Journal of Integrative Plant Biology 2013, 55 (11): 1069–1079

Research Article

Phylogeny and Classification of Prunus sensu lato (Rosaceae) Shuo Shi1,2, Jinlu Li1,3, Jiahui Sun1,2, Jing Yu1,2 and Shiliang Zhou1* 1

State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, China University of Chinese Academy of Sciences, Beijing 100043, China 3 College of Landscape Architecture, Northeast Forestry University, Harbin 150040, China
Correspondence author Tel: þ86 10 6283 6503; Fax: þ86 10 6259 0843; E‐mail: [email protected] Available online on 14 August 2013 at www.jipb.net and www.wileyonlinelibrary.com/journal/jipb doi: 10.1111/jipb.12095 2

Abstract The classification of the economically important genus Prunus L. sensu lato (s.l.) is controversial due to the high levels of convergent or the parallel evolution of morphological characters. In the present study, phylogenetic analyses of fifteen main segregates of Prunus s.l. represented by eighty‐four species were conducted with maximum parsimony and Bayesian approaches using twelve chloroplast regions (atpB‐ rbcL, matK, ndhF, psbA‐trnH, rbcL, rpL16, rpoC1, rps16, trnS‐G, trnL, trnL‐F and ycf1) and three nuclear genes (ITS, s6pdh and SbeI) to explore their infrageneric relationships. The results of these analyses were used to develop a new, phylogeny‐based classification of Prunus s.l. Our phylogenetic reconstructions resolved three main clades of Prunus s.l. with strong supports. We adopted a broad‐sensed genus, Prunus, and recognised three subgenera corresponding to the three main clades: subgenus Padus, subgenus Cerasus and subgenus Prunus. Seven sections of subgenus Prunus were recognised. The dwarf cherries, which were previously assigned to subgenus Cerasus, were included in this subgenus Prunus. One new section name, Prunus L. subgenus Prunus section Persicae (T. T. Yü & L. T. Lu) S. L. Zhou and one new species name, Prunus tianshanica (Pojarkov) S. Shi, were proposed. Keywords:

Amygdaloideae; phylogeny; Prunoideae; Prunus; taxonomy.

Shi S, Li J, Sun J, Yu J, Zhou S (2013) Phylogeny and classification of Prunus sensu lato (Rosaceae). J. Integr. Plant Biol. 55(11), 1069–1079.

Introduction Prunus L. sensu lato (s.l.) consists of more than 200 species that are mainly found in the temperate regions of the northern hemisphere and in subtropical and tropical regions (Rehder 1940; Yü et al. 1986). In a narrow sense, Prunus s.l. can be sorted into ten groups corresponding to ten genera, which are, alphabetically, Amygdalus L., Armeniaca Scop., Cerasus Mill., Emplectocladus Torr., Laurocerasus Duhamel, Maddenia Hook. f. & Thomson, Padus Mill., Persica Mill., Prunus L. sensu stricto (s.s.) and Pygeum Gaertn. (Chin et al. 2010). Some members of Prunus s.l. are common fruit or ornamental species of great economic importance, such as peaches (P. persica (L.) Batsch), plums (e.g., P. domestica L., P. cerasifera Ehrh.,

© 2013 Institute of Botany, Chinese Academy of Sciences

P. americana Marshall), apricots (P. armeniaca L.), almonds (Prunus communis (L.) Huds.) and cherries (e.g., P. avium (L.) L., P. pseudocerasus Lindl., P. serrulata Lindl.). Prunus s.l. together with other two small genera, Oemleria Rchb. and Princepia Royle, were traditionally classified into the Amygdaloideae (Prunoideae) (Robertson 1974; Goldblatt 1976; Zhang 1992; Morgan et al. 1994; Ghora and Panigrahi 1995; Takhtajan 1997; Zhou et al. 1999; Lee and Wen 2001). The members of the Amygdaloideae are also known as drupaceous plants because they all bear drupe fruits. However, recent molecular evidence suggested that the traditional Amygdaloideae should be divided into two non‐sister tribes under the newly circumscribed Spiraeoideae C. Agardh, in which Prunus s.l. belongs to the monotypic tribe Amygdaleae, while Oemleria,

Subg. Padus

Subg. Prunus sect. Prunus Gen. Prunus

Gen. Pygeum Gen. Pygeum –

–

Gen. Prunus sect. Prunus Gen. Prunus Plums

Pygeum

Gen. Prunus subg. Prunophora

Subg. Prunus sect. Emplectocladus

Subg. Padus Subg. Prunus sect. Persicae Gen. Maddenia Gen. Amygdalus

– –

– Gen. Amygdalus

Gen. Maddenia Gen. Prunus subg. Amygdalus

–

Gen. Maddenia Gen. Prunus sect. Amygdalopsis

– Emplectocladus

Maddenia Peaches

Subg. Padus Subg. Prunus sect. Microcerasus

Subg. Cerasus Gen. Cerasus

Gen. Laurocerasus Gen. Cerasus Gen. Prunus subg. Laurocerasus Gen. Prunus subg. Cerasus

Gen. Prunus subg. Cerasus Gen. Prunus sect. Cerasus

Gen. Prunus sect. Laurocerasus Gen. Prunus sect. Cerasoides

Gen. Prunus

Gen. Prunus –

Cherries

Cherry‐laurels Dwarf cherries

Subg. Prunus sect. Prunocerasus

Subg. Prunus sect. Amygdalus

Subg. Prunus sect. Armeniaca Subg. Padus Gen. Armeniaca Gen. Padus Gen. Prunus subg. Prunophora Gen. Prunus subg. Padus Gen. Prunus sect. Armeniaca Gen. Prunus sect. Laurocerasus Gen. Prunus Gen. Prunus Apricots Bird cherries

–

Gen. Amygdalus

– –

Gen. Prunus subg. Amygdalus Gen. Prunus sect. Amygdalus Gen. Amygdalus

American plums

Almonds

Gen. Prunus subg. Prunophora

This study Yü et al. (1986) Rehder (1940) Bentham and Hooker (1865)

Princepia and a non‐drupaceous‐genus, Exochorda Lindl., comprise the tribe Osmaronieae Rydb (Morgan et al. 1994; Potter et al. 2002; Potter et al. 2007). The circumscription of Prunus s.l. has varied significantly among taxonomists over the past 300 years (Table 1). Although six genera, Prunus, Armeniaca, Persica, Cerasus, Amygdalus and Laurocerasus, had been recognized in pre‐Linnaean times (Tournefort 1700), Linnaeus only accepted Amygdalus and Prunus in his work Species Plantarum (Linnaeus 1753). However, he divided Prunus into four genera, Padus (replaced Tournefort’s Laurocerasus), Armeniaca, Cerasus and Prunus, in his later work (Linnaeus 1764). After Linnaeus’ revision, some scholars chose to use micro‐genera and divided Prunus s.l. into five to eight genera (Miller 1754; Hutchinson 1964; Yü et al. 1986). There were a number of differences between the genera they proposed. For example, Laurocerasus and Padus were not distinguished initially (Tournefort 1700; Linnaeus 1753; Miller 1754) but were later treated as two different genera (Shishkin and Yuzeqchuk 1971; Yü et al. 1986), and these two genera were sometimes merged with the genus Cerasus (Jussieu 1789; de Candolle 1825). Amygdalus and Persica were either treated as separate genera (Miller 1754; de Candolle 1825; Shishkin and Yuzeqchuk 1971) or combined into one genus, Amygdalus (Jussieu 1789; Yü et al. 1986). Hutchinson (1964) included Tournefort’s genera Armeniaca and Cerasus in Prunus. Additionally, Microcerasus (Spach) Webb (Webb and Berthelot 1836) and Emplectocladus Torr. (Torrey 1851) were proposed but rarely accepted. Several small genera were adopted in regional flora (Shishkin and Yuzeqchuk 1971; Yü et al. 1986; Yasuhiro 2001; Fedorov 2002; Lu et al. 2003). Some taxonomists preferred to use a broad sense of genus containing all of the species of Prunus s.l. For example, Gray (1856) and Bentham and Hooker (1865) both proposed the inclusive genus Prunus and divided it into three and seven sections, respectively. Focke (1894) and Koehne (1893) also recognized the large genus Prunus but divided it into seven subgenera (which did not correspond with Bentham & Hooker’s seven sections). A single genus of Prunus s.l. was suggested in recent papers (McVaugh 1951; Kalkman 1965; Ghora and Panigrahi 1995; Chin et al. 2010). The distinction of Pygeum and Maddenia from Prunus s.l. has also been challenged based on morphology (Kalkman 1965) and molecular evidence (Chin et al. 2010; Shi et al. 2013). These treatments make the circumscription of Prunus s.l. even broader. The diverse inconsistency in the concept of the genus Prunus s.l. is largely due to a lack of knowledge regarding character convergence (Bortiri et al. 2006), reproductive isolation and phylogenetic relationships. Therefore, none of the treatments were convincing prior to the availability of molecular data. The early classifications were heavily based on the morphology of the fruits, and narrow‐sensed genera currently remain in use (Shishkin and Yuzeqchuk 1971; Lu et al. 2003). Recent

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Linnaeus (1753)

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Natural groups

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Table 1. Subdivisions of Prunus s.l. in some classifications

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molecular work revealed considerable homoplasy of the fruit morphology and supported the broad‐sensed genus (Bortiri et al. 2001; Lee and Wen 2001; Potter et al. 2007). Advances in biology of Prunus s.l. have also furthered our understanding of relationships within the group as follows. (1) Eurasian and American plums (Prunus s.s. and Prunocerasus) are separate and monophyletic groups (Shaw and Small 2004; Reales et al. 2010). (2) Dwarf cherries are reproductively more compatible and genetically closer to plums and peaches than to true cherries (Kataoka et al. 1988; Shimada et al. 2001). And (3) Maddenia has not diverged significantly from bird cherries (Padus) and is likely a member of Prunus s.l. (Chin et al. 2010; Shi et al. 2013). However, the problems related to resolving the phylogeny of Prunus s.l. are far from being solved because data from limited groups are not sufficient for the whole genus. The main goals of this study are to resolve the genetic relationships among the natural groups of species within Prunus s. l. and provide a formal taxonomic treatment of the subdivisions of Prunus s.l. under a phylogenetic framework. We sampled most of such groups, including Maddenia and Pygeum, to ensure high representations of every group within Prunus s.l. We developed a single copy nuclear marker of high resolution, SbeI, and integrated it with other two nuclear regions and twelve chloroplast regions to reconstruct the systematic relationships of all groups of Prunus s.l., and a refined classification of Prunus s.l. based on the phylogenetic relationships of its subdivisions is presented.

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Results The variability of the examined DNA regions and their maximum parsimony tree scores are shown in Table 2. The longest DNA region was ndhF, and the shortest was psbA‐trnH. The trnL‐F and ycf1 were more variable (p > 0.010), and atpB‐rbcL and rpoC1 are more conservative than other genes. Prior to the concatenation of all gene partitions into one for phylogenetic analyses, pairwise partition homogeneity tests (PHTs) were conducted and the p‐values (Table S1) indicated no significant conflict (p > 0.05) between the gene partitions. The concatenated data matrix of the 84 taxa included 13,203 nucleotides. There were 959 parsimony‐informative characters. MP searches yielded 1,180 most‐parsimonious trees with a consistency index (CI) of 0.804, a retention index (RI) of 0.846 and a tree length of 3,517. Consensus trees from the MP and BI analyses showed identical topologies (Figure 1). Prunus s.l. was shown to be monophyletic with strong bootstrap support (BS) and posterior probability (PP) (BS ¼ 100%, PP ¼ 1, Figure 1B) and is treated to be a single genus (see below). There were three major, monophyletic clades resolved under Prunus s.l. with good support (BS  99%, PP ¼ 1), which are treated to be three subgenera. Clade I (BS ¼ 99%, PP ¼ 1, subgenus Padus) consists of species belonging to groups Laurocerasus, Maddenia, Padus and Pygeum. Clade II (BS ¼ 100%, PP ¼ 1, subgenus Cerasus)

Table 2. Variability of twelve chloroplast regions and three nuclear genes in Prunus s.l. and their maximum parsimony tree scores

N Chloroplast genes atpB‐rbcL 26 matK 114 ndhF 39 psbA‐trnH 66 rbcL 111 rpL16 31 rpoC1 13 rps16 27 trnS‐G 82 trnL 101 trnL‐F 113 ycf1 30 Nuclear genes ITS 91 s6pdh 12 SbeI 29

La

Sc

S

NH

p

k

Vs

Is

L

CI

RI

760 817 1913 370 1043 771 487 946 1644 514 524 888

679 644 1800 167 487 656 487 663 575 344 296 853

23 90 92 17 53 32 6 20 53 31 67 72

16 52 32 17 31 16 5 10 42 31 41 21

0.004 0.0085 0.0102 0.0092 0.0096 0.0073 0.004 0.0068 0.0105 0.0093 0.023 0.0174

2.917 5.467 18.351 1.541 4.689 4.763 1.359 4.473 6.025 3.189 6.779 14.878

24 70 98 36 113 47 6 46 162 42 115 73

7 40 59 12 46 19 2 18 94 24 53 53

26 88 128 40 149 56 6 47 212 50 141 86

0.962 0.852 0.812 0.925 0.826 0.946 1 1 0.821 0.92 0.915 0.93

0.963 0.967 0.946 0.961 0.944 0.955 1 1 0.955 0.984 0.984 0.971

671 876 728

294 791 718

76 25 45

45 7 18

0.012 0.0094 0.01

3.646 7.47 7.458

256 34 48

147 15 26

618 40 58

0.547 0.9 0.845

0.81 0.889 0.898

N, number of species; La, aligned length; Sc, sites considered; S, number of polymorphic sites, excluding sites with missing data; NH, number of haplotypes; p, nucleotide diversity per site after exclusion of sites with missing data; k, average number of nucleotide differences; Vs, variable site; Is, parsimony‐information site; L, the tree length; CI, consistency index; and RI, retention index.

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Figure 1. Phylogenetic relationships of Prunus s.l. based on a concatenated dataset of chloroplast atpB‐rbcL, matK, ndhF, psbA‐trnH, rbcL, rpL16, rpoC1, rps16, trnS‐G, trnL, trnL‐F and ycf1, and nuclear ITS, s6pdh and SbeI. (A) Bayesian phylogram with branch length. (B) 50% majority‐rule Bayesian consensus tree with bootstrap supports. Values beside the branches are the bootstrap percentages for the branches in the maximum parsimony trees and Bayesian posterior probabilities. “‐” indicates the branch collapse in the parsimony tree.

Phylogeny and Classification of Prunus

includes the true cherry species and two species that were previously treated as bird cherries. Clade III (BS ¼ 100%, PP ¼ 1, subgenus Prunus) comprises almonds, apricots, dwarf cherries, peaches, and plums. The clade has diverged into seven monophyletic subclades which correspond to seven sections: section Amygdalus, section Armeniaca, section Emplectocladus, section Microcerasus, section Persicae, section Prunocerasus, and section Prunus. Section Emplectocladus has diverged the earliest. Section Amygdalus and section Persicae are closer each other than to any other sections.

Discussion Circumscription of the genus Prunus Whether a broad‐sensed Prunus genus or several narrow‐sensed genera should be used is a controversial topic and still under dispute. In recent decades, more scholars have preferred to use Prunus in the broad sense (Rehder 1940; Potter et al. 2007; Chin et al. 2010). We choose to use a broad‐sensed Prunus genus instead of a few genera according to Figure 1 for the following considerations. Firstly, Prunus s.l. is monophyletic (Lee and Wen 2001; Bortiri et al. 2006; Potter et al. 2007), which, similar to some of the other reasons provided below, is not a sufficient condition for this treatment, but a necessary one. The anatomical characters of woods suggested the monophyly of Prunus s.l. (Zhang 1992) as well. Secondly, the species of Prunus s.l. show high consistency in their morphology. All species in Prunus s.l. bear drupes and nearly every species has glands at the base of a blade or at the top of a petiole (Rehder 1940). The inflorescences of Prunus s.l. show continuous variation among different groups, ranging from racemes (Subgenus Padus) to a solitary flower (Subgenus Prunus). As noted by Kalkman (1965), sharp delimitation among groups of Prunus s.l. is lacking, and it is therefore preferable to adopt a broad sense of genus Prunus, with some subdivisions. Thirdly, the basic chromosome number of Prunus s.l. is x ¼ 8, the same number as the well‐diverged Exchorda, Oemleria and Prinsepia (Goldblatt and Johnson 1979– 2012) and natural hybridisation occurs between apricots, plums and dwarf cherries (Rehder 1940; Kataoka et al. 1988; Watkins 1995), indicating low genetic divergence among them. Fourthly and the most importantly, if narrow‐sensed genera of Prunus s.l. were to be adopted, many changes of names would be inevitable, which would bring troubles to users. On the contrary, when a broad‐sensed genus Prunus is used, there will be very few nomenclatural changes.

Three subgenera of Prunus The subdivisions of the genus Prunus were based on the following two considerations: (1) the monophyly of the subdivisions, and (2)

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the genetic divergence among subordinate classification groups. Separation of Clade I from Clade II þ Clade III is obvious and necessary because they have diverged significantly according to Figure 1. Clade I consists of tetraploid groups, such as Laurocerasus, Maddenia, Padus and Pygeum which are of paleopolypoid origins, while Clade II and Clade III consists of diploid and neopolyploid species. The basic inflorescence of species in Clade I is long racemes, but the flowers of the species in Clades II & III are solitary, clustered or in corymbs. Distinction of Clade II and Clade III is also considered appropriate when comparing the branch lengths of the two clades with those within each clade (Figure 1A). Clade III consists of almonds, apricots, dwarf cherries, peaches and plums which have flowers solitary or clustered, while Clade II consists of cherries which flowers are in corymbs. The three clades correspond to three subgenera, i.e. subgenus Padus, subgenus Cerasus, and subgenus Prunus. Such a subdivision of Prunus s.l. is completely different from any of the existing classifications (Table 1). Somewhat similar treatments can be found in classifications of Koehne (1893) or Focke (1894) who united Laurocerasus and Padus into subgenus Padus, grouped true cherries into subgenus Cerasus, and Focke classified dwarf cherries into subgenus Microcerasus. A significant difference of our proposal from the early classifications is the grouping of plums, peaches, almonds, apricots and dwarf cherries of singular flowers into a one subgenus, the true cherries of corymbs into the other subgenus, and the racemose groups into the third subgenus. We might be questioned why not to give all the monophyletic groups the generic ranks. The branching patterns and the branch lengths do tell us varying levels of genetic divergences which deserve corresponding taxonomic ranks. Judging from the branch lengths, the genetic divergences of the natural groups are not so significant when comparing to those of the three clades. Therefore, the natural groups of species are better sorted into sections rather than genera.

Relationships within subgenus Padus Subgenus Padus is a combination of four previously recognised genera: Laurocerasus, Maddenia, Padus and Pygeum. Pygeum and Laurocerasus form two strongly supported subclades under subgenus Padus. The monophyly of Maddenia is well supported but is nested within Padus, which is a polyphyletic group based on our results. Although these two taxa are readily distinguishable morphologically, they have not diverged into monophyletic lineages. The extensive morphological character parallelisms of Maddenia and Padus within the subgenus are most likely due to the multiple origins of these species. All of the species of this subgenus whose chromosome numbers have been counted to date are tetraploids (Goldblatt and Johnson 1979–2012), e.g., P. serotina Ehrh., P. padus L., P. grayana Maxim. and P. buergeriana Miq. The ancestors of the subgenus have not been documented. Collection

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of different species and further phylogenetic analysis will be necessary to explain the relationships of subgenus Padus. Therefore, subdividing this subgenus is premature.

Relationships within subgenus Cerasus The subgenus Cerasus consists only of true cherries. The dwarf cherries, Microcerasus, do not belong to this subgenus. True cherries bear only one winter bud at each leaf axils, while dwarf cherries bear three winter buds at each leaf axils (a character state observed on peaches and almonds). It had been realised that the dwarf cherries of North America are more similar to plums than to true cherries (Catling et al. 1999). It is easier to hybridise the dwarf cherries with plums, apricots or even peaches than with true cherries (Garley 1980; Kataoka et al. 1988). The dwarf cherries are more similar to plums in their fruit type. No solid evidence has been found so far to support the placement of dwarf cherries together with true cherries. Our molecular data support the membership of P. mahaleb L. and P. maackii Rupr. in subgenus Cerasus. These two species were sometimes treated as members of subgenus Padus because of elongated corymbs. Prunus mahaleb has been reported to be similar to true cherries (Krüssmann 1986) and is a diploid (González Zapatero et al. 1988), as the most species of subgenus Cerasus, while the examined species of subgenus Padus are all tetraploids (Goldblatt and Johnson 1979–2012). The similarity of P. maackii to cherries is supported by morphology (Li and Jiang 1998), and this species has been reported to be able form natural hybrids with Prunus maximowiczii Rupr., a species of subgenus Cerasus. The divergence of species in subgenus Cerasus appears to be insignificant, which may be due to quantum speciation or low resolutions of the markers. Interspecific hybridisations (Ohta et al. 2007) have complicated the taxonomy of this subgenus. Since the subgenus was not well represented in this study, more extensive species sampling and more variable genes are expected to be used to resolve phylogenetic relationships within the subgenus in the near future.

Seven sections of subgenus Prunus The subgenus Prunus is economically important and the phylogenetic relationships and taxonomic treatments of the subgenus are the major concern of this study. The phylogeny of this subgenus indicates that a radiation event occurred that gave birth to seven natural groups of species, which can be treated as different sections: section Amygdalus, section Armeniaca, section Emplectocladus, section Microcerasus, section Persicae, section Prunocerasus, and section Prunus. The American section Emplectocladus diverged the earliest within the subgenus, It was described as a distinct genus (Torrey 1851) and then treated as a section within Prunus

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(Gray 1874). The representative species P. fasciculata (Torr.) A. Gray displays unisexual flowers due to abortion of stamens or pistils (Mason 1913) and bears pubescent fruits. The section shows affinities to the sections Amygdalus and Prunus and we treat it as a section of subgenus Prunus. Section Amygdalus (almonds) and section Persica (peaches) are very similar in morphology but monophyletic groups (Figure 1; Bortiri et al. 2006). They were initially treated as two genera by Tournefort (1700) but were combined into one genus or subgenus by some later researchers (Linnaeus 1753; Rehder 1940; Yü et al. 1986; Yasuhiro 2001). The differences between the two sections are mainly found in their fruit: the mesocarp of almonds is dry and splits when ripe, while the mesocarp of peaches is flesh and does not split. Section Persicae is endemic to China and section Amygdalus has the richest species diversity in central Asia. Section Prunocerasus, the American plums, is distributed throughout North America and is very similar to section Prunus, the Eurasian plums. The two groups of plums were both found to form monophyletic groups in this study. The clade including these two groups of plums was not strongly supported (BS < 50%, PP ¼ 0.53), indicating that they should be treated as different sections. Prunus texana D. Dietr., a species was once treated as a member of Amygdalus because of its pubescent fruits (Wight 1913), was again confirmed to be in section Prunocerasus besides the earlier studies by Shaw and Small (2004, 2005). Apricots and dwarf cherries are grouped into section Armeniaca and section Microcerasus, respectively. Apricots are endemic to China. The French apricot, Prunus brigantina Vill. (¼P. brigantiaca Vill.) is actually a plum. The dwarf cherries are no doubt members of subgenus Prunus instead of subgenus Cerasus (Mowrey and Werner 1990; Bortiri et al. 2006). Therefore, we treated the dwarf cherries a separate section here. Although Flora of China was aimed to be a revised version of Flora Reipublicae Popularis Sinicae (Li 2008), many Chinese authors preferred to leave such tasks to next generations as articulated by L. T. Lu (an expert of Rosaceae taxonomy, personal communication). This work is a commitment to our predecessors and also outlines the classification of Prunus in the forthcoming Flora of Pan‐Himalayas.

Taxonomic treatments of the subdivisions of Prunus L. Prunus L., Sp. Pl. 1: 473. 1753. (Lectotype: Prunus domestica L.) Synonyms. Amygdalus L., Sp. Pl. 1: 472. 1753 Armeniaca Scop., Meth. Pl. 15. 1754 Cerasus Mill., Gard. Dict. Abr. (ed. 4) vol. 1. 1754 Padus Mill., Gard. Dict. Abr. (ed. 4) vol. 3. 1754 Persica Mill., Gard. Dict. Abr. (ed. 4) vol. 3. 1754 Laurocerasus Duhamel, Traité Arbr. Arbust. 1: 345. 1755

Phylogeny and Classification of Prunus

Pygeum Gaertn., Fruct. Sem. Pl. 1: 218. 1788 Prunophora Neck., Elem. Bot. 2:718. 1790 Microcerasus Webb & Berthel., Hist. Nat. Îles Canaries 3(2. 2): 19. 1842 Emplectocladus Torr., Proc. Amer. Assoc. Advancem. Sci. 4: 192. 1851 Maddenia Hook. f. & Thomson, Hooker’s J. Bot. Kew Gard. Misc. 6: 381. 1854 Subgenus 1. Prunus L. subgenus Padus (Mill.) Peterm., Deutschl. Fl. 159. 1846 Basionym. Padus Mill., Gard. Dict. Abr. (ed. 4) vol. 3. 1754 Synonym. Prunus L. subgenus Laurocerasus (Duhamel) Rehder, Man. Cult. Trees & Shrubs 478. 1927 Most of the species previously classified under Laurocerasus Duhamel, Maddenia Hook. f. & Thomson, Padus Mill. and Pygeum Gaertn. belong to this subgenus. Subgenus 2. Prunus L. subgenus Cerasus (Mill.) A. Gray, Manual (Gray), ed. 2. 112. 1856. p. p. Basionym. Cerasus Mill., Gard. Dict. Abr., ed. 4. 1754. p. p. Synonyms. Prunus L. subgenus Cerasus (Mill.) Focke in Engler & Prantl, Nat. Pflanzenfam. 3: 54. 1888. p. p. Prunus L. subgenus Cerasus (Mill.) Koehne, Sarg. Pl. Wils. 1:226. 1912. p. p. Subgenus Cerasus includes the true cherries, P. maackii Rupr. and P. mahaleb L. The dwarf cherries (Microcerasus) do not belong to this subgenus. Subgenus 3. Prunus L. subgenus Prunus Synonyms. Prunus L. subgenus Prunophora (Neck.) Focker in Engler & Prantl, Nat. Pflanzenfam. 3(3): 52. 1888, p. p. Prunus L. subgenus Amygdalus (L.) Focke in Engler & Prantl, Nat. Pflanzenfam. 3(3): 53. 1888 Prunus L. subgenus Microcerasus (Spach) Focke in Engler & Prantl, Nat. Pflanzenfam. 3(3): 54. 1888 Prunus L. subgenus Emplectocladus (Torr.) Sarg., Silva of North America. Boston, 4: 7. 1892 Prunus L. subgenus Armeniaca (Scop.) Nakai, Fl. Sylv. Kor. 5: 38. 1915 Prunus L. subgenus Lithocerasus Ingram, Ornamental Cherries. 1948 Section 1. Prunus L. subgenus Prunus section Prunus Synonyms. Prunus L. section Prunus Benth. & Hook. f., Gen. Pl., 1: 610. 1865 Prunus L. section Prunophora (Neck.) Fiori & Paoletti, Fl. Italia 12: 557. 1897. p. p.

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Section 2. Prunus L. subgenus Prunus section Amygdalus (L.) Benth. & Hook. f., Gen. Pl. 1: 610. 1865 Basionym. Amygdalus L., Sp. Pl. 1: 472. 1753 Synonym. Amygdalus L. subgenus Amygdalus section Amygdalus T. T. Yü & L. T. Lu, Fl. Reip. Pop. Sin. 38:11, 1986 Section Amygdalus includes the almonds, but Prunus texana, which was previously treated as an almond, does not belong to this section. Section 3. Prunus L. subgenus Prunus section Armeniaca (Scop.) Koch, Syn. Fl. Germ. Helv. 1: 205. 1837 Basionym. Armeniaca Scop., Meth. Pl. 15. (Mar.) 1754 Synonym. Prunus L. section Armeniaca (Mill.) Benth. et Hook. f., Gen. Pl. 1:610, 1865 Section 4. Prunus L. subgenus Prunus section Emplectocladus (Torr.) A. Gray, Proc. Amer. Acad. Arts 10: 70. 1874 Basionym. Emplectocladus Torr., Proc. Amer. Assoc. Advancem. Sci. 4: 192. 1851 Section 5. Prunus L. subgenus Prunus section Microcerasus (Spach) C. K. Schneid., Ill. Handb. Laubholzk. 1. 601. 1906 Basionym. Cerasus section Microcerasus Spach, Hist. Vég. I. 423. 1834 Synonym. Cerasus Mill. section Spiraeopsis Koehne, Deutsche Dendr. 306, 1893 Section 6. Prunus L. subgenus Prunus section Persicae (T. T. Yü & L. T. Lu) S. L. Zhou, com. nov. Basionym. Amygdalus subgenus Persica section Persicae T. T. Yü & L. T. Lu in Acta Phytotax. Sin. 23(3):209. 1985. Synonyms. Amygdalus subgenus Persica section Mirae T. T. Yü & L. T. Lu in Acta Phytotax. Sin. 23(3):209, 1985. Prunus L. subgenus Prunus section Persica (L.) S. L. Zhou & X. Quan in J. Syst. Evol. 49(2):138. 2011 Section Persicae includes peaches which are endemic to China. Section 7. Prunus L. subgenus Prunus section Prunocerasus Koehne, Deutsche dendrologie 302. Stuttgart: Verlag von Ferdinand Enke. 1893

Circumscription of the genus Prunus The name of the following taxon should be changed within Prunus according to the International Code of Nomenclature for algae, fungi, and plants. Prunus tianshanica (Pojarkov) S. Shi, comb. nov. Basionym. Cerasus tianshanica Pojarkov, Bot. Zhurn. S.S.S.R. 24(3): 242. 1939.

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Materials and Methods Sampling A total of eighty‐four species were collected: seventy‐seven species representing all ten groups of Prunus s.l., six species of other genera of Rosaceae and one species of Moraceae were chosen as outgroups (Table 3). Sequences of twelve chloroplast

regions, atpB‐rbcL, matK, ndhF, psbA‐trnH, rbcL, rpL16, rpoC1, rps16, trnS‐G, trnL, trnL‐F and ycf1, and three nuclear genes, ITS, s6pdh and SbeI, were to be sequenced in this study or downloaded from GenBank. The sequences of species not available in this study were downloaded from GenBank (Table S2). The accession numbers of the sequences determined in this study were also listed in Table S2. Voucher specimens of the species sequenced in this

Table 3. A list of samples and their vouchers of Prunus s.l. and outgroups sequenced in this study Taxon

Subgenus and section

Source

Voucher (PE)

Prunus armeniaca L.

Subg. Prunus sect. Armeniaca

Xinjiang: Urumchi Botanical Garden

S. L. Zhou BOP016969

Prunus armeniaca var. holosericea Batal.

Subg. Prunus sect. Armeniaca

Liaoning: Xiongyue

X. Quan 67

Prunus buergeriana Miq.

Subg. Padus

Yunnan: Weixi

S. Shi et al. SZ3264

Prunus cerasifera Ehrh.

Subg. Prunus sect. Prunus

Beijing Botanical Garden, CAS

S. L. Zhou et al. BOP010039 S. L. Zhou BOP016972

Prunus communis (L.) Hus.

Subg. Prunus sect. Amygdalus

Xinjiang: Urumchi Botanical Garden

Prunus conradinae Koehne

Subg. Cerasus

Guizhou: Guiyang

S. Shi et al. Z3022

Prunus conradinae Koehne

Subg. Cerasus

Hubei: Wuhan Botanical Garden

S. Shi et al. Z3087 S. L. Zhou et al. BOP010343

Prunus davidiana (Carrière) Franch.

Subg. Prunus sect. Persicae

Beijing Botanical Garden, CAS

Prunus davidiana(Carrière) Franch.

Subg. Prunus sect. Persicae

Beijing Botanical Garden, CAS

S. L. Zhou et al.BOP010009

Prunus ferganensis (Kost. & Rjab.) Kovalev & Kost.

Subg. Prunus sect. Persicae

Henan: Zhengzhou (from Xinjiang)

X. Quan 103

Prunus glandulosa Thunb.

Subg. Prunus sect. Microcerasus

Beijing Botanical Garden, CAS

S. L. Zhou et al. BOP010170

Prunus grayana Maxim.

Subg. Padus

Hunan: Xining

Z. C. Luo BOP002863

Prunus humilis Bunge

Subg. Prunus sect. Microcerasus

Hebei: Weichang

S. L. Zhou 090730‐3

Prunus hypoleuca (Koehne) J. Wen

Subg. Padus

Gansu: Wuning

S. L. Zhou BOP002508

Prunus hypoxantha (Koehne) J.Wen

Subg. Padus

Sichuan: Dujingyan

S. L. Zhou 053

Prunus incisoserrata (T.T.Yü & T.C.Ku) J. Wen

Subg. Padus

Shaanxi: Qinling

S. L. Zhou 054

Prunus japonica Thunb.

Subg. Prunus sect. Microcerasus

Beijing Botanical Garden, CAS

S. L. Zhou et al. BOP010337

Prunus kansuensis Rehd.

Subg. Prunus sect. Persicae

Gansu: Xihe

S. L. Zhou 51–63

Prunus mandshurica (Maxim.) Koehne

Subg. Prunus sect. Armeniaca

Heilongjiang: Harbin Botanical Garden

S. L. Zhou 135

Prunus mira Koehne

Subg. Prunus sect. Persicae

Tibet: Lasha, Norbu Lingka

S. L. Zhou 2001816

Prunus mume (Siebold) Siebold & Zucc.

Subg. Prunus sect. Armeniaca

Hubei: Wuhan Botanical Garden

X. Quan 127 S. L. Zhou et al. BOP017388

Prunus persica (L.) Batsch

Subg. Prunus sect. Persicae

Henan: Xinyang

Prunus phaeosticta (Hance) Maxim.

Subg. Padus

Hunan: Xinning

S. L. Zhou BOP002538

Prunus phaeosticta (Hance) Maxim.

Subg. Padus

Xinjiang: Urumchi Botanical Garden

S. L. Zhou BOP016970

Prunus salicina Lindl.

Subg. Prunus sect. Prunus

Gansu: Yuzhong

S. L. Zhou 090915E

Prunus sargentii Rehd.

Subg. Cerasus

Beijing Botanical Garden, CAS

S. L. Zhou et al. BOP010500

Prunus sibirica L.

Subg. Prunus sect. Armeniaca

Qinghai: Xining Botanical Garden

H. L. Sun S0042

Prunus spinulosa Siebold & Zucc.

Subg. Padus

Zhejiang: Hangzhou Botanical Garden

S. L. Zhou 050b

Prunus subhirtella var. pendula Tanaka

Subg. Cerasus

Beijing Botanical Garden, CAS

S. L. Zhou et al. BOP010112

Prunus tianshanica (Pojarkov) S. Shi

Subg. Prunus sect. Microcerasus

Xinjiang: Urumchi Botanical Garden

S. L. Zhou BOP016971

Prunus tomentosa Thunb.

Subg. Prunus

Beijing Botanical Garden, CAS

S. L. Zhou et al. BOP010008

Prunus triloba Lindl.

Subg. Prunus

Gansu: Xihe

S. L. Zhou 63–65

Prunus undulata (D. Don) Roem.

Subg. Padus

Hunan: Xinning

Z. C. Luo 121

Prunus ussuriensis Kovalev & Kostina

Subg. Prunus sect. Prunus

Beijing Botanical Garden, CAS

S. L. Zhou et al. BOP010329

Prunus yedoensis Matsum.

Subg. Cerasus

Beijing Botanical Garden, CAS

S. L. Zhou et al. BOP010038

Prunus zippeliana Miq.

Subg. Padus

Guangxi: Ziyuan

Z. C. Luo 118

Pygeum topengii Merr.

Subg. Padus

Guangdong: Zhaoqing

S. L. Zhou BOP002820

Aruncus sylvester Kostel. ex Maxim.

Outgroup

Heilongjiang: Tahe

S. L. Zhou & J. L. Li BOP016706

Exochorda racemosa (Lindl.) Rehd.

Outgroup

Beijing Botanical Garden, CAS

S. L. Zhou et al. BOP010047

Malus micromalus Makino

Outgroup

Beijing Botanical Garden, CAS

S. L. Zhou et al. BOP010089

Prinsepia sinensis (Oliv.) Oliv. ex Bean

Outgroup

Beijing Botanical Garden, CAS

S. L. Zhou et al. BOP002502

Prinsepia uniflora Batalin

Outgroup

Beijing Botanical Garden, CAS

S. L. Zhou et al. BOP010133

Rosa xanthina Lindl.

Outgroup

Beijing Botanical Garden, CAS

S. L. Zhou et al. BOP010007

The subgenera and sections the species belong are based on the taxonomic treatment in this study. GenBank accession numbers of the genes are listed in Table S2.

Phylogeny and Classification of Prunus

study were housed in the herbarium (PE) of the Institute of Botany, Chinese Academy of Sciences.

DNA extraction, amplification and sequencing DNA was extracted from fresh or dried leaves using the mCTAB (modified cetyltrimethyl ammonium bromide) approach described by Li et al. (2013). Approximately 20 mg of dried plant tissue was used per extraction. The primer sequences employed in this study can be found in previous reports: rbcL (Olmstead et al. 1992; Gastony and Rollo 1995), matK (Yu et al. 2011) and ndhF (Wen et al. 2008). Primers for the amplification of ycf1 are to be published. Primers for SbeI were SbeIF: 50 ‐GCT CCA CGA ATA TAT GAG GCA CAT G‐30 , SbeIR: 50 ‐TTC CAT GAA ATT TCC TTC ATT GAC CA‐30 ). Polymerase chain reaction amplification was carried out in a 25 mL volume with the following reagents: 20 ng DNA template, 200 mM dNTPs, 0.25 mM each primer, 1 unit of Taq polymerase and 1 Taq buffer (Takara Biotechnology Co., Dalian, China). The thermal cycling conditions were 3 min at 94° C, followed 35 cycles of 30 s at 94° C, 40 s at 52° C and 1.5 min at 72° C, with a final extension of 10 min at 72° C. The obtained PCR products were purified with PEG8000 and sequenced using ABI PRISM® BigDye™ Terminator Cycle Sequencing Kits v. 3.1 on an ABI 3730xl DNA Analyzer (Life Technologies, 5,791 Van Allen Way, Carlsbad, California 92008), following the manufacturer’s instructions.

Phylogenetic analyses The sequences of all Prunus s.l. species that were available in GenBank (Table S2) were downloaded. The newly generated sequences were edited and assembled using Sequencher v. 4.7 (Gene codes Corporation, Ann Arbor, Michigan, USA). The resulting sequences were combined with those downloaded from GenBank, aligned using the Clustal 2.0 (Thompson et al. 1997; Larkin et al. 2007) and manually adjusted using SE‐Al 2.0 (Rambaut 1996). Each dataset was then subjected to several rounds of phylogenetic evaluations to select reliable sequences. Sequences that were misidentified or exhibited large amounts of missing data were excluded from the datasets. To understand the phylogenetic performance of individual gene partitions, the variability of the genes was parameterised using DnaSP 5.0 (Librado and Rozas 2009) and PAUP 4.0b10 (Swofford 2003) based on factors such as nucleotide diversity and the number of polymorphic sites (Table 2). Prior to combining the dataset of each gene partition, pairwise partition homogeneity tests (PHTs) were performed on all fourteen datasets, and the datasets were finally concatenated using SquenceMatrix (Vaidya et al. 2011). Bayesian inference (BI) using the MrBayes ver. 3.2.1 (Ronquist and Huelsenbeck 2003) and maximum parsimony

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(MP) analyses using PAUP 4.0b10 (Swofford 2003), were carried out on single gene datasets and the concatenated dataset. In the MP analyses, all characters were unordered and equally weighted, and gaps were treated as missing data. Heuristic searches using 1,000 random sequence‐addition replicates were conducted with tree‐bisection‐reconnection (TBR) branch‐swapping. Bootstrap support (BS) percentages were calculated using 1,000 replicates, with ten trees being saved per replicate. In the BI analyses, the GTR þ I þ G model was selected by ModelTest 3.7 (Posada and Crandall 1998). Bayesian inference was run for 15,000,000 generations. Four chains, each starting with a random tree, and trees were sampled every 500 generations. Posterior probabilities (PP) were calculated from the majority consensus of all of the sampled trees when the standard deviation of the split frequencies (SDSF) permanently fell below 0.01, and the trees sampled during the burn‐in phase were discarded.

Acknowledgements This study was supported partly by National Natural Science Foundation of China (31270239) and the National High‐tech R&D Program (863 Program, 2012AA021602). Received 28 Mar. 2013 Accepted 22 Jul. 2013

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Table S1. P values from pair‐wise tests for partition homogeneity of fourteen data sets. P values smaller than 0.05 are painted yellow. Table S2. The sequences of all species belonging to drupaceous Rosaceae that were used in this study. Accessions paint yellow are new for this study.