
Two species, A. przewalskianum and A. negianum, of the subg. Rhizirideum, sect. Eduardia can be distinguished from A. stracheyi of the subg. Polyprason by using the following key.
Key to Allium negianum and related species Taxonomic delineation and relationship analysis using nuclear ITS sequence For taxonomic delineation and relationship analysis data set comprising 18 representative taxa from diverse subgenera were selected (Table 3; Fig. 4). The DNA sequence data set of nuclear Internal Transcribed Spacers (ITS) region used for phylogenetic analysis was generated for Allium negianum and other taxa used in the study. The generated ITS sequences and obtained ITS sequences from NCBI (Table 3) were used to construct the maximum likelihood tree. The tree with the highest log-likelihood is shown (Fig. 4). The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and selecting the topology with superior log likelihood value. The branch lengths measured in the number of substitutions per site. Two major clades were found within Allium, comprising subgen. Rhizirideum, on one side and second cluster had four subg. Butomissa, Allium, Polyprason and Cepa. on the other side. This former group was divided in two sister clades, with first clade having Allium przewalskianum, Allium negianum sp. nov. A. eduardii (all from section Eduardia); Allium subangulatum, A. polyrhizum from sect. Caespitosoprason; and A. nutans, A. prostratusm, A. spurium and A. spirale in sect. Rhizirideum. One of the taxon A. tenuissimum from sect. Tenuissima grouped separately. Second clade was divided into subgenera, namely Butomissa with one taxon, Allium tuberosum; subg. Allium, with Allium sativum and Allium ampeloprasum var. ampeloprasum; subg. Polyprason having Allium stracheyi; subg. Cepa that was the largest having four taxa, Allium cepa var. cepa, A. cepa var. aggregatum, A. oschaninii and A. schoenoprasum from distinct sections.
Based on the likelihood tree, the new Allium taxon was observed to be closely related to A. przewalskianum, both of Indian Himalayan origin along with a Chinese taxon, A. eduardii to form distinct cluster supporting the morphological resemblance of this taxa with section Eduardii under subg. Rhizirideum. The species from other sections under same genus were distantly placed in the phylogenetic tree. The species which are found in same geographical area belong to different subgenera viz. Allium, Cepa, Butomissa and Polyprason were distantly placed and used as outgroup in determining the integrity of newly described species Allium negianum.
The above findings indicated that the new taxon is a distinct species and is closely related to A. przewalskianum and belongs to sect. Eduardia under subg. Rhizideum. These findings supported the observations recorded using plant morphology, particularly the floral characters that were very distinct in both the taxa.
Recent advances in molecular phylogenetics have revolutionized our understanding of Allium taxonomy and evolution. However, the phylogenetic relationships in some Allium sections (such as the Allium sect. Eduardia) and the genetic bases of adaptative evolution remain poorly understood for the Indian taxa (Pandey et al. 2021). Molecular phylogeny study of the wild Allium in different centers of diversity (Nguyen et al. 2008;Xie et al. 2019;Jang et al. 2021) has helped in unlocking many aspects of the taxon relationships. The present study uncovered a new species relationship with its closest allied species and suggested that the selective habitat pressure has played an important role in the adaptation and evolution of Allium in this habitat which will facilitate uncover more taxa in the genus.

Conclusions
Allium negianum, a new species under the subg. Rhizirideum, is described using live and herbarium specimens. With the inclusion of this taxon, in the subg. Rhizirideum of the sect. Eduardia there are two taxa in India, and the latter one A. negianum was report-edly restricted to the Uttarakhand flora. Samples of this taxon collected during earlier explorations that remained unidentified will be designated with this new name and conserved as seed in the National Gene Bank (NGB), New Delhi and vegetative material will be maintained in the Field Gene Bank (FGB) at Bhowali, Uttarakhand, India.