﻿Molecular and morphological evidence for a new species of Stachys (Lamiaceae) from Hunan, China

﻿Abstract Stachysyingzuijieensis, a new species from western Hunan, China, is described and illustrated. Molecular phylogenetic analyses based on three nuclear ribosomal DNA loci (ETS, ITS and 5S-NTS) recovered S.yingzuijieensis within the Stachys clade and as a sister group of S.arrecta. The two species can be easily distinguished by the morphology of lamina, corolla and nutlet. A key to all species of Stachydeae from China is also provided.

A total of 18 species of Stachys are recorded from China and eight of them are endemic (Li and Hedge 1994).Except for Stachys, China also accommodates another three genera of Stachydeae, i.e.Chamaesphacos (1 sp.), Sideritis (2 spp.) and Suzukia (2 spp.) (Li and Hedge 1994;Liu and Zhang 2004).Recently, a potential new species of Stachys was discovered during our field investigation in western Hunan Province, China.By carrying out comprehensive molecular phylogenetic and morphological studies, we confirmed its status as a species new to science.It was, thus, named Stachys yingzuijieensis L. Wu & Y.P. Chen and described below.

Molecular phylogenetic analyses
The phylogenetic placement of the new species within Stachydeae was evaluated based on the framework of Salmaki et al. (2019).A total of 90 accessions representing 88 taxa from all 12 clades and 11 genera of Eurystachys, as well as Melittis melissophyllum L., were sampled as the ingroups.Two genera that are closely related to Stachydeae -Betonica L. and Galeopsis L. -were selected as the outgroups.Except for one accession of the new species and one accession for each of eight species of Stachys from China that were newly sequenced here, all remaining sequences were downloaded from GenBank.Voucher information for newly-sequenced samples and GenBank accession numbers for all sequences are listed in Appendix 1.
Total genomic DNA was extracted from silica-gel-dried leaf material using the modified CTAB method (Doyle and Doyle 1987).According to Salmaki et al. (2019), three nuclear ribosomal DNA loci, i.e. the internal and external transcribed spacers (ITS and ETS) and the 5S non-transcribed spacer (5S-NTS), were used to reconstruct the phylogenetic relationships.Polymerase chain reaction primers and protocols of ITS and ETS followed those used by Chen et al. (2019) and that of 5S-NTS followed Roy et al. (2013).
Trichomes on the lamina and calyx, as well as the nutlet and pollen morphology of the new species, were investigated using scanning electron microscopy (SEM).All materials were directly mounted on to stubs and sputter-coated with gold for 90 s at 20 mA.Micromorphological observations were conducted using a Zeiss EVO LS10 scanning electron microscope (Carl Zeiss NTS, Oberkochen, Germany) at 10 kV.Terminologies used for trichome, nutlet and pollen description followed those of Salmaki et al. (2008aSalmaki et al. ( , 2008bSalmaki et al. ( , 2009)), Karaismailoğlu andGüner (2019, 2021) and Totmaj and Salmaki (2022).

Phylogenetic results
The aligned length of the combined nuclear dataset was 1,381 bp (589 bp for ITS, 456 bp for ETS and 336 bp for 5S-NTS).The topologies of the BI and ML trees were largely consistent with each other, but the BI tree provided higher resolution.Thus, only the Bayesian 50% majority-rule consensus tree was presented (Fig. 1), the posterior probabilities (PP) and Bootstrap support (BS) values being superimposed on the nodes.

Morphological results
Stalked glandular and simple non-glandular trichomes were found on both surfaces of the lamina as well as the calyx of the new species (Fig. 2A-C).The abaxial surface of lamina and the outside surface of the calyx were more densely covered with longer trichomes.Pollen grains of Stachys yingzuijieensis were tricolpate with reticulate exine sculpturing (Fig. 2D-F), while nutlets were ovate with glabrous and reticulate surface (Fig. 2G-I).

Discussion
The backbone of Stachydeae in the present study and the 12 clades recovered in the Eurystachys clade (Fig. 1) were consistent with that of Salmaki et al. (2019).The Stachys clade, which was referred to as the "Stachys core clade" in Salmaki et al. (2013) and the Stachys s.s.clade in Lindqvist and Albert (2002), is one of the largest monophyletic groups in the Eurystachys clade and comprises five genera (Haplostachys, Phyllostegia, Stachys, Stenogyne and Suzukia) and over 100 species.No synapomorphy has been found for this clade due to large morphological and geographical diversity (Salmaki et al. 2019).Next-generation sequencing data and comprehensive morphological studies are needed to further clarify the synapomorphies and relationships within this taxonomically problematic and important group.
Only several representatives of Stachydeae from China had been included in previous molecular phylogenetic studies and no morphological study had been carried out for Chinese Stachys.In this study, nine species of Stachys from China were newly sequenced and included in the phylogenetic analyses.Our results showed that most species that were collected from or reported to be occurring in China were recovered within the Stachys clade, including the new species (Fig. 1).Stachys yingzuijieensis was further revealed to be sister to Stachys arrecta, a species distributed in the evergreen broad-leaved forests at altitudes of 1500-2000 m in central China.
Stachys yingzuijieensis differs from all other Chinese Stachydeae in its densely villosus and glandular pubescent plants, as well as the white corollas with tube included in the calyces (Figs 3, 4).For example, the corollas of Stachys arrecta are pink with purple spots and the corolla tubes are exerted from the calyces (Li and Hedge 1994).Except for above differences, Stachys yingzuijieensis also has oblong to oblong-lanceolate laminae with crenulate margin, whereas the laminae of Stachys arrecta are cordate with coarsely serrate margin.Moreover, they can be distinguished in the nutlet surface, which is smooth in the new species (Fig. 2), but verrucate in Stachys arrecta.More detailed differences between the two species are listed in Table 1.Here, we also provided a key to all species of Stachydeae from China below.Diagnosis.Stachys yingzuijieensis is most closely related to S. arrecta, but differs in its lamina oblong to elliptic-lanceolate (vs.cordate) with margin crenulate (vs.coarsely serrate), corolla white (vs.pink with purple spots) with tube included in calyx (vs.exerted from calyx) and nutlet surface smooth (vs.verrucate).
Phenology.Flowering from July to September, fruiting from August to October.Distribution and habitat.Currently, S. yingzuijieensis is only known from the Yingzhuijie National Nature Reserve and a total of 50 mature plants were found during our field investigation.The new species usually grows in shady and moist places in evergreen broad-leaved forests at an altitude of 300-800 m.
Etymology.The specific epithet is derived from the type locality of the new species, i.e. the Yingzuijie National Nature Reserve in western Hunan Province, China.

Figure 1 .
Figure 1.Bayesian 50% majority-rule consensus tree of Stachydeae based on combined nuclear (ITS, ETS and 5S-NTS) dataset.Support values ≥ 0.50 PP or 50% BS are displayed above the branches (an "*" indicates a support value = 1.00 PP or 100% BS and a "-" indicates a conflicting node in the BI and ML trees).Species marked in bold represent samples newly sequenced in the present study.Multiple accessions of the same species are numbered according to Appendix 1.

Figure 2 .
Figure 2. Trichome, pollen, and nutlet micromorphology of Stachys yingzuijieensis A trichomes on the adaxial surface of lamina B trichomes on the abaxial surface of lamina C trichomes on the outside surface of calyx D polar view of pollen E equatorial view of pollen F surface sculpturing of pollen G dorsal view of nutlet H ventral view of nutlet I surface sculpturing of nutlet.

Figure 3 .
Figure 3. Morphology of Stachys yingzuijieensis from the type locality A habitat B habit C-D inflorescence E adaxial view of lamina F abaxial view of lamina G roots and rhizomes H frontal view of corolla I lateral view of calyces J dissected corolla K nutlets (A-J photographed by Lei Wu K photographed by Ya-Ping Chen).