﻿Using scanning electron microscopy and molecular data to discover a new species from old herbarium collections: The case of Phlomoideshenryi (Lamiaceae, Lamioideae)

﻿Abstract Phlomoides is one of the largest genera of Lamiaceae with approximately 150–170 species distributed mainly in Eurasia. In this study, we describe and illustrate a new species, P.henryi, which was previously misidentified as P.bracteosa, from Yunnan Province, southwest China. Molecular phylogenetic analyses revealed that P.henryi is found within a clade in which most species lack basal leaves. In this clade, the new species is morphologically distinct from P.rotata in having an obvious stem and, from the rest, by having transparent to white trichomes inside the upper corolla lip. In addition, micro-features of trichomes on the calyx and leaf epidermis can differentiate the new species from other species grouped in the same clade and a key, based on trichome morphology for these species, is provided. The findings demonstrate that the use of scanning electron microscopy can reveal inconspicuous morphological affinities amongst morphologically similar species and play an important role in the taxonomic study of the genus Phlomoides.

As currently defined, Phlomoides consists of approximately 150-170 species and ranks the second largest genus within subfamily Lamioideae (Salmaki et al. 2012a, b;F Zhao et al. 2021).Species of Phlomoides are mainly distributed from central Europe to the Russian Far East, but highly diversified in three regions: Central Asia (59 spp.; Czerepanov (1995)), the Iranian highlands (ca.41 spp.; Salmaki et al. (2012a)) and China (58 spp.; Xiang et al. (2014); Zhao et al. (2021aZhao et al. ( , 2024))).In China, most species are found in the southwest region and 29 species and 11 varieties are endemic and geographically restricted (Li and Hedge 1994).The existing infrageneric classification of Chinese Phlomoides (= Phlomis section Phlomoides Briq.) was established by Hsuan (1977), who divided Chinese species into two subsections and 17 series, based on external morphology (e.g. the absence/presence of the basal leaves, shape of stem leaves, length and density of trichomes on stems and leaves etc.).However, most infrageneric categories were not recovered as monophyletic (Zhao et al. 2024) and those external and quantitative characters used for traditional taxonomy are highly variable amongst different species or at different populations for the same species.In contrast, some micro-features probably have taxonomic significance within Phlomoides.For example, Seyedi and Salmaki (2015) and Khosroshahi and Salmaki (2019) found trichome morphology to be important for the delimitation of sections and species of Phlomoides.In addition, trichome characters have significant taxonomic values in other genera of Lamiaceae (Gairola et al. 2009;Xiang et al. 2010;Hu et al. 2012;Yao et al. 2013).However, micro-features of trichomes and other characters of Chinese Phlomoides species are poorly known.
During the past ten years, phylogenetic and taxonomic studies have focused on Phlomoides from China (Xiang et al. 2014;Zhao et al. 2021aZhao et al. , b, 2023aZhao et al. , b, 2024) ) resolving some taxonomic puzzles.In the process of the continuing taxonomic study of the genus, two collections attracted our attention when investigating historical specimens.One collection with three sheets were collected by Augustine Henry in 1898 (A.bracts subulate, simple long trichomes on calyces, bracts and both sides of leaves) shown in these specimens are obviously different from those of P. bracteosa (upper floral leaves sessile, lower floral leaves with petioles up to 20 mm long, bracts lanceolate-linear, branched trichomes on calyces, bracts and both sides of leaves).Fortunately, we re-discovered the plant in the wild from the possible locality where specimens were collected by Henry, after more than 125 years since the first collection in 1887.Molecular phylogenetic analyses and macro-and micro-morphological studies demonstrate that the species is a new species, P. henryi and we describe and illustrate it in this study.

Taxon sampling
In total, we sampled 49 out of 58 (84.48%)Chinese species of Phlomoides for molecular phylogenetic analyses.Sampling is primarily based on previous molecular phylogenetic studies of Phlomoides (Zhao et al. 2024) and only samples of the potential new species and P. bracteosa were newly sequenced.Fresh leaves of the putative new species (P.henryi) were collected and dried with silica-gel in the field (Jianshui County, Yunnan Province) and herbarium materials of P. bracteosa were collected from the herbarium BM.
In addition, six species from the subclade comprising the potential new species, as well as P. bracteosa, were sampled to investigate macro-micro-features of trichomes on flora bracts and leaves.The list of sampled species and their origins are given in Table 1 and voucher specimens were deposited in the Herbarium of the Kunming Institute of Botany (KUN) and Institute of Botany (PE), Chinese Academy of Sciences.

DNA extraction, selection of markers and molecular phylogenetic analyses
Total genomic DNA was extracted using the CTAB method (Doyle and Doyle 1987).Previous studies revealed that plastid DNA phylogeny can better resolve relationships of Phlomoides than the tree inferred from the nuclear ribosomal internal and external transcribed spacer regions (nrITS and nrETS) (Zhao et al. 2023a, b;2024).In order to test systematic placement of the new species, nine plastid DNA regions (atpB-rbcL, psbA-trnH, rpl16, rpl32-trnL, rps16, trnK, trnL-trnF, trnS-trnG, trnT-L) were selected for phylogenetic reconstruction.Primers, polymerase chain reaction (PCR), sequencing and alignment followed those described in Zhao et al. (2024).The sequences newly generated in this study together with their GenBank accession numbers are listed in Appendix 1.

Morphological investigations
Species concept, definitions of characters and depiction generally follow Li and Hedge (1994).Type specimens and protologues for all species of Phlomoides in China were collated.Morphological features were based on herbarium as well as field investigations.Specimens at B, BM, C, CDBI, E, FI, GH, HIB, IBSC, K, KUN, LE, M, MA, MAO, MICH, MO, MW, NAS, P, PE, S, SG, TI, W, WUK and XJBI (herbarium acronyms followed Thiers 2022) and our collections from the field were examined for characterisation and morphological comparison.Additional morphological information (including habit, habitat, root, leaf, calyx, flower etc.) was taken from field observations, as well as literature (Hsuan 1977;Wu et al. 1977;Li 1985;Li and Hedge 1994).
Micro-features of leaf epidermis and floral bracts were investigated using Light Microscopy (LM) and Scanning Electron Microscopy (SEM).Photographs and morphological observations were taken using a Leica DM2500 light microscope (Leica Microsystems GmbH, Wetzlar, Germany).Mature leaves and floral bracts were taken from our collection (Table 1) for SEM investigation.Materials were mounted on to stubs and coated with gold, using a ZEISS EVO LS10 scanning electron microscope (Carl ZEISS NTS, Germany) with 10 kV voltage (Kunming Institute of Botany, Yunnan, China).Terminology of morphological characteristics of trichomes followed Khosroshahi and Salmaki (2019).

Molecular phylogeny and systematic placement of Phlomoides henryi
A total of 18 sequences were newly sequenced in the present study and they were submitted to GenBank under accession nos.OR674852-OR674869.The aligned length of the combined plastid dataset was 9259 bp (2380 bp for atpB-rbcL, 421 bp for psbA-trnH, 1361 bp for rpl16, 681 bp for rpl32-trnL, 967 bp for rps16, 958 bp for trnK, 868 bp for trnL-trnF, 831 bp for trnS-trnG and 792 bp for trnT-L), respectively.The topologies of the BI and ML trees were consistent with each other, only the Bayesian 50% majority-rule consensus tree being presented, with the posterior probabilities (PP) and Bootstrap support (BS) and values being superimposed near the nodes (Fig. 1).Monophyly of the genus Phlomoides was recovered (Fig. 1: PP =1.00/BS = 100%).The backbone topologies of Phlomoides recovered in present study are largely consistent with those of previous studies (Zhao et al. 2024), clade I is sister to Clade II with strong support values (Fig. 1: 1.00/100%), then sister to a large clade consisting of Clades III, IV, V and VI.Chinese Phlomoides species can subdivided into six clades (Fig. 1).
As shown in Fig. 1, the new species, Phlomoides henryi is distantly related to P. bracteosa.Instead, P. henryi is sister to a subclade (Fig. 1
Figs 3, 4 and Table 2 show the morphology and distribution of trichomes on leaves and bracts of the investigated taxa.Sub-sessile/sessile glandular trichomes occur widely in every part of each species of Phlomoides (Table 2).Short stalked glandular trichomes were observed on the abaxial leaf surface in five species and on the bracts of only one species, i.e.P. breviflora.Branched glandular trichomes were only recorded on the abaxial leaf surface of P. breviflora.
Simple short eglandular trichomes were observed in every species on leaf and bract surface, but were missing in the abaxial leaf of Phlomoides nyalamensis, since it was nearly glabrous (Fig. 4J).Adaxial leaf surfaces were often covered by simple eglandular trichomes, except for P. bracteosa (Fig. 4C), which has dense branched eglandular trichomes on the adaxial leaf surface.Simple long eglandular trichomes were most common on bracts (Fig. 3B, H, J, L, O).Abaxial leaf surfaces often had branched eglandular trichomes, but these are not present in the new species (Fig. 4B).
Trichomes were transparent to white or brown to black in Phlomoides.Trichomes inside the upper corolla lip of the new species (P.henryi), P. bracteosa and P. rotata were transparent to white, while the other five species were brown to black.Bract trichomes of P. tibetica and P. milingensis were brown to black (Fig. 3K, M), the other six species were transparent to white (Fig. 3A, C, E, G, I, O).

Discussion
Herbaria house millions of specimens that embody the plant diversity on the Earth.Many new species have been lurking in herbaria for many years before being published.Bebber et al. (2010) estimated that 84% of new species' descriptions were from old specimens collected more than five years prior to publication and 25% from specimens more than 50 years old.During the taxonomic review of some groups of Lamiaceae, we have also found some new species from old herbarium specimens (Chen et al. 2014;Dong et al. 2015), indicating taxonomic work, based on herbaria, is still a very important resource for the discovery of new taxa.
The resulting phylogenetic tree of Phlomoides in this study was similar to that in previous study (Zhao et al. 2024).The new species, P. henryi, was nested within Clade II and formed a separate branch (Fig. 1: 1.00/100%) that is sister to a subclade containing P. rotata and five species with brown to black trichomes on the upper corolla.Geographically, Phlomoides henryi is distributed in southern Yunnan, while the other six species in this subclade were mainly distributed in the Qinghai-Tibetan Plateau and Himalaya.The new species is morphologically distinct from the other six species in this subclade.For example, trichomes on the upper corolla lip of P. henryi and P. rotata are colourless and perceptually transparent to white, but brown to black in the other five species.Morphologically, P. rotata is distinct from all other species of Phlomoides by the very short stem producing a rosette of leaves with the plant often less than 10 cm high, while P. henryi is generally taller than 1 m.As we observed, all the species with trichomes brown to black were embedded within this subclade.The sister clade to that containing P. henryi contains 23 species that are mainly distributed in Hengduan Mountains.Phlomoides henryi is similar to other species in Clade I and Clade II in lacking basal leaves.Only four species have basal leaves in Clade II, i.e.P. rotata, P. tibetica, P. milingensis and P. atropurpurea, while all the species in Clades III-VI have basal leaves.
As above mentioned, we believe that the differences merit recognition of the new species and we describe it below.
Phlomoides is a morphologically diverse and taxonomically difficult group with many characters used for traditional taxonomy being highly variable.In this study, we investigated trichome micro-morphology on bracts and leaves of Phlomoides henryi and related species.We found that trichomes are a useful character to distinguish some morphologically similar species.Based on the colour of trichomes, we can separate two groups of those species.Phlomoides nyalamensis, P. macrophylla, P. tibetica, P. milingensis and P. breviflora have brown to black trichomes on the upper corolla lip, while the other species (P.rotata, P. bracteosa and the new species described here, P. henryi) have transparent to white trichomes on the upper corolla lip.Trichome density and bract trichome colour can separate P. tibetica from the similar P. milingensis.Both species are distributed in Xizang at an altitude from 3500-4500 m and Hsuan (1977) placed them within Series Tibeticae.Phlomoides tibetica has floral bracts with black simple trichomes and no branched trichomes, while P. milingensis has floral bracts with brown simple and branched trichomes.Similarly, the new species described here, P. henryi, can be distinguished from the six related species in the subclade by the absence of branched trichomes on the abaxial leaf surface (Fig. 5B).Phlomoides bracteosa can easily be separated from these six species by having branched trichomes on the adaxial leaves (Fig. 5C).Azizian and Cutler (1982) have found that adaxial and abaxial leaf surfaces have different trichome types, but in that work, Phlomoides was treated as a section of Phlomis and they only discussed the differences amongst Phlomis sect.Phlomis, Phlomis sect.Phlomoides and Eremostachys and not at the species level.Subsequent studies did not observe trichomes on different structures (Seyedi and Salmaki 2015;Khosroshahi and Salmaki 2019).However, here we found different structures were covered with significantly different trichomes and these differences can be used as evidence to separate morphologically similar species.Future studies should focus on micro-morphological investigation of trichomes and other characters (i.e.appendages, calyces, roots, mericarps) and those micro-features are probably helpful for taxonomy and species identification of Phlomoides species.
In order to distinguish those species grouped with the new species in the phylogenetic tree (Fig. 2), as well as P. bracteosa, we provide a key, mainly based on macro-and micro-morphological trichomes.Diagnosis.Within the subclade, Phlomoides henryi is morphologically similar to P. rotata for having transparent to white trichomes inside the upper corolla lip rather than brown to black and is distinct from all other species by lacking branched hairs.P. bracteosa has similar transparent to white trichomes inside the upper corolla lip, but with branched trichomes on both sides of leaves and floral bracts.The differences between P. henryi, P. rotata and P. bracteosa are listed in Table 3.

Figure 1 .
Figure 1.Phylogeny of Phlomoides inferred by Bayesian Inference (BI), based on the combined plastid dataset cpDNA.Support values displayed on the branches follow the order BI-PP/ML-BS (" * " indicates PP = 1.00 or BS = 100%, "-" indicates incongruent relationship between BI and ML tree.

Figure 3 .
Figure 3. Photos of bracts, SEM of bracts of Phlomoides henryi and related species A, B P. henryi C, D P. bracteosa E, F P. breviflora G, H P. macrophylla I, J P. nyalamensis K, L P. tibetica M, N P. milingensis O, P P. rotata.A, C, E, G, I, K, M, O photos of bracts B, D, F, H, J, L, N, P SEM of bracts.

Figure 4 .
Figure 4. SEM of both sides of leaves of Phlomoides henryi and related species A, B P. henryi C, D P. bracteosa E, F P. breviflora G, H P. macrophylla I, J P. nyalamensis K, L P. tibetica M, N P. milingensis O, P P. rotata A, C, E, G, I, K, M, O SEM of adaxial leaves B, D, F, H, J, L, N, P SEM of abaxial leaves.

Figure 5 .
Figure 5. Phlomoides henryi Y.Zhao & C.L.Xiang A habitat B plant with linear-tuberous roots C inflorescence D verticillaster E flowers F dissected flower G appendages at base of posterior filaments H fruiting calyces I dissected calyces J bracts K floral leaves L stem leaves.Photographs by Yue Zhao, except C by Li Chen.

Table 1 .
List of sampled Phlomoides species to investigate macro/micro features of trichomes and their voucher information.

Table 2 .
Distribution of different types of trichome in the examined Phlomoides spp.

Table 3 .
Morphological comparisons amongst Phlomoides henryi, P. rotata and P. bracteosa.No branched trichomes With branched trichomes on abaxial leaves With branched trichomes on both sides of leaves and bracts