﻿Elsholtziazhongyangii (Lamiaceae), a new species from Sichuan, China

﻿Abstract Elsholtziazhongyangii (Lamiaceae), a new species from Sichuan Province, China, is described and illustrated. The new species is morphologically similar to E.feddeif.feddei, but it can be easily distinguished from E.feddeif.feddei by smaller corolla (3.2–3.5 mm vs. 4.5–5.3 mm), bract indumentum (glabrous, except margin ciliate vs. villous, especially on veins abaxially, glabrous adaxially) and bract stalked (ca. 1.2 mm vs. sessile). Phylogenetic analyses, based on two nuclear ribosomal (ETS, ITS) and five plastid (rbcL, matK, trnL-F, ycf1, ycf1-rps15) regions, confirmed that the new species formed a monophyletic clade with robust support. The new species is currently known from western Sichuan.


Introduction
Elsholtzia Willdenow (Lamiaceae) is a member of the tribe Elsholtzieae (Harley et al. 2004). The genus is characterised by verticillasters in continuous or interrupted spikes or capitula, compact spikes cylindric or secund, and two-lipped corolla (Huang 1977) (Huang 1977). Elsholtzia is mainly distributed in East Asia, with 35 species found in China (Huang 1977;Li and Hedge 1994;Harley et al. 2004;Pu et al. 2012;Xiang and Liu 2012). Many species of Elsholtzia are important plant resources used both as medicine and flavouring. More than 60% of the Chinese species have been widely used as traditional medicinal herbs, nectar source plants, vegetables and spices (Huang 1977;Li and Hedge 1994;Editorial Board of Zhong Hua Ben Cao 1999).
During our botanical expedition to Yajiang County, Sichuan Province in September 2012, we discovered an unknown species of Elsholtzia. It is similar to Elsholtzia feddei f. feddei in calyx (villous, with two long and three short teeth with spinescent apices) and leaf apices acute, but differs from the latter by its smaller corolla (3.2-3.5 mm vs. 4.5-5.3 mm), bract glabrous, with a ciliate margin (vs. villous, especially on veins abaxially, glabrous adaxially) and bract stalked (ca. 1.2 mm vs. sessile). After carefully checking specimens and literature, together with evidence from molecular phylogenetic analyses, based on combined nrDNA (ETS, ITS) and combined ptDNA (rbcL, matK, trnL-F, ycf1, ycf1-rps15) datasets, we demonstrated that it is, indeed, a new species which is described and illustrated here.

Taxon sampling and molecular analyses
A total of 48 individuals of E. zhongyangii were collected from Yajiang County, Sichuan Province, China from September 2012 to December 2021 (Fig. 1). Voucher specimens were deposited in Wenzhou University (WZUH). The nuclear (ETS, ITS) and plastid (rbcL, matK, trnL-F, ycf1, ycf1-rps15) regions were used for reconstructing the phylogeny of the new species and its related taxa (Li et al. 2017). A total of 20 individuals, representing 16 species of Elsholtzia, were sampled, with Elsholtzia densa Benth. as the outgroup. The GenBank accession numbers are listed in Suppl. material 1. Most sequences were downloaded from GenBank, except for the new species, which was newly sequenced in the present study.
Two samples of the new species were sequenced using the genome skimming approach, then 14 sequences [each sample has two nrDNA (ETS, ITS) and five ptDNA (rbcL, matK, trnL-F, ycf1, ycf1-rps15) sequences as listed in Suppl. material 1] were mined for the phylogenetic analyses. Genomic DNA was extracted from approximately 20 mg of silica-gel-dried leaves using DNA Plantzol Reagent (Hangzhou Lifefeng Biotechnology Co., Ltd.) according to the manufacturer's protocol. For each species, genomic DNA (m > 1 μg, c > 12.5 ng/μl) was sheared (yielding < 800 bp fragments) and the quality of fragmentation was checked on an Agilent 2100 Bioanalyzer (Agilent Technologies). Preparation of short-insert (350 bp) paired-end libraries and sequencing were performed by the Beijing Genomics Institute (Shenzhen, China). These samples were pooled with others and run in a single lane of an Illumina HiSeq XTEN with a read length of 150 bp. For the two samples of the new species, we used the GetOrganelle pipeline (Jin et al. 2020) for de novo assembly of plant plastome and nuclear ribosomal RNA (ETS-18S-ITS1-5.8S-ITS2-26S-ETS). The nuclear (ETS, ITS) and chloroplast (rbcL, matK, trnL-F, ycf1, ycf1-rps15) segments were extracted from these assembled genomic sequences.
Maximum Likelihood (ML) analyses were performed in IQtree v.1.6.12 (Nguyen et al. 2015). The best-fitting substitution models (GTR+F+G4 for nrDNA and TVM+F+G4 for ptDNA) were selected by ModelFinder (Chernomor et al. 2016;Kalyaanamoorthy et al. 2017) according to the Bayesian Information Criterion (BIC). An ultrafast bootstrap (UFB) (Bui et al. 2013) of 1000 replications and the SH-aLRT test were used in the analysis to assess branch supports. Bayesian Inference analyses (BI) were conducted in MrBayes 3.2.6 online interface (Miller et al. 2010). MrModeltest 2.3 (Nylander 2004) was used to determine the appropriate DNA substitution model using the Akaike Information Criterion (AIC) and the results indicated that GTR+G (both for nrDNA and ptDNA) is the best-fit model. Four (one cold and three hot) simultaneous Markov chains were run for one million generations with sampling every 1000 th generation until the average deviation of split frequencies fell below 0.01. The posterior distribution of trees was summarised by the > 50% majority rule consensus tree after discarding the first 25% of samples as burn-in.
Incongruences amongst different datasets (combined ptDNA dataset, combined nrDNA dataset) were explored through visual comparison of tree topologies and support values. Hard incongruence was defined as BS ≥ 80% and/or PP ≥ 0.95 (Pelser et al. 2010).

Morphological comparison
Detailed morphological comparisons between the new species and four other sympatric or morphologically similar taxa are summarised in Table 1. In morphology, the putative new species is most similar to Elsholtzia feddei f. feddei, sharing features, such as two long and three short calyx teeth with spinescent apices, white villous outside of the calyx, acute leaves apex, obtuse serrate leave margin and villous leaves. However, the new species differs from the latter by smaller corolla (3.2-3.5 mm), bract stalked (ca. 1.2 mm vs. sessile) and bract glabrous, except margin ciliate (Table 1, Figs 3-4). The other four species could be easily distinguished from the new species by their larger corolla (4.5-7 mm vs. 3.2-3.5 mm) and sessile bract (sessile vs. stalked ca. 1.2 mm) ( Table 1, Fig. 4).
Phenology. Flowering and fruiting from September to December. Chinese name. Zhong-yang-xiang-ru (钟扬香薷). Etymology. The specific epithet is named in memory of Prof. Yang Zhong, a Chinese botanist who was dedicated to botanical research and education in Xizang (Tibet), China.

Discussion
Elsholtzia zhongyangii usually grows together with E. ciliata or other Elsholtzia species in grassland on mountainsides or even along roadsides, thus it must have been overlooked previously. We found that the plant is fragrant with dense glands. This species may be suitable as an aromatic plant, and thus it has the potential for development and application values.
There is one other Elsholtzia species with small corolla recorded in Sichuan, i.e. E. souliei. However, E. souliei is distantly related to the new species in the phylogenetic tree (Fig. 2) and the plant is much smaller (less than 10 cm tall).
Within the genus Elsholtzia, the taxonomy of the E. splendens-E. ciliata clade is the most problematic. For example, in the Flora Reipublicae Popularis Sinicae, Huang (1977) divided E. feddei into four different forms, which looks very different from each other. However, Li and Hedge (1994) treated all the forms as synonyms of E. feddei in the Flora of China. A recent molecular phylogenetic study (Li et al. 2017) included two forms of E. feddei and showed that they did not cluster together, with E. feddei f. robusta sister to E. souliei. Besides, they also found that E. splendens from China and South Korea did not form a clade, with the Chinese E. splendens sister to a sympatric E. saxatilis, and the Korean E. splendens sister to E. hallasanensis (endemic to Jeju Island). Our phylogenetic result (Fig. 2) is consistent with previous study. Together with the extremely variable morphology and controversial taxonomic treatments, these findings imply that the so-called "E. feddei" and "E. splendens" may contain some hidden diversity. Future studies based on comprehensive sampling and genomic data are needed to shed light on the systematics of this problematic group.