Tree Genetics & Genomes (2018) 14:6
https://doi.org/10.1007/s11295-017-1223-z

ORIGINAL ARTICLE

Phylogenetic relationships of Aurantioideae (Rutaceae)
based on RAD-Seq
Yukio Nagano 1 & Takashi Mimura 2 & Nobuhiro Kotoda 2 & Ryoji Matsumoto 2 & Atsushi J. Nagano 3,4,5 & Mie N. Honjo 3 &Hiroshi Kudoh 3 & Masashi Yamamoto 6
Received: 31 July 2017 / Revised: 27 November 2017 / Accepted: 25 December 2017
# Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
The economically and nutritionally important genus Citrus belongs to the subfamily Aurantioideae in the family Rutaceae.
Here, we analyzed the phylogenetic relationships of the subfamily Aurantioideae based on RAD-Seq. The RAD-Seq data
produced phylogenetic trees with high support values, clear discriminations based on branch length, and elucidations ofearly branching events. Our genetic classification corresponded well with the classical morphological classification systemand supported the subdivision of Citreae, one of two tribes of the Aurantioideae, into three subtribes—Triphasiinae,
Citrinae, and Balsamocitrinae. Additionally, it was largely consistent with the subdivision of Clauseneae, the other tribeof the Aurantioideae, into three subtribes—Micromelinae, Clauseninae, and Merrillinae; the exception was Murraya
paniculata. With the exception of members of primitive citrus fruit trees, namely, Severinia buxifolia and Hesperethusa
crenulata, lower-level morphological groupings under subtribes based on genetic and morphological classifications
corresponded well. The phylogenetic relationship between Asian Btrue citrus fruit trees^ (genera Citrus, Poncirus, andFortunella) and Australian/New Guinean citrus fruit trees (genera Microcitrus, Eremocitrus, and Clymenia) was inconsistent between present classification based mainly on the nuclear genome and the previous classification based on thechloroplast genome. This inconsistency may be explained by chloroplast capture. Our findings provide a valuable insight
into the genetic relationships of the subfamily Aurantioideae in the family Rutaceae.
Keywords Citrus . Aurantioideae . Rutaceae . RAD-Seq . Phylogeny . Genetic classification

Communicated by W.-W. Guo
Electronic supplementary material The online version of this article(https://doi.org/10.1007/s11295-017-1223-z) contains supplementarymaterial, which is available to authorized users.
* Yukio Nagano
nagano@cc.saga-u.ac.jp
1

Analytical Research Center for Experimental Sciences, Saga
University, 1 Honjo-machi, Saga 840-8502, Japan

2

Faculty of Agriculture, Saga University, 1 Honjo-machi,
Saga 840-8502, Japan

3

Center for Ecological Research, Kyoto University, 509-3 2-chome,
Hirano, Otsu, Shiga 520-2113, Japan

4

JST PRESTO, 4-1-8, Honcho, Kawaguchi, Saitama 332-0012, Japan

5

Faculty of Agriculture, Ryukoku University, 1-5 Yokotani, Seta
Oe-cho, Otsu, Shiga 520-2194, Japan

6

Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto,Kagoshima 890-0065, Japan

Introduction
Citrus is an economically and nutritionally important genus
and a member of the subfamily Aurantioideae in the family
Rutaceae. In addition to Citrus species, some other membersof Aurantioideae are used for various purposes such as foods,
spices, medicinal chemicals, cosmetics, and garden trees.
Therefore, it is important to understand the phylogenetic relationships of the subfamily Aurantioideae.
Classical morphology divided the subfamily
Aurantioideae into two tribes—Clauseneae and Citreae(Swingle and Reece 1967). Recent molecular classifications, using small numbers of DNA sequences, also clearly discriminated Citreae from Clauseneae (Bayer et al.
2009; Penjor et al. 2013; Schwartz et al. 2015).
Morphological classification subdivided each tribe into
three subtribes. The tribe Clauseneae consists ofMicromelinae, Clauseninae, and Merrillinae, and the tribe

6Page 2 of 11

C i t r e a e c o n s i s t s o f Tr i p h a s i i n a e , C i t r i n a e , a n d
Balsamocitrinae (Swingle and Reece 1967). However, recent molecular studies did not clearly reveal this level ofclassification (Bayer et al. 2009; Penjor et al. 2013;
Schwartz et al. 2015), probably because they did not havethe capability to determine earlier branching events.
The data size used in previous molecular analyses was
small, i.e., small numbers of markers or short DNA sequences. Therefore, these previous studies did not discriminate the earlier branching events that separated major
taxa. In addition, it was difficult to reveal genetic relationships among closely related species. Next-generation sequencing produces a large amount of data; hence, it isworthwhile using it to reveal the genetic relationships
within the subfamily Aurantioideae. In comparison withwhole genome sequencing, restriction site-associated
DNA sequencing (RAD-Seq) can analyze many samples
inexpensively and simultaneously, and it is suitable forstudying the relationships between genetically similar individuals (Baird et al. 2008). The original report of RADSeq (Baird et al. 2008) has more than 1000 Google
Scholar citations, and the technique has been applied tomany types of model and non-model organisms (Andrews
et al. 2016). In our case, we used this method to analyze
Citrus species (Penjor et al. 2014, 2016), which are genetically similar because of their cross-compatibilities.
The relationships between genetically distant individuals
at the genus and/or family level have been analyzed using
RAD-Seq data with the new software PyRAD (Eaton2014). The clustering method of PyRAD does not use
the reference genome and allows for lower similarity
thresholds and inclusion of indels. Therefore, it is possible
to analyze relationships within the subfamily
Aurantioideae using RAD-Seq. The RAD-Seq method
and the programs required to analyze the resulting data
were reviewed by Andrews et al. (2016).
In the present study, we analyzed the genetic relationships
within the subfamily Aurantioideae based on RAD-Seq. The
obtained phylogenetic trees elucidated branching order well.
Herein, we describe new findings and confirm previous findings. Our molecular classification is consistent with the classical morphological classification (Swingle and Reece 1967).

Materials and methods
Plant materials
Thirty-five species from 22 genera of the subfamily
Aurantioideae were used in this study (Table 1). The materials have been preserved at the Faculty of Agriculture,
Saga University, and the Faculty of Agriculture,
Kagoshima University.

Tree Genetics & Genomes (2018) 14:6

RAD-Seq analysis
The DNA purification procedure was identical to that describedby Penjor et al. (2014). The method used to create the libraryfor double-digest RAD-Seq was identical to that described by
Sakaguchi et al. (2015), which is a modification of the original
double-digest RAD-Seq (Peterson et al. 2012) as follows: BglII
was used as the first restriction site adjacent to the binding siteof the primer to read a single-end sequence, and EcoRI wasused as the second restriction site adjacent to the binding site toread an index sequence. The library was sequenced with 49 bp
single-end reads in one lane of an Illumina HiSeq2000(Illumina, San Diego, CA, USA) by BGI Hong Kong. At
BGI, the raw data were modified using the following two steps:(1) reads that were polluted by adapter sequences were deleted,and (2) reads that contained > 50% low-quality bases (quality
value ≤ 5) or > 10% N bases were removed.

Phylogenetic analysis based on the pyRAD program
Reads were further cleaned using the process_shortreads program of the Stacks package (version 1.46) with -c (clean data,
remove any read with an uncalled base) and -q (discard reads
with low-quality scores) options (Catchen et al. 2011, 2013).
For the construction of phylogenetic trees, multiple alignments
were created using the pyRAD program (version 3.0.66)(Eaton 2014). In multiple alignments c80m12, c85m12, andc90m12, Wclust (clustering threshold as a decimal) was set to0.80, 0.85, and 0.90, respectively, with Mindepth (min coverage for a cluster) = 6, NQual (max # sites with quality < 20) =4, MinCov (min samples in a final locus) = 12, and MaxSH(max inds with shared hetero site) = 3. In multiple alignments
c80m4, c85m4, and c90m4, Wclust was set to 0.80, 0.85, and0.90, respectively, with Mindepth = 6, NQual = 4, MinCov =4, and MaxSH = 3. In these calculations, the default parameterof maximum depth filtering was used. For all six multiplealignments, phylogenetic trees based on maximum likelihood
were constructed using the RAxML program (version 8.2.10)(Stamatakis 2014) (-f = a, -x = 12,345, -p = 12,345, -N (bootstrap value) = 1000, and -m = GTRGAMMA). For multiplealignments c80m12, c85m12, and c90m12, phylogenetic trees
based on Bayesian inference were constructed using the
MrBayes program (version 3.2.2) (Ronquist andHuelsenbeck 2003) (lset nst = 6, rates = invgamma, mcmc
ngen = 100,000, samplefreq = 1000, nchains = 4, andsavebrlens = yes). In each analysis, the midpoint was used asa root. The number of phylogenetically informative sites wascalculated using MEGA version 7 (Kumar et al. 2016).

Phylogenetic analysis based on the Stacks program
For the de novo analysis, the cleaned reads were analyzed
using the denovo_map.pl script of the Stacks package

Tree Genetics & Genomes (2018) 14:6
Table 1
Tribe

Page 3 of 11 6

Species used to analyze the genetic relationships within Aurantioideae
SubtribeGroup

Latin name (common name/accession name)

Clauseneae Micromelinae
Clauseninae

Citreae

Micromelum minutum (Forst.) Wt. & Arn.
Clausena anisata (Willd.) Hook. f.
Clausena harmandiana (Pierre) Guill.
Clausena lansium (Lour.) Skeels (wanpee)
Glycosmis citrifolia (Willd.) Lindl.
Glycosmis pentaphylla (Retz.) Correa (orangeberry)
Murraya koenigii (L.). Spreng. (curry tree)
Murraya paniculata (L.) Jack.
Merrillinae
Merrillia caloxylon (Ridl.) Swing.
Triphasiinae
Triphasia
Paramignya lobata Burkill
Triphasia trifolia (Burm. f.) P. Wils. f.
Balsamocitrinae Tabog
Swinglea glutinosa (Blanco) Merr
Bael fruit
Aegle marmelos (L.) Corr. (bael)
Afraegle paniculata (Schum.) Engl.
Wood apple
Feronia limonia (L.) Swing.
Feroniella oblata Swing.
Citrinae
Primitive citrus fruit trees Hesperethusa crenulata (Roxb.) Roem.
Near citrus fruit trees

True citrus fruit trees

Severinia buxifolia (Poir.) Tenore (Chinese box-orange)
Atalantia bilocularis (Roxb.) Wall. ex Skeels
Atalantia ceylanica (Arn.) Oliv.
Atalantia monophylla DC.
Atalantia roxburghiana Hook. f.
Atalantia spinosa (Willd.) Tanaka
Citropsis gabunensis (Engl.) Swing. & M. Kell.
Citropsis gilletiana Swing. & M. Kell.
Citropsis schweinfurthii (Engl.) Swing. & M. Kell.
Clymenia polyandra (Tan.) Swing.
Eremocitrus glauca (Lindl.) Swing.
Microcitrus australasica (F. Muell.) Swing.
Fortunella japonica (Thunb.) Swing. (round kumquat)
Poncirus trifoliata (L.) Raf. BStandard^ (trifoliate orange)
Citrus micrantha Wester (papeda/biasong)
Citrus medica L. (citron/Maru busshukan)
Citrus maxima (Burm.) Merr. (pummelo/Mato Buntan)
Citrus reticulata Blanco (mandarin/Yoshida Ponkan)(version 1.46) with the m (minimum number of identicalraw reads required to create a stack) option of 3. For thereference-based analysis, the cleaned reads were alignedwith the reference genomes using bowtie2 (Langmead andSalzberg 2012) with -q –no-unal -L 15 options, andaligned data were analyzed using the ref_map.pl scriptof the Stacks package with default parameters. As reference genomes, Citrus sinensis (sweet orange) (Xu et al.
2012) was used for the analysis of nine members of Btrue
citrus fruit^ trees, and Severinia buxifolia (Atalantia

SourceAccession no.

Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.

8650
8612
8613
8611
8601
8600
8622
8621
8640
8350
8500
8420
8400
8411
8450
8460
8320

Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Saga Univ.
Kagoshima
Univ.

8340
8312
8310
8314
8316
8315
8300
8302
8301
8280
8251
8203
8002
8100
7004
5001
3202
–

buxifolia) (Wang et al. 2017) was used for the analysis
of six members of Severinia and Atalantia. The populations program of the Stacks package was used to create
multiple alignments within the cluster using the options –phylip and –phylip_var. In this program, the -p (minimum
number of populations a locus must be present in to process a locus) option was set to 5 in the analysis of nine
members of Btrue citrus fruit trees^ and to 3 in the analysis of six members of Severinia and Atalantia. The phylogenetic trees were constructed as described above.

6

Page 4 of 11

Results
Phylogenetic trees of the subfamily Aurantioideae
We used double-digest RAD-Seq of 35 species to reveal the
relationships within the subfamily Aurantioideae (Table 1).
The library for double-digest RAD-Seq was created using
the restriction enzymes BglII and EcoRI. The region adjacentto BglII was sequenced with 49 bp single-end reads. Using thequality-filtered reads shown in Supplementary Table 1, the
PyRAD program created six multiple alignments by changing
the clustering thresholds (80, 85, or 90%) and number of samples (species) in a final locus (four or 12).
Based on these variables, the multiple alignments werenamed c80m4, c85m4, c90m4, c80m12, c85m12, andc90m12, and they contained 652,582; 641,156; 599,272;
189,271; 184,680; and 159,021 aligned sequences, respectively. The numbers of phylogenetically informative sites in multiple alignments c80m4, c85m4, c90m4, c80m12, c85m12,
and c90m12 were 32,999; 30,041; 19,717; 16,484; 15,258;
and 9979; respectively. Multiple alignments c80m4, c85m4,
and c90m4 extracted the locus conserved among only four
species; hence, they produced larger numbers of aligned sequences than did c80m12, c85m12, and c90m12, which extracted the locus conserved among 12 species. Analysis of thenucleotide compositions (Supplementary Tables 2–7) revealed that each alignment contained fewer heterozygous sites
and deletions than homologous sites.
Multiple alignments c80m12, c85m12, and c90m12 wereused to construct phylogenetic trees based on maximum likelihood (Supplementary Fig. 1, Supplementary Fig. 2, andFig. 1, respectively) and Bayesian inference (Supplementary
Fig. 3, Supplementary Fig. 4, and Fig. 2, respectively). Owing
to poor computer performance, c80m4, c85m4, and c90m4
were used to construct phylogenetic trees based on maximum
likelihood (Supplementary Fig. 5, Supplementary Fig. 6, andSupplementary Fig. 7, respectively) but not on Bayesian inference. It was difficult to include the RAD-Seq data of theoutgroup (distantly related species) in these phylogenetic analyses; hence, the midpoint was used as a root.
The obtained phylogenetic trees had similar topologies andgenerally shared several characteristics. (1) The subfamily
Aurantioideae was subdivided into two tribes—Clauseneae
and Citreae. (2) The tribe Citreae was subdivided into threesubtribes—Triphasiinae, Citrinae, and Balsamocitrinae. (3)The tribe Clauseneae was subdivided into three subtribes—
Micromelinae, Clauseninae, and Merrillinae—although
Murraya paniculata was an exception. (4) The subtribe
Balsamocitrinae was subdivided into three groups—wood apple, tabog, and bael fruit. (5) The subtribe Citrinae was
subdivided into three groups—a group containing the genera
Hesperethusa and Citropsis, a group containing the genera
Severinia and Atalantia, and the Btrue citrus fruit trees^—

Tree Genetics & Genomes (2018) 14:6

and the proposal to discriminate the two groups Bnear citrus
fruit trees^ and Bprimitive citrus fruit trees^ was not supported.
(7) BTrue citrus fruit trees^ formed a monophyletic group.
Comparison of the phylogenetic trees based on maximum
likelihood and Bayesian inference revealed no difference intree topology for each of the multiple alignments c80m12,
c85m12, and c90m12. However, comparison of the trees constructed using the six types of alignments (c80m4, c85m4, andc90m4; c80m12, c85m12, and c90m12) revealed some differences. (1) In all trees except the maximum-likelihood tree
based on c80m4 (Supplementary Fig. 5), Micromelinae wasan outgroup of a monophyletic clade containing the membersof Clauseninae; however, in the maximum-likelihood tree
based on c80m4 (Supplementary Fig. 5), Murraya koenigii(Bergera koenigii, commonly known as the curry tree) was
clustered together with Micromelinae (Micromelum
minutum). (2) In all trees except the maximum-likelihood trees
based on c90m4 (Supplementary Fig. 7), Murraya koenigii
was an outgroup of five species (two Glycosmis species andthree Clausena species); however, in the maximum-likelihood
tree based on c90m4 (Supplementary Fig. 7), two Glycosmis
species formed an outgroup of three Clausena species andMurraya koenigii, and Murraya koenigii was an outgroup of
three Clausena species. (3) In the maximum-likelihood trees
based on c90m12 and c90m4 (Fig. 1 and Supplementary
Fig. 7) and the Bayesian inference tree based on c90m12(Fig. 2), Triphasiinae was an outgroup of Balsamocitrinae
and Citrinae; however, in the maximum-likelihood trees basedon c80m12, c85m12, c80m4, and c85m4 (SupplementaryFigs. 1, 2, 5, and 6) and the Bayesian inference trees basedon c80m12 and c85m12 (Supplementary Figs. 3 and 4),
Balsamocitrinae was an outgroup of Triphasiinae andCitrinae. (4) In all trees except the maximum likelihood andBayesian inference trees based on c85m12 (SupplementaryFigs. 2 and 4), the branch containing wood apple, tabog, andCitrinae was subdivided into two sub-branches—one was
Citrinae, and the other consisted of wood apple and tabog;
in comparison, in the maximum likelihood and Bayesian inference trees based on c85m12 (Supplementary Figs. 2 and 4),
wood apple was an outgroup of the branch containing tabog
and Citrinae, and tabog was an outgroup of Citrinae. (5) There
was a conflict in the locations of Fortunella japonica (roundkumquat) and Citrus reticulata (mandarin); in all trees except
the maximum likelihood and Bayesian inference trees basedon c85m12 (Supplementary Figs. 2 and 4), these two species formed a single branch, and this branch was clustered
together with another branch containing Australian/New
Guinean Btrue citrus fruit trees^ (Clymenia, Eremocitrus,
and Microcitrus); in comparison, in the maximum likelihood and Bayesian inference trees based on c85m12(Supplementary Figs. 2 and 4), Citrus reticulata was an
outgroup of the branch containing Fortunella japonica
and Australian/New Guinean Btrue citrus fruit trees,^ andTree Genetics & Genomes (2018) 14:6

Page 5 of 11 6

Fig. 1 Phylogenetic tree based on maximum likelihood analysis of 35
species of the subfamily Aurantioideae. Numbers at the nodes indicate
bootstrap values (% over 1000 replicates). The scale bar shows thenumber of substitutions per site. The phylogenetic tree was calculated

based on multiple alignment c90m12. The multiple alignment wascalculated using the PyRAD program, with Wclust (clustering threshold
as a decimal) set to 0.90 and MinCov (min samples in a final locus) set to12Fortunella japonica was an outgroup of the branch containing Australian/New Guinean Btrue citrus fruit trees.^or hybrids of these four species (Nicolosi et al. 2000;
Froelicher et al. 2011; Garcia-Lor et al. 2013; Curk et al.
2014, 2015). We used these four species in our subsequent
analysis. One of the multiple alignments was created using ade novo method, and the other multiple alignment was createdby aligning the data to the reference genome of Citrus sinensis(sweet orange) (Xu et al. 2012). The former and latter calculations extracted 10,783 and 11,143 sites, respectively, and theseincluded 1003 and 1375 phylogenetically informative sites,
respectively. The nucleotide compositions of these alignments
are shown in Supplementary Tables 8 and 9. We constructed
phylogenetic trees based on maximum likelihood (Fig. 3) andBayesian inference (Supplementary Fig. 8). Comparison of thephylogenetic trees based on maximum likelihood andBayesian inference revealed no difference in the tree topology
for each multiple alignment. However, comparison of the trees
constructed using the two types of multiple alignment revealed
some differences in the locations of Fortunella japonica(round kumquat) and Citrus reticulata. In the trees based on
the de novo method, Fortunella japonica was an outgroup ofAustralian/New Guinean Btrue citrus fruit trees,^ and Citrus

Phylogenetic trees of closely related species
The pyRAD program is useful for creating multiple alignmentsof distantly related samples (Eaton 2014), and the HardyWeinberg principle is not expected to apply. However, in theanalysis of closely related samples such as cross-compatible
species, the Hardy-Weinberg principle should be considered.
The Stacks program is useful for analyzing the RAD-Seq dataof closely related samples (Catchen et al. 2011, 2013), and the
Hardy-Weinberg principle is expected to apply. The membersof Btrue citrus fruit trees^ (Citrus, Fortunella, Poncirus,
Eremocitrus, Microcitrus, and Clymenia) are closely related,and cross-compatibility was observed (Iwamasa et al. 1988).
Therefore, we analyzed the RAD-Seq data of Btrue citrus fruit
trees^ using the Stacks program. Citrus medica (citron), Citrus
micrantha (papeda), Citrus maxima (pummelo), and Citrus
reticulata (mandarin) are considered to be ancestral species,
and most other Citrus species are considered to be derivatives

6

Tree Genetics & Genomes (2018) 14:6

Page 6 of 11

Fig. 2 Phylogenetic tree based on Bayesian inference analysis of 35 species of the subfamily Aurantioideae. Numbers at the nodes indicate posterior
probabilities. The scale bar shows the number of substitutions per site. The phylogenetic tree was calculated based on multiple alignment c90m12

reticulata was an outgroup of the remaining three Citrus species (Fig. 3a and Supplementary Fig. 8A). However, in thetrees based on reference genome-based method, Fortunellajaponica and Citrus reticulata formed a single branch, and thisbranch was clustered together with another branch containing
Australian/New Guinean Btrue citrus fruit trees^; this finding
was consistent with most of the pyRAD-based phylogenetic
trees, except the maximum likelihood and Bayesian inference
trees based on c85m12 (Supplementary Figs. 2 and 4).
The pyRAD-based trees showed that Severinia buxifolia(Atalantia buxifolia) and five species of Atalantia (Atalantia
bilocularis, Atalantia ceylanica, Atalantia monophylla,
Atalantia roxburghiana, and Atalantia spinosa) are closely
related. Therefore, we analyzed the RAD-Seq data of thesesix species using the Stacks program, and we produced two
types of multiple alignments using the de novo and reference
genome-based methods. As a reference genome, we used the
genome sequence of Severinia buxifolia (Atalantia buxifolia)(Wang et al. 2017). The former and latter calculations extracted 7958 and 6836 sites, respectively, and these included 1479

and 1810 phylogenetically informative sites, respectively. The
nucleotide compositions of these alignments are shown in
Supplementary Tables 10 and 11. We constructed phylogenetic trees based on maximum likelihood (Supplementary Fig. 9)
and Bayesian inference (Supplementary Fig. 10). Comparisonof the phylogenetic trees based on maximum likelihood andBayesian inference revealed no difference in the topology of
the trees for each multiple alignment. Furthermore, there wasno conflict between the pyRAD-based trees and the Stacksbased trees.

Discussion
Relationships between the tribe Clauseneae
and the tribe Citreae
The trees constructed in our present study clearly discriminate
Citreae from Clauseneae, supporting previous molecular studies (Bayer et al. 2009; Penjor et al. 2013; Schwartz et al.

Tree Genetics & Genomes (2018) 14:6

2015). These previous studies showed that members ofClauseneae formed an outgroup of Citreae, and the membersof Clauseneae did not belong to the clade Citreae; the root was
located between the clade containing Murraya paniculata andMerrillia caloxylon and the clade containing the other members of Clauseneae. The root of the trees constructed in ourpresent study may be incorrect, because we did not include theoutgroup in the RAD-Seq analysis; hence, we placed the rootat the midpoint. In some previous studies, the polytomousclade containing Murraya paniculata and Merrillia caloxylon
was included in the tribe Citreae (Samuel et al. 2001; Morton
et al. 2003; Morton 2009; Oueslati et al. 2016); however, ourpresent study and other previous studies (Bayer et al. 2009;
Penjor et al. 2013; Schwartz et al. 2015) do not support theseobservations.

Page 7 of 11 6aRelationships within the tribe ClauseneaebClassical morphology subdivided the tribe Clauseneae into
three subtribes—Micromelinae, Clauseninae, and Merrillinae(Swingle and Reece 1967). In the present study, we analyzed
one member of Micromelinae, (Micromelum minutum), one
member of Merrillinae (Merrillia caloxylon; monotypic genus), and seven members of Clauseninae (two Murraya species, two Glycosmis species, and three Clausena species).
Without taking into consideration the position of Murraya
paniculata, in all trees except the maximum-likelihood tree
based on c80m4 (Supplementary Fig. 5), our molecular classification subdivides the tribe Clauseneae into three subtribes—Merrillinae is an outgroup of Micromelinae andClauseninae, and Micromelinae is an outgroup of monophyletic clade containing the members of Clauseninae; however,in the maximum-likelihood tree based on c80m4(Supplementary Fig. 5), Murraya koenigii (Bergera koenigii)
is clustered together with Micromelinae (Micromelum
minutum), although the support value is low. In previous molecular studies (Bayer et al. 2009; Penjor et al. 2013; Schwartz
et al. 2015), Micromelum minutum was nested in the subtribe
Clauseninae. Thus, our present molecular classification is consistent with the morphological classification (Swingle andReece 1967), in which the three subtribes were separated.
However, in the present study, we analyzed only one speciesof Micromelinae; hence, further studies with additional species are required.
Next, we compared our present classification within thetribe Clauseneae with that of our previous molecular study(Penjor et al. 2013). Some findings are consistent as follows:(1) three species of the genus Clausena—Clausena anisata,
Clausena harmandiana, and Clausena lansium (wanpee)—belong to a monophyletic clade; (2) two species of the genus
Glycosmis—Glycosmis citrifolia and Glycosmis pentaphylla(orangeberry)—belong to a monophyletic clade; and (3)Murraya paniculata is clustered with Merrillia caloxylon

Fig. 3 Phylogenetic trees based on maximum likelihood analysis of ninespecies of Btrue citrus fruit trees.^ Numbers at the nodes indicate bootstrap
values (% over 1000 replicates). The scale bar shows the number ofsubstitutions per site. The phylogenetic trees were calculated based on
multiple alignment created using de novo analysis (a) or reference
genome-based analysis (b) of the Stacks package

but not with Murraya koenigii. Similar groupings were observed in other previous studies (Samuel et al. 2001; Bayeret al. 2009; Schwartz et al. 2015); however, the species used inthese previous studies differed from those used in our present
and previous studies (Penjor et al. 2013).
It is interesting to consider the phylogenetic positions of thegenus Murraya. Genetic similarities between Murraya
paniculata and Merrillia caloxylon, and genetic differences
between Murraya paniculata and Murraya koenigii were described previously (Samuel et al. 2001; Bayer et al. 2009;
Penjor et al. 2013; Oueslati et al. 2016). Morphological similarities between Murraya paniculata and Merrillia caloxylon,
and morphological differences between Murraya paniculata
and Murraya koenigii, were discussed in our previous study

6Page 8 of 11(Penjor et al. 2013). Furthermore, previous phytochemical
analysis revealed similarities between Merrillia caloxylon
and some Murraya species, including Murraya paniculata
and Murraya exotica (Samuel et al. 2001). In addition, genetic
and karyotypic analyses showed that Murraya koenigii and
Murraya siamensis were isolated from the other Murraya species (Guerra et al. 2000). Our present study confirms theseobservations, i.e., Murraya paniculata should be groupedwith Merrillia caloxylon.
In our previous study (Penjor et al. 2013), the topology ofthe branch containing seven trees (three Clausena species, two
Glycosmis species, Micromelum minutum, and Murraya
koenigii) differed between neighbor-joining and maximumlikelihood trees. Furthermore, the topology of these fourgroups differed between several previous studies (Samuelet al. 2001; Bayer et al. 2009; Penjor et al. 2013). Our present
analysis shows that in all phylogenetic trees except the
maximum-likelihood trees based on c80m4 and c90m4(Supplementary Figs. 5 and 7), Micromelum minutum is an
outgroup of the remaining six species, and Murraya koenigii
is the outgroup of the clade containing two Glycosmis species
and three Clausena species, However, low support values arepresent in the clade containing these seven species. Therefore,
the branching order within the clade should be interpretedwith caution.
In comparison with our previous study (Penjor et al. 2013),
our present analysis more clearly elucidates the genetic relationships within the tribe Clauseneae. In the clade containing
three Clausena species, Clausena lansium is an outgroup of
the sub-clade containing Clausena anisata and Clausena
harmandiana; this was not clear in our previous study(Penjor et al. 2013).

Relationships within the tribe Citreae
Classical morphology subdivided the tribe Citreae into threesubtribes—Triphasiinae (minor citroid fruit trees), Citrinae(citrus fruit trees), and Balsamocitrinae (hard-shelled citroid
fruit trees) (Swingle and Reece 1967). Our present molecularstudy clearly discriminates these three subtribes as follows: (1)each of the subtribes Triphasiinae and Citrinae forms a monophyletic group, and (2) Balsamocitrinae does not form amonophyletic group and is an outgroup of Triphasiinae andCitrinae (Supplementary Figs. 1–6), or Triphasiinae is an
outgroup of Balsamocitrinae and Citrinae (Figs. 1 and 2, andSupplementary Fig. 7). Thus, our present analysis corresponds
well with the morphological classification (Swingle andReece 1967). Previous molecular analysis (Bayer et al.
2009; Penjor et al. 2013) was unable to elucidate the relationships among the three subtribes. For example, members of
Balsamocitrinae, Feronia limonia, Feroniella oblata, andSwinglea glutinosa were nested in the subtribe Citrinae(Bayer et al. 2009; Penjor et al. 2013). In a recent study

Tree Genetics & Genomes (2018) 14:6

(Schwartz et al. 2015), the monophyletic nature ofTriphasiinae was clearly shown; however, a member ofBalsamocitrinae (Swinglea) and a member of Citrinae(Citropsis) belonged to a single clade. Thus, our present analysis corresponds well with the morphologicalcharacterization.
Classical morphology subdivided the subtribe
Balsamocitrinae into three groups—wood apple, tabog(monotypic genus), and bael fruit (Swingle and Reece
1967). Our present analysis clearly separates these threegroups and corresponds well with the morphological observations (Swingle and Reece 1967). In all phylogenetic trees, baelfruit is an outgroup of a branch containing the subtribe
Citrinae and the other subtribes of Balsamocitrinae (woodapple and tabog). In all phylogenetic trees, except the maximum likelihood and Bayesian inference trees based on
c85m12 (Supplementary Figs. 2 and 4), this branch is
subdivided into two sub-branches—one is Citrinae, and theother consists of wood apple and tabog. In the maximum
likelihood and Bayesian inference trees based on c85m12(Supplementary Figs. 2 and 4), wood apple is an outgroupof the branch containing tabog and Citrinae, and tabog is an
outgroup of Citrinae.
Consistent with previous studies (Bayer et al. 2009; Penjor
et al. 2013), our present study shows that Aegle marmelos(Bael) and Afraegle paniculata—known as the Bael fruit
group of the subtribe Balsamocitrinae—form a single cluster.
It is important to note that Aegle marmelos is native to India,
and Afraegle paniculata is native to tropical Africa (Swingle
and Reece 1967). Our present study further shows that
Feronia limonia and Feroniella oblata, known as the woodapple group of the subtribe Balsamocitrinae (Swingle andReece 1967), belong to the same cluster; this observation isconsistent with the previous results (Morton et al. 2003;
Morton 2009; Penjor et al. 2010, 2013). Feroniella oblata is
not nested in Btrue citrus fruit trees,^ and this observation isinconsistent with the previous finding (Bayer et al. 2009).
Classical morphology subdivided the subtribe Citrinae into
three groups—Btrue citrus fruit trees,^ near citrus fruit trees,and primitive citrus fruit trees (Swingle and Reece 1967).
Members of the near citrus fruit trees were proposed to benearer to Citrus species than to the two subtribes
Balsamocitrinae and Triphasiinae; this observation is supported by our present analysis. Our present study shows that members of the primitive citrus fruit trees—Severinia buxifolia(Atalantia buxifolia, commonly known as Chinese box-orange) and Hesperethusa crenulata—are not clustered, andeach is nested in near citrus fruit trees. This observation isinconsistent with the morphological classification (Swingle
and Reece 1967).
Our present study shows that Severinia buxifolia (Atalantia
buxifolia) and five species of Atalantia (Atalantia bilocularis,
Atalantia ceylanica, Atalantia monophylla, Atalantia

Tree Genetics & Genomes (2018) 14:6

roxburghiana, and Atalantia spinosa) form a monophyletic
group; this finding is consistent with our previous observation(Penjor et al. 2013) and similar to those of other previous
studies (Morton et al. 2003; Bayer et al. 2009; Morton 2009;
Schwartz et al. 2015). In our present study, Hesperethusa
crenulata (Naringi crenulata) and three species of the genus
Citropsis (Citropsis gabunensis, Citropsis gilletiana, andCitropsis schweinfurthii) form a single cluster. Similar relationships between Hesperethusa and Citropsis were observedin previous studies (Morton et al. 2003; Bayer et al. 2009;
Morton 2009; Penjor et al. 2010, 2013; Schwartz et al.
2015). It is important to note that Citropsis is native toAfrica, and Hesperethusa crenulata is native to Southeast
Asia (Swingle and Reece 1967). Thus, although Severinia
buxifolia and Hesperethusa crenulata are members of primitive citrus fruit trees (Swingle and Reece 1967), our present
analysis shows that Severinia buxifolia is closely related to
Atalantia species, and Hesperethusa crenulata is closely related to Citropsis species.
Our present analysis shows that, in Citrinae, the group containing three Citropsis species with Hesperethusa crenulata as
an outgroup, and the group containing five Atalantia species
and Severinia buxifolia, are distinct from Btrue citrus fruit
trees^; in previous studies, it was difficult to detect these relationships (Morton et al. 2003; Bayer et al. 2009; Morton 2009;
Penjor et al. 2010, 2013; Oueslati et al. 2016).

Relationships within Btrue citrus fruit trees^
In the present study, the most economically important group,
Btrue citrus fruit trees,^ forms a monophyletic group and isclearly separated from the other species. With the exceptionof the relationships between Citrus reticulata (mandarin) and
Fortunella japonica (round kumquat) (compare Fig. 3a andSupplementary Figs. 2, 4, and 8A with Figs. 1, 2, and 3b, andSupplementary Figs. 1, 3, 5–7, and 8B), similar topologies
were obtained in all the phylogenetic trees. Further studies todetermine the precise locations of Citrus reticulata andFortunella japonica are required.
In our present analysis, we mainly used nuclear markers,
because the small portion of the whole genome is chloroplastor mitochondrial, and the maximum depth filtering in the
pyRAD program can remove these organellar sequences. In
concordance with a previous study (Schwartz et al. 2015)
using nuclear and chloroplast markers, our analysis showedthat Poncirus trifoliata (trifoliate orange) is an outgroup of theremaining Btrue citrus fruit trees.^ In previous studies using
chloroplast markers (Bayer et al. 2009; Penjor et al. 2013) orwhole chloroplast genome (Carbonell-Caballero et al. 2015),
Poncirus trifoliata was not an outgroup of the remaining Btrue
citrus fruit trees^; however, the analysis based on whole chloroplast genome indicated that the paternal parent of Poncirus
trifoliata may be a non-citrus plant (Carbonell-Caballero et al.

Page 9 of 11 62015). Therefore, paternal parentage of Poncirus trifoliata
contributes to the results, indicating that Poncirus trifoliata
is an outgroup of the remaining Btrue citrus fruit trees.^
Clymenia, Eremocitrus, and Microcitrus are native toAustralia/New Guinea, and the previous study discriminatedthese genera from Asian citrus trees, including Citrus,
Poncirus, and Fortunella (Penjor et al. 2013). However, ourprevious molecular analysis (Penjor et al. 2013) shows thatthese Australian/New Guinean citrus trees do not form a single clade—Clymenia was isolated from Eremocitrus,
Microcitrus, and Asian citrus trees. In our present analysis,
Clymenia, Eremocitrus, and Microcitrus form a monophyleticclade. In other previous studies, Clymenia, Eremocitrus, andMicrocitrus also formed a monophyletic clade, although
Citrus medica was nested in this clade (Bayer et al. 2009;
Schwartz et al. 2015). Geographic separation may explain
the genetic separation between Asian and Australian/New
Guinean citrus trees.
In concordance with a previous study (Schwartz et al.
2015) using nuclear and chloroplast markers, our present analysis, mainly using nuclear markers, shows that Australian/
New Guinean citrus trees are not an outgroup of Asian citrus
trees. In contrast, in previous studies using chloroplast
markers (Bayer et al. 2009; Penjor et al. 2013) or whole chloroplast genome (Carbonell-Caballero et al. 2015), Australian/
New Guinean citrus trees were an outgroup of Asian citrus
trees. Species belonging to five genera (Citrus, Fortunella,Poncirus, Eremocitrus, and Microcitrus) can cross-hybridize(Iwamasa et al. 1988). Cross-compatible features of these genera may contribute to the inconsistency between nuclearbased and chloroplast-based results. One possible explanation
for this inconsistency is chloroplast capture—the introgression of a chloroplast from one species into another(Rieseberg and Soltis 1991; Tsitrone et al. 2003). Probably,
ancestors of Australia/New Guinean citrus trees captured thechloroplast genome from ancestors of Asian citrus trees
through inter-species hybridization and subsequent
backcrosses.

Benefits and limitations of this study
In comparison with previously published studies using small
numbers of markers or short DNA sequences, RAD-Seq data
produced phylogenetic trees of the subfamily Aurantioideae
with higher support values, clear discriminations based on
longer branch length, and elucidations of earlier branching
events. Although this is a benefit of our study, there were also
some limitations. We obtained several types of multiple alignments, and there were some discrepancies between different
types of alignment. Currently, there is no means of generalizing the method used to create multiple alignments. As researchbased on large data size becomes more popular, improved
techniques for analyzing these types of data will be required.

6

Page 10 of 11

Additional limitations related to RAD-Seq have been
discussed previously (e.g., Andrews et al. 2016).
Acknowledgements We thank Ms. Akiko Sakai for technical assistance.
Part of this work was supported by a Grant-in-Aid for Scientific Research(26450039 to Y.N., 19405019 to M.Y) from the Japan Society for the
Promotion of Science. The RAD-Seq analysis was supported by the Joint
Usage/Research Program of the Center for Ecological Research, Kyoto
University. We would like to thank Editage for English language editing.
Author contributions Y.N., N.K., R.M., and M.Y. designed the research;
T.M., N.K., R.M., and M.Y. preserved the plants; T.M. extracted theDNA; A.N., M.H., and H.K. prepared the RAD-Seq library; Y.N. performed the bioinformatic analysis; Y.N., N.K., R.M., and M.Y. analyzed
the results; Y.N. wrote the manuscript; and N.K., R.M., and M.Y. revised
the manuscript; all authors approved the final version to be published.

Compliance with ethical standards
Conflict of interest The authors declare that they have no competinginterests.

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Data archiving statement
Sequences are available at the DDBJ Sequence Read Archive (http://
trace.ddbj.nig.ac.jp/dra/index_e.shtml; Accession no. DRA005954).
The Citrus species data were used in another study, and the accession
number is DRA004200 (Penjor et al. 2016).