Chemical Variability of Peel and Leaf Essential Oils in the Citrus Subgenus Papeda (Swingle) and Few Relatives

The Papeda Citrus subgenus includes several species belonging to two genetically distinct groups, containing mostly little-exploited wild forms of citrus. However, little is known about the potentially large and novel aromatic diversity contained in these wild citruses. In this study, we characterized and compared the essential oils obtained from peels and leaves from representatives of both Papeda groups, and three related hybrids. Using a combination of GC, GC-MS, and 13C-NMR spectrometry, we identified a total of 60 compounds in peel oils (PO), and 76 compounds in leaf oils (LO). Limonene was the major component in almost all citrus PO, except for C. micrantha and C. hystrix, where β-pinene dominated (around 35%). LO composition was more variable, with different major compounds among almost all samples, except for two citrus pairs: C. micrantha/C. hystrix and two accessions of C. ichangensis. In hybrid relatives, the profiles were largely consistent with their Citrus/Papeda parental lineage. This high chemical diversity, not only among the sections of the subgenus Papeda, but also between species and even at the intraspecific level, suggests that Papeda may be an important source of aroma diversity for future experimental crosses with field crop species.


Introduction
Citrus species are native to Southeast Asia, and their exceptional diversity is the result of both migration and geographical isolation over the course of their evolutionary history [1]. The current cultivated forms are the result of crosses between species that evolved in Southeast Asia [2][3][4][5]. Two other genera capable of crossing with Citrus species are also found in Asia: Fortunella and Poncirus. There are also Oceanian species known to hybridize with Citrus species, belonging to the genera Eremocitrus, Microcitrus, Clymenia, and Oxanthera. Together, these genera constitute the "true citrus" group as described by Swingle and Reece [6]. One of the main characteristics of Citrus is the presence of highly aromatic essential oils in tissue storage cells of the fruit, leaf, and flower (petals). These essential oils are complex mixtures that can contain hundreds of compounds with a very wide chemical diversity, which is prized by the aromatic and cosmetic industry [7]. The composition of essential oils in the majority of citrus fruits grown for consumption is very well documented [8]. However, much of the aromatic diversity found in other Citrus, including fruits of the Papeda subgenus, remains largely unknown.
Swingle recognized two subgenera in Citrus: Papeda and Citrus [9]. In the subgenus Papeda, he defined two sections (Papeda and Papedocitrus) with four species (C. hystrix D.C., C. macroptera Montrouz, C. micrantha Wester, and C. celebica Koord) listed in the former, and two species (C. ichangensis Swingle (Ichang papeda) and C. latipes (Swingle) Tanaka (Khasi papeda)) in the latter [9]. The section Papedocitrus is considered as intermediate between the two subgenera Papeda and Citrus. In the chapter entitled "Botany of citrus and its wild relatives", Swingle and Reece described the subgenus Papeda as follows: "pulpvesicles containing numerous droplets of acrid oil; petioles long and very broadly winged, but not cordate, often nearly as broad as the leaf blades; stamen usually free [..] flowers larger and petioles very long, 1.75-3 longer than broad" [6]. Recently, exploration of the Citrus genome by molecular markers and sequencing has demonstrated that Papeda is a non-homogeneous group actually consisting of two very distinct (polyphyletic) genetic groups. The first group includes C. micrantha as reference, and the second is represented by C. cavaleriei H. Lév. Ex Cavalerie (or C. ichangensis) [1,10]. These two genetic groups are considered to be two ancestral species, that have generated some cultivated varieties such as Yuzu (C. ichangensis × C. reticulata), Alemow and Mexican lime (C. micrantha × C. medica), Nasnaran mandarin (C. micrantha × C. reticulata), or Ichang lemon (C. maxima × C. junos) through outcrossing with other ancestral species (C. maxima (Burm.) Merr, C. reticulata Blanco, C. medica L.) [1,7,11,12].
Recently, Ollitrault et al. [13] proposed a new classification taking into account phylogenetic relationships and sexual compatibility, building on the former classifications of Tanaka [14], Swingle and Reece [6], and Zhang and Mabberley [15]. In the Papeda group, two true species are now recognized. The first, C. cavaleriei, originates from West-Central and Southwestern China, and includes C. ichangensis and C. latipes. The second, C. hystrix, originates from Southern Philippines and includes C. micrantha (with two varieties, micrantha (Biasong) and microcarpa (Samuyao), and appears to be very closely related to C. micrantha [15]. C. hystrix (Combava) also appears very closely related to C. micrantha [15]. This new classification partially confirms the work of Swingle and Reece, who had divided the Papeda group into two sections. However, it should be noted that the classification of Melanesian papeda (C. macroptera) has not yet been considered in this phylogenomic taxonomy. In terms of genetic diversity, there are very few studies concerning the Papeda group, though high intraspecific genetic diversity has been identified in C. macroptera [16] and C. cavaleriei (or C. ichangensis) [17].
Data concerning the chemical composition of peel and leaf oils from Citrus classified as Papeda are scarce; the literature is mainly focused on cultivated hybrids such as Yuzu [18,19]. Leaf oil composition was also reported in C. ichangensis [20][21][22]. C. hystrix is also well described in the literature [21][22][23][24], while C. macroptera leaf oil was described by Huang et al. [19] and Waikedre et al. [24]. C. latipes and C. macrophylla leaf oils were described only once, in the same publication [25]. The lack of data on C. macrophylla may be due to its sole use as a rootstock for citrus cultivation [26]. To our knowledge, there is no chemical data concerning C. micrantha in the literature.
The chemical composition of peel and leaf essential oils can be determined by (i) gas chromatography (GC) retention indices (RI) calculated for polar and apolar columns and (ii) gas chromatography-mass spectrometry (GC-MS). These methods provide abundant information, not only for metabolism-related research, but also for chemotaxonomy. Consequently, several studies on Citrus have used this approach [27,28]. The chemotaxonomy of Mangshanyegan (C. nobilis Lour.), was determined by comparison of volatile profiles of fruits and leaves and those of 29 other genotypes of Citrus, Poncirus, and Fortunella [20]. The chemical components identified in the peels of 66 citrus germplasms from four Citrus horticultural groups (mandarin, orange, grapefruit, and lemon) were also used for biomarker mining. Thirty potential biomarkers were identified, and four compounds (β-elemene, valencene, nootkatone, and limettin) were validated as biomarkers [29]. However, Luro et al. [30] found that the diversity based on leaf oil compositions from Citrus medica varieties did not agree with the molecular diversity and was therefore unsuitable for intraspecific phylogenetic studies.
In this context, the aim of this study was to investigate the diversity of chemical composition of peel and leaf oils from Citrus belonging to the subgenus Papeda present in the INRAE-CIRAD citrus germplasm bank (Corsica, France). All accessions are fully indexed in a plot with identical climatic and agronomic growing conditions [31]. These conditions are suitable to study the relationship between chemistry and taxonomy and to produce reference data for Papeda peel and leaf oils composition. We analyzed ten samples from eight species, including three Papeda species (C. hystrix, C. micrantha, and C. macroptera) and two Papedocitrus species (three accessions of C. ichangensis and one of C. latipes) to characterize the two sections of the subgenus Papeda, and three related hybrid species (C. junos, C. macrophylla, and C. wilsonii) in order to investigate the inheritance of chemical characters. To evaluate chemical diversity within and among species, we performed Principal Component Analysis on the peel and leaf oil data.

Peel Oils
Among the ten studied accessions, only nine peel oil samples were obtained by hydrodistillation of peels, because one accession of C. ichangensis did not produce a sufficient number of fruits. In total, 60 compounds were identified in peel oils, accounting for 91.6% to 99.9% of the total oil composition (Table 1).  Order of elution and relative percentages of individual components are given on an apolar column (BP-1) except those with an asterisk (*), for which percentages were taken on polar column (BP-20); RI A . RI P : retention indices measured on apolar and polar capillary columns, respectively; "-": absence of the component; tr: trace level (<0.05%); hys: C. hystrix, mic: C. micrantha, mapt: C. macroptera, lat: C. latipes, ich: C. ichangensis, wil: C. wilsonii, jun: C. junos, maph: C. macrophylla.
All of the peel oil samples were dominated by monoterpene hydrocarbons, mostly due to the abundance of limonene (20.7-81.4%), as described in the literature [23]. Despite this common characteristic, several chemical profiles were observed.
C. latipes peel oil composition differed from those of C. ichangensis by exceptionally high percentages of myrcene (18.8%) and γ-terpinene (16.2%). No chemical data were found about peel oil of C. latipes in the literature.
The chemical composition we observed for C. junos is similar than those described by Dugo and Di Giacomo [23] with the proportion of limonene varying between 60.4 and 82.4%, mainly associated with γ-terpinene (7.6-10.7%) and linalool (0.9-5.6%). No data were found about peel oil composition of C. macrophylla.
Yuzu and Ichang lemon, hybrids of C. ichangensis and other Citrus species, exhibited a significant amount of β-phellandrene. Such proportions at rates higher than 1% are rather unusual in Citrus, and could be inherited from a parent with a chemical profile close to the ich-3 accession of C. ichangensis, which expressed a higher amount of this compound (8.8%) than other species.
In our sampling, Papedocitrus peel oils constituted an intermediate between low amounts of limonene observed in Papeda section (around 25%) and high percentages (around 80%) in the related species. The variability of the chemical profiles is very large within the Papeda samples, as seen in Figure 1, a PCA in which the two principal axes accounted for 81.8% (70.9 and 10.7%; F1 and F2, respectively). This diversity is mainly due to three compounds (limonene, β-pinene, and sabinene) that separate the micrantha/hystrix pair from all other citrus fruits in general, but especially to C. macrophylla and C. junos, which show characteristics of their parent of the Citrus subgenus (citron and mandarin) ( Figure S1).

Leaf Oils
In total, 76 compounds were identified in leaf oils, accounting for 93.6% to 99.3% ( Table 2). The yields of the ten leaf oil samples varied drastically between 0.015 to 0.18% (Table 2). For example, the three C. ichangensis samples exhibited very different yields and strong intraspecific variability in composition.   Order of elution and relative percentages of individual components are given on an apolar column (BP-1) except those with an asterisk (*), for which percentages were taken on polar column (BP-20); RI A . RI P : retention indices measured on apolar and polar capillary columns, respectively; "-": absence of the component; tr: trace level (<0.05%); hys: C. hystrix, mic: C. micrantha, mapt: C. macroptera, ich: C. ichangensis, lat: C. latipes, wil: C. wilsonii, jun: C. junos, maph: C. macrophylla.
The ten leaf oil samples exhibited a chemical composition dominated by monoterpenes, as usually found in Citrus leaf essential oils [23]. However, we observed substantial quantitative variability among the major components: sabinene (0-44.6%), β-pinene C. hystrix, C. micrantha, and C. macrophylla leaf oils were dominated by oxygenated monoterpenes, whereas C. junos, C. ichangensis (three accessions), and C. macroptera were dominated by monoterpene hydrocarbons. The last species, C. latipes, exhibited a nearly 1:1 ratio between hydrocarbon/oxygenated terpenes.
In a recent study, Zhang et al. [22] reported the chemical composition of ten accessions of C. ichangensis. Five of ten oil samples exhibited the two aforementioned ocimenes as major components, in addition to linalyl acetate in four accessions and α-pinene in another one, whereas two of ten oil samples were dominated by sabinene, in addition to with γ-terpinene and limonene. In our sampling, a similar 2:1 ratio between these two compositions was observed. The three other accessions of C. ichangensis in Zhang et al. [22] were dominated by γ-terpinene in two samples, and linalyl acetate for the final sample.
The strong intraspecific diversity observed at the level of aromatic compounds is in agreement with the high genetic diversity of this taxa observed at the DNA level [17].

Related Species
The main differences between the three related species and the two sections Papeda and Papedocitrus leaf essential oils were in the proportions of γ-terpinene (6.2-28.2% vs. tr-0.2%, respectively) and p-cymene (4.3-11.4% vs. 0-3.5%), respectively. Thus, the three relatives were discriminated ( Figure 2). However, each has its own chemical characteristics.
This type of composition dominated by the association geranial/neral/limonene is usually found in leaf essential oils of citrons and some limes [37]. This characteristic of the chemical profile of Alemow might be inherited from its citron male paternal lineage.
The high diversity within the chemical composition of Citrus subgenus Papeda leaf essential oils is illustrated by a three-dimensional PCA ( Figure 2) where more than 80% of the global variability is represented by the three axes. We have also visualized the chemical compounds implicated in this representation ( Figure S2).

Plant Material
According to the systematics of Swingle and Reece (1967), ten accessions were selected to represent the diversity of the subgenus Papeda, including: three accessions of Ichang papeda (C. ichangensis Swing.) and one of Khasi papeda (C. latipes (Swing.) Tan.) for section Papedocitrus, Biasong (C. micrantha Wester), Combava (C. hystrix D.C.), and Melanesian papeda (C. macroptera Montr.) for section Papeda. Three other Citrus species related to Papeda have been added: Ichang lemon (C. wilsonii Tan.; C. maxima × C. junos), Alemow (C. macrophylla Wester; C. micrantha × C. medica), and Yuzu (C. junos Sieb. ex Tan.; C. ichangensis × C. reticulata) ( Table 3). All the trees are maintained in the INRAE-CIRAD citrus collection (certified as Biological Resource Center (BRC) citrus NF96-600) located in San Ghjulianu, Corsica (France): latitude 42 • 17' N; longitude 9 • 32' E; Mediterranean climate; average: rainfall and temperature 840 mm and 15.2 • C per annum, respectively; soil derived from alluvial deposits and classified as fersiallitic; pH range 6.0-6.6 [31]. About 100 g of fruit peels and 200 g of leaves were randomly collected all around the tree. The fresh materials underwent hydrodistillation for three hours using a Clevenger type apparatus. Since peel oil yields were influenced by the presence of variable amounts of albedo during the peeling of the epicarp, they were not calculated. Distillation yields of leaf oils were calculated using the weight of essential oil/weight of fresh leaves ratio. Each sample was analyzed by gas chromatography (GC) and gas chromatography coupled with mass spectrometry (GC-MS) in order to determine the chemical composition. To avoid any misidentification, some samples, selected on the basis of the chromatogram profile, were analyzed with carbon-13 nuclear magnetic resonance ( 13 C NMR) following a methodology developed in our laboratory [47].

Gas Chromatography (GC) Analysis
GC analyses were performed on a Clarus 500 FID gas chromatograph (PerkinElmer, Courtaboeuf, France) equipped with two fused silica gel capillary columns (50 m × 0.22 mm, film thickness 0.25 µm), BP-1 (polydimethylsiloxane) and BP-20 (polyethylene glycol). The oven temperature was programmed from 60 to 220 • C at 2 • C/min and then held isothermal at 220 • C for 20 min, injector temperature: 250 • C; detector temperature: 250 • C; carrier gas: hydrogen (1.0 mL/min); split: 1/60. The relative proportions of the oil constituents were expressed as percentages obtained by peak area normalization, without using correcting factors. Retention indices (RIs) were determined relative to the retention times of a series of n-alkanes (C 7 -C 28 ) with linear interpolation ("Target Compounds" software of PerkinElmer). The EOs samples (50 mg) were diluted in chloroform (1 mL).

NMR Analysis
13 C NMR analyses were performed on an AVANCE 400 Fourier Transform spectrometer (Bruker, Wissembourg, France) operating at 100.623 MHz for 13 C, equipped with a 5 mm probe, in CDCl 3 , with all shifts referred to internal tetramethylsilane (TMS). 13 C NMR spectra were recorded with the following parameters: pulse width (PW): 4 µs (flip angle 45 • ); acquisition time: 2.73 s for 128 K data table with a spectral width (SW) of 220.000 Hz (220 ppm); CPD mode decoupling; digital resolution 0.183 Hz/pt. The number of accumulated scans ranged from 2000-3000 for each sample (around 40 mg of oil in 0.5 mL of CDCl 3 ). Exponential line broadening multiplication (1.0 Hz) of the free induction decay was applied before Fourier Transformation.

Identification of Individual Components
Identification of the components was based on: (i) comparison of their GC retention indices (RIs) on polar and apolar columns, determined relative to the retention times of a series of n-alkanes with linear interpolation ("Target Compounds" software of Perkin Elmer, Courtaboeuf, France), with those of authentic compounds [48]; (ii) computer matching against commercial mass spectral libraries [49,50] and by comparison of spectra with literature data [51,52]; and (iii) comparison of the signals in the 13 C NMR spectra of EOs with those of reference spectra compiled in the laboratory spectral library, using custom-made software [47,53,54]. In the investigated samples, individual components were identified by NMR at contents as low as 0.5%.

Statistical Analysis
The data of investigated samples of peel and leaf essential oils were submitted to Principal Component Analysis (PCA) using PAST (Paleontological Statistics Software Package) 3.14 version software [55]. Only constituents in a proportion higher than 2% at least in one sample were used as variables for the PCA analysis.

Conclusions
We analyzed the chemical composition of peel and leaf essential oils of seven Citrus species belonging to two sections of the Papeda group. Among them, the major components of leaf essential oil were: citronellal for C. hystrix and C. micrantha; sabinene, linalool and β-pinene for C. macroptera; (E) and (Z)-ocimene for two accessions of C. ichangensis, and sabinene for the third accession; limonene, linalool, and citronellal for C. latipes. In the three related species, leaf oil profiles were dominated by γ-terpinene, geranial, neral, and β-pinene for C. wilsonii; γ-terpinene, β-phellandrene, and p-cymene for C. junos; and finally geranial, neral and limonene for C. macrophylla. Limonene was the major component in almost all peel oil samples, except in C. micrantha and C. hystrix oils, where β-pinene dominated. The two sections of Papeda are clearly distinguishable in both their leaf and fruit peel essential oil composition, supporting the classification of Swingle, who proposed two sections in Papeda. Furthermore, as stated in this classification, the section Papedocitrus is an intermediate between the two subgenera Papeda and Citrus, and certain aromatic compounds, such as limonene content, seem to confirm this status.
Substantial chemical diversity was also observed in leaf oils and peel oils between representatives of each section. However, some species (Biasong and Combava) have very similar chemical profiles, reflecting their close genetic relationship. On the other hand, the three representatives of C. ichangensis present very different profiles. These results suggest that Papeda may be an important source of aroma diversity, which may be uncovered by further surveys. The three Citrus × Papeda hybrids demonstrate that crosses between these two taxa can create high variability in the aromatic composition of essential oils. Future research may also be able to exploit this aromatic diversity by crossing these little-known citrus fruits with field crop species.