Phylogenetic incongruence in Cymbidium orchids
Guo-Qiang Zhanga,c,1, Gui-Zhen Chenb,c,1, Li-Jun Chenc, Jun-Wen Zhaib, Jie Huangb,c, Xin-Yi Wuc, Ming-He Lib, Dong-Hui Pengb, Wen-Hui Raoc, Zhong-Jian Liub, Si-Ren Lana,b,**     
a. Forestry College of Fujian Agriculture and Forestry University, Fuzhou, 350002, China;
b. Key Laboratory of National Forestry and Grassland Administration for Orchid Conservation and Utilization at College of Landscape Architecture, Fujian Agriculture and Forestry University, Fuzhou, 350002, China;
c. Shenzhen Key Laboratory for Orchid Conservation and Utilization, The National Orchid Conservation Center of China and the Orchid Conservation and Research Center of Shenzhen, Shenzhen, 518114, China
Abstract: Cymbidium, which includes approximately 80 species, is one of the most ornamental and cultivated orchid genera. However, a lack of markers and sparse sampling have posed great challenges to resolving the phylogenetic relationships within the genus. In the present study, we reconstructed the phylogenetic relationships by utilizing one nuclear DNA (nrITS) and seven plastid genes (rbcL, trnS, trnG, matK, trnL, psbA, and atpI) from 70 species (varieties) in Cymbidium. We also examined the occurrence of phylogenetic conflict between nuclear (nrITS) and plastid loci and investigated how phylogenetic conflict bears on taxonomic classification within the genus. We found that phylogenetic conflict and low support values may be explained by hybridization and a lack of informative characteristics. Our results do not support previous classification of the subgenera and sections within Cymbidium. Discordance between gene trees and network analysis indicate that reticulate evolution occurred in the genus Cymbidium. Overall, our study indicates that Cymbidium has undergone a complex evolution.
Keywords: Cymbidium    Phylogenetic conflict    nrITS    cpDNA    Reticulate evolution    
1. Introduction

The genus Cymbidium SW., with approximately 80 species, is primarily distributed throughout the subtropics and tropical areas of Asia and northern Australia (Du Puy and Cribb, 2007; Chen et al., 2009; Pridgeon et al., 2009). In China, more than 50 species are found according to the most recent revision and recently published new species (Liu and Chen, 1998, 2002, 2004, 2005; Long et al., 2003; Liu et al., 2006; Chen et al., 2009; Lan et al., 2018; Zhang et al., 2018). Cymbidium is one of the earliest orchid groups to be cultivated, making excellent potted plants and cut flowers due to its extremely high ornamental and economic value. Commercially important hybrids have been cultivated for a long time in China and adjacent regions (Liu et al., 2006). Despite its acknowledged importance, limited and often ambiguous morphological differences pose challenges to understanding intergeneric relationships within Cymbidium. The intergeneric relationships of Cymbidium remain an open question due to limited and often ambiguous morphological differences.

Since the establishment of Cymbidium by Swartz in 1799, various generic delimitations and infrageneric systems have been proposed based on morphological characters. Dressler (1981) places Cymbidium in Cymbidieae of Vandioideae, which contains all of the sympodial vandoid orchids, mostly with two pollinia. Schlechter (1924) proposes an infrageneric system of Cymbidium with eight sections, which is the basis of the modern infrageneric classification of Cymbidium, and most sections are still being recognized more or less in their original form. Hunt (1970) included Cyperorchis within Cymbidium and maintained Schlechter's sectional divisions. Seth and Cribb (1984) initially divided Cymbidium into three subgenera based on the number of pollinia and the state of fusion between lip and column: subgenus Cymbidium with two pollinia and free lip, subgenusCyperorchis with two pollinia and fusion of the lip and column base, and subgenus Jensoa with four pollinia and free lip. Puy and Cribb (1988) slightly modified this treatment and added section Borneensia for the recently described Cymbidium borneense Wood. Liu et al. (2006) followed the treatment of Puy and Cribb (1988) with some modifications and additions, added sections Nanula and Axillaria, transferred the section Borneensia from subgenus Cymbidium into the subgenus Jensoa, and reduced the section Maxillarianthe to synonymy of the section Jensoa.

Recently, the results of molecular analyses have shed new light on the taxonomy of Cymbidium (Cameron et al., 1999; Berg, 2002; Yukawa and Stern, 2002; Zhang et al., 2002; Sharma et al., 2012; Yang et al., 2013; Lan et al., 2018). The phylogeny of Cymbidium reconstructed by Zhang et al. (2002) based on the analyses of nrITS sequences in 30 taxa belonging to three subgenera showed that the genus was split into several clades and intermixed with the main subgenera, suggesting that the previous division among three subgenera should be evaluated with more data. Berg (2002) performed a molecular phylogenetic analysis of Cymbidium using one cpDNA marker (matK) and nrITS and indicated that two or three subgenera can potentially be defined within the genus and affirmed a southeast Asian origin for the genus.Yukawa and Stern. (2002) obtained the same result in their strict consensus tree based on nrITS and matK sequences. Assessment of phylogenetic interrelationships in the genus Cymbidium from northeast India based on nrITS showed that this genus was divided into three subgenera (Sharma et al., 2012). Du Puy and Cribb (2007) according to the DNA studies by Berg (2002) and Yukawa and Stern (2002), concluded that the subgenera of Cymbidium were not monophyletic, and they retained the sections but dispensed with subgenera.

All of the pre-DNA era classifications of Cymbidium were based on a relatively small set of morphological aspects and features, especially on the lip and pollinium numbers, which have led to considerable taxonomic uncertainty and debates (Dressler, 1993; Freudenstein and Rasmussen, 1999). As previous results of molecular systematics were largely based on sparse sampling across Cymbidium or mainly utilized either a single DNA marker (especially nrITS) or two markers (nrITS and matK), some conclusions and results were weakly supported or even without statistical support (Cameron et al., 1999; Yukawa and Stern, 2002; Zhang et al., 2002; Yang et al., 2013; Lan et al., 2018). Therefore, it is necessary to understand the relationships within Cymbidium and the delimitation of the infrageneric taxa to base the analyses on multiple DNA markers and a denser sampling acrossCymbidium.

One additional challenge to accurately reconstructing phylogenetic relationships is the possibility of phylogenetic incongruence between cpDNA and nuclear sequence data (Tu et al., 2008; Pelser et al., 2010; Guo et al., 2015; Tang et al., 2015). Phylogenetic incongruence may be a result of stochastic errors, systematic errors, incomplete lineage sorting (ILS), introgressive hybridization (IH), paralogous gene sampling, or horizontal gene transfer (HGT) Francine et al., 2017, Geuten et al., 2004, Richardson and Palmer, 2007, Russell et al., 2010, Yang et al., 2012.

In this study, we reconstructed the phylogeny of 70 representatives in three subgenera of Cymbidium using both plastid DNA (rbcL, trnS, trnG, matK, trnL, psbA, and atpI) and nrITS sequences. Our goals were to (1) establish a phylogeny based on the seven plastid DNA and one nuclear gene for the Cymbidium with significantly increased taxa sampling with an emphasis on Chinese species, (2) identify relationships that were inconsistent between nuclear and plastid trees, and (3) explore possible causes of the incongruence.

2. Material and methods 2.1. Taxon sampling

To assess phylogenetic relationships within Cymbidium, we used seven plastids (rbcL, trnS, trnG, matK, trnL, psbA and atpI) and one nuclear (nrITS) marker sampled from 70 species (varieties) of Cymbidium. Four species from the tribe Malaxideae Lindley (Lindley, 1826) and one species from the tribe Vandeae Lindley (Lindley, 1821) were used as outgroups. The selection of outgroups was based on the classification of Chase et al. (2015). In this study, 510 sequences (468 of which were newly sequenced) were obtained, the voucher information and GenBank accession numbers were listed in Table 1, and the specimen was deposited in the herbarium of the National Orchid Conservation Center of China (NOCC).

Table 1 Taxa studied, voucher information and GenBank accessions. A dash (−) indicates missing data, an asterisk (*) denotes sequences obtained in this study, and the remaining sequences are from GenBank.
Species Voucher nrITS matK rbcL trnL atpI trnS psbA trnG
Cymbidium aestivum Z.J.Liu 200254 MK439805* MK439758* MK439781* MK439712* MK439655* MK439667* MK439737* MK439688*
Cymbidium aloifolium Z.J.Liu 6591 MF861139* MF861054* MF861098* MF860930* MF860838* MF860955* MF860889*
Cymbidium atropurpureum Z.J.Liu 6592 MF861153* MF861069* MF861111* MF860945* MF860837* MF860986* MF861196* MF860902*
Cymbidium banaense Z.J.Liu 5331 MF861160* MF861076* MF861118* MF860952* MF860992* MF861201* MF860909*
Cymbidium baoshanense Z.J.Liu 2581 MK439807* MK439760* MK439783* MK439714* MK439662* MK439669* MK439739* MK439690*
Cymbidium bicolor AF284696 KX298601 FJ527762
Cymbidium candliculatum Z.J.Liu 6326 MF861161* MF861078* MF860850* MF860994* MF861168* MF860911*
Cymbidium changningense Z.J.Liu 6430 MF861126* MF861042* MF861085* MF860917* MF860865* MF860961* MF861173* MF860876*
Cymbidium chloranthum AF470499 HM137047 FJ527761
Cymbidium cochleare Z.J.Liu 2807 MF861130* MF861045* MF861089* MF860921* MF860846* MF860964* MF861176* MF860880*
Cymbidium cyperifolium Z.J.Liu 3205 MK439808* MK439761* MK439784* MK439715* MK439654* MK439670* MK439740* MK439691*
Cymbidium daweishanense Z.J.Liu 8663 MH59389 * MH593898* MH574772*
Cymbidium dayanum Z.J.Liu 6437 MF861122* MF861038* MF861081* MF860913* MF860831* MF860957* MF861169* MF860872*
Cymbidium defoliatum Z.J.Liu 2554 MF861135* MF861050* MF861094* MF860926* MF860859* MF860969* MF861181* MF860885*
Cymbidium devonianum Z.J.Liu 2693 MF861136* MF861051* MF861095* MF860927* MF860860* MF860970* MF861182* MF860886*
Cymbidium eburneum Z.J.Liu 2625 MF861124* MF861040* MF861083* MF860915* MF860833* MF860959* MF861171* MF860874*
Cymbidium eburneum var. longzhouense Z.J.Liu 3032 MF861144* MF861059* MF861103* MF860935* MF860864* MF860977* MF861162* MF860894*
Cymbidium elegans Z.J.Liu 6399 MF861147* MF861062* MF861106* MF860938* MF860870* MF860980* MF861190* MF860897*
Cymbidium ensifolium Z.J.Liu 6599 MF861138* MF861053* MF861097* MF860929* MF860849* MF860972* MF861184* MF860888*
Cymbidium erythraeum Z.J.Liu 2900 MK439809* MK439762* MK439785* MK439716* MK439651* MK439671* MK439741* MK439692*
Cymbidium erythraeum var. flavum Z.J.Liu 10140 MK439810* MK439763* MK439786* MK439717* MK439660* MK439672* MK439742* MK439693*
Cymbidium erythrostylum AF470524 AF470483
Cymbidium faberi Z.J.Liu 7071 MF861148* MF861063* MF861107* MF860939* MF860854* MF860981* MF861191* MF860898*
Cymbidium finlaysonianum AF470514 HM137048 FJ527763
Cymbidium floribundum Z.J.Liu 3256 MK439811* MK439764* MK439787* MK439718* MK439652* MK439673* MK439743 MK439694*
Cymbidium gaoligongense Z.J.Liu 6432 MF861142* MF861057* MF861101* MF860933* MF860840* MF860975* MF861187* MF860892*
Cymbidium goeringii Z.J.Liu 2522 MK439812 MK439765 MK439788 MK439719 MK439646 MK439674 MK439744 MK439695
Cymbidium haematodes Z.J.Liu 10160 MK439813 MK439766 MK439789 MK439720 MK439658 MK439675 MK439745 MK439696
Cymbidium hookerianum Z.J.Liu 6425 MF861143* MF861058* MF861102* MF860934* MF860863* MF860976* MF860893*
Cymbidium insigne Z.J.Liu 3251 MF861140* MF861055* MF861099* MF860931* MF860847* MF860973* MF861185* MF860890*
Cymbidium iridioides Z.J.Liu 6429 MF861141* MF861056* MF861100* MF860932* MF860845* MF860974* MF861186* MF860891*
Cymbidium kanran Z.J.Liu 2808 MK439814* MK439767* MK439790* MK439721* MK439659* MK439676* MK439746* MK439697*
Cymbidium lancifolium Z.J.Liu 7013 MF861137* MF861052* MF861096* MF860928* MF860839* MF860971* MF861183* MF860887*
Cymbidium lowianum var. iansonii Z.J.Liu 3029 MF861146* MF861061* MF861105* MF860937* MF860869* MF860979* MF861189* MF860896*
Cymbidium macrorhizon Z.J.Liu 200231 MK439815* MK439768* MK439791* MK439722* MK439666* MK439677* MK439698*
Cymbidium maguanense Z.J.Liu 3257 MF861125* MF861041* MF861084* MF860916* MF860834* MF860960* MF861172* MF860875*
Cymbidium mannii Z.J.Liu 6590 MF861121* MF861037* MF861080* MF860912* MF860830* MF860956* MF860871*
Cymbidium mastersii Z.J.Liu 2924 MK439816* MK439769* MK439792* MK439723* MK439653* MK439678* MK439747* MK439699
Cymbidium micranthum Z.J.Liu 2705 MF861149* MF861065* MF860941* MF860842* MF860982* MF861193*
Cymbidium multiradicatum Z.J.Liu 2614 MK439817* MK439793* MK439724* MK439644* MK439679* MK439748* MK439700*
Cymbidium nanulum Z.J.Liu 2562 MF861152* MF861068* MF860944* MF860862* MF860985* MF861195*
Cymbidium omeiense Z.J.Liu 3101 MF861064* MF861108* MF860940* MF860855* MF861192* MF860899*
Cymbidium paucifolium Z.J.Liu 2112 MF861151* MF861067* MF861110* MF860943* MF860844* MF860984* MF861166* MF860901*
Cymbidium puerense Z.J.Liu 10626 MG980600* MG980601* MG980602* MG980604* MG980599* MG980603*
Cymbidium pumilum AF284699
Cymbidium qiubeiense Z.J.Liu 2555 MF861158* MF861074* MF861116* MF860950* MF860853* MF860990* MF860907*
Cymbidium rectum AF470494 AF470463 FJ527767
Cymbidium rhizomatosum Z.J.Liu 2559 MF861150* MF861066* MF861109* MF860942* MF860843* MF860983* MF861194* MF860900*
Cymbidium schroederi Z.J.Liu 2837 MF861155* MF861071* MF861113* MF860947* MF860867* MF860988* MF861198* MF860904*
Cymbidium serratum Z.J.Liu 2575 MF861134* MF861049* MF861093* MF860925* MF860856* MF860968* MF861180* MF860884*
Cymbidium sichuanicum Z.J.Liu 3027 MF861154* MF861070* MF861112* MF860946* MF860857* MF860987* MF861197* MF860903*
Cymbidium sinense Z.J.Liu 2503 MF861159* MF861075* MF861117* MF860951* MF860861* MF860991* MF861163* MF860908*
Cymbidium sp.7066 Z.J.Liu 7066 MF861132* MF861047* MF861091* MF860923* MF860852* MF860966* MF861178* MF860882*
Cymbidium sp.5256 Z.J.Liu 5256 MK439820* MK439772* MK439796* MK439727* MK439645* MK439750* MK439703*
Cymbidium sp.10161 Z.J.Liu 10161 MK439821* MK439773* MK439797* MK439728* MK439661* MK439751* MK439704*
Cymbidium sp.5774 Z.J.Liu 5774 MK439822* MK439774* MK439798* MK439729* MK439663* MK439752* MK439705*
Cymbidium sp.10163 Z.J.Liu 10163 MK439823* MK439799* MK439730* MK439643* MK439681* MK439706*
Cymbidium sp.5828 Z.J.Liu 5828 MK439824* MK439775* MK439800* MK439731* MK439664* MK439682* MK439707*
Cymbidium sp.6016 Z.J.Liu 6016 MK439825* MK439776* MK439801* MK439732* MK439665* MK439683* MK439753* MK439708*
Cymbidium suavissimum Z.J.Liu 2881 MK439826* MK439777* MK439733* MK439649* MK439684* MK439754*
Cymbidium teretipetiolatum Z.J.Liu 2949 MK439827* MK439778* MK439802* MK439734* MK439647* MK439685* MK439755* MK439709*
Cymbidium tigrinum Z.J.Liu 10115 MK439828* MK439779* MK439803* MK439735* MK439648* MK439686* MK439756* MK439710*
Cymbidium tortisepalum Z.J.Liu 6403 MF861133* MF861048* MF861092* MF860924* MF860858* MF860967* MF861179* MF860883*
Cymbidium tortisepalum var. longibracteatum Z.J.Liu 7008 MF861131* MF861046* MF861090* MF860922* MF860851* MF860965* MF861177* MF860881*
Cymbidium tracyanum Z.J.Liu 6426 MF861123* MF861039* MF861082* MF860914* MF860832* MF860958* MF861170* MF860873*
Cymbidium wenshanense Z.J.Liu 6431 MF861128* MF861043* MF861087* MF860919* MF860835* MF861174* MF860878*
Cymbidium quinquelobum Z.J.Liu 10113 MK439829* MK439780* MK439804* MK439736* MK439650* MK439687* MK439757* MK439711*
Cymbidium whiteae AF470508 AF470474
Cymbidium wilsonii Z.J.Liu 7025 MF861156* MF861072* MF861114* MF860948* MF860868* MF860989* MF861199* MF860905*
Cymbidium dianla n Z.J.Liu 6039 MK319538* MK319536* MK319537*
Eulophia graminea AF284727 FJ565159 KF358040 FJ564680
Galeandra devoniana EU877142 KF660268 AF074171 EU877105
Paraholcoglossum amesianum KX29864 JN106350 JN106343 JX202637 JX202760 JX202707
Phalaenopsis lamelligera AY912233 EU179845 AY389387 AY265765
Tsiorchis kimballiana HQ404400 JN106345 HQ404490 HQ452931 JX202640 JX202763 HQ404450
2.2. Collection of DNA sequences

Total DNA was extracted from fresh material using a modified CTAB procedure of Doyle and Doyle (1987). DNA extraction, PCR amplification, and sequencing were performed according to Chen et al. (2017). The primers used for PCR analysis were listed in Table S1.

2.3. Sequence analysis and alignment

Both forward and reverse sequences referring to the corresponding chromatograms were edited and assembled into contig sequences using SeqMan v.7.1 (DNAStar, USA) with the default "Classic Assembler" parameters (Match Size = 12; Minimum Match Percentage = 80). DNA sequences were aligned with MEGA 5.05 under the Muscle model and manually adjusted to account for obvious or missing inserts (Tamura et al., 2011; Zhang et al., 2013).

2.4. Identification of incongruence

The congruence among the nuclear data (nrITS) and the combined chloroplast DNA data set (rbcL, trnS, trnG, matK, trnL, psbA and atpI) was tested using the incongruence length difference (ILD) test (Farris et al., 1995), implemented as the Partition Homogeneity test in PAUP* v.4.0b10, and followed procedures described by Li et al. (2015). Incongruence was also visually inspected for in trees that exhibited contrasting topologies when obtained from different data sets. The thresholds of hard incongruence followed those adopted by Pelser et al. (2010): bootstrap values ≥ 80 and/or PP ≥ 0.95, as well as ILD P < 0.01.

2.5. Phylogenetic analyses

Phylogenetic analyses were performed using Bayesian inference (BI) and maximum-likelihood (ML) methods. The evolutionary models for the ML and BI analyses were determined by jModelTest using the Akaike Information Criterion (AIC). ML analysis was performed using the CIPRES Science Gateway web server (RAxML-HPC2 on XSEDE 8.2.10) (Miller et al., 2010) with 1000 bootstrap replicates and settings that are described inEdgar. (2004). BI analysis was performed using the CIPRES Science Gateway web server (MrBayes 3.2.6 on XSEDE) (Stamatakis et al., 2008). The following settings were used: sampling frequency = 1000; tem = 0.1; burn-in = 2000; and number of Markov chain Monte Carlo generations = 10, 000, 000 (Li et al., 2015).

2.6. Network analyses

To visualize conflicts among gene trees, SPLITSTREE4 v.4 (Huson and Bryant, 2006) was used to generate a consensus tree (seven cpDNAs and one nuclear gene).

3. Results 3.1. Sequences and alignment

In the present study, 468 new Cymbidium sequences were obtained from 15 sections (except section Borneensia) of three subgenera, the division of sections within Cymbidium was adopted according to Liu et al. (2006). However, we were unable to amplify the chloroplast regions of a few accessions; we treated these as missing data. Aligned sequence length were as follows: 700 bp for the nrITS region (111 bp parsimony-uninformative and 189 bp parsimony-informative in the data set), 1293 bp for rbcL, 1626 bp for matK, 768 bp for trnG, 1027 bp for trnS, 1314 bp for trnL, 955 bp for psbA, and 838 bp for atpI. A total of 869 bp of the combined plastid regions (7524 bp) were parsimony-uninformative; 1004 bp were parsimony-informative. The numbers of variable and parsimony informative sites were listed in Table 2. Details pertinent to the best-fit model of molecular evolution could be found in Table 3.

Table 2 Statistics from the analyses.
Information nrITS Combined Plastid
No. of taxa 75 71
Aligned length (bp) 710 7805
No. parsimony-uninformative 111 869
No. parsimony-informative 189 1004
Tree length 563 4302
Consistency index 0.72 0.54
Retention index 0.55 0.65

Table 3 Best-fit model and parameter for the analysis data sets.
Region AIC select model Base frequencies Substitution model (rate matrix) I G
nrITS TIM3+G 0.1878 0.2905 0.3607 0.1609 0.6159 2.7987 1.0000 0.6159 6.5213 1.0000 0.0000 0.4760
cpDNA TVM+I+G 0.3227 0.1599 0.1699 0.3476 1.1776 1.2415 0.6934 0.2902 1.2414 1.0000 0.5340 0.3770
3.2. Phylogenetic analysis of the combined cpDNA data set

Our phylogenetic analyses of cpDNA focused on the combined data set. Seventy-one taxa were included in this cpDNA matrix, five of which were outgroups. Four major groupings within Cymbidium were recovered with moderate support (Fig. 1). Clade A consisted of one species of subgenus Cymbidium, three species of subgenus Jensoa and 23 species of subgenus Cyperorchis. Clade B was composed of one species of subgenus Cymbidium and one species of subgenus Jensoa, Cymbidium dayanum Rchb. f. and Cymbidium omeiense Y.S. Wu et S.C. Chen. Clade C was composed of nine species, seven species of subgenus Cymbidium, and two species of subgenus Cyperorchis (PP = 0.91). Clade D contained three species of subgenus Cymbidium, one species of subgenus Cyperorchis, and 24 species of subgenus Jensoa (PP = 0.91).

Fig. 1 Phylogenetic relationships of Cymbidium based on the plastid DNA (rbcL, trnS, trnG, matK, trnL, psbA, and atpI). The numbers near the nodes are bootstrap percentages (PP left, BS right). A dash (−) indicates values less than 50%.
3.3. Phylogenetic analysis of the nrITS data set

Seventy-five samples, including five species identified as outgroups, were sampled for the nrITS analysis, and three differentiation clades (clades A-C) were recovered (Fig. 2).

Fig. 2 Phylogenetic relationships of Cymbidium based on nrITS. The numbers near the nodes are bootstrap percentages (PP left, BS right). A dash (−) indicates values less than 50%. The classification follows Liu et al. (2006).

(1) Clade A included nine species from two sections of subgenus Cymbidium clustered in a single lineage (PP = 0.9, BS = 68): C. rectum Ridley., C. mannii Richb. f., C. paucifolium Z.J. Liu et S.C. Chen, C. bicolor Lindl., C. canaliculatum R.Br., and C. aloifolium (L.) Sw. C. finlaysonianum Lindl., C. atropurpureum (Lindl.) Rolfe, and C. puerense Z.J. Liu et S.R. Lan.

(2) Clade B consisted of 27 species from two subgenera and was poorly resolved with low support (PP = 0.55, BS = 52). It was subdivided into eight subclades (subclades 1–8). Subclade 1, which was sister to the other seven subclades, and included two subgenera: Cymbidium and Cyperorchis, with weak support. Subclade 2 contained eight species from five sections: Iridorchis, Eburnea, Annamaea, Cyperorchis, and Parishiella with weak support. The subclade 3 consisted of two species of section Cyperorchis: Cymbidium wenshanense and C. quinquelobum, with strong support (PP = 1, BS = 100). Subclade 4 only included one species of section Himantophyllum from subgenus Cymbidium, C. dayanum, with strong support (PP = 0.91). The subclade 5 (PP = 0.91, BS = 66) was composed of four species from section Iridorchis. Subclade 6 included two species from section Iridorchis. Subclade 7 was composed of two species of section Cyperorchis and one species of section Iridorchis and was sister to subclade 8, which consisted of four species of section Eburnea; however, the relationships had low support.

(3) Clade C (PP = 0.52, BS = 68) was composed of 27 species and seven un-identified species from two subgenera, subgenus Cymbidium and subgenus Jensoa. Six divergent subclades (subclades 9–14) were recovered in this clade. Subclade 9 included six species of subgenus Cymbidium, with strong support (PP = 1, BS = 92). The subclade 10 was composed of two species of section Geocymbidium and one species of section Pachyrhizon. Subclade 11 only included one species of section Pachyrhizon. Subclade 12 was composed of nine species from three sections (sections Axillaria, Jensoa, and Nanula) with strong support (PP = 1, BS = 97), but the interrelationships were poorly resolved with low support. Subclade 13 consisted of Cymbidium cyperifolium Wall. et Lindl., C. defoliatum Y.S. Wu et S.C. Chen, and C. faberi Rolfe, and one unidentified species was sister to the remaining members of this clade. Subclade 14 consisted of 11 species from two subgenera (Jensoa and Cymbidium), but the interrelationships were poorly resolved with low support.

3.4. Incongruence tests

Our molecular analyses using nrITS and a combined cpDNA data sets indicated many topological conflicts, some of which appear to be quite strong judging by support values. The phylogenetic tree based on the nrITS data was divided into three clades, but it could be divided into four clades based on the seven cpDNA sequences. The nuclear gene trees indicated that subgenusCymbidium diverged first, followed by subgenus Cyperorchises with two species of subgenus Cymbidium and finally the subgenus Jensoa with two species of subgenus Cymbidium and one species of subgenus Cyperorchis. However, the cpDNA gene trees indicated that subgenus Cyperorchis diverged first with one species of subgenus Cymbidium and three species of subgenus Jensoa, followed by the clade C. dayanum-C. omeiense, followed by the subgenus Cymbidium with two species of subgenus Cyperorchis, and finally the subgenus Jensoa with three species of subgenus Cymbidium and one species of subgenus Cyperorchis.

Moreover, there were many topological conflicts within sections. For example, the section Floribunda was monophyletic in the nrITS gene tree, with strong support (PP = 0.99, BS = 97). This section was subdivided into two clades in the cpDNA tree. The section Cymbidium was monophyletic in the cpDNA tree and was sister with Cymbidium canaliculatum. However, in the nrITS gene tree, C. canaliculatum nested within this section, creating a polyphyletic group. There were also many topological conflicts in species, such as Cymbidium faberi, Cymbidium aestivum, and Cymbidium daweishanense. The ILD test for the nrDNA and combined cpDNA data resulted in P < 0.01 and indicated incongruence between the two data sets; therefore, we did not concatenate these two data sets.

3.5. Network analysis

The generated networks revealed inter- and intrasectional reticulations in Cymbidium (Fig. 4). The three subgenera (Cymbidium, Cyperorchis, and Jensoa) that exhibited discordant phylogenetic positions in the separate gene trees formed complex networks, suggesting that hybridization events occurred between these three subgenera (Fig. 4a). When some species with highly diverged alleles were excluded, the network became much simpler, but reticulation was still observed (Fig. 4b).

Fig. 3 ITS (left) and combined plastid (right) phylogenies of Cymbidium. Bayesian consensus cladograms were generated based on data of the nrITS (left) and the seven plastid loci (matK, rbcL, trnL, trnS, trnG, psbA, and atpI) (right). The classification follows Liu et al. (2006).

Fig. 4 Filtered super-networks constructed from separate cpDNA and nuclear gene trees. (a) All species were included. (b) Twenty-four species were excluded.
4. Discussion 4.1. The phylogeny of the Cymbidium

In the present study, an updated phylogeny of Cymbidium was proposed based on comprehensive sampling of 75 species (varieties) (five of which were outgroup) and 468 new DNA sequences. The overall results of the phylogenetic analysis of Cymbidium were consistent with previously published results (Berg, 2002, Yang et al., 2012, Yukawa and Stern, 2002). Based on the nuclear DNA (nrITS) data set, Cymbidium was found to be composed of three major clades, while it was divided into four clades in the cpDNA tree.

We investigated the relationships among the subgenera of Cymbidium. The monophyly of subgenus Cymbidium was broken, divided into five clades, four of which were nested in two other subgenera (Cyperorchis and Jensoa). The monophyly of subgenus Jensoa was broken by the nested position of subgenus Cymbidium section Himantophyllum, C. aestivum, and subgenus Cyperorchis, C. daweishanense. The principal synapomorphic character of the subgenus Jensoa is having four pollinia (Du Puy and Cribb, 2007, Liu et al., 2006); however, C. aestivum and C. daweishanense do not share this character. If it were not for the positions of Cymbidium devonianum and C. dayanum, subgenus Cyperorchis would be monophyletic. Transfer of C. devonianum and C. dayanum to a member of subgenus Cyperorchis resulted in the loss of a prominent synapomorphic character of subgenus Cyperorchis, that is, a fused basal part of the lip with the column. Taking the morphological characteristics and phylogenetic relationships into account, subdivisions of Cymbidium at the subgeneric level were not useful. We did not find any stable synapomorphic characters that are featured in each subgenus clarified in this study. The delimitation of sections within Cymbidium were also problematic, and most sections were found to be polyphyletic. In summary, the currently defined subgenera and sections of Cymbidium are not monophyletic. Furthermore, the phylogenetic relationships among Cymbidium have not been resolved. Additional markers or second- or third-generation sequencing may be required for further study.

4.2. Reticulate evolution in Cymbidium

The evolutionary relationships at the species level and above could be idealistically represented with bifurcating phylogenetic trees based on the theory of universal common descent. Some evolutionary events, such as horizontal gene transfer, lineage sorting, rapid radiation, hybridization and introgression, may result in conflicts among gene trees, and therefore, phylogenetic networks are more suitable to model the real relationships among species (Geuten et al., 2004; Russell et al., 2010; Yang et al., 2012; Guo et al., 2015).

Phylogenies of Cymbidium obtained from nrITS and cpDNA sequences were concordant in certain respects and discordant in others (Fig. 3). Both phylogenies support non-monophyly of each of the three subgenera of Cymbidium, as well as sections. The generated networks revealed inter- and intrasectional reticulations in Cymbidium (Fig. 4). The three subgenera (Cymbidium, Cyperorchis, and Jensoa) that exhibited discordant phylogenetic positions in the separate gene trees formed complex networks, suggesting that hybridization events occurred between these three subgenera (Fig. 4a). When we deleted the 24 highly divergent alleles, the reticulation relationship among Cymbidium was still very complex, indicating that species within Cymbidium underwent complex reticulate evolution (Fig. 4b). Actually, due to the sympatric distribution, weak reproductive isolation of the species, and synchronous flowering, the natural interspecific hybridization within orchids occurs commonly (Cribb, 1998; Liu et al., 2009; Guo et al., 2015). Moreover, there have been 15 natural hybrid species reported in the Cymbidium ( Furthermore, thousands of artificial interspecific hybrids are listed by the Royal Horticultural Society ( This phenomenon, which is caused by absence of strong interspecific reproductive barriers and hybrid zones, has also been found in other orchid genera, such as Orchis Tourn. ex L. (Bateman et al., 2008), Ophrys L. (Cortis et al., 2009), Epidendrum Pav. ex Lindl (Pinheiro et al., 2010), and Paphiopedilum Pfitzer (Guo et al., 2015). Accordingly, hybridization may play an important role in orchid speciation.

4.3. The possible causes of tree incongruence and the low value

When comparing the nuclear and combined plastid trees (Fig. 3), we immediately observed conflicting branches, but the most branches did not have strong support. The ILD test for the nrDNA and combined cpDNA data resulted in P < 0.01, indicating incongruence between the two data sets. In addition, to further determine incongruence of phylogeny tree, we generated the network, which showed that there were complex networks inCymbidium. Tree incongruence and low support values may be explained by hybridization inCymbidium and the lack of informative characters.

4.3.1. Hybridization

Hybridization has long been reported in plant lineages and appreciated to be a key mechanism in plant evolution, as many extant taxa have likely recently originated from hybridizations (Zhang et al., 2012; Guo et al., 2015; Francine et al., 2017). Sequences from different genomes of hybrid species usually reflect different lines of inheritance (e.g., mitochondrion genes from the paternal line, plastid genes from the maternal line, and nuclear genes from both parental lines), which could result in the incongruence between these different data sources (Yu et al., 2013; Zhai et al., 2014; Guo et al., 2015; Kanzi et al., 2020). In Cymbidium, we speculated that hybridization may be a cause of tree incongruence. Good examples are described taxa such as Cymbidium × nishiuchianum, Cymbidium × purpuratum, Cymbidium × latifolium, Cymbidium × uniflorum, Cymbidium × oblancifolium and Cymbidium × nujiangense. This phenomenon is also found in Calanthe (Zhai et al., 2014), Epidendrum (Pinheiro et al., 2010), and Paphiopedilum (Guo et al., 2015).

4.3.2. The lack of informative characters

Although we used eight genes to construct the phylogenetic relationships ofCymbidium, the informative characters available in these eight genes were limited, and they might represent only a small piece of the evolutionary story in Cymbidium. Moreover, the support values for most clades were low. The lack of informative characteristics may be one cause of topological incongruence between the plastid and nuclear phylogenies (Tang et al., 2015).

5. Conclusion

The present work clarified the phylogenetic relationships within Cymbidium through molecular evaluations. There were incongruent results in the topology of the combined chloroplast and nrITS trees, and the support values of clades were low. These findings may be the result of natural hybridization and a lack of informative characters. In addition, we detected reticulate evolution in Cymbidium. These results add valuable insights into the evolution of Cymbidium. Additional studies based on second- or third-generation sequencing are needed, with a focus on geographic and ecological patterns and the tempo and mode of evolution in the genus. This genus is mainly distributed in subtropical and tropical areas of Asia, and the investigation of diversification patterns of this genus will shed light on biodiversity evolution in this region.

Author contributions

GQZ and JWZ designed the experiments. JH and XYW performed the experiments. GQZ and GZC contributed to the data analysis and molecular system construction. LJC drew the figures. GZC wrote the manuscript. WHR contributed to the collection and morphological identification of some samples. ZJL, SRL, DHP, and MHL provided suggestions on the experimental design and discussion sections. All the authors read and approved the final manuscript.

Declaration of competing interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.


This research was funded by The National Key Research and Development Program of China (Nos. 2018YFD1000401 and 2018YFD1000400), the Natural Science Foundation of Guangdong Province (No. 2017A030312004), and the Science and Technology Plan Project of Shenzhen (No. JCYJ20170817151501595). The authors thank Xu-Hui Chen and Wei-Rong Liu for their support in data collection and the morphological identification of some samples and Fu-Ye Wen for the daily management of samples.

Appendix A. Supplementary data

Supplementary data to this article can be found online at

Bateman, R.M., Smith, R.J., Fay, M.F., 2008. Morphometric and population genetic analyses elucidate the origin, evolutionary significance and conservation implications of Orchis angusticruris (O. purpurea, O. simia), a hybrid orchid new to Britain. Bot. J. Linn. Soc., 157: 687-711. DOI:10.1111/j.1095-8339.2008.00856.x
Berg, V.D., 2002. Molecular phylogenetics of Cymbidium (Orchidaceae: Maxillarieae) sequence data from internal transcribed spacers (ITS) of nuclear ribosomal DNA and plastid matK. Lindleyana, 17: 102-111.
Cameron, K.M., Chase, M.W., Whitten, W.M., et al, 1999. A phylogenetic analysis of the Orchidaceae: evidence from rbcL nucleotide sequences. Am. J. Bot., 86: 208-224. DOI:10.2307/2656938
Chase, M.W., Cameron, K.M., Freudenstein, J.V., et al, 2015. An updated classification of Orchidaceae. Bot. J. Linn. Soc., 177: 151-174. DOI:10.1111/boj.12234
Chen, S.C., Liu, Z.J., Zhu, G.H., et al., 2009. Orchidaceae, Flora of China, vol. 25. Science Press, Beijing & Missouri Botanical Garden Press, Beijing.
Chen, G.Z., Huang, J., Zhang, G.Q., et al, 2017. New subtribe Pachitinae (Orchideae) of Orchidaceae: evidence from morphological and molecular analyses. Phytotaxa, 329: 114-126. DOI:10.11646/phytotaxa.329.2.2
Cortis, P., Vereecken, N.J., Schiestl, F.P., et al, 2009. Pollinator convergence and the nature of species' boundaries in sympatric Sardinian Ophrys (Orchidaceae). Ann. Bot., 104: 497-506. DOI:10.1093/aob/mcn219
Cribb, P.J., 1998. The genus Paphiopedilum. second ed. Natural History Publications, Kota Kinabalu and Kew.
Doyle, J.J., Doyle, J.L., 1987. A rapid isolation procedure from small quantities of fresh leaf tissue. Phytochem. Bull., 19: 11-15.
Dressler, R.L., 1981. The Orchids: Natural History and Classification. New York: Cambridge Harvard University Press.
Dressler, R.L., 1993. Phylogeny and Classification of the Orchid Family. Cambridge University Press.
Du Puy, D.J., Cribb, P.J., 2007. The Genus Cymbidium. Royal Botanic Garden, Kew, Richmond.
Edgar, R.C., 2004. Muscle: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res., 32: 1792-1797. DOI:10.1093/nar/gkh340
Farris, J.S., Källersjö, M., Kluge, A.G., 1995. Constructing a significance test for incongruence. Syst. Biol., 44: 570-572. DOI:10.1093/sysbio/44.4.570
Francine, C., De Ré, Robe, L.J., et al, 2017. Inferring the phylogenetic position of the Drosophila flavopilosa group: incongruence within and between mitochondrial and nuclear multilocus datasets. J. Zool. Syst. Evol. Res., 55: 208-221.
Freudenstein, J.V., Rasmussen, F.N., 1999. What does morphology tell us about orchid relationships? A cladistic analysis. Am. J. Bot., 86: 225-248. DOI:10.2307/2656939
Geuten, K., Smets, E., Schols, P., et al, 2004. Conflicting phylogenies of Balsaminoid families and the polytomy in Ericales: combining data in a Bayesian framework. Mol. Phylogenet. Evol., 31: 711-729. DOI:10.1016/j.ympev.2003.09.014
Guo, Y.Y., Luo, Y.B., Liu, Z.J., et al, 2015. Reticulate evolution and sea-level fluctuations together drove species diversification of slipper orchids (Paphiopedilum) in South-East Asia. Mol. Ecol., 24: 2838-2855. DOI:10.1111/mec.13189
Hunt, P.F., 1970. Notes on Asiatic orchis 5. Kew Bulletin, 24: 93-94.
Huson, D.H., Bryant, D., 2006. Application of phylogenetic networks in evolutionary studies. Mol. Biol. Evol., 23: 254-267. DOI:10.1093/molbev/msj030
Kanzi, A.M., Trollip, C., Wingfield, M.J., et al, 2020. Phylogenomic incongruence in Ceratocystis: a clue to speciation. BMC Genom., 21: 362. DOI:10.1186/s12864-020-6772-0
Lan, S.R., Chen, L.J., Chen, G.Z., et al, 2018. Cymbidium densiflorum (Orchidaceae: Epidendroideae: Cymbidieae): a new orchid species from China based on morphological and molecular evidence. Phytotaxa, 345: 51-58. DOI:10.11646/phytotaxa.345.1.6
Li, M.H., Zhang, G.Q., Lan, S.R., et al, 2015. A molecular phylogeny of Chinese orchids. J. Systemat. Evol., 54: 349-362. DOI:10.1002/cplx.21570
Lindley, J., 1821. Eulophia. Botan. Reg., 7: 573.
Lindley, J., 1826. Epidendreae. Collect. Bot., 17: 16.
Liu, Z.J., Chen, X.Q., 1998. Five new species of Cymbidium from Asia. J. S. China Agric. Univ., 19: 114-118.
Liu, Z.J., Chen, X.Q., 2002. Cymbidium paucifolium, a new species of Orchidaceae from China. J. Wuhan Bot. Res., 20: 350-352.
Liu, Z.J., Chen, X.Q., 2004. Cymbidium micranthum, a new orchid from Yunnan, China. J. Wuhan Bot. Res., 22: 500-502.
Liu, Z.J., Chen, X.Q., 2005. Cymbidium rhizomatosum, a new species of Orchidaceae from Yunnan, China. J. Wuhan Bot. Res., 27: 378-380.
Liu, Z.J., Chen, S.C., Chen, L.J., Lei, S.P., 2009. The Genus Paphiopedilum in China. Beijing: Science Press.
Liu, Z.J., Chen, X.Q., Ru, Z.Z., et al., 2006. The Genus Cymbidium in China. Beijing: Science Press.
Long, C.L., Dao, Z.L., Li, H., 2003. A new species of Cymbidium (Orchidaceae) from Tibet (Xizang), China. Novon, 13: 203-205. DOI:10.2307/3393520
Miller, M.A., Pfeiffer, W., Schwartz T., 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE). New Orleans, LA, pp. 1-8.
Pelser, P.B., Kennedy, A.H., Tepe, E.J., et al, 2010. Patterns and causes of incongruence between plastid and nuclear Senecioneae (Asteraceae) phylogenies. Am. J. Bot, 97: 856-873. DOI:10.3732/ajb.0900287
Pinheiro, F., de Barros, F., Palma-Silva, C., et al, 2010. Hybridization and introgression across different ploidy levels in the Neotropical orchids Epidendrum fulgens and E. puniceoluteum (Orchidaceae). Mol. Ecol., 19: 3981-3994. DOI:10.1111/j.1365-294X.2010.04780.x
Pridgeon, A.M., Cribb, P.J., Chase, M.W., et al., 2009. Genera Orchidacearum, 5. Oxford University Press, Oxford. Epidendroideae (Part 2).
Puy, D.D., Cribb, P., 1988. The Genus Cymbidium. USA, Christopher Helm: Timber Press.
Richardson, A.O., Palmer, J.D., 2007. Horizontal gene transfer in plants. J. Exp. Bot., 58: 1-9. DOI:10.1007/s10142-013-0345-0
Russell, A., Samuel, R., Klejna, V., et al, 2010. Reticulate evolution in diploid and tetraploid species of Polystachya (Orchidaceae) as shown by plastid DNA sequences and low-copy nuclear genes. Ann. Bot., 106: 37-56. DOI:10.1093/aob/mcq092
Schlechter, R., 1924. Die Gattungen Cymbidium Sw. und Cyperorchis Bl. Repert. Sp. Nov. Regni., 20: 96-110. DOI:10.1002/fedr.19240200605
Seth, C.J., Cribb, P.J., 1984. A reassessment of the sectional limits in the genus Cymbidium Swartz. In Orchid Biology, Reviews and Prospectives 3, ed. Cornell Univ. Press, Ithaca and London.
Sharma, S.K., Dkhar, J., Kumaria, S., et al, 2012. Assessment of phylogenetic inter-relationships in the genus Cymbidium (Orchidaceae) based on internal transcribed spacer region of rDNA. Gene, 495: 10-15. DOI:10.5120/6106-8314
Stamatakis, A., Hoover, P., Rougemont, J., 2008. A rapid bootstrap algorithm for the RAxML web–servers. Syst. Biol., 75: 758-771. DOI:10.1080/10635150802429642
Tamura, K., Peterson, D., Peterson, et al, 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and Maximum Parsimony methods. Mol. Biol. Evol., 28: 2731. DOI:10.1093/molbev/msr121
Tang, Y., Yukawa, T., Bateman, R.M., et al, 2015. Phylogeny and classification of the east Asian Amitostigma alliance (Orchidaceae: Orchideae) based on six DNA markers. BMC Evol. Biol., 15: 96. DOI:10.1186/s12862-015-0376-3
Tu, T., Dillon, M.O., Sun, H., et al, 2008. Phylogeny of Nolana (Solanaceae) of the Atacama and Peruvian deserts inferred from sequences of four plastid markers and the nuclear LEAFY second intron. Mol. Phylogenet. Evol., 49: 561-573. DOI:10.1016/j.ympev.2008.07.018
Yang, Z.Y., Ran, J.H., Wang, X.Q., 2012. Three genome-based phylogeny of Cupressaceae s. l. : further evidence for the evolution of gymnosperms and Southern Hemisphere biogeography. Mol. Phylogenet. Evol., 64: 452-470. DOI:10.1186/1471-2148-13-84
Yang, J.B., Tang, M., Li, H.T., et al, 2013. Complete chloroplast genome of the genus Cymbidium: lights into the species identification, phylogenetic implications and population genetic analyses. BMC Evol. Biol., 13: 84. DOI:10.1186/1471-2148-13-84
Yu, W.B., Huang, P.H., Li, D.Z., et al, 2013. Incongruence between nuclear and chloroplast DNA phylogenies in Pedicularis section Cyathophora (Orobanchaceae). PLoS One, 8: e74828. DOI:10.1371/journal.pone.0074828
Yukawa, T., Stern, W.L., 2002. Comparative vegetative anatomy and systematics of Cymbidium (Cymbidieae: Orchidaceae). Bot. J. Linn. Soc., 138: 383-419. DOI:10.1046/j.1095-8339.2002.00038.x
Zhai, J.W., Zhang, G.Q., Li, L., et al, 2014. A new phylogenetic analysis sheds new light on the relationships in the Calanthe alliance (Orchidaceae) in China. Mol. Phylogenet. Evol., 77: 216-222. DOI:10.1016/j.ympev.2014.04.005
Zhang, G.Q., Chen, G.Z., Liu, Z.J., et al, 2018. Cymbidium daweishanense (Orchidaceae; Epidendroideae), a new species from China: evidence from morphological and molecular analyses. Phytotaxa, 374: 249-256. DOI:10.11646/phytotaxa.374.3.6
Zhang, G.Q., Liu, K.W., Chen, L.J., et al, 2013. A new molecular phylogeny and a new genus, Pendulorchis, of the AeridesVanda alliance (Orchidaceae: Epidendroideae). PLoS One, 8: e60097. DOI:10.1371/journal.pone.0060097
Zhang, M.Y., Yun, S.C., Gang, H., et al, 2002. A preliminary analysis of phylogenetic relationships in Cymbidium (Orchidaceae) based on nrITS sequence data. Acta Bot. Sin., 44: 588-592.
Zhang, Y.X., Zeng, C.X., Li, D.Z., 2012. Complex evolution in Arundinarieae (Poaceae: Bambusoideae): incongruence between plastid and nuclear GBSSI gene phylogenies. Mol. Phylogenet. Evol., 63: 777-797. DOI:10.1016/j.ympev.2012.02.023