Botanical Studies (2012) 53: 151-164.

SYSTEMATICS


	Generic limits of Pyrinae: Insights from nuclear ribosomal DNA sequences
	Qing-Yan LI¹, Wei GUO¹, Wen-Bo LIAO¹, James A. MACKLIN², and Jian-Hua LI³
	¹Sun Yat-sen University, School of Life Sciences, Guangdong Key Laboratory of Plant Resources, Guangzhou, Guangdong, 510275, P.R. China ²Harvard University Herbaria, Organismal and Evolutionary Biology, 22 Divinity Avenue, Cambridge, Massachusetts, 02138, USA ³Biology Department, Hope College, MI 49423, USA
	(Received August 23, 2010; Accepted October 6, 2011)
	ABSTRACT. The subtribe Pyrinae, formerly the Maloideae, is a monophyletic group of about 1,000 species that includes well known fruit crops such as apple (Malus), pear (Pyrus), quince (C^ydonia), loquat (Eriobotrya), chokeberry (Aronia), and serviceberry (Amelanchier). Generic limits have been fluid in Pyrinae, especially in Malus, Sorbus and Photinia. This study evaluated the generic limits of 180 samples of multiple species or accessions from each of the traditional genera using sequences of the nrDNA ITS region. The ITS data recognized 24 genera, including Amelanchier, Aria (including Micromeles), Aronia, Chaenomeles, Chamaemespilus, Chamaemeles, Cormus, Cotoneaster, Crataegus, Cydonia, Dichotomanthes, Eriobotrya, Hesperomeles, Mala-comeles, Malus (including Chloromeles, Docynia, Docyniopsis, and Eriolobus), Mespilus, Osteomeles, Pera-phyllum, Pourthiaea, Pseudocydonia, Pyrus, Rhaphiolepis, Sorbus, and Torminalis. However, both Photinia and Pyracantha are polyphyletic. Photinia is separated into different clades, one of which contains species of Heteromeles and Stranvaesia. Asian species of Pyracantha do not form a clade with P. coccinea of southern Europe and Iran. Our results support the close relationship of Amelanchier, Malacomeles, and Peraphyllum, and of Crataegus and Mespilus, and for the first time recognize the sister relationship of the South American genus Hesperomeles with the Crataegus-Mespilus clade.
	Keywords: Generic limits; Hesperomeles; Maloideae; nrDNA ITS Pyrinae.


	INTRODUCTION
	The Rosaceae subtribe Pyrinae, formerly subfamily Maloideae (Potter et al., 2007), contains about 1000 species (Phipps et al., 1990), many of which are economically important, such as apple (Malus domestica Borkh.), pear (Pyrus pyrifolia Nakai), loquat (Eriobotrya Lindl.), and chokeberry (Aronia Mitchell). The Pyrinae is defined by several synapomorphic characters: the pome fruit, base number of chromosomes x=17 (Phipps et al., 1991), rust parasites (Savile, 1979), and gametophytic apomixis (Campbell et al., 1991). Menz (1964) divided Pyrinae into two tribes: Crataegeae, with fruit called polypyre-nous drupes (Kalkman, 1988; Baird and Thieret, 1989), in which most or the entire ovary wall becomes hard, and each carpel forms a separate nutlet or pyrene (Rohrer et al., 1991), as with Crataegus and Pyracantha; and Sor-beae (Maleae), with connate endocarps, a membranous to cartilaginous inner ovary wall, and connate carpels form-

	*Corresponding author: E-mail: li@hope.edu, Tel: +001616-395-7460, Fax: +001-616-395-7125 (Jianhua LI); E-mail: lsslwb@yahoo.com.cn, Tel: +86-020-84115882 (Wenbo LIAO).


	ing a single multilocular core (Rohrer et al., 1991), as with Malus and Pyrus. Although Rohrer et al. (1991) studied Pyrinae fruit structure and could not substantiate its division based on core textures, the circumscription of Pyrinae has never been seriously challenged. Minor changes have included the removal of Dichotomanthes S. Kurz by Glad-kova (1969), the inclusion of Vauquelinia Correa ex Bonpl in the subfamily by Goldblatt (1976), the inclusion of Vauquelinia, Limdleya and Kageneckia in two tribes of Py-roideae, and the division of Pyrinae into Maleae and Cra-taegeae by Takhtajan (1997). Recent phylogenetic studies, however, support the placement of Dichotomanthes in the Pyrinae and the sister relationship Pyrinae has with Vau-quelinia, Lindleya and Kageneckia (Evans et al., 2000).
	Generic limits within Pyrinae, however, have been controversial (Linnaeus, 1753; Lindley, 1822; de Candolle, 1825; Lindley, 1837; Decaisne, 1874; Focke, 1888; Koeh-ne, 1891; Fritsch, 1898; Fritsch, 1899; Lindley, 1845; Ro-emer, 1847; Wenzig, 1883; Rehder, 1940; Rehder, 1949; Robertson et al., 1991). The center of the controversy lies in the circumscriptions of Sorbus L., Malus Mill., and Photinia Lindl. There are two concepts of Sorbus. Wenzig (1883) used Sorbus broadly and included Chamaemespi-

152

Botanical Studies, Vol. 53, 2012


	lus, Aria, Torminaria M. Roem., Aronia, Eriolobus, Cor-mus, and Sorbus species. Roemer (1847) and Robertson et al. (1991), however, gave them all generic status. Chlo-romeles (Decne.) Decne. was placed with Malus Wenzig (1883), and Robertson et al. (1991). Stanvaesia Lindl. is morphologically very similar to Photinia Lindl. and the two genera have sometimes been merged under Photinia Lindl. (Vidal, 1965; Kalkman, 1973); Aronia Medik. has been considered a close relative of Photinia, and is sometimes listed with that genus (Robertson et al., 1991), Pourithiaea Decaisne is sometimes considered morphologically or anatomically distinct from Photinia (Iketani and Ohashi, 1991; Zhang, 1992; Lu et al., 2003).
	A cladistic analysis of morphological characters by Phipps et al. (1991) concluded that while the genera formed clusters, the consistency was low, such that adding a few OTUs changed the placement of the genera. The low consistency reflects the fact that Pyrinae genera hybridize easily (Robertson et al., 1991), as with Crataegus x Sorbus, Cotoneaster x Sorbus, Crataegus x P-yrus, Pyracantha x Osteomeles, Pyracamtha x Cotoneaster, Cydonia x Pyrus, Cydonia x Malus, Malus x Sorbus. Only a few of these hybrids, such as Sorbus x Aria, Sorbus x Torminalis and Sorbus x Chamaemespilus, however, regularly occur in nature, and this has been taken to indicate close relationships among these genera.
	Although several molecular phylogenetic analyses of the Pyrinae have been conducted in recent years (Morgan et al., 1994; Campbell et al., 1995; Evans et al., 2000; Evans and Campbell, 2002; Campbell et al., 2007; Potter et al., 2007), intergeneric relationships remain unresolved. The lack of phylogenetic information may be partially due to possible rapid radiation of the Pyrinae generic lineages (Campbell et al., 2007). To date, no molecular phyloge-netic analyses have focused on the generic limits of the Pyrinae. Our objective was thus to evaluate these generic limits by sampling multiple species or accessions from each of the possible genera. We used sequences from the internal transcribed spacer regions of nuclear ribosomal DNA (nrDNA ITS), as is common in phylogenetic reconstructions of flowering plants, including Rosaceae (Campbell et al., 1995; Campbell et al., 1997; Oh and Potter, 2003; Lo et al., 2007).
	MATERIALS AND METHODS
	This study included 180 samples representing the geographic distribution and morphological diversity of all genera of the Pyrinae (81 samples, representing 73 species, are studied here for the first time) (Table 1). Lindleya Kunth, Kageneckia Ruiz & Pav., and Vauquelinia were used for rooting purposes since they are most closely related to the Pyrinae (Campbell et al., 1995; Evans and Campbell, 2002; Morgan et al., 1994).
	Genomic DNAs were extracted from fresh or silica gel-dried leaf material using a DNeasy Plant Mini Kit following manufacturer's instructions (Qiagen, Valencia, CA).


	Polymerase chain reactions (PCR) were conducted using a MJ Research Thermocycler or an Eppendorf Mastercycler in a 25 fil reaction system. The PCR protocols and thermo-cycler programs followed Li (2008). PCR products of the expected size were cut from 1% agarose gels and purified using a Qiagen Gel Purification Kit. Direct sequencing of the purified PCR products was done using an ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA polymerase, FS. Sequences were obtained using an ABI 3730 Automated Genetic Analyzer and edited in Sequencher (version 4.0, Ann Arbor Gene Code, Inc.). Sequences were aligned using the MUSCLE program (Edgar, 2004), available freely at http://www.drive5.com/muscle/ download3.6.html, with a slight manual adjustment. Ambiguously aligned regions, where indels could be inserted in more than one site, were excluded from phylogenetic analyses.
	Both maximum parsimony (MP) and Bayesian inference (BI) analyses were used to reconstruct phylogenetic trees of the Pyrinae. Characters were equally weighted and their states were unordered. MP analyses were done in PAUP* (version 4.0b) (Swofford, 2002) using the heuristic tree search algorithm with the following options: random sequence addition of 5000 replicates with one tree held per replicate, MAXTREES set to 20,000, TBR branch swapping, MULTREES on, and STEEPEST DESCENT off. Bootstrap analyses of 10,000 replicates were performed to evaluate support for individual clades (Felsenstein, 1985) using the FAST STEPWISE ADDITION search in PAUP* due to the large data set size. Bayesian analyses were conducted for two runs using the MRBAYES computer program (Ronquist and Huelsenbeck, 2003) and the best-fit model selected by the hierarchical likelihood ratio test in the MODELTEST (Posada and Crandall, 1998). Four MCMC chains were run for 2,000,000 generations with trees sampled every 1,000 generations. The likelihood scores were plotted against the number of generations; all generations prior to likelihood stationarity were discarded as burn-in. The 50% majority rule consensus of the re-maining 4001 trees was used to calculate posterior prob-abilities (pp) for individual clades.
	RESULTS
	The ITS data set had a total of 180 sequences, including 81 sequences reported here for the first time. The sequence alignment included 710 sites, 385 of which were variable and 247 of which were parsimony informative. A 63-bp fragment in the ITS-1 was excluded from analyses due to alignment ambiguity. The MP analyses produced 20, 000 trees (the limit set on Maxtrees) with a consistency index of 0.39 and retention index of 0.76. The best-fit evolutionary model of the nrDNA ITS data was the TVM+I+G model as selected by the MODELTEST. In the BI analyses, the maximum likelihood scores reached plateau in 148,000 generations; thus the first 148 trees were discard-ed as burn-in, and the remaining trees were used to obtain the pp for individual clades.

LI et al. ― Generic limits of Pyrinae

153


Table 1. Species, their source, and vouchers used in Pyrinae phylogenetic analyses.
Species	Source	Voucher	GenBank accessions
Amelanchier Medikus
Amelanchier arborea			EF127041
Amelanchier bartramiana			U15191
Aria (Persoon) Host
Aria coronata	Yunnan, China	Qingyan Li YN-003	FJ810012
Aria hemsleyi	Arnold Arboretum, U.S.A.	Qingyan Li 1771-80C	FJ810010
Aria yuana	Arnold Arboretum, U.S.A.	Qingyan Li 1539-80C	FJ810007
Aronia Mitchell
Aronia pyrifolia 1			U16199
Aronia pyrifolia 2			U16199
Aronia sp.			EF127043
Aronia arbutifolia	Arnold Arboretum, U.S.A.	Qingyan Li1905-81	FJ796911
Aronia melanocarpa	Arnold Arboretum, U.S.A.	Qingyan Li 1906-81MASS	FJ810003
Aronia prunifolia	Arnold Arboretum, U.S.A.	Qingyan Li 1389-83C	FJ810001
Chaenomeles Lindley
Chaenomeles cathayensis			U16186
Chamaemeles Lindley
Chaenomeles speciosa			AF186530
Chamaemeles coriacea			DQ811768
Chamaemespilus Medikus
Chamaemespilus alpina			DQ811769
Chamaemespilus alpina	Arnold Arboretum, U.S.A.	Qingyan Li 1110-65A	FJ810045
Chloromeles (Decaisne) Decaisne
Chloromeles coronaria			AF186524
Chloromeles coronaria			AF186525
Chloromeles ioensis			AF186526
Chloromeles angustifolia			AF186523
Cormus Spach
Cormus domestica 1			U16187
Cormus domestica 2	Arnold Arboretum, U.S.A.	Qingyan Li 1043-64A	FJ810017
Cotoneaster Medikus
Cotoneaster acutifdius	Arnold Arboretum, U.S.A.	Qingyan Li 00165718	FJ796931
Cotoneaster acutinatus	Arnold Arboretum, U.S.A.	Qingyan Li 00191805;00191728	FJ796921
Cotoneaster atropurpureus	Arnold Arboretum, U.S.A.	Qingyan Li 00166599	FJ796922
Cotoneaster przewalskii	Arnold Arboretum, U.S.A.	Qingyan Li 00223832	FJ796903
Cotoneaster adpressus	Arnold Arboretum, U.S.A.	Qingyan Li 00191505	FJ796933
Cotoneaster conspicuus	Arnold Arboretum, U.S.A.	Qingyan Li 00191716	FJ796937
Cotoneaster dielelanus	Arnold Arboretum, U.S.A.	Qingyan Li 00166620	FJ796919
Cotoneaster apiculatus	Arnold Arboretum, U.S.A.	Qingyan Li 7275A	FJ796933
Cotoneaster dielsianus	Arnold Arboretum, U.S.A.	Qingyan Li 13428B	FJ796920
Cotoneaster integerrimus	Xinjiang, China	Qingyan Li 780074	FJ796948
Cotoneaster melanocarpus	Xinjiang, China	Qingyan Li 780006	FJ796949
Cotoneaster melanocarpus	Arnold Arboretum, U.S.A.	Qingyan Li 00223183;00191532	FJ796946
Cotoneaster morrisonensis	Arnold Arboretum, U.S.A.	Qingyan Li 271-98A	FJ796941

154

Botanical Studies, Vol. 53, 2012


Table 1. (Continued)
Species	Source	Voucher	GenBank accessions
Cotoneaster perpusillus	Arnold Arboretum, U.S.A.	Qingyan Li 7157C	FJ796928
Cotoneaster procumbens	Arnold Arboretum, U.S.A.	Qingyan Li 1979-0164 A	FJ796938
Cotoneaster rotundifolius	Nanjing, China	Qingyan Li 0706-014	FJ796950
Cotoneaster soongoricus	Xi'an, China	Qingyan Li 780177	FJ796936
Cotoneaster verruculosus	Yunnan, China	Lihua Zhou GLGS 22004	FJ796935
Crataegus L.
Crataegus aestivalis			EF127023
Crataegus brachyacantha			EF127032
Crataegus calpodendron			EF127018
Crataegus chlorosarca			EF127009
Crataegus crusgalli			EF127010
Crataegus dahurica			EF127028
Crataegus heldreichii			EF127016
Crataegus hupehensis			EF127038
Crataegus kansuensis			EF127029
Crataegus laevigata			EF127015
Crataegus marshallii			EF127037
Crataegus maximowiczii			EF127030
Crataegus mollis 1			U16190
Crataegus mollis 2			EF127012
Crataegus monogyna			EF127014
Crataegus nigra			EF127007
Crataegus opaca			EF127022
Crataegus pentagyna			EF127035
Crataegus phaenopyrum			EF127034
Crataegus pubescens			EF127021
Crataegus punctata			EF127011
Crataegus saligna			EF127031
Crataegus sanguinea			EF127027
Crataegus songarica			EF127036
Crataegus spathulata			EF127033
Crataegus suksdorfii 1			EF127025
Crataegus suksdorfii 2			EF127026
Crataegus triflora			EF127019
Crataegus uniflora			EF127020
Crataegus viridis			EF127013
Crataegus wilsonii			EF127008
Crataegus lassa			EF127024
Cydonia Miller
Cydonia oblonga 1			U16189
Cydonia oblonga 2			AF186531
Dichotomanthes Kurz
Dichotomanthes tristanicarpa 1			DQ811770
Dichotomanthes tristanicarpa	Yunnan, China	Wei Guo 8305	FJ796909

LI et al. ― Generic limits of Pyrinae

155


Table 1. (Continued)
Species	Source	Voucher	GenBank accessions
Docynia Decaisne
Docynia delavayi	Yunnan, China	Lihua Zhou GLGS19031	FJ796912
Docyniopsis (C. K. Schneider) Koidzumi
Docyniopsis prattii			AF186511
Docyniopsis tschonoskii 1			AF186527
Docyniopsis tschonoskii 2			DQ811771
Docyniopsis yunnanensis			AF186508
Eriobotrya Lindley
Eriobotrya cavaleriei	Guangxi, China	Xiaomin Fu,1060435	FJ810022
Eriobotrya sp.	Yunnan, China	Qiang Fan Q6002	FJ810023
Eriobotrya fragrans	Guangdong, China	Xiaomin, Fu, 6050113	FJ810024
Eriobotrya fragrans	Guangxi, China	Wei Guo7236	FJ810025
Eriobotrya japonica			U16192
Eriobotrya tengyuehensis	Yunnan, China	Lihua Zhou GLGS 24171	FJ796915
Eriolobus (A. P. de Candolle) M. J. Roemer
Eriolobus trilobatus			AF186521
Hesperomeles Lindley
Hesperomeles palcensis		Paniagua 5770 (MOBOT)	FJ796914
Hesperomeles latifolia		Paniagua 5764 (MOBOT)	FJ810044
Heteromeles M. J. Roemer
Heteromeles arbutifolia			U16193
Malacomeles (Decaisne) Engler
Malacomeles denticulata			U16194
Malus Miller
Malus asiatica			EF442030
Malus asiatica			AF186494
Malus baccata			AF186501
Malus domestica			U16195
Malus doumeri			AF186529
Malus florentina			AF186520
Malus floribunda			EF493836
Malus fusca			AF186514
Malus halliana			AF186502
Malus honanensis			AF186510
Malus hupehensis			AF186503
Malus kansuensis			AF186512
Malus mandshurica			AF186504
Malus neidzwetzkyana			AF186495
Malus ombrophila			AF186513
Malus orientalis			AF186498
Malus orientalis			AF186499
Malus prunifolia			AF186500

156

Botanical Studies, Vol. 53, 2012


Table 1. (Continued)
Species	Source	Voucher	GenBank accessions
Malus sargentii			AF186507
Malus sieboldii			AF186505
Malus toringoides			AF186517
Malus transitoria			AF186518
Mespilus L.
Mespilus canescens			EF127039
Mespilus germanica 1			U16196
Mespilus germanica 2			EF127040
Micromeles Decaisne
Micromeles alnifolia 1			U16185
Micromeles alnifolia 2	Arnold Arboretum, U.S.A.	Qingyan Li 00160096--00160099	FJ796908
Micromeles alnifolia 3	Arnold Arboretum, U.S.A.	Qingyan Li 413-94-B	FJ810006
Micromeles caloneura	Yunnan, China	Wei Guo YN-019	FJ810008
Micromeles thomsonill	Jiangxi, China	Wei Guo SQ0809502	FJ810009
Micromeles tsinglingenis	Arnold Arboretum, U.S.A.	Qingyan Li 544-88E	FJ810011
Osteomeles Lindley
Osteomeles anthyllidifolia			AY864895
Osteomeles schwerinae 1			U16197
Osteomeles schwerinae 2	Yunnan, China	Wei Guo YN-30	FJ796910
Peraphyllum Nuttal ex Torrey & A. Gray
Peraphyllum ramosissimum			U16198
Photinia Lindley
Photinia davidsoniae	Nanjing, China	Qingyan Li, 0706019	FJ810005
Photinia glabra	Jiangxi, China	Wei Guo 10218	FJ796905
Photinia nussia	Arnold Arboretum, U.S.A.	Jianhua Li 1974-5668	FJ810004
Photinia prinophylla	Yunnan, China	Wei Guo YN-35	FJ810018
Photinia prunifolia	Zhejiang, China	Qingyan Li 8230	FJ810019
Photinia serralata	Jiangxi, China	Wei Guo 8564	FJ810021
Photinia tushanensis	Guangxi, China	Wei Guo 70722003	FJ810020
Pourthiaea Decaisne
Pourthiaea beauverdiana	Zhejiang, China	Qingyan Li 0706003	FJ796907
Purthiaea benthamiana			FJ810013
Pourthiaea benthamiana	Guangdong, China	Wei Guo 0013	FJ810014
Pourthiaea parvifolia	Jiangxi, China	Wei Guo 20120	FJ810015
Pourthiaea villosa	Guizhou, China	Wei Guo 283-82B	FJ810016
Pseudocydonia (C. K. Schneider) C. K. Schneider
Pseudocydonia sinensis			U16201
Pyracantha Roemer
Pyracantha angustifolia	Zhejiang, China	Qingyan Li H0706-006	FJ796916
Pyracantha coccineae 1			DQ811772
Pyracantha coccineae	Brooklyn Botanic Garden, U.S.A.	Jinshuang Ma BBG67068	FJ821024
Pyracantha fortuneaena	Zhejiang, China	Qingyan Li 706003	FJ810049

LI et al. ― Generic limits of Pyrinae

157


Table 1. (Continued)
Species	Source	Voucher	GenBank accessions
Pyrus L.
Pyrus calleryana			U16202
Pyrus caucasica	Arnold Arboretum, U.S.A.	Qingyan Li 1335-80B	FJ796917
Pyrus elaeagnifolia	Arnold Arboretum, U.S.A.	Qingyan Li 00186151	FJ810046
Pyrus pyrifolia 1	Arnold Arboretum, U.S.A.	Qingyan Li 00223812;00190718	FJ810047
Pyrus pyrifolia 2			AF287246
Pyrus salicifolia			AF186532
Pyrus ussuriensis	Arnold Arboretum, U.S.A.	Qingyan Li 00223291	FJ810050
Rhaphiolepis Lindley
Rhaphiolepis indica			GU947645
Rhaphiolepis indica			U16203
Sorbus L.
Sorbus acuparia	Arnold Arboretum, U.S.A.	Qingyan Li 1257-84A	FJ796913
Sorbus amabilis	Jiangxi, China	Wei Guo SQ0809501	FJ810033
Sorbus americana	Arnold Arboretum, U.S.A.	Qingyan Li 1845-66A	FJ810037
Sorbus aronioides	Yunnan, China	Wei Guo YN-013	FJ810031
Sorbus aucuparia			U16204
Sorbus discolor	Arnold Arboretum, U.S.A.	Qingyan Li 136-79A	FJ810026
Sorbus dumisa	Arnold Arboretum, U.S.A.	Qingyan Li 423-88A	FJ810041
Sorbus forrestii	Arnold Arboretum, U.S.A.	Qingyan Li 814-77-D	FJ810028
Sorbus huphensis	Arnold Arboretum, U.S.A.	Qingyan Li 1675-80C	FJ810027
Sorbus intermedia	Arnold Arboretum, U.S.A.	Qingyan Li 136-56A	FJ810036
Sorbus koehneana	Arnold Arboretum, U.S.A.	Qingyan Li 1693-80B	FJ810029
Sorbus pohuashanensis	Arnold Arboretum, U.S.A.	Qingyan Li 477-80B	FJ810034
Sorbus prattii	Yunnan, China	Lihua Zhou GLGS20390	FJ810032
Sorbus pteridophylla	Yunnan, China	Lihua Zhou GLGS20376	FJ810030
Sorbus rufo-ferruginea	Arnold Arboretum, U.S.A.	Qingyan Li 367-80A	FJ810038
Sorbus sambucifolia 1	Arnold Arboretum, U.S.A.	Qingyan Li 1730-77A	FJ810042
Sorbus sambucifolia 2	Arnold Arboretum, U.S.A.	Qingyan Li 1730-77A	FJ810048
Sorbus scopulina	Arnold Arboretum, U.S.A.	Qingyan Li 310-75A	FJ810039
Sorbus tianschanica	Xinjiang, China	Qingyan Li 0780061	FJ810043
Sorbus vilmorinii	Arnold Arboretum, U.S.A.	Qingyan Li 151-87B	FJ810040
Sorbus wilfordii	Arnold Arboretum, U.S.A.	Qingyan Li 326-86A	FJ810035
Stranvaesia
Stranvesia davidiana	Yunnan, China	Lihua Zhou GLGS22604	FJ796906
Torminalis Medikus
Torminalis clusii 1	Arnold Arboretum, U.S.A.	Qingyan Li 246-98C	FJ796918
Torminalis clusii 2			DQ811773
Torminalis clusii 3			AF186533
Vauquelinia
Vauquelinia californica			DQ811766
Vauquelinia corymbosa			DQ811767
Kageneckia angustifolia			DQ811764
Lindleya mespiloides			DQ811765

158

Botanical Studies, Vol. 53, 2012


	The MP (Figure 1) and BI (Figure 2) trees were largely congruent, with a few minor differences. In the MP tree (Figure 1), Amelanchier Medik., Malacomeles (Decne.) Engl. and Peraphyllum Nutt. formed a clade (bootstrap, bs=63%) that was sister to the remainder of the tribe (bs=89%). In the BI tree (Figure 2), however, Amelanch-ier, Malacomeles, Peraphyllum, Crataegus L., Mespilus L., and Hesperomeles Lindl. formed a robust clade (posterior probability, pp=100%). Crataegus and Mespilus each formed their own clades, and were sister to each other in both the MP and BI trees (Figures 1-2). Hesperomeles formed a clade (bs=pp=100%) but its sister relationship to the Mespilus-Crataegus clade was also poorly supported (pp=51%). Sorbus was moderately supported in the MP tree (bs=75%). In the BI tree, however, Sorbus had strong support (pp=99%).
	The ITS data provided moderate to strong support in both MP and BI trees (Figures 1-2) for monophyly of several genera, including Aronia (bs=98%, pp=100%), Chaenomeles Lindl. (bs=68%, pp=70%), Eriobotrya (bs=87%, pp=100%), Osteomeles Lindl. (bs=71%, pp=98%), Pourthiaea Decne (bs=93%, pp=100%), and Pyrus (bs=95%, pp=100%). Monophyly of Cotoneas-ter Rupp. was weakly supported by the MP analyses (bs=50%), but had strong support from the BI analyses (pp=95%). Pyracantha M. Roem. did not form a clade in either MP or BI tree (Figures 1-2). Neither Aria nor Micromeles Decne. formed its own clade. Nevertheless, together they formed a robust clade in both MP and BI trees (pp=100%, Figures 1-2). Malus was paraphyletic to Chloromeles, Eriolobus, Docynia Decne., and Docyni-opsis (C.K. Schneid.) Koidz. (Figures 1-2). Rhaphiolepis Lindl. was sister to Eriobotrya (bs=83%, pp=100%), while Photinia species appeared in different clades: some with Heteromeles M. Roem. and Stranvaesia Lindl., and others with unclear affinities. Nevertheless, the support for the relationships among Photinia, Heteromeles, Stranvaesia, and other genera was weak (Figures 1-2).
	DISCUSSION
	Generic limits have been controversial in the Pyrinae. Since Linnaeus's (1753, 1754) recognition of only four genera, many new Pyrinae genera have been proposed. The number of currently recognized genera is 28. Many genera (e.g., Cotoneaster, Crataegus, Osteomeles, Rhaphi-olepis, Eriobotrya, Pyrus) can be explicitly circumscribed by morphological characters, but the limits of some genera, (e.g., Amelanchier, Sorbus, Photinia, Malus, and Stranvaesia) have remained unclear. Relationships at the genus and species levels have been successfully resolved in Rosaceae using sequences of nrDNA ITS (Campbell et al., 1995; Lo et al., 2007). Incomplete concerted evolution may lead to the existence of paralogous copies within a single species and the failure to sample all copies may result in erroneous relationships. The paralogy of the ITS region is probably only a minor issue in our analysis because


	multiple individuals of the same species formed clades and our focus was on testing generic limits. To our knowledge, this study provides the first molecular evaluation of the generic limits of the Pyrinae with a comprehensive taxon sampling of each genus.
	Amelanchier is a disjunct genus between Eurasia and North America with most species in North America and only a few in Asia (Campbell et al., 1997). Apomixis, polyploidy and hybridization have caused the number of recognized species in the genus to range from six to thirty-three (Landery, 1975, Phipps et al., 1991). Amelanchier is easily distinguished from other Pyrinae genera by a combination of characters including racemose inflorescence, narrow petals, false locular septa in each locule, and pseudoberries (Robertson et al., 1991). Peraphyllum and Malacomeles share fruit characters with Amelanchier. However, Peraphyllum, a monotypic genus, has narrow, fascicled leaves, reduced inflorescences, and orange-colored fruits, while Malacomeles, with three species, has a xeromorphic habit and barely connate carpels. In the ITS trees, Amelanchier is monophyletic and closely related to Peraphyllum and Malacomeles, as reported in previous studies (Campbell et al., 1995; Campbell et al., 2007).
	Crataegus is a shrub or small tree genus of 186-256 species with a distribution in Eurasia, as well as North America (Phipps et al., 1990). The genus usually possesses lobed leaves, small fruits containing from one to five hard pyrenes, and most species have thorns, which do not occur in any other Pyrinae genera. Our broad analysis of the ITS data supports the monophyly of Crataegus. Mespilus was separated from Crataegus by Medikus in 1793 in light of the fact that its pyrenes are covered while Crataegus' are exposed. It is a small genus comprised of two species, one in southern Europe (m. germanica L.) and the other in Arkansas (m. canescens J.B. Phipps). They form a clade with weak support (Figures 1-2). Mespilus differs from Crataegus in having entire or sub-entire leaves, large flowers with flattish hypanthia, and pomes with wide-spreading persistent sepals giving a "hollow" appearance to the fruit. Nonetheless, both genera have thorns and two superposed seeds per locule. Their sister relationship is well supported (Figures 1-2). However, a recent study, based on two nuclear (ribosomal ITS and LEAFY intron2) and four plastid intergenic regions (trnS-trnG, psbA-trnH, trnH-rpl12, and rpl20-rps12) has pointed out that M. canescens might be a hybrid species between Mespilus and Crataegus (Lo et al., 2007).
	Eriobotrya has a confined distribution in the subtropical and tropical regions of southern and eastern Asia, and is an evergreen taxon with 26 species (Phipps et al., 1990; Robertson et al., 1991). Our ITS data support the mono-phyly of Eriobotya. Rhaphiolepis indica, distributed.in eastern and southern Asia, is sister to the Eriobotrya clade. Their close relationship has been suggested based on various shared morphological traits including the coreless fruit with a large seed and thin endocarp (Robertson et al., 1991).

LI et al. ― Generic limits of Pyrinae

159

Figure 1. Strict consensus of 20,000 trees based on parsimony analyses of nrDNA ITS sequences (CI=0.39, RI=0.76). Numbers above and below branches are branch lengths and bootstrap percentages, respectively.

160

Botanical Studies, Vol. 53, 2012

Figure 2. Majority-rule consensus of 3852 trees based on the Bayesian inference with 4,000,000 generations. Numbers at branches indicate posterior probability percentages.

LI et al. ― Generic limits of Pyrinae

161


	Hesperomeles is an evergreen genus, with or without thorns, having simple leaves, generally 1-4 flowered inflorescences, and small, pinkish flowers. It has five carpels, is fully adnate, has a free style, one ovule per carpel, red to black fruits, and very hard pyrenes separated by a fleshy layer (Robertson et al. 1991). Hesperomeles is comprised of 11 species, endemic to the Andes Mountains of South America. It shares solitary ovules and hard pyrenes with Osteomeles (Rohrer et al., 1991). But Robertson et al. (1991) considered Hesperomeles to be a distinct genus based on the simple leaves and reduced inflorescences. As the first molecular evaluation of Hesperomeles, our ITS data suggest that Hesperomeles is distantly related to Os-teomeles and may be sister to the clade of Crataegus and Mespilus (Figures 1-2). The three genera share morphologically similar simple leaves, possible thorns, and a hard core. Most species of Crataegus are distributed in North America, but the distribution of Crataegus mexicana DC. extends to Guatemala; and in the Hesperomeles genus, Hesperomeles obovata (Pittier) Standi, and Hesperomeles heterophylla (Ruiz. & Pav.) Hook can be found in Costa Rica. Based on the distribution of the two genera, Phipps (1983) suggested that Hesperomeles may have originated from Crataegus mexicana or an extinct relative. Our data provide weak support for the close relationship of Amelanchier-Malacomeles-Peraphyllum and Crataegus-Mespilus-Hesperomeles, as shown in Campbell et al., 2007, but do not support the derivation of Hesperomeles from within Crataegus.
	Osteomeles has three species in eastern Asia and Hawaii, is the only evergreen genus, and has compound leaves and hard pyrenes (Robertson et al., 1991). It is supported as a clade in the MP tree (71%, Figure 1). In the BI tree, however, Osteomeles forms a clade with Di-chotomanthes with strong support (99%) in the BI tree. Dichotomanthes is a monotypic genus found in limited areas of Yunnan and Sichuan provinces of China, and is unique with its single carpel and oblique style that is not adnate to the hypanthium. Nevertheless, evidence from cytology, flavonoid chemistry and wood anatomy indicates a strong affinity between Dichotomanthes and the remaining genera of Pyrinae (Rohrer et al. 1994). Albeit without morphological synapomorphy, Dichotomanthes seems to be most closely related to Osteomeles (pp=99% in the BI tree).
	Chamaemeles is a monotypic genus endemic to Madeira. Although with a single carpel as in Dichotomanthes, Chamaemeles has carpels almost fully inferior. In the ITS trees (Figures 1-2) and Campbell et al.'s study (2007), the relationship of Chamaemeles is unresolved.
	Cydonia Mill. is a monotypic genus in southwestern and central Asia, and shares multiple ovules per locule with Pseudocydonia C.K. Schneid., another monotypic genus in Asia. Chaenomeles Lindley is distributed in eastern Asia. In both our MP and BI trees (Figures 1-2), Cy-donia and Psuedocydonia form a weak clade that is sister to Cotoneaster (pp=59%). Cotoneaster is a species-rich


	genus with over 250 species. Two subgenera have been recognized based on petal characters: subg. Chaenopeta-lum with white, spreading petals; subg. Cotoneaster with pinkish flowers and erect petals (Robertson et al., 1991). In the ITS trees, although neither of the two subgenera is monophyletic, together they form a robust clade (pp=95%, Figures 1-2). Morphologically, Cotoneaster is distinguished from other genera by a combination of characters including lack of thorns, simple and entire leaves with camptodromous venation, 2-3 carpels, 2/3 adnation, no connation, free styles, fruits with hard pyrenes, and calyx lobe flesh, incurved, and persistent.
	Pyracantha consists of nine species in Eurasia and has occasionally been included in Cotoneaster (Focke, 1888; Wenzig, 1883). Pyracantha differs from Cotoneaster in having thorns, toothed leaves, and five carpels. Albeit with poor resolution, our ITS data and Campbell et al.'s (2007) results do not support the close relationship of Cotoneaster and Pyracantha (Figures 1-2). Asian species of Pyracan-tha form a clade, but they do not show a close relationship with P. coccinea of southern Europe and Iran (Figures 1-2). Therefore, Pyracantha may not be monophyle.
	Photinia differs from other genera of the Pyrinae in having the combination of simple leaves, calyx lobes that are persistent, incurved, and fleshy and a soft to leathery core. However, other characters are diverse: unarmed or armed with thorns, toothed or entire leaves, red, black, or purple fruits. Therefore, several segregate genera have been recognized from Photinia: Stranvaesia, Pourthiaea, and Aronia. Stranvaesia is separated from Photinia due to its dehiscent carpels (Lindley, 1837). However, this diagnostic character may have resulted from the artificial pressing of herbarium specimens (Kalman, 1973) and is confidently rejected today. In the ITS tree (Figures 1-2), Stranvaesia is sister to the clade containing Heteromeles, Photinia glabra (Thunb.) Franch. & Sav., P. prunifolia Lindl., and P. tushanensis T.T. Yu. The support, however, is weak. Stranvaesia and Photinia do not form a clade in either cpDNA or nuclear DNA trees (Campbell et al., 2007). Pourthiaea forms a well-supported clade in our ITS trees, indicating that it may be recognized as a separate genus. The potential synapomorphy of Pourthiaea is the deciduous habit. Aronia is different from Photinia in having glands along the upper midribs of the leaves, a feature also present in other Pyrinae genera (Robertson, 1992). Nevertheless, the monophyly of Aronia is supported by our molecular data (Figures 1-2) and by Guo et al. (2010).
	Pyrus consists of 73 species with corymbose-racemose inflorescences, 2 ovules per locule, free styles, a cartilaginous endocarp and dense sclereids in the fruits (Robertson et al., 1991). In our ITS trees (Figures 1-2), Pyrus forms a well supported clade.
	Malus and Sorbus are the most controversial genera in the Pyrinae. The disagreement on the generic limits of Malus rests on whether or not to recognize several mono-typic or small genera: Chloromeles, Eriolobus, Docynia, and Docyniopsis. Chloromeles differs from other species

162

Botanical Studies, Vol. 53, 2012


	of Malus in having greenish, fragrant, often waxy fruits with a dense layer of sclereids around the core and just under the skin. Eriolobus, with a single species in the eastern Mediterranean, is unique in having deeply lobed simple leaves, incomplete adnation of carpels, and abundant sclereids in fruits. Docynia has two species, one in the Himalayas and from Assam to Vietnam and the other in southwestern China. Docyniopsis consists of four species, all in eastern Asia, and differs from Docynia in having only two ovules per locule (vs. 3-10 in Docynia). Nevertheless, the two genera share similar flavonoids chemistry (Williams, 1982). In the phylogenies, Docynia delavayi C.K. Schneid. is closely related to Malus doumeri A. Chev., M. florentina C.K. Schneid. and Eriolobus. Docyni-opsis tschonoskii (Maxim.) Koidz., D. prattii (C.K. Sch-neid.) Koidz., and D. yunnanensis (C.K. Schneid.) Koidz. do not form a clade, and the latter two species are closely allied with Malus honanensis Rehder, M. kansuensis (Batalin) C.K. Scheid., and M. ombrophila Hand.-Mazz. (bs=86%, pp=100%). Chloromeles forms a clade, but its relationship with other clades within Malus remains unresolved. Similarly, Docyniopsis, Eriolobus and Malus form a robust clade (95%) in Campbell et al.'s (2007) GBSSI-2B tree, but their relationships are unresolved. Therefore, it is appropriate to circumscribe Malus broadly, containing Chloromeles, Docynia, Docyniopsis, Eriolobus, and Malus.
	Some authors recognize Sorbus in the broad sense, while others divide it into five genera (Robertson et al., 1991): Sorbus, Aria, Cormus, Torminalis, and Chamae-mespilus. A major reason that taxonomists in Europe and western Asia include these other genera in Sorbus is the large number of apomictic microspecies intermediate between them in those regions (McAllister H. 2005). Robertson et al. (1991) cited several examples of intergeneric hybrids involving Sorbus and other genera of the Pyrinae, such as xSorbocotoneaster, xSorbaronia, xAmelosorbus and x Crataegosorbus, and concluded that "the extensive hybridization between genera and subgeneric groups seems to reflect weak overall barriers to hybridization rather than indicating evolutionary relationships", and "it seems best to discount intergeneric hybridization when setting generic limits."
	Cormus and Sorbus have pinnately compound leaves, Torminalis leaves are pinnately lobed, and those of Chamaemespilus are simple and toothed with campto-dromous venation. However, Aria is diverse in leaf morphology; some species have coarsely toothed leaves with craspedodromous venation, while others have simple leaves and camptodromous venation (Robertson, 1992). Kovanda and Challice (1981) segregated species with deciduous calyx lobes into Micromeles. However, the calyx feature is inconsistent in the Pyrinae, and thus Micromeles should not be recognized (Robertson, 1992; Rohrer et al., 1991). In the ITS trees, Micromeles species are intermixed with those of Aria (Figures 1-2), while Cormus, Tormina-lis, Chamaemespilus each form their own clades. Our ITS


	data thus support their generic status in the Pyrinae.
	CONCLUSIONS
	Our ITS data, from multiple species representing the diversity of traditionally recognized genera, support recognition of 24 genera that are resolved as monophyl-etic: Amelanchier, Aria (including Micromeles), Aronia, Chaenomeles, Chamaemespilus, Chamaemeles, Cormus, Cotoneaster, Crataegus, Cydonia, Dichotomanthes, Eriobotrya, Hesperomeles, Malacomeles, Malus (including Chloromeles, Docynia, Docyniopsis, and Eriolobus), Mespilus, Osteomeles, Peraphyllum, Pourthiaea, Pseudo-cydonia, Pyrus, Rhaphiolepis, Sorbus, and Torminalis. Most of these genera are essentially in agreement with recent works (Robertson et al., 1991). Among those genera, Aronia and Pourthiaea are separated from Photinia as independent genera, and Pourthiaea is for the first time supported by molecular data as a genus; Hesperomeles is also examined for the first time using molecular data and may have a close relationship to Crataegus-mespilus instead of Osteomeles. Our data support the inclusion in Malus of Chloromeles, Docynia, and Docyniopsis and suggest that Pyracantha may be polyphyletic. Photinia is found to be polyphyletic and possibly closely related to Heteromeles and Stranvaesia. However, more extensive sampling is needed to determine the generic limits of Py-racantha, Photinia, and Stranvaesia.
	Acknowledgements. We thank Kyle Port, Kathryn Richardson and Eric Youngerman for their help in collecting plant material from the Arnold Arboretum, Jim Solomon of Missouri Botanical Garden for providing Hesperome-les leaf material, and Margaret Frank for lab assistance. Qingyan Li is grateful to the China Scholarship Council for a foreign study scholarship. This project was partially supported by grants from the National Natural Science Foundation of China (#30670141, #31170202) and the National Infrastructure of Natural Resources for Science and Technology (2005DKA21403) to Wenbo Liao.
	LITERATURE CITED
	Campbell, C.S., C.W. Greene, and T.A. Dickinson. 1991. Reproductive biology in subfam. Maloideae, Rosaceae. Syst. Bot. 16: 333-349.
	Campbell, C.S., M.J. Donoghue, B.G. Baldwin, and M.F. Wojciechowski. 1995. Phylogenetic-Relationships in Maloideae (Rosaceae) - Evidence from Sequences of the In-ternal Transcribed Spacers of Nuclear Ribosomal DNA and Its Congruence with Morphology. Amer. J. Bot. 82: 903-918.
	Campbell, C.S., M.F. Wojciechowski, B.G. Baldwin, L.A. Alice, and M.J. Donoghue. 1997. Persistent nuclear ribosomal DNA sequence polymorphism in the Amelanchier agamic complex (Rosaceae). Mol. Biol. Evol. 14: 81-90.

LI et al. ― Generic limits of Pyrinae

163


	Campbell, C.S., R.C. Evans, D.R. Morgan, T.A. Dickinson, and M.P. Arsenault. 2007. Phylogeny of subtribe Pyrinae (formerly the Maloideae, Rosaceae): Limited resolution of a complex evolutionary history. Plant Syst. Evol. 266: 119145.
	de Candolle, A.P. 1825. Rosaceae. Prodromus 2: 525-639.
	Decaisne, M.J. 1874. Memoirs sur le famile des Pomacees. Nouv. Arch. Mus. Hist. Nat. 10: 113-192.
	Edgar, R.C. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucl. Acids Res. 32: 1792-1797.
	Evans, R.C. and C. Campbell. 2002. The origin of the apple subfamily (Maloideae; Rosaceae) is clarified by DNA sequence data from duplicated GBSSI genes. Am. J. Bot. 89: 14781484.
	Evans, R.C., L.A. Alice, C.S. Campbell, E.A. Kellogg, and T.A. Dickinson. 2000. The granule-bound starch synthase (GB-SSI) gene in the Rosaceae: Multiple loci and phylogenetic utility. Molec. Phylog. Evol. 17: 388-400.
	Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791.
	Focke, W.O. 1888. Rosaceae Nat. Pflanzenfam. III., 1-61, Lepig. pp. 1-61.
	Fritsch, K. 1898. Zur Systematik der Gattung Sorbus. Osterr. Bot. Z. 48: 1-4, 47-49, 167-171.
	Fritsch, K. 1899. Zur Systematik der Gattung Sorbus. Osterr. Bot. Z. 49: 381-385, 426-429.
	Gladkova, V.N. 1969. On the systematic position of the genus Dichotomanthes. Bot. Zhur. 54: 421-436.
	Goldblatt, P. 1976. Cytotaxonomic studies in the tribe Quillajeae (Rosaceae). Ann. Missouri Bot. Gard. 63: 200-206.
	Kalman, C. 1973. The Malesian species of the subfamily Maloideae (Rosaceae). Blumea 21: 413-442.
	Koehne, E. 1891. Die Gattugngen der Pomaceen. Gartenflora 40: 4-7, 35-38, 59-61.
	Kovanda, M. and J.S. Challice. 1981. The genus Micromeles revisted. Folia Geobotanica Phytotaxonomica 16: 181-193.
	Li, J. 2008. Phylogeny of Catalpa (Bignoniaceae) inferred from sequences of chloroplast ndhF and nuclear ribosomal DNA. J. Syst. Evol. 46: 341-348.
	Lindley, J. 1822. Observations on the natural group of plants called Pomaceae. Trans. Linn. Soc. London 13: 88-106.
	Lindley, J. 1837. Stranvaesia glaucescens. Bot. Reg. 23: t.1956.
	Lindley, J. 1845. Nagelia denticulata. Bot. Reg. 31 (misc.): 40.
	Linnaeus, C. 1753. Sp. Pl. pp.
	Lo, E.Y.Y., S. Stefanovic, and T.A. Dickinson. 2007. Molecular reappraisal of relationships between Crataegus and Mespi-lus (Rosaceae, Pyreae) - two genera or one? Syst. Bot. 32: 596-616.
	Morgan, D.R., D.E. Soltis, and K.R. Robertson. 1994. System-


	atic and Evolutionary Implications of Rbcl Sequence Variation in Rosaceae. Amer. J. Bot. 81: 890-903.
	Oh, S.H. and D. Potter. 2003. Phylogenetic utility of the second intron of LEAFY in Neillia and Stephanandra (Rosaceae) and implications for the origin of Stephanandra. Mol. Phy- logenetics Evol. 29: 203-215.
	Phipps, J.B., K.R. Robertson, P.G. Smith, and J.R. Rhohrer. 1990. A checklist of the subfamily Maloideae (Rosaceae). Canad. J. Bot. 68: 2209-2269.
	Phipps, J.B., K.R. Robertson, J.R. Rohrer, and P.G. Smith. 1991. Origin and evolution of subfam. Maloideae (Rosaceae). Syst. Bot. 16: 303-332.
	Posada, D. and K.A. Crandall. 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics Application Note 14: 817-818.
	Potter, D., T. Eriksson, R.C. Evans, S. Oh, J.E.E. Smedmark, D.R. Morgan, M. Kerr, K.R. Robertson, M. Arsenault, T.A. Dickinson, and C.S. Campbell. 2007. Phylogeny and classification of Rosaceae. Plant Syst. Evol. 266: 5-43.
	Rehder, A. 1940. Manual of cultivated trees and shrubs hardy in North America exclusive of the subtropical and warmer temperature regions. 2nd edition ed. Macmillan, New York, pp.
	Rehder, A. 1949. bibliography of cultivated trees and shrubs hardy in the cooler temperature regions of the northern hemisphere Arnold Arboretum of Harvard University, Jamaica Plain, MA.
	Robertson, K.R. 1992. Summary of leaves in the genera of Maloideae (Rosaceae). Ann. Miss. Bot. Gard. 79: 81-94.
	Robertson, K.R., J.B. Phipps, J.R. Rohrer, and P.G. Smith. 1991. A synopsis of genera in Maloideae (Rosaceae). Syst. Bot. 16: 376-394.
	Roemer, M.J. 1847. Familiarum naturalium regni vegetabilis synopses monographicae. III. Rosiflorae. Amygdalacearum et Pomacearum Landes-Industrie-Comptoir, Weimar.
	Rohrer, J.R., K.R. Robertson, and J.B. Phipps. 1991. Variation in structure among fruits of Maloideae (Rosaceae). Amer. J. Bot. 78: 1617-1635.
	Ronquist, F. and J. Huelsenbeck. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572-1574.
	Savile, D.B.O. 1979. Fungi as aids in higher plant classification. Bot. Rev. (Lancaster) 45: 377-503.
	Swofford, D丄.，2002. PAUP. Phylogenetic analysis using parsimony ( and other methods), 4.0b10 ed. Sinauer Associates, Sunderland.
	Wenzig, T. 1883. Die Pomaceen. Charaktere der Gattungen und Arten. Jahrb. Konigl. Bot. Gart. Berlin 2: 287-307.
	Williams, A.H. 1982. Chemical evidence from the flavonoids relevant to the classification of Malus species. J. Linn. Soc. Bot. 84: 31-39.

164

Botanical Studies, Vol. 53, 2012


	基於核基因序列探討蘋果亞科的屬間界限
	李慶豔¹ 郭微¹ 廖文波^1,4 James A. MACKLIN² 李建華^3,4
	¹中國中山大學生命科學學院廣東省熱帶亞熱帶植物資源與利用重點實驗室
	² Harvard University Herbaria, Organismal and Evolutionary Biology, 22 Divinity Avenue, Cambridge, Massachusetts, 02138, USA
	³Biology Department, Hope College, MI 49423, USA
	Pyrinae亞族（原蘋果亞科）為單系類群，約具1,000個種。本亞族包括了許多著名的水果：如蘋果、梨、榲椁、枇杷、野櫻莓、唐棣。本亞族的分類一直很混亂，特別是對蘋果屬、花楸屬和石楠屬的分類一直存在爭議。本次研究共包括180個ITS序列，代表了本亞族內所有的屬，本文即利用ITS分子序列分析來研究Pyrinae亞族的屬間關係。ITS序列分析結果顯示，以下24屬得到確認，即： Amelanchier、Aria (包括Micromeles) 、 Aronia 、 Chaenomeles 、 Chamaemespilus 、 Chamaemeles 、 Cormus 、 Cotoneaster 、 Crataegus 、 Cydonia 、 Dichotomanthes 、 Eriobotrya 、 Hesperomeles 、 Malacomeles 、 Malus (包括Chloromeles, Docynia, Docyniopsis 和 Eriolobus) 、 Mespilus 、 Osteomeles 、 Peraphyllum 、 Pourthiaea 、 Pseudocydonia 、 Pyrus 、 Rhaphiolepis 、 Sorbus和 Torminalis 。但石楠屬和火棘屬則顯示為多起源，包括了 Heteromeles 和 Stranvaesia ，所以它們與亞族內其它屬的關係尚未解決。研究結果支持唐棣屬與Malacomeles 、 Peraphyllum具有較近的親緣關係，山楂屬和歐楂屬具有較近的親緣關係，並第一次確定了南美的Hesperomeles與Crataegus-Mespilus具較近的親緣關係。
	關鍵詞：蘋果亞科；Pyrinae ； nrDNA ITS ；屬間關係；Hesperomeles 。