Phylogeny of Maleae (Rosaceae) Based on Multiple

Research ArticlePhylogeny of Maleae (Rosaceae) Based on MultipleChloroplast Regions Implications to Genera Circumscription

Jiahui Sun 12 Shuo Shi 123 Jinlu Li14 Jing Yu1 LingWang4 Xueying Yang5

Ling Guo 6 and Shiliang Zhou 12

1State Key Laboratory of Systematic and Evolutionary Botany Institute of Botany Chinese Academy of Sciences Beijing 100093 China2University of the Chinese Academy of Sciences Beijing 100043 China3College of Life Science Hebei Normal University Shijiazhuang 050024 China4The Department of Landscape Architecture Northeast Forestry University Harbin 150040 China5Key Laboratory of Forensic Genetics Institute of Forensic Science Ministry of Public Security Beijing 100038 China6Beijing Botanical Garden Beijing 100093 China

Correspondence should be addressed to Ling Guo guolingbeijingbgcom and Shiliang Zhou slzhouibcasaccn

Received 21 September 2017 Revised 11 December 2017 Accepted 2 January 2018 Published 19 March 2018

Academic Editor Fengjie Sun

Copyright copy 2018 Jiahui Sun et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

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

1 Introduction

Rosaceae or rose family consisting of approximately 4828species in 91 genera [1] is of great economic and ecologicalimportance Many species are cultivated for their fruits oras ornamentals The monophyly of the family is impliedby the presence of unique floral structures and stronglysupported by rbcL phylogeny [2] However the rose familydisplays a considerable diversity inmorphology and anatomyand it had been generally subdivided into four subfamiliesthat is Rosoideae Spiraeoideae Amygdaloideae (incorrectlyPrunoideae) andMaloideae Such a subdivision was recentlychallenged bymolecular phylogenies ofmatK and trnL-F [3]six nuclear and four chloroplast regions [4] and hundredsof nuclear genes [5] A formal three-subfamily classificationwas proposed Dryadoideae Rosoideae and Amygdaloideae

(incorrectly Spiraeoideae in [4]) Dryadoideae was sepa-rated from Rosoideae and Spiraeoideae and Maloideae weremerged with Amygdaloideae (incorrectly Spiraeoideae)

One of the most striking changes in the new classifica-tion is that traditional Maloideae became subtribe Malinae(incorrectly Pyrinae) Here we use tribe Maleae insteadof supertribe Pyrodae sensu [4] to include the traditionalpome-bearing Maloideae plus Gillenia Moench (=Porteran-thus Britton) Kageneckia Ruiz amp Pav Lindleya Kunth andVauquelinia Correa ex Bonpl in traditional Spiraeoideae [46ndash9]

Maleae consists of about 1000 species occurring mostlyin the temperate Northern Hemisphere The tribe includesmany well-known fruit crops such as apple (Malus pumilaMill) pear (Pyrus spp) loquat (Eriobotrya japonica (Thunb)Lindl) and black chokeberry (Aronia melanocarpa (Michx)

HindawiBioMed Research InternationalVolume 2018 Article ID 7627191 10 pageshttpsdoiorg10115520187627191

2 BioMed Research International

Elliott) as well as many ornamentals The core Maleae ischaracterized by a synapomorphic pome a type of accessoryfruit that does not occur in other Rosaceae plants [10] anda basal chromosome number 119909 = 17 With the addition ofGillenia Kageneckia Lindleya and Vauquelinia the tribe hasdrupaceous or follicle fruits and 119909 = 9 (or 15) as well[2 4 6 9 11ndash13]

The origin of core Maleae (119909 = 17) has long beenconsidered an example of allopolyploidization between thespecies with 119909 = 9 in traditional Spiraeoideae and the specieswith 119909 = 8 in traditional Amygdaloideae [14ndash17] Howeverrecently discovered genomic data suggested an origin viaautopolyploidization followed by aneuploidization around 50million years ago [18 19] In contrast the allopolyploid natureof core Maleae was confirmed by GBSSI which had fourcopies in the core Maleae but only two copies in other groups[6]

Polyploidization affects systematics at both generic andspecific levels It is unlikely to resolve the polytomy of ances-tral populations that have just a few closely related speciesinvolved in historical speciation and subsequent diversifica-tion owing to the lack of phylogenetically informative signalsand incomplete lineage sorting The merging of severalgenomes into one species enriches the pool of available ge-netic combinations and the survival of recombinants isovercome by apomixis Maleae is one such species-rich tribeof genera but is difficult to classify

The pome-bearing plants have been generally subdividedinto two groups one with connate endocarps and the otherwith polypyrenous drupes [10 20ndash22] However such sub-divisions of the core Maleae do not receive support frommolecular data Considerable controversies exist on cir-cumscriptions of genera based on morphology or anatomy[21 23ndash37] Sorbus L is considered to include both thepinnate-leaved species (Sorbus ss and Cormus Spach) andthe simple-leaved species (Aria (Pers) Host MicromelesDecne ChamaemespilusMedik and TorminalisMedik) [2134 38 39]AroniaMedikHeteromelesM Roem PourthiaeaDecne and Stranvaesia Lindl are either merged togetherwith Photinia Lindl or accepted as distinct genera [37 4041] Varying opinions are also found among Malus MillDocyniopsis Koidz and Eriolobus (DC) M Roem as wellas among Pseudocydonia (C K Schneid) C K SchneidCydonia Mill and Chaenomeles Lindl [22 27 37] Lack ofconsensus treatment among these genera is a result of uncer-tainty about genetic relationships among all entities (or gen-era in the narrow sense) and molecular data have been usedto reveal their phylogenies Both chloroplast DNA sequences[2 4 7 9 42] and nuclear DNA sequences ([4 6ndash8 12 43] Loet al 2012) have been tried and it is clear that themonophylyof each entity is highly supported the major unsolvedproblem is the phylogeny among entities To elucidate theintricate relationships among these entities (the generallyrecognized genera in the narrow sense within Maleae) wesampled representative species for each of them including theoccasionally accepted genera and subgenera and constructedtheir phylogeny using 15 chloroplast regions Our results areexpected to be helpful for the correct circumscription ofgenera in Maleae

2 Materials and Methods

21 Taxon Sampling A total of 41 species were examined35 species representing all generally recognized genera ofMaleae and five species representing other major lineagesof Amygdaloideae and Rosa rugosa Thunb as an outgroup(Table 1) All samples were collected under appropriatepermits Herbarium Institute of Botany CAS Beijing Botan-ical Garden CAS Kunming Botanical Garden CAS andXishuangbanna Tropical Botanical Garden CAS and otherplaces listed in Table 1

22 DNA Extraction Amplification and Sequencing Totalgenomic DNA was extracted using the mCTAB method[44] and purified using the Wizard DNA Clean-Up Sys-tem (A7280 Promega Corporation Madison WI) Fifteenchloroplast regions that is atpB-rbcLmatK ndhF petA-psbJpsbA-trnH psbM-trnD rbcL rpl16 rpl20-rps12 rps16 trnC-ycf6 trnH-rpl2 trnL-trnF trnS-trnG and ycf1 were usedin this study Fourteen primer pairs published in Dong etal [45 46] were synthesized by Sangon Biotech (Shanghai)Co Ltd (Beijing China) and used to amplify and sequencethese regions (Table 2) Sequences of rps16 were downloadedfromGenBankThe polymerase chain reaction (PCR) ampli-fications were carried out in a mixture volume of 20120583Lcontaining 1x Taq buffer (1molL KCl 20mmol Tris-HClpH 90 1 Triton X-100) 20 120583L dNTPs (2mmolL) 10 120583Leach of the primers (5 120583molL) 20 ng of genomic DNA and1 unit of Taq polymerase PCR was conducted using a C1000Thermal Cycler (Bio-Rad Laboratories Hercules CA USA)Theprogram started at 94∘C for 3min followed by 35 cycles at94∘C for 30 s 50∘C for 30 s and 72∘C for 2min and ended at72∘C for 5minThePCRproducts were purifiedwith an equalmixture of 40 PEG 8000 and 5molL NaCl followed by awashing step with 80 ethanol The PCR products were thenSanger sequenced for both strands on an ABI 3730xl DNAAnalyzer using BigDye Terminator cycle sequencing kit v31(Applied Biosystems Foster City CAS USA) following themanufacturerrsquos instructions

23 Sequence Data Preparation and Evaluation The newlygenerated sequences were checked and assembled usingSequencer 47 (Gene codes Corporation Ann Arbor Michi-gan USA) The resulting sequences (Supplementary TableS2) were combined with the published sequences of Maleaespecies downloaded from GenBank (Supplementary TableS1) aligned with Clustal X [47 48] and then manuallyadjustedwith Se-Al 20 [49] Each individual gene dataset wasthen subjected to several rounds of phylogenetic evaluationsto select reliable sequences Sequences thatweremisidentifiedor exhibited large amounts of missing data were excludedfrom the datasets To predict the phylogenetic performanceof individual gene partitions the variability of genes wasparameterized by DnaSP 50 [50] and PAUP 40b10 [51] usingnucleotide diversity the number of polymorphic sites andso forth Prior to concatenating the dataset of each markerincongruence length difference (ILD) tests were performedon all 15 datasets The datasets were finally concatenated

BioMed Research International 3

Table 1 Representative species of Maleae other major lineages of Amygdaloideae and an outgroup used in this study together with samplevouchers and sampling localities

Taxon Voucher LocalityAmelanchier arborea (F Michx) Fernald S L Zhou BOP022147 Herbarium Institute of Botany CAS (PE)Aria niveaHost 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 CASChamaemeles coriacea Lindl K R Roberson 4775 Illinois Natural History Survey Herbarium (ILLS)Chamaemespilus alpina (Mill) K R Robertson amp 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 CASCrataegus kansuensis E H Wilson S L Zhou BOP010010 Beijing Botanical Garden CASCydonia oblongaMill S L Zhou BOP010020 Beijing Botanical Garden CASDichotomanthes tristaniicarpa Kurz S L Zhou BOP027700 Kunming Botanical Garden CASDocynia delavayi (Franch) C K Schneid S L Zhou BOP027738 Xishuangbanna Tropical Botanical Garden CASDocyniopsis tschonoskii (Maxim) Koidz S L Zhou BOP022164 Herbarium Institute of Botany CAS (PE)Eriobotrya japonica (Thunb) Lindl S L Zhou BOP003044 Beijing Botanical Garden CASEriolobus kansuensis (Batalin) C K Schneid S L Zhou BOP011038 Huludao Liaoning CHNGillenia 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 CASMespilus germanica L S L Zhou BOP022165 Herbarium Institute of Botany CAS (PE)Micromeles folgneri C K Schneid S L Zhou BOP017025 Harbin Heilongjiang CHNOsteomeles schwerinae C K Schneid S L Zhou BOP027697 Kunming Botanical Garden CASPeraphyllum ramosissimum Nutt ex Torr amp A Gray S L Zhou BOP022168 Herbarium Institute of Botany CAS (PE)Photinia serratifolia (Desf) Kalkman S L Zhou BOP027633 Beijing Botanical Garden CASPourthiaea 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 CASPyracantha fortuneana (Maxim) H L Li S L Zhou BOP003043 Beijing Botanical Garden CASPyrus bretschneideri Rehder S L Zhou BOP010065 Beijing Botanical Garden CASRhaphiolepis indica (L) Lindl S L Zhou BOP016354 Kunming Botanical Garden CASSorbus aucuparia L S L Zhou BOP016569 Harbin Heilongjiang CHNStranvaesia davidiana Decne S L Zhou BOP027698 Kunming Botanical Garden CASTorminalis clusii (MRoem) K R Robertson amp J B Phipps K R Roberson 5275 Illinois Natural History Survey Herbarium (ILLS)Vauquelinia corymbosa Correa ex Humb amp Bonpl S L Zhou BOP022177 Herbarium Institute of Botany CAS (PE)Other lineagesPhysocarpus amurensis (Maxim) Maxim S L Zhou BOP010043 Beijing Botanical Garden CASPrinsepia sinensis (Oliv) Oliv ex Bean S L Zhou BOP010133 Beijing Botanical Garden CASRhodotypos scandens (Thunb) Makino S L Zhou BOP010022 Beijing Botanical Garden CASSorbaria sorbifolia (L) A Braun S L Zhou BOP016568 Beijing Botanical Garden CASSpiraea pubescens Turcz S L Zhou BOP010042 Beijing Botanical Garden CASOutgroupRosa rugosaThunb S L Zhou BOP010536 Beijing Botanical Garden CAS

into one final data matrix using SequenceMatrix [52] forphylogenetic analyses

24 Phylogenetic Analysis Phylogenetic analyses were per-formed on single gene datasets and the concatenated dataset

using PAUP 40b10 [51] for maximum parsimony (MP)RAxML v8124 [53] for maximum likelihood (ML) andMrBayes 322 [54] for Bayesian inference (BI) The MPanalysis used a heuristic search that treats all characters asequally weighted and unordered obtaining the starting trees


Table 2 Primers used to amplify and sequence 15 regions of chloroplast genome of Rosaceae

Gene regions Forward primer (51015840-31015840) Reverse primer (51015840-31015840) ReferenceatpB-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 studypsbA-trnH GAACCCGCGCATGGTGGATTCAC TGGCTCCCTATTCAGTGCTATGC This studypsbM-trnD GTTTTTACGTATATTATAAGTA GTTCAAATCCAGCTCGGCCCA This studyrbcL 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]

Table 3 Variability of 15 chloroplast regions among species in Maleae and the maximum parsimony tree scores of all taxa

Gene regions 119873lowast La Sc S Is NH 120587 k 119871lowast CIlowast RIlowast

atpB-rbcL 39 796 659 33 9 25 000422 415 177 0921 0781matK 41 2045 1761 167 47 32 000879 817 663 0741 0538ndhF 39 1973 1821 148 38 29 000937 75 694 0745 0567petA-psbJ 34 1370 1072 155 36 27 001046 1131 554 0791 0629psbA-trnH 37 283 222 34 10 15 002547 1201 196 0789 0436psbM-trnD 34 1503 1225 126 33 27 000901 838 589 081 0666rbcL 38 1353 1293 60 25 25 000539 443 296 0547 0442rpl16 40 1178 940 124 31 32 000804 1053 463 0689 0518rpl20-rps12 37 743 677 42 15 30 000710 565 230 0752 0617rps16 39 964 772 84 22 28 000999 871 378 0746 0639trnC-ycf6 36 1032 810 90 30 28 001084 872 525 0785 0616trnH-rpl2 36 205 189 11 5 5 000391 537 196 0789 0436trnL-trnF 41 1088 944 90 24 26 000783 827 420 0757 0612trnS-trnG 36 895 661 102 20 27 001011 114 470 0781 0528ycf1 34 866 742 88 21 28 001210 1016 489 0765 0595119873 number of species La aligned length Sc sites considered 119878 number of polymorphic sites excluding sites with missing data Is parsimony-informativesite NH number of haplotypes 120587 nucleotide diversity 119896 average number of nucleotide differences 119871 the tree length CI consistency index RI retentionindex lowastIncluding all taxa

by stepwise addition random stepwise addition of 100 repli-cates TBR and MulTrees enabled Branch support for MPtrees was assessed with 1000 bootstrap replicates and alltrees were saved at each replicate ML analysis was performedwith 1000 nonparametric bootstrap replicates (BP) Thesuitable substitution model (GTR + I + G) for BI analysiswas inferred by using ModelTest version 37 [55] under theAkaike information criterion Default settings were used fortheMrBayes run and 2 times four chains were run for tenmilliongenerations sampled every 1000 generations Posterior prob-abilities (PP) were calculated from the majority consensus ofall of the sampled trees when the standard deviation of thesplit frequencies (SDSF) permanently fell below 001 and thetrees sampled during the burn-in phase were discarded

3 Results

31 Sequence Variability withinMaleae The ILD tests did notsuggest serious conflicts between datasets of the 15 chloro-plast regions (119901 = 011) The variability of the 15 examinedDNA regions within Maleae are given in Table 3 togetherwith the MP tree scores of all taxa The longest DNA regionwas matK (La = 2045) and the shortest was trnH-rpl2(La = 205) Of the 15 chloroplast regions psbA-trnH was themost variable fragment with a 120587 (nucleotide diversity) valueof 002547 whereas trnH-rpl2 was the least variable fragmentwith a 120587 value of 000391 The psbA-trnH petA-psbJ ycf1trnS-trnG and trnC-ycf6 gene regions were more variable(120587 gt 0010) than other genes


The concatenated data matrix of the 41 taxa reached analigned length of 17554 bp with 1266 parsimony-informativecharacters MP searches yielded one best tree with a consis-tency index (CI) of 0779 a retention index (RI) of 0637 anda tree length of 5974

32 Phylogenetic Relationships of the Basal Maleae Poly-tomies were observed in all the 15 best trees based on eachchloroplast region However the tree topologies were sim-ilar we thus concatenated them to build best resolvedphylogenetic trees using MP ML and BI methods Theconsensus trees from the MP ML and BI analyses showedsubstantially identical topologies and the monophyly ofMaleae was recovered (Figure 1) A long branch leads tothe crown taxa of Maleae including Gillenia LindleyaKageneckia and Vauquelinia (Figure 1(A)) and this branchis strongly supported (Figure 1(B)) (PB = 100 BP = 100PP = 1)

An earliest and remarkable divergence of Gillenia isclearly indicated in Figure 1 Lindleya and Kageneckia forma monophyletic clade and they diverged slightly earlier thanVauquelinia The divergence among Lindleya + KageneckiaVauquelinia and the core Maleae happened within a veryshort span of time as indicated by the very short branches Allthe three branches are well supported The basal branchingpattern of Maleae may serve as the foundation of subtribaldivision within the tribe if necessary

33 Phylogenetic Relationships within the Core Maleae Themonophyly of the core Maleae is clearly indicated (Fig-ure 1(A)) and well supported (Figure 1(B)) The genera inthe core Maleae seem to be from the second radiation eventNevertheless three multigenus clades within the core Maleaeare recognizable Clade I consists of Amelanchier MedikCrataegus L Cydonia Mill Malacomeles (Decne) DecneMespilus L and Peraphyllum Nutt and is well supported(PB = 95 BP = 90 PP = 1) A very close relationshipbetween Crataegus andMespilus is indicated Clade II (PB =65 BP = 79 PP = 1) consists of four Photinia-relatedthree Sorbus-related and three distinct entitiesThere are fourpairs of genera in this clade but only two of them are highlysupported (PB = 100 BP = 100 PP = 1) namely EriobotryaLindl versus Rhaphiolepis Lindl and Micromeles Decneversus Sorbus L Stranvaesia Lindl was closer to CotoneasterMecik rather than to Photinia Lindl Clade III consists of fourMalus-related onePhotinia-related three Sorbus-related andfive distinct entities and is weakly supported (PB = 50BP = 67 PP = 091) Very close relationships were revealedbetween Malus-related Docyniopsis Koidz and Malus Mill(PB = 100 BP = 100 PP = 1) and among the Sorbus-relatedAria (Pers) Host Chamaemespilus Medik and TorminalisMedik (PB = 72 BP = 94 PP = 1) Docynia Decneand Eriolobus (DC) Roem also fall in the same clade asDocyniopsis andMalus but the clade is onlyweakly supported(PB = 56 BP = 67 PP = 1) Chaenomeles Lindl is related toPseudocydonia (C K Schneid) C K Schneid (BP = 52PP = 1)

4 Discussion

41 Taxa and Gene Sampling Strategies It is often a challengeto reconstruct the phylogeny of taxa from recent radiationevents due to unclear relationships among subdivisions andlow resolution of markers Maleae is such a taxon and that iswhy early studies failed to establish solid phylogenetic rela-tionships among its subdivisions In ldquoA checklist of thesubfamily Maloideae (Rosaceae)rdquo [21] only 23 genera wereaccepted Six Sorbus-related entities that isAriaChamaeme-spilusCormusMicromeles Sorbus andTorminalis were pro-posed to be merged To test the distinctness of these taxa weincluded all of them in this studyMalus-related andPhotinia-related entities have similar taxonomic problems Inclusion ofrepresentative species from other lineages of Amygdaloideaewas to test the monophyly of Maleae Considering that themonophyly of the genera in the narrowest sense has been wellverified 35 entities with one representative species eachwere sampled for being the most inclusive and economicallyaffordable

Sampling chloroplast regions as molecular markers forMaleae is another challenge The tribe was found to be quiteyoung and the core Maleae was even younger Chloroplastmarkers have showed very low resolutions in previous studies[2 4 7 9 42] Therefore the most variable regions suggestedby Dong et al [56] were used together with the four conven-tional DNA barcodes matK psbA-trnH rbcL and ycf1 [46]By doing so the major groups in Maleae were well resolved

42 Phylogenetic Relationships among Major Groups ofMaleae Thephylogeny (Figure 1) strongly suggests inclusionof Gillenia Kageneckia Lindleya and Vauquelinia in Maleae(incorrectly Pyrodae in [7] and in [4]) Gillenia seemed todiverge slightly early andKageneckia+ LindleyaVauqueliniaand the coreMaleae are quite probably from the first radiationevent Although the inclusion of Kageneckia Lindleya andVauquelinia into Maleae seems disagreeable with respect tofruit types their basal chromosome number (119909 = 15 or 17)suggests that they had probably experienced similar specia-tion eventsThey bridge the gap between the coreMaleae andtrue spiraeoid Gillenia The inclusion of Gillenia in Maleaeverifies that the core Maleae is from spiraeoid memberswithin the tribe or other tribes in Amygdaloideae Unfortu-nately transcriptome data of nuclear genes did not providemore information for the issue of origin of the core Maleaebecause Figure 1 is substantially similar to the tree topologiesbased on transcriptome data in [5] Given that Gillenia is theonly diploid member in Maleae the ancestor of Gillenia orits close relatives must be the maternal parent of the extantMaleae

43 Phylogenetic Relationships within the Core Maleae Thecore Maleae is a natural group with several synapomorphiccharacters such as syncarpous ovaries epigynous flowersand fleshy fruits derived from the hypanthial ovary [57ndash59]It could be better classified as subtribe Malinae (incorrectlyPyrinae) if necessary The Malinae diverged into genera byradiation which is the second radiation event in Malese The


001 changesite

Sorbaria sorbifolia

Eriobotrya

Gillenia

Chaenomeles

Cydonia

Osteomeles

Stranvaesia

Malacomeles

Sorbus

Peraphyllum

Torminalis

Chamaemeles

Crataegus

Heteromeles

Docyniopsis

Docynia

Micromeles

Amelanchier

Vauquelinia

Cotoneaster

Chamaemespilus

Physocarpus amurensis

Pyracantha

Photinia

Cormus

Mespilus

Eriolobus

Pyrus

Pourthiaea

Spiraea pubescens

Aria

Lindleya

Rosa rugosa

DichotomanthesAronia

Pseudocydonia

Rhaphiolepis

Kageneckia

Malus

Prinsepia sinensisRhodotypos scandens

Malus-relatedPhotinia-related

Sorbus-related

Clad

e ICl

ade I

ICl

ade I

II

72941

1001001

--059

--099

56671

--091

-521

61691

5067091

65791

555509357521

--1

--079--08

-540871001001

--087

1001001

95961

95901

661001

1001001

94961

10010011001001

7775096

85971

-811

68601

981001

1001001

Maleae

(A)

(B)

Maleae

Figure 1 Phylogenetic relationships of Maleae based on a concatenated dataset of 15 chloroplast gene regions (A) Bayesian phylogramwith branch lengths (B) 50 majority-rule Bayesian consensus tree with bootstrap supports Values beside the branches are the bootstrappercentages from maximum parsimony analysis maximum likelihood analysis and Bayesian posterior probabilities ldquo-rdquo indicates a branchcollapse in the maximum parsimony and maximum likelihood trees

genera are so closely related that it is unnecessary to subdividethis subtribe further

431 Generic Pairs and Their Taxonomic Status There arefour generic pairs for which the taxonomic status needs to be

clarified Firstly a close genetic relationship between Cratae-gus and Mespilus has been revealed by Lo and Donoghue[9] in detail which is confirmed here again IfMespilus wereto be accepted as a distinct genus Crataegus would becomea paraphyletic group Although Mespilus was merged with


Crataegus its distinction was still recognized by giving it thetaxonomic rank of section in Crataegus [9] Secondly reduc-tion of Pseudocydonia to Chaenomeles was done by Guand Spongberg [60] and their treatment is supported byCampbell et al [7] However their close relationship doesnot receive high support in this study (BP = 52 PP = 1)They have diverged for some time because the clade does notshow remarkable branch length They had better be consid-ered distinct genera Thirdly Stranvaesia is morphologicallysimilar to both Photinia and Cotoneaster and sometimessubmerged into Photinia [61] Phylogeny based on chloro-plast regions done by Campbell et al [7] indicated a closerelationship between Stranvaesia and Pyracantha Howeverour data support a close relationship between Stranvaesiaand Cotoneaster a relationship also supported by GBSSI-1B[7] Fourthly although very close morphological and geneticrelationships have been revealed in almost all studies involv-ingEriobotrya andRhaphiolepis there has been no suggestionyet to merge them into one despite the existence of theirhybrids [62] Genetic divergence between them is veryrecently because their clade has a relatively long branch

432 Sorbus-Related Genera So far no well-sampled phy-logeny of all Sorbus-related taxa is available owing to thegenetic complexity of the group and difficulties in samplingThere are ca 250 species belonging tomainly temperate areasin the Northern Hemisphere [21] and six narrowly definedgenera or subgenera under Sorbus in the broadest sense areusually accepted Aria Chamaemespilus Cormus Microme-les Sorbus and Torminalis [21 37ndash39 43 58 63ndash72] Thesetaxa fall into two clades in this study (Figure 1) Sorbus sensustricto and Cormus with compound leaves nested in Clade Iand Aria Chamaemespilus and Torminalis with lobed orunlobed simple leaves nested inClade IIIMicromeles a genuscreated to contain the species without persistent calyx lobesis not a natural entity and is therefore unacceptable [8]Micromeles folgneri in this study is a synonym of Sorbusfolgneri AlthoughCormus had been considered a synonymofSorbus its taxonomic position remains to be determinedAriaChamaemespilus and Torminalis in Clade III are closelyrelated and they had better merge into one separate fromSorbus

Sorbus-related entities are notorious in taxonomy due tocomplexity in phenotypes resulting from interspecifichybridization and facultative agamospermy [39 69 70]Apomictic microspecies confound systematic resolution ofagamic complexes using nuclear markers [71] Phylogeniesbased on nuclear genes may suffer from paralogue problemsand chloroplast markers would work better at the verybeginning when no clear ideas are available for classification

433 Photinia-Related Genera Four genera in their narrow-est sense Aronia Heteromeles Pourthiaea and Stranvaesiaare considered related to Photinia [24 25 29 31 37] Thisstudy demonstrates that all the five genera are superficiallysimilar but actually distinct groups Heteromeles possesses asoft pyrene while Photinia possesses a soft core [16 37 58]Stranvaesia is distinguishable from Photinia by the dehiscentfruits at maturity Although Guo et al [73] stated with

quite certainty that Stranvaesiamust bemerged into Photiniawe believe that further studies are needed for a reliableconclusion Pourthiaea belongs to Clade III instead of CladeII as other genera It is readily separable from other generaby characters such as deciduous habit warty peduncles andpedicels and a pulp structure in the fruits [41] The dis-tinction of Pourthiaea is further supported by leaf epidermisand wood anatomy [74 75] Aronia is not a sister group ofPourthiaea because the clade is poorly supported a conclu-sion that is similar to the earlier studies ([8 9 73])

434 Malus-Related Genera The four genera DocyniaDocyniopsis Eriolobus andMalus really have very close rela-tionships among them especially between Docyniopsis andMalus Some species have been transferred among the fourgenera by different authors The monophyly of a clade com-prising the four genera is indicated by our chloroplast data(Figure 1(B)) There are only two species in Docynia twospecies in Docyniopsis and one species in Eriolobus Consid-ering that these species are nested within Malus [18] thesegenera could better be merged withMalus

5 Conclusions

This molecular phylogenetic study was conducted on allthe genera of Maleae by using 15 variable chloroplast generegions Four major clades are well resolved and the branchesare well supported These clades could be classified into foursubtribes The first radiation event gave birth to Lindleya +KageneckiaVauquelinia and the ancestor of the coreMaleaeand the second radiation event triggered the divergence ofthe genera in the core Maleae within a short time periodWithin the coreMaleae the fourMalus-related genera shouldbetter be merged into one genus the six Sorbus-relatedgenera would be better classified into at least two generaand all Photinia-related genera should be accepted as distinctgenera For a more reliable classification phylogeny basedon the whole chloroplast genomes of representative speciesfrom each genus should be used Such a strategy has beenpracticed on many seed plants (eg [76ndash83]) Besides thenuclear genes especially the single copy nuclear genes indiploid species such as starch-branching enzyme (Sbe [84])should be considered with priority to document the originsofMaleae as thematernally transmittedmarkers can only tellone aspect of the whole story Application of nuclear genomeinformation in phylogenetic reconstruction of Maleae alsoseems feasible because the genomes of several species in thetribe have been sequenced and the resequencing of manyspecies is underway We are expecting a completely resolvedphylogeny of Maleae and that is why we do not provide aformal taxonomic treatment in this study

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper


Supplementary Materials

Supplementary Table S1 sequences downloaded from Gen-Bank in this study with accession numbers SupplementaryTable S2 newly generated sequences withGenBank accessionnumbers in this study (Supplementary Material)

References

[1] M J M Christenhusz and J W Byng ldquoThe number of knownplants species in the world and its annual increaserdquo Phytotaxavol 261 no 3 pp 201ndash217 2016

[2] D R Morgan D E Soltis and K R Robertson ldquoSystematicand evolutionary implications of RBCL sequence variation inRosaceaerdquo American Journal of Botany vol 81 no 7 pp 890ndash903 1994

[3] D Potter F Gao P E Bortiri S-H Oh and S Baggett ldquoPhylo-genetic relationships in Rosaceae inferred from chloroplast matK and trn L-trn F nucleotide sequence datardquo Plant Systematicsand Evolution vol 231 no 1ndash4 pp 77ndash89 2002

[4] D Potter T Eriksson R C Evans et al ldquoPhylogeny andclassification of Rosaceaerdquo Plant Systematics and Evolution vol266 no 1-2 pp 5ndash43 2007

[5] Y Xiang C Huang Y Hu et al ldquoEvolution of Rosaceae FruitTypes Based onNuclear Phylogeny in the Context of GeologicalTimes and Genome DuplicationrdquoMolecular Biology and Evolu-tion 2016

[6] R C Evans and C S Campbell ldquoThe origin of the apple sub-family (Maloideae Rosaceae) is clarified byDNA sequence datafrom duplicated GBSSI genesrdquo American Journal of Botany vol89 no 9 pp 1478ndash1484 2002

[7] C S Campbell R C Evans D R Morgan T A Dickinsonand M P Arsenault ldquoPhylogeny of subtribe Pyrinae (formerlythe Maloideae Rosaceae) Limited resolution of a complexevolutionary historyrdquo Plant Systematics and Evolution vol 266no 1-2 pp 119ndash145 2007

[8] Q-Y Li W Guo W-B Liao J A Macklin and J-H LildquoGeneric limits of Pyrinae Insights from nuclear ribosomalDNA sequencesrdquo Botanical Studies vol 53 no 1 pp 151ndash1642012

[9] E Y Y Lo and M J Donoghue ldquoExpanded phylogenetic anddating analyses of the apples and their relatives (PyreaeRosaceae)rdquo Molecular Phylogenetics and Evolution vol 63 no2 pp 230ndash243 2012

[10] G K Schulze-Menz ldquoRosaceaerdquo in Englerrsquos Syllabus der Pflan-zenfamilien H Melchior Ed vol II pp 209ndash218 GebruderBorntraeger Berlin 1964

[11] P Goldblatt ldquoCytotaxonomic Studies in the tribe quillajeae(rosaceae)rdquoAnnals of theMissouri Botanical Garden vol 63 no1 pp 200ndash206 1976

[12] R C Evans L A Alice C S Campbell E A Kellogg and T ADickinson ldquoThe granule-bound starch synthase (GBSSI) genein the Rosaceae Multiple loci and phylogenetic utilityrdquoMolecu-lar Phylogenetics and Evolution vol 17 no 3 pp 388ndash400 2000

[13] R C Evans and T A Dickinson ldquoFloral ontogeny and mor-phology in Gillenia (ldquoSpiraeoideaerdquo) and subfamily Maloideaec weber (Rosaceae)rdquo International Journal of Plant Sciences vol166 no 3 pp 427ndash447 2005

[14] E Chevreau Y Lespinasse andMGallet ldquoInheritance of pollenenzymes and polyploid origin of apple (Malus x domesticaBorkh)rdquo Theoretical and Applied Genetics vol 71 no 2 pp268ndash277 1985

[15] N F Weeden and R C Lamb ldquoGenetics and linkage analysis of19 isozyme loci in Applerdquo Journal of the American Society forHorticultural Science vol 112 pp 865ndash872 1987

[16] J B Phipps K R Robertson J R Rohrer and P G SmithldquoOrigins and Evolution of Subfam Maloideae (Rosaceae)rdquoSystematic Botany vol 16 no 2 p 303 1991

[17] O Raspe A-L Jacquemart and J De Sloover ldquoIsozymes inSorbus aucuparia (Rosaceae Maloideae) Genetic analysis andevolutionary significance of zymogramsrdquo International Journalof Plant Sciences vol 159 no 4 pp 627ndash636 1998

[18] R Velasco A Zharkikh and J Affourtit ldquoThe genome of thedomesticated apple (Malus times domestica Borkh)rdquoNature Genet-ics vol 42 no 10 pp 833ndash839 2010

[19] M J Considine Y Wan M F DrsquoAntuono et al ldquoMoleculargenetic features of polyploidization and aneuploidization revealunique patterns for genome duplication in diploidMalusrdquo PLoSONE vol 7 no 1 Article ID e29449 2012

[20] J HutchinsonTheGenera of Flowering Plants vol 1 ClarendonPress UK 1964

[21] J B Phipps K R Robertson P G Smith and J R Rohrer ldquoAchecklist of the subfamily Maloideae (Rosaceae)rdquo Botany vol68 no 10 pp 2209ndash2269 1990

[22] C Kalkman ldquoRosaceaerdquo inThe Families and Genera of VascularPlants K Kubitzki Ed pp 343ndash386 Springer Berlin 2004

[23] C Linnaeus Species Plantarum Stockholm Sweden 1753[24] J Lindley ldquoXI Observations on the natural Group of Plants

called PomaceaerdquoTransactions of the Linnean Society of Londonvol 13 no 1 pp 88ndash106 1821

[25] A P De Candolle ldquoRosaceaerdquo in Prodromus Systematais Nat-uralis Regni Vegetabilis vol 2 pp 525ndash639 Treuttel amp WurtzParis 1825

[26] J Lindley ldquoPhotinia argutardquo Botanical Register vol 23 1956[27] M J Roemer ldquoRosiflorae Amygdalacearum et Pomacearumrdquo

in Familiarum Naturalium Regni Vegetabilis Synopses Mono-graphicae Weimar Landes-Industrie-Comptoir 1847

[28] M J Decaisne ldquoMemoirs sur le famile des PomaceesrdquoNouvellesArchives du Museum drsquoHistoire Naturelle vol 10 pp 113ndash1921874

[29] T Wenzig ldquoDie Pomaceenrdquo in Charaktere der Gattungen undArten vol 2 pp 287ndash307 Jahrbuch des Konigl BotanischenGartens Berlin 1883

[30] WO Focke ldquoRosaceaerdquo inDieNaturlichen Pflanzenfamilien AEngler andK Prantl Eds vol 3 ofEngelmann pp 1ndash61 LeipzigGermany 1894

[31] E Koehne ldquoDie Gattugngen der Pomaceenrdquo Gartenflora vol40 p 61 1891

[32] K Fritsch ldquoZur Systematik der Gattung Sorbusrdquo Plant System-atics and Evolution vol 48 no 2 p 1-4 47-49 167-171 1898

[33] K Fritsch ldquoZur Systematik der Gattung Sorbusrdquo Plant System-atics and Evolution vol 49 no 12 381-385 426-429 pages 1899

[34] A Rehder Manual of Cultivated Trees and Shrubs Hardy inNorth America Exclusive of the Subtropical andWarmer temper-ate Regions MacMillan NY USA 2nd edition 1940

[35] A Rehder Bibliography of cultivated trees and shrubs hardyin the cooler temperate regions of the Northern HemisphereHarvard Univ 1949

[36] C S Campbell and T A Dickinson ldquoApomixis Patterns ofMorphological Variation and Species Concepts in subfamMaloideae (Rosaceae)rdquo Systematic Botany vol 15 no 1 pp 124ndash135 1990


[37] K R Robertson J B Phipps J R Rohrer and P G SmithldquoA Synopsis of Genera in Maloideae (Rosaceae)rdquo SystematicBotany vol 16 no 2 p 376 1991

[38] T T Yu ldquoRosaceae (1) Spiraeoideae-Maloideaerdquo in FloraReipublicae Popularis Sinicae T T Yu Ed vol 36 SciencePress Beijing China 1974

[39] J J Aldasoro C Aedo C Navarro and F M Garmendia ldquoThegenus sorbus (maloideae rosaceae) in Europe and in NorthAfrica Morphological analysis and systematicsrdquo SystematicBotany vol 23 no 2 pp 189ndash212 1998

[40] C Kalkman ldquoTheMalesian species of the subfamily MaloideaeRosaceaerdquo Blumea vol 21 pp 413ndash442 1973

[41] H Iketani andH Ohashi ldquoPourthiaea (Rosaceae) distinct fromPhotiniardquo Journal of Japanese Botany vol 66 pp 352ndash355 1991

[42] R C Evans andTADickinson ldquoFloral ontogeny andmorphol-ogy in subfamily Spiraeoideae endl (Rosaceae)rdquo InternationalJournal of Plant Sciences vol 160 no 5 pp 981ndash1012 1999

[43] C S Campbell M J Donoghue B G Baldwin and MF Wojciechowski ldquoPhylogenetic relationships in Maloideae(Rosaceae) Evidence from sequences of the internal tran-scribed spacers of nuclear ribosomal DNA and its congruencewithmorphologyrdquoAmerican Journal of Botany vol 82 no 7 pp903ndash918 1995

[44] L Jinlu W Shuo Y Jing W Ling and Z Shiliang ldquoA ModifiedCTAB Protocol for Plant DNA Extractionrdquo Chinese Bulletin ofBotany vol 48 no 1 pp 72ndash78 2013

[45] W Dong C Xu T Cheng K Lin and S Zhou ldquoSequencingangiosperm plastid genomes made easy A complete set of uni-versal primers and a case study on the phylogeny of saxifra-galesrdquo Genome Biology and Evolution vol 5 no 5 pp 989ndash9972013

[46] W Dong C Xu C Li et al ldquoycf1 the most promising plastidDNA barcode of land plantsrdquo Scientific Reports vol 5 p 83482015

[47] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997

[48] M A Larkin G Blackshields N P Brown et al ldquoClustalW andclustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947-2948 2007

[49] A Rambaut ldquoSe-Al Sequence alignment editor ver 20rdquoAvailable from httptreebioedacuksoftwareseal 1996

[50] P Librado and J Rozas ldquoDnaSP v5 a software for comprehen-sive analysis of DNA polymorphism datardquo Bioinformatics vol25 no 11 pp 1451-1452 2009

[51] D L Swofford PAUP Phylogenetic Analysis Using Parsimony(and Other Methods) Sinauer Associates Sunderland Version40b10 edition 2003

[52] G Vaidya D J Lohman and R Meier ldquoSequenceMatrixConcatenation software for the fast assembly of multi-genedatasets with character set and codon informationrdquo Cladisticsvol 27 no 2 pp 171ndash180 2011

[53] A Stamatakis ldquoRAxML version 8 a tool for phylogeneticanalysis and post-analysis of large phylogeniesrdquo Bioinformaticsvol 30 no 9 pp 1312-1313 2014

[54] F Ronquist and J P Huelsenbeck ldquoMrBayes 3 bayesian phylo-genetic inference under mixed modelsrdquo Bioinformatics vol 19no 12 pp 1572ndash1574 2003

[55] D Posada andK A Crandall ldquoMODELTEST testing themodelof DNA substitutionrdquo Bioinformatics vol 14 no 9 pp 817-8181998

[56] W Dong J Liu J Yu L Wang S Zhou and A MoustafaldquoHighly Variable Chloroplast Markers for Evaluating PlantPhylogeny at Low Taxonomic Levels and for DNA BarcodingrdquoPLoS ONE vol 7 no 4 p e35071 2012

[57] P LeinsThe Carpel in Superior and Inferior Gynoecia vol 85 ofBerichteder Deutschen Botanischen Gesellschaft 1972

[58] J R Rohrer K R Robertson and J B Phipps ldquoFloral mor-phology of Maloideae (Rosaceae) and its systematic relevancerdquoAmerican Journal of Botany vol 81 no 5 pp 574ndash581 1994

[59] W B Zomlefer Guide to Flowering Plant Families Chapel HillUniversity of North Carolina Press 1994

[60] C Gu and S Spongberg ldquoChaenomelesrdquo in Flora of China ZWu and P Raven Eds pp 46ndash434 Science Press BeijingChina Missouri Botanical Garden Press St Louis MissouriUSA 2003

[61] J E Vidal ldquoNotes sur quelques Rosacees Asiatique (II) (Pho-tinia Stranvaesia)rdquo Adansonia vol 5 pp 221ndash237 1965

[62] J J Aldasoro C Aedo and C Navarro ldquoPhylogenetic andphytogeographical relationships inmaloideae (Rosaceae) basedon morphological and anatomical charactersrdquo Blumea Journalof Plant Taxonomy and Plant Geography vol 50 no 1 pp 3ndash322005

[63] T Hedlund ldquoMonographie der Gattung SorbusrdquoKonigl SvenskaVetenskaps-Akademiens Handlingar vol 35 pp 1ndash147 1901

[64] A Rehder ldquoSorbus Lrdquo inPlantaeWilsonianae C S Sargent Edvol 2 pp 266ndash279 Cambridge University Press CambridgeNY USA 1915

[65] M Kovanda ldquoTaxonomical studies in Sorbus subg AriardquoDendrologicky Sbornık vol 3 pp 23ndash70 1961

[66] J E Vidal ldquoSorbus Lrdquo in Flore du Cambodge du Laos and duVietnam Eds vol 6 pp 23ndash34 Museum National drsquoHistoireNaturelle 1968

[67] E Gabrielian ldquoThe Genus Sorbus Lrdquo in Eastern Asia and theHimalayas Armenian Academy of Sciences Yerevan Armenia1978

[68] M Kovanda and J Challice ldquoThe genus Micromeles revisitedrdquoFolia Geobotanica et Phytotaxonomica vol 16 no 2 pp 181ndash1931981

[69] A Jankun ldquoEvolutionary significance of apomixis in the genusSorbus Rosaceaerdquo Fragmenta Floristica et Geobotanica vol 38no 2 pp 627ndash686 1993

[70] G Aas J Maier M Baltisberger and S Metzger ldquoMorphol-ogy isozyme variation cytology and reproduction of hybridsbetween Sorbus aria (L) Crantz and Storminalis (L) CrantzrdquoBotanica Helvetica vol 104 no 2 pp 195ndash214 1994

[71] C S Campbell W AWright T F Vining andW A HaltemenldquoMorphological variation in sexual and agamospermous Ame-lanchier (Rosaceae)rdquo Canadian Journal of Botany vol 75 no 7pp 1166ndash1173 1997

[72] J J Aldasoro C Aedo F M Garmendia F P Hoz and CNavarro ldquoRevision of Sorbus Subgenera Aria and Torminaria(Rosaceae-Maloideae)rdquo Systematic Botany Monographs vol 69pp 1ndash148 2004

[73] W Guo Y Yu R-J Shen W-B Liao S-W Chin and D PotterldquoA phylogeny of Photinia sensu lato (Rosaceae) and relatedgenera based on nrITS and cpDNA analysisrdquo Plant Systematicsand Evolution vol 291 no 1 pp 91ndash102 2011


[74] L T Lu Z L Wang and G Li ldquoThe significance of the leafepidermis in the taxonomy of the Photinia complex (RosaceaeMaloideae)rdquo Cathaya vol 3 pp 93ndash108 1991

[75] S Zhang and P Baas ldquoWood anatomy of trees and shrubs fromchina III rosaceaerdquo IAWA Journal vol 13 no 1 pp 21ndash91 1992

[76] R C Haberle Phylogeny and Comparative Chloroplast Ge-nomics of the Campanulaceae [PhD thesis] Austin Universityof Texas 2006

[77] E Bortiri D Coleman-Derr G R Lazo O D Andersonand Y Q Gu ldquoThe complete chloroplast genome sequence ofBrachypodium distachyon sequence comparison and phyloge-netic analysis of eight grass plastomesrdquo BMC Research Notesvol 1 article 61 2008

[78] M Parks R Cronn and A Liston ldquoIncreasing phylogeneticresolution at low taxonomic levels using massively parallelsequencing of chloroplast genomesrdquo BMC Biology vol 7 articleno 84 2009

[79] J-H Xue W-P Dong T Cheng and S-L Zhou ldquoNelum-bonaceae Systematic position and species diversification re-vealed by the complete chloroplast genomerdquo Journal of System-atics and Evolution vol 50 no 6 pp 477ndash487 2012

[80] S V Nikiforova D Cavalieri R Velasco and V GoremykinldquoPhylogenetic analysis of 47 chloroplast genomes clarifies thecontribution of wild species to the domesticated apple maternallinerdquo Molecular Biology and Evolution vol 30 no 8 pp 1751ndash1760 2013

[81] J L Cotton W P Wysocki L G Clark et al ldquoResolving deeprelationships of PACMAD grasses A phylogenomic approachrdquoBMC Plant Biology vol 15 no 1 article no 178 2015

[82] T G Ross C F Barrett M Soto Gomez et al ldquoPlastid phyloge-nomics and molecular evolution of Alismatalesrdquo Cladistics vol32 no 2 pp 160ndash178 2016

[83] Y Sun M J Moore S Zhang et al ldquoPhylogenomic andstructural analyses of 18 complete plastomes across nearly allfamilies of early-diverging eudicots including an angiosperm-wide analysis of IR gene content evolutionrdquoMolecular Phyloge-netics and Evolution vol 96 pp 93ndash101 2016

[84] Y Han K Gasic F Sun M Xu and S S Korban ldquoA geneencoding starch branching enzyme I (SBEI) in apple (Malus timesdomestica Rosaceae) and its phylogenetic relationship to Sbegenes from other angiospermsrdquo Molecular Phylogenetics andEvolution vol 43 no 3 pp 852ndash863 2007

[85] B Oxelman M Liden and D Berglund ldquoChloroplast rps16intron phylogeny of the tribe Sileneae (Caryophyllaceae)rdquo PlantSystematics and Evolution vol 206 no 1ndash4 pp 393ndash410 1997

Hindawiwwwhindawicom

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Zoology

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Anatomy Research International

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Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom


Elliott) as well as many ornamentals The core Maleae ischaracterized by a synapomorphic pome a type of accessoryfruit that does not occur in other Rosaceae plants [10] anda basal chromosome number 119909 = 17 With the addition ofGillenia Kageneckia Lindleya and Vauquelinia the tribe hasdrupaceous or follicle fruits and 119909 = 9 (or 15) as well[2 4 6 9 11ndash13]

The origin of core Maleae (119909 = 17) has long beenconsidered an example of allopolyploidization between thespecies with 119909 = 9 in traditional Spiraeoideae and the specieswith 119909 = 8 in traditional Amygdaloideae [14ndash17] Howeverrecently discovered genomic data suggested an origin viaautopolyploidization followed by aneuploidization around 50million years ago [18 19] In contrast the allopolyploid natureof core Maleae was confirmed by GBSSI which had fourcopies in the core Maleae but only two copies in other groups[6]

Polyploidization affects systematics at both generic andspecific levels It is unlikely to resolve the polytomy of ances-tral populations that have just a few closely related speciesinvolved in historical speciation and subsequent diversifica-tion owing to the lack of phylogenetically informative signalsand incomplete lineage sorting The merging of severalgenomes into one species enriches the pool of available ge-netic combinations and the survival of recombinants isovercome by apomixis Maleae is one such species-rich tribeof genera but is difficult to classify

The pome-bearing plants have been generally subdividedinto two groups one with connate endocarps and the otherwith polypyrenous drupes [10 20ndash22] However such sub-divisions of the core Maleae do not receive support frommolecular data Considerable controversies exist on cir-cumscriptions of genera based on morphology or anatomy[21 23ndash37] Sorbus L is considered to include both thepinnate-leaved species (Sorbus ss and Cormus Spach) andthe simple-leaved species (Aria (Pers) Host MicromelesDecne ChamaemespilusMedik and TorminalisMedik) [2134 38 39]AroniaMedikHeteromelesM Roem PourthiaeaDecne and Stranvaesia Lindl are either merged togetherwith Photinia Lindl or accepted as distinct genera [37 4041] Varying opinions are also found among Malus MillDocyniopsis Koidz and Eriolobus (DC) M Roem as wellas among Pseudocydonia (C K Schneid) C K SchneidCydonia Mill and Chaenomeles Lindl [22 27 37] Lack ofconsensus treatment among these genera is a result of uncer-tainty about genetic relationships among all entities (or gen-era in the narrow sense) and molecular data have been usedto reveal their phylogenies Both chloroplast DNA sequences[2 4 7 9 42] and nuclear DNA sequences ([4 6ndash8 12 43] Loet al 2012) have been tried and it is clear that themonophylyof each entity is highly supported the major unsolvedproblem is the phylogeny among entities To elucidate theintricate relationships among these entities (the generallyrecognized genera in the narrow sense within Maleae) wesampled representative species for each of them including theoccasionally accepted genera and subgenera and constructedtheir phylogeny using 15 chloroplast regions Our results areexpected to be helpful for the correct circumscription ofgenera in Maleae

2 Materials and Methods

21 Taxon Sampling A total of 41 species were examined35 species representing all generally recognized genera ofMaleae and five species representing other major lineagesof Amygdaloideae and Rosa rugosa Thunb as an outgroup(Table 1) All samples were collected under appropriatepermits Herbarium Institute of Botany CAS Beijing Botan-ical Garden CAS Kunming Botanical Garden CAS andXishuangbanna Tropical Botanical Garden CAS and otherplaces listed in Table 1

22 DNA Extraction Amplification and Sequencing Totalgenomic DNA was extracted using the mCTAB method[44] and purified using the Wizard DNA Clean-Up Sys-tem (A7280 Promega Corporation Madison WI) Fifteenchloroplast regions that is atpB-rbcLmatK ndhF petA-psbJpsbA-trnH psbM-trnD rbcL rpl16 rpl20-rps12 rps16 trnC-ycf6 trnH-rpl2 trnL-trnF trnS-trnG and ycf1 were usedin this study Fourteen primer pairs published in Dong etal [45 46] were synthesized by Sangon Biotech (Shanghai)Co Ltd (Beijing China) and used to amplify and sequencethese regions (Table 2) Sequences of rps16 were downloadedfromGenBankThe polymerase chain reaction (PCR) ampli-fications were carried out in a mixture volume of 20120583Lcontaining 1x Taq buffer (1molL KCl 20mmol Tris-HClpH 90 1 Triton X-100) 20 120583L dNTPs (2mmolL) 10 120583Leach of the primers (5 120583molL) 20 ng of genomic DNA and1 unit of Taq polymerase PCR was conducted using a C1000Thermal Cycler (Bio-Rad Laboratories Hercules CA USA)Theprogram started at 94∘C for 3min followed by 35 cycles at94∘C for 30 s 50∘C for 30 s and 72∘C for 2min and ended at72∘C for 5minThePCRproducts were purifiedwith an equalmixture of 40 PEG 8000 and 5molL NaCl followed by awashing step with 80 ethanol The PCR products were thenSanger sequenced for both strands on an ABI 3730xl DNAAnalyzer using BigDye Terminator cycle sequencing kit v31(Applied Biosystems Foster City CAS USA) following themanufacturerrsquos instructions

23 Sequence Data Preparation and Evaluation The newlygenerated sequences were checked and assembled usingSequencer 47 (Gene codes Corporation Ann Arbor Michi-gan USA) The resulting sequences (Supplementary TableS2) were combined with the published sequences of Maleaespecies downloaded from GenBank (Supplementary TableS1) aligned with Clustal X [47 48] and then manuallyadjustedwith Se-Al 20 [49] Each individual gene dataset wasthen subjected to several rounds of phylogenetic evaluationsto select reliable sequences Sequences thatweremisidentifiedor exhibited large amounts of missing data were excludedfrom the datasets To predict the phylogenetic performanceof individual gene partitions the variability of genes wasparameterized by DnaSP 50 [50] and PAUP 40b10 [51] usingnucleotide diversity the number of polymorphic sites andso forth Prior to concatenating the dataset of each markerincongruence length difference (ILD) tests were performedon all 15 datasets The datasets were finally concatenated














3 Results







4 Discussion






001 changesite

Sorbaria sorbifolia

Eriobotrya

Gillenia

Chaenomeles

Cydonia

Osteomeles

Stranvaesia

Malacomeles

Sorbus

Peraphyllum

Torminalis

Chamaemeles

Crataegus

Heteromeles

Docyniopsis

Docynia

Micromeles

Amelanchier

Vauquelinia

Cotoneaster

Chamaemespilus


Pyracantha

Photinia

Cormus

Mespilus

Eriolobus

Pyrus

Pourthiaea

Spiraea pubescens

Aria

Lindleya

Rosa rugosa


Pseudocydonia

Rhaphiolepis

Kageneckia

Malus



Sorbus-related

Clad

e ICl

ade I

ICl

ade I

II

72941

1001001

--059

--099

56671

--091

-521

61691

5067091

65791

555509357521

--1

--079--08

-540871001001

--087

1001001

95961

95901

661001

1001001

94961

10010011001001

7775096

85971

-811

68601

981001

1001001

Maleae

(A)

(B)

Maleae












5 Conclusions







References


























































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018















3 Results







4 Discussion






001 changesite

Sorbaria sorbifolia

Eriobotrya

Gillenia

Chaenomeles

Cydonia

Osteomeles

Stranvaesia

Malacomeles

Sorbus

Peraphyllum

Torminalis

Chamaemeles

Crataegus

Heteromeles

Docyniopsis

Docynia

Micromeles

Amelanchier

Vauquelinia

Cotoneaster

Chamaemespilus


Pyracantha

Photinia

Cormus

Mespilus

Eriolobus

Pyrus

Pourthiaea

Spiraea pubescens

Aria

Lindleya

Rosa rugosa


Pseudocydonia

Rhaphiolepis

Kageneckia

Malus



Sorbus-related

Clad

e ICl

ade I

ICl

ade I

II

72941

1001001

--059

--099

56671

--091

-521

61691

5067091

65791

555509357521

--1

--079--08

-540871001001

--087

1001001

95961

95901

661001

1001001

94961

10010011001001

7775096

85971

-811

68601

981001

1001001

Maleae

(A)

(B)

Maleae












5 Conclusions







References


























































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018









3 Results







4 Discussion






001 changesite

Sorbaria sorbifolia

Eriobotrya

Gillenia

Chaenomeles

Cydonia

Osteomeles

Stranvaesia

Malacomeles

Sorbus

Peraphyllum

Torminalis

Chamaemeles

Crataegus

Heteromeles

Docyniopsis

Docynia

Micromeles

Amelanchier

Vauquelinia

Cotoneaster

Chamaemespilus


Pyracantha

Photinia

Cormus

Mespilus

Eriolobus

Pyrus

Pourthiaea

Spiraea pubescens

Aria

Lindleya

Rosa rugosa


Pseudocydonia

Rhaphiolepis

Kageneckia

Malus



Sorbus-related

Clad

e ICl

ade I

ICl

ade I

II

72941

1001001

--059

--099

56671

--091

-521

61691

5067091

65791

555509357521

--1

--079--08

-540871001001

--087

1001001

95961

95901

661001

1001001

94961

10010011001001

7775096

85971

-811

68601

981001

1001001

Maleae

(A)

(B)

Maleae












5 Conclusions







References


























































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018







4 Discussion






001 changesite

Sorbaria sorbifolia

Eriobotrya

Gillenia

Chaenomeles

Cydonia

Osteomeles

Stranvaesia

Malacomeles

Sorbus

Peraphyllum

Torminalis

Chamaemeles

Crataegus

Heteromeles

Docyniopsis

Docynia

Micromeles

Amelanchier

Vauquelinia

Cotoneaster

Chamaemespilus


Pyracantha

Photinia

Cormus

Mespilus

Eriolobus

Pyrus

Pourthiaea

Spiraea pubescens

Aria

Lindleya

Rosa rugosa


Pseudocydonia

Rhaphiolepis

Kageneckia

Malus



Sorbus-related

Clad

e ICl

ade I

ICl

ade I

II

72941

1001001

--059

--099

56671

--091

-521

61691

5067091

65791

555509357521

--1

--079--08

-540871001001

--087

1001001

95961

95901

661001

1001001

94961

10010011001001

7775096

85971

-811

68601

981001

1001001

Maleae

(A)

(B)

Maleae












5 Conclusions







References


























































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



001 changesite

Sorbaria sorbifolia

Eriobotrya

Gillenia

Chaenomeles

Cydonia

Osteomeles

Stranvaesia

Malacomeles

Sorbus

Peraphyllum

Torminalis

Chamaemeles

Crataegus

Heteromeles

Docyniopsis

Docynia

Micromeles

Amelanchier

Vauquelinia

Cotoneaster

Chamaemespilus


Pyracantha

Photinia

Cormus

Mespilus

Eriolobus

Pyrus

Pourthiaea

Spiraea pubescens

Aria

Lindleya

Rosa rugosa


Pseudocydonia

Rhaphiolepis

Kageneckia

Malus



Sorbus-related

Clad

e ICl

ade I

ICl

ade I

II

72941

1001001

--059

--099

56671

--091

-521

61691

5067091

65791

555509357521

--1

--079--08

-540871001001

--087

1001001

95961

95901

661001

1001001

94961

10010011001001

7775096

85971

-811

68601

981001

1001001

Maleae

(A)

(B)

Maleae












5 Conclusions







References


























































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018









5 Conclusions







References


























































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





References


























































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018

















Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Documents

Phylogeny of Maleae (Rosaceae) Based on Multiple