Pak. J. Bot., 49(1): 215-225, 2017.

COMPLEX PHYLOGENETIC PLACEMENT OF ILEX SPECIES (AQUIFOLIACEAE): A CASE STUDY OF MOLECULAR PHYLOGENY

FAN YI1,2, LE SUN1,2, DA-CHENG HAO3, YONG PENG1,2,* FENG-MING HAN4,* AND PEI-GEN XIAO1,2

1Institute of Medicinal Development, Chinese Academy of Medical Sciences, Beijing 100193, China 2Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Beijing 100193, China 3Biotechnology Institute, School of Environment and Chemical Engineering, Dalian Jiaotong University, Dalian 116028, China 4Beijing Biomarker Technologies Co., LTD, Beijing 101300, China *Corresponding author’s email: [email protected]; [email protected]

Abstract

To investigate the phylogenetic relationships among Ilex species distributed in China, we analyzed two alignments including 4,698 characters corresponding to six plastid sequences (matK, rbcL, atpB-rbcL, trnL-F, psbA-trnH, and rpl32- trnL) and 1,748 characters corresponding to two nuclear sequences (ITS and nepGS). Using different partitioning strategies and approaches (i.e., Bayesian inference, maximum likelihood, and maximum parsimony) for phylogeny reconstruction, different topologies and clade supports were determined. A total of 18 Ilex species was divided into two major groups (group I and II) in both plastid and nuclear phylogenies with some incongruences. Potential hybridization events may account, in part, for those phylogenetic uncertainties. The analyses, together with previously identified sequences, indicated that all 18 species were recovered within Eurasia or Asia/North America groups based on plastid data. Meanwhile, the species in group II in the nuclear phylogeny were placed in the Aquifolium clade, as inferred from traditional classification, whereas the species in group I belonged to several other clades. The divergence time of most of the 18 Ilex species was estimated to be not more than 10 million years ago. Based on the results of this study, we concluded that paleogeographical events and past climate changes during the same period might have played important roles in these diversifications.

Key words: Dating, Ilex, Molecular phylogeny, Phylogeography.

Introduction Loizeau et al. (2005) recently found the floral

The genus Ilex L., which belongs to the family morphologies of the Ilex species are uniform among Aquifoliaceae Bartl., comprises deciduous and evergreen species but leaf morphologies show high variability bushes or distributed throughout temperate and tropical within the species, making it difficult to discriminate regions worldwide (Galle, 1997), many of which have between different species. Many phylogenetic studies health-promoting and/or therapeutic values in traditional on the genus Ilex have shown that the plastid and Chinese medicine and many other ethnomedicine (Hao et al., nuclear phylogenies are often incongruent based on 2013b). Most of these species in Australia, Europe, and plastid markers (psbA-trnH, rbcL, atpB-rbcL, and trnL- Africa became extinct (Manen, 2004), while Ilex continues F), nuclear markers [the nuclear ribosomal internal to exist with high diversity in East Asia, North America, and transcribed spacer (ITS), the 5S spacer, and the nuclear South America (Manen et al., 2010). This genus is also well- encoded plastid glutamine synthetase gene (nepGS)], as represented in China. Two hundred and four species, of well as amplified fragment length polymorphism which 149 species are endemic, are identified and are (AFLP) analyses (Cuénoud et al., 2000; Gottlieb et al., distributing in south regions of the Yangtze River and 2005; Selbach-Schnadelbach et al., 2009; Setoguchi & Qinling Mountains, mainly in South and South-west China Watanabe, 2000; Munir, et al., 2015; Gürkanli et al., (www.eFloras.org). 2016). For example, the plastid phylogenies The oldest pollen record of the Ilex species dates determined using the plastid RFLPs and trnL-trnF back to the Turonian or earliest upper Cretaceous period spacer were found to agree well with the in Australia, approximately 90 million years ago (mya) morphological for the Ilex species in the (Martin, 1977; Savaroglu et al., 2016; Rasheed et al., Bonin Islands and the Ryukyu Islands of Japan, 2016), but the lineage is thought to be cosmopolitan by whereas the ITS phylogenies were unrelated (Setoguchi the late Cretaceous period, at about 70–85 mya (Lobreau- & Watanabe, 2000). Additionally, within two distantly Callen, 1975; Manen et al., 2002; Muller, 1981). related sympatric species collected in Tenerife (Canary However, theses fossil records remain controversial Island), I. perado and I. canariensis, the plastid (Manen et al., 2002). markers were also found to correlate well with species Although the first worldwide investigation of identity (Manen, 2004). However, a recent study morphological taxonomy was already conducted over a showed that the nuclear phylogeny was more century ago (Loesener, 1901), the relationships among compatible with current taxonomic classification and various Ilex species have long been disputed. Baas the plastid phylogeny was strongly correlated with the (1973; 1975) concluded that Ilex species could not be geographic distribution (Manen et al., 2010). distinguished according to wood and leaf anatomy. Therefore, Manen et al. (2002) concluded that the Ilex Later, Lobreau-Callen (1975) found that Ilex species species most likely experienced a more complex could not be separated based on pollen characteristics. evolutionary path. 216 FAN YI ET AL.,

In the present study, we analyzed the phylogenetic using the Plant Genomic DNA Kit (TianGen, Beijing, relationships among the 18 Ilex species collected in a China) according to the manufacturer's instructions. small region of China, mainly from Hunan Province of Polymerase chain reaction (PCR) was performed using an central south China (Fig. 1), using different partitioning MJ Research PTC-200 Peltier Thermal Cycler (MJ strategies and approaches. Both plastid markers (matK, Research, Watertown, MA), and amplifications were rbcL, trnL-F, atpB-rbcL, psbA-trnH, and rpl32-trnL) and conducted in a 50 μL reaction system containing 15 ng nuclear markers (ITS and nepGS) were used in the genomic DNA, 5 pmol each primer (for sequences see analysis. Among them the rpl32-trnL sequence was first Table 3), 0.2 mM dNTPs, 2 mM MgCl2, and 1 U DNA used in the phylogenetic reconstruction of Ilex species polymerase (Takara in Beijing, China). Briefly, after a (Deng et al., 2015). The rpl32-trnL spacer is located in denaturation step at 94°C for 2 min, amplification was the small single-copy (SSC) region of the plastid genome carried out with 36 cycles at a melting temperature of (Shaw et al., 2007), with an average length of 1,018 bp, 94°C for 30 sec, an annealing temperature of 50–55°C for ranging from 543–1,417 bp in different species. This 30 sec, and an extension temperature of 72°C for 1 min. spacer has recently been widely used in phylogenetic Finally, an extra extension step at 72°C for 10 min was analyses (Calvente et al., 2011; Gutiérrez-Rodríguez et included. For sequences that were difficult to amplify al., 2011; Zecca et al., 2012). In combination of using the normal PCR procedure, a “slow and cold” additional published molecular data, the phylogenetic program described by Shaw et al. (2005) was used. positions of the 18 species in the Ilex genus were Six plastid regions (matK, rbcL, atpB-rbcL, trnL-F, proposed. Finally, we examined the divergence times and psbA-trnH, and rpl32-trnL) and two nuclear regions (ITS causes of diversification for the 18 Ilex species. and nepGS) were amplified. If two ITS bands appeared, the longer band was used in the analysis. The ITS region Materials and Methods includes the partial internally transcribed spacer 1 (ITS1), 5S rRNA