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

in Journal of the American Society for Horticultural Science
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  • 1 MOE Key Laboratory of Bio-Resources and Eco-Environment, College of Life Science, Sichuan University, 29 Wangjiang Road, Chengdu 610064, China
  • 2 State Key Laboratory of Grassland Agro-Ecosystem, School of Life Sciences, Lanzhou University, Lanzhou 730000, China
  • 3 MOE Key Laboratory of Bio-Resources and Eco-Environment, College of Life Science, Sichuan University, 29 Wangjiang Road, Chengdu 610064, China
  • 4 College of Life Sciences, Northwest University, Xi’an 710069, China
  • 5 The Shippen, Ashill, Cullompton, Devon, EX15 3NL, U.K.
  • 6 Biology Department, Baylor University, P.O. Box 97388, Waco, TX 76798

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).

Abstract

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 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).

Fig. 1.
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.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 139, 5; 10.21273/JASHS.139.5.558

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 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.

Table 1.

Collecting sites, voucher, and documented history of alaska cypress, monterey cypress, and leyland cypress samples that were adopted in this study (see also Adams et al., 2006).

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 25-μL volume containing 20 μL of sterile water, 2.5 μL of 10 × PCR buffer, 0.25 μL of 10 mm dNTPs, 1 μL of 5 μM each primer, 0.25 μL of 5 U·μL−1 rTaq DNA polymerase enzyme (TakaRa, Dalian, China), and 1 μL of extracted DNA (20 to 40 ng·μL−1). 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−1 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) 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).

Results

For nuclear DNA regions, alignments of molecular cloning DNA sequences (eight to 10 per individual) from the six leyland cypress cultivars as well as their putative parent species, alaska cypress and monterey cypress, generated data matrices of 1019, 818, and 882 bp for ITS, leafy, and needly, respectively. A total of 19 variable sites and four indels (insertions/deletions) were detected in the ITS data matrix, among which 18 sites and four indels are heterozygous in the six putative hybrids (Fig. 2A). Twenty-five variable sites and four indels were detected in the leafy data matrix, among which 23 sites and four indels are heterozygous in the six putative hybrids (Fig. 2B). Twenty-six variable sites and one indel were detected in the needly data matrix, among which 13 sites and one indel are heterozygous in the six putative hybrids (Fig. 2C). In these, there are 21 sites (indels), 13 sites (indels), and one site in ITS, leafy, and needly, respectively, in which the six leyland cypress cultivars showed heterozygous sites, which likely originated from homozygous sites in alaska cypress and monterey cypress.

Fig. 2.
Fig. 2.

A summary of variable nucleotide sites in DNA sequences of the three nuclear DNA regions: (A) internal transcribed spacer (ITS), (B) leafy, and (C) needly among sampled trees of alaska cypress, monterey cypress, and leyland cypress. Each cell represents a nucleotide site in a certain DNA sequence of an individual tree; the proportion of black and white colors in each cell represents the proportion of two different nucleotides at a certain site among all DNA sequences, which were generated from successful molecular cloning of each individual tree. Numbers of nucleotide sites are labeled on the left of each subfigure and abbreviated voucher numbers (collector’s name was omitted) are marked at the top of each subfigure.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 139, 5; 10.21273/JASHS.139.5.558

Meanwhile, alignment of the two cp DNA regions of the six leyland cypress individuals, alaska cypress and monterey cypress, generated data matrices of 964 and 959 bp for rbcL and matK, respectively. Eleven nucleotide differences were found between alaska cypress and monterey cypress (rbcL, nine; matK, two).

According to the alignment of the three mt DNA regions, several differences were found between the species: atpA, 784 bp, two differences; coxI, 858 bp, one difference; and rps3, 937 bp, one difference. No intraspecific variation was detected in either alaska cypress or monterey cypress. Because common inheritance without recombination can be assumed for both cp and mt genomes in plants, the two cp DNA regions were concatenated into one unit for analysis; so did the three mt DNA regions.

Discussion

Nuclear DNA regions.

In contrast with the Yamaguchi et al. (2000) study that used only a single heterozygous site in the 18S rDNA to examine the hybrid origin of leyland cypress, in the present study, three nuclear DNA regions and more abundant variable sites were used to analyze the hybrid/backcross origins of leyland cypress cultivars.

Combinations of variable sites (indels) in each nuclear DNA region lead to one to three genotypes in each individual. Neighbor network analyses suggested three points. First, most nuclear genotypes of leyland cypress were identical with (or very close to) nuclear genotypes of either alaska cypress or monterey cypress [ITS: 10 of 14, leafy: 11 of 13, needly: eight of 13 (Fig. 3)], whereas the other nuclear genotypes of leyland cypress were most likely recombinants between/among nuclear genotypes of putative parents as inferred from their intermediate phylogenetic positions (Fig. 3). Jackson and Dallimore (1926) reported that fertile offspring have been raised from seeds taken from leyland cypress cultivars. Alleles in F1 hybrids from both parent species may have experienced recombination during meiosis when they generate pollens or ovules, so F1 hybrids usually contribute recombinant alleles (genotypes) to their offspring (e.g., backcrosses and F2 hybrids). Thus, leyland cypress cultivars that possess nuclear genotypes identical to those of either alaska cypress and monterey cypress are likely F1 hybrids; meanwhile, leyland cypress cultivars that possess recombinant genotypes are most likely backcrosses or F2 hybrids.

Fig. 3.
Fig. 3.

Neighbor network among genotypes (alleles) of sampled trees of alaska cypress, monterey cypress, and leyland cypress according to DNA sequences of the three nuclear DNA regions: (A) internal transcribed spacer (ITS), (B) leafy, and (C) needly. Genotypes (alleles) that were detected from alaska cypress and monterey cypress are presented in black color, and alaska cypress and monterey cypress samples are marked with black triangular and black hexagon, respectively; genotypes (alleles) of leyland cypress cultivar samples were presented in gray color; those cultivars whose maternal parent were documented as alaska cypress and monterey cypress are marked with gray triangular and gray hexagon, respectively.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 139, 5; 10.21273/JASHS.139.5.558

It would be interesting to compare classifications of the six leyland cypress putative hybrids (Table 2). The ITS sequence data classify ‘Green Spire’, ‘Haggerston Grey’, and ‘Naylor’s Blue’ as hybrids between alaska cypress and monterey cypress. ‘Castlewellan’ and ‘Galway Gold’ are classified as backcrosses to alaska cypress and ‘Leighton Green’ is classified as a backcross to monterey cypress (Table 2).

Table 2.

Classification of hybrid form for the six leyland cypresses as revealed by three different nuclear DNA markers, internal transcribed spacer (ITS), leafy, and needly.

Table 2.

In contrast, the leafy sequence data classify ‘Green Spire’, ‘Haggerston Grey’, ‘Naylor’s Blue’, ‘Castlewellan’, and ‘Galway Gold’ as hybrids between alaska cypress and monterey cypress and ‘Leighton Green’ as a possible backcross to alaska cypress (Table 2).

A different classification was found by the needly sequence data. ‘Castlewellan’ and ‘Galway Gold’ were classified as putative hybrids and ‘Green Spire’, ‘Haggerston Grey’, and ‘Naylor’s Blue’ as possible backcrosses to alaska cypress (Table 2). ‘Leighton Green’ appeared to be an F2 generation hybrid (Table 2).

Neighbor network analyses (Fig. 3) suggested leyland cypress cultivars consist of both F1 hybrids between alaska cypress and monterey cypress, and backcrosses as well as F2 hybrids. Note that different nuclear DNA regions may have experienced different evolutionary histories; therefore, all available nuclear DNA regions should better be considered so as to precisely determine the property of hybrids.

Second, among the three nuclear DNA regions, ITS performed the best in identifying hybrids. Notice that phylogenetic relationships among genotypes using the ITS sequence data are simpler (Fig. 3A) than these using the other two nuclear DNA regions (Fig. 3B–C). In addition, the intraspecific genetic distances between ITS genotypes of either putative parental species are much shorter than intraspecific genetic distance between/among leafy and needly genotypes (Fig. 3). Usually, the nuclear ITS regions in plants are composed of multiple (reiterated) copies and are subject to concerted evolution (Alvarez and Wendel, 2003). During this process, different copies become homogenized to the same DNA sequence or at least become almost identical as a result of various mechanisms such as gene conversion or high-frequency unequal crossing over (Alvarez and Wendel, 2003). The nuclear ITS region, therefore, experiences a faster lineage sorting process than most nuclear DNA regions that are of single- or low-copy. As a result of these characteristics, the nuclear ITS is not only a highly efficient marker in delimitating closely related species (e.g., Wang et al., 2011), but also an effective marker in identifying recently originated hybrids (e.g., Feng et al., 2013).

Lastly, the six leyland cypress cultivars most likely originated from alaska cypress individuals that possess few intraspecific genetic variations. As shown in Figure 3, all genotypes (ITS and leafy) or most genotypes (needly) of alaska cypress are very closely related to each other. Both alaska cypress individuals were homozygous and they shared the same leafy genotype with each other as well as with all hybrid individuals. The ITS genotypes of both alaska cypress individuals are homozygous and they differ with each other by one mutation, and each of them shares the same ITS genotype with two hybrid individuals. One ITS genotype of another hybrid individual was closely related to these two ITS genotypes of alaska cypress. High intraspecific variation was found among the three needly genotypes of alaska cypress, but only one genotype was shared between this species and all hybrid individuals. In contrast, high-level genetic variation among genotypes of leafy and needly was detected in monterey cypress (Fig. 3).

Cytoplasmic DNA regions.

Chloroplast and mitochondrial genomes of most Cupressaceae species have been shown to be paternally inherited (Kondo et al., 1998; Mogensen, 1996; Neale et al., 1989, 1991; Sakaguchi et al., 2014; Whittle and Johnston, 2002); this holds true for alaska cypress and monterey cypress.

In the present study, all six leyland cypress individuals shared the same cp and mt haplotype. These were identical to the cp and mt haplotype of alaska cypress. Therefore, it is reasonable to propose that the cp genome and mt genome were inherited through the same method (e.g., paternally or maternally) in leyland cypress hybrids.

A summary of the historical literature concerning reports of the maternal parentage of leyland cypress cultivars (see the review in Adams et al., 2006) is given in Table 3. The literature reports that seed cones were collected from monterey cypress for ‘Castlewellan’, ‘Galway Gold’, ‘Leighton Green’, and ‘Naylor’s Blue’ (Adams et al., 2006). For ‘Green Spire’ and ‘Haggerston Grey’, Owens et al. (1964) reported that in 1888, some seeds were collected from alaska cypress growing at Leighton Hall and sown. Six of the resulting seedlings had different foliage from the other seedlings. C.J. Leyland took the six unusual plantlets (called clones 1 to 6; note that they were not clonally derived but siblings from the same maternal source) and planted them at his home, Haggerston Castle. Plant 2 (clone 2) was later named ‘Haggerston Grey’ and is extensively planted. Plant 1 (clone 1) was named ‘Green Spire’ and is also widely cultivated. From this account, it seems credible that the maternal parent of ‘Green Spire’ and ‘Haggerston Grey’ was alaska cypress, but of course there is always a possibility that these records have become confused. If ‘Green Spire’ and ‘Haggerston Grey’ were from alaska cypress seed (Table 3) and have cp and mt DNA of alaska cypress, then it appears that inheritance was maternal. This could be caused by maternal leakage. As Mogensen (1996) shows, both maternal and paternal cytoplasmic organelles may be present in the fertilized zygote; then either maternal or paternal organelles are eliminated. However, the process may not be 100% effective as Owens and Morris (1991) note that in the Cupressaceae, “presumably, most of the proembryo cytoplasm is of paternal origin, but some maternal organelles may be included during the migration.” Wagner et al. (1991) found paternal leakage of mt DNA in 125 seedlings from a cross of jack pine (Pinus banksiana Lamb.) and lodgepole pine (P. contorta Douglas ex Loudon): 119 seedlings displayed maternal inheritance, but six seedlings displayed paternal inheritance. Cato and Richardson (1996) reported 99% paternal and 1% maternal inheritance of cp DNA in monterey pine (Pinus radiata D. Don). Grivet et al. (1999) reported 94 offspring with paternal mt DNA and two with maternal DNA in norway spruce [Picea abies (L.) H. Karst.]. Shiraishi et al. (2001) found the cp genome exhibited paternal inheritance in 97.5% (352) and maternal in 2.5% (nine) of the progeny of a cross within japanese cypress [Chamaecyparis obtusa (Siebold & Zucc.) Endl.]. It appears that maternal or paternal leakage may be small but not uncommon. In summary, the appearance of ‘Green Spire’ and ‘Haggerston Grey’ from the same maternal tree source [alaska cypress (Table 3)] may be the result of maternal leakage, but confusion in the literature records cannot be eliminated. Additional research is needed to clarify the matter.

Table 3.

Putative origin of the chloroplast (cp) and mitochondrial (mt) genomes of leyland cypress cultivars that were inferred based on DNA sequences and documentary history.

Table 3.

Evolutionary implications.

Molecular dating analyses revealed that alaska cypress and monterey cypress shared a common ancestor ≈46 million years ago (Mya) [credible interval: 72 to 21 Mya (Mao et al., 2012)]. In this study, our results clearly support leyland cypress as hybrids between alaska cypress and monterey cypress. Most importantly, some leyland cypress individuals were identified as putative backcrosses or F2 hybrids, suggesting that hybrids between these two parent species are fertile. Under natural conditions, such hybridization events among conifer species will lead to the formation of hybrid zones (e.g., Little, 2004), interspecific gene flow or introgression (Li et al., 2012), and even new hybrid species (e.g., Ren et al., 2012; Sun et al., 2014). Hybridizations usually lead to merging or combining of different evolutionary lineages. When reconstructing phylogenetic trees based on multiple loci that have undergone different inheritance pathways, species or lineages that experienced hybridization events usually exhibit incongruences of phylogenetic placements among different gene trees (e.g., Guggisberg et al., 2009).

Hybridization events may have played an important role in the evolutionary history of Cupressaceae (Wang and Ran, 2014; Yang et al., 2012). Molecular phylogenetic studies suggested that historical hybridization events may have occurred in the redwood subfamily (Sequoioideae) and the Southern Hemisphere cypress subfamily (Callitroideae) (Yang et al., 2012) and are very common in the cypress subfamily (Cupressoideae) (e.g., Peng and Wang, 2008; Yang et al., 2012). In the last subfamily, phylogenetic incongruences have been observed among genera: Cupresssus (the Old World cypresses), Xanthocyparis–CallitropsisHesperocyparis (vietnamese golden cypress, alaska cypress, the New World cypresses), and junipers (Little, 2006); among subgenera and clades in junipers (Mao et al., 2010); and among species in Thuja L. (arborvitaes) and Thujopsis Siebold & Zucc. ex Endl. (asunaro) (e.g., Peng and Wang, 2008), Chamaecyparis (false cypresses) and Fokienia A. Henry & H. H. Thomas (fujian cypress) (e.g., Liao et al., 2010). Given the fact that two Cupressaceae species that diverged with each other ≈46 Mya can still generate fertile hybrids, we expect that more hybridization events among genera, (intrageneric) clades, and taxa will be discovered in the Cupressaceae as well as other conifer families considering the fast development of sequencing technology (Twyford and Ennos, 2012).

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  • Christenhusz, M., Reveal, J., Farjon, A., Gardner, M.F., Mill, R.R. & Chase, M.W. 2011 A new classification and linear sequence of extant gymnosperms Phytotaxa 19 55 70

    • Search Google Scholar
    • Export Citation
  • De Laubenfels, D.J. 2009 Nomenclatural actions for the New World cypresses (Cupressaceae) Novon 19 300 306

  • Doyle, J.J. & Doyle, J.L. 1987 A rapid DNA isolation procedure for small quantities of fresh leaf tissue Phytochem. Bul. 19 11 15

  • Farjon, A. 2005 A monograph of Cupressaceae and Sciadopitys. Royal Bot. Gardens, Kew, London, UK

  • Farjon, A., Nguyen, T.H., Harder, D.K., Phan, K.L. & Averyanov, L. 2002 A new genus and species in Cupressaceae (Coniferales) from northern Vietnam, Xanthocyparis vietnamensis Novon 12 179 189

    • Search Google Scholar
    • Export Citation
  • Feng, J.J., Jiang, D.C., Shang, H.Y., Dong, M., Wang, G.N., He, X.Y., Zhao, C.M. & Mao, K.S. 2013 Barcoding poplars (Populus L.) from western China PLoS One 8 e71710

    • Search Google Scholar
    • Export Citation
  • Garland, M.A. & Moore, G. 2012 ×Hesperotropsis, a new nothogenus for intergeneric crosses between Hesperocyparis and Callitropsis (Cupressaceae), and a review of the complicated nomenclatural history of the leyland cypress Taxon 61 667 670

    • Search Google Scholar
    • Export Citation
  • Grivet, D., Jeandroz, S. & Favre, J.M. 1999 Nad1 b/c intron polymorphism reveals maternal inheritance of the mitochondrial genome in Picea abies Theor. Appl. Genet. 99 346 349

    • Search Google Scholar
    • Export Citation
  • Guggisberg, A., Mansion, G. & Conti, E. 2009 Disentangling reticulate evolution in an Arctic–alpine polyploid complex Syst. Biol. 58 55 73

  • Hinesley, L.E., Hardy, D., Cleveland, B. & Myers, J. 2008 Soil pH and fertility affect growth of leyland cypress Christmas trees J. Environ. Hort. 26 4 8

    • Search Google Scholar
    • Export Citation
  • Huson, D.H. & Bryant, D. 2006 Application of phylogenetic networks in evolutionary studies Mol. Biol. Evol. 23 254 267

  • Jackson, A.B. & Dallimore, W. 1926 A new hybrid conifer Bul. Misc. Info. Royal Gardens Kew 3 113 115

  • James, A. 2011 The fertility of leyland cypress Plantsman (Lond., Engl.) 10 254 256

  • Kimura, M. 1980 A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences J. Mol. Evol. 16 111 120

    • Search Google Scholar
    • Export Citation
  • Kondo, T., Tsumura, Y., Kawahara, T. & Okamura, M. 1998 Paternal inheritance of chloroplast and mitochondrial DNA in interspecific hybrids of Chamaecyparis spp Breed. Sci. 48 177 179

    • Search Google Scholar
    • Export Citation
  • Li, Z.H., Zou, J.B., Mao, K.S., Lin, K., Li, H.P., Liu, J.Q. & Lascoux, M. 2012 Population genetic evidence for complex evolutionary histories of four high altitude juniper species in the Qinghai–Tibetan Plateau Evolution 66 831 845

    • Search Google Scholar
    • Export Citation
  • Liao, P.C., Lin, T.P. & Hwang, S.Y. 2010 Reexamination of the pattern of geographical disjunction of Chamaecyparis (Cupressaceae) in North America and East Asia Bot. Stud. (Taipei, Taiwan) 51 511 520

    • Search Google Scholar
    • Export Citation
  • Librado, P. & Rozas, J. 2009 DnaSP v5: A software for comprehensive analysis of DNA polymorphism data Bioinformatics 25 1451 1452

  • Lindstrom, O.M. 1992 Transplant date influences cold hardiness of leyland cypress following transplanting into the field HortScience 27 217 219

    • Search Google Scholar
    • Export Citation
  • Little, D.P. 2004 Documentation of hybridization between Californian cypresses: Cupressus macnabiana × sargentii Syst. Biol. 29 825 833

  • Little, D.P. 2006 Evolution and circumscription of the true cypress (Cupressaceae: Cupressus) Syst. Biol. 31 461 480

  • Little, D.P., Schwarzbach, A.E., Adams, R.P. & Hsieh, C.F. 2004 The circumscription and phylogenetic relationships of Callitropsis and the newly described genus Xanthocyparis (Cupressaceae) Amer. J. Bot. 91 1872 1881

    • Search Google Scholar
    • Export Citation
  • Mao, K.S., Hao, G., Liu, J.Q., Adams, R.P. & Milne, R.I. 2010 Diversification and biogeography of Juniperus (Cupressaceae): Variable diversification rates and multiple intercontinental dispersals New Phytol. 188 254 272

    • Search Google Scholar
    • Export Citation
  • Mao, K.S., Milne, R.I., Zhang, L.B., Peng, Y.L., Liu, J.Q., Thomas, P., Mill, R.R. & Renner, S.S. 2012 Distribution of living Cupressaceae reflects the breakup of Pangea Proc. Natl. Acad. Sci. USA 109 7793 7798

    • Search Google Scholar
    • Export Citation
  • McNeill, J., Barrie, F.R., Buck, W.R., Demoulin, V., Greuter, W., Hawksworth, D.L., Herendeen, P.S., Knapp, S., Marhold, K., Prado, J., Prud'homme Van Reine, W.F., Smith, G.F., Wiersema, J.H. & Turland, N.J. 2012 International code of nomenclature for algae, fungi, and plants (Melbourne Code) adopted by the Eighteenth international botanical congress, Melbourne, Australia, July 2011. Regnum Vegetable 154. Koeltz Scientific Books, Koenigstein, Germany

  • Mill, R.R. & Farjon, A. 2006 (1710) Proposal to conserve the name Xanthocyparis against Callitropsis Oerst. (Cupressaceae) Taxon 55 229 231

  • Mitchell, A.F. 1996 Alan Mitchell's trees of Britain. Harper Collins, London, UK

  • Mogensen, H.L. 1996 The hows and whys of cytoplasmic inheritance in seed plants Amer. J. Bot. 83 383 404

  • Neale, D.B., Marshall, K.A. & Harry, D.E. 1991 Inheritance of chloroplast and mitochondrial DNA in incense-cedar (Calocedrus decurrens) Can. J. For. Res. 21 717 720

    • Search Google Scholar
    • Export Citation
  • Neale, D.B., Marshall, K.A. & Sederoff, R.R. 1989 Chloroplast and mitochondrial DNA are paternally inherited in Sequoia sempervirens D. Don Endl Proc. Natl. Acad. Sci. USA 86 9347 9349

    • Search Google Scholar
    • Export Citation
  • Owens, H., Blight, W. & Mitchell, A.F. 1964 The clones of leyland cypress Qrtly. J. For. 58 8 19

  • Owens, J.N. & Morris, S.J. 1991 Cytological basis for cytoplasmic inheritance in Pseudotsuga menziesii. II. Fertilization and proembryo development Amer. J. Bot. 78 1515 1527

    • Search Google Scholar
    • Export Citation
  • Peng, D. & Wang, X.Q. 2008 Reticulate evolution in Thuja inferred from multiple gene sequences: Implications for the study of biogeographical disjunction between eastern Asia and North America Mol. Phylogenet. Evol. 47 1190 1202

    • Search Google Scholar
    • Export Citation
  • Ran, J.H., Gao, H. & Wang, X.Q. 2010 Fast evolution of the retroprocessed mitochondrial rsp3 gene in Conifer II and further evidence for the phylogeny of gymnosperms Mol. Phylogenet. Evol. 54 136 149

    • Search Google Scholar
    • Export Citation
  • Ren, G.P., Abbott, R.J., Zhou, Y.F., Zhang, L.R., Peng, Y.L. & Liu, J.Q. 2012 Genetic divergence, range expansion and possible homoploid hybrid speciation among pine species in northeast China Heredity 108 552 562

    • Search Google Scholar
    • Export Citation
  • Sakaguchi, S., Tsumura, Y., Crisp, M.D., Bowman, D.M.J.S. & Isagi, Y. 2014 Genetic evidence for paternal inheritance of the chloroplast in four Australian Callitris species (Cupressaceae) J. For. Res. 19 244 248

    • Search Google Scholar
    • Export Citation
  • Shiraishi, S., Maeda, H., Toda, T., Seido, K. & Sasaki, Y. 2001 Incomplete paternal inheritance of chloroplast DNA recognized in Chamaecyparis obtusa using an intraspecific polymorphism of the trnD-trnY intergenic spacer region Theor. Appl. Genet. 102 935 941

    • Search Google Scholar
    • Export Citation
  • Sturrock, J. 1989 Leyland cypress introduction to New Zealand N.Z. Forestry 34 18 20

  • Sun, Y.S., Abbott, R.J., Li, L.L., Li, L., Zou, J.B. & Liu, J.Q. 2014 Evolutionary history of purple cone spruce (Picea purpurea) in the Qinghai–Tibet Plateau: Homoploid hybrid origin and Pleistocene expansion Mol. Ecol. 23 343 359

    • Search Google Scholar
    • Export Citation
  • Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. 2011 MEGA 5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods Mol. Biol. Evol. 28 2731 2739

    • Search Google Scholar
    • Export Citation
  • Terry, R.G., Bartel, J.A. & Adams, R.P. 2012 Phylogenetic relationships among the New World cypresses (Hesperocyparis, Cupressaceae): Evidence from noncoding chloroplast DNA sequences Plant Syst. Evol. 298 1987 2000

    • Search Google Scholar
    • Export Citation
  • Thompson, J.D., Gibson, T.J., Plewniak, F., Eanmougin, F. & Higgins, D.G. 1997 The Clustal_X Windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools Nucleic Acids Res. 25 4876 4882

    • Search Google Scholar
    • Export Citation
  • Twyford, A.D. & Ennos, R.A. 2012 Next-generation hybridization and introgression Heredity 108 179 189

  • Wagner, D.B., Dong, J., Carlson, M.R. & Yanchuk, A.D. 1991 Paternal leakage of mitochondrial DNA in Pinus Theor. Appl. Genet. 82 510 514

  • Wang, Q., Yu, Q.S. & Liu, J.Q. 2011 Are nuclear loci ideal for barcoding plants? A case study of genetic delimitation of two sister species using multiple loci and multiple intraspecific individuals J. Systematics Evol. 49 182 188

    • Search Google Scholar
    • Export Citation
  • Wang, X.Q. & Ran, J.H. 2014 Evolution and biogeography of gymnosperms Mol. Phylogenet. Evol. 75 24 40

  • Whittle, C.A. & Johnston, M.O. 2002 Male-driven evolution of mitochondrial and chloroplastidial DNA sequences in plants Mol. Biol. Evol. 19 938 949

    • Search Google Scholar
    • Export Citation
  • Yamaguchi, N., Shiraishi, S., Sasaki, Y. & Yamamoto, C. 2000 Molecular evidence of the hybrid origins of leyland cypress (×Cupressocyparis leylandii) J. For. Res. 5 35 38

    • Search Google Scholar
    • Export Citation
  • Yang, Z.Y., Ran, J.H. & Wang, X.Q. 2012 Three genome-based phylogeny of Cupressaceae s.l.: Further evidence for the evolution of gymnosperms and Southern Hemisphere biogeography Mol. Phylogenet. Evol. 64 452 470

    • Search Google Scholar
    • Export Citation

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Contributor Notes

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: maokangshan@163.com.

  • View in gallery

    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.

  • View in gallery

    A summary of variable nucleotide sites in DNA sequences of the three nuclear DNA regions: (A) internal transcribed spacer (ITS), (B) leafy, and (C) needly among sampled trees of alaska cypress, monterey cypress, and leyland cypress. Each cell represents a nucleotide site in a certain DNA sequence of an individual tree; the proportion of black and white colors in each cell represents the proportion of two different nucleotides at a certain site among all DNA sequences, which were generated from successful molecular cloning of each individual tree. Numbers of nucleotide sites are labeled on the left of each subfigure and abbreviated voucher numbers (collector’s name was omitted) are marked at the top of each subfigure.

  • View in gallery

    Neighbor network among genotypes (alleles) of sampled trees of alaska cypress, monterey cypress, and leyland cypress according to DNA sequences of the three nuclear DNA regions: (A) internal transcribed spacer (ITS), (B) leafy, and (C) needly. Genotypes (alleles) that were detected from alaska cypress and monterey cypress are presented in black color, and alaska cypress and monterey cypress samples are marked with black triangular and black hexagon, respectively; genotypes (alleles) of leyland cypress cultivar samples were presented in gray color; those cultivars whose maternal parent were documented as alaska cypress and monterey cypress are marked with gray triangular and gray hexagon, respectively.

  • Adams, R.P., Bartel, J.A. & Price, R.A. 2009 A new genus, Hesperocyparis, for the cypresses of the Western Hemisphere (Cupressaceae) Phytologia 91 160 185

    • Search Google Scholar
    • Export Citation
  • Adams, R.P., Rushforth, K. & Trimble, S.N. 2006 The origins of leyland’s cypresses (×Cupressocyparis leylandii) based on DNA data Phytologia 88 1 16

    • Search Google Scholar
    • Export Citation
  • Alvarez, I. & Wendel, J.F. 2003 Ribosomal ITS sequences and plant phylogenetic inference Mol. Phylogenet. Evol. 29 417 434

  • Baldwin B.G., Goldman D.H., Keil D.J., Patterson R., Rosatti T.J. & Wilken D.H. 2012 The Jepson manual: Vascular plants of California. 2nd Ed. Univ. of California Press, Berkeley, CA

  • Cato, S.A. & Richardson, T.E. 1996 Inter- and intraspecific polymorphism at chloroplast SSR loci and the inheritance of plasmids in Pinus radiata D. Don Theor. Appl. Genet. 93 587 592

    • Search Google Scholar
    • Export Citation
  • Christenhusz, M., Reveal, J., Farjon, A., Gardner, M.F., Mill, R.R. & Chase, M.W. 2011 A new classification and linear sequence of extant gymnosperms Phytotaxa 19 55 70

    • Search Google Scholar
    • Export Citation
  • De Laubenfels, D.J. 2009 Nomenclatural actions for the New World cypresses (Cupressaceae) Novon 19 300 306

  • Doyle, J.J. & Doyle, J.L. 1987 A rapid DNA isolation procedure for small quantities of fresh leaf tissue Phytochem. Bul. 19 11 15

  • Farjon, A. 2005 A monograph of Cupressaceae and Sciadopitys. Royal Bot. Gardens, Kew, London, UK

  • Farjon, A., Nguyen, T.H., Harder, D.K., Phan, K.L. & Averyanov, L. 2002 A new genus and species in Cupressaceae (Coniferales) from northern Vietnam, Xanthocyparis vietnamensis Novon 12 179 189

    • Search Google Scholar
    • Export Citation
  • Feng, J.J., Jiang, D.C., Shang, H.Y., Dong, M., Wang, G.N., He, X.Y., Zhao, C.M. & Mao, K.S. 2013 Barcoding poplars (Populus L.) from western China PLoS One 8 e71710

    • Search Google Scholar
    • Export Citation
  • Garland, M.A. & Moore, G. 2012 ×Hesperotropsis, a new nothogenus for intergeneric crosses between Hesperocyparis and Callitropsis (Cupressaceae), and a review of the complicated nomenclatural history of the leyland cypress Taxon 61 667 670

    • Search Google Scholar
    • Export Citation
  • Grivet, D., Jeandroz, S. & Favre, J.M. 1999 Nad1 b/c intron polymorphism reveals maternal inheritance of the mitochondrial genome in Picea abies Theor. Appl. Genet. 99 346 349

    • Search Google Scholar
    • Export Citation
  • Guggisberg, A., Mansion, G. & Conti, E. 2009 Disentangling reticulate evolution in an Arctic–alpine polyploid complex Syst. Biol. 58 55 73

  • Hinesley, L.E., Hardy, D., Cleveland, B. & Myers, J. 2008 Soil pH and fertility affect growth of leyland cypress Christmas trees J. Environ. Hort. 26 4 8

    • Search Google Scholar
    • Export Citation
  • Huson, D.H. & Bryant, D. 2006 Application of phylogenetic networks in evolutionary studies Mol. Biol. Evol. 23 254 267

  • Jackson, A.B. & Dallimore, W. 1926 A new hybrid conifer Bul. Misc. Info. Royal Gardens Kew 3 113 115

  • James, A. 2011 The fertility of leyland cypress Plantsman (Lond., Engl.) 10 254 256

  • Kimura, M. 1980 A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences J. Mol. Evol. 16 111 120

    • Search Google Scholar
    • Export Citation
  • Kondo, T., Tsumura, Y., Kawahara, T. & Okamura, M. 1998 Paternal inheritance of chloroplast and mitochondrial DNA in interspecific hybrids of Chamaecyparis spp Breed. Sci. 48 177 179

    • Search Google Scholar
    • Export Citation
  • Li, Z.H., Zou, J.B., Mao, K.S., Lin, K., Li, H.P., Liu, J.Q. & Lascoux, M. 2012 Population genetic evidence for complex evolutionary histories of four high altitude juniper species in the Qinghai–Tibetan Plateau Evolution 66 831 845

    • Search Google Scholar
    • Export Citation
  • Liao, P.C., Lin, T.P. & Hwang, S.Y. 2010 Reexamination of the pattern of geographical disjunction of Chamaecyparis (Cupressaceae) in North America and East Asia Bot. Stud. (Taipei, Taiwan) 51 511 520

    • Search Google Scholar
    • Export Citation
  • Librado, P. & Rozas, J. 2009 DnaSP v5: A software for comprehensive analysis of DNA polymorphism data Bioinformatics 25 1451 1452

  • Lindstrom, O.M. 1992 Transplant date influences cold hardiness of leyland cypress following transplanting into the field HortScience 27 217 219

    • Search Google Scholar
    • Export Citation
  • Little, D.P. 2004 Documentation of hybridization between Californian cypresses: Cupressus macnabiana × sargentii Syst. Biol. 29 825 833

  • Little, D.P. 2006 Evolution and circumscription of the true cypress (Cupressaceae: Cupressus) Syst. Biol. 31 461 480

  • Little, D.P., Schwarzbach, A.E., Adams, R.P. & Hsieh, C.F. 2004 The circumscription and phylogenetic relationships of Callitropsis and the newly described genus Xanthocyparis (Cupressaceae) Amer. J. Bot. 91 1872 1881

    • Search Google Scholar
    • Export Citation
  • Mao, K.S., Hao, G., Liu, J.Q., Adams, R.P. & Milne, R.I. 2010 Diversification and biogeography of Juniperus (Cupressaceae): Variable diversification rates and multiple intercontinental dispersals New Phytol. 188 254 272

    • Search Google Scholar
    • Export Citation
  • Mao, K.S., Milne, R.I., Zhang, L.B., Peng, Y.L., Liu, J.Q., Thomas, P., Mill, R.R. & Renner, S.S. 2012 Distribution of living Cupressaceae reflects the breakup of Pangea Proc. Natl. Acad. Sci. USA 109 7793 7798

    • Search Google Scholar
    • Export Citation
  • McNeill, J., Barrie, F.R., Buck, W.R., Demoulin, V., Greuter, W., Hawksworth, D.L., Herendeen, P.S., Knapp, S., Marhold, K., Prado, J., Prud'homme Van Reine, W.F., Smith, G.F., Wiersema, J.H. & Turland, N.J. 2012 International code of nomenclature for algae, fungi, and plants (Melbourne Code) adopted by the Eighteenth international botanical congress, Melbourne, Australia, July 2011. Regnum Vegetable 154. Koeltz Scientific Books, Koenigstein, Germany

  • Mill, R.R. & Farjon, A. 2006 (1710) Proposal to conserve the name Xanthocyparis against Callitropsis Oerst. (Cupressaceae) Taxon 55 229 231

  • Mitchell, A.F. 1996 Alan Mitchell's trees of Britain. Harper Collins, London, UK

  • Mogensen, H.L. 1996 The hows and whys of cytoplasmic inheritance in seed plants Amer. J. Bot. 83 383 404

  • Neale, D.B., Marshall, K.A. & Harry, D.E. 1991 Inheritance of chloroplast and mitochondrial DNA in incense-cedar (Calocedrus decurrens) Can. J. For. Res. 21 717 720

    • Search Google Scholar
    • Export Citation
  • Neale, D.B., Marshall, K.A. & Sederoff, R.R. 1989 Chloroplast and mitochondrial DNA are paternally inherited in Sequoia sempervirens D. Don Endl Proc. Natl. Acad. Sci. USA 86 9347 9349

    • Search Google Scholar
    • Export Citation
  • Owens, H., Blight, W. & Mitchell, A.F. 1964 The clones of leyland cypress Qrtly. J. For. 58 8 19

  • Owens, J.N. & Morris, S.J. 1991 Cytological basis for cytoplasmic inheritance in Pseudotsuga menziesii. II. Fertilization and proembryo development Amer. J. Bot. 78 1515 1527

    • Search Google Scholar
    • Export Citation
  • Peng, D. & Wang, X.Q. 2008 Reticulate evolution in Thuja inferred from multiple gene sequences: Implications for the study of biogeographical disjunction between eastern Asia and North America Mol. Phylogenet. Evol. 47 1190 1202

    • Search Google Scholar
    • Export Citation
  • Ran, J.H., Gao, H. & Wang, X.Q. 2010 Fast evolution of the retroprocessed mitochondrial rsp3 gene in Conifer II and further evidence for the phylogeny of gymnosperms Mol. Phylogenet. Evol. 54 136 149

    • Search Google Scholar
    • Export Citation
  • Ren, G.P., Abbott, R.J., Zhou, Y.F., Zhang, L.R., Peng, Y.L. & Liu, J.Q. 2012 Genetic divergence, range expansion and possible homoploid hybrid speciation among pine species in northeast China Heredity 108 552 562

    • Search Google Scholar
    • Export Citation
  • Sakaguchi, S., Tsumura, Y., Crisp, M.D., Bowman, D.M.J.S. & Isagi, Y. 2014 Genetic evidence for paternal inheritance of the chloroplast in four Australian Callitris species (Cupressaceae) J. For. Res. 19 244 248

    • Search Google Scholar
    • Export Citation
  • Shiraishi, S., Maeda, H., Toda, T., Seido, K. & Sasaki, Y. 2001 Incomplete paternal inheritance of chloroplast DNA recognized in Chamaecyparis obtusa using an intraspecific polymorphism of the trnD-trnY intergenic spacer region Theor. Appl. Genet. 102 935 941

    • Search Google Scholar
    • Export Citation
  • Sturrock, J. 1989 Leyland cypress introduction to New Zealand N.Z. Forestry 34 18 20

  • Sun, Y.S., Abbott, R.J., Li, L.L., Li, L., Zou, J.B. & Liu, J.Q. 2014 Evolutionary history of purple cone spruce (Picea purpurea) in the Qinghai–Tibet Plateau: Homoploid hybrid origin and Pleistocene expansion Mol. Ecol. 23 343 359

    • Search Google Scholar
    • Export Citation
  • Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. 2011 MEGA 5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods Mol. Biol. Evol. 28 2731 2739

    • Search Google Scholar
    • Export Citation
  • Terry, R.G., Bartel, J.A. & Adams, R.P. 2012 Phylogenetic relationships among the New World cypresses (Hesperocyparis, Cupressaceae): Evidence from noncoding chloroplast DNA sequences Plant Syst. Evol. 298 1987 2000

    • Search Google Scholar
    • Export Citation
  • Thompson, J.D., Gibson, T.J., Plewniak, F., Eanmougin, F. & Higgins, D.G. 1997 The Clustal_X Windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools Nucleic Acids Res. 25 4876 4882

    • Search Google Scholar
    • Export Citation
  • Twyford, A.D. & Ennos, R.A. 2012 Next-generation hybridization and introgression Heredity 108 179 189

  • Wagner, D.B., Dong, J., Carlson, M.R. & Yanchuk, A.D. 1991 Paternal leakage of mitochondrial DNA in Pinus Theor. Appl. Genet. 82 510 514

  • Wang, Q., Yu, Q.S. & Liu, J.Q. 2011 Are nuclear loci ideal for barcoding plants? A case study of genetic delimitation of two sister species using multiple loci and multiple intraspecific individuals J. Systematics Evol. 49 182 188

    • Search Google Scholar
    • Export Citation
  • Wang, X.Q. & Ran, J.H. 2014 Evolution and biogeography of gymnosperms Mol. Phylogenet. Evol. 75 24 40

  • Whittle, C.A. & Johnston, M.O. 2002 Male-driven evolution of mitochondrial and chloroplastidial DNA sequences in plants Mol. Biol. Evol. 19 938 949

    • Search Google Scholar
    • Export Citation
  • Yamaguchi, N., Shiraishi, S., Sasaki, Y. & Yamamoto, C. 2000 Molecular evidence of the hybrid origins of leyland cypress (×Cupressocyparis leylandii) J. For. Res. 5 35 38

    • Search Google Scholar
    • Export Citation
  • Yang, Z.Y., Ran, J.H. & Wang, X.Q. 2012 Three genome-based phylogeny of Cupressaceae s.l.: Further evidence for the evolution of gymnosperms and Southern Hemisphere biogeography Mol. Phylogenet. Evol. 64 452 470

    • Search Google Scholar
    • Export Citation
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