Nuclear and Cytoplasmic DNA Sequence Data Further Illuminate the Genetic Composition of Leyland Cypresses

Leyland cypress [·Hesperotropsis leylandii (A.B. Jacks. & Dallim.) Garland & G. Moore, Cupressaceae] is a well-known horticultural evergreen conifer in the United Kingdom, United States, Australia, New Zealand, and other countries. As demonstrated by previous studies, this taxon is a hybrid between alaska (nootka) cypress [Callitropsis nootkatensis (D. Don) Oerst. ex D.P. Little] and monterey cypress [Hesperocyparis macrocarpa (Hartw. ex Gordon) Bartel]. However, the genetic background of leyland cypress cultivars is unclear. Are they F1 or F2 hybrids or backcrosses? In this study, six individuals that represent major leyland cypress cultivars and two individuals each of its two putative parental species were collected, and three nuclear DNA regions (internal transcribed spacer, leafy and needly), three mitochondrial (mt) DNA regions (coxI, atpA, and rps3), and two chloroplast (cp) DNA regions (matK and rbcL) were sequenced and analyzed. Sequencing results of nuclear DNA regions revealed that leyland cypress cultivars consist of putative F1 and F2 hybrids as well as backcrosses. Analysis of the cp and mt DNA from six cultivars of leyland cypress revealed that their cytoplasmic (cp and mt) genomes came from alaska cypress. Our findings will provide important instructions and background knowledge on the management of these major leyland cypress cultivars as well as future studies. Meanwhile, alaska cypress and monterey cypress may have diverged with each other’46 million years ago. The fact that they can produce fertile hybrids indicates that hybridization events may have played an important role in the evolutionary history of the cypress family (Cupressaceae). Leyland cypress, a rapidly growing hybrid conifer that possesses important ornamental and economic values, is widely cultivated in the United Kingdom, United States, Australia, New Zealand, and many other countries (Hinesley et al., 2008; James, 2011; Lindstrom, 1992; Mitchell, 1996; Sturrock, 1989). Leyland cypress cultivars have been postulated to be spontaneous hybrids between monterey cypress and alaska (nootka) cypress (known by many common names including alaska cypress, nootka cypress, yellow cypress, alaska yellow cypress, alaska cedar, nootka cedar, yellow cedar, and alaska yellow cedar) (Farjon, 2005; Mitchell, 1996; Owens et al., 1964). Leyland cypresses were first raised through spontaneous hybridization in the United Kingdom when alaska cypress and monterey cypress were imported and planted together (see Adams et al., 2006, for a brief review); during the period from 1888 to 1911, many popular cultivars were generated there (Adams et al., 2006; Mitchell, 1996; Owens et al., 1964). Leyland cypresses have morphological traits such as the size of the cone, the number of scales, and the number of seeds that are intermediate between alaska cypress and monterey cypress (Yamaguchi et al., 2000). Leyland cypresses exhibit over-parent heterosis. They grow taller and faster than both parent species and Received for publication 21 Apr. 2014. Accepted for publication 4 July 2014. This research was financially supported by the National Natural Science Foundation of China (No. 31370261 and 31100488) and funds from Sichuan University and Baylor University. We appreciated permissions from Alice Holt and Castlewellan Forest Parks, U.K., on collecting plant samples. We are deeply indebted to Didier Maerki, Hugh McAllister, and Jian-Quan Liu for their constructive suggestions. These authors contributed equally to this work. Corresponding author. E-mail: [email protected]. 558 J. AMER. SOC. HORT. SCI. 139(5):558–566. 2014. tolerate a wide range of soil type (e.g., clay, sand, acid, alkaline, etc.), different light conditions (full sunlight or partial shade), and various sites from mesic to semiarid (Yamaguchi et al., 2000). Thus, many cultivars have been selected that differ in coloration and growth habit for use in shelterbelts, hedges, landscape plantings, wood production, and Christmas tree plantations in the United Kingdom, United States, Australia, New Zealand, and other countries (Mitchell, 1996; Yamaguchi et al., 2000). There are more than 40 cultivars of leyland cypresses. Six of the most popular cultivars are Castlewellan, Galway Gold, Green Spire, Haggerston Grey, Leighton Green, and Naylor’s Blue (Adams et al., 2006; James, 2011; Mitchell, 1996). Although leyland cypress has long been regarded as a hybrid between monterey cypress and alaska cypress, it was only at the end of the last century when this hypothesis was tested using DNA data. Generally, nuclear DNA regions are biparentally inherited, whereas cp and mt DNA regions are paternally inherited in the cypress family, especially in the subfamily (Cupressioideae) that monterey cypress and alaska cypress belongs to (Kondo et al., 1998; Mogensen, 1996; Neale et al., 1989, 1991; Sakaguchi et al., 2014; Whittle and Johnston, 2002). Yamaguchi et al. (2000) examined the 152-bp nuclear ribosomal DNA (nrDNA) sequences (18S rDNA) and 436-bp cp DNA (rbcL) sequences that were generated from a single leyland cypress tree, a single alaska cypress tree, and two monterey cypress trees. One base difference in the nrDNA sequences was found between alaska cypress (A) and monterey cypress (T). The leyland cypress tree had nucleotides of both species at this site (A and T). Two bases were found to differ between alaska cypress and monterey cypress in cp DNA (rbcL) sequences. The rbcL sequence of leyland cypress was identical to that of alaska cypress. They proposed a hybrid origin of this leyland cypress tree with monterey cypress and alaska cypress as parental species and alaska cypress as the likely cp genome donor to leyland cypress (Yamaguchi et al., 2000). Subsequently, Adams et al. (2006) investigated the hybrid origin of leyland cypresses by surveying 25 leyland cypress, three monterey cypress, and two alaska cypress plants using random amplified polymorphic DNA (RAPD). Principal coordinates analysis of a total of 77 RAPD bands revealed that leyland cypress individuals were ordinated in an intermediate position between monterey cypress and alaska cypress; and several complementary bands from either monterey cypress or alaska cypress were found in leyland cypress. Combining with preliminary intersimple sequence repeat results, which showed a similar pattern to RAPD results, both of these dominant markers suggested the hybrid origin of leyland cypress from parental species, monterey cypress and alaska cypress (Adams et al., 2006). Art. H.6.2 of the International Code of Nomenclature for algae, fungi, and plants [Melbourne Code (McNeill et al., 2012)] requires that nothogeneric name of a bigeneric hybrids must be a combination of the names of the parental genera. However, the generic name for leyland cypress became unstable because the Latin names for the genera of their parent species, alaska cypress and monterey cypress, are controversial (for a detailed review on this issue, see Garland and Moore, 2012). In previous publications, alaska cypress has been assigned to four different genera (Cupressus L., Chamaecyparis Spach, Callitropsis Oerst., Xanthocyparis Farjon & T.H.Nguyên) as has monterey cypress (Cupressus, Callitropsis, Hesperocyparis Bartel & R.A.Price, and Neocupressus de Laub.) (De Laubenfels, 2009). According to recent molecular phylogenetic researches (Little, 2006; Little et al., 2004; Mao et al., 2010, 2012; Terry et al., 2012) and nomenclature publications (Adams et al., 2009; Garland and Moore, 2012; Little, 2006; Little et al., 2004; Mill and Farjon, 2006), the Old World cypresses (Fig. 1) clustered into a monophyletic clade, and the New World cypresses (Fig. 1), together with alaska cypress and vietnamese golden cypress (Xanthocyparis vietnamensis Farjon & Hiep), formed another monophyletic clade (Fig. 1); these two clades were supposed to be sisters with each other (Mao et al., 2010, 2012) or that either of them were sisters to junipers (Juniperus L.) (Little, 2006; Little et al., 2004). Several taxonomic treatments have been proposed for these taxa (Adams et al., 2009; Christenhusz et al., 2011; Farjon, 2005; Farjon et al., 2002; Little, 2006) [for details, see Fig. 1 (K. Rushforth prefers to treat both alaska cypress and monterey cypress as part of Cupressus but is of the opinion that the generic status of the parent species is not relevant to the science reported here)], but a recent taxonomic treatment (Adams et al., 2009; Fig. 1D) represented major contributions of previous works well and has been accepted by a broad botanical community (e.g., Baldwin et al., 2012; Garland and Moore, 2012; Mao et al., 2012; Yang et al., 2012). Therefore, the latest legitimate nomenclature of leyland cypress was proposed as ·Hesperotropsis leylandii (Garland and Moore, 2012). There is still little known about the detailed genetic background of leyland cypress. It has not been well substantiated whether all forms of this taxon are F1 hybrids as early cultivation history documented or if some of the cultivars are F2 hybrids or backcrosses to the parental species. The origin of their cytoplasmic (cp and mt) genome is still not known with certainty. In this study, we analyzed six leyland cypress cultivars, two alaska cypress putative parents and two monterey cypress putative parents, by sequencing three nuclear DNA regions [internal transcribed spacer (ITS), leafy and needly], two cp DNA regions (matK and rbcL) as well as three mt DNA regions (atpA, coxI, and rps3). Our purpose was to confirm the hybrid/backcross origin of leyland cypress cultivars using multiple DNA regions from all three plant genomes and further investigate the genetic background of leyland cypress. Materials and Methods PLANT MATERIALS. Leaf samples used in a previous study (Adams et al., 2006) were obtained for six leyland cypress cultivars. These cultivars were Green Spire and Haggerston Grey (whose maternal parent have been said to be alaska cypress), Leighton Green and Naylor’s Blue (reported to have monterey cypress as the maternal parent), Castlewellan [reputedly from seed in a cone from monterey cypress (cv. Lutea) growing near an alaska cypress (cv. Aurea)], and ‘Galway Gold’ (of unknown origin but often thought to be a renaming of ‘Castlewellan’ or a sister seedling) (Adams et al., 2006). Leaf materials were obtained from the two putative parent species of leyland cypress, monterey cypress, and alaska cypress, which were used in Adams et al. (2006). These two species are both endemic to the west coast of North America, but because leyland cypress was originally raised in the United Kingdom, samples of two accessions for each of the two species were obtained from Botanic gardens in the United Kingdom (including from both putative parent trees of ‘Castlewellan’ and possibly ‘Galway Gold’). Adams and Rushforth (Adams et al., 2006) collected fresh foliage from living trees and placed the J. AMER. SOC. HORT. SCI. 139(5):558–566. 2014. 559 leaves in silica gel, transported them back to the laboratory, and subsequently stored them at –20 C. Detailed information of each accession is listed in Table 1. DATA COLLECTION (MOLECULAR SEQUENCES). Total genomic DNA was extracted from 20 mg dried leaf using a modified CTAB method (Doyle and Doyle, 1987). Using these primers from previous studies, three nuclear DNA regions [ITS, leafy and needly (Little et al., 2004; Yang et al., 2012)], three mt DNA regions [coxI, atpA, and rps3 (Mao et al., 2012; Ran et al., 2010)], and two cp DNA regions [matK and rbcL (Mao et al., 2010)] were amplified and sequenced. Polymerase chain reactions (PCRs) were performed in a 25mL volume containing 20 mL of sterile water, 2.5 mL of 10 · PCR buffer, 0.25 mL of 10 mM dNTPs, 1 mL of 5 mM each primer, 0.25 mL of 5 U mL rTaq DNA polymerase enzyme (TakaRa, Dalian, China), and 1 mL of extracted DNA (20 to 40 ng mL). The PCR protocols used to amplify were as follows: initial denaturation at 95 C for 5 min, 37 cycles of 40 s denaturation at 95 C, annealing at 55 to 60 C for 1 min (55 C for ITS, leafy and rbcL; 58 C for needly and matK; 60 C for coxI, atpA, and rps3), and elongation at 72 C for 1 min 10 s followed by a final elongation period at 72 C for 7 min. Following previous studies, PCR products of nuclear DNA regions were purified using an Agarose Gel DNA Purification kit, and then cloned using pMD19-T vector following the recommended protocol (TakaRa) and transformed into competent Escherichia coli (Migula) Castellani & Chalmers strain JM109 at 42 C. The transformed bacteria were screened on solid Luria Broth media with 150 mg mL ampicillin at 37 C overnight, and five positive clones were amplified using universal primers (M13-47 and RV-M) for each nuclear DNA region of each individual. PCR product purifications, sequencing reactions, and successive purifications were performed and capillary analyses were run on a DNA sequencer (ABI 3130XL; Applied Biosystems, Foster City, CA) following the manufacturer’s protocols. All sequences that were determined in this study were submitted to National Center for Biotechnology Information GenBank (KJ849621–KJ849731). DATA ANALYSIS. DNA sequence alignments were conducted using Clustal_X 1.83 (Thompson et al., 1997) and then manually modified in MEGA 5.0 (Tamura et al., 2011) according to the original chromatogram. Subsequently, haplotypes (genotypes) and variable sites of mt and cp DNA sequences as well as each of the three nuclear DNA regions were detected using DnaSP Version 5 (Librado and Rozas, 2009). To investigate the hybridization events in the cultivation history of leyland cypress, the Neighbor network method as implemented in Splitstree 4.11.3 (Huson and Bryant, 2006) was used to reconstruct reticulate networks based on sequences of each nuclear DNA region. For distance calculations, we excluded insertions/deletions (indels) Fig. 1. A cladogram that shows phylogenetic relationships among the Old World cypresses, the New World cypresses, alaska cypress, vietnamese golden cypress, and junipers (courtesy of Mao et al., 2012: Fig. S1), and taxonomic treatments of different authors. Black bars to the right of the cladogram illustrate common names of all involved taxa, (A) the taxonomic treatment of Farjon (2005), (B) the taxonomic treatment of Christenhusz et al. (2011), (C) the taxonomic treatment of Little (2006), and (D) the taxonomic treatment of Adams et al. (2009). Brach lengths represent genetic distances as calculated using maximum likelihood approach, black and gray asterisks above branch indicate strong and moderate bootstrap support values, respectively: A. = alaska cypress; V. = vietnamese golden cypress; C. = Callitropsis nootkatensis; X. = Xanthocyparis vietnamensis; Xan. = Xanthocyparis. 560 J. AMER. SOC. HORT. SCI. 139(5):558–566. 2014. and used the K2P model (Kimura, 1980). The relative robustness of the clades was estimated by performing 1000 bootstrap replicates based on which a 95% confidence network was constructed for each data set (Huson and Bryant, 2006).