Molecular Identification and Allopatric Divergence of the White Pine
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Biochemical Systematics and Ecology 61 (2015) 161e168 Contents lists available at ScienceDirect Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco Molecular identification and allopatric divergence of the white pine species in China based on the cytoplasmic DNA variation * Zhong-Hu Li a, b, c, , 1, Chen Yang b, 1, Kang-Shan Mao b, Ya-Zhen Ma b, Jie Liu c, Zhan-Lin Liu a, Tuan-Tuan Deng a, Gui-Fang Zhao a a Key Laboratory of Resource Biology and Biotechnology in Western China (Ministry of Education), College of Life Sciences, Northwest University, Xi'an 710069, China b Molecular Ecology Group, State Key Laboratory of Grassland Agro-Ecosystem, College of Life Sciences, Lanzhou University, Lanzhou 730000, Gansu, China c Key Laboratory of Biodiversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China article info abstract Article history: Molecular identification of plant species may be highly related to the geographic isolation Received 11 December 2014 and speciation stages among species. In this study, we examined these possibilities in a Received in revised form 15 May 2015 group of white pines in China. We sampled 449 individuals from 60 natural populations of Accepted 11 June 2015 seven species from sect. Quinquefoliae subsect. Strobus. We sequenced four chloroplast Available online xxx DNA regions (around 3100 bp in length) and two mitochondrial DNAs (around 1000 bp in length). We identified 21 chlorotypes and 10 mitotypes. Both chlorotypes and mitotypes Keywords: recovered from four species with long disjunction and restricted distributions in northern Allopatric divergence Geographic isolation or northwestern China, Pinus sibirica, Pinus koraiensis, Pinus wallichiana and Pinus pumila fi Speciation stage are species-speci c, suggesting that these cytoplasmic DNAs can distinguish them from the Species identification close relatives. Allopatric isolations should have contributed greatly to their genetic di- White pines vergences. However, both chlorotypes and mitotypes recovered for Pinus dabeshanensis and Pinus fenzeliana distributed in southeastern and southern China are shared or closely related to those found in the widely distributed Pinus armandii. These two species may have diverged or derived from P. armandii recently. All of our findings together suggest that the discrimination power of the molecular identifications based on the cytoplasmic DNA barcodes may show variable discriminability depending on geographic isolation and speciation stages among the sampled species. © 2015 Elsevier Ltd. All rights reserved. 1. Introduction Species identification of plant materials is critical in diverse fields, especially related to biodiversity. Traditional identi- fication depend exclusively on morphological traits. Both well-trained taxonomists and the presence of the key morpho- logical traits (e.g. flowers and seeds) are necessary in identifying plant samples into species. However, molecular * Corresponding author. Key Laboratory of Resource Biology and Biotechnology in Western China (Ministry of Education), College of Life Sciences, Northwest University, Xi'an 710069, China. E-mail address: [email protected] (Z.-H. Li). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.bse.2015.06.002 0305-1978/© 2015 Elsevier Ltd. All rights reserved. 162 Z.-H. Li et al. / Biochemical Systematics and Ecology 61 (2015) 161e168 identification based on the standard DNA barcodes and special DNA fragments provides rapid and accurate species identi- fication (Kress et al., 2005; Hollingsworth et al., 2009, 2011). Cytoplasmic DNA fragments, especially chloroplast (cp) DNA regions (e.g., rbcL and matK), are suggested as the suitable candidates for identifying plant species (Kress et al., 2005; CBOL Plant Working Group, 2009). However, the commonly used cpDNA fragments (e.g., matK and rbcL) failed to develop species- specific mutations in gymnosperm groups (Liu et al., 2012a). Although some nuclear DNA regions in conifers may be useful for identifying plant species, it is difficult to amplify and sequence these fragments. Therefore, other cpDNAs as well as the mitochondrial (mt) DNAs with moderate mutations, were suggested to identify species in conifers (Liu et al., 2012a; Hao et al., 2015). In addition to looking for candidate DNA regions in barcoding species of numerous families or a special group (CBOL Plant Working Group, 2009; Hollingsworth et al., 2009; China Plant BOL Group, 2011), the recommended barcodes were also used for diverse applications, for example, to construct biodiversity inventory (Lahaye et al., 2008) and identify cryptic species (Yassin et al., 2008; Ragupathy et al., 2009; Liu et al., 2011). However, few researches were designed to examine discrimi- nation powers of these barcodes in identifying the closely related species with different speciation ages and allopatric iso- lations. Here we conduct such an example study to address this question using seven species of the white pine species (sect. Quinquefoliae subsect. Strobus) as a system. Around 11 white pine species are recorded in China, Pinus armandii Franchet, Pinus bhutanica Grierson & al., Pinus dabeshanensis Cheng et Law, Pinus pumila (Pallas) Regel in Kuester & al., Pinus fenzeliana Handel-Mazzetti, Pinus koraiensis Siebold & Zuccarini, Pinus kwantungensis Chun & Tsiang, Pinus morrisonicola Hayata, Pinus sibirica Du Tour in Deterville, Pinus wallichiana A. B. Jackson and Pinus wangii Hu & W. C. Cheng (Fu et al., 1999; Eckert et al., 2013). Among them, P. armandii is widely distributed from western to southeast China with altitudes ranged from 1000 to 3300 m. Six other species, Pinus bhutanica, Pinus kwantungensis, Pinus fenzeliana, Pinus dabeshanensis, Pinus wangii and Pinus morrisonicola were closely related to P. armandii. All of these seven species show short-distance disjunctions to each another. The other five species occur in southern China and some of them are narrowly restricted to a small range, for example, P. morrisonicola endemic to Taiwan and P. dabeshanensis to the Dabieshan Mountains. Recent studies suggested that P. armandii shared genetic variant with the other six species at the examined loci, indicating no complete lineage sorting among them (Liu et al., 2014a; Hao et al., 2015). However, Pinus wallichiana is found at higher altitude (1600e3300 m) along temperate forests in the Himalaya while P. koraiensis has a wide distribution in northeastern China. The other two species, P. pumila and P. sibirica, are found in the northeastern and western China, respectively. These four species are closely related to each another than to P. armandii complex although they are distributed with long-distance disjunctions based on morphological and molecular evidences (Fu et al., 1999; Eckert et al., 2013). Furthermore, the sequence variations from the commonly used cpDNAs could not diagnose them effectively (Hernandez-Le on et al., 2013; Liu et al., 2014a; Hao et al., 2015). In this study, we sequenced four cpDNA fragments and two mtDNAs for 449 individuals from 60 natural populations of seven species from two tentative groups of sect. Quinquefoliae subsect. Strobus. One group comprises P. armandii, P. dabeshanensis and P. fenzeliana and distributional ranges between them are allopatric, but with short-distance. The other group consists of P. sibirica, P. koraiensis, P. wallichiana and P. pumila with long-distance allopatric distributions. We hypothesized that with the more cpDNA fragments sequenced, it is likely to distinguish these species. However, discrimination power for species identification based on sequence variations will change dependent on the geographic isolations and possible speciation ages. In addition to test this hypothesis, our range-wide sampling and two large population genetic datasets based on cytoplasmic DNA variation undoubtedly will aid to understand the interspecific relationship of these species and their response to the historical climate changes (Liu et al., 2012b, 2014b; Hao et al., 2015). 2. Materials and methods 2.1. Plant material and sampling design We collected 449 individuals from 60 natural populations of Pinus armandi, P. dabeshanensis, P. fenzeliana, P. koraiensis, P. pumila, P. sibirica and P. wallichiana. For both P. armandi and P. dabeshanensis, our samples cover their entire distributional ranges. We collected samples for P. koraiensis, P. pumila, P. sibirica and P. wallichiana from most recorded counties in China. For most populations, 5e10 individuals were sampled and needle leaves were taken and preserved in silica gel until DNA extraction. The geographic information of each sampling site, which includes the latitude, longitude and altitude, were recorded by an Extrex GPS (Garmin, Olathe, USA). A population of Pinus tabulaeformis was sampled as outgroup (Table 1, Figs. 1 and 2). 2.2. DNA extraction, amplification and sequencing We extracted total genomic DNA from needle leaves using the modified 2Â cetyltrimethylammonium bromide (CTAB) procedure (Doyle and Doyle, 1987). For all sampled individuals, four cpDNA non-coding regions (trnL-F, trnS-fM, trnS-G, rpl16) and two mtDNA non-coding regions (nad5 intron1, nad7 intron1) were amplified and sequenced using universal primers (see primer sequences and references in Table 2). All PCR amplifications were conducted in a total volume of 25 mL on a Gene Amp PCR system 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA), using 10e40 ng genomic DNA.