﻿Mosladadoensis (Lamiaceae), a new species from the southern islands of South Korea

﻿Abstract Mosladadoensis (Lamiaceae), a new species from the southern islands of South Korea, is described and illustrated. The new species is morphologically similar to M.chinensis, but is distinguished from the latter by having two types of hairs on its stems, wider leaf blades, longer corolla length, and ellipsoid nutlets with a narrowly U-shaped extended area of abscission scar. Mosladadoensis is also distinguished from the Chinese narrow endemic M.hangchouensis by having an included pistil to the corolla, smaller ellipsoid nutlets, and later flowering and fruiting season. Phylogenetic analyses, based on two nuclear ribosomal (ETS, ITS) and three chloroplast (rbcL, matK, trnL-F) DNA regions, confirmed that the new species was constructed as monophyletic, and that M.dadoensis and M.hangchouensis form a sister group with robust support. We hereby provide a detailed morphological description of M.dadoensis with its corresponding geographical distributions, and comparison tables of related taxa.


Introduction
Mosla (Benth.) Buch.-Ham. ex Maxim. is a genus within the sixth largest family, Lamiaceae (the mint family). Although Mosla is a small genus of approximately 20 species, it is the second largest genus in the tribe Elsholtzieae (Wu and Li 1977a, b;Li and Hedge 1994;Harley et al. 2004). Elsholtzieae contains eight genera and roughly 70 species and is the smallest tribe within the most species-rich subfamily Nepetoideae (105 genera and about 3,400 spp.; Zhao et al. 2021). Mosla is mainly distributed in China, Japan, and Korea; however, M. dianthera (Buch.-Hamilt. ex Roxb.) Maxim. occurs in eastern Russia, the western Himalayas, and some Southeast Asian countries (Wu and Li 1977b;Li and Hedge 1994;Zhou et al. 1997;Govaerts et al. 2022). Phylogenetically, Mosla is nested within the eastern Asian Mosla-Keiskea-Perilla clade, and the monophyly of Mosla is strongly supported by previous morphological and taxonomic studies (Zhou 1995;Zhou et al. 1997;Li et al. 2017). Four fertile stamens are common in Elsholtzieae, and subequal or anterior pairs are normally longer, except in Mosla, which is characterized by two posterior fertile stamens (Harley et al. 2004;Kim 2018).
In Korea, four species of Mosla are recognized, namely M. chinensis Maxim., M. dianthera, M. japonica (Benth. ex Oliv.) Maxim., and M. scabra (Thunb.) C.W.Wu & H.W.Li (Seo and Park 2018;Korea National Arboretum 2020). The main diagnostic characteristics for species identification are the different leaf and calyx shapes. Leaves with a linear to linear-lanceolate shape are found only in M. chinensis and M. japonica, characterized by a subequal 5-toothed calyx. Although M. scabra and M. dianthera both have a 2-labiate calyx, the apex of the calyx lobe differs and is acute in M. scabra and obtuse in M. dianthera (Kim 2018).
During general floristic study in the southern part of Korea during October 2021, we found an unusual species which is restricted to the southern islands. This species is readily distinguished from previously known Mosla species in Korea by a considerably longer corolla. M. chinensis could be the closest ally, but the leaf shapes and flower features are significantly different. After a thorough literature survey and investigation of the relevant specimens, we designate M. dadoensis K.K.Jeong, M.J.Nam & H.J.Choi as a new species of Mosla from the southern islands of Korea. To clarify the systematic status of M. dadoensis we also conducted barcoding analysis based on nuclear ribosomal (nr) and chloroplast (cp) DNA regions, and observed detailed nutlet morphology, which is well known as a systematically important characteristic in Lamiaceae (Moon et al. 2009;Ryding 2010;Jeon et al. 2020). A detailed morphological description of M. dadoensis and its geographical distribution is also provided.

Morphological characters
Morphological descriptions were based on specimens from the KB, KH (abbreviations are according to the Index Herbariorum [http://sweetgum.nybg.org/science/ih/]), and the herbarium of Changwon National University. Field surveys were also conducted from October 2021 to February 2022. Materials preserved in 70% ethanol were used for observation and measurement of floral parts. For quantitative characters, measurements were based on at least 50 samples.

Microscopic analysis
For morphological observations and size measurements, the nutlets were first examined using a stereomicroscope (SM; Olympus SZX16, Olympus, Tokyo, Japan). Nutlet sizes were measured using at least 30 randomly chosen individuals from each species. Prior to scanning electron microscopic observations, all the dried nutlets were rehydrated overnight using the wetting agent Agepon (Agfa-Gevaert, Leverkusen, Germany) and distilled water (1:200) at 37-40 °C. The rehydrated materials were dehydrated through an ethanol series (50%, 70%, 90%, 95%, and 100%) at room temperature for 1 h each. The completely dehydrated materials were immersed in liquid carbon dioxide (CO 2 ) for critical point-drying (CPD; SPI-13200J-AB, SPI Supplies, West Chester, PA, USA). For the micromorphological observations, selected nutlets were mounted on aluminum stubs using a doublesided adhesive conductive carbon disk (05073-BA, SPI Supplies, West Chester, PA, USA). Specimens were coated with gold using an ion-sputtering device (208HR, Cressington Scientific Instruments Ltd., Watford, UK), and then observed using a low-voltage field emission scanning electron microscope (FE-SEM; JSM-7600F, JEOL, Tokyo, Japan) at an accelerating voltage of 10 kV and a working distance of 8-10 mm (Song and Hong 2020).

Phylogenetic analysis
To confirm the systematic placement of the putative new species within the genus Mosla, molecular phylogenetic analyses were conducted. The combined cpDNA dataset (rbcL, matK, and trnL-trnF) and nrDNA dataset (ITS, ETS) used in Li et al. (2017) were employed with the addition of three individuals (H.J.Choi 210923-001_1-3) of the putative new species (Table 1). Keiskea japonica Miq. was selected as the outgroup since it is a member of Elsholtzieae placed within the sister clade to Mosla (Li et al. 2017). Details of voucher information and GenBank accession numbers of the species used in this study are provided in Table 2.
Total genomic DNA of M. dadoensis was extracted from silica gel-dried leaf materials using a DNeasy Plant Mini Kit (Qiagen Ltd., Crawley, West Sussex, UK). We conducted PCR with a ProFlex 96-Well PCR System (Applied Biosystems, Foster City, CA, USA). Each reaction mixture contained AccuPower PCR PreMix (Bioneer, Daejeon, South Korea), ca. 10 ng (1 μL) of genomic DNA, and 100 pM of primers in a total volume of 20 μL. Conditions included an initial denaturation at 95 °C for 5 min, followed by 40 amplification cycles comprising 95 °C for 30 sec, 50 °C for 30 sec, and 72 °C for 1 min, with a final extension at 72 °C for 5 min. After the PCR products were visualized on 2% agarose gels, they were treated with a MG PCR Purification kit (MGmed), and sequenced with the ABI 3730xl Analyzer, using the ABI BigDye Terminator v3.1 Cycle Sequencing Kits (Applied Biosystems, Foster City, CA, USA).

Taxonomic treatment
Phenology. Flowering and fruiting from August to November.

Distribution and habitat.
Endemic to southern coastal regions of Korea (Fig. 5). Open rocky area near the coast; at altitudes of 8-500 m.
Etymology. The specific epithet, "dadoensis", is based on the name of location, the Dadohae southern coastal region of Korea, where Mosla dadoensis was discovered.
Vernacular name. The Korean name of the new species is "Da-do-hae-san-deulkkae (다도해산들깨)".
Morphological assessment. Mosla dadoensis is morphologically similar to M. chinensis, from which it is clearly differentiated by the hairs on its stems [white recurved hairs and intermixed with white villous (Fig. 1B) vs. white recurved hairs only], shape and size of leaf blades [narrowly lanceolate to lance-ovate, 1-3 cm × 4-10 mm (Figs 1D, E) vs. linear to linear-lanceolate, 1-5 cm × 1.3-4 mm], length of corolla [8-9 mm (Figs 1G, H) vs. 5-6 mm], and shape of nutlets [ellipsoid with narrowly U-shaped extended area of abscission scar (Figs 1J, 3A, C, 4A, C) vs. globose to subglobose with widely U-shaped extended area of abscission scar (Figs 3B, D, 4B, D)]. Mosla dadoensis is also distinguished from M. chinensis by its distinctive tetragonal to hexagonal nutlet surface cells with straight and thin anticlinal walls (Table 3; Fig. 3). In addition, this new species is morphologically similar to Chinese narrow endemic M. hangchouensis Matsuda. However, it is easily distinguished by its length of corolla [8-9 mm and ca. twice as long as calyx (Fig. 1F) vs. ca. 10 mm and ca. three times longer than calyx], relative length of pistil to the corolla [included (Figs 1B, F, G) vs. clearly exserted], shape and size of nutlets [ellipsoid, 0.9-1.3 mm in diam. (Figs 1J, 3A, C) vs. globose to subglobose, ca. 2.1 mm in diam], and later flowering and fruiting season (August to November vs. June to September). The major characters of the new species are compared to those of the related M. chinensis and M. hangchouensis in Table 3.
Phylogenetic analysis. The combined dataset has 12 aligned sequences comprising 2,910 bp (609 bp for ITS, 371 bp for ETS, 439 bp for rbcL, 736 bp for matK, and 755 bp for trnL-F), of which 102 occupied variable positions (3.51%). Our phylogenetic tree (Fig. 6) revealed a similar topology to that obtained in the previous study (Li et al. 2017). Mosla species were constructed as monophyletic, and M. dadoensis was classified as a clade independent from other members of Mosla on the ML tree. M. dadoensis was distinguished from M. chinensis, a related species distributed in China and Korea. Instead, the tree is shown to form a clade closer to M. dadoensis in Korea and M. hangchouensis in China (Fig. 6).