Sedum formosanum subsp. miyakojimense (Crassulaceae), a new subspecies from Miyako-jima Island of the Ryukyu Islands, Japan

Abstract We re-examined the taxonomic status of plants treated as Sedum formosanum (Crassulaceae) from Miyako-jima Island of the Ryukyu Islands, Japan, using morphological comparison and molecular phylogenetic analyses with related species. In morphology, plants from Miyako-jima Island bore a close resemblance to the other plants of S. formosanum, but differed in being perennial, polycarpic, and having lateral axillary branches. Molecular analyses based on ITS of nrDNA and six regions of cpDNA sequencing indicated that the Miyako-jima plants formed a distinct subclade. This subclade was part of a polytomy with three other subclades comprising nine taxa endemic to Taiwan and S. formosanum from other areas, including the type locality. Therefore, we propose and describe the Miyako-jima plants as a new subspecies, Sedum formosanum subsp. miyakojimense.


Introduction
The genus Sedum L. (Crassulaceae) comprises about 470 succulent herbaceous species (Thiede and Eggli 2007). Species within this genus are widely distributed in the Northern Hemisphere, and are most diverse in the Mediterranean Sea, Central America, the Himalayas, and East Asia (Stephenson 1994;Thiede and Eggli 2007). A previous phylogenetic study indicated that Sedum is a polyphyletic group within seven American genera (Carrillo-Reyes et al. 2009). However, in East Asia, Sedum has been shown to be monophyletic (Mayuzumi and Ohba 2004;Carrillo-Reyes et al. 2009). The Flora of China (Fu and Ohba 2001) divides East Asian Sedum species into three sections (sects.); Sedum, Oreades (Fröderström) K.T. Fu, and Filipes (Fröderström) S.H. Fu. Section Sedum is distinguished from sects. Oreades and Filipes by adaxially gibbous carpels and follicles, and sect. Oreades is differentiated from sect. Filipes by the absence of spurred leaves at the base. Additionally, species of sect. Oreades generally have yellow or purple-red (rarely red) petals, whereas members of sect. Filipes have white or reddish purple (rarely yellow) petals (Fu and Ohba 2001). Seventeen species of Sedum are reported from Japan, including four subspecies and four varieties within sect. Sedum, and one species within sect. Filipes (Ohba 2001).  Table 2 for abbreviations for collection localities). Sedum formosanum N. E. Brown, described based on a type specimen collected from Taiwan (Brown 1885), occurs on rocky seashore slopes in the southern part of Kyushu in the Ryukyu Islands of Japan, in Taiwan, and on Batan Island in the Philippines (Hatusima 1975;Lin 1999;Ohba 2001;Hotta 2013;Shiuchi and Hotta 2015;Ryukyu Plant Research Group 2018). Sedum formosanum, a monocarpic biennial herb, is one of the few species of East Asian Sedum characterized by a trichotomous branching form (Ohba 2001). In Japan, populations of S. formosanum are scattered on the Ryukyu Islands (the Ryukyus), which comprise approximately 140 islands in a 1,300-km-long stretch between Kyushu and Taiwan (Fig. 1). Owing to its scarcity, this species is classified as 'Near Threatened' (NT) on the Red List of Threatened Species of Japan (Japanese Ministry of the Environment 2019). However, accurate identification of Sedum species can be hindered by high morphological similarity and plasticity. Therefore, there is a lack of clarity in the taxonomic identity of S. formosanum (Ito et al. 2017a). In fact, Sedum plants distributed on the Danjo Islands, Japan, which had historically been treated as S. formosanum, were recently described as a distinct taxon, S. danjoense Takuro Ito, H. Nakanishi & G. Kokub. (Ito et al. 2017a).
Based on previous field surveys, we noted that plants treated as S. formosanum on Miyako-jima of the Ryukyus differed morphologically from other populations. In this study, we conducted morphological comparisons and molecular phylogenetic analyses to elucidate the taxonomic status of plants treated as S. formosanum on Miyako-jima Island.

DNA Sample collection
The plants treated as S. formosanum are only known from one locality on Miyako-jima Island. We collected two individuals of the plants from the island for DNA samples. To clarify the phylogenetic position of S. formosanum growing on Miyako-jima Island, we utilized ITS (Internal Transcribed Spacer region of nuclear ribosomal DNA) sequences of 50 taxa (72 accessions) of Sedum in Asia including S. formosanum from 20 localities in Kyushu, the Ryukyus, Taiwan and the Philippines as ingroup reported by previous study (Mayuzumi and Ohba 2004;Ito et al. 2014Ito et al. , 2017aIto et al. , 2017b (Tables 1, 2). Additionally, we sequenced one species of the eastern Asian species, S. emarginatum (Table  1). Following previously reported phylogenetic study of Crassulaceae (Mayuzumi and Ohba 2004), Aeonium castello-paivae Bolle, A. gomerense Praeger, A. lancerottense Praeger, A. viscatum Bolle, and Greenovia aizoon Bolle, which were collected by Mort et al. (2002) and stored in GenBank were selected as outgroups (Table 1). In total, 80 operational taxonomic units (OTUs) were included in our molecular phylogenetic analysis based on ITS (Tables 1, 2). Subsequently, we conducted molecular phylogenetic analysis based on six cpDNA (Chloroplast DNA) regions with S. formosanum and its close relatives to clarify the detailed phylogenetic relationships. Following Ito et al. (2017b), nine Taiwanese taxa were selected as ingroup, S. alfredii Hance, and S. sekiteiense Yamam.  Mort et al. (2002). and S. tricarpum Makino were selected as outgroups (Table 3). In total, 27 OTUs were included in our molecular phylogenetic analysis based on cpDNA (Tables 2, 3). Taxonomic treatments tentatively followed Ohba (2001) and Ito et al. (2018) for Japanese taxa, Lin (1999) and Lu et al. (2019) for Taiwanese taxa, and Fu and Ohba (2001) for Chinese taxa. Voucher specimens for our collections were primarily deposited in the herbarium of the National Museum of Nature and Science, Japan (TNS).
DNA extraction, PCR amplification, and sequencing DNA was extracted from dried leaves using a DNeasy Plant Mini Kit (Qiagen, Valencia, CA), in accordance with the manufacturer's protocols. The ITS region containing the ITS1, 5.8S rDNA, and ITS2 and six regions of cpDNA (matK-trnK, ndhA, psbM-ycf6, rpS16, trnD-psbM and trnL-F) sequences were amplified by polymerase chain reaction (PCR) with an iCycler (Bio-Rad, Hercules, CA, USA). The ITS and six regions of cpDNA sequences were amplified using EmeraldAmp PCR Master Mix dye (Takara, Otsu, Japan) and the following forward and reverse primers, respectively: ITS, primers ITS1 and ITS4 (White et al. 1990); matK-trnK intron primers matKAF and trnK2R; ndhA intron, primers ndh×1 and ndh×2 (Shaw et al. 2007); psbM-ycf6 intron, primers psbMR and ycf6F; rpS16 intron, primers rpS16F and rp-S16R; trnD-psbM intron, primers psbMF and trnD (Shaw et al., 2005); and trnL-F, primers trnLc and trnFf (Taberlet et al. 1991) by an iCycler (Bio-Rad, Hercules, CA). The PCR profile consisted of an initial 3 min at 94°C followed by 35 cycles of 30 s at 94°C, 30 s at 50°C for the ITS sequence or 55°C for the cpDNA sequence, and 90 s at 72°C. The PCR product were purified by ExoStar clean-up kit (USB, Cleveland, OH). Cycle sequencing was performed using a BigDye Terminator Cycle Sequencing Kit ver. 3.1 (Applied Biosystems, Foster City, CA) and the PCR primers mentioned above for the ITS and cpDNA sequences. The Sanger sequencing products were then purified by ethanol precipitation. Automated sequencing was carried out with an Applied Biosystems 3130xl Genetic Analyzer. The electropherograms were assembled using ATGC ver. 6 (GENETYX, Tokyo, Japan). The sequence data obtained in this study were deposited in the DDBJ/EMBL/GenBank database (http://www.ncbi. nlm.nih.gov/gquery/).

Phylogenetic analysis using ITS and cpDNA sequences
The ITS and cpDNA sequences were aligned using ClustalW 1.8 (Thompson et al. 1994) and then adjusted manually. Phylogenetic analyses were conducted with a Bayesian approach using MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) and maximum-likelihood (ML) phylogenetic analysis using RAxML (Stamatakis 2014). In the Bayesian phylogenetic analysis, we used Akaike's Information Criterion (AIC) implemented in MrModeltest 2.2 (Nylander 2004) to obtain an appropriate evo- Table 2.
Plant materials of Sedum formosanum with their collection locality, voucher information, and accession numbers of ITS and cpDNA sequences. lutionary model of nucleotide substitutions. And then we performed two separate runs of Metropolis-coupled Markov chain Monte Carlo (MCMCMC) analysis, each with a random starting tree and four chains (one cold and three hot) based on the selected model. The MCMCMC length was one million generations, and the chain was sampled every one hundredth generation from the cold chain. The first 2,500 sample trees (25% of the total 10,000 sample trees) were discarded as burn-in after checking that the average standard deviation of split frequencies (ASDSF) reached a stationary state at < 0.01 thereafter. A 50% majority consensus tree of the output tree file from MrBayes was generated using FigTree ver. 1.3.1 (Rambaut 2009). The ML phylogenetic analyses were implemented in RAxML 8 (Stamatakis 2014) with a GTRGAMMA substitution model. The ML bootstrap proportions (BPs) and trees were obtained by simultaneously running rapid bootstrapping with 1,000 iterations followed by a search for the most likely tree.

Intraspecific morphological comparison
The plants known as S. formosanum from Miyako-jima Island (T. Ito 1115Ito , 1120Ito , 2402 and 2408, TNS) were used for morphological comparisons. Herbarium specimens of S. formosanum deposited in the Kagoshima University Museum (KAG), the University of the Ryukyus (RYU), the National Museum of Nature and Science (TNS), the National Taiwan University (TAI) and the Taiwan Forestry Research Institute (TAIF) were examined. By field survey, the phenotypic plasticity of leaf shape in response to environmental changes was observed. Therefore, we also have cultivated the plants from Miyako-jima Island and from Taiwan, where the type locality of the species is, in Tsukuba Botanical Garden to compare their leaf shape and life cycle during 2015-2017.

Phylogenetic analyses using ITS and cpDNA
We used 80 operational taxonomic units (OTUs), including 75 as ingroup accessions and 5 as outgroup accessions in the Bayesian and ML analyses based on ITS sequences (Tables 1, 2). Following alignment, we obtained a matrix of 629 base pairs (bp) and selected GTR+I+G for the Bayesian analysis. The 50% majority rule consensus tree of all post burn-in trees is shown with Bayesian posterior probabilities (PPs) in Fig. 2A.
The topology of the ML tree was highly compatible with that of the Bayesian tree ( Fig.  2A). In both the Bayesian and ML analyses based on ITS sequences, S. formosanum and nine taxa endemic to Taiwan formed a well-supported clade (PP/BS = 1.00/93 We used 29 OTUs, including 26 accessions as ingroups and 3 as outgroups in the Bayesian and ML analyses based on combined six regions of cpDNA sequence (Tables  2, 3). Following alignment, we obtained a matrix of 5,115 bp. In the resulting Bayesian and ML phylogenetic trees, we observed a topology similar to the trees formed using ITS data. We again observed strong evidence that S. formosanum and nine taxa endemic to Taiwan formed a well-supported clade with four subclades (1.00/100; Fig. 2B). However, these four subclades formed a polytomy that differed from that suggested by the ITS tree. Although S. formosanum from Miyako-jima Island was again supported as forming a subclade (1.00/100, Clade Bll), we found that the nine Taiwanese endemics were divided into two subclades (1.00/93, Clade All-l; 0.95/61, Clade All-ll), and S. formosanum on Izena Island and Iheya Island formed a subclade with the 18 accessions from Japan (excluding Miyako-jima Island), Taiwan and the Philippines (1.00/99, Clade Cll).

Morphological comparison
We observed a similar flower morphology among the herbarium specimens from Miyako-jima Island (TNS; T. Ito 1115Ito , 1120Ito , 2402Ito , and 2408 and those from other regions in Japan, Taiwan, and the Philippines. Generally, S. formosanum displays trichotomous branching at the shoot tip and does not produce lateral branches. The Miyako-jima plants also displayed trichotomous branching at the shoot tips, but they often developed lateral branches in the leaf axils of long shoots. Additionally, we found similar plants of S. formosanum that also produce axillary lateral branches on Ishigaki Island, part of the Yaeyama Islands, on Gaja-jima Island and Akuseki-jima Island in the Tokara Islands, and on Yoron Island in the Amami Islands by specimen survey. In terms of leaf morphology, we observed high variation and no clear difference between the Miyako-jima plants and those from other locations. To remove the potentially confounding influence of environmental factors on leaf morphology, we cultivated plants from both Miyako-jima Island and Taiwan (obtained from the type locality) and compared them. Using this approach, we detected slight differences in leaf shape. Plants from Miyako-jima Island had spatulate to oblanceolate leaves, whereas plants from Taiwan had leaves that were spatulate to widely obovate. Most notably, plants from Miyako-jima Island were perennial and polycarpic, whereas plants from Taiwan were biennial and monocarpic.

Intraspecific taxonomy of S. formosanum
The molecular phylogenetic analyses based on both ITS and cpDNA indicated that the Sedum species from Miyako-jima Island, which are currently considered as S. formosanum, formed a well-supported clade. This clade was distinct from that of S. formosanum collected from other regions of Japan, Taiwan (including the type locality), and the Philippines (Fig. 2). Morphologically, plants from Miyako-jima Island were distinguishable from plants from other areas due to the presence of axillary lateral branches and by life cycle, i.e., perennial and polycarpic versus biennial and monocarpic (Figs 3, 4). Leaf shape differed slightly between the Miyako-jima plants and those from other locations, i.e., spatulate to oblanceolate versus spatulate to widely obovate (Figs 3, 4). Therefore, we concluded that S. formosanum from Miyako-jima Island should be considered a distinct taxonomic entity and have thus described a new subspecies in this study.
Description. Perennial herb, fleshy, glabrous. First year stem stout, erect, partly woody, 1-5 lateral branches in the leaf axils, 3-10 cm tall, with lax rosettes; rosettes 2.5-6 cm wide with 7-15 leaves. Flowering stems fleshy, 10-20 cm tall, base ca. 5 mm broad, yellowish green, erect or sprawling and creeping at base. Roots fibrous, sometimes adventitious at the leaf scar. Leaves alternate, occasionally verticillate, sessile, green or yellowish, flattish, ± thick, spatulate to oblanceolate, 1.1-3.1 cm long, 0.3-1.0 cm wide, apex rounded, base long, attenuate, margins entire. Inflorescences terminal, cymes, basically trifurcate with 3 primary axes, sometimes with 2, 4, or 5 primary axes; primary axis The Ryukyu Islands, including Miyako-jima Island, experienced extensive land configuration changes throughout the Neogene and the Quaternary as a result of tectonic movements and sea level fluctuations induced by climatic oscillations (Kimura 2002;Osozawa et al. 2011;Furukawa and Fujitani 2014). Miyako-jima Island was likely originally located at the eastern margin of the continent, based on evidence of deposits derived from the continent during the late Miocene to Pliocene (Osozawa et al. 2011). The highest point on Miyako-jima Island is only 100 m above sea level; therefore, the entire island was likely submerged in the past under higher sea levels. Furthermore, the mud-dominant Shimajiri Group is mostly overlaid by the Ryukyu Group, which is composed of Pleistocene reef-complex deposits (Shokita et al. 2006). Although some endemic freshwater and terrestrial organisms, such as the Miyako toad (Bufo gargarizans miyakonis Okada) and the potamid crab (Geothelphusa miyakoensis Shokita, Naruse & Fujii) are reported from Miyako-jima Island (Shokita et al. 2006). Oshiro and Nohara (2000) suggested that the island likely reconnected to the Yaeyama Islands, located in the southern Ryukyus, during the last glacial period. However, these endemic species and their close relatives are not distributed in the Yaeyama Islands, and it is highly unlikely that they experienced long-range dispersal. Therefore, if these islands were connected during the last glacial period, it is unlikely that migration occurred from the Yaeyama Islands via a land bridge. Interestingly, the Shimajiri Group is partly exposed to the surface on the eastern portion of Miyako-jima Island (Shokita et al. 2006). This suggests that some areas of the island may have remained above water during sea level fluctuations, and freshwater species such as G. miyakoensis, freshwater red alga (Thorea gaudichaudii C. Agardh), and oriental weatherfish (Misgurnus anguillicaudatus Cantor) are only distributed in this area (Shokita et al. , 2006. Collectively, this suggests that some organisms may have survived in isolation as relict populations, and further implies that the island may not have been entirely submerged in the past or, potentially, the existence of an ancient landmass adjacent to the island after its division from the continent (Shokita et al. 2006;Furukawa and Fujitani 2014). Previous molecular dating of East Asian Sedum species reported that S. formosanum diverged from the endemic Taiwanese species during the Pleistocene 1.41 Ma (0.79-2.25 Ma) (Ito et al. 2017b). Thus, it is reasonable to assume that S. formosanum subsp. miyakojimense may have diverged during the Pleistocene and has long since been genetically isolated from other species. Furthermore, S. formosanum subsp. miyakojimense is distributed in a restricted area on the eastern part of the island, in a similar location as the aforementioned endemic freshwater organisms. The discovery of a new endemic plant taxon, S. formosanum subsp. miyakojimense, on Miyako-jima Island is biogeographically important because it may imply that portions of the island remained above water over long time periods.