﻿A new species and a new record of Phlomoides (Lamiaceae) from Xizang, China

﻿Abstract Phlomoidesbomiensis, a new species in Bomi County, Xizang, China, was described and illustrated. In addition, Phlomoideslongidentata, previously only known from Nepal and Bhutan, is newly recorded from Dingri County, Xizang, China. The phylogenetic placement of both species within the genus was analysed using nine plastid DNA markers (atpB-rbcL, psbA-trnH, rpl16, rpl32-trnL, rps16, trnK, trnL-trnF, trnS-trnG, trnT-trnL). Both species have brown-black trichomes inside the upper corolla lip and nested within the same subclade of Clade II. A diagnostic key to the Phlomoides species belonging to this subclade is provided.

China is one of the three diversity centres of Phlomoides, boasting 60 species and 17 varieties, of which 39 species are distributed in the Tibetan Plateau, Himalaya and Hengduan Mountains (Wu and Li 1977;Li and Hedge 1994;Zhao et al. 2021aZhao et al. , b, 2022Zhao et al. , 2023aZhao et al. , 2024a, b, c), b, c).However, the diversity of Phlomoides in these areas remains understudied.Recent discoveries have shed light on this hidden species diversity, with the description of four species new to science within these  (Pendry 2021;Zhao et al. 2021aZhao et al. , 2024a, b), b).
During our field trip in Xizang, we found one new species of Phlomoides in Bomi County and one new record of the genus in Dingri County.Both species have brownblack trichomes inside the upper corolla lip.In this study, we provide detailed descriptions and illustrations of both the new and newly-recorded species.In addition, we make detailed morphological comparisons with other species which have brown-black trichomes in the upper corolla lip.This comparative analysis aims to facilitate accurate identification and classification within the genus Phlomoides.

Taxon sampling
Taxon sampling is primarily based on our previous molecular phylogenetic study (Zhao et al. 2024a).For this particular study, the putative new species

DNA extraction, selection of markers and molecular phylogenetic analyses
The CTAB method was used to extract total genomic DNA from silica gel dried leaf materials (Doyle and Doyle 1987).Sequences of nine plastid DNA markers (atpB-rbcL, psbA-trnH, rpl16, rpl32-trnL, rps16, trnK, trnL-trnF, trnS-trnG, trnT-trnL) were chosen for the phylogenetic reconstruction.Primers, polymerase chain reaction (PCR), sequencing and alignment were carried out according to procedures used in Zhao et al. (2024a).The sequences, newly generated in this study together with their GenBank accession numbers, are listed in Suppl.material 1.
Bayesian Inference (BI) and Maximum Likelihood (ML) were used for phylogenetic reconstruction.Detailed settings for BI and ML analyses followed Zhao et al. (2024a).TreeGraph2 (Stöver and Müller 2010) was applied to visualise and edit all trees.

Morphological and taxonomy study
We thoroughly examined herbarium specimens or their digital images from the following Herbaria: B, BM, C, CDBI, E, FI, GH, HIB, IBSC, K, KUN, KYO, L, LE, M, MA, MAO, MO, MW, NAS, P, PE and TI.During our field investigations, we observed and documented important diagnostic characteristics of Phlomoides species.These observations were complemented by high-resolution photographs taken in their natural habitats.Trichome morphology was observed and measured under a Leica DM2500 optical microscope (Leica Microsystems GmbH, Wetzlar, Germany).

Sequence characterisation
In total, 558 sequences were included for phylogenetic analyses, of which 63 sequences were newly sequenced in this study and they were submitted to Gen-Bank (Suppl.material 1).The aligned cpDNA dataset was 9,222 nucleotides in length (2,382 bp for atpB-rbcL, 439 bp for psbA-trnH, 1,365 bp for rpl16, 677 bp for rpl32-trnL, 968 bp for rps16, 954 bp for trnK, 869 bp for trnL-trnF, 825 bp for trnS-trnG and 743 bp for trnT-L, respectively), of which 883 bp (9.57%) are variable.Characteristics for all datasets are listed in Table 1.

Phylogenetic reconstruction
The phylogenetic analyses using both Bayesian Inference (BI) and Maximum Likelihood (ML) methods yielded largely congruent tree topologies.Therefore, only the Bayesian 50% majority rule consensus tree is presented, with posterior probabilities (PP) and bootstrap values (BS) indicated near nodes (Fig. 1).
Consistent with our previous molecular phylogenetic analyses (Zhao et al. 2024a), six well-supported clades can be recognised (Fig. 1).Clade II includes the majority of species characterised by having linear-tuberous roots, without persistent basal leaves and glabrous nutlets (Zhao et al. 2024a).In the present study, we found that Clade II can be divided into two major subclades with strong support values.Subclade IIa (Fig. 1: PP = 1.00/BS = 100%) contains the putative new species (P.bomiensis), as well as P. longidentata.Most species in this subclade are characterised by brown-black trichomes inside the upper corolla lip, except for P. rotata and P. henryi (Zhao et al. 2024c)   Phlomoides longidentata is sister to P. rotata which is represented by four individuals (Fig. 1: 1.00/93%) and these species are sisters to P. breviflora (Fig. 1: 0.98/76%).P. bomiensis is sister to all three species (Fig. 1: 0.99/62%).

Phlomoides bomiensis
Phenology.Flowering from August to September, fruiting from October to November.
Distribution and habitat.Based on our field collections and previously collected specimens, P. bomiensis is known to occur in Bomi County and Motuo County, Xizang (Tibet), China.It grows in forests and forest margins at altitudes between 3400 and 4200 m (Fig. 3).
Etymology.The specific epithet refers to the name of the Bomi County in Xizang Autonomous Region, where the new species was discovered.
Chinese name (assigned here).bō mì cǎo cāo sū (波密草糙苏) Additional specimen examined (paratypes).China.Phlomoides bomiensis was first collected more than 40 years ago (S.Z.Cheng & B.S. Li 00315; PE 00923558!, PE 00832483!, PE 00832484!), but the specimens were then identified as Phlomoides umbrosa (Turcz.)Kamelin & Makhm.var.australis (Hemsl.)C.L.Xiang & H.Peng.However, Phlomoides umbrosa var.australis was distinguished from P. bomiensis by having white or transparent trichomes on upper corolla and subsessile floral leaves (vs.brownblack trichomes on upper corolla and floral leaves with petioles 0.5-7 cm long).Another specimen of P. bomiensis was collected in 2010 (South Tibet Expedition STET1237; PE 02328210!), but it was misidentified as Phlomoides tibetica.The differences between Phlomoides tibetica and P. bomiensis are provided in Table 2.  Diagnosis.Phlomoides longidentata is a recently described species from Bhutan and Nepal (Pendry 2021).Specimens of P. longidentata were collected by Chinese collectors as early as 1959 (Anonymous 752; PE 00832482!), when it was identified as P. umbrosa and P. umbrosa var.australis.During our field investigations in Rongxia Town, we rediscovered this species in the wild.It rather looks like a perennial herb, but not annual as described by Pendry (2021).Here we provide description of this species.
Phenology.Flowering from July to September, fruiting from October to December.
Distribution and habitat.China, Bhutan and Nepal; new record for China found in Xizang (see below).It grows in forests and forest margins at altitudes between 2000 and 3800 m (Fig. 3).

Figure 2 .
Figure 2. Phlomoides bomiensis C.L.Xiang & Y.Zhao A habitat B, C verticillaster with floral leaves D plant E verticillaster F flower G bracts H dissected flower I appendages at base of posterior filaments (arrow) J calyx and dissected calyx K floral leaves L Stem leaves (Photographed by Yue Zhao).

Figure 4 .
Figure 4. Phlomoides longidentata Pendry A habitat B, D plant C, E verticillaster F bracts G flower H dissected calyces I dissected flower J appendages at base of posterior filaments (arrow) K floral leaves L stem leaves (Photographed by Yue Zhao).

Table 1 .
All the species in subclade IIb (Fig.1:1.00/98%)have white and transparent trichomes inside the upper corolla lip.The statistics of all datasets for phylogenetic analysis.