Saussurea talungensis (Asteraceae), a new species from Humla, Nepal Himalayas

Abstract A new species Saussurea talungensis S.K.Ghimire & H.K.Rana, sp. nov. (sect. Strictae), from Talung valley of Humla district, Nepal, is described and illustrated. Morphologically, this species resembles Saussurea roylei and Saussurea lanata in habit, though it can be distinguished in having longer leaf petioles, purplish leaf margin, 1 or 3 capitula, shorter phyllaries, shorter receptacle bristles and the same anthers, comparatively shorter corolla with shorter lobes. Phylogenomic analysis also supports S. talungensis as a distinct species of Saussurea. Here, we provide taxonomic note, distribution map and phylogenomic inference to distinguish the new species and its allied members.

open vegetation types elsewhere in the Northern Hemisphere (Wang et al. 2009). Chen (2015) reported ca. 458 species and von Raab-Straube (2017) approximated the species number to be 493. Among them, Nepal represents 44 species including 8 endemics (Rana et al. 2018;Tiwari et al. 2019). Recently, a number of new species were discovered from the Himalaya and Central/East Asia, including S. ramchaudharyi S.K.Ghimire & H.K. Rana A distinct population of Saussurea ( Fig. 1) was recognized in 2012, during a botanical expedition to the alpine region of Humla district (NW Nepal). Based on habit, this population of Saussurea was initially considered as S. roylei (DC.) Sch.Bip. with some extent of resemblance with Saussurea lanata Y.L.Chen & S.Y.Liang. However, the population from Talung valley could not be ascribed to any known species of the genus Saussurea and may represent a new species. Therefore, in order to clarify this presumption, we characterized its morphology and clarify its genetic distinctness using chloroplast genome sequence. In particular, with the advent in the next-generation sequencing (NGS) technology, DNA-barcoding provides a rapid and precise solution for honing plant taxonomies when combined with more traditional, morphology-based approaches (Kress et al. 2005;Hajibabaei et al. 2007;Hollingsworth 2011;Ji et al. 2020). Integratively, morphological observations and molecular analysis led us to conclude that this population belongs to a new previously undescribed species of Saussurea (see below). We thus formally presented the description of the new species, Saussurea talungensis S.K.Ghimire & H.K.Rana.

Methods
Three Saussurea specimen were collected in September 2012 from the type locality, Talung valley, Humla district in NW Nepal ( Fig. 1A-D). The specimens were used for morphological and phylogenomic inference. A distribution map was produced using the type locality coordinates (Fig. 1A, B).

Morphological observations
Morphological characteristics were described based on both observation and measurement collected with a ruler, calipers and electronic digital compound microscope. For the comparative morphological characteristics of allied taxa (S. roylei and S. lanata), related literatures (Chen et al. 1981;Shi and von Raab-Straube 2011;Chen 2014Chen , 2015, live plant images and herbarium, and digitized specimen images from E, GH, K, KATH, PE, BM (acronyms following Thiers 2020, continuously updated) and CVH (www.cvh.ac.cn) were consulted.

Plastome sequencing, assembly, annotation and phylogenomic analyses
Total genomic DNA was extracted from ~20 mg herbarium leaf tissue using a modified cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1987). A 500 bp DNA TrueSeq Illumina (Illumina Inc., San Diego, CA, USA) sequencing pair-end libraries were constructed using 3-5 μg sonicated DNA, according to the manufacturer's instructions. The libraries were pair-end sequenced on the Illumina HiSeq 2000 platform. Raw reads were subsequently filtered to remove the low-quality reads and adaptors using the NGS QC Toolkit (Patel and Jain 2012), setting the cut off value for percentage read length to 80 and Phred quality score to 30. Remaining high-quality reads were assembled de novo to generate complete plastome with GetOrganelle pipeline developed by Jin et al. (2020). All the reads were then reference-assembled against the plastome of S. hookeri C.B.Clarke (MK952740) to check if the genomes were correctly assembled. The consensus sequence was annotated using S. hookeri as a reference in GENEIOUS v.7.0.2 (Kearse et al. 2012) and then corrected manually for the start/stop codons and intron/exon boundaries. Finally, the annotated plastid genome was submitted to GenBank (MW524864) and a physical map of the circular plastome was visualized with OrgannellarGenomeDRAW (OGDRAW: Lohse et al. 2013). To determine the phylogenetic position of the new species within the genus Saussurea, 64 plastome and a rbcl sequence (of S. roylei) of the genus Saussurea, plus one each for Hemisteptia lyrata (Bunge) Fisch. & C.A.Mey. and Aucklandia costus Falc. were accessed from GenBank (Table 1) and aligned with the newly generated sequence of S. talungensis in MAFT-WIN v.7.221 (Katoh and Standley 2013;Yamada et al. 2016). From the initial alignment, we selected conserved blocks with GBLOCK  (Castresana 2000). We used GTR+I+G as the best fitting substitution model based on the Akaike information criterion (AIC) using JMODELTEST v.2.1.6 (Posada and Crandall 1998). We performed Bayesian phylogenetic inference (BI) analysis in MRBAYES v.3.2.6 (Ronquist and Huelsenbeck 2003) on online CIPRES Science Gateway v.3.3 (Miller et al. 2010; https://www.phylo.org). For BI, two independent analyses of four Markov Chain Monte Carlo (MCMC) chains were run for 5 × 10 7 generations each with sampling every 1,000 generations. We assessed the stationarity of the runs using TRACER v.1.7 (Rambaut et al. 2018) and generated a majority rule consensus after removing a 20% burn-in. Maximum Likelihood (ML) analysis was performed using the graphical front-end RAXML GUI v.1.5b2 (Silvestro and Michalak 2012) in RAXML v.8.2.x (Stamatakis 2014) with 1,000 rapid bootstraps with 10,000 maximum number of trees. The Bayesian posterior probability (PP) from BI and Likelihood bootstrap support (BS) from ML of each branch was obtained. Nodes with PP ≥ 0.95 (Ronquist and Huelsenbeck 2003) and BS ≥ 75% (Hillis and Bull 1993) were considered well-supported.
Phenology. Flowering and fruiting from July to September. Etymology. The specific epithet is derived with reference to the type locality of Saussurea talungensis, Talung valley, Humla district, NW Nepal.

Distribution and habitat.
Saussurea talungensis is currently recorded only from the type locality in Talung valley (between Nyalu Pass and Ning Tsho), Humla district, NW Nepal (Fig. 1A-D). It grows on the alpine open gravelly or stony slopes at an elevation ca. 4300 m a.s.l. (Fig. 1C, D).
Conservation status. Saussurea talungensis is restricted to a single mountain and is represented by ~50 mature individuals within an area of < 500 m 2 and appears to be local endemic. Moreover, its habitat and the population are subjected to high anthropogenic pressure, due to livestock grazing, and harvesting of caterpillar fungus and other aromatic plants used in medicine. Owing to population size, isolated distribution and observed constraints on the habitat due to anthropogenic pressure, Saussurea talungensis should be categorized as Critically Endangered [CR; B1ab (iii), B2ab (iii) and D] according to the IUCN Standards and Petitions Committee (2019).
Morphological affinities. Critical examination of collected specimens, comparison with type material of allied taxa and relevant taxonomic literature revealed that S. talungensis is a new member of Saussurea (sect. Strictae). Based on morphology, distribution and ecology, this population of Saussurea was initially considered as S. roylei from sect. Strictae. To a certain extent it also resembles S. lanata in being a perennial herb with well-developed leafy stem, leaf blade undivided but lanceolate, many series phyllaries, campanulate involucres with more than 1 cm diameter, lanate anther tails, ribbed and glabrous achenes, and two rows of pale brown pappus. However, it differs from its allied taxa in having a number of qualitative and quantitative characters (see Table 2; Figs 2, 3). Furthermore, in the western Himalayan alpine region, S. roylei is considered to have diverse morphological variations but this proposed new species owned peculiar affinities which undoubtedly differentiates it from the stated and other Saussurea species. 1.2-2.5 cm (10-13 mm / 6-9 mm with 4-5 mm lobes) 1.2-1.6 cm (4-8 mm / 6-8 mm with ca. 3 mm long lobes) Anther ca. 5 mm with 1.2 mm tail ca. 8 mm with ca. 1.5 mm tail ca. 6.5 mm with ca. 1.8 mm tail Achene size 3.5-4.5 × 1.5-2.0 mm 5-6 mm 4-5 mm

Molecular affinities.
The typical quadripartite structure of the newly sequenced plastome has size of 152,355 bp (37.7% GC content) consisting of a large single copy (LSC: 83,371 bp, 35.8% GC content), a small single copy (SSC: 18,562 bp, 31.4% GC content), inverted repeats (IRs: 25,211 bp, 43.1% GC contents each of IRA and IRB) (Fig. 1E). The newly sequenced chloroplast genome was used to determine the phylogenomic relationship of S. talungensis with its allied species and infer its position within Saussurea. The molecular phylogeny through BI and ML tree revealed that S. talungensis is nested within a clade comprising S. roylei, S. lanata, S. hookeri, S.  Fig. 4). It is more evident that S. talungensis is a sister to S. roylei (an allied species), and is supported by Bayesian posterior probability (PP = 1) and Likelihood bootstrap support (BS = 98%) (Fig. 4). Also, complete chloroplast genome structure is conservative in overall size and the order and size of each gene and intergenic region (Fig. 1E). The identical BI and ML phylogenomic tree using plastome sequence revealed that S. talungensis is most closely related to S. roylei (Fig. 4), which is in congruence with the morphological observations.