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-
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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




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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




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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




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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).
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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.
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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
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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.
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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
180ITS序列,代表了本亞族內所有的屬,本文即利
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,
Amelanchier、Aria (包括Micromeles) Aronia Chaenomeles Chamaemespilus Chamaemeles
Cormus Cotoneaster Crataegus Cydonia Dichotomanthes Eriobotrya Hesperomeles
Malacomeles Malus (包括Chloromeles, Docynia, Docyniopsis Eriolobus) Mespilus Osteomeles
Peraphyllum Pourthiaea Pseudocydonia Pyrus Rhaphiolepis Sorbus Torminalis 。但石楠屬和火棘
屬則顯示為多起源,包括了
HeteromelesStranvaesia ,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HesperomelesCrataegus-Mespilus具較近的親緣關係。
關鍵詞:蘋果亞科;Pyrinae nrDNA ITS ;屬間關係;Hesperomeles