A new record of critically endangered Saussurea bogedaensis (Asteraceae) from Dzungarian Gobi, Mongolia

Abstract A species in the family Asteraceae, Saussurea bogedaensis, was newly described from Bogeda Mountain in Xinjiang, China and is a critically endangered species in China. Morphological and genetic characteristics confirm the presence of this species in Mongolia, as it was found in Baitag Bogd Mountain (in the Dzungarian Gobi). In addition, the distribution and conservation status of S. bogedaensis are provided.


Introduction
Saussurea DC. is one of the largest genera in the tribe Cardueae (Asteraceae) and comprises ~500 species, classified into six subgenera and 20 sections (Lipschitz 1979;Raab-Straube 2017). The genus is distributed throughout the Northern Hemisphere, with diverse species in Central Asia (Wang et al. 2009). Saussurea spp. occur in a wide range of habitats, especially at higher altitudes with cold and dry conditions, but they also grow in lowlands. However, Saussurea spp. have a tendency towards habitat specificity (Butola MA is likely to be S. orgaadayi. Thus, inconsistencies in the distribution range of these three Saussurea species, which could have been misidentified in Mongolia as well, motivated us to conduct an in-depth taxonomic assessment. In addition, Chen et al. (2019) recommended the use of nuclear ribosomal (nr) DNA ITS and chloroplast (cp) DNA regions of rbcL and trnH-psbA as candidate DNA barcode markers for species in the subg. Amphilaena. Using these three markers, it was possible to discriminate the Saussurea species that are morphologically similar and separated very recently.
The main objectives of the present study were to (1) re-identify the above mentioned Saussurea species recorded in western Mongolia and (2) newly report S. bogedaensis and describe its distribution and conservation status in the Mongolian flora.

Herbarium and field research
The basic distribution data and photographs of the target Saussurea species, which had been known as S. involucrata and S. orgaadayi in Mongolia, were collected during our fieldwork from 2013 to 2019 in western Mongolia. We also included herbarium materials kept at UBA, UBU, OSBU and MW (abbreviations are according to Thiers 2019+).

DNA barcoding
In this study, we investigated the application of combined nrDNA region of ITS and cpDNA regions of trnK, trnH-psbA and rbcL in barcoding analyses of two Mongolian Saussurea species. Additionally, a total of 36 sequences, based on four markers of three species (S. bogedaensis, S. orgaadayi and S. involucrata), which were used by Chen and Wang (2018) to evaluate the phylogenetic relationships between these species, were obtained from NCBI GenBank (Table 1). Jurinea multiflora (L.) B.Fedtsch. was selected as an outgroup based on Chen and Wang (2018) and Chen et al. (2019). Detailed information on sample collection, voucher specimens, Genbank accession numbers and references of each sample is provided in Table 1.
Total genomic DNA was extracted from silica gel-dried leaf materials following the CTAB method (Doyle and Doyle 1987). The PCR reaction was performed in a 50 µl volume, containing approximately 200 ng DNA, 1.5 mM MgCl 2 , 0.2 mM dNTP, 1 µM of each primer and 0.75 units of Taq DNA polymerase. Initial template denaturation was programmed at 94 °C for 4 min and then followed by 30 cycles of 94 °C for 1 min, annealing at 50-56 °C for 1 min and extension at 72 °C for 1 min, with a final extension step of 72 °C for 7 min. Markers used for the amplification and sequencing are listed in Table 2. PCR products were sent to ZanaaSPX, Mongolia (www.hangal.mn) for commercial sequencing. Sequences were aligned using MEGA 7 (Kumar et al. 2016), with the default settings and manual adjustments were made using SnapGene Viewer 4.2.6. Sequences were edited manually using SnapGene Sequence Alignment Editor (GSL Biotech LLC). Ambiguous nucleotide bases were corrected using the corresponding base of the sequence that was obtained by the reverse primer. Multiple sequences were aligned using ClustalW with its default parameters (Thompson et al. 1994) and consensus sequences were created for each species. For the combined dataset, the genetic   Olmstead et al. (1992) trnH CGCGCATGGTGGATTCACAATCC divergences were calculated using DNASP v.6 (Julio et al. 2017) and used to determine whether a barcoding gap was present. The DNA sequences generated in this study have been deposited in GenBank (Table 1). The phylogenetic analyses were conducted using Bayesian Inference (BI), Maximum Likelihood (ML) and Maximum Parsimony (MP). For BI analysis, the best close fit model of evolution for each partition neighbour joining (NJ) tree was estimated using MEGA 7 (Kumar et al. 2016). Posterior probability was determined by Markov Chain Monte Carlo sampling (MCMC) with the programme MrBayes v. 3.2.6 (Huelsenberk and Ronquist 2001;Ronquist and Huelsenberk 2003), as implemented in Geneious v. 10.2.2 (Kearse et al. 2012), using the estimated models of evolution. For each dataset, four simulation Markov chains were run for 1 million generations and trees were sampled every 100 th generation. The ML analysis was performed using RAxML v. 8.2.11 (Stamatakis 2006(Stamatakis , 2014 as implemented in Geneious v. 10.2.2 (Kearse et al. 2012), using the GTRGAMMA model with rapid bootstrapping and a search for the best-scoring ML tree algorithm, including 1,000 bootstrap replicates. The MP analyses were performed with MEGA 7 (Kumar et al. 2016), using treebisection-reconnection (TBR) as the branch-swapping algorithm. The robustness of the tree was evaluated using 1,000 bootstrap replication indices and the consistency index, retention index and composite index were calculated.

Results
We discovered S. bogedaensis from Baitag Bogd Mt in the DzG region of Mongolia. This species is newly documented in the Mongolian flora. Detailed data on morphological and genetic identification, geographical distribution and conservation status of the S. bogedaensis are provided below.

Saussurea bogedaensis Yu J.Wang & J.Chen, PloS ONE 13(7): e0199416 (12) (2018) Figs 1, 3
Morphological identification. Saussurea bogedaensis (Fig. 1) was recently discovered on Bogeda Mt in Xinjiang, China by Chen and Wang (2018) (Fig. 3). This species is very similar to S. involucrata and S. orgaadayi (Fig. 2), but several morphological characteristics of the bracts, involucres and phyllaries differentiate them (Chen and Wang 2018). In particular, S. bogedaensis differs by having elliptic, apically obtuse stem leaves (Fig. 1C) vs. lanceolate, long-acuminate stem leaves in S. orgaadayi ( Fig. 2A); dirty white pappus colour (Fig. 1D) vs. straw-coloured pappi in S. orgaadayi (Fig. 2D); densely pubescent phyllaries (Fig. 1E) vs. glabrous phyllaries in S. involucrata; and campanulate involucres in S. bogedaensis vs. hemispherical involucres in S. involucrata. Genetic identification. The combined sequence dataset consisted of 15 samples, including the outgroup, Jurinea multiflora. The sequence dataset comprised 2,315 characteristics, of which 20 were parsimony-informative, 108 were variable and 2,169 were constant. The gene boundaries on the ITS -trnK -trnH-psbA -rbcL multi-locus alignment were as follows: ITS: 1-656, trnK: 657-1,284, trnH-psbA: 1,285-1,680 and rbcL: 1,681-2,315. The final ML optimisation likelihood of ML analysis was: Inl = -3650.7353. A single most parsimonious tree was generated by MP analysis with a tree length of 105 steps, consistency index: 1.0, retention index: 1.0 and composite index: 1.0. The BI phylogeny, including BI posterior probability values, as well as ML and MP bootstrap support values, are provided in Fig. 4. Our genetic identification revealed a similar topology to that of Chen and Wang (2018) and confirms each distinct clade of S. bogedaensis, S. involucrata and S. orgaadayi, respectively (Fig. 4). Three individuals of newly-revealed Saussurea specimens from Baitag Bogd Mt formed one cluster with the Chinese S. bogedaensis with high support: BI/ML/MP = 1/100/99. Additionally, sequence divergence amongst the three species was 0-0.002% in our S. bogedaensis specimens, whereas there was 3.02% sequence divergence in S. involucrata and 2.04% sequence divergence in S. orgaadayi. Sequence alignment revealed that the Mongolian and Chinese S. bogedaensis share several specific nucleotide residues that are different from those of other Saussurea species (Fig. 5). The other three samples (Fig. 2) from Munkhkhairkhan Mt in the MA region clustered with S. orgaadayi from China (BI/ML/MP = 1/100/99). Therefore, our study proves that the Saussurea samples from the DzG and MA regions are S. bogedaensis (Fig. 1) and S. orgaadayi (Fig. 2), respectively. Our genetic results provide only the genetic differences between the three related species in the subg. Amphilaena and not a true phylogeny of all related Saussurea species.
General distribution and habitat. Mongolia (Dzungarian Gobi, Baitag Bogd Mt) and China (Xinjiang, Bogeda Mt). In Mongolia, S. bogedaensis grows on high mountain rocky slopes, screes, boulders and river banks in the alpine belt at altitudes of 2400-3300 m a.s.l. This species is closely related to S. involucrata and S. orgaadayi. However, the three species are geographically isolated: S. bogedaensis occurs in the Dzungarian basin and the eastern Chinese Tien-Shan Mts and S. involucrata occurs in the Tien-Shan Mts (which cover parts of China and Central Asian states), whereas S. orgaadayi is present in the Altai Mts (which cover parts of China, Mongolia and Russia) (Fig. 3), according to Raab-Straube (2017) and Chen and Wang (2018).
Conservation status. Saussurea bogedaensis is new to the Mongolian flora and occurs in the Baitag Bogd Mt in the DzG region. Individuals of the species were found in a few  locations, namely in Baitag Bogd Mt and Altan Ovoo in the DzG region (Fig. 3). During our field surveys, we detected two different populations, which in total, accounted for fewer than 600 individuals in this region. This species is under threat, particularly owing to human interference and random cutting. Thus, S. bogedaensis has been assessed as Critically Endangered [CR C2a(i)] in Mongolia according to the IUCN Red List categories and criteria (IUCN 2019). This species was also evaluated as critically endangered in China (Chen and Wang 2018). In situ studies on the reproductive biology of S. bogedaensis are needed to more accurately assess the conservation status of this species in Mongolia.

Discussion
Saussurea bogedaensis, S. orgaadayi and S. involucrata belong to the taxonomically complicated Saussurea subg. Amphilaena (Raab-Straube 2017). Despite their similar morphological characteristics and habitats, there are clear morphological differences, geographically isolated distributions and genetic identities that make these species recognisable with an in-depth investigation (Figs 1-5; Chen and Wang 2018;Chen et al. 2019). There are some distribution records of S. involucrata from the regions of Khovd and the Depression of Great Lake in Mongolia (Urgamal et al. 2014). Due to limited numbers of samples and surveyed areas of the MA and DzG regions in this study, data on Mongolian S. involucrata are still unclear. Hence, correct identification based on this study will provide an important basis for future studies on the taxonomic identity of Mongolian S. involucrata. Figure 5. Multiple sequence alignment of combined nr DNA (ITS) and cpDNA (trnK, trnH-psbA and rbcL) sequences. ITS region shows more differences than cpDNA regions amongst those closely related species. (*) -no differences found between species.
improving the English writing of manuscript. Finally, the authors thank Dr. Yu-Jin Wang (Lanzhou University, China) for his careful review with valuable suggestions and personal discussion on the first draft.