Phylogenetic and taxonomic status of Citrus halimii B.C. Stone determined by genotyping complemented by chemical analysis of leaf and fruit rind essential oils

Highlights

• Citrus halimii is not an interspecific hybrid but a true wild citrus species.

• C. halimii shares a commune ancestor with kumquats (Fortunella sp.).

• The essential oils of C. halimii present original compounds never described in citrus.

• Germacrene d-8-one is specific to C. halimii representing 8.7% of total leaf essential oil.

Abstract

Citrus phylogeny is currently based on genome analysis using molecular markers and sequencing.  The 7 pure genetic groups that gave rise to all cultivated citrus underlie the diversity of citrus accessions originating from Asia. However, there are wild citrus forms whose phylogenetic position is unknown, such as mountain citron (Citrus halimii B.C. Stone) that was discovered in Malaysia in the early 1970s. We sought to elucidate its status by determining its genetic profile with 30 SSR and InDel markers distributed on the 9 chromosomes of the citrus reference genome as compared to those of the 7 pure genetic groups represented by 4 or 5 varieties each. The genetic study was supplemented by a comparison of the composition of essential oils obtained by fruit peel and leaf hydrodistillation to those of the citrus fruits used for genotyping. The genetic study demonstrated that C. halimii is not an interspecific hybrid (low heterozygosity) but rather a true species that shares a common ancestor with kumquats (Fortunella sp.), which would have evolved separately. The fruit aromatic profiles confirmed this kumquat/mountain citron relationship but also highlighted the uniqueness of C. halimii due to the presence of high proportions of compounds that have never been observed in other citrus fruits, such as germacrene d-8-one (accounting for 8.7% of the leaf essential oil).

Introduction

Morphological descriptors were widely used before the 1980s in numerical taxonomy studies to elucidate plant genetic diversity and relationships between various species (Ollitrault et al., 2020). Based on these descriptors, Barrett and Rhodes (1976) were the first to put forward a hypothesis on Citrus phylogeny. They suggested that all cultivated citrus originated from three basic taxa (C. maxima, C. medica, and C. reticulata). Later on, Scora (1988) used essential oil and polyphenol chemical compositions in citrus taxonomic investigations and revealed four true Citrus species (C. halimii, C. maxima, C. medica, and C. reticulata). DNA polymorphism techniques, which have been widely implemented since the nineties, contributed to highlighting the phylogenetic structures of citrus (Nicolosi et al., 2000; Barkley et al., 2006; Garcia-Lor et al., 2012; 2013; Curk et al., 2016; Shimizu et al., 2016). These studies revealed a phylogeny of the Citrus genus based on 4 ancestral species: C. maxima, C. reticulata, C. medica and C. micrantha. These four species appear to be the ancestors of most cultivated citrus, often with few recombination events, such as sour orange (C. aurantium), which is a direct hybrid of pummelo and mandarin, and Tahitian lime (C. latifolia), a third-generation hybrid whose genome is an admixture of the four ancestral species (Ahmed et al., 2019).

The first complete reference sequences of the citrus genome were posted in Phytozome from 2011 (Wu et al., 2014). Since then, genome sequencing has enhanced the phylogenomic profile by providing precise information on the genomes structure of and the meiotic events that generated them (Ollitrault et al., 2020). Phylogenetic hypotheses based on DNA markers, such as SSRs and SNPs, were validated, thereby indicating that many mandarin cultivars were not pure mandarins because they introgressed small parts of the pummelo genome during their evolution (Oueslati et al., 2017; Wu et al., 2018). Recent phylogenomic data confirmed the existence of five pure Citrus species: C. cavaleriei H. Lev. (including C. ichangensis Swingle and C. latipes (Swingle) Tanaka), C. micrantha Wester, C. maxima (Burm.) Merr., C. medica L. and C. reticulata Blanco (Ollitrault et al., 2020). Fortunella taxa (kumquats) and Poncirus taxa (trifoliate oranges), both originating from northern China (Swingle and Reece, 1967), should now be added to the list of pure species or genetic groups of Asian citrus. In a phylogenetic analysis, Garcia-Lor et al. (2013) observed that C. reticulata, also originating from northern China, constituted a single clade with Poncirus and Fortunella. The different flowering seasons of the three genera could probably explain the differentiation between these three taxa, which evolved in sympatry. A major part of the actual phenotypic diversity of edible citrus should be related to the differentiation between these pure species prior to reticulation and introgression processes (Ollitrault et al., 2020). A close correlation between the genetic and phenotypic diversity was thus observed irrespective of the traits, such as the fruit juice chemical composition in primary metabolites (Luro et al., 2011), carotenoids (Fanciullino et al., 2005) or leaves and fruit rind aromatic compounds (Liu et al., 2013).

The volatile composition of citrus peel oils is generally a mixture of the dominant limonene, other monoterpenes and sesquiterpenes, as well as many oxygenated derivatives (Dugo and Mondello, 2010). Despite this common general profile, each Citrus species has a unique organoleptic signature due to a balanced mixture of major constituents and to the presence of minor components such as neral and geranial in lemon (Lota et al., 2002) or nootkatone in grapefruit (Paoli et al., 2016). Chemotaxonomic analyses based on volatile compounds in both fruit peel and leaves have been shown to be suitable for interspecies phylogenetic studies in various Citrus species (Liu et al., 2013; Zhang et al., 2017; 2019).

Several Citrus species could still exist as wild plants or little-altered landraces growing in natural conditions (Bayer et al., 2009). Mountain citron (C. halimii B.C. Stone), or so called limau kadangsa in Malay language, is one of them (Fig. 1).

It was discovered in Malaysia and Thailand in the 1970s (Stone et al., 1973) and its classification and genetic origin has yet to be totally elucidated. Based on morphological and phytochemical data, it was first suggested to be a citron/kumquat hybrid (Scora et al., 1976) Stone et al. (1973). and Ogawa et al. (2001) proposed that C. halimii is a natural hybrid between the Citrus and Fortunella genera, although a RFLP marker analysis did not find any association between C. halimii and Fortunella (Federici et al., 1998). In other studies, C. halimii was found to be clustered with Fortunella based on isozyme (Herrero et al., 1996), SSR (Barkley et al., 2006) and AFLP (Pang et al., 2007) marker studies. This relation was supported by cpDNA analysis findings (Bayer et al., 2009) Barkley et al., 2006. and Bayer et al. (2019) rejected the ancestral species status and supported the wild hybrid status because they had not observed any unique alleles in C. halimii and concluded that it was an admixture between the kumquat and citron groups, with the majority of its genetic makeup being derived from kumquat. However, Oueslati et al. (2017) identified four specific SNPs for C. halimii as compared to 78 other accessions of the Aurantioideae subfamily, including Fortunella species, based on the sequence of eight plastid genomic regions published by Bayer et al. (2019).

C. halimii has seldom been studied from a chemotaxonomical perspective and little information is available on the chemical composition of its tissues, only on leaf cuticle wax (Gulz et al., 1987), and its leaf and rind essential oils have only been briefly described (Scora et al., 1976). These studies highlighted the highly original chemical features of this citrus fruit, but the comparisons were only made on citrus samples that just included a few cultivated varieties, which were not representative of the genetic diversity of Asian citrus.

Our study aimed to assess the taxonomy and genetic status of C. halimii based on codominant DNA molecular markers and the essential oil composition of fruit and leaves. We thus selected varieties from the 7 pure genetic groups representative of the diversity of Asian citrus for comparison with mountain citron.