Rediscovery of Pogostemon dielsianus (Lamiaceae, Lamioideae), a rare endemic species from southwestern China, after one century

Abstract Pogostemon dielsianus (Lamiaceae) was described in 1913 based on a single gathering from northwestern Yunnan of China collected in 1905, and thereafter no further collections were observed until 2019. We rediscovered the rare endemic species in Lushui County, Yunnan. Molecular phylogenetic analyses based on four cpDNA markers (rbcL, rps16, psbA-trnH, and trnL-trnF) and the nuclear ribosomal internal transcribed spacer (ITS) region confirmed its infrageneric placement within subg. Pogostemon. Based on observations of the rediscovered population of P. dielsianus, we updated its morphological description, provided an illustration, and discussed its distribution. Under IUCN criteria, the species was categorized as “Critically Endangered (CR)”.

introduction Pogostemon Desf. is the largest genus of tribe Pogostemoneae of subfamily Lamioideae in Lamiaceae (Bendiksby et al. 2011;Li et al. 2016). After combining with Dysophylla Blume based on the molecular phylogenetic analyses (Bendiksby et al. 2011;Yao et al. 2015Yao et al. , 2016, Pogostemon, as currently circumscribed in a broad sense, contains approximately 80 species. On the basis of molecular and morphological evidence, Pogostemon was divided into two subgenera: subg. Pogostemon and subg. Dysophyllus (Bl.) Bhatti & Ingr. ex G. Yao, Y.F. Deng & X.J. Ge (Yao et al. 2016). The genus can be easily distinguished from other Lamiaceae genera by possessing moniliform hairs at the middle of the staminal filaments. Pogostemon is distributed mainly in tropical and subtropical regions of Asia with a few species in tropical Africa, Northern Australia, Japan and the Korea Peninsula (Bhatti and Ingrouille 1997;Harley et al. 2004;Yao et al. 2015).
In China, 27 species and two varieties were recorded, of which 10 species and one variety are endemic (Yao et al. 2015;Yao and Ge 2018). Pogostemon dielsianus was described in 1913 based on a gathering (G. Forrest 875) with two specimens deposited at E and K respectively from Fugong County, northwest Yunnan, China and was not collected again since over the following 100 years. When conducting a taxonomic revision of Chinese Pogostemon, Yao et al. (2015) noted that only the type specimens of P. dielsianus were examined, and the species was unable to be included in the subsequent molecular phylogenetic analysis (Yao et al. 2016).
During a scientific field trip in Nujiang Canyon, northwestern Yunnan of China in November 2019, a population of Pogostemon was discovered in thickets near a tributary of Nujiang River (also known as Salween River). After scrutiny of the data available (Wu and Huang 1977;Li and Hedge 1994;Bhatti and Ingrouille 1997;Yao et al. 2015), we rediscovered Pogostemon dielsianus after 106 years. This finding allowed us to update its morphological description, discuss its geographic distribution, assess its conservation status, and infer its phylogenetic position within Pogostemon.

Materials and methods
Taxon sampling, DNA extraction, amplification and sequencing Following the latest phylogenetic study (Yao et al. 2016), a total of 28 species (including Pogostemon dielsianus) were sampled from both subgenera of Pogostemon to explore the phylogenetic position of P. dielsianus (Table 1). In addition, three species of its sister genus Anisomeles R. Brown were selected as outgroups based on previous studies (Li et al. 2016;Yao et al. 2016). Except for the newly generated sequences of P. dielsianus, all other data were downloaded from GenBank.
Total genomic DNA of Pogostemon dielsianus was extracted from silica gel-dried leaf material following the modified CTAB method of Doyle and Doyle (1987). The nuclear ribosomal internal transcribed spacer (ITS) region was amplified using primers table 1. Voucher information and GenBank accession numbers for taxa used in this study. *indicates the new sequences, and "-" indicates missing data. ITS5 and ITS4 (White et al. 1990). Four chloroplast DNA markers were employed to make phylogenetic analyses and the rbcL was amplified with primers of Z1F and 51R (Soltis et al. 1992), the rps16 with rps-LamF and rps-LamR (Bendiksby et al. 2011), the psbA-trnH with psbAF and trnHR (Sang et al. 1997), and the trnL-trnF with trnc and trn-f (Taberlet et al. 1991). All makers were amplified and sequenced with the same conditions following Hu et al. (2020).

Sequence alignment and phylogenetic analyses
Sequences were checked and assembled employing Sequencher v.4.1.4 (Gene Codes, Ann Arbor, Michigan, USA) and then aligned Mafft-win v7.221 (Katoh and Standley 2013) by default. The final alignments were manually adjusted in PhyDE v.0.9971 (Müller et al. 2010). Nuclear dataset (ITS) and plastid matrix (consisting of rbcL, rps16, psbA-trnH, and trnL-trnF) were analyzed separately using maximum likelihood (ML) and Bayesian inference (BI). ML analyses were performed using RAxML-HPC2 on XSEDE v.8.2.12 (Stamatakis 2014) under the GTRCAT model on the CIPRES science gateway portal (http://www.phylo.org/) (Miller et al. 2010). Except for setting the bootstrap iterations (-# | -N) to 1000, other parameters followed default. BI analysis was performed in MrBayes v3.2.6 (Ronquist et al. 2012) as implemented in PhyloSuite (Zhang et al. 2020) with the ModelFinder used to select the best model (Kalyaanamoorthy et al. 2017). Under the Akaike information criterion (AIC), the GTR+F+G4 model was selected for nrDNA dataset and the GTR+F+I+G4 for cp-DNA matrix. In each analysis, four Markov chain Monte Carlo (MCMC) chains were run simultaneously for 20 million generations, starting with one random tree and sampling one tree every 1000 th generation. Convergence of runs was reached when the average standard deviation of split frequencies (ASDSF) fell below 0.01. After discarding the first 25% of the resulting trees as burn-in, the remaining trees were used to assess posterior probabilities (PP) in a majority-rule consensus tree.
Distribution and habitat. The type locality of Pogostemon dielsianus was recorded in Fugong County, northwestern Yunnan of China, which is the only historical known site until our new discovery. As coordinate information of the collection is incomplete due to the lack of longitude data, the precise situation of type specimen is unclear. Based on the latitude provided in the original record, the type specimen is more likely to be collected in the north of Lushui County, a neighboring county of Fugong (Fig. 5). Although the recently collected population was also discovered in north Lushui County, distribution of the two populations does not overlap because they are located on different sides of Nujiang River (Fig. 5). In accordance with type specimen record, P. dielsianus grows amongst thickets on dry rocky hillsides with elevations ranging from 1524-1829 m. The finding that the newly recorded population grows on the riverside indicates that P. dielsianus is more likely to occur in humid areas of dry hillsides. Actually, a similar habitat can also be found elsewhere in Nujiang Canyon. Potential populations of this species, therefore, may be discovered through further field investigation in this region.
Phenology. Flowering and fruiting from November to December. Conservation status and preliminary IUCN assessment. Pogostemon dielsianus is historically known from only two specimens collected from the type locality (Fugong, Yunnan, China) in 1905, and it has not been recollected for the past 114 years until our expedition to Nujiang Canyon in 2019. In the newly recorded locality (Lushui, Yunnan, China), only about 10 mature individuals have been discovered. Due to the lack of exact geographical information of the type locality, it is difficult to confirm the number of individuals there. Based on current investigations and historical records, we inferred that mature individuals of this species may be fewer than 250, and no subpopulation contains more than 50 mature individuals. Therefore, under the IUCN criteria C2a(i) (IUCN 2012), we propose that P. dielsianus should be classified as "Critically Endangered (CR)".
Additional specimens   Notes. Pogostemon dielsianus is morphologically similar to P. elsholtzioides and P. griffithii in having lanceolate leaves. However, P. dielsianus can be easily distinguished from P. elsholtzioides and P. griffithii by its longer and tubular calyx, smaller ratio of the length of calyx teeth and calyx tube and longer corolla, filament and style (Table 2). In addition, the geographical distribution of these three species is also different in that P. dielsianus is endemic to NW Yunnan, China, P. elsholtzioides is widely distributed in the Himalayan regions (Bhutan, India, and SE Xizang, China), and P. griffithii is endemic to Myanmar (Bhatti and Ingrouille 1997;Yao et al. 2015;Yao and Ge 2018).
In the protologue, Dunn (1913) did not designate a type for the name Pogostemon dielsianus Dunn. Bhatti and Ingrouille (1997) indicated the specimen deposited in E and K as holotype and isotype, respectively. In fact, they effectively chose the lectotype for the name and the term"holotype" and "isotype" can be corrected as "lectotype" and "isolectotype" according to Article 9.10 of the International Code of Nomenclature for algae, fungi, and plants (Shenzhen Code) (Turland et al. 2018).