Phylogenetic Position of the Chinese Endemic Genus Heterolamium: a Close Relative of Subtribe Nepetinae (Lamiaceae)
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J. Jpn. Bot. 92(1): 12–19 (2017) Phylogenetic Position of the Chinese Endemic Genus Heterolamium: A Close Relative of Subtribe Nepetinae (Lamiaceae) a,b,d b,d c b Jing-Chao LI , Jian-Wen ZHANG , Dai-Gui ZHANG , Tao DENG , b b, a, Sergey VOLIS , Hang SUN * and Zhi-Min LI ** aSchool of Life Science, Yunnan Normal University, Kunming, Yunnan, 650500 CHINA; bKey Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan, 650201 CHINA; cKey Laboratory of Plant Resources Conservation and Utilization, Jishou University, Jishou, Hunan, 416000 CHINA; dThese authors contributed equally to the work *Corresponding author: [email protected] **Corresponding author: [email protected] (Accepted on September 1, 2016) The phylogenetic position of Heterolamium, an endemic genus from China, is still a controversy. Based on its distinct morphological features, it was previously regarded as the only genus of tribe Heterolamieae, sister to the tribes Stachydeae and Saturejeae of subfamily Lamioideae (Lamiaceae). However, recent palynological studies identified this genus as sister to the rest of subtribe Nepetinae (Lamiaceae). To resolve the dispute, we used comprehensive sampling and DNA sequence data from both the nuclear (ITS and ETS) and the plastid (rbcL, rps16, trnL–F, and rpl32–trnL) genomes to reconstruct a molecular phylogeny of Heterolamium and identify the generic relationship with subtribe Nepetinae. Maximum parsimony and Bayesian inference were used to analyze sequence data. Our results strongly support the conclusion that Heterolamium is a member of Nepetinae. There are two clades inferred in subtribe Nepetinae: Clade I including Heterolamium, Hymenocrater, Marmoritis, Nepeta, Drepanocaryum and Lophanthus; Clade II including Meehania, Lallemantia, Dracocephalum, Glechoma, Agastache, and Hyssopus. Furthermore, some typical Heterolamium morphological traits, such as 15-nerved calyx, long-exserted stamens and so on, are similar with species in subtribe Nepetinae, which provide powerful support for our conclusion as well. Key words: Heterolamium, Lamiaceae, molecular phylogeny, Nepetinae. Heterolamium C. Y. Wu is endemic to Heterolamium plants are slender perennial China and restricted to central and southwestern herbs. The leaves are cordate and toothed with China, growing in somewhat open places in long petioles; inflorescence 2–6-flowered, forests, hillsides and streamsides (1500–2700 pedunculate, in narrow, secund, terminal m a.s.l.) (Li and Hedge 1994). It is a member panicles; calyx 15-nerved, mid-lobe of posterior of the mint family (Lamiaceae or Labiatae) calyx-lip broad and slightly decurrent at base, with a distribution in Sichuan, Hunan, Shanxi, posterior corolla-lip with 4 short, rounded lobes, Hubei and Yunnan (Wu and Chow 1965). stamens long-exserted (Harley et al. 2004, Li and —12— February 2017 The Journal of Japanese Botany Vol. 92 No. 1 13 Hedge 1994). The shape of leaf-teeth and color has not been a subject of molecular systematic of flowers are remarkably diverse and are used studies. According to the most recent as major taxonomic characters to distinguish classification of Lamiaceae (Harley et al. lower taxa within the genus. Although the 2004), Heterolamium belongs to the subfamily traditional view recognizes a single species H. Nepetoideae tribe Mentheae. However, the debile (Hemsl.) C. Y. Wu with two varieties, H. results of palynology (Moon et al. 2008a, debile var. tochauense (Kudô) C. Y. Wu and H. 2008b) strongly support placing Heterolamium debile var. cardiophyllum (Hemsl.) C. Y. Wu within subtribe Nepetinae. Genetic analysis of (Harley et al. 2004, Li and Hedge 1994) in the a single individual identified as Heterolamium genus, some authors treat the latter variety as debile (Zhiduan 960093), was placed within another species belonging to the same genus (H. Meehania with high support (Drew and Sytsma flaviflorum (Z. Y. Zhu) L. Wei) (Li and Shi 2007) 2011). However, careful examination of the or even as the monotypic genus Changruicaoia gross morphology of the accession, along Z. Y. Zhu (C. flaviflora Z. Y. Zhu) (Li 2002, Zhu with examination of the herbarium record, 2001). Comparing with the typical variety, var. revealed misidentification of that individual, cardiophyllum has less acute and slender leaf- “Zhiduan, 960093” (Deng et al. 2015). Thus, teeth, leaf-blade purple beneath and flowers deep the phylogenetic position of Heterolamium red to purple and blue, and var. tochauense has should be reconsidered preferably with multiple wide-cuneate and spikier leaf base, sparser leaf- representatives of this taxon and related taxa. teeth, and flowers uniformly red (Wu and Chow Our study employed comprehensive 1965). sampling and DNA sequence data from both Heterolamium debile was first described and the nuclear (ITS and ETS) and the plastid assigned to Orthosiphon Benth., as O. debile genomes (rbcL, rps16, trnL–F, and rpl32– Hemsl. (Forbes and Hemsley 1890) and then trnL) to reconstruct the molecular phylogeny of recognized as a new genus Heterolamium by Heterolamium and its phylogenetic relationships Wu and Chow (1965), based on its distinct with members of Nepetinae. anther and corolla characters, subsequently adopted in all following taxonomic revisions Materials and Methods (e.g., Li and Hedge 1994, Harley et al. 2004). Taxon sampling However, the phylogenetic position and status Based on a preliminary analysis 10 of Heterolamium remains uncertain (Harley et accessions representing three varieties of al. 2004). Wu and Chow (1965) recognized this Heterolamium debile and 12 accessions from genus as an independent tribe Heterolamieae that Nepetinae, consisiting of the putative closest is transitional between the tribes Lamieae and outgroups to Heterolamium, were used. In Saturejeae of the subfamily Lamioideae. Some addition, sequences of Agastache rugosa authors consider the genus to have the ancestral (Fisch. & C. A. Mey.) Kuntze, Dracocephalum position in the subfamily Plectranthoideae (Wu parviflorum Nutt. and Lallemantia canescens et al. 2007). Fisch. & C. A. Mey. from GenBank were Despite significant progress in molecular included in the analysis. Cedronella canariensis phylogeny at the familial, subfamilial, tribal (L.) Webb & Berth and Lycopus uniflorus and generic levels of Lamiaceae (Agostini et al. Michx. of the tribe Mentheae were used as 2012, Conn et al. 2009, Drew and Sytsma 2011, outgroups. Voucher information and GenBank Drew and Sytsma 2012, Drew and Sytsma 2013, accession numbers for all specimens used in this Lindqvist et al. 2010, Ryding 2007, Ryding et study are listed in Table 1. al. 2011, Bendiksby et al. 2011), Heterolamium 14 Table 1. Samples, vouchers, DNA numbers and GenBank accession DNA GenBank accession Taxa Voucher no. ITS ETS rbcL rps16 trnL–F rpl32–trnL HM590067/ Agastache rugosa – – JQ669076 JQ669145 – JQ669022 JQ669275 FJ513154 Cedronella canariensis – – JQ669079 JQ669148 HM849871 – JF301360 JQ669281 Dracocephalum bullatum – – JQ669096 JQ669167 – – JF301366 JQ669303 Dracocephalum parviflorum – – JQ669097 JQ669168 – – JQ669038 JQ669304 Drepanocaryum sewerzowii – – DQ667328 JQ669169 – – DQ667517 JQ669305 植物研究雑誌 第 Glechoma longitubae Deng 415 (KUN) dt157 KM886722 KM886687 KM886784 KM886654 KX289297 KM886817 Heterolamium debile var. tochauense zdg 7112 (KUN) dt558 KX289269 KX298129 KX289277 KX289293 KX289304 KX289285 Heterolamium debile var. tochauense Zhangqinglong 64128 (KUN) dt261 – KX298126 – – KX289300 – Heterolamium debile var. tochauense Chuangjingda 2551 (KUN) dt262 KX289266 – – – KX289301 – Heterolamium debile var. cardiophyllum SNJ Exped. 20110718079 (KUN) dt158 KX289264 KX298124 KX289273 KX289289 KX289298 KX289281 Heterolamium debile var. cardiophyllum Deng 2440 (KUN) dt710 KX289272 KX298132 KX289280 KX289296 KX289307 KX289288 92 Heterolamium debile var. cardiophyllum Liuende 2181 (KUN) dt264 KX289267 KX298127 KX289275 KX289291 KX289302 KX289283 巻 第 Heterolamium debile var. cardiophyllum Zhang & Shen 50989 (KUN) dt265 KX289268 KX298128 KX289276 KX289292 KX289303 KX289284 Heterolamium debile var. cardiophyllum YIF-0398 (KUN) dt648 KX289271 KX298131 KX289279 KX289295 KX289306 KX289287 1 Heterolamium debile var. debile zdg 6261 (KUN) dt559 KX289270 KX298130 KX289278 KX289294 KX289305 KX289286 号 Heterolamium debile var. debile SNJ Exped. 20110923004 (KUN) dt255 KX289265 KX298125 KX289274 KX289290 KX289299 KX289282 Hymenocrater bituminosus – – JQ669105 JQ669179 – – JQ669045 JQ669316 Hyssopus officinalis – – JQ669106 JQ669180 Z37395 – JF301371 JQ669318 Lallemantia canescens – – JQ669108 JQ669182 – – JF301373 JQ669321 Lophanthus lipskyanus – – JQ669109 JQ669183 – – JF301384 JQ669328 Lycopus uniflorus – – DQ667302 JQ669185 – – DQ667488 JQ669330 Marmoritis complanata Deng 2359 (KUN) – KM886731 – KM886793 KM886663 KM886627 KM886829 Meehania cordata dt 101 (KUN) – KM886732 – KM886794 KM886664 KM886628 KM886830 Nepeta cataria – – JQ669126 JQ669202 Z37421 – JF301391 JQ669349 2017 年 2 月 February 2017 The Journal of Japanese Botany Vol. 92 No. 1 15 DNA isolation, amplification and sequencing inference. The MP analyses were conducted Total genomic DNA was isolated from using PAUP* version 4.0b10 (Swofford silica gel-dried leaf material using a Universal 2002). All characters were weighted equally Genomic DNA Extraction Kit (Takara, Dalian, and unordered. Most parsimonious trees were China). Four chloroplast (rbcL, rps16 intron, searched with a heuristic algorithm comprising trnL–F region and rpl32–trnL