﻿Alliumsunhangii – a new species from section Brevidentia F.O.Khass. & Iengal. (Amaryllidaceae) from Southern Pamir-Alay, Uzbekistan

﻿Abstract A new species, Alliumsunhangiisp. nov., of the Middle Asiatic section Brevidentia F.O.Khass. & Iengal., (subgenusAllium, tribe Allioideae, Amaryllidaceae) is described. The species is a small plant from the Babatag Ridge in the Surkhandarya province of Uzbekistan. It is morphologically close to Alliumbrevidens Vved. in having initially dark violet filaments and three-cuspidate inner filaments, but differs by its small size and visibly unequal tepals as well as in the phylogenetic analysis based on ITS data.


Introduction
Allium Linnaeus (1753), one of the largest genera in the Amaryllidaceae (Friesen et al. 2006;Li et al. 2010), has more than 1100 species worldwide (Govaerts et al. 2021). Members of the genus, such as garlic, leek, onion and shallot, are used as food, medicine and ornament (Herden et al. 2016) and are characterized by bulbs enclosed in a membranous, fibrous or reticulate tunic, free or basally connate tepals and often a subgynobasic style (Friesen et al. 2006). Allium has two probable diversity centers, one in South-Western and Middle Asia and in the Mediterranean region, and a smaller center is in western North America (Friesen et al. 2006;Nguyen et al. 2008). The most recent classification of Allium, by Friesen et al. (2006), based on molecular phylogenetic analyses, includes 15 subgenera and 72 sections.
Subgenus Allium, with more than 375 species and 35 subspecies, is the largest subgenus within Allium, and is one of three main evolutionary lines within the genus (Friesen et al. 2006;Fritsch and Friesen 2002). Subgenus Allium consists of two main groups (Hanelt 1992;Friesen et al. 2006); one has simple inner filaments while the other has three-cuspidate inner filaments. The newly described sections are supported by nuclear molecular data (Friesen et al. 2006) and have revealed the presence of centers of recent speciation in the Middle Asia, Pakistan, Iran, Afghanistan and the Middle East (Khassanov 2018). Also, results from whole chloroplast genome analyses are continuing and being compared with morphology to determine whether morphologybased taxonomy corresponds well to molecular data (Munavvarov et al. 2022) Section Brividentia F.O.Khass. & Iengal. was previously treated as a part of section Allium of subgenus Allium. Khassanov et al. (1997)  ). According to the last revised and updated classification of subgenus Allium (Khassanov 2018), section Brevidentia includes 12 species, most of which are in Middle Asia and adjacent areas. The main characteristics are purple filaments, the inner ones three-cuspidate, as well as a rounded purplish ovary with pocket-like mounds of the nectary tubes. Most species show S-to U-type, U-type anticlinal walls and (globular) convex periclinal walls of seeds .
In 2021, during grid mapping of the flora of the Surkhandarya province (Babatag Ridge, Zarkasa peak in Uzbekistan), we collected an interesting species of Allium. Comparisons of molecular and morphological characteristics showed it as a member of sect. Brevidentia. Morphologically, it resembles A. brevidens in its purple, three-cuspidate inner filaments, but differs in unequal tepals, which showed that it was a previously unknown characteristic for the species of Allium. Here, we propose it as new species and provide a comprehensive description based on morphological and molecular approaches.

Plant material
A total of 14 specimens were collected in the summer of 2021. Material from the new species was collected in the Zarkasa (Babatag Ridge) peak, Surkhandarya province, Uzbekistan.

DNA extraction, PCR amplification and sequencing
Leaves for molecular analysis were dried in silica gel upon collecting. Total DNA was isolated by the CTAB protocol (Doyle and Doyle 1987) from 1 g of well-dried leaves. ITS1 and ITS4 primers were from White et al. (1990). Polymerase chain reaction (PCR) was performed under the following conditions: 5 min of initial denaturation at 94 °C, 35 cycles of denaturation for 45 secs at 94 °C, annealing for 45 secs at 55 °C, and extension for 1-1.5 min at 72 °C, then a final extension at 72 °C for 5 min. PCR products were visualized using electrophoresis on 1.5% agarose TAE gel and sent to Beijing Genomics Institute (Shenzhen, China) for sequencing.

Phylogenetic analyses
To assemble and edit complementary strands, we used Sequencher 4.1.4 software (Burland 2000). Clustal X (Jeanmougin et al. 1998) was used to align DNA sequences, which were then manually adjusted using MEGA 7.0 (Kumar et al. 2016). Analysis of parsimony was conducted in PAUP* 4.0b10 (Swofford and Sullivan 2003) using heuristic searches with TBR and 1000 random addition sequence replicates. Bootstrap support (BS) was estimated with 1000 replicates, each with 100 random addition sequence searches according to Felsenstein (1985). The major consensus trees constructed from a maximum of 1000 trees were saved. RAxML v 8.2.8 (Stamatakis 2014). The best-fitting nucleotide substitution model GTR + G model was determined for each dataset and 1000 bootstrap replicates were used for performing Maximum Likelihood (ML) analyses. Based on the Akaike information criterion (AIC) implemented in jModelTest2 on XSEDE (www.phylo.org). For Bayesian inference (BI) analyses, MrBayes version 3.1.2 (Huelsenbeck and Ronquist 2001) was utilized, with 10,000,000 generations with random trees sampled every 1000 generations. In the latter analysis, after discarding the first 25% of trees as burn-in, and in order to estimate posterior probabilities (PP) we constructed a 50% majority-rule consensus tree from the remaining trees.

Taxonomic treatment
Diagnosis. This species is most similar to Allium brevidens Vved. (Fig. 1), from which it differs in a more compact habit, remaining small spathe with a short beak, unequal tepals and strongly exserted, dark violet filaments (Fig. 2).
Distribution and habitat. Allium sunhangii is known from one population occurring to the south in the northwestern part of the Zarkasa peak, at 2251 m a.s.l. (Figs 3, 4). New species grows in continental and drier Juniperus forests (Fig. 4B 1  Etymology. Allium sunhangii is named after Prof. Sun Hang, one of the leading botanists at the Kunming Institute of Botany, Chinese Academy of Sciences, China, who actively promotes several projects within Central Asia. Phenology. Allium sunhangii was flowering ( Fig. 4 A 1 -A 2 ) on 22 June, 2021 when we found its fruits began to mature at the same time. It is supposed that flowering starts in about late May and/or early June. As we visited this area only once, we are not sure when fruiting finishes.
Conservation status. Allium sunhangii is so far only known from two closely spaced localities. The total distribution area of this species is around 5 km 2 . The total

Phylogenetic analysis
Allium sunhangii was placed in the section Brevidentia (subgen. Allium) in all phylogenetic analyses (MP, ML and BI) (Fig. 5). Phylogenetic tree based on ITS data suggests that the new species closely related to A. brevidens.

Discussion
Allium sunhangii is morphologically close to A. brevidens in having initially dark violet filaments. However, it differs in a more compact habit, remaining small spathe with a short beak, unequal tepals and strongly exserted, dark violet filaments. Compared to all known species of Allium sect. Brevidentia, the new species differs by having leaves longer than scape, spathe with rather small beak ca. 3 mm long; tepals whitish with greenish midvein. Most significantly, the new species has lax (vs dense) and umbellate (vs globose) inflorescence, and also fewer flowers, 10-25 (vs 30-50) ( Table 1)  et al. 2010). Accordingly, our phylogenetic analysis was also consistent with those phylogenetic analyses. However, A. sunhangii can be distinguished morphologically and geographically from the representatives of sect. Allium. In consistence of morphologic evidence, the position of A. sunhangii and A. brevidens in the phylogenetic tree supports that they are most relative and the new species belongs to sect. Brevidentia. Also, the distribution of the new species and the related species may also slightly support this arrangement. Thus, current molecular and morphological data support the recognition of A. sunhangii as a new species of Allium sect. Brevidentia.