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Home , Agastache, Glechoma, Hymenocrater, Lallemantia, Lophanthus, Marmoritis

J. Jpn. Bot. 92(1): 12–19 (2017)

Phylogenetic Position of the Chinese Endemic Heterolamium: A Close Relative of Subtribe Nepetinae ()

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 Diversity and Biogeography of East , 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 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, , , , Drepanocaryum and ; Clade II including , , , , , and . Furthermore, some typical Heterolamium morphological traits, such as 15-nerved calyx, long-exserted and so on, are similar with 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 are slender perennial China and restricted to central and southwestern . The leaves are cordate and toothed with China, growing in somewhat open places in long petioles; 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 (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

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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. tribe . 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 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. 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 植物研究雑誌 第 92 巻 第 1 号 2017 年 2 月 trnL – rpl32 JQ669275 JQ669281 JQ669303 JQ669304 JQ669305 KM886817 KX289285 – – KX289281 KX289288 KX289283 KX289284 KX289287 KX289286 KX289282 JQ669316 JQ669316 JQ669318 JQ669321 JQ669328 JQ669330 KM886829 KM886830 JQ669349 F – trnL JQ669022 JF301360 JF301366 JQ669038 DQ667517 KX289297 KX289304 KX289300 KX289301 KX289298 KX289307 KX289302 KX289303 KX289306 KX289305 KX289299 JQ669045 JF301371 JF301373 JF301384 DQ667488 KM886627 KM886628 JF301391 rps16 – – – – – KM886654 KX289293 – – KX289289 KX289296 KX289291 KX289292 KX289295 KX289294 KX289290 – – – – – KM886663 KM886664 – GenBank accession GenBank rbcL HM590067/ FJ513154 HM849871 – – – KM886784 KX289277 – – KX289273 KX289280 KX289275 KX289276 KX289279 KX289278 KX289274 – Z37395 – – – KM886793 KM886794 Z37421 ETS JQ669145 JQ669148 JQ669167 JQ669168 JQ669169 KM886687 KX298129 KX298126 – KX298124 KX298132 KX298127 KX298128 KX298131 KX298130 KX298125 JQ669179 JQ669180 JQ669182 JQ669183 JQ669185 – – JQ669202 ITS JQ669076 JQ669079 JQ669096 JQ669097 DQ667328 KM886722 KX289269 – KX289266 KX289264 KX289272 KX289267 KX289268 KX289271 KX289270 KX289265 JQ669105 JQ669106 JQ669108 JQ669109 DQ667302 KM886731 KM886732 JQ669126 no. DNA DNA – – – – – dt157 dt558 dt261 dt262 dt158 dt710 dt264 dt265 dt648 dt559 dt255 – – – – – – – – Voucher – – – – – Deng 415 (KUN) 415 Deng zdg 7112 (KUN) 7112 zdg Zhangqinglong 64128 (KUN) 64128 Zhangqinglong Chuangjingda 2551 (KUN) 2551 Chuangjingda SNJ Exped. 20110718079 (KUN) 20110718079 Exped. SNJ Deng 2440 (KUN) 2440 Deng Liuende 2181 (KUN) 2181 Liuende Zhang & Shen 50989 (KUN) 50989 Shen & Zhang YIF-0398 (KUN) YIF-0398 zdg 6261 (KUN) 6261 zdg SNJ Exped. 20110923004 (KUN) 20110923004 Exped. SNJ – – – – – Deng 2359 (KUN) 2359 Deng dt 101 (KUN) 101 dt – tochauense tochauense tochauense cardiophyllum cardiophyllum cardiophyllum cardiophyllum cardiophyllum debile debile var. var. var. var. var. var. var. var. var. var. var. var. var. var. var. var. var. var. var. var. Taxa Table 1. Samples, vouchers, DNA numbers and GenBank accession GenBank and numbers DNA vouchers, Samples, 1. Table Agastache rugosa Agastache Cedronella canariensis Cedronella Dracocephalum bullatum Dracocephalum Dracocephalum parviflorum parviflorum Dracocephalum Drepanocaryum sewerzowii Drepanocaryum Glechoma longitubae Glechoma Heterolamium debile debile Heterolamium Heterolamium debile debile Heterolamium Heterolamium debile debile Heterolamium Heterolamium debile debile Heterolamium Heterolamium debile debile Heterolamium Heterolamium debile debile Heterolamium Heterolamium debile debile Heterolamium Heterolamium debile debile Heterolamium Heterolamium debile debile Heterolamium Heterolamium debile debile Heterolamium Hymenocrater bituminosus Hymenocrater Hyssopus Lallemantia canescens canescens Lallemantia Lophanthus lipskyanus Lophanthus Lycopus uniflorus Lycopus Marmoritis complanata Marmoritis Meehania Nepeta cataria Nepeta 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 intergenic spacer) tree bisection-reconnection, branch swapping, and two nuclear ribosomal regions (ITS and MULPARS, and the alternative character state. ETS) were selected for phylogenetic inference. Strict consensus trees were constructed from Primers used for amplification and sequencing the most parsimonious trees. Bootstrap analyses were Z1 and 1204R for rbcL (Zurawski et (BP; 1000 pseudoreplicates) were conducted al. 1981), F and 2R for the rps16 (Oxelman to examine the relative level of support for et al. 1997), and tabc and tabf (Taberlet et al. individual clades on the cladograms of each 1991) for trnL–F. The rpl32–trnL spacers search (Felsenstein 1985). The consistency index were amplified using the primers as described (CI; Kluge and Farris 1969) and retention index by Shaw et al. (2007). ITS was amplified and (RI; Farris 1989) were calculated to measure the sequenced using the primers ITS1 and ITS4 amount of homoplasy in the data set. (White et al. 1990), and ETS was amplified and In Bayesian inference, nucleotide substitution sequenced as described in Drew and Sytsma model parameters were determined for cpDNA (2011). Amplified DNA samples were analyzed and nrDNA data sets using Modeltest version by electrophoresis on 1.4% agarose gel, run 2.3 (Nylander 2004, Posada and Buckley 2004), in a 0.5×TBE buffer and detected by ethidium a tree search was conducted using MrBayes bromide staining. The PCR products were version 3.2.1 (Huelsenbeck and Ronquist 2001, then purified using a QiaQuick gel extraction Ronquist and Huelsenbeck 2003) with the model kit (Qiagen, Inc., Valencia, California, USA) parameters determined from Modeltest. The and directly sequenced in both directions using chosen models were used as follows: GTR+I for the amplification primers on an the ABI 3730 each plastid marker, and GTR+I+G for the ITS automated sequencer (Applied Biosystems, data. The Markov chain Monte Carlo (MCMC) Forster City, California, USA). algorithm was run for 3,000,000 generations with one cold and three heated chains, starting Sequence alignment and phylogenetic analyses from random trees and sampling one out of The chromatograms were checked and every 300 generations. Runs were repeated contiguous sequences edited in DNA Baser v. 3 twice to confirm results. After discarding the (http://www.DnaBaser.com). Multiple-sequence trees saved prior to the burn-in point (ca. 15%), alignment was performed using MAFFT v.6 the remaining trees were imported into PAUP (Kotoh et al. 2008; available at http://www. and a 50% majority-rule consensus tree was genome.jp/tools/mafft) and Se-Al v2.0a11 produced to obtain posterior probabilities (PP) of (http://tree.bio.ed.ac.uk/software/seal/) with the the clades. default alignment parameters. Gaps were coded As the ILD test (P = 0.42 > 0.05) suggested as missing. The incongruence length difference congruence between the two data sets, we (ILD) test (Farris 1994) was used to evaluate performed the phylogenetic analyses on congruence between nrDNA and cpDNA data combined nrDNA and cpDNA data. sets. For all ILD tests, 100 replications were performed using PAUP*. Results Phylogenetic trees were constructed using Phylogenetic analyses maximum-parsimony (MP) and Bayesian The combined nrDNA (ITS and ETS) 16 植物研究雑誌 第 92 巻 第 1 号 2017 年 2 月

Fig. 1. The Bayesian 50% majority-rule consensus tree of Heterolamium and closely related taxa inferred from analyses using combined nuclear ribosomal DNA regions (ITS and ETS) and chloroplast DNA regions (rbcL, rps16, trnL– F, and rpl32–trnL). Bootstrap values for maximum likelihood/maximum parsimony analyses are indicated below the branches (bootstrap scores > 50% are shown) and the Bayesian Markov chain Monte Carlo (MCMC) posterior probabilities higher than 95% are above the branches. The ‘‘–’’ symbol indicates support of less than 50% bootstrap value or 0.95 posterior probability. data matrix had 1071 characters, of which shows monophyly of Heterolamium debile (PP 175 characters were variable and parsimony- = 1.00, BP = 100). The subtribe Nepetinae is informative. The parsimony strict consensus also monophyletic and comprises two major tree was largely congruent with the Bayesian evolutionary clades. Clade I is formed from consensus tree, especially concerning the Heterolamium, Hymenocrater, Marmoritis, backbone of the Heterolamium phylogeny. Lophanthus, Nepeta and Drepanocaryum. The combined cpDNA (rbcL, rps16 intron, Clade II includes Hyssopus, Dracocephalum, trnL–F, and rpl32–trnL) matrix consisted of Lallemantia, Meehania, Glechoma and 5081 characters, of which 447 characters were Agastache. variable and parsimony-informative. Topologies from the parsimony strict consensus tree and the Discussion Bayesian tree were largely congruent, and the Heterolamium is nested within subtribe Bayesian tree produced from combined nrDNA Nepetinae (Fig. 1), being sister to a clade and cpDNA data is shown in Fig. 1. The tree comprising Hymenocrater, Marmoritis, February 2017 The Journal of Japanese Botany Vol. 92 No. 1 17

Lophanthus, Nepeta and Drepanocaryum. These (XDB03030106), NSFC-Yunnan Natural results are based on better genus representation Science Foundation United Project (Grant no. which reject the misidentification and therefore U1136601) and The CAS/SAFEA International are more reliable. The detected position of Partnership Program for Creative Research Heterolamium within Nepetinae is supported Teams, Hundred Talents Program of the Chinese by nutlet morphology (Moon et al. 2008) and Academy of Sciences (2011312D11022). other traits. Nepetinae (Clade I and Clade II), with all these characters, looks similar to species References of Heterolamium (Fig. 1). For example, the two Agostini G., Echeverrigaray S. and Souza-Chies T. T. 2012. anther cells divaricate in most genera of this A preliminary phylogeny of the genus Cunila D. Royen subtribe (except for some species of Lophanthus ex L. (Lamiaceae) based on ITS rDNA and trnL–F regions. Mol. Phylogenet. Evol. 65: 739–747. and Glechoma) and Heterolamium. A notable Bendiksby M., Thorbek L., Scheen A. C., Lindqvist C. feature of Heterolamium is calyx tube with 15 and Ryding O. 2011. An updated phylogeny and nerves, however, the genus is not unique in this classification of Lamiaceae subfamily Lamioideae. trait, since it occurs in other species of Nepetinae. Taxon 60: 471–484. Conn B. J., Streiber N., Brown E. A., Heywood M. J. Harley et al. (2004) also mentioned this. Within and Olmstead R. G. 2009. Infrageneric phylogeny of Clade I, other characters of Heterolamium such Chloantheae (Lamiaceae) based on chloroplast ndhF as puberulent annulus inside calyx also found and nuclear ITS sequence data. Austral. Syst. Bot. 22: in sister genera Lophanthus and Marmoritis. 243–256. Furthermore, Moon et al. (2008b) found the Deng T., Nie Z. L., Drew B. T., Volis S., Kim C., Xiang C. L., Zhang J. W., Wang Y. H. and Sun H. 2015. Does pollen exine ornamentation of Heterolamium the Arcto-Tertiary biogeographic hypothesis explain to be most similar to those of members of the disjunct distribution of Northern Hemisphere Drepanocaryum, Lophanthus and Nepeta. In herbaceous plants? The case of Meehania (Lamiaceae). addition, nutlets elliptic to oblong, hairless PLoS ONE 10: e0117171. Drew B. T. and Sytsma K. J. 2011. Testing the monophyly with ridge are found in both Heterolamium and placement of Lepechinia in the tribe Mentheae and Fedtschenkiella Kudr. As a consequence, (Lamiaceae). Syst. Bot. 36: 1038–1049. not only the molecular phylogenetic evidence Drew B. T. and Sytsma K. J. 2012. Phylogenetics, but also the morphological traits reveal biogeography, and staminal in the tribe that Heterolamium is a member of subtribe Mentheae (Lamiaceae). Amer. J. Bot. 99: 933–953. Drew B. T. and Sytsma K. J. 2013. The South American Nepetinae. radiation of Lepechinia (Lamiaceae): phylogenetics, Morphological variation in Heterolamium divergence times and evolution of dioecy. Bot. J. Linn. used to distinguish three varieties (Wu and Soc. 171: 171–190. Chow 1965) got no molecular support in our Farris J. S. 1989. The retention index and rescaled consistency index. 5: 417–419. study. This, however, might be derived from Farris J. S., Källersjö M., Kluge A. G. and Bult C. 1994. insufficient genetic sorting from ancestral Testing significance of incongruence. Cladistics 10: genetic diversity, which does not mean that the 315–319. observed variation has no genetic basis and of no Felsenstein J. 1985. Confidence limits on phylogenies: an taxonomic value. Additional studies are needed approach using the bootstrap. Evolution 39: 783–791. Forbes F. B. and Hemsley W. B. 1890. An enumeration to investigate whether the observed varieties of all the plants known from China Proper, Formosa, resulted from adaptation to local environmental Hainan, Corea, the Luchu Archipelago, and the Island conditions or geographic isolation. of Hongkong, together with their distribution and synonymy. J. Linn. Soc., Bot. 26: 267–268. Harley R. M., Atkins S., Budantsev A. L., Cantino P. D., This study was supported by Grants-in- Conn B. J., Grayer R., Harley M. M., Kok R. D., Aid from the Strategic Priority Research Krestovskaja T., Morales R., Paton A. J., Ryding O. and Program of the Chinese Academy of Sciences Upson T. 2004. Labiatae. In: Kubitzki K. (ed.), The 18 植物研究雑誌 第 92 巻 第 1 号 2017 年 2 月

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J.-C. Lia,b．J.-W. Zhangb，D.-G. Zhangc，T. Dengb，S. b b a Volis ，H. Sun ，Z.-M. Li ：中国固有の Heterolamium 属（シソ科）はハッカ連イヌハッカ亜連に属する 中国固有の Heterolamium 属（シソ科）の系統的位 こで，核と色素体のゲノムを用いる分子系統学研究に 置については今もなお議論が絶えない．本属は，形 よって，本属とハッカ連イヌハッカ亜連との関係を調 態的特徴にもとづき，オドリコソウ亜科 Lamioideae べた．その結果，Heterolamium 属はイヌハッカ亜連の の Stachydeae 連 と Saturejeae 連 の 姉 妹 群 で あ る 一員であるという結論を得た．イヌハッカ亜連には二 Heterolamieae 連の唯一の属と考えられてきた．しか つのクレードがあり，クレード I には Heterolamium 属 し，花粉学的解析によると，本属はイヌハッカ亜連 やイヌハッカ属など 6 属が含まれ，クレード II には Nepetinae の残りの属と姉妹群を構成するとされた．そ ラショウモンカズラ属 Meehania，ムシャリンドウ属 February 2017 The Journal of Japanese Botany Vol. 92 No. 1 19

Dracocephalum，カキドオシ属 Glechoma，カワミドリ 支持している． 属 Agastacheなど 6属が含まれることが明らかになった． （a中国・雲南師範大学生命科学学院， さらに，萼筒に 15 脈があることや雄蕊が花冠から長く b中国・中国科学院昆明植物研究所 突き出すことなど，いくつかの形態的特徴はイヌハッカ 東亜植物多様性与生物地理学重点実験室， 亜連の種と似ていた．このことは我々が得た結論を強く c中国・吉首大学植物資源保護与利用湖南省重点実験室）