Merging Maddenia with the Morphologically Diverse Prunus

Botanical Journal of the Linnean Society, 2010, 164, 236–245. With 2 ﬁgures

Merging Maddenia with the morphologically diverse

Prunus (Rosaceae)boj_1083 236..245

SIEW-WAI CHIN1*, JUN WEN2, GABRIEL JOHNSON2 and DAN POTTER1 Downloaded from https://academic.oup.com/botlinnean/article-abstract/164/3/236/2418483 by guest on 01 March 2020 1Department of Plant Sciences, MS2, University of California, Davis, CA 95616, USA 2Department of Botany, National Museum of Natural History, MRC 166, Smithsonian Institution, Washington DC, 20013-7012, USA

Received 29 June 2010; revised 27 July 2010; accepted for publication 20 August 2010

Maddenia (Rosaceae) has been distinguished from Prunus on the basis of its tepaloid perianth and one- to two-carpellate gynoecium. These distinctive morphological traits nonetheless overlap with several Prunus spp. Maddenia has previously been shown to be nested within Prunus, more specifically within a clade containing members of subgenera Laurocerasus and Padus, but its phylogenetic position within that clade has not been defined precisely. This study clarifies the position of Maddenia within Prunus through phylogenetic analyses of nuclear ribosomal internal transcribed spacer (ITS) and plastid ndhF sequences, with an expanded sampling of tropical species of subgenus Laurocerasus and the inclusion of three Maddenia spp. The monophyly of Maddenia is supported by both the ITS and ndhF analyses, but both datasets support the inclusion of Maddenia in Prunus. All trees from the ITS analysis and some trees from the ndhF analysis also support a close alliance of Maddenia with a clade comprising temperate species of subgenera Laurocerasus and Padus. On the basis of these results, all recognized species of Maddenia are herein formally transferred to Prunus, which requires four new combinations and one new name: Prunus fujianensis (Y.T.Chang) J.Wen, comb. nov.; Prunus himalayana J.Wen, nom. nov.; Prunus hypoleuca (Koehne) J.Wen, comb. nov.; Prunus hypoxantha (Koehne) J.Wen, comb. nov.; and Prunus incisoserrata (T.T.Yü & T.C.Ku) J.Wen, comb. nov. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 164, 236–245.

ADDITIONAL KEYWORDS: ITS – Laurocerasus – ndhF – Padus – phylogeny.

INTRODUCTION sepia Royle, Maddenia Hook.f. & Thoms., Oemleria Rchb. and Pygeum Gaertn.), was previously included Prunus L. (Rosaceae) consists of over 200 species of in subfamily Amygdaloideae Arn. (Takhtajan, 1997), trees and shrubs with many economically important but this grouping was not supported by a recent members, including peach [P. persica (L.) Batsch], treatment of Rosaceae based on molecular phyloge- apricot (P. armeniaca L.), almond [P. dulcis (Mill.) D. netic analyses (Potter et al., 2007). In this treatment, A. Webb] and sweet cherry [P. avium (L.) L.]. Prunus Prunus, Maddenia and Pygeum comprise tribe spp. are widely distributed in the temperate zone of Amygdaleae Juss. of the newly circumscribed subfam- the Northern Hemisphere. In addition, there are over ily Spiraeoideae C.Agardh, whereas Oemleria and two 80 less well-known species distributed in tropical Asia other genera comprise tribe Osmaronieae Rydb. and the Americas. Some of these species [e.g. P. afri- The most widely accepted classiﬁcation for Prunus cana (Hook.f.) Kalkman and P. arborea (Blume) is that of Rehder (1940), who recognized ﬁve subgen- Kalkman] contain useful phytoesterols that have era: Amygdalus (L.) Focke, Prunus, Cerasus Pers., been applied in the treatment of prostate disorders Laurocerasus Koehne and Padus (Moench) Koehne. (Cuervo, Francia & Fraile, 1978; Andro & Riffaud, Mason (1913) described an additional subgenus, 1995). Prunus, together with four other genera (Prin- Emplectocladus (Torr.) Sargent, comprising a small group of shrubby species distributed in the deserts of *Corresponding author. E-mail: [email protected] south-western North America, but this group was

236 © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 164, 236–245 MERGING MADDENIA WITH PRUNUS (ROSACEAE) 237 classified under subgenus Amygdalus by Rehder ill-differentiated perianth found in Pygeum (subgenus (1940). Several molecular phylogenetic studies Laurocerasus). Sterling (1964) pointed out that the (Mowrey & Werner, 1990; Bortiri et al., 2001; Lee & anatomical structures of the carpel in fruits of Mad- Wen, 2001; Bortiri, Vanden Heuvel & Potter, 2006; denia and Pygeum are similar. Moreover, dioecy in Wen et al., 2008) have been conducted to investigate Maddenia is not without precedent in Prunus,as the evolutionary relationships among the species and andromonoecy has been reported in P. caroliniana subgenera of Prunus. In analyses to date, plastid data Aiton (subgenus Padus) (Wolfe & Drapalik, 1999) and (trnL–trnF, trnS–trnG–trnG and ndhF) have sup- in several members of the Pygeum group, including P. ported two main groups within Prunus, one including ceylanica (Wight) Miq., P. dolichobotrys (Laut. & subgenera Padus, Laurocerasus and Pygeum and the K.Sch.) Kalkman and P. polystachya (Hook.f.) Downloaded from https://academic.oup.com/botlinnean/article-abstract/164/3/236/2418483 by guest on 01 March 2020 other consisting of subgenera Amygdalus, Prunus, Kalkman (Kalkman, 1965). Emplectocladus and Cerasus. However, nuclear It has been a decade since the first report based regions [ribosomal internal transcribed spacer (ITS) on molecular evidence (Lee & Wen, 2001) suggesting and s6pdh] have supported a different tree topology, the questionable generic status of Maddenia.The also with two main groups in the genus, but with one primary objectives of this study were: (1) to confirm group consisting of Cerasus–Padus–Laurocerasus– and establish more precisely the phylogenetic position Pygeum and the second group composed of of Maddenia within Prunus based on phylogenetic Amygdalus–Prunus–Emplectocladus and section analyses of both nuclear and plastid sequences and Microcerasus Webb. (subgenus Cerasus). The incon- using a wider sampling of Maddenia spp. and gruent phylogenetic position of subgenus Cerasus members of Prunus subgenus Laurocerasus, espe- (excluding section Microcerasus) in plastid and cially the tropical Pygeum and the Neotropical nuclear analyses has led to the suggestion that this species; and (2) to provide the formal taxonomic group of species may be derived from ancient hybrid- nomenclatural changes that are required, based on ization events between members of the two clades the results of objective (1), to maintain the monophyly (Bortiri et al., 2006; Wen et al., 2008). of Prunus. Maddenia is a small genus with about six species distributed in temperate regions of the Himalayas and eastern to western China (Rehder, 1940; Yü et al., MATERIAL AND METHODS 1986). It has been distinguished from Prunus by its TAXA UNDER STUDY polygamo-dioecious sexual system (Hooker & The taxa sampled for this study are listed in Table 1. Thomson, 1854; Sterling, 1964) with ten tepaloid peri- Twenty-eight species of Prunus, including three anth segments and its often two-carpellate gynoecium species of Maddenia, were sampled as ingroup taxa (Focke, 1894). The variable number of indistinguish- for this study. Representatives of three other tribes of able perianth segments, i.e. petals not distinct from subfamily Spiraeoideae, closely related to Prunus sepals (Backer & Bakhuizen van den Brink, 1963), (Potter et al., 2007), were selected as outgroup taxa. has been used as a diagnostic character for the 40 We only included a smaller sampling of taxa from palaeotropical Pygeum spp. However, Kalkman (1965) subgenera Amygdalus, Prunus, Emplectocladus and considered the undifferentiated perianth in Pygeum Cerasus because our primary goal was to clarify the as ‘an overlapping’ character with Prunus, which phylogenetic position of Maddenia within the sub- prompted him to transfer Pygeum to Prunus subge- genus Laurocerasus–Padus clade. nus Laurocerasus sections Mesopygeum Kalkman and Laurocerasus. Lee & Wen (2001), using nuclear ITS data, showed MOLECULAR DATA that Maddenia is nested within Prunus and is closely DNA extraction and PCR amplification of ITS and related to subgenera Laurocerasus and Padus. This ndhF were performed largely according to Wen et al. result was supported subsequently by other phyloge- (2008). The 1F and 15F ndhF primers employed in netic studies of both nuclear and plastid sequences Olmstead & Sweeney (1994) failed to amplify most of (Bortiri et al., 2006; Wen et al., 2008). In all of these the tropical species sampled in this study. We thus studies, Maddenia has been represented by only one designed ndhf-15FPrun (5′-ATGGAAYAKACATATM species: M. hypoleuca Koehne. The inclusion of Mad- AATAYKSRTGG-3′). PCR amplicons were purified denia, with unisexual or bisexual flowers bearing ten using the polyethylene glycol (PEG) precipitation pro- tepaloid perianth parts, in Prunus is not all that cedure following Sambrook, Fritsch & Maniatis anomalous, as Maddenia shares several morphologi- (1989). Cycle sequencing was conducted using BigDye cal characters with members of Laurocerasus and 3.1 reagents and the following profile: 35 cycles of Padus. These include a leafy raceme that is similar to 97 °C for 15 s, 50 °C for 5 s and 60 °C for 4 min. The Padus and a perianth that closely resembles the products of cycle-sequencing reactions were cleaned

Table 1. List of taxa sampled for this study

GenBank accession Taxon Source and voucher (ITS; ndhF)

Subgenus Prunus Section Prunus P. spinosa L. USA, cult. – Wen 7307 (US) *EU669100; *EU669167 P. salicina Lindl. China, Zhejiang. – Wen 3020 (CS) *AF179486-87; *EU669143 Section Prunocerasus Koehne

P. angustifolia Marsh. USA, Florida – Gholson s.n. (CS) *AF179490; *EU669131 Downloaded from https://academic.oup.com/botlinnean/article-abstract/164/3/236/2418483 by guest on 01 March 2020 P. umbellata Ell. USA, Florida – Gholson s.n. (CS) *AF179493; *EU669120 Subgenus Amygdalus (L.) Focke P. persica (L.) Batsch. China, Zhejiang – Wen 3017 (CS) *AF179562; *EU669129 P. dulcis (Mill.) Webb. USA, cult. – HM Davis s.n. (CS) *EU669085; *EU669146 Subgenus Emplectocladus (Torr.) Sargent P. fasciculata (Torr.) A.Gray USA, California – M Beck s.n. (CS) *EU669086; *EU669153 P. havardii Mason USA, cult. – Wen s.n. (US) *EU669096; *EU669165 Subgenus Cerasus Pers. Section Cerasus P. avium (L.) L. USA, Virginia – Wen 8660 (US) HM856805; HM856816 Section Mahaleb Focke P. mahaleb (Dougl.) L. USA, cult. – Lee & Wen 4015 (CS) *AF179523-24; *EU669134 Section Phyllomahaleb Koehne P. maximowiczii Rupr. USA, cult. – Lee & Wen 4079 (CS) *AF179526; *EU669124 Section Pseudocerasus Koehne P. nipponica Matsum. var. nipponica USA, cult. – Lee & Wen 4077 (CS) *AF179507-08; *EU669144 Subgenus Padus (Moench) Koehne P. padus L. var. commutata Dipp. USA, cult. – Lee & Wen 4027 (CS) *AF179527; *EU669132 P. virginiana L. var. virginiana USA, Colorado-Lee & Wen 4022 (CS) *AF179536-37; *EU669126 Subgenus Laurocerasus Koehne P. laurocerasus L. Cult. – Lee & Wen 5001 (CS) *AF179545-46; NA P. laurocerasus L. Russia, Krashodarsky Krai – NA, HM856817 Wen 10366 (US) P. lusitanica L. Cult. – Bortiri 90 (DAV) †AY100698; NA P. wallichii Steud. Vietnam, Vinh Phuc Prov. – HM856806; HM856818 Wen 10808 (US) P. phaeosticta (Hance) Maxim. China, Taiwan – H-Y Liu s.n. (F) *EU669095; *EU669163 P. javanica (Teijsm. & Binn.) Miq. Indonesia, W. Java – Wen 10150 (US) HM856807; HM856819 P. zippeliana Miq. Vietnam, Lao Cai – Wen 6030 (US) HM856808; *EU669170 P. tucumanensis Lillo Bolivia – Nee & Wen 53882 (US) HM856809; HM856820 P. oleifolia Koehne Bolivia – Nee & Wen 53831 (US) HM856810; HM856821 P. integrifolia Sarg. Peru, Prov. Oxapampa – Wen 8620 (US) HM856811; HM856822 P. lancilimba (Merr.) Kalkman Vietnam, Lao Cai – Wen 10851 (US) HM856812; HM856823 P. malayana Kalkman Malaysia, Pahang – Wen 8366 (US) *EU669107; *EU669176 P. dolichobotrys (Laut. & K.Sch.) Kalkman Indonesia, Papua Prov. – Wen 10703 (US) HM856813; HM856824 Maddenia hypoleuca Koehne USA, cult. – Lee & Wen 5005 (CS) *AF179549-50; *EU669117 Maddenia himalaica Hook.f. & Thomson China, Tibet – Wen et al. 2612 (US) HM856814; HM856825 Maddenia incisoserrata T.T.Yü & T.C.Ku China, Zhejiang – Wen 11291 (US) HM856815; HM856826 Oemleria cerasiformis (Hook. & Arn.) Landon USA, cult. – Lee & Wen 5002 (CS) *AF179553-54; *EU669115 Physocarpus monogynus (Torr.) Coult USA, Colorado – Owens 205 (CS) NA; *EU669112 Physocarpus opulifolius (L.) Maxim. – ‡AY555326; NA Spiraea densiﬂora Nutt. USA – Potter 970619-02 (DAV) ‡DQ88362; NA Spiraea cantoniensis Lour. USA, cult. NA; ‡DQ851556

Classiﬁcation scheme follows Rehder (1940), Mason (1913) and Kalkman (1965). CS, Colorado State University Herbarium; DAV, Herbarium of the University of California, Davis; F, Field Museum of Natural History Herbarium; US, US National Herbarium. *Sequences reported in Wen et al. (2008). †Sequences reported in Bortiri et al. (2002). ‡Sequences reported in Potter et al. (2007).

© 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 164, 236–245 MERGING MADDENIA WITH PRUNUS (ROSACEAE) 239 using Sephadex columns (Amersham Pharmacia informative. The number of potentially parsimony Biotech, Piscataway, NJ, USA). The sequences were informative characters excluding outgroups was 66. generated on an ABI PRISM 3730xl capillary The ITS data matrix had a total aligned length of sequencer (Applied Biosystems, Foster City, CA, 737 bp, including 256 variable characters and 135 USA). All the sequences generated and employed in potentially parsimony informative characters. One this study are listed as GenBank accession numbers hundred and six characters were potentially parsi- in Table 1. The generated DNA sequences were edited mony informative among the ingroup taxa. The in Sequencher™ 4.1.2 (Gene Codes Corporation, Ann Kimura two-parameter genetic distances were in the Arbor, MI, USA) and aligned using Clustal X (Thomp- range 0.00611–0.08661 and 0.00103–0.02171 among son et al., 1997). Minor manual editing of the aligned Prunus spp. for the ITS and ndhF data matrices, Downloaded from https://academic.oup.com/botlinnean/article-abstract/164/3/236/2418483 by guest on 01 March 2020 sequences was performed using Se-Al 2.0 (Rambaut, respectively. The average genetic distance observed 1996). was 0.045615 for the ITS data and 0.01137 for the ndhF data. For the ITS matrix, the maximum genetic distance (0.08661) was observed between P. dolicho- PHYLOGENETIC RECONSTRUCTION botrys (‘Pygeum’) and P. havardii Mason (subgenus Gaps present in both ndhF and ITS datasets were Emplectocladus), whereas, in the plastid matrix mostly autapomorphic and not found among members (0.02171), this corresponded to the pair P. dolichobot- of subgenera Laurocerasus and Padus; therefore, they rys and P. mahaleb (Dougl.) L. (subgenus Cerasus). were treated as uninformative and were not coded separately. Phylogenetic analyses using maximum parsimony were performed in PAUP* 4.0b10 (Swof- PHYLOGENETIC ANALYSIS ford, 2001). Heuristic searches for the most parsimo- The parsimony analysis of nuclear ITS generated nious trees (MPTs) were conducted with the tree eight MPTs of 580 steps, CI = 0.46 and RI = 0.63. One bisection and reconnection (TBR) branch-swapping of these is shown in Figure 1. The MPT consisted algorithm, with 100 random taxon addition sequence mainly of two well-supported clades. One contained repetitions holding 10 trees at each step. Homoplasy members of subgenera Amygdalus, Prunus and in each dataset was estimated by consistency and Emplectocladus. Relationships within this clade were retention indices (CI and RI, respectively). Parsimony poorly resolved, but each subgenus was supported as bootstrap support (BS) for each node was estimated monophyletic. In the second clade, the species of from 500 replicates, simple taxon addition sequence subgenus Laurocerasus sampled in this study did not and TBR, and Max Trees set at 20 100. Pairwise form a monophyletic group. Instead, the Neotropical genetic distances were calculated using the Kimura species (‘Neotropica’) and the palaeotropical Pygeum two-parameter model (Kimura, 1980). The optimal spp. each formed a monophyletic group. The three model of molecular evolution was determined by the Maddenia spp. were supported together as monophyl- Aikaike Information Criterion (AIC) using Modeltest etic (BS = 97%), and were included with strong ver. 3.7 (Posada & Crandall, 1998; Posada & Buckley, support (BS = 78%) in a clade with some members of 2004). Bayesian inference was conducted with subgenera Laurocerasus and Padus. This group, MrBayes ver. 3.1.2 (Huelsenbeck & Ronquist, 2001) named the ‘Maddenia composite’ hereafter, was sister with four independent chains that were set to run for (BS = 80%) to subgenus Cerasus. The relationships one million generations and sampled every 100 gen- among the Maddenia composite and the tropical erations. On the basis of plots of the likelihood scores species were not resolved. of the trees to locate the stable likelihood plateau, the Maximum parsimony analysis of the ndhF initial samples of 1200–1600 trees were discarded as sequences produced 29 MPTs, with a length of 342 burn-in, and the 50% majority-rule consensus tree steps, CI = 0.68 and RI = 0.86, and the overall topol- was constructed from the remaining trees. Bayesian ogy was best represented by one of the 29 MPTs in posterior probability (PP; also known as clade cred- Figure 2. Two major clades were formed, one includ- ibility) values were estimated on the basis of the ing members of subgenera Amygdalus, Cerasus, frequency of occurrence of each clade. Emplectocladus and Prunus, and the second including members of Maddenia and subgenera Laurocerasus RESULTS and Padus. Within the ﬁrst clade, subgenera Cerasus and Emplectocladus were both supported as mono- CHARACTERISTICS OF THE NUCLEAR AND PLASTID phyletic, whereas neither the monophyly of sub- DATA MATRICES genus Amygdalus nor that of subgenus Prunus was The total aligned length of ndhF sequences, including supported. indels (gaps), was 1952 bp, 1703 characters of which Within the second clade, the three Maddenia spp. were constant and 110 were potentially parsimony were supported as a monophyletic group (BS = 62%)

© 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 164, 236–245 240 S.-W. CHIN ET AL. Downloaded from https://academic.oup.com/botlinnean/article-abstract/164/3/236/2418483 by guest on 01 March 2020

Figure 1. One of eight maximally parsimonious trees of Prunus and Maddenia based on nuclear internal transcribed spacer (ITS) sequence data [L = 580, consistency index (CI) excluding informative characters, 0.46; retention index (RI), 0.63]. Numbers above the branches are bootstrap values (> 70%), and numbers below the branches represent Bayesian posterior probability values (> 90%). Asterisks (*) indicate the clades that collapse in the strict consensus tree. Abbreviations for subgenera: Amyg., Amygdalus; Cs., Cerasus; Emp., Emplectocladus; Lc., Laurocerasus; Pd., Padus; Pru., Prunus. and the Maddenia composite was not supported. majority-rule consensus trees resulting from Baye- Among the species of subgenus Laurocerasus, the sian analyses of both ITS and ndhF data were largely ‘Neotropica’ and Pygeum groups were both shown to similar in topology to the MPTs inferred by maximum be monophyletic. parsimony, but the support values were generally The evolutionary model selected by AIC for both the higher (Figs 1, 2). In contrast with the low BS for the nuclear and plastid data was TRN + I + G. The 50% monophyly of Maddenia spp. in the ndhF dataset,

© 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 164, 236–245 MERGING MADDENIA WITH PRUNUS (ROSACEAE) 241 Downloaded from https://academic.oup.com/botlinnean/article-abstract/164/3/236/2418483 by guest on 01 March 2020

Figure 2. One of 29 maximally parsimonious trees of Prunus and Maddenia based on the plastid ndhF sequence data [L = 342, consistency index (CI) excluding informative characters, 0.69; retention index (RI), 0.86]. Numbers above the branches are bootstrap values (> 70%), and numbers below the branches represent Bayesian posterior probability values (> 90%). Asterisks (*) indicate clades that collapse in the strict consensus tree. Abbreviations for subgenera: Amyg., Amygdalus; Cs., Cerasus; Emp., Emplectocladus; Lc., Laurocerasus; Pd., Padus; Pru., Prunus. this group was strongly supported as monophyletic by ported to be monophyletic (PP = 0.99). However, the Bayesian reconstruction (PP = 1.0). The Maddenia sister relationship of subgenus Cerasus to the Mad- composite recovered from nuclear ITS was also sup- denia composite was not well supported (PP = 0.54).

As a result of the conﬂicting placement of subgenus therefore, the monophyly of Maddenia has never been Cerasus in analyses of nuclear vs. plastid genes assessed. In our study, the three Maddenia spp. were observed in this and other studies, supported by a strongly supported as monophyletic in both nuclear previous signiﬁcant partition homogeneity test ITS (BS = 97%, PP = 1.0) and plastid ndhF (PP = 1.0) (P = 0.01) conducted by Wen et al. (2008) in their analyses. This result, combined with the fact that all ndhF and ITS analyses, the two datasets from ITS six species of Maddenia share many morphological and ndhF were not combined for further analysis. similarities, including the absence of two differentiated perianth whorls, suggests a single evolutionary origin of the Maddenia group.

DISCUSSION The phylogenetic position of Maddenia was poorly Downloaded from https://academic.oup.com/botlinnean/article-abstract/164/3/236/2418483 by guest on 01 March 2020 This study extends the analysis by Wen et al. (2008), resolved in all previous analyses. It was first shown adding more samples of species of Maddenia and as sister to subgenus Prunus (Lee & Wen, 2001), and tropical species of Laurocerasus in order to under- was subsequently shown to be nested within the clade stand better the phylogenetic position of Maddenia of subgenera Laurocerasus and Padus (Bortiri et al., within the clade formed by subgenera Laurocerasus 2006; Wen et al., 2008). The additional sampling of and Padus. The poor resolution of our plastid phylo- the tropical species of Laurocerasus in our study has genetic analysis was evidently a result of the conser- more precisely defined the position of Maddenia vative nature of the protein coding genes, which within the Laurocerasus–Padus clade. It is also inevitably leads to a low phylogenetic signal present strongly indicated here that Maddenia has a close in the data matrix. In the ndhF dataset, there were phylogenetic relationship with a group of temperate 66 potentially parsimony informative characters Laurocerasus–Padus species (Maddenia composite). among ingroup Prunus taxa, compared with 106 in Moreover, Maddenia was also found to be more the nuclear ITS data, and the average pairwise closely allied to the temperate Laurocerasus (P. lau- genetic distance between ingroup taxa was 0.01 for rocerasus L., P. lusitanica L and P. wallichii) than to ndhF compared with 0.04 for the four-fold more diver- the species of Padus. All species of Maddenia and gent ITS sequences. Laurocerasus in the Maddenia composite share similar gross fruit morphology of a rounded base with an acute apex, in contrast with the spherical to ellip- PHYLOGENETIC RELATIONSHIP OF MADDENIA soid fruits found in most other species of Prunus.The WITH PRUNUS determination of whether or not this morphological Maddenia is nested within a clade of temperate taxa trait is a synapomorphy for this clade will require a of subgenera Laurocerasus and Padus as supported wider survey in the future. (BS = 78%; PP = 0.99) by our nuclear ITS phylogenetic analysis. Although the Maddenia composite clade was not recovered in the strict consensus tree PECULIAR MORPHOLOGY OF MADDENIA for the ndhF analysis, the group was recovered in Maddenia has always been treated as a distinct eight of the 29 MPTs obtained (Fig. 2). In addition, genus, closely related to Prunus. The presence of the the genetic distances between M. hypoleuca and the predominantly one- but sometimes two-carpellate non-Maddenia members of the composite clade gynoecium, the absence of two distinct perianth ranged from 0.00568 (P. wallichii Steud.) to 0.00621 whorls and the apparent lack of the diagnostic (P. virginiana L. var. virginiana), whereas the genetic ‘Prunus’ gland, a type of petiolar gland seen in cherry distances between M. hypoleuca and P. lancilimba and peach, have been cited as characters distinguish- (Merr.) Kalkman (‘Pygeum’) and P. oleifolia Koehne ing Maddenia from Prunus. However, the inclusion (‘Neotropica’) were 0.00878 and 0.0093, respectively. of species of the former Pygeum, also characterized Hence, the Maddenia composite clade was probably by its ill-differentiated perianth segments, into not supported in all trees from our ndhF analysis Prunus subgenus Laurocerasus (Kalkman, 1965) because of the low sampling of informative charac- makes the inclusion of Maddenia in Prunus no longer ters. The following discussion is therefore based on an anomalous. a priori assumption that the Maddenia composite is On the basis of the seemingly identical floral trait monophyletic. (absence of differentiated perianth), most authors The generic status of Maddenia was first ques- (Bortiri et al., 2006; Wen et al., 2008) have posited a tioned through a nuclear ITS phylogenetic analysis by close relationship of Maddenia with the Pygeum Lee & Wen (2001), in which it was shown to be nested group. This relationship is nonetheless remote, as within Prunus, and its recognition would render the Maddenia forms its closest alliance with the temper- latter genus paraphyletic. However, only one species ate species, whereas Pygeum is more closely associ- (M. hypoleuca) of six was included in their analysis; ated with the tropical species. Thus, the loss of the

© 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 164, 236–245 MERGING MADDENIA WITH PRUNUS (ROSACEAE) 243 distinction between sepals and petals seems to have specimens (M. himalaica Hook.f. & Thomson occurred twice in Prunus (in Maddenia and Pygeum). US#3105861, Bartholomew 1552; M. hypoleuca The ancestral floral traits for Prunus have been Koehne US#1331826, JF Rock 12531; M. hypoxantha shown to be ‘Padus-like’, i.e. biseriate flowers borne Koehne US#1245429, RC Ching 391; and M. wilsonii on a leafy raceme (Bortiri et al., 2006). On the basis of Koehne (= M. hypoxantha) US#1626817, WC Cheng this assumption, it is most parsimonious to suggest 705) revealed that these resemble the depressed- that the floral traits in Maddenia and Pygeum are disciform apical glandular teeth along the leaf margin derived within Prunus. of Cerasus discadenia (Koehne) C. L. Li & S. Y. Jiang (US#1332895, JF Rock 8694). Consequently, on the

basis of our molecular phylogenetic analyses and mor- Downloaded from https://academic.oup.com/botlinnean/article-abstract/164/3/236/2418483 by guest on 01 March 2020 THE MADDENIA COMPOSITE AS A PLAUSIBLE POLLEN phological observations, we conclude that the pater- SOURCE FOR THE HYBRID ORIGIN OF CHERRY nal lineage that gave rise to subgenus Cerasus (SUBGENUS CERASUS) probably belonged to the Maddenia composite clade. The incongruent position of subgenus Cerasus,as observed in separate phylogenetic analyses of plastid and nuclear sequences, as shown here and in previous TAXONOMIC SYNOPSIS OF THE MADDENIA GROUP studies (Bortiri et al., 2001, 2006; Wen et al., 2008), OF PRUNUS has been hypothesized to be the result of an ancient This study is part of a larger comprehensive system- hybridization event between two major clades: Clade atic study of Prunus, with one of the main goals being A containing subgenera Amygdalus, Prunus and to provide a new infrageneric classification for the Emplectocladus and Clade B comprising subgenera genus. As that study is still in progress, it is premature Laurocerasus and Padus. to say whether Maddenia and/or the Maddenia com- The plastid genome is maternally inherited in posite clade will be given formal taxonomic recogni- nearly all angiosperms, including Prunus, e.g. Prunus tion, and, if so, at which rank(s) (subgenus, section, cerasus (Brettin et al., 2000). The maternal lineage etc.). Nevertheless, our results so far have firmly for subgenus Cerasus has been unambiguously indi- placed Maddenia within Prunus. We provide here the cated to be derived from Clade A as pointed out in the nomenclatural changes concerning the merging of plastid phylogenetic analyses. All phylogenetic analy- Maddenia with Prunus. A detailed taxonomic revision ses with nuclear markers, e.g. nuclear ribosomal ITS of the group is in preparation and will include more (Bortiri et al., 2001, 2006; Lee & Wen, 2001 and Wen detailed information concerning species’ delimitation et al., 2008) and sorbitol 6 dehydrogenase (s6pdh) criteria, descriptions and taxonomic keys. genes (Bortiri et al., 2002), have supported subgenus Cerasus as sister to Clade B but, because of the low resolution within this clade, the direct paternal 1. Prunus fujianensis (Y.T.Chang) J.Wen, comb. lineage could not be satisfactorily inferred. Our nov. results provide a first indication that the paternal Basionym: Maddenia fujianensis Y.T.Chang, Guihaia lineage is likely to have been part of or closely related 5: 25. 1985. to the Maddenia composite clade, as shown by the Distribution: east China. strong support for the sister relationship of subgenus 2. Prunus himalayana J.Wen, nom. nov. Cerasus to that group (BS = 80%). Basionym: Maddenia himalaica Hook.f. & Thomson, In addition to the molecular evidence that supports Hooker’s J. Bot. Kew Gard. Misc. 6: 381. 1854, non this relationship, several lines of morphological evi- Prunus himalaica Kitam. dence are apparent. A wood anatomical study across Distribution: Bhutan, Nepal, north India and west Rosaceae, conducted by Zhang (1992), revealed that China. Padus, together with a group designated as Lauro- Notes: Maddenia himalaica var. glabrifolia H.Hara is cerasus A, was closer to Amygdalus, Prunus and not conspecific with Prunus himalayana and repre- Cerasus, whereas a group designated as Laurocerasus sents a yet-to-be described species (J.Wen, unpubl. B was closer to the Pygeum group. The Laurocerasus data). A members were all temperate species, including P. 3. Prunus hypoleuca (Koehne) J.Wen, comb. nov. laurocerasus and P. lusitanica, and the Padus species Basionym: Maddenia hypoleuca Koehne, in were P. padus and P. virginiana;theLaurocerasus B C.S.Sargent, Pl. Wilson. 1: 56. 1911. group included P. phaeosticta (Hance) Maxim. and P. Distribution: central China. zippeliana Miq. Laurocerasus A and Padus in Zhang’s 4. Prunus hypoxantha (Koehne) J.Wen, comb. nov. study belonged to the Maddenia composite in our Basionym: Maddenia hypoxantha Koehne, in study. Furthermore, examination of the glandular- C.S.Sargent, Pl. Wilson. 1: 57. 1911. tipped teeth along the leaf margin in four Maddenia Distribution: central and west China.

Notes: Maddenia wilsonii Koehne may be best treated Bortiri E, Oh S-H, Jiang J-G, Baggett S, Granger A, as a synonym of Prunus hypoxantha (J.Wen, unpubl. Weeks C, Buckingham M, Potter D, Parfitt DE. data) and therefore its name is not changed in the 2001. Phylogeny and systematics of Prunus (Rosaceae) present article. as determined by sequences analysis of ITS and the chlo- 5. Prunus incisoserrata (T.T.Yü & T.C.Ku) J.Wen, roplast trnL-trnF spacer DNA. Systematic Botany 26: 797– comb. nov. 807. Basionym: Maddenia incisoserrata T.T.Yü & T.C.Ku, Bortiri E, Vanden Heuvel B, Potter D. 2006. Phylogenetic Acta Phytotax. Sin. 23(3): 214. 1985. analysis of morphology in Prunus reveals extensive homoplasy. Plant Systematics and Evolution 259: 53–71. Distribution: east and central China. Brettin TS, Karle R, Crowe EL, Iezzoni AF. 2000. Chlo- Maddenia pedicellata Hook.f. has been treated as Downloaded from https://academic.oup.com/botlinnean/article-abstract/164/3/236/2418483 by guest on 01 March 2020 roplast inheritance and DNA variation in Sweet, Sour, and belonging to the Cerasus group of Prunus, specifically Ground Cherry. Journal of Heredity 91: 75–79. as a synonym of Prunus cerasoides Buchanan- Cuervo BE, Francia BA, Fraile GB. 1978. Clinical study of Hamilton ex D.Don (see Li, Jiang & Bartholomew, a phytoesterol extract of Prunus arborea and 3 amino acids: 2003). glycine, alanine and glutamic acid. Archivos Españoles de Urologia 31: 97–98. CONCLUSION Focke WO. 1894. Rosaceae. In: Engler A, Prantl K, eds. Die natürlichen Pflanzenfamilien, Vol. VIII. Leipzig: Engel- The monophyly of the Maddenia group and its inclu- mann, 1–61. sion in Prunus are supported by our results. More- Hooker JD, Thomson T. 1854. On Maddenia and Dia- over, both the plastid and nuclear phylogenetic plarche, new genera of Himalayan plants. Hooker’s Journal analyses support Maddenia as closely allied with a of Botany 6: 380–384. group of temperate species of subgenera Laurocerasus Huelsenbeck JP, Ronquist R. 2001. MrBAYES: Bayesian and Padus, and Maddenia appears to be closer to the inference of phylogenetic trees. Bioinformatics 17: 754–755. members of subgenus Laurocerasus. Tropical Kalkman C. 1965. The Old World species of Prunus subgenus members of subgenus Laurocerasus form a lineage Laurocerasus including those formerly referred to Pygeum. distinct from the temperate members and, among the Blumea 13: 1–115. tropical species, the palaeotropical Pygeum and neo- Kimura M. 1980. A simple method for estimating evolution- tropical Laurocerasus species are phylogenetically ary rates of base substitution through comparative studies distinct. The previous hypothesis of Maddenia of nucleotide sequences. Journal of Molecular Evolution 16: sharing a closer relationship to Pygeum based on 111–120. their similar floral traits is refuted by our study. Lee S, Wen J. 2001. A phylogenetic analysis of Prunus and the Amygdaloideae (Rosaceae) using ITS sequences of Formal nomenclatural changes are made here to nuclear ribosomal DNA. American Journal of Botany 88: transfer Maddenia into Prunus. 150–160. Li C-L, Jiang S-Y, Bartholomew B. 2003. Cerasus.In:Wu ACKNOWLEDGEMENTS C-Y, Hong D, Raven PH, eds. Flora of China. v. 9. Beijing & St. Louis: Science Press & Missouri Botanical Garden, 404– This study was supported by NSF Award number 420. 0515431 and the Smithsonian Endowment Program. Mason SC. 1913. The pubescent-fruited species of Prunus of New sequences were generated at the Laboratory of the Southwestern States. Journal of Agricultural Research Analytical Biology of the Smithsonian Institution. 1: 147–179. Field assistance was provided by M. Dillon, C.-X. Fu, Mowrey BD, Werner DJ. 1990. Phylogenetic relationships N. T. Hiep, N. Q. Hieu, M. Nee, Z.-L. Nie, E. Ortiz, among species of Prunus as inferred by isozyme markers. Y.-X. Qiu and E. Widjaja. Theoretical and Applied Genetics 80: 129–133. Olmstead RG, Sweeney JA. 1994. Combining data in phylogenetic systematics: an empirical approach using three REFERENCES molecular data sets in Solanaceae. Systematic Biology 43: Andro MC, Riffaud JP. 1995. Pygeum africana extract for 467–481. the treatment of patients with benign prostatic hyperplasia: Posada D, Buckley TR. 2004. Model selection and model a review of 25 years of published experience. Current Thera- averaging in phylogenetics: advantages of the AIC and peutic Research 56: 796–817. Bayesian approaches over likelihood ratio tests. Systematic Backer CA, Bakhuizen van den Brink RC. 1963. Flora of Biology 53: 793–808. Java, v.I. Groningen: P. Noordhoff. Posada D, Crandall KA. 1998. Modeltest: testing the model Bortiri E, Oh S-H, Gao F-Y, Potter D. 2002. The phyloge- of DNA substitution. Bioinformatics 14: 817–818. netic utility of nucleotide sequences of sorbitol 6-phosphate Potter D, Eriksson T, Evans RC, Oh S-H, Smedmark dehydrogenase in Prunus (Rosaceae). American Journal of JEE, Morgan DR, Kerr M, Robertson KR, Arsenault M, Botany 89: 1697–1708. Dickinson TA, Campbell CS. 2007. Phylogeny and

classification of Rosaceae. Plant Systematics and Evolution Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, 266: 5–43. Higgins DG. 1997. The ClustalX windows interface: flex- Rambaut A. 1996. Sequence alignment editor 2.0. Available ible strategies for multiple sequence alignment aided by at http://tree.bio.ed.ac.uk/software/seal/ quality analysis tools. Nucleic Acids Research 25: 4876– Rehder A. 1940. Manual of cultivated trees and shrubs hardy 4882. in North America exclusive of the subtropical and warmer Wen J, Berggren ST, Lee C-H, Ickert-Bond S, Yi T-S, Yoo temperate regions, 2nd edn. New York: MacMillan. K-O, Xie L, Shaw J, Potter D. 2008. Phylogenetic infer- Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular ences in Prunus (Rosaceae) using chloroplast ndhF and cloning: a laboratory manual, 2nd edn. New York: Cold ribosomal ITS sequences. Journal of Systematics and Evo- Spring Harbor Laboratory Press. lution 46: 322–332. Downloaded from https://academic.oup.com/botlinnean/article-abstract/164/3/236/2418483 by guest on 01 March 2020 Sterling C. 1964. Comparative morphology of the carpel in Wolfe LM, Drapalik DJ. 1999. Variation in the degree of the Rosaceae. II. Amygdaloideae: Maddenia, Pygeum, andromonoecy in Prunus caroliniana. Castanea 64: 259– Osmaronia. American Journal of Botany 51: 354–360. 262. Swofford DL. 2001. PAUP*. Phylogenetic analysis using par- Yü T-T, Lu L-T, Ku T-C, Li C-L, Chen S-X. 1986. Rosaceae simony (*and other methods), version 4. Sunderland, MA: (3) Prunoideae. In: Yü T-T, ed. Flora reipublicae popularis Sinauer. sinicae, Vol. 38. Beijing: Science Press, 1–133. Takhtajan A. 1997. Diversity and classification of flowering Zhang S-Y. 1992. Systematic wood anatomy of the Rosaceae. plants. New York: Columbia University Press. Blumea 37: 81–158.