# Phylogeny of Maleae (Rosaceae) Based on Multiple Chloroplast Regions: Implications to Genera Circumscription

Authors: Jiahui Sun; Shuo Shi; Jinlu Li; Jing Yu; Ling Wang; Xueying Yang; Ling Guo; Shiliang Zhou
Journal: BioMed Research International
 (2018)

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## Abstract

Maleae consists of economically and ecologically important plants. However, there are considerable disputes on generic circumscription due to the lack of a reliable phylogeny at generic level. In this study, molecular phylogeny of 35 generally accepted genera in Maleae is established using 15 chloroplast regions.Gillenia is the most basal clade of Maleae, followed byKageneckia +Lindleya,Vauquelinia, and a typical radiation clade, the core Maleae, suggesting that the proposal of four subtribes is reasonable. In the core Maleae including 31 genera, chloroplast gene data support that the fourMalus-related genera should better be merged into one genus and the sixSorbus-related genera would be classified into two genera, whereas allPhotinia-related genera should be accepted as distinct genera. Although the phylogenetic relationships among the genera in Maleae are much clearer than before, it is still premature to make a formal taxonomic treatment for these genera.


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## Body

## 1. Introduction

Rosaceae or rose family, consisting of approximately 4,828 species in 91 genera [1], is of great economic and ecological importance. Many species are cultivated for their fruits or as ornamentals. The monophyly of the family is implied by the presence of unique floral structures and strongly supported byrbcL phylogeny [2]. However, the rose family displays a considerable diversity in morphology and anatomy, and it had been generally subdivided into four subfamilies, that is, Rosoideae, Spiraeoideae, Amygdaloideae (incorrectly Prunoideae), and Maloideae. Such a subdivision was recently challenged by molecular phylogenies ofmatK andtrnL-F [3], six nuclear and four chloroplast regions [4], and hundreds of nuclear genes [5]. A formal three-subfamily classification was proposed: Dryadoideae, Rosoideae, and Amygdaloideae (incorrectly Spiraeoideae in [4]). Dryadoideae was separated from Rosoideae, and Spiraeoideae and Maloideae were merged with Amygdaloideae (incorrectly Spiraeoideae).One of the most striking changes in the new classification is that traditional Maloideae became subtribe Malinae (incorrectly Pyrinae). Here we use tribe Maleae instead of supertribe Pyrodae sensu [4] to include the traditional pome-bearing Maloideae plusGillenia Moench (=Porteranthus Britton),Kageneckia Ruiz & Pav.,Lindleya Kunth, andVauquelinia Corrêa ex Bonpl. in traditional Spiraeoideae [4, 6–9].Maleae consists of about 1,000 species, occurring mostly in the temperate Northern Hemisphere. The tribe includes many well-known fruit crops such as apple (Malus pumila Mill.), pear(Pyrus spp.), loquat (Eriobotrya japonica (Thunb.) Lindl.), and black chokeberry (Aronia melanocarpa (Michx.) Elliott), as well as many ornamentals. The core Maleae is characterized by a synapomorphic pome, a type of accessory fruit that does not occur in other Rosaceae plants [10], and a basal chromosome number, x=17. With the addition ofGillenia,Kageneckia,Lindleya, andVauquelinia, the tribe has drupaceous or follicle fruits and x=9 (or 15) as well [2, 4, 6, 9, 11–13].The origin of core Maleae(x=17) has long been considered an example of allopolyploidization between the species with x=9 in traditional Spiraeoideae and the species with x=8 in traditional Amygdaloideae [14–17]. However, recently discovered genomic data suggested an origin via autopolyploidization followed by aneuploidization around 50 million years ago [18, 19]. In contrast, the allopolyploid nature of core Maleae was confirmed by GBSSI, which had four copies in the core Maleae but only two copies in other groups [6].Polyploidization affects systematics at both generic and specific levels. It is unlikely to resolve the polytomy of ancestral populations that have just a few closely related species involved in historical speciation and subsequent diversification, owing to the lack of phylogenetically informative signals and incomplete lineage sorting. The merging of several genomes into one species enriches the pool of available genetic combinations and the survival of recombinants is overcome by apomixis. Maleae is one such species-rich tribe of genera but is difficult to classify.The pome-bearing plants have been generally subdivided into two groups: one with connate endocarps and the other with polypyrenous drupes [10, 20–22]. However, such subdivisions of the core Maleae do not receive support from molecular data. Considerable controversies exist on circumscriptions of genera based on morphology or anatomy  [21, 23–37]. Sorbus L. is considered to include both the pinnate-leaved species (Sorbus s.s. andCormusSpach) and the simple-leaved species (Aria (Pers.) Host,Micromeles Decne.,Chamaemespilus Medik., andTorminalis Medik.) [21, 34, 38, 39].AroniaMedik.,Heteromeles M. Roem.,Pourthiaea Decne., andStranvaesiaLindl. are either merged together withPhotinia Lindl. or accepted as distinct genera [37, 40, 41]. Varying opinions are also found amongMalus Mill.,Docyniopsis Koidz., andEriolobus (DC.) M. Roem., as well as amongPseudocydonia(C. K. Schneid.) C. K. Schneid.,Cydonia Mill., andChaenomeles Lindl. [22, 27, 37]. Lack of consensus treatment among these genera is a result of uncertainty about genetic relationships among all entities (or genera in the narrow sense) and molecular data have been used to reveal their phylogenies. Both chloroplast DNA sequences [2, 4, 7, 9, 42] and nuclear DNA sequences ([4, 6–8, 12, 43]; Lo et al., 2012) have been tried, and it is clear that the monophyly of each entity is highly supported; the major unsolved problem is the phylogeny among entities. To elucidate the intricate relationships among these entities (the generally recognized genera in the narrow sense within Maleae), we sampled representative species for each of them, including the occasionally accepted genera and subgenera, and constructed their phylogeny using 15 chloroplast regions. Our results are expected to be helpful for the correct circumscription of genera in Maleae.


## 2. Materials and Methods

### 2.1. Taxon Sampling

A total of 41 species were examined: 35 species representing all generally recognized genera of Maleae and five species representing other major lineages of Amygdaloideae andRosa rugosa Thunb. as an outgroup (Table 1). All samples were collected under appropriate permits: Herbarium, Institute of Botany, CAS; Beijing Botanical Garden, CAS; Kunming Botanical Garden, CAS; and Xishuangbanna Tropical Botanical Garden, CAS; and other places listed in Table 1.Table 1
Representative species of Maleae, other major lineages of Amygdaloideae, and an outgroup used in this study, together with sample vouchers and sampling localities.
Taxon Voucher Locality Amelanchier arborea (F. Michx.) Fernald S. L. Zhou BOP022147 Herbarium, Institute of Botany, CAS (PE) Aria nivea Host S. L. Zhou BOP022174 Herbarium, Institute of Botany, CAS (PE) Aronia melanocarpa (Michx.) Elliott S. L. Zhou BOP022150 Herbarium, Institute of Botany, CAS (PE) Chaenomeles speciosa (Sweet) Nakai S. L. Zhou BOP010027 Beijing Botanical Garden, CAS Chamaemeles coriacea Lindl. K. R. Roberson 4775 Illinois Natural History Survey Herbarium (ILLS) Chamaemespilus alpina (Mill.) K. R. Robertson & J. B. Phipps K. R. Roberson 5276 Illinois Natural History Survey Herbarium (ILLS) Cormus domestica (L.) Spach S. L. Zhou BOP022163 Herbarium, Institute of Botany, CAS (PE) Cotoneaster multiflorus Bunge S. L. Zhou BOP010016 Beijing Botanical Garden, CAS Crataegus kansuensis E. H. Wilson S. L. Zhou BOP010010 Beijing Botanical Garden, CAS Cydonia oblonga Mill. S. L. Zhou BOP010020 Beijing Botanical Garden, CAS Dichotomanthes tristaniicarpa Kurz S. L. Zhou BOP027700 Kunming Botanical Garden, CAS Docynia delavayi (Franch.) C. K. Schneid. S. L. Zhou BOP027738 Xishuangbanna Tropical Botanical Garden, CAS Docyniopsis tschonoskii (Maxim.) Koidz. S. L. Zhou BOP022164 Herbarium, Institute of Botany, CAS (PE) Eriobotrya japonica (Thunb.) Lindl. S. L. Zhou BOP003044 Beijing Botanical Garden, CAS Eriolobus kansuensis (Batalin) C. K. Schneid. S. L. Zhou BOP011038 Huludao, Liaoning, CHN Gillenia trifoliata(L.) Moench S. L. Zhou BOP022159 Herbarium, Institute of Botany, CAS (PE) Heteromeles arbutifolia (Dryand.) M. Roem. S. L. Zhou BOP022160 Herbarium, Institute of Botany, CAS (PE) Kageneckia  crataegifolia Lindl. S. L. Zhou BOP022176 Herbarium, Institute of Botany, CAS (PE) Lindleyella schiedeana (Schltdl.) Rydb. S. L. Zhou BOP022161 Herbarium, Institute of Botany, CAS (PE) Malacomeles denticulata (Kunth) G. N. Jones S. L. Zhou BOP022179 Herbarium, Institute of Botany, CAS (PE) Malus baccata(L.) Borkh. S. L. Zhou BOP016421 Beijing Botanical Garden, CAS Mespilus germanica L. S. L. Zhou BOP022165 Herbarium, Institute of Botany, CAS (PE) Micromeles folgneriC. K. Schneid. S. L. Zhou BOP017025 Harbin, Heilongjiang, CHN Osteomeles schwerinaeC. K. Schneid. S. L. Zhou BOP027697 Kunming Botanical Garden, CAS Peraphyllum ramosissimum Nutt. ex Torr. & A. Gray S. L. Zhou BOP022168 Herbarium, Institute of Botany, CAS (PE) Photinia serratifolia (Desf.) Kalkman S. L. Zhou BOP027633 Beijing Botanical Garden, CAS Pourthiaea arguta var. salicifolia (Decne.) Hook. f. S. L. Zhou BOP022178 Herbarium, Institute of Botany, CAS (PE) Pseudocydonia sinensis(Dum. Cours.) C. K. Schneid. S. L. Zhou BOP010349 Beijing Botanical Garden, CAS Pyracantha fortuneana (Maxim.) H. L. Li S. L. Zhou BOP003043 Beijing Botanical Garden, CAS Pyrus  bretschneideri Rehder S. L. Zhou BOP010065 Beijing Botanical Garden, CAS Rhaphiolepis indica (L.) Lindl. S. L. Zhou BOP016354 Kunming Botanical Garden, CAS Sorbus aucupariaL. S. L. Zhou BOP016569 Harbin, Heilongjiang, CHN Stranvaesia davidiana Decne. S. L. Zhou BOP027698 Kunming Botanical Garden, CAS Torminalis clusii (M.Roem.) K. R. Robertson & J. B. Phipps K. R. Roberson 5275 Illinois Natural History Survey Herbarium (ILLS) Vauquelinia corymbosa Corrêa ex Humb. & Bonpl. S. L. Zhou BOP022177 Herbarium, Institute of Botany, CAS (PE) Other lineages Physocarpus amurensis (Maxim.) Maxim. S. L. Zhou BOP010043 Beijing Botanical Garden, CAS Prinsepia sinensis (Oliv.) Oliv. ex Bean S. L. Zhou BOP010133 Beijing Botanical Garden, CAS Rhodotypos scandens (Thunb.) Makino S. L. Zhou BOP010022 Beijing Botanical Garden, CAS Sorbaria sorbifolia (L.) A. Braun S. L. Zhou BOP016568 Beijing Botanical Garden, CAS Spiraea pubescens Turcz. S. L. Zhou BOP010042 Beijing Botanical Garden, CAS Outgroup Rosa rugosaThunb. S. L. Zhou BOP010536 Beijing Botanical Garden, CAS


### 2.2. DNA Extraction, Amplification, and Sequencing

Total genomic DNA was extracted using the mCTAB method [44] and purified using the Wizard DNA Clean-Up System (A7280, Promega Corporation, Madison, WI). Fifteen chloroplast regions, that is,atpB-rbcL,matK,ndhF,petA-psbJ,psbA-trnH,psbM-trnD,rbcL,rpl16,rpl20-rps12,rps16,trnC-ycf6,trnH-rpl2,trnL-trnF,trnS-trnG, andycf1, were used in this study. Fourteen primer pairs published in Dong et al. [45, 46] were synthesized by Sangon Biotech (Shanghai) Co. Ltd. (Beijing, China) and used to amplify and sequence these regions (Table 2). Sequences ofrps16 were downloaded from GenBank. The polymerase chain reaction (PCR) amplifications were carried out in a mixture volume of 20 μL, containing 1x Taq buffer (1 mol/L KCl; 20 mmol Tris-HCl pH 9.0; 1% Triton X-100), 2.0 μL dNTPs (2 mmol/L), 1.0 μL each of the primers (5 μmol/L), 20 ng of genomic DNA, and 1 unit of Taq polymerase. PCR was conducted using a C1000 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). The program started at 94°C for 3 min, followed by 35 cycles at 94°C for 30 s, 50°C for 30 s, and 72°C for 2 min, and ended at 72°C for 5 min. The PCR products were purified with an equal mixture of 40% PEG 8000 and 5 mol/L NaCl, followed by a washing step with 80% ethanol. The PCR products were then Sanger sequenced for both strands on an ABI 3730xl DNA Analyzer using BigDye Terminator cycle sequencing kit v3.1 (Applied Biosystems, Foster City, CAS, USA) following the manufacturer’s instructions.Table 2
Primers used to amplify and sequence 15 regions of chloroplast genome of Rosaceae.
Gene regions Forward primer (5′-3′) Reverse primer (5′-3′) Reference atpB-rbcL TTTCCTAATAATTGCTGTACC ACAGTTGTCCATGTACCAGTAG Dong et al., 2013 [45] matK TCTAGCACACGAAAGTCGAAGT CGATCTATTCATTCAATATTTC Dong et al., 2013 [45] ndhF ACACCAACGCCATTCGTAATGCCATC AAGATGAAATTCTTAATGATAGTTGG Dong et al., 2013 [45] petA-psbJ CTAATGTGGGTGGATTTGGTCA ATGGCCGATACTACTGGAAGG This study psbA-trnH GAACCCGCGCATGGTGGATTCAC TGGCTCCCTATTCAGTGCTATGC This study psbM-trnD GTTTTTACGTATATTATAAGTA GTTCAAATCCAGCTCGGCCCA This study rbcL ATGTCACCACAAACAGAGACTAAAGC TTCCATACTTCACAAGCAGCAGCTAG Dong et al., 2013 [45] rpl16 TCCCGAAATAATGAATTGAGTTCG TCAGAGAAGGTAGGGTTCCCCT Dong et al., 2013 [45] rpl20-rps12 ATGATCTCATTGGAAATCATATAAAG AGGGTAATGATCCATCAACCGGC Dong et al., 2013 [45] rps16 GTGGTAGAAAGCAACGTGCGACTT TCGGGATCGAACATCAATTGCAAC Oxelman et al., 1997 [85] trnC-ycf6 GGCGGCATGGCCGAGTGGTAAGGC TCCACTTCTTCCCCATACTACGA Dong et al., 2013 [45] trnH-rpl2 AGCCAACACTTAGATCCGGCTCTAC GATTTGTGAATCCACCATGCGCG Dong et al., 2013 [45] trnL-trnF GTTCAAGTCCCTCTATCCCCA GATTTTCAAGAACGGGAATCTTA Dong et al., 2013 [45] trnS-trnG AAACTCTTCGTTTACACAGTAGTGA CTTTTACCACTAAACTATACCCGC Dong et al., 2013 [45] ycf1 TCTCGACGAAAATCAGATTGTTGTGAAT ATACATGTCAAAGTGATGGAAAA Dong et al., 2015 [46]


### 2.3. Sequence Data Preparation and Evaluation

The newly generated sequences were checked and assembled using Sequencer 4.7 (Gene codes Corporation, Ann Arbor, Michigan, USA). The resulting sequences (Supplementary TableS2) were combined with the published sequences of Maleae species downloaded from GenBank (Supplementary Table S1), aligned with Clustal X [47, 48], and then manually adjusted with Se-Al 2.0 [49]. Each individual gene dataset was then subjected to several rounds of phylogenetic evaluations to select reliable sequences. Sequences that were misidentified or exhibited large amounts of missing data were excluded from the datasets. To predict the phylogenetic performance of individual gene partitions, the variability of genes was parameterized by DnaSP 5.0 [50] and PAUP 4.0b10 [51] using nucleotide diversity, the number of polymorphic sites, and so forth. Prior to concatenating the dataset of each marker, incongruence length difference (ILD) tests were performed on all 15 datasets. The datasets were finally concatenated into one final data matrix using SequenceMatrix [52] for phylogenetic analyses.


### 2.4. Phylogenetic Analysis

Phylogenetic analyses were performed on single gene datasets and the concatenated dataset, using PAUP 4.0b10 [51] for maximum parsimony (MP), RAxML v.8.1.24 [53] for maximum likelihood (ML), and MrBayes 3.2.2 [54] for Bayesian inference (BI). The MP analysis used a heuristic search that treats all characters as equally weighted and unordered, obtaining the starting trees by stepwise addition, random stepwise addition of 100 replicates, TBR, and MulTrees enabled. Branch support for MP trees was assessed with 1,000 bootstrap replicates and all trees were saved at each replicate. ML analysis was performed with 1,000 nonparametric bootstrap replicates (BP). The suitable substitution model (GTR + I + G) for BI analysis was inferred by using ModelTest version 3.7 [55] under the Akaike information criterion. Default settings were used for the MrBayes run, and 2 × four chains were run for ten million generations, sampled every 1,000 generations. Posterior probabilities (PP) were calculated from the majority consensus of all of the sampled trees when the standard deviation of the split frequencies (SDSF) permanently fell below 0.01, and the trees sampled during the burn-in phase were discarded.


## 2.1. Taxon Sampling

A total of 41 species were examined: 35 species representing all generally recognized genera of Maleae and five species representing other major lineages of Amygdaloideae andRosa rugosa Thunb. as an outgroup (Table 1). All samples were collected under appropriate permits: Herbarium, Institute of Botany, CAS; Beijing Botanical Garden, CAS; Kunming Botanical Garden, CAS; and Xishuangbanna Tropical Botanical Garden, CAS; and other places listed in Table 1.Table 1
Representative species of Maleae, other major lineages of Amygdaloideae, and an outgroup used in this study, together with sample vouchers and sampling localities.
Taxon Voucher Locality Amelanchier arborea (F. Michx.) Fernald S. L. Zhou BOP022147 Herbarium, Institute of Botany, CAS (PE) Aria nivea Host S. L. Zhou BOP022174 Herbarium, Institute of Botany, CAS (PE) Aronia melanocarpa (Michx.) Elliott S. L. Zhou BOP022150 Herbarium, Institute of Botany, CAS (PE) Chaenomeles speciosa (Sweet) Nakai S. L. Zhou BOP010027 Beijing Botanical Garden, CAS Chamaemeles coriacea Lindl. K. R. Roberson 4775 Illinois Natural History Survey Herbarium (ILLS) Chamaemespilus alpina (Mill.) K. R. Robertson & J. B. Phipps K. R. Roberson 5276 Illinois Natural History Survey Herbarium (ILLS) Cormus domestica (L.) Spach S. L. Zhou BOP022163 Herbarium, Institute of Botany, CAS (PE) Cotoneaster multiflorus Bunge S. L. Zhou BOP010016 Beijing Botanical Garden, CAS Crataegus kansuensis E. H. Wilson S. L. Zhou BOP010010 Beijing Botanical Garden, CAS Cydonia oblonga Mill. S. L. Zhou BOP010020 Beijing Botanical Garden, CAS Dichotomanthes tristaniicarpa Kurz S. L. Zhou BOP027700 Kunming Botanical Garden, CAS Docynia delavayi (Franch.) C. K. Schneid. S. L. Zhou BOP027738 Xishuangbanna Tropical Botanical Garden, CAS Docyniopsis tschonoskii (Maxim.) Koidz. S. L. Zhou BOP022164 Herbarium, Institute of Botany, CAS (PE) Eriobotrya japonica (Thunb.) Lindl. S. L. Zhou BOP003044 Beijing Botanical Garden, CAS Eriolobus kansuensis (Batalin) C. K. Schneid. S. L. Zhou BOP011038 Huludao, Liaoning, CHN Gillenia trifoliata(L.) Moench S. L. Zhou BOP022159 Herbarium, Institute of Botany, CAS (PE) Heteromeles arbutifolia (Dryand.) M. Roem. S. L. Zhou BOP022160 Herbarium, Institute of Botany, CAS (PE) Kageneckia  crataegifolia Lindl. S. L. Zhou BOP022176 Herbarium, Institute of Botany, CAS (PE) Lindleyella schiedeana (Schltdl.) Rydb. S. L. Zhou BOP022161 Herbarium, Institute of Botany, CAS (PE) Malacomeles denticulata (Kunth) G. N. Jones S. L. Zhou BOP022179 Herbarium, Institute of Botany, CAS (PE) Malus baccata(L.) Borkh. S. L. Zhou BOP016421 Beijing Botanical Garden, CAS Mespilus germanica L. S. L. Zhou BOP022165 Herbarium, Institute of Botany, CAS (PE) Micromeles folgneriC. K. Schneid. S. L. Zhou BOP017025 Harbin, Heilongjiang, CHN Osteomeles schwerinaeC. K. Schneid. S. L. Zhou BOP027697 Kunming Botanical Garden, CAS Peraphyllum ramosissimum Nutt. ex Torr. & A. Gray S. L. Zhou BOP022168 Herbarium, Institute of Botany, CAS (PE) Photinia serratifolia (Desf.) Kalkman S. L. Zhou BOP027633 Beijing Botanical Garden, CAS Pourthiaea arguta var. salicifolia (Decne.) Hook. f. S. L. Zhou BOP022178 Herbarium, Institute of Botany, CAS (PE) Pseudocydonia sinensis(Dum. Cours.) C. K. Schneid. S. L. Zhou BOP010349 Beijing Botanical Garden, CAS Pyracantha fortuneana (Maxim.) H. L. Li S. L. Zhou BOP003043 Beijing Botanical Garden, CAS Pyrus  bretschneideri Rehder S. L. Zhou BOP010065 Beijing Botanical Garden, CAS Rhaphiolepis indica (L.) Lindl. S. L. Zhou BOP016354 Kunming Botanical Garden, CAS Sorbus aucupariaL. S. L. Zhou BOP016569 Harbin, Heilongjiang, CHN Stranvaesia davidiana Decne. S. L. Zhou BOP027698 Kunming Botanical Garden, CAS Torminalis clusii (M.Roem.) K. R. Robertson & J. B. Phipps K. R. Roberson 5275 Illinois Natural History Survey Herbarium (ILLS) Vauquelinia corymbosa Corrêa ex Humb. & Bonpl. S. L. Zhou BOP022177 Herbarium, Institute of Botany, CAS (PE) Other lineages Physocarpus amurensis (Maxim.) Maxim. S. L. Zhou BOP010043 Beijing Botanical Garden, CAS Prinsepia sinensis (Oliv.) Oliv. ex Bean S. L. Zhou BOP010133 Beijing Botanical Garden, CAS Rhodotypos scandens (Thunb.) Makino S. L. Zhou BOP010022 Beijing Botanical Garden, CAS Sorbaria sorbifolia (L.) A. Braun S. L. Zhou BOP016568 Beijing Botanical Garden, CAS Spiraea pubescens Turcz. S. L. Zhou BOP010042 Beijing Botanical Garden, CAS Outgroup Rosa rugosaThunb. S. L. Zhou BOP010536 Beijing Botanical Garden, CAS


## 2.2. DNA Extraction, Amplification, and Sequencing

Total genomic DNA was extracted using the mCTAB method [44] and purified using the Wizard DNA Clean-Up System (A7280, Promega Corporation, Madison, WI). Fifteen chloroplast regions, that is,atpB-rbcL,matK,ndhF,petA-psbJ,psbA-trnH,psbM-trnD,rbcL,rpl16,rpl20-rps12,rps16,trnC-ycf6,trnH-rpl2,trnL-trnF,trnS-trnG, andycf1, were used in this study. Fourteen primer pairs published in Dong et al. [45, 46] were synthesized by Sangon Biotech (Shanghai) Co. Ltd. (Beijing, China) and used to amplify and sequence these regions (Table 2). Sequences ofrps16 were downloaded from GenBank. The polymerase chain reaction (PCR) amplifications were carried out in a mixture volume of 20 μL, containing 1x Taq buffer (1 mol/L KCl; 20 mmol Tris-HCl pH 9.0; 1% Triton X-100), 2.0 μL dNTPs (2 mmol/L), 1.0 μL each of the primers (5 μmol/L), 20 ng of genomic DNA, and 1 unit of Taq polymerase. PCR was conducted using a C1000 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). The program started at 94°C for 3 min, followed by 35 cycles at 94°C for 30 s, 50°C for 30 s, and 72°C for 2 min, and ended at 72°C for 5 min. The PCR products were purified with an equal mixture of 40% PEG 8000 and 5 mol/L NaCl, followed by a washing step with 80% ethanol. The PCR products were then Sanger sequenced for both strands on an ABI 3730xl DNA Analyzer using BigDye Terminator cycle sequencing kit v3.1 (Applied Biosystems, Foster City, CAS, USA) following the manufacturer’s instructions.Table 2
Primers used to amplify and sequence 15 regions of chloroplast genome of Rosaceae.
Gene regions Forward primer (5′-3′) Reverse primer (5′-3′) Reference atpB-rbcL TTTCCTAATAATTGCTGTACC ACAGTTGTCCATGTACCAGTAG Dong et al., 2013 [45] matK TCTAGCACACGAAAGTCGAAGT CGATCTATTCATTCAATATTTC Dong et al., 2013 [45] ndhF ACACCAACGCCATTCGTAATGCCATC AAGATGAAATTCTTAATGATAGTTGG Dong et al., 2013 [45] petA-psbJ CTAATGTGGGTGGATTTGGTCA ATGGCCGATACTACTGGAAGG This study psbA-trnH GAACCCGCGCATGGTGGATTCAC TGGCTCCCTATTCAGTGCTATGC This study psbM-trnD GTTTTTACGTATATTATAAGTA GTTCAAATCCAGCTCGGCCCA This study rbcL ATGTCACCACAAACAGAGACTAAAGC TTCCATACTTCACAAGCAGCAGCTAG Dong et al., 2013 [45] rpl16 TCCCGAAATAATGAATTGAGTTCG TCAGAGAAGGTAGGGTTCCCCT Dong et al., 2013 [45] rpl20-rps12 ATGATCTCATTGGAAATCATATAAAG AGGGTAATGATCCATCAACCGGC Dong et al., 2013 [45] rps16 GTGGTAGAAAGCAACGTGCGACTT TCGGGATCGAACATCAATTGCAAC Oxelman et al., 1997 [85] trnC-ycf6 GGCGGCATGGCCGAGTGGTAAGGC TCCACTTCTTCCCCATACTACGA Dong et al., 2013 [45] trnH-rpl2 AGCCAACACTTAGATCCGGCTCTAC GATTTGTGAATCCACCATGCGCG Dong et al., 2013 [45] trnL-trnF GTTCAAGTCCCTCTATCCCCA GATTTTCAAGAACGGGAATCTTA Dong et al., 2013 [45] trnS-trnG AAACTCTTCGTTTACACAGTAGTGA CTTTTACCACTAAACTATACCCGC Dong et al., 2013 [45] ycf1 TCTCGACGAAAATCAGATTGTTGTGAAT ATACATGTCAAAGTGATGGAAAA Dong et al., 2015 [46]


## 2.3. Sequence Data Preparation and Evaluation

The newly generated sequences were checked and assembled using Sequencer 4.7 (Gene codes Corporation, Ann Arbor, Michigan, USA). The resulting sequences (Supplementary TableS2) were combined with the published sequences of Maleae species downloaded from GenBank (Supplementary Table S1), aligned with Clustal X [47, 48], and then manually adjusted with Se-Al 2.0 [49]. Each individual gene dataset was then subjected to several rounds of phylogenetic evaluations to select reliable sequences. Sequences that were misidentified or exhibited large amounts of missing data were excluded from the datasets. To predict the phylogenetic performance of individual gene partitions, the variability of genes was parameterized by DnaSP 5.0 [50] and PAUP 4.0b10 [51] using nucleotide diversity, the number of polymorphic sites, and so forth. Prior to concatenating the dataset of each marker, incongruence length difference (ILD) tests were performed on all 15 datasets. The datasets were finally concatenated into one final data matrix using SequenceMatrix [52] for phylogenetic analyses.


## 2.4. Phylogenetic Analysis

Phylogenetic analyses were performed on single gene datasets and the concatenated dataset, using PAUP 4.0b10 [51] for maximum parsimony (MP), RAxML v.8.1.24 [53] for maximum likelihood (ML), and MrBayes 3.2.2 [54] for Bayesian inference (BI). The MP analysis used a heuristic search that treats all characters as equally weighted and unordered, obtaining the starting trees by stepwise addition, random stepwise addition of 100 replicates, TBR, and MulTrees enabled. Branch support for MP trees was assessed with 1,000 bootstrap replicates and all trees were saved at each replicate. ML analysis was performed with 1,000 nonparametric bootstrap replicates (BP). The suitable substitution model (GTR + I + G) for BI analysis was inferred by using ModelTest version 3.7 [55] under the Akaike information criterion. Default settings were used for the MrBayes run, and 2 × four chains were run for ten million generations, sampled every 1,000 generations. Posterior probabilities (PP) were calculated from the majority consensus of all of the sampled trees when the standard deviation of the split frequencies (SDSF) permanently fell below 0.01, and the trees sampled during the burn-in phase were discarded.


## 3. Results

### 3.1. Sequence Variability within Maleae

The ILD tests did not suggest serious conflicts between datasets of the 15 chloroplast regions (p=0.11). The variability of the 15 examined DNA regions within Maleae are given in Table 3, together with the MP tree scores of all taxa. The longest DNA region wasmatK(La=2045), and the shortest wastrnH-rpl2(La=205). Of the 15 chloroplast regions,psbA-trnHwas the most variable fragment with a π (nucleotide diversity) value of 0.02547, whereastrnH-rpl2 was the least variable fragment with a π value of 0.00391. ThepsbA-trnH,petA-psbJ,ycf1,trnS-trnG, andtrnC-ycf6 gene regions were more variable (π>0.010) than other genes.Table 3
Variability of 15 chloroplast regions among species in Maleae and the maximum parsimony tree scores of all taxa.
Gene regions N ∗ La Sc S Is NH π k L ∗ C I ∗ R I ∗ atpB-rbcL 39 796 659 33 9 25 0.00422 4.15 177 0.921 0.781 matK 41 2045 1761 167 47 32 0.00879 8.17 663 0.741 0.538 ndhF 39 1973 1821 148 38 29 0.00937 7.5 694 0.745 0.567 petA-psbJ 34 1370 1072 155 36 27 0.01046 11.31 554 0.791 0.629 psbA-trnH 37 283 222 34 10 15 0.02547 12.01 196 0.789 0.436 psbM-trnD 34 1503 1225 126 33 27 0.00901 8.38 589 0.81 0.666 rbcL 38 1353 1293 60 25 25 0.00539 4.43 296 0.547 0.442 rpl16 40 1178 940 124 31 32 0.00804 10.53 463 0.689 0.518 rpl20-rps12 37 743 677 42 15 30 0.00710 5.65 230 0.752 0.617 rps16 39 964 772 84 22 28 0.00999 8.71 378 0.746 0.639 trnC-ycf6 36 1032 810 90 30 28 0.01084 8.72 525 0.785 0.616 trnH-rpl2 36 205 189 11 5 5 0.00391 5.37 196 0.789 0.436 trnL-trnF 41 1088 944 90 24 26 0.00783 8.27 420 0.757 0.612 trnS-trnG 36 895 661 102 20 27 0.01011 11.4 470 0.781 0.528 ycf1 34 866 742 88 21 28 0.01210 10.16 489 0.765 0.595 N, number of species; La, aligned length; Sc, sites considered; S, number of polymorphic sites, excluding sites with missing data; Is, parsimony-informative site; NH, number of haplotypes; π, nucleotide diversity; k, average number of nucleotide differences; L, the tree length; CI, consistency index; RI, retention index. ∗Including all taxa.The concatenated data matrix of the 41 taxa reached an aligned length of 17,554 bp with 1,266 parsimony-informative characters. MP searches yielded one best tree with a consistency index (CI) of 0.779, a retention index (RI) of 0.637, and a tree length of 5,974.


### 3.2. Phylogenetic Relationships of the Basal Maleae

Polytomies were observed in all the 15 best trees based on each chloroplast region. However, the tree topologies were similar; we thus concatenated them to build best resolved phylogenetic trees using MP, ML, and BI methods. The consensus trees from the MP, ML, and BI analyses showed substantially identical topologies and the monophyly of Maleae was recovered (Figure1). A long branch leads to the crown taxa of Maleae, includingGillenia,Lindleya,Kageneckia, andVauquelinia (Figure 1(A)), and this branch is strongly supported (Figure 1(B)) (PB=100, BP=100, PP=1).Figure 1
Phylogenetic relationships of Maleae based on a concatenated dataset of 15 chloroplast gene regions. (A) Bayesian phylogram with branch lengths. (B) 50% majority-rule Bayesian consensus tree with bootstrap supports. Values beside the branches are the bootstrap percentages from maximum parsimony analysis, maximum likelihood analysis, and Bayesian posterior probabilities. “-” indicates a branch collapse in the maximum parsimony and maximum likelihood trees.An earliest and remarkable divergence ofGillenia is clearly indicated in Figure 1.Lindleya andKageneckia form a monophyletic clade and they diverged slightly earlier thanVauquelinia. The divergence amongLindleya +Kageneckia,Vauquelinia, and the core Maleae happened within a very short span of time, as indicated by the very short branches. All the three branches are well supported. The basal branching pattern of Maleae may serve as the foundation of subtribal division within the tribe if necessary.


### 3.3. Phylogenetic Relationships within the Core Maleae

The monophyly of the core Maleae is clearly indicated (Figure1(A)) and well supported (Figure 1(B)). The genera in the core Maleae seem to be from the second radiation event. Nevertheless, three multigenus clades within the core Maleae are recognizable. Clade I consists ofAmelanchier Medik.,Crataegus L.,Cydonia Mill.,Malacomeles (Decne.) Decne.,Mespilus L., andPeraphyllum Nutt. and is well supported (PB=95, BP=90, PP=1). A very close relationship betweenCrataegus andMespilus is indicated. Clade II (PB=65, BP=79, PP=1) consists of fourPhotinia-related, threeSorbus-related, and three distinct entities. There are four pairs of genera in this clade but only two of them are highly supported (PB=100, BP=100, PP=1), namely,Eriobotrya Lindl. versusRhaphiolepisLindl. andMicromeles Decne. versusSorbus L.StranvaesiaLindl. was closer toCotoneaster Mecik. rather than toPhotiniaLindl. Clade III consists of fourMalus-related, onePhotinia-related, threeSorbus-related, and five distinct entities and is weakly supported (PB=50, BP=67, PP=0.91). Very close relationships were revealed betweenMalus-relatedDocyniopsisKoidz. andMalus Mill. (PB=100, BP=100, PP=1) and among theSorbus-relatedAria(Pers.) Host,ChamaemespilusMedik., andTorminalisMedik. (PB=72,BP=94,PP=1).Docynia Decne. andEriolobus (DC.) Roem. also fall in the same clade asDocyniopsisandMalus, but the clade is only weakly supported (PB=56, BP=67, PP=1).Chaenomeles Lindl. is related toPseudocydonia (C. K. Schneid.) C. K. Schneid. (BP=52, PP=1).


## 3.1. Sequence Variability within Maleae

The ILD tests did not suggest serious conflicts between datasets of the 15 chloroplast regions (p=0.11). The variability of the 15 examined DNA regions within Maleae are given in Table 3, together with the MP tree scores of all taxa. The longest DNA region wasmatK(La=2045), and the shortest wastrnH-rpl2(La=205). Of the 15 chloroplast regions,psbA-trnHwas the most variable fragment with a π (nucleotide diversity) value of 0.02547, whereastrnH-rpl2 was the least variable fragment with a π value of 0.00391. ThepsbA-trnH,petA-psbJ,ycf1,trnS-trnG, andtrnC-ycf6 gene regions were more variable (π>0.010) than other genes.Table 3
Variability of 15 chloroplast regions among species in Maleae and the maximum parsimony tree scores of all taxa.
Gene regions N ∗ La Sc S Is NH π k L ∗ C I ∗ R I ∗ atpB-rbcL 39 796 659 33 9 25 0.00422 4.15 177 0.921 0.781 matK 41 2045 1761 167 47 32 0.00879 8.17 663 0.741 0.538 ndhF 39 1973 1821 148 38 29 0.00937 7.5 694 0.745 0.567 petA-psbJ 34 1370 1072 155 36 27 0.01046 11.31 554 0.791 0.629 psbA-trnH 37 283 222 34 10 15 0.02547 12.01 196 0.789 0.436 psbM-trnD 34 1503 1225 126 33 27 0.00901 8.38 589 0.81 0.666 rbcL 38 1353 1293 60 25 25 0.00539 4.43 296 0.547 0.442 rpl16 40 1178 940 124 31 32 0.00804 10.53 463 0.689 0.518 rpl20-rps12 37 743 677 42 15 30 0.00710 5.65 230 0.752 0.617 rps16 39 964 772 84 22 28 0.00999 8.71 378 0.746 0.639 trnC-ycf6 36 1032 810 90 30 28 0.01084 8.72 525 0.785 0.616 trnH-rpl2 36 205 189 11 5 5 0.00391 5.37 196 0.789 0.436 trnL-trnF 41 1088 944 90 24 26 0.00783 8.27 420 0.757 0.612 trnS-trnG 36 895 661 102 20 27 0.01011 11.4 470 0.781 0.528 ycf1 34 866 742 88 21 28 0.01210 10.16 489 0.765 0.595 N, number of species; La, aligned length; Sc, sites considered; S, number of polymorphic sites, excluding sites with missing data; Is, parsimony-informative site; NH, number of haplotypes; π, nucleotide diversity; k, average number of nucleotide differences; L, the tree length; CI, consistency index; RI, retention index. ∗Including all taxa.The concatenated data matrix of the 41 taxa reached an aligned length of 17,554 bp with 1,266 parsimony-informative characters. MP searches yielded one best tree with a consistency index (CI) of 0.779, a retention index (RI) of 0.637, and a tree length of 5,974.


## 3.2. Phylogenetic Relationships of the Basal Maleae

Polytomies were observed in all the 15 best trees based on each chloroplast region. However, the tree topologies were similar; we thus concatenated them to build best resolved phylogenetic trees using MP, ML, and BI methods. The consensus trees from the MP, ML, and BI analyses showed substantially identical topologies and the monophyly of Maleae was recovered (Figure1). A long branch leads to the crown taxa of Maleae, includingGillenia,Lindleya,Kageneckia, andVauquelinia (Figure 1(A)), and this branch is strongly supported (Figure 1(B)) (PB=100, BP=100, PP=1).Figure 1
Phylogenetic relationships of Maleae based on a concatenated dataset of 15 chloroplast gene regions. (A) Bayesian phylogram with branch lengths. (B) 50% majority-rule Bayesian consensus tree with bootstrap supports. Values beside the branches are the bootstrap percentages from maximum parsimony analysis, maximum likelihood analysis, and Bayesian posterior probabilities. “-” indicates a branch collapse in the maximum parsimony and maximum likelihood trees.An earliest and remarkable divergence ofGillenia is clearly indicated in Figure 1.Lindleya andKageneckia form a monophyletic clade and they diverged slightly earlier thanVauquelinia. The divergence amongLindleya +Kageneckia,Vauquelinia, and the core Maleae happened within a very short span of time, as indicated by the very short branches. All the three branches are well supported. The basal branching pattern of Maleae may serve as the foundation of subtribal division within the tribe if necessary.


## 3.3. Phylogenetic Relationships within the Core Maleae

The monophyly of the core Maleae is clearly indicated (Figure1(A)) and well supported (Figure 1(B)). The genera in the core Maleae seem to be from the second radiation event. Nevertheless, three multigenus clades within the core Maleae are recognizable. Clade I consists ofAmelanchier Medik.,Crataegus L.,Cydonia Mill.,Malacomeles (Decne.) Decne.,Mespilus L., andPeraphyllum Nutt. and is well supported (PB=95, BP=90, PP=1). A very close relationship betweenCrataegus andMespilus is indicated. Clade II (PB=65, BP=79, PP=1) consists of fourPhotinia-related, threeSorbus-related, and three distinct entities. There are four pairs of genera in this clade but only two of them are highly supported (PB=100, BP=100, PP=1), namely,Eriobotrya Lindl. versusRhaphiolepisLindl. andMicromeles Decne. versusSorbus L.StranvaesiaLindl. was closer toCotoneaster Mecik. rather than toPhotiniaLindl. Clade III consists of fourMalus-related, onePhotinia-related, threeSorbus-related, and five distinct entities and is weakly supported (PB=50, BP=67, PP=0.91). Very close relationships were revealed betweenMalus-relatedDocyniopsisKoidz. andMalus Mill. (PB=100, BP=100, PP=1) and among theSorbus-relatedAria(Pers.) Host,ChamaemespilusMedik., andTorminalisMedik. (PB=72,BP=94,PP=1).Docynia Decne. andEriolobus (DC.) Roem. also fall in the same clade asDocyniopsisandMalus, but the clade is only weakly supported (PB=56, BP=67, PP=1).Chaenomeles Lindl. is related toPseudocydonia (C. K. Schneid.) C. K. Schneid. (BP=52, PP=1).


## 4. Discussion

### 4.1. Taxa and Gene Sampling Strategies

It is often a challenge to reconstruct the phylogeny of taxa from recent radiation events due to unclear relationships among subdivisions and low resolution of markers. Maleae is such a taxon and that is why early studies failed to establish solid phylogenetic relationships among its subdivisions. In “A checklist of the subfamily Maloideae (Rosaceae)” [21], only 23 genera were accepted. SixSorbus-related entities, that is,Aria,Chamaemespilus,Cormus,Micromeles,Sorbus, andTorminalis, were proposed to be merged. To test the distinctness of these taxa, we included all of them in this study.Malus-related andPhotinia-related entities have similar taxonomic problems. Inclusion of representative species from other lineages of Amygdaloideae was to test the monophyly of Maleae. Considering that the monophyly of the genera in the narrowest sense has been well verified, 35 entities with one representative species each were sampled for being the most inclusive and economically affordable.Sampling chloroplast regions as molecular markers for Maleae is another challenge. The tribe was found to be quite young and the core Maleae was even younger. Chloroplast markers have showed very low resolutions in previous studies [2, 4, 7, 9, 42]. Therefore, the most variable regions suggested by Dong et al. [56] were used together with the four conventional DNA barcodes,matK,psbA-trnH,rbcL, andycf1 [46]. By doing so, the major groups in Maleae were well resolved.


### 4.2. Phylogenetic Relationships among Major Groups of Maleae

The phylogeny (Figure1) strongly suggests inclusion ofGillenia,Kageneckia,Lindleya, andVauquelinia in Maleae (incorrectly Pyrodae in [7] and in [4]).Gillenia seemed to diverge slightly early, andKageneckia+Lindleya,Vauquelinia, and the core Maleae are quite probably from the first radiation event. Although the inclusion ofKageneckia,Lindleya, andVauquelinia into Maleae seems disagreeable with respect to fruit types, their basal chromosome number (x=15 or 17) suggests that they had probably experienced similar speciation events. They bridge the gap between the core Maleae and true spiraeoidGillenia. The inclusion ofGillenia in Maleae verifies that the core Maleae is from spiraeoid members within the tribe or other tribes in Amygdaloideae. Unfortunately, transcriptome data of nuclear genes did not provide more information for the issue of origin of the core Maleae because Figure 1 is substantially similar to the tree topologies based on transcriptome data in [5]. Given thatGillenia is the only diploid member in Maleae, the ancestor ofGillenia or its close relatives must be the maternal parent of the extant Maleae.


### 4.3. Phylogenetic Relationships within the Core Maleae

The core Maleae is a natural group with several synapomorphic characters, such as syncarpous ovaries, epigynous flowers, and fleshy fruits derived from the hypanthial ovary [57–59]. It could be better classified as subtribe Malinae (incorrectly Pyrinae) if necessary. The Malinae diverged into genera by radiation which is the second radiation event in Malese. The genera are so closely related that it is unnecessary to subdivide this subtribe further.


#### 4.3.1. Generic Pairs and Their Taxonomic Status

There are four generic pairs, for which the taxonomic status needs to be clarified. Firstly, a close genetic relationship betweenCrataegus andMespilus has been revealed by Lo and Donoghue [9] in detail, which is confirmed here again. IfMespilus were to be accepted as a distinct genus,Crataegus would become a paraphyletic group. AlthoughMespilus was merged withCrataegus, its distinction was still recognized by giving it the taxonomic rank of section inCrataegus[9]. Secondly, reduction ofPseudocydonia toChaenomeles was done by Gu and Spongberg [60], and their treatment is supported by Campbell et al. [7]. However, their close relationship does not receive high support in this study (BP=52, PP=1). They have diverged for some time because the clade does not show remarkable branch length. They had better be considered distinct genera. Thirdly,Stranvaesia is morphologically similar to bothPhotinia andCotoneaster and sometimes submerged intoPhotinia [61]. Phylogeny based on chloroplast regions done by Campbell et al. [7] indicated a close relationship betweenStranvaesia andPyracantha. However, our data support a close relationship betweenStranvaesia andCotoneaster, a relationship also supported by GBSSI-1B [7]. Fourthly, although very close morphological and genetic relationships have been revealed in almost all studies involvingEriobotrya andRhaphiolepis, there has been no suggestion yet to merge them into one, despite the existence of their hybrids [62]. Genetic divergence between them is very recently because their clade has a relatively long branch.


#### 4.3.2. Sorbus-Related Genera

So far, no well-sampled phylogeny of allSorbus-related taxa is available, owing to the genetic complexity of the group and difficulties in sampling. There are ca. 250 species belonging to mainly temperate areas in the Northern Hemisphere [21], and six narrowly defined genera or subgenera underSorbus in the broadest sense are usually accepted:Aria,Chamaemespilus,Cormus,Micromeles,Sorbus, andTorminalis [21, 37–39, 43, 58, 63–72]. These taxa fall into two clades in this study (Figure 1).Sorbus sensu stricto andCormus with compound leaves nested in Clade I, andAria,Chamaemespilus, andTorminalis with lobed or unlobed simple leaves nested in Clade III.Micromeles, a genus created to contain the species without persistent calyx lobes, is not a natural entity and is, therefore, unacceptable [8].Micromeles folgneri in this study is a synonym ofSorbus folgneri. AlthoughCormus had been considered a synonym ofSorbus, its taxonomic position remains to be determined.Aria,Chamaemespilus, andTorminalis in Clade III are closely related and they had better merge into one separate fromSorbus.Sorbus-related entities are notorious in taxonomy due to complexity in phenotypes resulting from interspecific hybridization and facultative agamospermy [39, 69, 70]. Apomictic microspecies confound systematic resolution of agamic complexes using nuclear markers [71]. Phylogenies based on nuclear genes may suffer from paralogue problems and chloroplast markers would work better at the very beginning when no clear ideas are available for classification.


#### 4.3.3. Photinia-Related Genera

Four genera in their narrowest sense,Aronia,Heteromeles, Pourthiaea, andStranvaesia, are considered related toPhotinia [24, 25, 29, 31, 37].  This study demonstrates that all the five genera are superficially similar but actually distinct groups.Heteromeles possesses a soft pyrene, whilePhotinia possesses a soft core [16, 37, 58].Stranvaesia is distinguishable fromPhotinia by the dehiscent fruits at maturity. Although Guo et al. [73] stated with quite certainty thatStranvaesia must be merged intoPhotinia, we believe that further studies are needed for a reliable conclusion.Pourthiaea belongs to Clade III instead of Clade II as other genera. It is readily separable from other genera by characters such as deciduous habit, warty peduncles and pedicels, and a pulp structure in the fruits [41]. The distinction ofPourthiaea is further supported by leaf epidermis and wood anatomy [74, 75].Aronia is not a sister group ofPourthiaea because the clade is poorly supported, a conclusion that is similar to the earlier studies ([8, 9, 73]).


#### 4.3.4. Malus-Related Genera

The four genera,Docynia,Docyniopsis,Eriolobus, andMalus, really have very close relationships among them, especially betweenDocyniopsis andMalus. Some species have been transferred among the four genera by different authors. The monophyly of a clade comprising the four genera is indicated by our chloroplast data (Figure 1(B)). There are only two species inDocynia, two species inDocyniopsis, and one species inEriolobus. Considering that these species are nested withinMalus [18], these genera could better be merged withMalus.


## 4.1. Taxa and Gene Sampling Strategies

It is often a challenge to reconstruct the phylogeny of taxa from recent radiation events due to unclear relationships among subdivisions and low resolution of markers. Maleae is such a taxon and that is why early studies failed to establish solid phylogenetic relationships among its subdivisions. In “A checklist of the subfamily Maloideae (Rosaceae)” [21], only 23 genera were accepted. SixSorbus-related entities, that is,Aria,Chamaemespilus,Cormus,Micromeles,Sorbus, andTorminalis, were proposed to be merged. To test the distinctness of these taxa, we included all of them in this study.Malus-related andPhotinia-related entities have similar taxonomic problems. Inclusion of representative species from other lineages of Amygdaloideae was to test the monophyly of Maleae. Considering that the monophyly of the genera in the narrowest sense has been well verified, 35 entities with one representative species each were sampled for being the most inclusive and economically affordable.Sampling chloroplast regions as molecular markers for Maleae is another challenge. The tribe was found to be quite young and the core Maleae was even younger. Chloroplast markers have showed very low resolutions in previous studies [2, 4, 7, 9, 42]. Therefore, the most variable regions suggested by Dong et al. [56] were used together with the four conventional DNA barcodes,matK,psbA-trnH,rbcL, andycf1 [46]. By doing so, the major groups in Maleae were well resolved.


## 4.2. Phylogenetic Relationships among Major Groups of Maleae

The phylogeny (Figure1) strongly suggests inclusion ofGillenia,Kageneckia,Lindleya, andVauquelinia in Maleae (incorrectly Pyrodae in [7] and in [4]).Gillenia seemed to diverge slightly early, andKageneckia+Lindleya,Vauquelinia, and the core Maleae are quite probably from the first radiation event. Although the inclusion ofKageneckia,Lindleya, andVauquelinia into Maleae seems disagreeable with respect to fruit types, their basal chromosome number (x=15 or 17) suggests that they had probably experienced similar speciation events. They bridge the gap between the core Maleae and true spiraeoidGillenia. The inclusion ofGillenia in Maleae verifies that the core Maleae is from spiraeoid members within the tribe or other tribes in Amygdaloideae. Unfortunately, transcriptome data of nuclear genes did not provide more information for the issue of origin of the core Maleae because Figure 1 is substantially similar to the tree topologies based on transcriptome data in [5]. Given thatGillenia is the only diploid member in Maleae, the ancestor ofGillenia or its close relatives must be the maternal parent of the extant Maleae.


## 4.3. Phylogenetic Relationships within the Core Maleae

The core Maleae is a natural group with several synapomorphic characters, such as syncarpous ovaries, epigynous flowers, and fleshy fruits derived from the hypanthial ovary [57–59]. It could be better classified as subtribe Malinae (incorrectly Pyrinae) if necessary. The Malinae diverged into genera by radiation which is the second radiation event in Malese. The genera are so closely related that it is unnecessary to subdivide this subtribe further.


### 4.3.1. Generic Pairs and Their Taxonomic Status

There are four generic pairs, for which the taxonomic status needs to be clarified. Firstly, a close genetic relationship betweenCrataegus andMespilus has been revealed by Lo and Donoghue [9] in detail, which is confirmed here again. IfMespilus were to be accepted as a distinct genus,Crataegus would become a paraphyletic group. AlthoughMespilus was merged withCrataegus, its distinction was still recognized by giving it the taxonomic rank of section inCrataegus[9]. Secondly, reduction ofPseudocydonia toChaenomeles was done by Gu and Spongberg [60], and their treatment is supported by Campbell et al. [7]. However, their close relationship does not receive high support in this study (BP=52, PP=1). They have diverged for some time because the clade does not show remarkable branch length. They had better be considered distinct genera. Thirdly,Stranvaesia is morphologically similar to bothPhotinia andCotoneaster and sometimes submerged intoPhotinia [61]. Phylogeny based on chloroplast regions done by Campbell et al. [7] indicated a close relationship betweenStranvaesia andPyracantha. However, our data support a close relationship betweenStranvaesia andCotoneaster, a relationship also supported by GBSSI-1B [7]. Fourthly, although very close morphological and genetic relationships have been revealed in almost all studies involvingEriobotrya andRhaphiolepis, there has been no suggestion yet to merge them into one, despite the existence of their hybrids [62]. Genetic divergence between them is very recently because their clade has a relatively long branch.


### 4.3.2. Sorbus-Related Genera

So far, no well-sampled phylogeny of allSorbus-related taxa is available, owing to the genetic complexity of the group and difficulties in sampling. There are ca. 250 species belonging to mainly temperate areas in the Northern Hemisphere [21], and six narrowly defined genera or subgenera underSorbus in the broadest sense are usually accepted:Aria,Chamaemespilus,Cormus,Micromeles,Sorbus, andTorminalis [21, 37–39, 43, 58, 63–72]. These taxa fall into two clades in this study (Figure 1).Sorbus sensu stricto andCormus with compound leaves nested in Clade I, andAria,Chamaemespilus, andTorminalis with lobed or unlobed simple leaves nested in Clade III.Micromeles, a genus created to contain the species without persistent calyx lobes, is not a natural entity and is, therefore, unacceptable [8].Micromeles folgneri in this study is a synonym ofSorbus folgneri. AlthoughCormus had been considered a synonym ofSorbus, its taxonomic position remains to be determined.Aria,Chamaemespilus, andTorminalis in Clade III are closely related and they had better merge into one separate fromSorbus.Sorbus-related entities are notorious in taxonomy due to complexity in phenotypes resulting from interspecific hybridization and facultative agamospermy [39, 69, 70]. Apomictic microspecies confound systematic resolution of agamic complexes using nuclear markers [71]. Phylogenies based on nuclear genes may suffer from paralogue problems and chloroplast markers would work better at the very beginning when no clear ideas are available for classification.


### 4.3.3. Photinia-Related Genera

Four genera in their narrowest sense,Aronia,Heteromeles, Pourthiaea, andStranvaesia, are considered related toPhotinia [24, 25, 29, 31, 37].  This study demonstrates that all the five genera are superficially similar but actually distinct groups.Heteromeles possesses a soft pyrene, whilePhotinia possesses a soft core [16, 37, 58].Stranvaesia is distinguishable fromPhotinia by the dehiscent fruits at maturity. Although Guo et al. [73] stated with quite certainty thatStranvaesia must be merged intoPhotinia, we believe that further studies are needed for a reliable conclusion.Pourthiaea belongs to Clade III instead of Clade II as other genera. It is readily separable from other genera by characters such as deciduous habit, warty peduncles and pedicels, and a pulp structure in the fruits [41]. The distinction ofPourthiaea is further supported by leaf epidermis and wood anatomy [74, 75].Aronia is not a sister group ofPourthiaea because the clade is poorly supported, a conclusion that is similar to the earlier studies ([8, 9, 73]).


### 4.3.4. Malus-Related Genera

The four genera,Docynia,Docyniopsis,Eriolobus, andMalus, really have very close relationships among them, especially betweenDocyniopsis andMalus. Some species have been transferred among the four genera by different authors. The monophyly of a clade comprising the four genera is indicated by our chloroplast data (Figure 1(B)). There are only two species inDocynia, two species inDocyniopsis, and one species inEriolobus. Considering that these species are nested withinMalus [18], these genera could better be merged withMalus.


## 4.3.1. Generic Pairs and Their Taxonomic Status

There are four generic pairs, for which the taxonomic status needs to be clarified. Firstly, a close genetic relationship betweenCrataegus andMespilus has been revealed by Lo and Donoghue [9] in detail, which is confirmed here again. IfMespilus were to be accepted as a distinct genus,Crataegus would become a paraphyletic group. AlthoughMespilus was merged withCrataegus, its distinction was still recognized by giving it the taxonomic rank of section inCrataegus[9]. Secondly, reduction ofPseudocydonia toChaenomeles was done by Gu and Spongberg [60], and their treatment is supported by Campbell et al. [7]. However, their close relationship does not receive high support in this study (BP=52, PP=1). They have diverged for some time because the clade does not show remarkable branch length. They had better be considered distinct genera. Thirdly,Stranvaesia is morphologically similar to bothPhotinia andCotoneaster and sometimes submerged intoPhotinia [61]. Phylogeny based on chloroplast regions done by Campbell et al. [7] indicated a close relationship betweenStranvaesia andPyracantha. However, our data support a close relationship betweenStranvaesia andCotoneaster, a relationship also supported by GBSSI-1B [7]. Fourthly, although very close morphological and genetic relationships have been revealed in almost all studies involvingEriobotrya andRhaphiolepis, there has been no suggestion yet to merge them into one, despite the existence of their hybrids [62]. Genetic divergence between them is very recently because their clade has a relatively long branch.


## 4.3.2. Sorbus-Related Genera

So far, no well-sampled phylogeny of allSorbus-related taxa is available, owing to the genetic complexity of the group and difficulties in sampling. There are ca. 250 species belonging to mainly temperate areas in the Northern Hemisphere [21], and six narrowly defined genera or subgenera underSorbus in the broadest sense are usually accepted:Aria,Chamaemespilus,Cormus,Micromeles,Sorbus, andTorminalis [21, 37–39, 43, 58, 63–72]. These taxa fall into two clades in this study (Figure 1).Sorbus sensu stricto andCormus with compound leaves nested in Clade I, andAria,Chamaemespilus, andTorminalis with lobed or unlobed simple leaves nested in Clade III.Micromeles, a genus created to contain the species without persistent calyx lobes, is not a natural entity and is, therefore, unacceptable [8].Micromeles folgneri in this study is a synonym ofSorbus folgneri. AlthoughCormus had been considered a synonym ofSorbus, its taxonomic position remains to be determined.Aria,Chamaemespilus, andTorminalis in Clade III are closely related and they had better merge into one separate fromSorbus.Sorbus-related entities are notorious in taxonomy due to complexity in phenotypes resulting from interspecific hybridization and facultative agamospermy [39, 69, 70]. Apomictic microspecies confound systematic resolution of agamic complexes using nuclear markers [71]. Phylogenies based on nuclear genes may suffer from paralogue problems and chloroplast markers would work better at the very beginning when no clear ideas are available for classification.


## 4.3.3. Photinia-Related Genera

Four genera in their narrowest sense,Aronia,Heteromeles, Pourthiaea, andStranvaesia, are considered related toPhotinia [24, 25, 29, 31, 37].  This study demonstrates that all the five genera are superficially similar but actually distinct groups.Heteromeles possesses a soft pyrene, whilePhotinia possesses a soft core [16, 37, 58].Stranvaesia is distinguishable fromPhotinia by the dehiscent fruits at maturity. Although Guo et al. [73] stated with quite certainty thatStranvaesia must be merged intoPhotinia, we believe that further studies are needed for a reliable conclusion.Pourthiaea belongs to Clade III instead of Clade II as other genera. It is readily separable from other genera by characters such as deciduous habit, warty peduncles and pedicels, and a pulp structure in the fruits [41]. The distinction ofPourthiaea is further supported by leaf epidermis and wood anatomy [74, 75].Aronia is not a sister group ofPourthiaea because the clade is poorly supported, a conclusion that is similar to the earlier studies ([8, 9, 73]).


## 4.3.4. Malus-Related Genera

The four genera,Docynia,Docyniopsis,Eriolobus, andMalus, really have very close relationships among them, especially betweenDocyniopsis andMalus. Some species have been transferred among the four genera by different authors. The monophyly of a clade comprising the four genera is indicated by our chloroplast data (Figure 1(B)). There are only two species inDocynia, two species inDocyniopsis, and one species inEriolobus. Considering that these species are nested withinMalus [18], these genera could better be merged withMalus.


## 5. Conclusions

This molecular phylogenetic study was conducted on all the genera of Maleae, by using 15 variable chloroplast gene regions. Four major clades are well resolved and the branches are well supported. These clades could be classified into four subtribes. The first radiation event gave birth toLindleya +Kageneckia,Vauquelinia, and the ancestor of the core Maleae, and the second radiation event triggered the divergence of the genera in the core Maleae within a short time period. Within the core Maleae the fourMalus-related genera should better be merged into one genus; the sixSorbus-related genera would be better classified into at least two genera; and allPhotinia-related genera should be accepted as distinct genera. For a more reliable classification, phylogeny based on the whole chloroplast genomes of representative species from each genus should be used. Such a strategy has been practiced on many seed plants (e.g., [76–83]). Besides, the nuclear genes, especially the single copy nuclear genes in diploid species such as starch-branching enzyme (Sbe; [84]), should be considered with priority to document the origins of Maleae, as the maternally transmitted markers can only tell one aspect of the whole story. Application of nuclear genome information in phylogenetic reconstruction of Maleae also seems feasible, because the genomes of several species in the tribe have been sequenced, and the resequencing of many species is underway. We are expecting a completely resolved phylogeny of Maleae, and that is why we do not provide a formal taxonomic treatment in this study.



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*Source: 7627191-2018-03-19.xml*