PLoS ONE
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Chloroplast genomes of Rubiaceae: Comparative genomics and molecular phylogeny in subfamily Ixoroideae
Volume: 15 , Issue: 4
Doi: 10.1371/journal.pone.0232295
Abstract

In Rubiaceae phylogenetics, the number of markers often proved a limitation with authors failing to provide well-supported trees at tribal and generic levels. A robust phylogeny is a prerequisite to study the evolutionary patterns of traits at different taxonomic levels. Advances in next-generation sequencing technologies have revolutionized biology by providing, at reduced cost, huge amounts of data for an increased number of species. Due to their highly conserved structure, generally recombination-free, and mostly uniparental inheritance, chloroplast DNA sequences have long been used as choice markers for plant phylogeny reconstruction. The main objectives of this study are: 1) to gain insight in chloroplast genome evolution in the Rubiaceae (Ixoroideae) through efficient methodology for de novo assembly of plastid genomes; and, 2) to test the efficiency of mining SNPs in the nuclear genome of Ixoroideae based on the use of a coffee reference genome to produce well-supported nuclear trees. We assembled whole chloroplast genome sequences for 27 species of the Rubiaceae subfamily Ixoroideae using next-generation sequences. Analysis of the plastid genome structure reveals a relatively good conservation of gene content and order. Generally, low variation was observed between taxa in the boundary regions with the exception of the inverted repeat at both the large and short single copy junctions for some taxa. An average of 79% of the SNP determined in the Coffea genus are transferable to Ixoroideae, with variation ranging from 35% to 96%. In general, the plastid and the nuclear genome phylogenies are congruent with each other. They are well-resolved with well-supported branches. Generally, the tribes form well-identified clades but the tribe Sherbournieae is shown to be polyphyletic. The results are discussed relative to the methodology used and the chloroplast genome features in Rubiaceae and compared to previous Rubiaceae phylogenies.

Ly, Garavito, De Block, Asselman, Guyeux, Charr, Janssens, Mouly, Hamon, Guyot, and Chen: Chloroplast genomes of Rubiaceae: Comparative genomics and molecular phylogeny in subfamily Ixoroideae

Introduction

Rubiaceae (coffee family) belongs to Gentianales in the eudicots. It is the fourth most species-rich and diverse family in the flowering plants [1, 2, https://stateoftheworldsplants.org/2017/], comprising ca. 13,600 species grouped in ca. 620 genera and ca. 60 tribes [2, 3]. Rubiaceae are mainly tropical trees and shrubs, and less often annual or perennial herbs [4]. They occupy a large range of ecological niches from desert to evergreen humid forests and from sea level to high altitudes (above 4,000 m [5]). While some herbaceous species reached the temperate regions, Rubiaceae are especially abundant (species diversity and biomass) in lowland humid tropical forest, where they often are the most species-abundant of the woody plant families [2]. The Rubiaceae are divided into two subfamilies, Rubioideae and Cinchonoideae by [1], whereas Bremer and Eriksson [6] recognized three subfamilies, splitting the Cinchonoideae into Ixoroideae and Cinchonoideae.

The pantropical Ixoroideae subfamily comprises ca. 4,000 species [7], distributed into 27 tribes [7, 8, 9], and several well-known genera, i.e. the economically important Coffea and the horticulturally important Gardenia and Ixora [10], besides other less economically important genera such as Vangueria, Alibertia and Duroia L.f.

Molecular phylogenetic analyses of Rubiaceae have been carried out using either nuclear sequences (ETS, ITS, 5S-NTS, pep-C large, pep-V small, PI, Tpi), plastid DNA sequences (accD-psa1, atpB-rbcL, ndhF, matK, petD, rbcL, rpl16, rpl32-trnL, rps16, trnG, trnH-psbA, trnL-F, trnT-L, trnS-G ) or a combination of both [7, 11, 12, 13, 14]. Altogether, more than twenty markers (fourteen from cpDNA and seven nuclear) have been used for Rubiaceae phylogeny reconstruction, the most popular being ITS and rbcL . However, the actual number of amplicons used in individual studies is much lower, e.g. for the dating of the family, subfamily and tribes based on fossils, only five plastid sequences were used [6]. The number of markers used often proved a limitation at tribal and generic levels as authors failed to provide well-supported trees [15, 16]. For instance, Maurin and coworkers [15], using four plastid regions and the internal transcribed spacer (ITS) region of nuclear rDNA (ITS 1/5.8S/ITS 2), failed to get a robust molecular tree for Coffea . Similarly, using one plastid and one nuclear marker, Khan and coworkers [17] re-circumscribed the Sabiceeae tribe but were unable to perform a proper biogeographic analysis, given that the molecular tree was largely unresolved. The availability of a robust phylogeny is a prerequisite to accurately study trait evolution at different taxonomic levels (family, subfamily, tribe or genus). This is the case, for instance, when mapping morphological and functional traits in Gardenieae [9], when investigating the evolution of sexual systems and growth habit in Mussaenda [18], or when studying the evolution of caffeine content in Coffea [19]. Since two decades, advances in next-generation sequencing (NGS) technologies have revolutionized the field of biology by providing, at reduced cost, huge amounts of data for an increased number of plant species. Among them, short-read sequencing technologies occupy an important place as they need relatively small amounts of DNA (from 600 ng to 1 μg), which allows the use of limited quantities of initial material, such as from herbarium samples [20]. Sequencing on total DNA permits to reconstruct whole chloroplast (cp) genome sequences of around 150–170 kb [21, 22, 23, 24], which can be used to construct robust phylogenies.

Chloroplasts are derived from endosymbiosis between independent living cyanobacteria and a non-photosynthetic eukaryotic host [25, 26]. Most flowering plants, including Coffea species [24] and Emmenopterys henryi [23, 27, 28] have a quadripartite circular chloroplast structure with two copies of Inverted Repeat (IR) regions (further called IRA and IRB) separating two regions of unique DNA sequence named large single copy (LSC) and small single copy (SSC) according to their length and gene composition [21]. The comparison of the structure and gene composition in cp genomes in broad sets of organisms permits to better understand their origin and function [29]. Due to their highly conserved structure, generally recombination-free, and mostly uniparental inheritance, cp DNA sequences have long been used as choice markers for plant phylogeny [30, 31, 32]. However, the low degree of polymorphism among the regular DNA markers used for Rubiaceae phylogenetics often does not resolve relationships at genus level in case of recent speciation [10]. In such conditions, the use of whole cp genome sequences could be a good alternative. In Rubiaceae, complete cp genomes are available for three species of two tribes in subfamily Cinchonoideae (tribe Naucleeae: Mitragyna speciosa Korth. [33], Neolamarckia cadamba (Roxb.) Bosser [34]; tribe Guettardeae: Antirhea chinensis (Champ. ex Benth.) Benth. & Hook.f. ex F.B.Forbes & Hemsl. [35]), five species belonging to at least three tribes in subfamily Rubioideae (tribe Spermacoceae: Hedyotis ovata Thunb. ex Maxim. [36]; insertae sedis: Paralasianthus hainanensis (Merr.) H.Zhu (as Saprosma merrillii H.S.Lo; [37]; tribe Rubieae: Galium mollugo L. (NC_028009); tribe Morindeae: Gynochthodes officinalis(F.C.How) Razafim. & B.Bremer (as Morinda officinalis F.C.How; NC_028009), Gynochthodes nanlingensis (Y.Z.Ruan) Razafim. & B.Bremer (NC_028614)], and two species belonging to subfamily Ixoroideae (tribe Condamineae: Emmenopterys henryi [23] and tribe Scyphiphoreae: Scyphiphora hydrophyllaceae [38]). However, large projects aiming to develop a library of plastid genomes (including Rubiaceae) are ongoing [33 (GenomeTrakrCP project), 39]).

Nuclear genomic raw data can be assembled into short contigs and used to mine Single Nucleotide Polymorphisms (SNPs) to study the genetic diversity within and between populations and species [40], the evolution of traits of interest [19] or the dynamics of transposable elements [41]. Methodologies based upon short read sequencing such as Genotyping-By-Sequencing (GBS) using a reference genome, permit to define sets of nuclear SNPs for high numbers of genotypes (convenient for multiples of 96 well-plates) as was done for Coffea species [19]. This is possible even with low nuclear genome coverage sequencing (about 10 x coverage). The combination of independent whole genome short read sequencing and bioinformatics tools permit to search these SNPs in different sets of species.

The main objectives of this study were i)- to develop efficient methodology to obtain complete de novo assembled cp genomes permitting comparative genomics and a robust molecular phylogenetic tree, ii)- to test the efficiency of mining SNPs in the nuclear genome of non-coffee Rubiaceae based on the use of a coffee reference genome in order to produce a well-supported nuclear tree, and, iii)- to gain insight in chloroplast genome evolution in the Rubiaceae.

Material and methods

Material

For this study, we have limited the sampling to subfamily Ixoroideae, to which also Coffea belongs. We included 27 taxa representing 10 tribes (Coffeeae, Condamineae, Cordiereae, Gardenieae, Ixoreae, Mussaendeae, Octotropideae, Pavetteae, Sherbournieae and Vanguerieae) plus Emmenopterys henryi [23], the complete cp sequence of which was retrieved from NCBI. Detailed information on sampling is given in Table 1.

Table 1
1 according to [1]; 2 according to [6].
Taxa studied (species name, tribe, geographic origin, voucher).
TribeGenusSpeciesCountryVoucher (collector, collector number, herbarium)Barcode of herbarium voucher or silica collection (*); accession number of living plant (**) or sequence (***)
CoffeeaeBelonophora Hook.f.B. coffeoides Hook.f.CameroonDessein et al. 2554 (BR)BR0000005094424
CoffeeaeCoffea L.C. arabica L.EthiopiaNAET39**, BRC Bassin-Martin, Reunion
CoffeeaeCoffea L.C. canephora PierreDR CongoNADH200-94**, BRC Bassin-Martin, Reunion
CoffeeaeCoffea L.C. sessiliflora BridsonTanzaniaNAPA60**, BRC Bassin-Martin, Reunion
CoffeeaeEmpogona Hook.f.E. congesta (Oliv.) HiernZambiaDessein et al. 1103 (BR)BR6202001591004*
CoffeeaePsilanthus Hook.f.P. ebracteolatus HiernIvory CoastNAPSI11**, BRC Bassin-Martin, Reunion
CoffeeaeTricalysia A.Rich. ex DC.T. hensii De Wild.DR CongoBoyekoli Ebale 708 (BR)BR00000012568055
CoffeeaeTricalysia A.Rich. ex DC.T. lasiodelphys (K.Schum. & K.Krause) A.Chev.CameroonDessein & Sonké 1462 (BR)BR0000009955950
CoffeeaeTricalysia A.Rich. ex DC.T. semidecidua BridsonZambiaDessein et al. 1093 (BR)BR6202001590007*
Coffeeae1 Bertiereae2Bertiera Aubl.B. breviflora HiernGabonChampluvier 6182 (BR)BR0000009043350
Coffeeae1 Bertiereae2Bertiera Aubl.B. iturensis K.KrauseGabonChampluvier 6118 (BR)BR0000009043206
Coffeeae1 Bertiereae2Bertiera Aubl.B. laxa Benth.CameroonDessein et al. 2754 (BR)BR0000005335817
CondamineaeEmmenopterys Oliv.E. henryi Oliv.AsiaNANC 036300.1***
CondamineaePentagonia Benth.P. tinajita Seem.Costa RicaVan Caekenberghe 252 (BR)BR0000009807754
CordiereaeAlibertia A.Rich. ex DC.A. edulis (Rich.) A.Rich.BrazilVan Caekenberghe 485 (BR)20121070–69**
GardenieaeAtractocarpus Schltr. & K.KrauseA. fitzalanii (F.Muell.) PuttockAustraliaVan Caekenberghe 330 (BR)BR0000005036035
GardenieaeEuclinia Salisb.E. longiflora Salisb.AfricaVan Caekenberghe 348 (BR)BR0000005036790
GardenieaeGardenia J.EllisG. sp.AfricaVan Caekenberghe 509 (BR)20121077–76**
GardenieaeSchumanniophyton HarmsS. magnificum (K.Schum.) HarmsAfricaVan Caekenberghe 499 (BR)20090453–07**
GardenieaeSherbournia G.DonS. buccularia N.HalléCameroonLachenaud et al. 736 (BR)BR0000005336715
IxoreaeIxora L.I. chinensis Lam.AsiaVan Caekenberghe 316 (BR)BR00009959309
MussaendeaeMussaenda Burm. ex L.M. pubescens Dryand.AsiaVan Caekenberghe 450 (BR)20111010–00**
MussaendeaePseudomussaenda WernhamP. stenocarpa (Hiern) E.M.A.PetitDR CongoVan Caekenberghe 500 (BR)20100295–52**
OctotropideaeFeretia DelileF. aeruginescens StapfZambiaDessein et al. 912 (BR)BR0000009819672
PavetteaeLeptactina Hook.f.L. leopoldi-secundi BüttnerCongoChampluvier 5428 (BR)BR0000008566447
PavetteaePavetta L.P. schumanniana F.Hoffm. ex K.Schum.DR CongoMalaisse 13702 (BR)BR0000006430252
PavetteaeTarenna Gaertn.T. grevei (Drake) HomolleMadagascarDe Block et al. 959 (BR)BR0000009125964
SherbournieaeMitriostigma Hochst.M. axillare Hochst.AfricaVan Caekenberghe 44 (BR)BR0000006429812
VanguerieaeVangueria Juss.V. infausta Burch.ZambiaDessein et al. 879 (BR)BR6202005552001*

Our analyses resulted in a single sample, Sherbournia, with a phylogenetic position different from what was expected. In order to verify the identity of this sample, TrnL-F and rps16 sequences were blasted in GenBank. Blasting was then repeated with sequences from other Sherbournia samples obtained with Sanger sequencing. Samples used were S. bignoniiflora (Welw.) Hua [Boyekoli Ebale 283 (BR)], S. buccularia [Lachenaud et al. 730 (BR)] and S. zenkeri Hua [Dessein et al. 1428 (BR)].

Authors of genus and species names of the studied taxa are given in Table 1; for other taxa they are given in the text upon first use.

Genome sequencing

Whole genomic DNA was isolated from silica or living plant material following a modified cetyltrimethylammonium bromide (CTAB) method [42]. A total of 25 mg (silica dried) or 100 mg (fresh) leaf material was ground into a fine powder. To eliminate secondary metabolites, two consecutive chloroform cleaning steps were carried out. DNA was lysed either in elution buffer (10 mM Tris-HCl, pH 8.0–8.5) or sterile PCR-grade water. The use of EDTA in elution buffer should be avoided to circumvent possible enzymatic inhibition in downstream applications which may lead to lower library quality. The short-read sequencing was done using the BGI-seq 500 platform, 2x100 bp paired-end. The quality of reads was verified using the Java software FASTQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The raw data were checked in order to detect potential contamination using Kraken and Krona tools [43]. Raw reads were cleaned when necessary using Trimmomatic [44].

Chloroplast genome assembly and annotation

Cp genomes were de novo assembled using NOVOplasty software [45] from the sorted cp raw sequences obtained. Good quality raw reads were split into two sets corresponding to forward (F) and reverse (R) reads. With the aim to sort only cp data, the two sets of data (F and R) were mapped against the Coffea arabica cp reference genome [46] using Bowtie2 [47]. The choice of C. arabica (the genetically best-known species among Rubiaceae) at this step is justified since one objective of this study is to test the transferability of tools and methodology developed for Coffea to other members of Rubiaceae. Then, in silico filtered reads were considered for the cp genome de novo assembly using NOVOplasty. The recalcitrant cp genomes were re-assembled using Abyss [48]. At the end of the assembly process, the cp genomes were compared with the reference C. arabica genome using Gepard [49] in order to check for incongruences of the assembly. To confirm the overall structure, the pair-end illumina reads are mapped back to the assembly using Bowtie2 [47] and BAM files are used to display the read coverage using Artemis (https://www.sanger.ac.uk/science/tools/artemis-comparison-tool-act). The cp genomes were then annotated using Geseq [50] as recommended by Guyuex et al., [24]. The circular visualization of each cp genome was obtained using the OrganellarGenomeDRAW tool (OGDRAW) [51]. The linear gene order comparison was obtained using ACT (Artemis Comparison Tool [52]).

Sequence divergence and junction sequences divergence

Given that Coffea arabica could not be used as outgroup in the phylogeny, we decided to use the annotated genomes of Antirhea chinensis [35], Mitragyna speciosa [33] and Neolamarckia cadamba [34] belonging to the Cinchonoideae subfamily as reference genomes and outgroup taxa for the cp genome analyses. The alignments of the complete chloroplast genome sequences of the 28 studied Rubiaceae were visualized using mVISTA [53] in order to show global interspecific variation and variation within the tribes.

Taking into account data obtained from for each taxon (length of regions LSC, SSC, IR and gene annotation), we calculated the distance between boundaries and the nearest gene to visualize junction sequence divergence between species and within tribes.

Phylogenetic relationships

The plastid phylogeny was produced using a total of 28 cp sequences and one of three outgroups belonging to the Cinchonoideae subfamily (Antirhea chinensis, Mitragyna speciosa and Neolamarckia cadamba , retrieved from GenBank). The sequences were first aligned using MAFFT 7.305 with the following parameters BLOSUM62 and 200PAM/ k = 2 [54].

The nuclear tree was produced using a total of 27 taxa (no data available for Emmenopterys henryi). No non-Ixoroideae data were available, so the trees were rooted midpoint. The 28,800 SNPs used for Coffea [19] were mined according to the methodology developed by these authors. In a second step, in order to reduce the amount of missing data, rare or too common sites were removed using Tassel ver. 5.0 [55] with the following parameters: minimum count = 18, minimum frequency = 0.2, maximum frequency = 0.8. A total of 1,726 sites (SNPs) were kept.

MAFFT alignment of cp sequences and nuclear SNP concatenation were used to infer the phylogenetic trees. The phylogenetic reconstructions were done using RAxML version 8 [56] under the General Time Reversible nucleotide substitution model with gamma distributed rate variation among sites (GTR+G), ML estimate of alpha-parameter, BFGS method to optimize GTR rate parameters and Felsenstein’s bootstraps option autoMRE as recommended by the author). The trees were then edited with FigTree ver. 1.3.1 [57] and Inkscape (https://inkscape.org/fr/release/0.91/).

Results and discussion

Chloroplast genome features in Ixoroideae

Among the 28 studied samples 25 exhibited the classical quadripartite structure but three had an apparent tripartite structure with only one IR (Mussaenda pubescens, Feretia aeruginescens and Pavetta schumanniana). These latter belong to three different tribes (Mussaendeae, Octotropideae and Pavetteae, respectively) but the tripartite structure is not present in all representatives of these tribes.

Regarding the quadripartite genomes, total length ranges from 153,056 bp for Bertiera breviflora to 155,328 bp for Sherbournia buccularia. Similar length differences are observed among the tripartite genomes (from 127,396 bp for Pavetta schumanniana to 129,508 bp for Mussaenda pubescens). Length variations were also noted for the different regions: from 83,406 bp (Vangueria infausta) to 85,461 bp (Belonophora coffeoides) for LSC, from 17,915 bp (Mitriostigma axillare) to 18,245 bp (Emmenopterys henryi) for SSC and from 24,855 bp (Bertiera laxa) to 25,978 bp (Mussaenda pubescens ) for IR. In all species, GC content was similar for the complete cp genome (ca. 37%) as well as for each of the cp subregions (LSC: ca. 35%; SSC: ca. 31%; IR: ca. 43%). Individual information is summarized in Table 2.

Table 2
Chloroplast genome main features of the 28 studied taxa ordered alphabetically within 10 tribes.
Species namenumber of IRLength in bp
GenomeLSCSSCIR
Coffeeae
Belonophora coffeoides2155190854611813525797
Coffea arabica2155186851571813925945
Coffea canephora2154982851092129724288
Coffea sessiliflora2155010851001811025900
Empogona congesta2154672851061818225692
Psilanthus ebracteolatus2155084851341814225904
Tricalysia hensii2154953854071816625690
Tricalysia lasiodelphys2154898854181813825665
Tricalysia semidecidua2154816853381816625656
Coffeeae/Bertiereae
Bertiera breviflora2153055852312197422925
Bertiera iturensis2154675853991817225552
Bertiera laxa2153778854691798125164
Condamineae
Emmenopterys henryi2155379855541824525790
Pentagonia tinajita2153604848221810625338
Cordiereae
Alibertia edulis2154508846921813825839
Gardenieae
Atractocarpus fitzalanii2154627849911793025853
Euclinia longiflora2155182853631818125819
Gardenia sp.2155294854751812725846
Schumanniophyton magnificum2155081853861811525790
Sherbournia buccularia2155328855291817125814
Ixoreae
Ixora chinensis2154665848741815725817
Mussaendeae
Mussaenda pubescens1129508854111811825979
Pseudomussaenda stenocarpa2155057851891801825925
Octotropideae
Feretia aeruginescens1129434852851821225937
Pavetteae
Leptactina leopoldi-secundi2154462849361822225652
Pavetta schumanniana1127401835691803325796
Tarenna grevei2154164844201812425810
Sherbournieae
Mitriostigma axillare2153606849671791525362
Vanguerieae
Vangueria infausta2152987834061801925781

We annotated a total of 118 different genes belonging to 14 functional categories and present in all the genomes with the exception of Tarenna grevei which has lost trnH-GUG. One hundred genes were present as single copy, 17 were duplicated and one (rps12) was triplicated in IR. The LSC, SSC and IR regions contained 87, 13 and 18 genes respectively. Among the 80 protein-coding genes identified, only nine include introns. Seven of these contain one intron (atpF, ndhA & B, rpoC1, rps12, rps16, rpl2) whereas clpP and ycf3 have two introns. Complete infA and pbf1 genes were present in all studied species. For the three taxa showing only one IR, the corresponding genes were present in only one copy.

The annotated cp genome sequences permitted to compare the length of the junctions of the main regions LSC, IR and SSC among the studied Rubiaceae (Table 3). Generally, low variation was observed between the taxa in the boundary regions. However, while the distance for LSC/IRB junctions was 88 bp for most species, variation was noted in Emmenopterys henryi (Condamineae) with 30 bp, in Psilanthus ebracteolatus (Coffeeae) with 358 bp, in Coffea sessiliflora (Coffeeae) with 157 bp and in Sherbournia buccularia (Gardenieae) with 148 bp. A similar variation was obtained for the IRB/SSC junction. (S1 Table)

Table 3
Estimations were done from data extracted from [33, 34, 35, 59] or calculated from data extracted from GenBank for Morinda officinalis (NC_028009) and Gynochtodes nanlingensis (NC_028614).
Plastid genomes features of three taxa from the Rubioideae and three taxa from the Cinchonoideae subfamilies.
Species namenumber of IRLength in bpGC content (%)
total genomeLSCSSCIROverallLSCSSCIR
Rubioideae subfamily
Morinda officinalis215339884011178552576636353143
Gynochtodes nanlingensis215408684329181152582137353143
Galium mollugo215367784471170562607537353143
Cinchonoideae subfamily 
Antirhea chinensis215561686252179842569038363143
Mitragyna speciosa215560086213182012559337353243
Neolamarckia cadamba2154,99985880178512563438353243

Data obtained for other Rubiaceae such as Hedyotis ovata [36] and Paralasianthus hainanensis (as Saprosma merrillii ; [37]) from the Rubioideae subfamily and Antirhea chinensis [35] from the Cinchonoideae subfamily indicate a quadripartite structure and a total of 114 genes (eight duplicated genes counted once and eight genes missing in Rubiaceae, see below) of which 80 are unique protein-coding genes. For Neolamarckia cadamba (Cinchonoideae), [34] revealed a total of 130 genes, 79 of which are protein-coding. Data obtained from GenBank for three Rubioideae (accession numbers NC_036970 for Galium mollugo, NC_028009 for Morinda officinalis and NC_028614 for Gynochtodes nanlenginsis ) and from literature for two Cinchonoideae [34, 35] permitted us to determine their plastid features (Table 3). All have the classical quadripartite structure. In the Rubioideae, the total cp length varies from 153,398 to 154,086 bp; LSC from 84,011 to 84,471 bp; SSC from 17,056 to 18,115 bp and IR from 25,766 to 26,075 bp. In the Cinchonoideae, total cp length varies from 154,999 to 155,616 bp; LSC from 85,880 to 86,252 bp; SSC from 17,851 to 17,984 bp and IR from 25,634 to 25,690 bp. The GC content for the total sequence and for the different regions are similar to our results in Ixoroideae. The chloroplast genome features of the Ixoroideae, and of the Rubiaceae as a whole, are in the ranges reported for most flowering plants [29, 30, 58].

The tripartite genome structure was not yet reported in the Rubiaceae but was recorded for Fabaceae [60], Geraniaceae [61], Pinaceae [62], Cactaceae [63], Arecaceae [64] and Passifloraceae [65]. Within the Ixoroideae, the chloroplast assembly of three species showed a tripartite genome structure. Besides frequent inversions, duplications, or losses of fragments [65, 66], IR expansion/contraction and even IR absence contributed to substantial variation in cp genome length [67]. However, the robustness of our assemblies of species showing only one IR were tested. All reads were mapped on the assembly and the read coverage was displayed. The read coverage showed an increase at the IR region of the assembly suggesting that two IR regions may be present but assembled into only one IR (S1 Fig). So, the assembly of these chloroplast sequences (Mussaenda pubescens, Feretia aeruginescens and Pavetta schumanniana) and the IR absence should be considered with caution since this event is very rare in most other plant families [68] and since we cannot exclude that the assembly process collapsed the IR regions into only one. Increasing the number of taxa investigated and using long read sequencing techniques, such as PacBio and Oxford Nanopore may demonstrate it to be a not so rare event in the Rubiaceae, which would lead to questions on the role of two or one IR in the evolution of land plants.

Circular visualization of the Bertiera breviflora cp genome is given in Fig 1 as an example. Gene order and orientation from pairwise comparisons were generally well-conserved although some gene orientations were different (S2 Fig).

Circular visualization of annotated Rubiaceae genomes showing the quadripartite structure of Bertiera breviflora (similar to 25 taxa studied here).
Fig 1
Circular visualization of annotated Rubiaceae genomes showing the quadripartite structure of Bertiera breviflora (similar to 25 taxa studied here).

Sequence divergence was visualized using mVISTA with Coffea arabica as the reference annotated genome. The choice of Coffea arabica instead of Antirhea chinensis (outgroup used for the plastid phylogeny) was justified by the level of divergence between Cinchonideae and Ixoroideae. Globally, sequence divergence among all taxa was relatively high and mainly concentrated in conserved non-coding sequences and in Untranslated Transcribed Regions (UTR). However, variation among species seemed to be negligible for UTRs located in the IR region (rpl2, ndhB and rps12 genes). Substitutions were more frequent but indels were observed as well, even in the ycf2 exon. Four (the conserved non-coding regions between matK and atpA, rpoB and psbD, rps4 and ndhJ and ndhC and atpE), and two (ndhF—ccsA and ycf1 ) hypervariable regions were identified in the LSC and SSC regions respectively. A representation of sequence divergence is given for a selected set of taxa (Fig 2). In total, 31.5% sites of the complete alignment included indels.

Sequence identity plot comparing nine species of subfamily Ixoroideae with Coffea arabica as the annotated reference genome using mVISTA.
Fig 2
The location and orientation of the genes are indicated on the top. Exons and UTRs are in purple and turquoise respectively. Conserved non-coding regions are in orange. The y-axis ranges from 100% (top) to 50% identity between each sequence and the reference. The order of the taxa used from top to bottom is: Bertiera iturensis (Bertiereae), Psilanthus ebracteolatus (Coffeeae), Empogona congesta (Coffeeae), Euclinia longiflora (Gardenieae), Pavetta schumanniana (Pavetteae), Pseudomussaenda stenocarpa (Mussaendeae), Feretia aeruginescens (Octotropideae), Mitriostigma axillare (Sherbournieae) and Alibertia edulis (Cordiereae). Feretia aeruginescens and Pavetta schumanniana showing only one IR in the current assembly.Sequence identity plot comparing nine species of subfamily Ixoroideae with Coffea arabica as the annotated reference genome using mVISTA.

In Ixoroideae, plotting the length variation of the different regions relative to the smallest cp genome (here Vangueria infausta and considering only the quadripartite cp genomes) , showed the pattern of variation given in Fig 3. The length of the different regions did not increase simultaneously to the total cp length except for the smallest four cp genomes. The increase in size seems to be mainly due to increase in length of LSC and possibly to gene and/or intron length increases. For Bertiera breviflora and Bertiera laxa, the increase in cp size is mainly due to the increase in length of LSC associated with a decrease in length of IR. Variation in the length of SSC has only limited impact on cp size variation. Regarding the four Cinchonoideae and the five Rubioideae species, similar patterns of variation are observed with the exception of Galium mollugo for which a decrease in length of SSC is notable. Therefore, with the exception of a few species, it seems that length variation in LSC is the main contributor to cp size variation. Among eudicots, the progressive expansion of the IR has been documented in Pelargonium L'Hér. ex Aiton [69] and Passiflora L. [65], and a similar molecular mechanism driving the IR evolution in these two unrelated lineages could be a possibility.

Variation in the length of the different regions [y-axis values are minus data for the smallest cp genome total length (Vangueria infausta)].
Fig 3
The taxa are ordered in increasing total cp genome size in each subfamily (24 Ixoroideae, four Cinchonideae in blue box marked with one asterisk and five Rubioideae in red box marked with two asterisks). Data for taxa indicated with asterisk(s) was retrieved from literature [23, 33, 34, 35, 36, 37] or calculated from data extracted from GenBank for Morinda officinalis (NC_028009), Gallium mollugo (NC_036970), Gynochtodes nanlingensis (NC_028614).Variation in the length of the different regions [y-axis values are minus data for the smallest cp genome total length (Vangueria infausta)].

Angiosperm cp genomes exhibit a remarkably conserved gene content and order as observed for instance within Fagaceae [21, 70, 71, 72] and more specifically for Quercus L. [22]. Likewise, gene content and order were nearly identical in the Ixoroideae representatives studied as well as in representatives of the two other Rubiaceae subfamilies.

Recorded in tobacco and in most others members of Solanaceae as a pseudogene [46], infA was intact in all Ixoroideae species of this study and in the Rubioideae and Cinchonoideae species for which whole cp genomes are available. Similarly, putatively involved in photosystem I and II biogenesis, pbf1 (psbN in Coffea arabica , [46]) was present in all Ixoroideae as well as in the Rubioideae and Cinchonoideae. In all Ixoroideae of this study and in the species of the other subfamilies, a fragment of rps19 appeared duplicated at the IR/LSC boundaries as reported in Solanaceae with the exception of tobacco [73]. Eight genes (CHLB, CHLL, CHLN, CYSA, CYST, MBPX, PSAM, and RPL21) were absent in the study of 16 wild coffee trees [24]. This study showed their absence in all Ixoroideae and the other Rubioideae and Cinchonoideae tested. Finally, despite minor changes in gene content, orientation and order, Ixoroideae plastid genomes are well conserved within and between tribes. This was also the case in the available Cinchonoideae and Rubioideae species and, therefore, could be true for the whole family. However, sequence divergence within and between tribes was observed and at much higher level (Fig 2) than reported in Quercus [22].

Plastid molecular phylogeny and comparison to previous Rubiaceae phylogenies

The complete cp genome-based phylogeny included 28 Ixoroideae taxa and Antirhea chinensis (Cinchonoideae subfamily) as outgroup. Maximum Likelihood analyses resulted in a generally well-resolved topology with highly supported branches, except for four lineages: the branch between Empogona and the Belonophora/Tricalysia clade, the branch between Leptactina and Pavetta/Tarenna within the Pavetteae tribe, the branch towards the Cordiereae/Octotropideae clade and the branch towards the Mussaendeae/Condamineae clade (BS < 80%, Fig 4). The ingroup has three main clades: Mussaendeae (Fig 4; in green), Condamineae (Fig 4; in red) and a large clade comprising all other taxa. The Mussaendeae and Condamineae are well-supported as distinct monophyletic lineages (BS = 100) but their mutual relationship and their relationship with the rest of the ingroup remain unclear. The rest of the ingroup forms a well-supported clade (BS = 100) and comprises two well-supported subclades (BS = 100) that correspond to the Vanguerieae alliance (Ixora and Vangueria) and the Coffeeae alliance. Within the Coffeeae alliance, the tribe Pavetteae (Fig 4; in pink), the Coffeeae/Bertiereae lineage with Bertiera sister to the Coffeeae (Fig 4; in blue) and the clade comprising Schumanniophyton, Gardenia, Sherbournia, Euclina and Atractocarpus (Gardenieae, Fig 4; in brown) are supported as monophyletic groups (BS = 100). All tribes represented by at least two representatives are retrieved as monophyletic with the exception of the Sherbournieae. Sherbournia does not form a clade with Mitriostigma but is firmly embedded in Gardenieae. The same phylogeny was obtained with Mitragyna speciosa (Cinchonoideae) as outgroup. However, when using Neolamarckia cadamba as outgroup, a slightly different phylogenetic tree was obtained (data not shown).

Maximum likelihood plastid tree (RAxML with GTR model of substitution) based on the whole cp sequences of 28 Ixoroideae (with Antirhea chinensis as outgroup) and bootstrap values to estimate the branch support.
Fig 4
Four well-supported clades are marked in green for Mussaendeae, red for Condamineae, pink for Pavetteae, brown for Gardenieae and blue for Coffeeae/Bertiereae.Maximum likelihood plastid tree (RAxML with GTR model of substitution) based on the whole cp sequences of 28 Ixoroideae (with Antirhea chinensis as outgroup) and bootstrap values to estimate the branch support.

This chloroplast phylogeny concurs well with previously published phylogenetic trees based on Sanger sequencing of several markers [1, 6, 7]. Within the Ixoroideae (Ixoridinae sensu Robbrecht and Manen), Robbrecht and Manen [1] recognized a basal clade (basal Ixoridinae; not represented in our analysis) and two main evolutionary lineages Ixoridinae I and Ixoridinae II. Ixoridineae I is essentially neotropical and represented here by the tribe Condamineae. Ixoridineae II is mainly paleotropical and includes all other members of the ingroup. Unlike the super-tree of Robbrecht and Manen [1], our plastid phylogeny is not resolved at the base and does not clearly separate Ixoridineae I and II, since Mussaendeae is considered part of Ixoridineae II by [1]. Bremer and Eriksson [6] did not distinguish lineages within the subfamily Ixoroideae, probably because the base of their phylogenetic tree is unresolved. Kainulainen et al., [7] recognized within the Ixoroideae a basal grade (here represented by Condamineae and Mussaendeae) and a clade of core Ixoroideae. Our analysis confirms the subfamilial classification of Kainulainen et al., [7] rather than that of Robbrecht and Manen [1]. Within the core Ixoroideae, Kainulainen et al., [7] differentiated between the Vanguerieae alliance and the Coffeeae alliance. These two clades are also retrieved in our analysis. Our analysis confirms the sister relationships between Ixoreae and Vanguerieae [1, 6, 7] and between Coffeeae and Bertiereae [1, 6, 7, 9].

Gardenieae is retrieved as monophyletic only with the inclusion of Sherbournia, which has been considered part of the tribe Sherbournieae [9]. The Sherbournieae were recently instated [9] to include the former Gardenieae genera Sherbournia, Mitriostigma, Atractogyne and Oxyanthus, the last two of which are not included in our analysis. With the exception of Sherbournia, these genera are characterized by pollen grains in tetrads [73]. Persson and more recently Bremer and Eriksson [74, 6] also retrieved this group of three genera with pollen in tetrads as monophyletic. However, the inclusion of Sherbournia makes the tribe morphologically heterogeneous as regards to pollen characters (pollen in monads). In order to check the identity of our Sherbournia sample we separated TrnL-F and rps16 sequences from the whole genomes sequence and blasted them in GenBank, where they showed more similarity with Rothmannia Thunb. than with the Sherbournia sequences present there. This was repeated with sequences from other Sherbournia species obtained with Sanger sequencing with the same results. We are therefore confident that Sherbournia does not form part of the tribe Sherbournieae as delimited by Darwin [9], but belongs to the Gardenieae. It should be noted that also in the phylogeny of Persson [74], Sherbournia groups with Rothmannia. The tribe Gardenieae has been demonstrated in several studies to be polyphyletic [1, 7, 9]. The fact that it is not so in our analysis is the result of the small number of representatives included, notably five genera out of over fifty [9]. The five genera making up the Gardenieae clade in our analysis are not generally considered closely related. Atractocarpus is part of Gardenieae IV in [1] and of the Porterandia group in [9], Gardenia is part of Gardenieae II [1] and the Gardenia group [9], Euclinia belongs to Gardenieae III [1] and the Randia group [9], Schumanniophyton belongs to Gardenieae I [1] and remains unplaced in [9] and Sherbournia is unplaced in [1] and belongs to the Sherbournieae tribe in [9].

Nuclear SNP mining and Efficiency of transferability of methods from Coffea to Ixoroideae

The genome of Coffea canephora Pierre ex A.Froehner was used as reference genome to mine SNPs as described in Hamon et al. (2017). This methodology was efficient despite unequal results between taxa. No outgroup from another subfamily of the Rubiaceae was available so the tree was rooted midpoint. In this analysis Coffea canephora was added but Emmenopterys henryi could not be included since no nuclear genome data was available.

An average of 22,906 SNPs was sorted with the extremes ranging from 10,335 in Pentagonia tinajita to 27,642 in Tricalysia lasiodelphys. Among the 806,400 individual data expected (28 x 28,800), excluding all Coffea species, the average percentage of missing data was 31% ranging from 10% in Tricalysia hensii to 77% in Ixora chinensis and Pentagonia tinajita. The percentage of heterozygotes was 0.6% on average but varied from 0.25% in Atractocarpus fitzalanii to 3.2% in Psilanthus ebracteolatus . The nucleotide percentage was 29.1% for A, 29.3% for T, 20.5% for G and 20.2% for C (S1 Appendix). So, the SNP transferability from Coffea [19] to non-coffee Rubiaceae belonging to ten tribes of subfamily Ixoroideae can be considered as successful. Interestingly, the phylogenetically most distant species are those with the fewest orthologous sequences.

The species relationships obtained with the complete dataset (28,800 sites) are shown in Fig 5. The Maximum Likelihood tree shows a majority of well-supported branches (BS of 86–100%). The ingroup shows two main, well-supported clades, the first comprising the Coffeeae alliance and the second comprising the Vanguerieae alliance, the Condamineae and the Mussaendeae. The Mussaendeae (Fig 5; in green), Pavetteae (Fig 5; in pink), Coffeeae/Bertiereae (Fig 5; in blue) and Gardenieae (Fig 5; in brown) are supported as monophyletic with high branch support values. Bertiera is sister to the Coffeeae. The following results are in contrast to the results of the plastid phylogeny: the Gardenieae clade (Fig 5; in brown) does not include Schumanniophyton; Ixora and Vangueria (Vanguerieae alliance) do not form a monophyletic group. The monophyly of the Condamineae cannot be evaluated because only a single representative is present in this analysis (no data for Emmenopterys henryi). The tribe Sherbournieae (Sherbournia and Mitriostigma) is not retrieved as monophyletic and it is embedded in the Gardenieae clade.

Maximum likelihood nuclear tree of 28 Ixoroideae based on 28,800 SNPs (RAxML with GTR model of substitution) and bootstrap values to estimate branch support.
Fig 5
Colored clades indicated well-defined tribes. The tree is rooted midpoint as no outgroup is available. Green for Mussaendeae; pink for Pavetteae; brown for Gardenieae; blue for Coffeeae and turquoise for Coffeeae/Bertiereae.Maximum likelihood nuclear tree of 28 Ixoroideae based on 28,800 SNPs (RAxML with GTR model of substitution) and bootstrap values to estimate branch support.

The phylogenetic tree resulting from the SNP mining of the nuclear genome is similar to the chloroplast based phylogenetic tree with the same clades (Mussaendeae, Pavetteae, Bertiereae, Gardenieae, Coffeeae and Coffeeae/Bertiereae) being retrieved and highly supported even though the position of individual taxa within the clades may be different. Other relationships, such as the sister relationship between Ixoreae and Vanguerieae and between the Vanguerieae alliance and the Coffeeae alliance, are not retrieved in the nuclear phylogeny.

With the aim to use a dataset with less missing data, SNPs were filtered leading to a total of 1,726 sites (SNPs) retained for further analysis. The resulting tree (S3 Fig) shows a long branch for the Psilanthus-Coffea clade that may indicate a highly divergent evolution between these species and the rest of the ingroup. The tree further differs from the one based on 28.800 SNPs in that Bertiera is not sister to the Coffeeae but to the ingroup clade consisting of all species except for Coffeeae. Similarly to the 28,800 SNPs-based tree, branch support values are generally high. The clades Pavetteae, Mussaendeae, Gardenieae (excluding Schumanniophyton), Bertiereae and Coffeeae are supported as monophyletic. However, while the main clades are similar, their relative position is not the same in the two analyses. Sherbournieae are not retrieved as monophyletic, indicating that the reduction of the number of SNPs should be done with care due to possible bias in markers genomic distribution.

Conclusions

In this study we reported and analyzed the chloroplast genome sequences for 27 species of the Rubiaceae subfamily Ixoroideae using next-generation sequences (NGS. Plastid and nuclear genome phylogenies are well congruent with each other with an overall well-supported branch. Generally, the tribes form well-identified clades but the tribe Sherbournieae is shown to be polyphyletic. With continuously dropping prices and an increasing output and efficiency of bioinformatic tools, NGS appears to be now the best choice to study difficult or neglected plant families, tribes or genera. Our methodology used here combined plastid genome reconstruction and SNP mining of the nuclear genome and was successful for Ixoroideae. The same methodology should be extended to the two other Rubiaceae subfamilies (Cinchonoideae and Rubioideae). This would permit to clarify the relationships between Rubiaceae taxa and to better understand genome evolution in the family in relation to adaptive traits. The increased availability of more reference genomes other than Coffea genomes will facilitate and speed up this process.

Acknowledgements

We thank the gardeners, Mr. H. Leqeux, Mr. J. Van Eeckhoudt, and the scientific curators, Ms. V. leyman and Dr. M. Reynders, for their care for the living Rubiaceae collection at Meise Botanic Garden. We thank Mr. F. Van Caekenberghe for help with the collection of plant material. We are grateful to the manager of the molecular laboratory at Meise Botanic Garden, Mr. W. Baert, for his goodwill towards this project. We thank Meise Botanic Garden for funding sequencing work, travel and publication fees. We thank the Institut de Recherche pour le Développement (IRD) for funding sequencing work and for providing a fellowship for S.N. Ly. The Laboratory Chrono-Environment, Université de Bourgogne Franche-Comté, also funded part of the sequencing.

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18 Mar 2020

PONE-D-19-35274

Chloroplast genomes of Rubiaceae: comparative genomics and molecular phylogeny in subfamily Ixoroideae

PLOS ONE

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: N/A

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The research article is overall good, but many minor errors need to be removed prior to publishing. Tables including supplementary tables contains may space and other spelling, scientific nomenclature errors and the tables format. Therefore, my suggestion is to revise the manuscript thoroughly.

Abstract should be rewritten according to the PLos ONE.

Many spelling errors should be revised, here only give some examples:

Line 3 of introduction: ‘......13.600 species......’ should be ‘.......13,600 species.......’?

‘......such as Vangueria, Alibertia and Duroia L.f.’: scientific nomenclature should be consistent.

Reviewer #2: the authors provided whole cp genome sequences for 27 species of the Rubiaceae subfamily Ixoroideae which sequeced using high-throughput genome sequencing method, analyzed the plastid genome structure and the chloroplast genome evolution in the Rubiaceae through efficient methodology for de novo assembly of plastid genomes. this work is meaningful for the molecular phylogeny analysis in Rubiaceae subfamily Ixoroideae.

Some suggestion:

1) What is the role played by the second main objectives of this paper, where "to test the efficiency of mining SNPs in the nuclear genome of Ixoroideae based on the use of a coffee reference genome to produce well-supported nuclear trees",support the chloroplast genome results ?

2) The content of the discussion part and the result part are repeated, so it is suggested that merged these two parts.

**********

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Reviewer #1: No

Reviewer #2: No

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25 Mar 2020

PONE-D-19-35274 Responses to Reviewers.

Thanks to reviewers for their valuable suggestions. Please find below our answers.

Reviewer #1: “The research article is overall good, but many minor errors need to be removed prior to publishing. Tables including supplementary tables contains may space and other spelling, scientific nomenclature errors and the tables format. Therefore, my suggestion is to revise the manuscript thoroughly”.

Ans: The manuscript has been revised thoroughly.

“Abstract should be rewritten according to the PLos ONE”.

Ans: Done: Describe the main objective(s) of the study -> OK ; Explain how the study was done, including any model organisms used, without methodological detail -> Ok ; Summarize the most important results and their significance -> Ok ; Not exceed 300 words -> OK ; Abstracts should not include Citations -> Ok

“Many spelling errors should be revised, here only give some examples: Line 3 of introduction: ‘......13.600 species......’ should be ‘.......13,600 species.......’? ‘......such as Vangueria, Alibertia and Duroia L.f.’: scientific nomenclature should be consistent.”

Ans: Done. The changes have been highlighter along the manuscript.

Reviewer #2: “the authors provided whole cp genome sequences for 27 species of the Rubiaceae subfamily Ixoroideae which sequeced using high-throughput genome sequencing method, analyzed the plastid genome structure and the chloroplast genome evolution in the Rubiaceae through efficient methodology for de novo assembly of plastid genomes. this work is meaningful for the molecular phylogeny analysis in Rubiaceae subfamily Ixoroideae.”

Some suggestion:

“1) What is the role played by the second main objectives of this paper, where "to test the efficiency of mining SNPs in the nuclear genome of Ixoroideae based on the use of a coffee reference genome to produce well-supported nuclear trees",support the chloroplast genome results?”

Ans: Mining SNP using the C. canephora genome has been tested (Hamon et al., 2017) and was successful. An average of 79% of the SNP determined in the Coffea genus are transferable to Ixoroideae, with variation ranging from 35% to 96%. A sentence has been introduced in the Abstract and a chapter called “Nuclear SNP mining and Efficiency of transferability of methods from Coffea to Ixoroideae” in Results and Discussion“ has been reorganized to explain better this part.

“2) The content of the discussion part and the result part are repeated, so it is suggested that merged these two parts.”

Ans: Done. We reorganize the manuscript with a Results and Discussion chapter.

Submitted filename: Responses-to-reviewers.docx

13 Apr 2020

Chloroplast genomes of Rubiaceae: comparative genomics and molecular phylogeny in subfamily Ixoroideae

PONE-D-19-35274R1

Dear Dr. guyot,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Shilin Chen

Academic Editor

PLOS ONE

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No


17 Apr 2020

PONE-D-19-35274R1

Chloroplast genomes of Rubiaceae: comparative genomics and molecular phylogeny in subfamily Ixoroideae

Dear Dr. guyot:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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PLOS ONE

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