New taxa of Rhododendron tschonoskii alliance (Ericaceae) from East Asia

Abstract Three new taxa, Rhododendron sohayakiense Y.Watan. & T.Yukawa (Ericaceae), and its two varieties, var. kiusianum Y.Watan., T.Yukawa & T.Minamitani and var. koreanum Y.Watan. & T.Yukawa are described and illustrated from Japan and South Korea. They can be distinguished from each other and from the other members of the R. tschonoskii alliance, i.e. R. tschonoskii, R. tetramerum, R. trinerve and R. tsusiophyllum, through their combination of leaf size, leaf morphologies including lateral nerves on abaxial leaf surface, corolla morphologies including number of corolla lobes, style length and anther form. Phylogenetic inferences based on chloroplast DNA and genome-wide sequences revealed that each of the three new taxa is monophyletic and they further form a clade. Distributions of the three taxa are also clearly separated from each other and also from the other members of the R. tschonoskii alliance.


Introduction
The genus Rhododendron L. (Ericaceae) is morphologically diverse, comprising about 1,000 woody species (Chamberlain et al. 1996). The genus is mostly distributed across the Northern Hemisphere and members of subgenus Vireya extend into the South-ern Hemisphere via the Indo-Australian Archipelago. The subgenus Tsutsusi is mostly found in East Asia (Yamazaki 1996;Kron and Powell 2009). Although most species of this subgenus occur in warm-temperate to subtropical regions, some are present in cold and alpine regions.
Rhododendron tschonoskii Maxim., R. tetramerum (Makino) Nakai, R. trinerve Franch. ex Boisser and R. tsusiophyllum Sugim. are closely related species placed within the subgenus Tsutsusi, which grow on exposed rocks or open sites in slopes and ridges on mountains. Among them, R. tsusiophyllum was originally described as a species of an independent genus Tsusiophyllum tanakae Maxim. (1870), because anthers of this species open through longitudinal slits while anthers of all other species in Rhododendron open through apical pores. The first taxonomic reappraisal of this group except R. tsusiophyllum was conducted by Takahashi (1975) in which he recognized the aforementioned four taxa at species rank. Subsequently, Yamazaki (1996) treated these two taxa as varieties of R. tschonoskii, i.e. R. tschonoskii var. tetramerum (Makino) Komatsu and R. tschonoskii var. trinerve (Franch. ex Boisser) Makino. In this study, we tentatively adopt Takahashi's (1975) concept.
Rhododendron tschonoskii sensu Takahashi (1975) is widely distributed across the Japanese Archipelago and extends to the southern part of the Korean Peninsula. Despite the wide distribution, the species is absent from the central part of the Japanese Archipelago. As pointed out by Takahashi (1975) and Minamitani (1993), morphological characters of the species are distinct between eastern and western parts of the Japanese Archipelago. To evaluate these previous observations, we investigate morphological and macromolecular characters of R. tschonoskii and its related species by using the samples covering the entire species ranges.

Methods
The morphological characters were observed and measured based on living materials in the field and herbarium specimens listed in the sections "Additional specimens examined".
Samples for DNA analyses were collected from three individuals for Rhododendron tschonoskii, R. tetramerum, R. trinerve, R. tsusiophyllum and new entities, respectively. Three samples for each species and two entities and two samples for one entity (see Results and Discussion) were selected for covering entire range, and a holotype for each entity was included (Table 1). In addition, one individual for each species was collected from other relatives, i.e. R. dilatatum, R. kaempferi, R. macrosepalum, R. reticulatum, R. serpyllifolium and R. tashiroi belonging subgenus Tsutsusi (Kron and Powell 2009). For phylogenetic analysis, genomic DNA was extracted from silica-dried leaf samples using a DNeasy Plant mini kit (Qiagen, Hilden, Germany) after treatment with sorbitol buffer (Wagner et al. 1987). Five non-coding regions of chloroplast DNA (trnL-F, trnL intron, trnS-G, trnG intron and rpl32-trnL) were amplified and sequenced following the protocols described in Yoichi et al. (2017). The sequences were assembled Haplotype, Haplotype codes detected by chloroplast DNA sequences, corresponding to those in Fig. 3.
Specimens of all of the analysed samples were deposited in TNS.
In addition, genome-wide SNPs were identified from two double digest restriction-site associated DNA (ddRAD) libraries using Peterson et al. (2012) protocol with some modifications. To fragment DNA sequences, 10 ng of genomic DNA was digested with EcoRI and BglII. Digestion and ligation were performed at 37 °C for 16 h in a 10 μL volume containing 20-40 ng of genomic DNA, 0.5 μL of each 10U/μL EcoRI and BglII enzyme (Takara, Kyoto, Japan), 1 μL of ×10 NEB buffer 2, 0.1 μL of ×100 BSA (New England Biolabs, Ipswich, USA), 0.4 μL of each 5 μM EcoRI and BglII adapter, 0.1 μL of 100 mM ATP and 0.5 μL of T4 DNA ligase (Enzymatics, Beverly, USA). The ligated product was purified with AMPure XP (Beckman Coulter, Brea, USA). The purified adaptor-ligated DNA was subsequently amplified by PCR. The PCR was performed in a 10 μL volume containing 2 μL of adaptor-ligated DNA, 2 μL of 5 μM index primer including 6-mer variable sequences for identifying different samples, 1 μL of 10 μM TruSeq universal primer, 5 μL of ×2 KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington, USA). The PCR was performed with an initial denaturation for 4 mins at 94 °C, followed by 20 cycles of 10 s at 98 °C, 15 s at 65 °C and 15 s at 68 °C. The PCR products from different samples were pooled and purified again with AMPure XP. Fragments of 350-400 bp in the purified DNA solution were retrieved by electrophoresis using a 2.0% of E-Gel SizeSelect (Life Technologies, Carlsbad, USA). After quantity assessment using a QuantiFluor dsDNA System (Promega, Madison, USA) and quality assessment using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA), the libraries were sequenced with 51-bp single-end reads in two lanes of an Illumina HiSeq2000 (Illumina, San Diego, USA). After removing reads containing low-quality bases and adaptor sequences from the raw data using Trimmomatic v. 0.33 (Bolger et al. 2014), sequences with polymorphic SNPs were assembled by pyRAD v. 3.0 (Eaton 2014). Parameters for the assembly were set as follows: the minimum depth coverage for creating a cluster from reads was set to 6, the similarity threshold of clusters within and across individuals was set to 0.85, the maximum number of samples with shared heterozygous sites in a locus for filtering potential paralogs was set to 3, and polymorphic loci sequenced in more than half of the samples were finally exported as consensus sequences (Eaton 2014).
The phylogenetic relationships were inferred from two data sets, which were obtained from chloroplast DNA sequences and RAD-seq, based on the maximum likelihood method using RAxML v. 8.2.0 (Stamatakis 2014). In the analyses, the GTR-GAMMA model was used as a substitution model, and node supports were assessed by bootstrap analysis with 1000 replicates. Phylogenetic relationships among individuals for Rhododendron tschonoskii, R. tetramerum, R. trinerve, R. tsusiophyllum and new entities based on RAD-seq were further evaluated by constructing a neighbor-net based on p-distance using SplitTree4 (Huson and Bryant 2006).

Morphological differences
We found three new entities (Types 1, 2 and 3) that have previously been included within Rhododendron tschonoskii (Fig. 2). However, corolla lobes for the three types, R. trinerve and R. tetramerum are tetramerous; contrastingly R. tschonoskii and R. tsusiophyllum are pentamerous. It is noteworthy to mention that the number of corolla lobes is sometimes variable within individuals. Further, they are distinguished from each other and from the other members of the R. tschonoskii alliance, i.e. R. tschonoskii, R. tetramerum, R. trinerve and R. tsusiophyllum through their leaf size, leaf morphologies including lateral nerves on abaxial leaf surface, corolla morphologies and style length ( Table 2, Figs 1, 2). Leaf sizes of Types 1, 2 and R. tschonoskii are medium (5-30 mm long), while Type 3 and R. trinerve are large (10-50 mm long). Lateral nerves on the abaxial surface of the leaf are pinnate but obscure for Type 1, while 1-3 pairs of pin- nate lateral nerves are raised for Types 2 and 3. Corolla tube lengths of Types 1, 3, R. tschonoskii and R. trinerve are short (2-4 mm long), while Type 2, R. tetramerum and R. tsusiophyllum are long (3.5-7 mm long). Styles of Types 1, 3, R. tschonoskii and R. trinerve are longer than the corolla tube and exserted from the corolla, while those of Type 2, R. tetramerum and R. tsusiophyllum are similar or shorter than the corolla tube and included within the corolla.
The three types share the following combination of characters as commonly derived character states, which can be distinguished from the other members of the R. tschonoskii alliance. The corolla form of the three types are tubular-funnelform and corolla lobes are tetramerous; in addition, lateral nerves on abaxial leaf surface are raised or obscure raised, and not prominent, such as R. trinerve.

Phylogenetic relationships
Phylogenetic relationships of the R. tschonoskii alliance based on chloroplast DNA sequences (2,847 bp with 76 polymorphic sites) and genome-wide sequences (RAD-seq, 316,455 bp with 37266 SNPs) were almost concordant including outgroup species (Fig. 3). The R. tschonoskii alliance formed a monophyletic group (95% for chloroplast DNA and 100% for RAD-seq). The chloroplast DNA sequences identified four haplotypes from the three types, Types 1 and 3 had one haplotype respectively and Type 2 had two haplotypes. Although the monophyly of the clade comprising Types 1, 2 and 3 was supported with high bootstrap probability (80%), the monophyly of two haplotypes in Type 2 was not supported. The monophyly of the clade comprising Types 1, 2 and 3 and the monophyly of each type were supported with the highest bootstrap probabilities (100%) based on RAD-seq. The neighbor-net for a data set, which in-   cluded only the R. tschonoskii alliance, identified three groups corresponding to Types 1, 2 and 3, which can be clearly distinguished from each other (Fig. 4). Thus the three types can be distinguished from each other and also from the other members of the R. tschonoskii alliance. The results indicate that the three types should be treated as different taxa but these are more closely related than the others in the R. tschonoskii alliance. Since we confirmed the independent state of the three types from morphological and phylogenetic characteristics, we hereby describe them as a new species and its two varieties.    Description. Much branched semi-evergreen shrubs 1-1.5 m tall. Branchlets and petioles with dense appressed flattened brownish strigose hairs. Spring leaves scattered or crowded on upper branchlets; petioles 0.5-1 mm long; blade thick chartaceous, oblong, 10-20 mm long (at maximum within each individual), 4-7 mm wide, apex acute and terminating in a gland, base acute, adaxial surface green, abaxial surface pale green, densely strigose on adaxial surface, glabrous or sparsely strigose on abaxial surface without midrib; midrib prominent abaxially; lateral nerves pinnate, 2-3 paired, obscure raised abaxially. Summer leaves oblanceolate, 5-10 mm long, 1-6 mm wide, densely strigose on both surfaces. Flower buds terminal, single, broadly ovoid, acute, ca. 2 mm long, 2 mm wide; scales widely ovate, densely strigose on upper outer surface. Inflorescences umbel-like, 2-4 flowers. Pedicel 2-4 mm long at flowering, densely appressed hirsute. Calyx saucer-shaped, ca. 1.5 mm in diam., densely strigose, shallowly 4-lobed; lobes semiorbiculate, ca. 0.5 mm long. Corolla white, no blotches, openly tubularfunnelform, 8-12 mm long and wide, dissected 1/2 corolla length into 4 lobes; tube 2-3 mm long, ca. 2 mm wide, glabrous outside, pilose on upper inside; lobes elliptic to oblong, rounded, 3-5 mm long, 2-4 mm wide. Stamens 4, subequal, 5-8 mm long, exserted; filaments densely pilose on lower three-quarters; anthers yellow, oblong, ca. 1 mm long. Ovary ovoid, densely soft strigose, ca. 1.5 mm. Style 4-10 mm long, exserted, glabrous. Capsule ovoid, 2-5 mm long, 2-3 mm wide, densely strigose.

Rhododendron sohayakiense
Distribution. JAPAN: Honshu (Kii Peninsula), Shikoku. Ecology. The plants inhabit sunny places and grow on mountain ridges and slopes at altitudes over 1000 m above sea level. In such places, there are few trees and established communities of shrubs and dwarf bamboos (Sasa sp.). Flowering specimens have been collected from July to August; fruiting specimens have been collected from October to November. Bumblebees are frequent visitors to the flowers, suggesting that they are pollinators of the species.
Ecology. The plants inhabit sunny and rocky mountain ridges and slopes at altitudes over 1000 m above sea level. Flowering specimens have been collected from July to August; fruiting specimens have been collected from October to November.
Etymology. The specific epithet refers to 'Kyushu' where the new variety is distributed. Note. Although the style of this variety is included within the corolla, this part is exserted from the corolla in individuals from Mt. Ichifusa. Minamitani, but is distinguished by its large leaf size. Further, it differs from the former through its raised lateral nerves on the abaxial leaf surface and from the latter through its shorter corolla tube and by its longer style exserted from the corolla.
Distribution. SOUTH KOREA: Jeollabuk-do, Gyeongsangnam-do. Ecology. The plants inhabit mountain ridges and slopes at altitudes over 800 m above sea level. Flowering specimens have been collected from June to August; fruiting specimens have been collected from October. Honeybees are frequent visitors to the flowers, suggesting that they are pollinators of the variety.
Etymology. The specific epithet refers to 'Korea' where the new variety is distributed.