The genus Citrus,
comprising some of the most widely cultivated fruit crops worldwide,
includes an uncertain number of species. Here we describe ten natural
citrus species, using genomic, phylogenetic and biogeographic analyses
of 60 accessions representing diverse citrus germ plasms, and propose
that citrus diversified during the late Miocene epoch through a rapid
southeast Asian radiation that correlates with a marked weakening of the
monsoons. A second radiation enabled by migration across the Wallace
line gave rise to the Australian limes in the early Pliocene epoch.
Further identification and analyses of hybrids and admixed genomes
provides insights into the genealogy of major commercial cultivars of
citrus. Among mandarins and sweet orange, we find an extensive network
of relatedness that illuminates the domestication of these groups.
Widespread pummelo admixture among these mandarins and its correlation
with fruit size and acidity suggests a plausible role of pummelo
introgression in the selection of palatable mandarins. This work
provides a new evolutionary framework for the genus Citrus.
Main
The genus Citrus and related genera (Fortunella, Poncirus, Eremocitrus and Microcitrus)
belong to the angiosperm subfamily Aurantioideae of the Rutaceae
family, which is widely distributed across the monsoon region from west
Pakistan to north-central China and south through the East Indian
Archipelago to New Guinea and the Bismarck Archipelago, northeastern
Australia, New Caledonia, Melanesia and the western Polynesian islands1. Native habitats of citrus and related genera roughly extend throughout this broad area (Extended Data Fig. 1a and Supplementary Table 1),
although the geographical origin, timing and dispersal of citrus
species across southeast Asia remain unclear. A major obstacle to
resolving these uncertainties is our poor understanding of the genealogy
of complex admixture in cultivated citrus, as has recently been shown2. Some citrus are clonally propagated apomictically3
through nucellar embryony, that is, the development of non-sexual
embryos originating in the maternal nucellar tissue of the ovule, and
this natural process may have been co-opted during domestication;
grafting is a relatively recent phenomenon4.
Both modes of clonal propagation have led to the domestication of fixed
(desirable) genotypes, including interspecific hybrids, such as
oranges, limes, lemons, grapefruits and other types.
Under this scenario, it is not surprising that the current chaotic citrus taxonomy—based on long-standing, conflicting proposals5,6—requires
a solid reformulation consistent with a full understanding of the
hybrid and/or admixture nature of cultivated citrus species. Here we
analyse genome sequences of diverse citrus to characterize the diversity
and evolution of citrus at the species level and identify citrus
admixtures and interspecific hybrids. We further examine the network of
relatedness among mandarins and sweet orange, as well as the pattern of
the introgression of pummelos among mandarins for clues to the early
stages of citrus domestication.
Diversity and evolution of the genus Citrus
To
investigate the genetic diversity and evolutionary history of citrus,
we analysed the genomes of 58 citrus accessions and two outgroup genera (Poncirus and Severinia) that were sequenced to high coverage, including recently published sequences2,3,7
as well as 30 new genome sequences described here. For our purpose, we
do not include accessions related by somatic mutations. These sequences
represent a diverse sampling of citrus species, their admixtures and
hybrids (Supplementary Tables 2, 3 and Supplementary Notes 1, 2).
Our collection includes accessions from eight previously unsequenced
and/or unexamined citrus species, such as pure mandarins (Citrus reticulata), citron (Citrus medica), Citrus micrantha (a wild species from within the subgenus Papeda), Nagami kumquat (Fortunella margarita, also known as Citrus japonica var. margarita), and Citrus ichangensis (also known as Citrus cavaleriei; this species is also considered a Papeda), as well as three Australian citrus species (Supplementary Notes 3, 4). For each species, we have sequenced one or more pure accessions without interspecific admixture.
Local
segmental ancestry of each accession can be delineated for both admixed
and hybrid genotypes, based on genome-wide ancestry-informative
single-nucleotide polymorphisms (Supplementary Note 5). Comparative genome analysis further identified shared haplotypes among the accessions (Supplementary Notes 6, 7).
In particular, we demonstrate the F1 interspecific hybrid nature of
Rangpur lime and red rough lemon (two different mandarin–citron
hybrids), Mexican lime (a micrantha–citron hybrid) and calamondin (a
kumquat–mandarin hybrid), and confirm, using whole-genome sequence data,
the origins of grapefruit (a pummelo–sweet orange hybrid), lemon (a
sour orange–citron hybrid) and eremorange (a sweet orange and Eremocitrus glauca (also known as Citrus glauca)
hybrid). We also verified the parentage of Cocktail grapefruit, with
low-acid pummelo as the seed parent and King and Dancy mandarins as the
two grandparents on the paternal side. The origin of the Ambersweet
orange is similarly confirmed to be a mandarin–sweet orange hybrid with
Clementine as a grandparent. We have previously shown that sour orange
(cv. Seville) (Citrus aurantium) is a pummelo–mandarin hybrid, and have analysed the more complex origin of sweet orange (Citrus sinensis)2. Re-analysing sequences from ten cultivars of sweet orange3 shows that they are all derived from the same genome by somatic mutations, and were thus not included in our study.
We identified ten progenitor citrus species (Supplementary Note 4.1) by combining diversity analysis (Extended Data Table 1), multidimensional scaling and chloroplast genome phylogeny (Extended Data Fig. 1b). The first two principal coordinates in the multidimensional scaling (Fig. 1a) separate three ancestral (sometimes called ‘fundamental’) Citrus species associated with commercially important types8,9—citrons (C. medica), mandarins (C. reticulata) and pummelos (Citrus maxima)—and
display lemons, limes, oranges and grapefruits as hybrids involving
these three species. The nucleotide diversity distributions (Fig. 1b)
show distinct scales for interspecific divergence and intraspecific
variation, and reflect the genetic origin of each accession. Hybrid
accessions (sour orange, calamondin, lemon and non-Australian limes)
with ancestry from two or more citrus species are readily identified on
the basis of their higher segmental heterozygosity (1.5–2.4%) relative
to intraspecific diversity (0.1–0.6%). Other citrus accessions show
bimodal distributions in heterozygosity (sweet orange, grapefruits and
some highly heterozygous mandarins) due to interspecific admixture, a
process that generally involves complex backcrosses. Among the pure
genotypes without interspecific admixture, citrons show significantly
lower intraspecific diversity (around 0.1%) than the other species
(0.3–0.6%). The reduced heterozygosity of citrons, a mono-embryonic
species, is probably due to the cleistogamy of its flowers10, a mechanism that promotes pollination and self-fertilization in unopened flower buds, which in turn reduces heterozygosity.
Figure 1: Genetic structure, heterozygosity and phylogeny of Citrus species.
a,
Principal coordinate analysis of 58 citrus accessions based on pairwise
nuclear genome distances and metric multidimensional scaling. The first
two axes separate the three main citrus groups (citrons, pummelos and
mandarins) with interspecific hybrids (oranges, grapefruit, lemon and
limes) situated at intermediate positions relative to their parental
genotypes. b, Violin plots of the heterozygosity distribution in
58 citrus accessions, representing 10 taxonomic groups as well as 2
related genera, Poncirus (Poncirus trifoliata, also known as Citrus trifoliata) and Chinese box orange (Severinia).
White dot, median; bar limits, upper and lower quartiles; whiskers,
1.5× interquartile range. The bimodal separation of intraspecies (light
blue) and interspecies (light pink) genetic diversity is manifested
among the admixed mandarins and across different genotypes including
interspecific hybrids. Three-letter codes are listed in parenthesis with
additional descriptions in Supplementary Table 2. c,
Chronogram of citrus speciation. Two distinct and temporally
well-separated phases of species radiation are apparent, with the
southeast Asian citrus radiation followed by the Australian citrus
diversification. Age calibration is based on the citrus fossil C. linczangensis16
from the Late Miocene (denoted by a filled red circle). The 95%
confidence intervals are derived from 200 bootstraps. Bayesian posterior
probability is 1.0 for all nodes. d, Proposed origin of citrus
and ancient dispersal routes. Arrows suggest plausible migration
directions of the ancestral citrus species from the centre of origin—the
triangle formed by northeastern India, northern Myanmar and
northwestern Yunnan. The proposal is compatible with citrus
biogeography, phylogenetic relationships, the inferred timing of
diversification and the paleogeography of the region, especially the
geological history of Wallacea and Japan. The red star marks the fossil
location of C. linczangensis. Citrus fruit images in c and d are not drawn to scale.
The
identification of a set of pure citrus species provides new insights
into the phylogeny of citrus, their origins, evolution and dispersal.
Citrus phylogeny is controversial1,5,6,11,12,
in part owing to the difficulty of identifying pure or wild progenitor
species, because of substantial interspecific hybridization that has
resulted in several clonally propagated and cultivated accessions. Some
authors assign separate binomial species designations to clonally
propagated genotypes1,6.
Our nuclear genome-based phylogeny, which is derived from 362,748
single-nucleotide polymorphisms in non-genic and non-pericentromeric
genomic regions, reveals that citrus species are a monophyletic group
and establishes well-defined relationships among its lineages (Fig. 1c and Supplementary Note 8). Notably, the nuclear genome-derived phylogeny differs in detail from the chloroplast-derived phylogeny (Extended Data Fig. 1).
This is not unexpected, as chloroplast DNA is a single, non-recombining
unit and is unlikely to show perfect lineage sorting during rapid
radiation (Supplementary Note 8.3).
The origin of citrus has generally been considered to be in southeast Asia1, a biodiversity hotspot13 with a climate that has been influenced by both east and south Asian monsoons14 (Supplementary Note 9). Specific regions include the Yunnan province of southwest China15, Myanmar and northeastern India in the Himalayan foothills1. A fossil specimen from the late Miocene epoch of Lincang in Yunnan, Citrus linczangensis16,
has traits that are characteristic of current major citrus groups, and
provides definite evidence for the existence of a common Citrus ancestor within the Yunnan province approximately 8 million years ago (Ma).
Our analysis establishes a relatively rapid Asian radiation of citrus species in the late Miocene (6–8 Ma; Fig. 1c, d), a period coincident with an extensive weakening of monsoons and a pronounced climate transition from wet to drier conditions17. In southeast Asia, this marked climate alteration caused major changes in biota, including the migration of mammals18 and rapid radiation of various plant lineages19,20. Australian citrus species form a distinct clade that was proposed to be nested with citrons12, although distinct generic names (Eremocitrus and Microcitrus) were assigned in botanical classifications by Swingle1,5. Both molecular dating analysis21 and our whole-genome phylogenetic analysis do not support an Australian origin for citrus22.
Rather, citrus species spread from southeast Asia to Australasia,
probably via transoceanic dispersals. Our genomic analysis indicates
that the Australian radiation occurred during the early Pliocene epoch,
around 4 Ma. This is contemporaneous with other west-to-east angiosperm
migrations from southeast Asia23,24, presumably taking advantage of the elevation of Malesia and Wallacea in the late Miocene and Pliocene25,26 (Supplementary Note 9).
The
nuclear and chloroplast genome phylogenies indicate that there are
three Australian species in our collection. One of the two Australian
finger limes shows clear signs of admixture with round limes (Supplementary Note 5.4). The closest relative to Australian citrus is Fortunella, a species that has been reported to grow in the wild in southern China27.
Australian citrus species are diverse, and found natively in both dry
and rainforest environments in northeast Australia, depending on the
species28.
Our phylogeny shows that the progenitor citrus probably migrated across
the Wallace line, a natural barrier for species dispersal from
southeast Asia to Australasia, and later adapted to these diverse
climates.
The results also show that the Tachibana mandarin, naturally found in Taiwan, the Ryukyu archipelago and Japan29, split from mainland Asian mandarins (Fig. 1c, d) during the early Pleistocene (around 2 Ma), a geological epoch with strong glacial maxima30. Tachibana, as did other flora and fauna in the region, very probably arrived in these islands from the adjacent mainland31 during the drop in the sea level of the South China Sea and the emergence of land bridges32,33, a process promoted by the expansion of ice sheets that repetitively occurred during glacial maxima (Supplementary Note 9).
Although Tachibana5,6 has been assigned its own species (Citrus tachibana), sequence analysis reveals that it has a close affinity to C. reticulata34,35 and does not support its taxonomic position as a separate species (Supplementary Note 4.1). However, both chloroplast genome phylogeny (Extended Data Fig. 1b) and nuclear genome clustering (Fig. 1a)
clearly distinguish Tachibana from the mainland Asian mandarins. This
suggests that Tachibana should be designated a subspecies of C. reticulata. By contrast, the wild Mangshan ‘mandarin’ (Citrus mangshanensis)7 represents a distinct species, with comparable distances to C. reticulata, pummelo and citron2 (Extended Data Table 1).
Pattern of pummelo admixture in the mandarins
Using 588,583 ancestry-informative single-nucleotide polymorphisms derived from three species, C. medica, C. maxima and C. reticulata, we delineate the segmental ancestry of 46 citrus accessions (Extended Data Fig. 2 and Supplementary Note 5).
Pummelo admixture is found in all but 5 of the 28 sequenced mandarins,
and the amount and pattern of pummelo admixture, as identified by phased
pummelo haplotypes (Fig. 2a and Supplementary Note 6), suggests the classification of the mandarins into three types.
Figure 2: Admixture proportion and citrus genealogy.
a, Allelic proportion of five progenitor citrus species in 50 accessions. CI, C. medica; FO, Fortunella; MA, C. reticulata; MC, C. micrantha; PU, C. maxima;
UNK, unknown. The pummelos and citrons represent pure citrus species,
whereas in the heterogeneous set of mandarins, the degree of pummelo
introgression subdivides the group into pure (type-1) and admixed
(type-2 and -3) mandarins. Three-letter code as in Fig. 1, see Supplementary Table 2 for details. b,
Genealogy of major citrus genotypes. The five progenitor species are
shown at the top. Blue lines represent simple crosses between two
parental genotypes, whereas red lines represent more complex processes
involving multiple individuals, generations and/or backcrosses. Whereas
type-1 mandarins are pure species, type-2 (early-admixture) mandarins
contain a small amount of pummelo admixture that can be traced back to a
common pummelo ancestor (with P1 or P2 haplotypes). Later, additional
pummelo introgressions into type-2 mandarins gave rise to both type-3
(late-admixture) mandarins and sweet orange. Further breeding between
sweet orange and mandarins or within late-admixture mandarins produced
additional modern mandarins. Fruit images are not to scale and represent
the most popular citrus types. See Supplementary Note 1.1 for nomenclature usage.
Type-1 mandarins represent pure C. reticulata with no evidence of interspecific admixture and include Tachibana, three unnamed Chinese mandarins (M01, M02, M04)3
and the ancient Chinese cultivar Sun Chu Sha Kat reported here, a small
tart mandarin commonly grown in China and Japan, and also found in
Assam. This cultivar is likely described in Han Yen-Chih’s ad 1178 monograph ‘Chü Lu’36, which includes references to citrus cultivated during the reign of Emperor Ta Yu (2205–2197 bc).
Sixteen of the twenty-eight mandarins belong to type-2 mandarins, which
have a small amount of pummelo admixture (1–10% of the length of the
genetic map; Fig. 2a),
usually in the form of a few short segments distributed across the
genome. Although the lengths and locations of these admixed segments may
be distinct in different mandarins, they share one or two common
pummelo haplotypes (designated as P1 and P2) (Extended Data Fig. 3). By contrast, the seven remaining mandarins (type-3) contain higher proportions of pummelo alleles (12–38%; Fig. 2a)
in longer segments. Although the P1 and P2 pummelo haplotypes are also
detectable among type-3 mandarins, other more extensive pummelo
haplotypes dominate the pummelo admixture in type-3 mandarins (Fig. 2b and Extended Data Table 2).
These
observations suggest that the initial pummelo introgression into the
mandarin gene pool may have involved as few as one pummelo tree
(carrying both P1 and P2 haplotypes), the contribution of which was
diluted by repeated backcrosses with mandarins (Supplementary Note 6.3). The introgressed pummelo haplotypes became widespread and gave rise to type-2 (early-admixture) mandarins (Fig. 2b).
We propose that later, additional pummelo introgressions gave rise to
type-3 (late-admixture) mandarins and sweet orange, and that some modern
type-3 mandarins were derived from hybridizations among existing
mandarins and sweet orange. This late-admixture model for type-3
mandarins is consistent with the historical records for Clementine and
Kiyomi (both mandarin–sweet orange hybrids), and for W. Murcott, Wilking
and Fallglo (hybrids involving other type-3 mandarins), whereas
definitive records for the remaining two late-admixture mandarins (King
and Satsuma) are not available.
Domestication of mandarins and sweet orange
Citrus
domestication probably began with the identification and asexual
propagation of selected, possibly hybrid or admixed individuals, rather
than recurrent selection from a breeding population as for annual crops37,38.
Additional diversity was obtained by capturing somatic mutations that
occur within a relatively few basic genotypes. Therefore, conventional
approaches to identifying selective pressures under recurrent breeding39
cannot be applied. We can, however, use genome sequences to infer some
features of the early stages of citrus domestication. Here we focus on
mandarins, a class of citrus comprising small and easily peeled fruits
that are of high commercial value.
All 28 mandarin accessions,
except for Tachibana, exhibit an extensive network of relatedness (with a
coefficient of relatedness, r > 1/8), and all but four
mandarins (three of the four are pure or type-1 mandarins) show second
degree or higher relatedness (r > 1/4) to at least one (mean = 7) other mandarin (Fig. 3a and Supplementary Note 7).
By contrast, sequenced pummelos and citrons appear to be independent
selections from relatively large populations. In the absence of
historical records for most mandarins, the actual kinships are difficult
to infer, owing to extensive haplotype sharing among the ancestors,
although some parent–child pairs can be identified. In addition to
confirming, using the whole-genome sequence, the parentage of Wilking
(King–Willowleaf), Kiyomi (Satsuma–sweet orange) and Fallglo (one
grandparent is Clementine), we find parent–child relationships between
two pairs of mandarins (Ponkan is a parent of Dancy; Huanglingmiao (a
somatic mutant of Kishu) is a parent of Satsuma)34, in addition to the previously established parent–child pair of Willowleaf and Clementine mandarins2. Additional parent–child pairs involving the recently sequenced Chinese mandarins3 are also identified (Supplementary Note 7.3).
A few cultivar types in this network (Satsuma, Dancy, Clementine,
Kiyomi, Fallgo and the Chinese cultivar BTJ mandarins) have marked signs
of inbreeding, indicated by runs of homozygosity (Extended Data Fig. 4a)
as a result of shared haplotypes between their parents. The high degree
of relatedness among mandarins implies extensive sharing of C. reticulata haplotypes.
Figure 3: Citrus relatedness network and haplotype sharing with sweet orange.
a,
Genetic relatedness among 48 citrus accessions derived from four
progenitor species including citrons, pummelos, pure mandarins and
micrantha. Solid lines connect pairs with coefficient of relatedness r > 0.45,
with parent–child pairs denoted by arrows pointing from parent to
child. Dashed and dotted lines correspond to 0.35 <r <0 .45="" 0.25="" and="" i="">r
<0 .35="" a="" an="" ancestral="" are="" as="" by="" code="" distinguished="" extensive="" from="" gene="" groups="" haplotypes="" href="https://www.nature.com/articles/nature25447/figures/1" in="" indicating="" mandarins="" network="" other="" pool.="" relatedness="" respectively.="" shared="" taxonomic="" the="" three-letter="">Fig. 1, see Supplementary Table 2 for details. b, Shown in decreasing order are the values of coefficient of relatedness between sweet orange and other accessions, with C. maxima (rP) and C. reticulata (rM)
components in green and light salmon, respectively.There is significant
haplotype sharing between sweet orange and all mandarins, except for
three of the type-1 mandarins. Five accessions (Clementine and Kiyomi
mandarins, eremorange, Marsh grapefruit, and Ambersweet orange) have
sweet orange as the male parent.
Sweet orange also shows extensive haplotype sharing at the level of r > 0.1 with 25 of the 28 sequenced mandarins (except for three pure or type-1 mandarins; Fig. 3b and Extended Data Fig. 4b).
Two late-admixture mandarins (Clementine and Kiyomi) are direct
offspring of sweet orange. Among the early-admixture (type-2) mandarins,
Ponkan shows the highest affinity to sweet orange2 with r ≈ 0.36. Even the pure mandarin, Sun Chu Sha Kat has r ≈ 0.23,
equivalent to second degree relatedness to sweet orange. We can rule
out the scenario that sweet orange is the common ancestor of the
mandarins, because of a lack of pummelo haplotypes (derived from sweet
orange) among the mandarins. Rather, the extensive C. reticulata
haplotype sharing between sweet orange and mandarins suggests that the
mandarin parent of sweet orange was part of an expansive network of
relatedness among mandarins.
Because our collection of mandarins
represents a diverse set of both ancient and modern varieties, including
economically important accessions with mostly unknown parentage, the
presence of an extensive relatedness network was not anticipated a
priori. The shared C. reticulata haplotypes are suggestive of and
consistent with signatures of the human selection process, during which
mandarins with desirable traits were necessarily maintained through
clonal propagation (nucellar polyembryony or grafting). Although one
cannot preclude the possibility that the relatedness network was
initiated before domestication from a small number of founder trees,
human selection of accessions resulting from natural hybridization
probably had a key role in the process of domestication that eventually
led to the extensive relatedness network observed today. For example,
modern mandarins, such as Clementine and W. Murcott, are known to be
selections from chance seedlings found in Algeria40 and Morocco2, at the onset and middle of the last century, respectively.
Pummelo
admixture is correlated with fruit size and acidity, suggesting a role
for pummelo introgression in citrus domestication. As both fruit size
and acidity profile for the most recently sequenced accessions3
are not described, we used 37 citrus accessions in this analysis. We
find that the fruit sizes of mandarins, oranges, grapefruit and pummelos
show a strong positive correlation (Pearson correlation coefficient r = 0.88) with pummelo admixture proportion (Extended Data Fig. 5a, b and Supplementary Note 10.1).
In addition to fruit size, a pivotal driver of fruit domestication is
palatability, a characteristic that in citrus requires low to moderate
levels of acidity. In mandarins, palatability appears to be linked to
pummelo introgression at a major locus at the start of chromosome 8
(0.3–2.2 Mb), where all nine known palatable mandarins, but none of the
four known acidic mandarins, show pummelo admixture in at least part of
the genomic region (Extended Data Fig. 3). This locus is also found to be significant in a genome scan for palatability association (Extended Data Fig. 5c, d and Extended Data Table 3) and contains several potentially relevant genes (Supplementary Note 10.2). Among these genes is a gene encoding the mitochondrial NAD+-dependent isocitrate dehydrogenase (IDH) which regulates citric-acid synthesis41 (Extended Data Table 4).
Our
study finds that domesticated citrus fruit crops, such as mandarins and
sweet orange, experienced a complex history of admixture, conceptually
similar to those well-recognized in annual crops, such as rice42 and maize43, and in other fruit trees, such as apple44 and grape45,
for which the current genomic diversity is linked to widespread ancient
introgression. Other cultivated citrus groups, the interspecific F1
hybrids in particular, originated from hybridizations of two pure
parental species. Several of these involve C. medica (citron), including limes and lemons10. A unique and critical characteristic of the three pivotal species (C. maxima, C. reticulata and C. medica) that gave rise to most cultivated citrus fruits is the occurrence of a complex floral anatomy (Extended Data Fig. 6), thus leading to the development of more complex fruit. Other species were also involved in hybridizations, including Fortunella and C. micrantha.
Distinct from the mandarin lineages, these hybrids are characterized by
their acidic fruit, and their selection must have been made on the
basis of other characteristics, such as a sweet edible peel and aroma2, respectively.
Conclusion
On
the basis of genomic, phylogenetic and biogeographic analyses of 60
diverse citrus and related accessions, we propose that the centre of
origin of citrus species was the southeast foothills of the Himalayas,
in a region that includes the eastern area of Assam, northern Myanmar
and western Yunnan. Our analyses suggest that the ancestral citrus
species underwent a sudden speciation event during the late Miocene.
This radiation coincided with a pronounced transition from wet monsoon
conditions to a drier climate, as observed in nearby areas in many other
plant and animal lineages. The Australian citrus species and Tachibana,
a native Japanese mandarin, split later from mainland citrus during the
early Pliocene and Pleistocene, respectively. By distinguishing between
pure species, hybrids and admixtures, we could trace the genealogy and
genetic origin of the major citrus commercial cultivars. Both the
extensive relatedness network among mandarins and sweet orange, and the
association of pummelo admixture with desirable fruit traits suggest a
complex domestication process.
Our work challenges previous proposals for citrus taxonomy. For example, we find that several named genera (Fortunella, Eremocitrus and Microcitrus)
are in fact nested within the citrus clade. These and other distinct
clades that we have identified are therefore more appropriately
considered species within the genus Citrus, on a par with those that formerly were referred to as the three ‘true’ or ‘biological’ species (C. reticulata, C. maxima and C. medica). Additionally, the related genus, Poncirus, a subject of continuous controversy since it was originally proposed to be within the genus Citrus12,46, is clearly a distinct clade that is separate from Citrus based on sequence divergence and whole-genome phylogeny.
In
summary, this work presents insights into the origin, evolution and
domestication of citrus, and the genealogy of the most important wild
and cultivated varieties. Taken together, these findings draw a new
evolutionary framework for these fruit crops, a scenario that challenges
current taxonomic and phylogenetic thoughts, and points towards a
reformulation of the genus Citrus.
Methods
Sample collection and sequencing
Whole-genome
sequences from a total of 60 accessions were analysed: 58 citrus
accessions with different geographical origins and two representative
outgroup genera. Twelve of these genomes, including five mandarins, four
pummelos, two oranges and a wild Mangshan mandarin (C. mangshanensis) were reanalysed from previous works2,7.
We also reanalysed 19 genomes from Chinese collections, including 15
unnamed mandarins, 2 Chinese sour oranges, Ambersweet orange and
Cocktail grapefruit (a hybrid resembling grapefruit) that have been
previously reported3.
The
30 accessions that were newly sequenced came from citrus germ-plasm
banks and collections at IVIA, Valencia, Spain; SRA, Corse, France; UCR,
Riverside and FDACS/DPI, Florida and included nine mandarins, two
limes, one rough lemon, one grapefruit, one lemon, four citrons, one
Australian desert lime, one eremorange, two Australian finger limes, two
Australian round limes, one kumquat, one calamondin, one micrantha, one
Ichang papeda, one trifoliate orange and one Chinese box orange (Supplementary Note 1).
DNA
libraries were constructed using standard protocols with some
modifications. Library insert sizes range from 325 to 500 bp. Sequencing
was performed on HiSeq2000/2500 instruments using 100-bp paired-end
reads. Primary analysis of the data included quality control on the
Illumina RTA sequence analysis pipeline (Supplementary Note 2).
Variant calls and Citrus species diversity
Illumina paired-end reads were aligned to the haploid Clementine reference sequence2 and the sweet orange chloroplast genome assembly47 using bwa-mem48. PCR duplicates were removed using Picard. Raw variants were called using GATK HaplotypeCaller49 with subsequent filtering based on read map quality score, base quality score, read depth and so on (Supplementary Note 3.1).
Interspecific
admixtures versus pure citrus species were distinguished based on
sliding window analysis of heterozygosity and pairwise genetic distance D
(Supplementary Note 4).
Genome-wide ancestry informative markers for the progenitor species
were derived using pure accessions. Admixture analysis was carried out
in sliding windows using ancestry informative markers (Supplementary Notes 5).
Citrus relatedness and haplotype sharing
Interspecific
phasing was used to extract admixed haplotypes. Identical-by-descent
sharing was calculated for each of the non-overlapping sliding windows
across the genome and used to estimate coefficient of relatedness among
citrus accessions (Supplementary Notes 6, 7).
Phylogeny and speciation dating
We used Chinese box orange (genus Severinia) as an outgroup. Time calibration is based on the C. linczangensis16 fossil from Lincang, Yunnan, China. MrBayes50 was used for whole genome Bayesian phylogenetic inference, and corroborated with a PhyML51 reconstructed maximum likelihood tree. A penalized likelihood method52 as implemented in APE53 was used to construct the chronogram (Supplementary Note 8).
Genome scan of palatability association
We used a mixed linear model as implemented in gemma54
for a case–control study of citrus acidity and palatability with 37
citrus accessions. A conservative Bonferroni correction was used to
select significant genomic loci, with subsequent manual examination of
each candidate variant in all accessions to identify most discriminatory
loci for fruit palatability (Supplementary Note 10).
Data availability
Whole-genome shotgun-sequencing data generated in this study have been deposited at NCBI under BioProject PRJNA414519.
Prior resequencing data analysed here can be accessed under BioProject
accession numbers PRJNA320985 (mandarins) and PRJNA321100 (oranges), and
also under the NCBI Sequence Read Archive accession codes SRX372786
(sour orange), SRX372703 (sweet orange), SRX372702 (low-acid pummelo),
SRX372688 (Chandler pummelo), SRX372685 (Willowleaf mandarin), SRX372687
(W. Murcott mandarin), SRX372665 (Ponkan mandarin) and SRX371962
(Clementine mandarin). The Clementine reference sequence used here is
available at https://phytozome.jgi.doe.gov/.
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US Department of Energy Joint Genome Institute, Walnut Creek, California, USA
Guohong Albert Wu
& Daniel S. Rokhsar
Centro de Genómica, Instituto Valenciano de Investigaciones Agrarias (IVIA), Moncada, Valencia, Spain
Javier Terol
, Victoria Ibanez
, Antonio López-García
, Estela Pérez-Román
, Carles Borredá
, Concha Domingo
, Francisco R. Tadeo
& Manuel Talon
Computational Genomics Department, Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain
Jose Carbonell-Caballero
, Roberto Alonso
& Joaquin Dopazo
AGAP Research Unit, Institut National de la Recherche Agronomique (INRA), San Giuliano, France
Franck Curk
Citrus
Research and Education Center (CREC), Institute of Food and
Agricultural Sciences (IFAS), University of Florida, Lake Alfred,
Florida, USA
Dongliang Du
& Frederick G. Gmitter
AGAP
Research Unit, Centre de Coopération Internationale en Recherche
Agronomique pour le Développement (CIRAD), Petit-Bourg, Guadeloupe,
France
Patrick Ollitrault
Department of Botany and Plant Sciences, University of California, Riverside, Riverside, California, USA.
Mikeal L. Roose
Functional Genomics Node, Spanish National Institute of Bioinformatics (ELIXIR-es) at CIPF, Valencia, Spain
Joaquin Dopazo
Department
of Molecular and Cell Biology and Center for Integrative Genomics,
University of California, Berkeley, Berkeley, California, USA
Daniel S. Rokhsar
Molecular Genetics Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa 904-0495, Japan
Daniel S. Rokhsar
Contributions
M.T.,
D.S.R. and G.A.W. developed the project and acted as project
coordinators and provided scientific leadership; G.A.W. developed
methods for admixture analysis and interspecific phasing, and performed
comparative genome analysis. J.T., V.I., A.L.-G., E.P.-R., C.B., C.D.,
F.R.T., J.C.-C., R.A., J.D. and M.T. contributed 26 genomes; J.T.,
J.C.-C., R.A. and J.D. provided bioinformatics support; J.T. and E.P.-R.
contributed to the study of the IDH gene; V.I., E.P.-R. and C.B.
contributed to the variant analysis of candidate genes using
genome-wide association studies; A.L.-G. and C.B. assisted in the
biogeographic study; A.L.-G. and F.G.G. contributed to the description
of citrus accessions and discriminatory characteristics; P.O. and F.C.
contributed to germplasm, admixture analysis and hypothesis on the
origin of cultivated citrus species; D.D. and F.G.G. contributed one
citrus genome; M.L.R. contributed seven citrus genomes; F.G.G.
contributed perspective garnered from more than 35 years of experience
working on the genetic improvement of citrus; G.A.W., M.T., D.S.R. and
F.G.G. wrote the manuscript; G.A.W. and M.T. contributed the hypothesis
on the origin and dispersal of citrus.
Competing interests
The authors declare no competing financial interests.
Corresponding authors
Correspondence to Guohong Albert Wu or Daniel S. Rokhsar or Manuel Talon. Reviewer InformationNature thanks J. Ross-Ibarra, P. Wincker and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Publisher's
note: Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.
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