Taxonomic Reconsideration of Chinese Lespedeza Maximowiczii (Fabaceae) Based on Morphological and Genetic Features, and Recommendation As the Independent Species L
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pISSN 1225-8318 − Korean J. Pl. Taxon. 48(3): 153 162 (2018) eISSN 2466-1546 https://doi.org/10.11110/kjpt.2018.48.3.153 Korean Journal of RESEARCH ARTICLE Plant Taxonomy Taxonomic reconsideration of Chinese Lespedeza maximowiczii (Fabaceae) based on morphological and genetic features, and recommendation as the independent species L. pseudomaximowiczii Dong-Pil JIN, Bo XU1 and Byoung-Hee CHOI* Department of Biological Sciences, Inha University, Incheon 22212, Korea 1The ECORES Lab, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China (Received 16 August 2018; Revised 15 September 2018; Accepted 20 September 2018) ABSTRACT: Lespedeza maximowiczii C. K. Schneid. (Fabaceae) is a deciduous shrub which is known to be dis- tributed in the temperate forests of China, Korea and on Tsushima Island of Japan. Due to severe morphological variations within species, numerous examinations have been conducted for Korean L. maximowiczii. However, the morphology of Chinese plants has not been studied as thoroughly, despite doubts about their taxonomy. To clarify this taxonomic issue, we investigated morphological characters and undertook a Bayesian clustering analysis with microsatellite markers. The morphological and genetic traits of Chinese individuals varied considerably from those of typical L. maximowiczii growing in Korea. For example, petals of the former had a different shape and bore long claws, while the calyx lobes were diverged above the middle and the upper surface of the leaflet was pubescent. Their terete buds and spirally arranged bud scales were distinct from those within the series/section Heterolespe- deza, which includes L. maximowiczii. Our Bayesian clustering analysis additionally included L. buergeri as an out- group. Those results indicated that the Chinese samples clustered into a lineage separated from L. maximowiczii (optimum cluster, K = 2), despite the fact that the latter is grouped into the same lineage with L. buergeri. There- fore, we treat those Chinese plants as a new species with the name L. pseudomaximowiczii. Keywords: Bayesian clustering analysis, Lespedeza maximowiczii, Lespedeza pseudomaximowiczii, microsat- ellite, morphology, new species Lespedeza maximowiczii C. K. Schneid. is a deciduous shrub synonym of section Macrolespedeza (Ohashi and Nemoto, in the family Fabaceae (Choi, 2007). This species is 2014). First described based on Faurie’s collection (Schneider, traditionally considered to belong to section (Nakai, 1939) or 1907), L. maximowiczii was sampled from Quen-san (= series (Akiyama, 1988) Heterolespedeza Nakai, which is Wonsan-si), Hamgyeongnam-do, Korea. The origins of its local characterized by flattened buds and distichously arranged bud name were reviewed by Chang et al. (2004). Now, this species scales (Nakai, 1939; Akiyama, 1988). In a recent revision of is known to be distributed in the temperate forests of Korea, the taxonomic system for Lespedeza Michx. (Ohashi and China, and Japan (Akiyama, 1988; Choi, 2007; Ohashi et al., Nemoto, 2014), Heterolespedeza was placed in section 2009). Plants grow abundantly on the mountains in all Korean Macrolespedeza Maxim., reflecting the results of molecular provinces (Choi, 2007), and are also found in Anhui, Henan phylogenetic studies (Han et al., 2010; Xu et al., 2012). and Zhejiang provinces of central China (Ohashi et al., 2009). Simultaneously, series Formosae S. Akiyama & H. Ohba In Japan, this species is restricted to the northern part of Isl. (including L. thunbergii (DC.) Nakai, L. davidii Franch., L. Tsushima (Akiyama, 1988), close to the Korean Peninsula. homoloba Nakai, and L. patens Nakai) was treated as a Therefore, the highest concentrations of plants are in China *Author for correspondence: [email protected] http://e-kjpt.org, © 2018 the Korean Society of Plant Taxonomists. This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 153 154 Dong-Pil JIN, Bo XU and Byoung-Hee CHOI and Korea, two regions separated by ca. 1170 km. morphological and genetic traits of typical L. maximowiczii The morphological characters of L. maximowiczii vary with those of Chinese samples currently identified as L. widely, with many intra taxa being described, such as var. maximowiczii and (2) clarify the taxonomic entity of plants tomentella, var. elongata, and f. alba (Nakai, 1927; Akiyama, growing in China. 1988). To clarify the circumscription on this species, those characters have been examined several times for Korean and Materials and Methods Japanese individuals (Lee, 1965; Hatusima, 1967; Akiyama, 1988), and the taxonomic rank of this species and intra taxa Morphological examination and survey of has also been changed by researchers. The Japanese individuals geographical distribution are regarded as typical forms even though some have smaller To compare the morphological characters of typical Lespedeza calyces (ca. 2 mm long) (Hatusima, 1967; Akiyama, 1988). maximowiczii with those of Chinese plants identified the same However, only a few morphological investigations have been way, we collected specimens from both countries and deposited performed with Chinese L. maximowiczii. Although Ohashi et them in the Herbarium of Inha University (IUI). Additional al. (2009) and Huang et al. (2010) revised Chinese morphological examination was conducted by observing Macrolespedeza, shrub forms of Lespedeza, they did not specimen from the following herbaria: the Korea National consider L. maximowiczii when observing those specimens. Arboretum (KH) and the Chinese Academy of Sciences, Beijing Flower characters are key to distinguishing among Lespedeza (PE). We also gathered more information throughout the Chinese species (Akiyama, 1988) but have not been described in minute Virtual Herbarium (CVH) (http://www.cvh.ac.cn/); and by detail. If plants of that species have been geographically looking at photograph and geographical distribution of Chinese isolated between China and Korea/Japan for a long time, it is specimens held at the Hangzhou Botanical Garden (HHBG); the possible that Chinese individuals show distinct morphological Institute of Botany, Jiangsu Province and Chinese Academy of traits and should be treated as intra taxa or a new species. In Sciences (NAS); and the Kunming Institution of Botany (KUN). the case of the related L. buergeri Miq., which belongs to the All observations of morphological characters were made with a same series/section Heterolespedeza, plants in the Korean stereomicroscope (Leica MZ8; Wetzlar, Germany), and criteria population differ from those of Chinese and Japanese for floral measurements were mainly those stipulated by populations because of their smaller bracteoles (Jin et al., Akiyama (1988). 2016b). Hence, morphological examination of Chinese individuals is required to confirm its taxonomic entity. Sampling, DNA extraction, and microsatellite Several phylogenetic studies on Lespedeza have utilized polymerase chain reaction molecular markers such as cpDNA and nrITS (Han et al., 2010; Genetic traits were studied using leaves sampled from typical Xu et al., 2012), and nrITS and nuclear gene PGK (Xu et al, forms (Korean, n = 87) and Chinese individuals (n = 38) which 2017). However, Chinese and Korean L. maximowiczii have were identified as L. maximowiczii (Table 1). Plants of L. never been sequenced in parallel. It is difficult to delimit maximowiczii also grow, albeit only in small numbers, on Isl. species of Macrolespedeza because the resolution of markers Tsushima (Akiyama, 1988). Because of that scarcity, we did is low due to frequent hybridization, reticulate evolution, and not include them in this molecular examination. As the rapid diversification (Han et al., 2010; Xu et al., 2012, 2017). outgroup, we analyzed one population each from China and The PGK markers show relatively high resolution for section Japan of the related species L. buergeri (n = 14 and n = 11, Junceae (Maxim.) H. Ohashi & T. Nemoto but not for respectively). Macrolespedeza. Therefore, other markers are needed. We Genomic DNA (3 μg) was extracted from silica-dried leaves employed microsatellite markers developed from this genus with an MG Plant Genomic DNA Extraction SV Miniprep Kit (Jin et al., 2016a). Because they are highly polymorphic and (MGmed, Seoul, Korea), according to the manufacturer’s distributed across multiple loci (Duminil et al., 2012), they can instructions. For Bayesian cluster analysis, we used the be used to reveal genetic diversity among populations following eight microsatellite loci (Jin et al., 2016a): LMS3, (Sunnucks, 2000) as well as to delimit closely related taxa in LMS18, LMS28, LMS39, LMS45, LMS47, LMS53, and various groups, such as those within the genera of Carapa LMS58. The polymerase chain reaction (PCR) protocol utilized Aubl., Phoenix L. and Quercus L. (Pintaud et al., 2010; those markers with a GeneAmp PCR System 2700 Thermal Duminil et al., 2012; Lee et al., 2014). Cycler (Applied Biosystems, Foster City, CA, USA). Each Here, our research aims were to (1) compare the reaction mixture (10 μL total volume) contained 5 ng of DNA, Korean Journal of Plant Taxonomy Vol. 48 No. 3 (2018) Taxonomic reconsideration of Chinese Lespedeza maximowiczii (Fabaceae) 155 Table 1. Sampling information for Lespedeza species used in this study. No.