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Sequencing of whole plastid genomes and nuclear ribosomal DNA of Diospyros
species (Ebenaceae) endemic to New Caledonia: many species, little divergence
Barbara Turner
1,
*, Ovidiu Paun
1
,Je´roˆme Munzinger
2
, Mark W. Chase
3,4
and Rosabelle Samuel
1
1
Department of Botany and Biodiversity Research, Faculty of Life Sciences, University of Vienna, Rennweg 14, 1030
Wien, Austria,
2
IRD, UMR AMAP, TA A51/PS2, 34398 Montpellier Cedex 5, France,
3
Jodrell Laboratory,
Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK and
4
School of Plant Biology,
The University of Western Australia, Crawley, WA 6009, Australia
* For correspondence. Present address: Department of Integrative Biology and Biodiversity Research, University of Natural
Resources and Life Sciences, Vienna, Gregor-Mendel-Straße 33, 1180 Wien, Austria. E-mail barbara.turner@univie.ac.at
Received: 23 October 2015 Returned for revision: 10 February 2016 Accepted: 26 February 2016
Background and Aims Some plant groups, especially on islands, have been shaped by strong ancestral bottle-
necks and rapid, recent radiation of phenotypic characters. Single molecular markers are often not informative
enough for phylogenetic reconstruction in such plant groups. Whole plastid genomes and nuclear ribosomal DNA
(nrDNA) are viewed by many researchers as sources of information for phylogenetic reconstruction of groups in
which expected levels of divergence in standard markers are low. Here we evaluate the usefulness of these data
types to resolve phylogenetic relationships among closely related Diospyros species.
Methods Twenty-two closely related Diospyros species from New Caledonia were investigated using whole plas-
tid genomes and nrDNA data from low-coverage next-generation sequencing (NGS). Phylogenetic trees were
inferred using maximum parsimony, maximum likelihood and Bayesian inference on separate plastid and nrDNA
and combined matrices.
Key Results The plastid and nrDNA sequences were, singly and together, unable to provide well supported
phylogenetic relationships among the closely related New Caledonian Diospyros species. In the nrDNA, a 6-fold
greater percentage of parsimony-informative characters compared with plastid DNA was found, but the total num-
ber of informative sites was greater for the much larger plastid DNA genomes. Combining the plastid and nuclear
data improved resolution. Plastid results showed a trend towards geographical clustering of accessions rather than
following taxonomic species.
Conclusions In plant groups in which multiple plastid markers are not sufficiently informative, an investigation at
the level of the entire plastid genome may also not be sufficient for detailed phylogenetic reconstruction.
Sequencing of complete plastid genomes and nrDNA repeats seems to clarify some relationships among the New
Caledonian Diospyros species, but the higher percentage of parsimony-informative characters in nrDNA compared
with plastid DNA did not help to resolve the phylogenetic tree because the total number of variable sites was much
lower than in the entire plastid genome. The geographical clustering of the individuals against a background of
overall low sequence divergence could indicate transfer of plastid genomes due to hybridization and introgression
following secondary contact.
Key words: Diospyros, genome skimming, island floras, New Caledonia, next-generation sequencing, nuclear
ribosomal DNA, rapid radiation, complete plastid genomes.
INTRODUCTION
New Caledonia comprises an archipelago in the southern
Pacific known for its characteristic, rich endemic flora
(Lowry, 1998;Morat et al., 2012). Due to its complex geolo-
gical history, New Caledonia features a mosaic of soil types
(Pelletier, 2006;Maurizot and Vende´-Leclerc, 2009), which, in
combination with its elevational and climatic heterogeneity, re-
sults in many different habitats. One of the genera that has
adapted to a wide range of these habitats is Diospyros
(Ebenaceae).
Diospyros is a large genus of woody dioecious plants found
worldwide in the tropics and subtropics, including 31 species in
New Caledonia. Previous studies based on plastid markers
(Duangjai et al.,2009)showedthatDiospyros colonized New
Caledonia at least four times via long-distance dispersal.
Two of the successful dispersal events each resulted in a single
species that still persists; an additional dispersal event led to a
small clade comprising five species; and yet another event gave
rise to a putatively rapidly radiating group of 24 endemic spe-
cies. These 24 species have been shown to be highly similar
genetically using low-copy nuclear and plastid markers
(Duangjai et al., 2009;Turner et al., 2013a). Most of these
closely related species are morphologically and ecologically
clearly differentiated, and current species delimitations (White,
1993) have been generally confirmed by analyses of amplified
fragment length polymorphisms (AFLPs; Turner et al., 2013b)
and restriction site-associated DNA sequencing (RADseq; Paun
et al.,2016).
V
CThe Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/
by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Annals of Botany Page 1 of 11
doi:10.1093/aob/mcw060, available online at www.aob.oxfordjournals.org
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On New Caledonia, Diospyros species are found in many
habitats, but they often grow in proximity to each other. At
some localities, several species are microsympatric, which
allows interspecific gene flow if reproductive isolation is still
incomplete. Dating analysis based on four plastid and two low-
copy nuclear DNA regions showed that the ancestors of this
group of New Caledonian Diospyros species arrived in New
Caledonia around 9 million years ago (Turner et al.,2013a).
Given that Diospyros includes long-lived perennial plants, it be-
comes obvious that they have evolved relatively recently.
Resolving the phylogenetic relationships in such a young group
of rapidly radiating and potentially hybridizing taxa poses sig-
nificant challenges (Glor, 2010). We test here the usefulness of
next-generation sequencing (NGS)-based genome skimming to
obtain phylogenetic data, in particular by sequencing whole
plastomes and the full-length nuclear ribosomal DNA region
(i.e. nrDNA).
The plastid genome has proved useful for molecular phylo-
genetic investigations of plants at different taxonomic levels. In
the past two decades, sequences from the plastid genome have
been extensively used to infer phylogenetic relationships among
plants (e.g. Chase et al., 1993;Barfuss et al.,2005;Duangjai
et al., 2009;Russell et al.,2010). Uniparental inheritance, low
mutation rates and high copy number are well-known features
of plastid genomes and the basis for their standard usage in
plant systematics. Due to the slow rate of evolution of the plas-
tid genome, the level of variation is often low compared with
nuclear DNA in general and mitochondrial markers in animals
(Schaal et al.,1998). The high level of conservation has pre-
vented the development of a universally applicable single bar-
coding region in plants (CBOL Plant Working Group, 2009).
Recently, whole-plastid genome sequencing has become afford-
able, and this has been employed to generate more highly
resolved phylogenetic trees (e.g. Ku et al., 2013;Yang et al.,
2013;Barrett et al., 2014;Male´et al., 2014).
Genes coding for nuclear ribosomal RNA are found in the
genome in multiple copies arranged in tandem repeats, and
therefore it is feasible to obtain full-length sequences from low-
coverage NGS approaches. Because of concerted evolution,
these thousands of copies mostly behave as single-copy genes,
and potential evidence of hybridization is normally eliminated
within a few generations (Chase et al., 2003). Each nrDNA re-
peat consists of coding and non-coding elements. In plants, the
four rRNA genes are arranged in two clusters. One of these
clusters comprises three genes (18S, 58S and 26S) separated
by two internal transcribed spacers (ITSs), the external tran-
scribed spacer (ETS) and the non-transcribed spacer (NTS).
The second cluster includes the coding region of one rRNA
(5S) and a spacer between the repeats. The nrDNA genes are
relatively conserved but contain enough variation that they
have been used for phylogenetic reconstructions at higher taxo-
nomic levels (Maia et al.,2014). The non-coding spacers, ITS
and ETS, between the genes are much more variable and repre-
sent a useful source of phylogenetic information among closely
related species (e.g. Devos et al.,2006;Sanz et al.,2008;
Akhani et al.,2013;Nu¨rk et al.,2013;Zhu et al.,2013).
Here we aimed to use whole plastid genomes as well as com-
plete sequences of the nrDNA for phylogenetic analyses for
species of the New Caledonian Diospyros to investigate
whether this approach would produce an improved estimate of
phylogenetic relationships in this putatively rapidly radiating
group.
MATERIALS AND METHODS
Leaf material from New Caledonian Diospyros species was col-
lected on the main island, Grande Terre, and on one smaller is-
land in the south, ^
Ile des Pins (Fig. 1). For the widespread
species, at least two representative individuals were sequenced
(Table 1). In total we included here 36 individuals of New
Caledonian Diospyros species (corresponding to 21 identified
and one unidentified species) as well as one individual of
Diospyros olen (from New Caledonia, but not closely related to
the other New Caledonian Diospyros species) and one individ-
ual of Diospyros ferrea from Thailand. Diospyros olen and D.
ferrea were used as outgroups. Wherever possible, we used the
exact same individuals for which Sanger sequence data are
available from previous studies (Turner et al.,2013a). However,
because of poor DNA quality (unsuitable for NGS) or unavail-
ability for some of the accessions, we had to include in this study
another accession from the same population or locality.
DNA was extracted from silica gel-dried leaf material using
a modified sorbitol/high-salt cetyltrimethylammonium bromide
(CTAB) method (Tel-Zur et al., 1999). Extracts were purified
using the NucleoSpin gDNA Clean-up kit (Marcherey-Nagel,
Germany), according to the manufacturer’s protocol.
From each sample, 300 ng of DNA was sheared (in two
cycles of 45 s with 30 s break between the two shearing runs)
using an ultrasonicator (Bioruptor Pico, Diagenode, Belgium),
targeting a mean fragment size of 400 bp. Library preparation
was performed using the NEBNext Ultra DNA Library Prep
Kit for Illumina (New England Biolabs, USA) according to the
manufacturer’s protocol. All individuals were barcoded and
pooled to reach an equal representation of each individual in
the final libraries. In total, two libraries (containing 14 and 24
samples per library, respectively) were prepared for the 38 sam-
ples used. The two libraries were sequenced on an Illumina
HiSeq as 100-bp paired-end reads at the VBCF (Vienna
Biocenter Core Facilities, Vienna, Austria; http://www.vbcf.
ac.at/facilities/next-generation-sequencing/). Demultiplexing of
the raw data was performed, allowing for a maximum of one
mismatch using the Picard BamIndexDecoder (included in the
Picard Illumina2bam package; available from https://github.
com/wtsi-npg/illumina2bam). The number and quality of raw
reads obtained from each individual were evaluated with
FastQC (Andrews, 2010).
Assembling and annotating plastid genomes
Reads originating from the plastid genome (pt DNA) were
filtered using a multistep and iterative in-house established
pipeline. First, the individual raw files were imported into the
CLC GENOMIC WORKBENCH v. 6.5 (Qiagen) and trimmed
by quality at P<005, retaining reads of at least 30 bp. Then
the reads of D. ferrea were mapped on the complete plastid
genome of Camellia sinensis (Theaceae, Ericales, GenBank:
KC143082.1). For this initial mapping of both coding and non-
coding regions (the latter comprising introns and intergenic spa-
cers), a mismatch cost of 2 and insertion and deletion cost of 3
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were used, requiring at least 80 % of a read to be at least 90 %
similar to the target for each successful mapping. With these
settings, 403 980 reads of D. ferrea mapped to the Camellia
plastome. We re-extracted the initial paired-end read data cor-
responding to these reads using FastQ.filter.pl (Rodriguez-R
and Konstantinidis, 2016, available at https://github.com/lmro
driguezr/enveomics/) and have assembled them de novo in the
CLC GENOMIC WORKBENCH, with automatic optimization
of the word and bubble sizes and updating the contigs after
mapping back the reads. We obtained three contigs, which have
been concatenated by aligning them to the C. sinensis reference
sequence manually in the program BioEdit v. 7.2.5 (Hall,
1999). From the coverage information and by comparison with
the C. sinensis reference we were able to identify both inverted
repeats and confirm their presence in the plastid genome of
Diospyros. Both inverted repeats were reconstructed together
and duplicated to represent a complete plastid genome. The
base composition of the assembled contigs was extracted from
the alignments using BioEdit v. 7.2.5 (Hall, 1999).
The plastid genomes of the rest of the Diospyros species
were obtained in a similar way, but the initial mapping step was
performed on the assembled D. ferrea genome. Finally, annota-
tion of coding regions was performed using DOGMA (Wyman
et al.,2004) using only the D. vieillardii BT025 plastid
genome. The circular plastid genome map (Fig. 2) was visual-
ized with OGDRAW (Lohse et al., 2007).
Due to the difficulties with assembling the mitochondrial
genome of plants as well as the low level of phylogenetic infor-
mation in plant mtDNA (Male´et al.,2014), we did not attempt
to assemble the mitochondrial genome of Diospyros.
Assembly of nrDNA repeats
Reads containing sequences from the nrDNA region were
collected and assembled using the program MITObim v. 1.7
(Hahn et al., 2013). As initial seed, we used previously gener-
ated Sanger sequences from the ITS region of Diospyros vieil-
lardii BT025 (B. Turner, unpubl. res.). Characteristics of
assemblies such as the number of reads assembled in each con-
tig and coverage were inspected from the MITObim output
files. Assembled sequences were aligned using Muscle v. 3.8
(Edgar, 2004). The alignment was manually inspected using the
program BioEdit v. 7.2.5 (Hall, 1999). The beginning and end
of each coding region were estimated by comparing the
Diospyros alignment with annotated sequences of Solanum
lycopersicum (GenBank: AY366529, AY552528). We also ex-
tracted the 5S nrDNA region using the procedure described
100km
25
26
24
5
4
3
12
19 18 19
7
11
12
20
23
7
89
10
21
22
13
14
15
16
Grande Terre
Île des Pins
1 Nakutakoin
2 Dumbea
3 Between Plum and Prony
4 Col de Prony
5 Kuébini
6 La Roche Percée
7 Pindai/Népoui
8 Voh
9 Between Koné and Poindimié
10 Conservàtoire botanique de Tiéa
11 Aoupinié
12 Nétéa
13 Mandjelia
14 Koumac
15 Koumac
16 Paagoumène
17 Baie de Gadji
18 Yahoué
19 Forêt de la Superbe (Montagne des Sources)
20 Moindah
21 Plateau de Tiéa
22 Plateau de Tango
23 Népoui
24 Île Kuébini
25 Île des Pins, Kanuméra
26 Île des Pins, Pic N‘ga
02550
FIG. 1. Map of New Caledonia indicating the 26 sampling localities for this study. Numbered dots indicate sampling sites (see also Table 1). Dots are coloured ac-
cording to sampling region (north, green; middle, blue; south, red).
Turner et al. — Plastid genomes and nrDNA of Diospyros species from New Caledonia Page 3 of 11
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above. As initial seed we used the 120-bp coding fragment of
5S nrDNA sequences of Actinidia chinensis (GenBank:
AF394578).
Phylogenetic analyses
For the phylogenetic analyses of the plastid sequence data,
only one copy of the inverted repeat was included in the final
alignment. For analyses of the nrDNA sequences, only the tran-
scribed regions were used.
Parsimony analyses including bootstrapping were performed
using PAUP* v. 4b10 (Swofford, 2003). They were run using a
heuristic search with stepwise addition, random sequence add-
ition (1000 replicates) and tree bisection–reconnection. Gaps
were treated as missing data. To estimate clade support, boot-
strapping with 1000 replicates was performed using the same
settings as above (including 1000 random replicates per boot-
strap replicate). Estimation of consistency index (CI) and reten-
tion index (RI) was done with PAUP. Likelihood analysis
including bootstrapping was performed using RAxML v. 8.1.3
(Stamatakis, 2014). We used the Broyden–Fletcher–Goldfarb–
Shanno (BFGS) method to optimize generalized time reversible
(GTR) rate parameters, the gamma model of rate heterogeneity
and 1000 rapid bootstrap inferences with a subsequent thorough
maximum likelihood (ML) search. The results were visualized
with FigTree 14 (available from http://tree.bio.ed.ac.uk/soft
ware/figtree/). We rooted the trees obtained with D. olen ac-
cording to earlier results (Turner et al.,2013a).
To reduce the file size and speed up analyses in the com-
bined analyses, D. olen and constant positions were removed
using Mesquite v. 3.01 (Maddison and Maddison, 2014). In
trees conducted from the combined data set D. ferrea was used
as outgroup. Parsimony and likelihood analyses were per-
formed as described for the individual data sets. In addition, we
conducted a Bayesian analysis for the combined data set using
BEAST v. 1.8.1 (Drummond et al.,2012). The best evolution-
ary models for the two subsets (ptDNA and nrDNA) were eval-
uated using jModeltest v. 2.1.6 (Guindon and Gascuel, 2003;
Darriba et al., 2012). For the plastid partition, the transversional
TABLE 1. Table of accessions including all individuals used in this study. The identification numbers of sampling localities are given in
Fig. 1. Voucher codes: JMXXXX: collection number J. Munzinger; Tree No. XXXXX: Tree of New Caledonian Plant Inventory and
Permanent Plot Network (NC-PIPPN, Ibanez et al., 2014); KUFF, Herbarium of the Faculty of Forestry Kasertsat University
Bangkok; MPU, Herbarium of the University of Montpellier; NOU, Herbarium of IRD Noume´ a; P, Herbarium of the Natural History
Museum Paris; WU, Herbarium of the University of Vienna
Taxon Accession number Sampling location Voucher
D. calciphila F.White BT313 25 MPU026746
D. cherrieri F.White BT293 23 NOU079547
D. erudita F.White BT280 21 WU062858
D. ferrea (Wild.) Bakh. Eb045 Duangjai 106 (KUFF)
D. flavocarpa (Vieill. ex P.Parm.) F.White BT130 9 MPU026741
BT156 11 MPU026737
D. glans F.White BT093 5 Turner et al. 093 (MPU)
D. impolita F.White BT102 6 NOU019538
D. inexplorata F.White BT308 24 NOU005818
D. labillardierei F.White BT122 9 NOU052188
D. minimifolia F.White BT135 10 NOU019556
BT233 17 NOU019554
BT263 20 NOU079549, WU062872
D. olen Hiern BT001 1 NOU052191
D. pancheri Kosterm. BT028 3 MPU026742
BT031 3 MPU026742
D. parviflora (Schltr.) Bakh. BT041 4 Turner et al. s.n. (NOU)
BT090 5 NOU2519
BT147 10 NOU052175
BT187 13 NOU031409
BT250 19 Tree no. 23109
BT290 22 NOU079550
D. perplexa F.White BT004 1 MPU026738
D. pustulata F.White BT111 7 NOU019572
BT140 10 NOU052177
BT261 20 NOU079544
D. revolutissima F.White BT120 8 NOU023189
BT221 16 NOU084762
D. tridentata F.White BT207 14 NOU052179
D. trisulca F.White BT185 13 NOU031344
D. umbrosa F.White BT176 12 JM6635 (NOU)
BT247 19 NOU023234
D. veillonii F.White BT227 17 NOU019582
D. vieillardii (Hiern) Kosterm. BT025 2 Turner et al. s.n. (NOU)
BT100 5 NOU006676
BT215 15 NOU023242
D. yahouensis (Schltr.) Kosterm. BT238 18 P00057340
D. sp. Pic N’ga BT318 26 NOU054315
Page 4 of 11 Turner et al. — Plastid genomes and nrDNA of Diospyros species from New Caledonia
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model with equal frequencies (TVMef; Posada, 2003) showed
the best fit to the data, and for the nrDNA partition Tamura and
Nei’s model (TrN; Tamura and Nei, 1993) with among-site rate
variation modelled with a gamma distribution (TrNþC)showed
the best fit. Base frequencies (uniform), substitution rates
among bases (gamma shape 10) and alpha (gamma shape 10)
were inferred by jModeltest for each data set. For flexibility,
we used a relaxed uncorrelated log-normal clock model
(Drummond et al., 2006). As a speciation model, we used a
Yule model because the investigated group is so young that we
expected a low proportion of lineage extinction (Yule, 1925;
Gernhard, 2008). For further details regarding the parameters
used see Supplementary Data Fig. S1. Two independent
Metropolis-coupled Markov chain Monte Carlo (MCMC)
rpl2
rpl23
trnI (CAU)
ycf2
ycf15
trnL (CAA)
ndhB
rps7
rps12_3end
trnV (GAC)
rrn16
trnI (GAU)
ycf68
trnA (UGC)
orf42
orf56
rrn23
rrn4.5
rrn5
trnR (ACG)
trnN (GUU)
ycf1
rps15
ndhH
ndhA
I
h
d
n
G
h
dn
ndhE
psaC
ndhD
ccsA
trnL (UAG)
rpl32
ndhF
ycf1
trnN (GUU)
trnR (ACG)
rrn5
rrn4.5
rrn23
trnA (UGC)
orf56
orf42
trnI (GAU)
ycf68
rrn16
trnV (GAC)
rps12_3end
rps7
ndhB
trnL (CAA)
ycf15
ycf2
trnH (CAU)
rpl23
rpl2
rps19
rpl22
rps3
rpl16
rpl14
rps8
infA
rpl36
rps11
rpoA
petD
petB
psbH
psbN
psbT
psbB
clpP
rps12
rpl20
rps18
rpl33
psaJ
trnP (UGG)
trnW (CCA)
petG
petL
psbE
psbF
psbL
psbJ
petA
cemA
ycf4
psaI
accD
rbcL
atpB
atpE
trnM (CAU)
trnV (UAC)
ndhC
ndhK
ndhJ
trnF (GAA)
trnL (UAA)
trnT (UGU)
rps4
trnS (GGA)
ycf3
psaA
psaB
rps14
tRNA-fM (CAU)
trnG (GCC)
psbZ
trnS (UGA)
psbC
ps
bD
trnT (GGU)
trnE (UUC)
trnY (GUA)
trnD (GUC)
psbM
petN
trnC (GCA)
rpoB
rpoC1
rpoC2
rps2
atpI
atpH
atpF
atpA
trnR (UCU)
trnG (UCC)
trnS (GCU)
psbI
psbK
trnQ (UUG)
rps16
matK
psbA
trnH (GUG)
Diospyros vieillardii
plastid genome
157,535 bp
Introns
Ribosomal RNAs
Transfer RNAs
ORFs
H
y
pothetical chloroplast readin
g
frames (
y
cf)
Other genes
clpP, matK
Ribosomal proteins (LSU)
Ribosomal proteins (SSU)
RNA polymerase
RubisCO large subunit
NADH dehydrogenase
ATP s
y
nthase
Cytochrome b/f complex
Photosystem II
Photosystem I
I
R
A
I
R
B
S
S
C
L
S
C
FIG. 2. Graphic representation of the annotated plastid genome of Diospyros vieillardii.
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analyses, each with 20 million generations, were run, sampling
each 1000th generation. The initial 10 % of trees obtained from
each MCMC run were removed as burn-in; the remaining trees
of both runs were used to calculate the maximum clade cred-
ibility tree.
For directly comparing the results of the present study with
previous ones (Turner et al., 2013a) based on four plastid
markers (atpB,rbcL,trnK-matK,trnS-trnG; 5979 bp) and two
low-copy nuclear markers (ncpGS, 717 bp; PHYA, 1189 bp), a
subset of those data corresponding to the individuals included
in this study was analysed in the same way as described above
for the Bayesian analysis. In most cases, to represent each spe-
cies we used either the same accession or individuals from the
same population. In the few cases where no data from individ-
uals of the same population were available, then the geograph-
ically closest individual assigned to the same species was used.
Due to these differences, results of the previous studies are not
strictly comparable to those produced here, but we have com-
pared them nonetheless due to their overall similarity.
A general pattern of geographical clustering was visually
observed in the resulting trees, in particular in the plastid data.
Based on the geographical coordinates of samples and the dis-
tance matrix of pairwise uncorrected P-values (calculated with
SplitsTree), we tested the significance of geographical cluster-
ing of the samples in the trees using a Mantel test. We estimated
the correlation of geographical and genetic discontinuities in
the data by performing this analysis on the plastid and the
combined data sets with Isolation by Distance web service
(IBDWS) (Jensen et al., 2005).
To investigate the relationships between populations we con-
structed a neighbour network based on the plastid markers,
using SplitsTree4 v. 4.13.1 (Hudson and Bryant, 2006) and un-
corrected Pdistance estimates. Based on the results from the
Mantel test [Table 2 andRADseqresults(Paun et al.,2016)],
we excluded samples of D. olen,D. ferrea,D. vieillardii,
D. umbrosa,D. flavocarpa,D. cherrieri and D. veillonii from
this analysis to get a clearer picture of the relationships within
the recently and rapidly radiated species group.
RESULTS
After the demultiplexing step, the number of raw Illumina se-
quences ranged from 10 to 29 million paired-end reads per indi-
vidual. Details of sample characteristics are given in Table 3.
Plastid genomes
We obtained between 75 262 (47average coverage, for
D. parviflora BT250) and 857 039 (531coverage, for D. sp.
Pic N’ga BT318) pairs of reads per individual that mapped to
the plastid genome (for details see Table 3). The GC content of
the plastid genomes of Diospyros (37 %) is similar to those of
many other angiosperms [average 37 %; e.g. Ardisia (Ku
et al., 2013); Camellia (Yang et al.,2013); Potentilla (Ferrarini
et al.,2013); Musa (Martin et al., 2013); Zingiberales (Barrett
et al.,2014)].
Thesize(157 kb) and gene order of the plastid genome of
D. vieillardii (Fig. 2) is similar to that of C. sinensis (GenBank:
KC143082.1). This plastid genome is the first fully sequenced
plastid genome of Ebenaceae reported in the literature.
The plastid matrix of Diospyros used for phylogenetic ana-
lyses (including only one of the inverted repeats) included
133 210 characters, of which 1295 variable positions were
parsimony-uninformative and 384 (03 %) were potentially par-
simony-informative. Phylogenetic reconstruction using parsi-
mony produced 1127 equally parsimonious trees (results not
shown) with a CI of 093 and RI of 087. Phylogenetic relation-
ships between the species were in many cases not well sup-
ported, and in several cases individuals of the same species
failed to form well-supported clusters. Similar results were
obtained using maximum likelihood (Supplementary Data
Fig. S2).
Nuclear ribosomal DNA
Between 008 % (D. impolita BT102) and 051 % (D. ferrea
Eb045) of the total reads pertained to the nrDNA region (for de-
tails see Table 3). The contigs obtained had average coverages
ranging from 209(for D. impolita BT102) to 1159(for
D. vieillardii BT025). The NTS of the intergenic spacer was
difficult to align and therefore excluded from further analyses.
The aligned nrDNA matrix of Diospyros included 7233 charac-
ters, of which 368 variable positions were parsimony-unin-
formative and 141 (19 %) were potentially parsimony-
informative. The parsimony phylogenetic reconstruction with
only the nrDNA sequences produced 84 equally parsimonious
trees (results not shown) with a CI of 066 and RI of 053.
Phylogenetic relationships generally disagreed with results ob-
tained from other markers, but these incongruent relationships
are all weakly supported (results not shown). Several species
TABLE 2. The extent of geographical clustering in the data, as evidenced with Mantel tests performed on IBDWS
Dataset Mantel’s rSignificance
Plastid data
Including all individuals, except the outgroups D. olen and D. ferrea 0103 P¼009
Excluding D. olen,D. ferrea and all D. vieillardii 0242 P<0001
Excluding D. olen,D. ferrea,D. vieillardii,D. umbrosa and D. flavocarpa 0383 P<0001
Excluding D. olen,D. ferrea,D. vieillardii,D. umbrosa,D. flavocarpa,D. cherrieri and D. veillonii 0427 P<0001
Combined
Including all individuals except the outgroups D. olen and D. ferrea 0079 P¼010
Excluding D. olen,D. ferrea and all D. vieillardii 0121 P<005
Excluding D. olen,D. ferrea,D. vieillardii,D. umbrosa and D. flavocarpa 0302 P<0001
Excluding D. olen,D. ferrea,D. vieillardii,D. umbrosa,D. flavocarpa,D. cherrieri and D. veillonii 0374 P<0001
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failed to form unique groups. Similar results were obtained
using maximum likelihood (Supplementary Data Fig. S3).
The assembled 5S nrDNA region containing coding and non-
coding parts was short (less than 500 bp) and therefore did not
contain many informative characters. Phylogenetic trees based
on this fragment were poorly resolved, and therefore the trees
are not presented or discussed further. The 5S nrDNA region
was also not included in the combined analysis.
Analyses of the combined data set
In addition to the individual analyses of the two data sets, we
also combined them to determine whether this approach pro-
vides better resolution/support.
The combined matrix (i.e. reduced to variable positions as
explained in Materials and methods) included 1136 characters,
of which 437 were potentially parsimony-informative.
Phylogenetic reconstruction using parsimony produced a single
most parsimonious tree of 1580 steps (Supplementary Data Fig.
S4) with a CI of 073 and RI of 052. Phylogenetic relationships
depicted in the combined analysis were better resolved than in
the trees obtained from the individual analyses. A comparable
pattern was found in the Bayesian analysis of the combined
data set (Fig. 3). All three individuals of D. vieillardii
clustered together and were sister to the rest of the New
Caledonian endemic species group. Individuals from
D. flavocarpa and D. umbrosa formed unique groups and were
sister to the rest of the remaining accessions, among which
D. cherrieri and D. veillonii were sister to the rest. The results
from the combined analysis were also in agreement with earlier
results based on plastid and nuclear markers (Turner et al.,
2013a).
There is a trend of geographical clustering visible in the
Bayesian tree (Fig. 3) and in the neighbour network
(Supplementary Data Fig. S5). The neighbour network (Fig.
S5) clearly shows that individuals and populations from the
south and the middle of New Caledonia clustered according to
their sampling region. The Mantel test (Table 2) confirmed
a significant geographical clustering of the genetic informa-
tion (Fig. 3), in particular in the plastid data across the crown
group.
TABLE 3. Details of samples used here for sequencing of whole plastid genomes and nrDNA
Raw reads Plastid genome Nuclear ribosomal DNA
Percentage of
reads mapping
Coverage () GC (%) Percentage of
reads mapping
Coverage () GC (%)
D. calciphila BT313 19549968 134 162 3737 017 444 5924
D. cherrieri BT293 15164658 330 309 3736 011 229 5852
D. erudita BT280 12612118 303 236 3736 015 246 5936
D. ferrea Eb045 9943646 481 300 3734 051 559 5846
D. flavocarpa BT130 10129514 099 62 3736 029 331 5707
D. flavocarpa BT156 15656792 053 51 3737 010 219 5853
D. glans BT093 14436316 091 81 3736 019 386 5848
D. impolita BT102 18636398 114 131 3736 008 209 5926
D. inexplorata BT308 16201056 106 106 3736 024 511 5965
D. labillardierei BT122 26574012 095 156 3736 015 538 586
D. minimifolia BT135 26685314 119 199 3736 009 314 5882
D. minimifolia BT233 25526086 084 134 3735 013 384 5824
D. minimifolia BT263 16154630 195 198 3735 029 616 5914
D. olen BT001 24966688 151 236 3744 038 1084 5832
D. pancheri BT028 27590124 080 136 3735 009 316 5938
D. pancheri BT031 29453086 071 130 3736 009 361 5898
D. parviflora BT041 27178316 083 140 3736 009 356 5854
D. parviflora BT090 25432978 040 63 3737 019 656 5934
D. parviflora BT147 17887304 065 71 3736 028 690 5869
D. parviflora BT187 26648984 045 74 3736 021 690 566
D. parviflora BT250 17588828 043 47 3736 037 877 5902
D. parviflora BT290 19187356 039 47 3736 028 737 5908
D. perplexa BT004 18085506 074 82 3736 030 595 5743
D. pustulata BT111 15958418 246 242 3736 028 467 5769
D. pustulata BT140 17029506 180 188 3736 026 580 5952
D. pustulata BT261 15585090 119 114 3736 037 745 5994
D. revolutissima BT221 20867576 150 193 3737 020 551 5798
D. revolutissima BT120 17111770 146 154 3735 021 466 5916
D. tridentata BT207 14190292 089 77 3735 021 356 5779
D. trisulca BT185 17297816 075 80 3736 029 710 5867
D. umbrosa BT176 18856642 085 98 3738 048 632 5592
D. umbrosa BT247 28845756 059 104 3736 010 385 5919
D. veillonii BT227 26594158 180 297 3736 030 1056 5865
D. vieillardii BT025 26595776 091 152 3734 038 1160 5829
D. vieillardii BT100 22649344 210 297 3733 011 327 5852
D. vieillardii BT215 27710030 080 138 3732 014 501 5894
D. yahouensis BT238 29242716 080 144 3736 015 578 5968
D. sp. Pic N’ga BT318 18515350 463 531 3736 021 500 5937
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0·002
D. parviflora BT187
D. perplexa BT004
D. pancheri BT028
D. pustulata BT140
D. calciphila BT313
D. parviflora BT147
D. tridentata BT207
D. ferrea Eb045
D. umbrosa BT176
D. trisulca BT185
D. parviflora BT250
D. glans BT093
D. sp. Pic N‘
g
a BT318
D. vieillardii BT100
D. flavocarpa BT156
D. inexplorata BT308
D. vieillardii BT215
D. minimifolia BT233
D. parviflora BT041
D. labillardierei BT122
D. flavocarpa BT130
D. revolutissima BT120
D. pustulata BT111
D. revolutissima BT221
D. pustulata BT261
D. parviflora BT090
D. vieillardii BT025
D. erudita BT280
D. yahouensis BT238
D. umbrosa BT247
D. cherrieri BT293
D. veillonii BT227
D. parviflora BT290
D. minimifolia BT135
D. impolita BT102
D. minimifolia BT263
D. pancheri BT031
1·00
1·00
1·00
1·00
1·00
1·00
1·00
1·00
1·00
1·00
0·98
1·00
1·00
1·00
1·00
1·00
1·00
1·00
1·00
1·00
1·00
1·00
0·99
D. parviflora BT147
D. veillonii BT229
D. yahouensis BT238
D. pustulata BT137
D. impolita BT102
D. calciphila BT314
D. glans BT093
D. parviflora M2037
D. perplexa BT004
D. minimifolia BT231
D. parviflora M2338
D. minimifolia BT264
D. vieillardii BT213
D. minimifolia BT133
D. sp. Pic N‘ga BT318
D. flavocarpa BT126
D. erudita BT287
D. umbrosa M2265
D. revolutissima BT116
D. ferrea Eb045
D. parviflora M2071
D. inexplorata BT311
D. vieillardii BT100
D. parviflora BT187
D. pancheri BT028
D. labillardierei BT122
D. cherrieri BT297
D. vieillardii BT025
D. pustulata BT259
D. umbrosa BT247
D. pancheri BT031
D. revolutissima BT219
D. trisulca BT185
D. pustulata BT113
D. parviflora BT040
D. flavocarpa BT156
D. tridentata BT203
0·98
1·00
1·00
1·00
1·00
Whole plastid genome and nrDNA
B
Four plastid and two low copy nuclear markers
A
FIG. 3. Phylogenetic trees resulting from the Bayesian analysis of the (A) combined data set of the present study (variable nucleotide positions in the whole plastid genome and nrDNA) and (B) combined data
set of a previous study including four plastid and two low-copy nuclear markers (Turner et al.,2013a). The numbers indicate posterior probabilities >90. Trees are scaled to the same branch length. Samples are
coloured according to sampling region (see Fig. 1).
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DISCUSSION
Previous standard approaches to phylogenetic analysis of
Diospyros species including samples from New Caledonia used
nine (Duangjai et al.,2009) and four (Turner et al., 2013a)plas-
tid markers (alignment length >8and64 kb, respectively).
They demonstrated low levels of sequence divergence among
these species, indicating a fairly recent and rapid radiation.
Inclusion of low-copy nuclear markers, which have been shown
to be highly informative and useful for resolving phylogenetic
relationships at lower taxonomic levels in some taxa [e.g.
Paeonia (Tank and Sang, 2001)andPassiflora (Yockteng and
Nadot, 2004)], only partly improved resolution among the New
Caledonian Diospyros species (Turner et al.,2013a). Similar
results have been observed in two genera of Cunoniaceae from
New Caledonia [Spiraeanthemum (Pillon et al.,2009a)and
Codia (Pillon et al.,2009b)].
AFLP markers are typically used at low taxonomic level in
closely related species and population analyses (e.g. Despre´
et al.,2003;Tremetsberger et al.,2006;Gaudeul et al., 2012).
In the case of the New Caledonian Diospyros species, we used
AFLPs to evaluate species limits (Turner et al., 2013b), and in
most cases we found congruence with the species concepts of
White (1993). However, the AFLP approach was not useful in
resolving phylogenetic relationships among these species
(Turner et al., 2013b) because there were few of the individual
AFLP fragments shared by two or more species. It would ap-
pear from the AFLP results that fragmentation of an original
widespread population occurred in many regions of New
Caledonia more or less simultaneously, resulting in genetically
distinct populations that correspond to the morphologically
based species delimitations of White (1993) without leaving
much evidence for interspecific relationships.
The phylogenetic tree obtained here based on the whole plas-
tid genome (Fig. S2) is similar to the phylogenetic tree previ-
ously based on four plastid markers (Turner et al., 2013a). The
combined tree of this study (Fig. 3A)is similar both in reso-
lution and structure to the phylogenetic tree based on four plas-
tid and two low-copy nuclear markers (Fig. 3B). Although not
always represented by the same individuals, general relation-
ships of species are in agreement.
The nuclear ribosomal region included a higher percentage
of parsimony-informative characters (19versus03%)than
the plastid DNA, which is in agreement with findings in other
plant groups (Hamby and Zimmer, 1992;Doyle, 1993;Male´
et al., 2014). Despite this variability, nrDNA was still too short
to contain enough variation (141 versus 384 informative sites)
and failed to resolve phylogenetic relationships among the New
Caledonian Diospyros species (Fig. S3).
Our results clearly show that for this group of species, in
which standard plastid and nuclear markers were not helpful for
resolving the phylogenetic relationships, using the whole plas-
tid genome does not greatly increase resolution and support.
There are only a few other studies available in which whole
plastid genomes have been used to resolve phylogenetic rela-
tionships at the intraspecific level among closely related spe-
cies. Some studies [e.g. in Chrysobalanaceae (Male´et al.,
2014) and eucalypts (Myrtaceae) (Bayly et al.,2013)] have re-
vealed that this approach can be useful for resolving phylogen-
etic relationships among genera, but they failed to resolve
phylogenetic relationships among closely related species and to
group together individuals of the same taxonomic species. In
cases of recently radiating species groups, in particular follow-
ing an extreme bottleneck associated with a long-distance dis-
persal event such as the arrival of Diospyros in New Caledonia,
plastid genomes appear to be insufficient for inference of
phylogenetic relationships. The basis of the rapid radiation of
Diospyros in New Caledonia is not yet clear, but it has been
speculated that it is an adaptive origin associated with different
soil types (Paun et al., 2016), as recently shown for the genus
Geissois in Cunoniaceae (Pillon et al., 2014).
The individuals of D. vieillardii,D. umbrosa,D. flavocarpa,
D. cherrieri and D. veillonii form a minimally isolated group in
the combined analyses (Fig. 3). These species form clusters that
are successively sister to the rest of the taxa, which are well
supported collectively but form a highly unresolved central
cluster. This unresolved central cluster is less than 6 million
years old (Paun et al.,2016) and could be the result of two lin-
eages that developed in isolation and then subsequently colon-
ized some of the same habitats. This too could be the result of
simultaneous parallel divergence in different parts of New
Caledonia combined with effects of local and more recent hy-
bridization (as was indicated in the AFLP study; Turner et al.,
2013b). Retention of ancient polymorphisms present in the ori-
ginal colonizing population, which probably also underwent a
severe bottleneck, could not produce such a geographically
structured pattern.
Comparisons of the phylogenetic tree based on plastid se-
quences (Fig. S2) with the tree derived from RAD data [Paun
et al., 2016 (Supplementary Data Fig. S6)] showed several clus-
ters of individuals (D. trisulca and D. parviflora from L13, D.
pustulata and D. minimifolia from L20, D. pancheri and D. par-
viflora from L3 and 4; Table 1,Fig. 1) that occur with high stat-
istical support in the plastid results, but are not present in the
nuclear tree. These clusters consist of individuals found in the
same or very nearby locations, which could indicate introgres-
sive hybridization and transfer of plastid genomes as a relevant
phenomenon (Naciri and Linder, 2015, and references therein).
Geographical rather than taxonomic clustering was observed
for all populations of D. minimifolia and D. parviflora that also
failed to form unique groups in nuclear results [AFLP (Turner
et al., 2013b)RAD(Paun et al.,2016)]. Phenomena like intro-
gressive hybridization and transfer of plastid genomes could
also explain the geographical clustering of individuals observed
in the plastid data set (Figs S2 and S5;Table 2), whereas in nu-
clear-based data sets [AFLP (Turner et al., 2013b), RAD (Paun
et al., 2016)] no such geographical clustering was observed.
Similar geographical clustering for plastid results has previ-
ously been reported in other plant groups [e.g. Nothofagus
(Acosta and Premoli, 2010)].
Although there are more than 140 kb of DNA sequence
included in this study, it effectively corresponds to only two
markers (plastid genome and rDNA region). As not all genes
evolve in the same mode and at the same tempo, phylogenies
based on different genes might show different phylogenetic re-
lationships (Heled and Drummond, 2010). It is therefore im-
portant to use many phylogenetic markers for reconstruction of
relationships to overcome the limitations of individual genes
and produce results as close as possible to the real species tree.
Phylogenetic trees based on plastid data should always be
Turner et al. — Plastid genomes and nrDNA of Diospyros species from New Caledonia Page 9 of 11
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viewed as gene trees because of the typical evolutionary path-
ways of these organelles (Naciri and Linder, 2015). The trees
presented here may hence show the evolutionary history of the
particular region investigated, and may differ from the species
trees. In the case of the New Caledonian Diospyros species, we
consider the SNP data derived from RAD (Paun et al.,2016)as
the best approximation of the species tree, because it involves
nearly 8500 independent markers from the nuclear genome.
CONCLUSIONS
Although New Caledonian Diospyros are morphologically and
ecologically diverse, they show little genetic divergence. For
these rapidly radiating Diospyros species, in which standard
plastid and nuclear markers were not helpful for resolving the
phylogenetic relationships, using the whole plastid genome
does not greatly increase resolution and support. Plastid
markers grouped accessions according to geographical proven-
ance, which could result from local transfer of plastid genomes
due to hybridization and introgression following secondary
contact.
We are now conducting additional nuclear genome studies
(both coding and non-coding regions) to determine whether
other approaches could help us determine the potential adaptive
nature of this radiation, which has thus far defeated our at-
tempts using standard and next-generation methods.
SUPPLEMENTARY DATA
Supplementary data are available online at www.aob.oxfordjour
nals.org and consist of the following. Figure S1: BEAST input
file for the Bayesian analysis of the combined data set including
all settings and priors of both data sets. Figure S2: maximum
likelihood tree based on plastid sequences. Figure S3: max-
imum likelihood tree based on nrDNA sequences. Figure S4:
single most parsimonious tree based on the combined data ma-
trix. Figure S5: neighbour network based on the plastid data set.
Individuals are coloured according to their sampling region.
Figure S6: maximum likelihood tree based on RAD SNP data.
ACKNOWLEDGEMENTS
We thank Emiliano Trucchi for his help and ideas concerning
data analysis and IRD’s Noume´a team for assistance with field
work, especially Ce´line Chambrey. Voucher specimens are
deposited in the herbaria of Noumea (NOU), University of
Montpellier (MPU), University of Vienna (WU) and the
Faculty of Forestry of the Kasertsat University Bangkok
(KUFF). This work was supported by the Austrian Science
Fund (P 22159-B16 to R. S.).
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