Abstact：【Objective】Evergreen trifoliate orange is a special member of Citrus s.l., with its trifoliolate similar to those of C. trifoliata, yet its evergreen features resembles that of species within Citrus s.s. Currently, two types of evergreen trifoliate orange have been established as independent species, including C. × pubinervia and C. × polytrifolia. Evergreen trifoliate orange possess strong cold resistance and are excellent citrus rootstock resources. However, their taxonomic status has long been disputed since their discovery, hindering research on their conservation and utilization. 【Methods】Considering the impacts of hybridization and incomplete lineage sorting, four nDNA segments (CTV.4, HYB, LGT, P12) that show no significant linkage relationship were selected for direct or cloning sequencing in C. × polytrifolia, and C. × pubinervia, and C. cavaleriei to reconstruct the phylogenetic tree of the Citrus s.l. and, further exploring the taxonomic status of these two evergreen trifoliate orange. 【Results】The phylogenetic tree based on HYB gene sequences reveals that C. trifoliata, C. × polytrifolia, and C. × pubinervia form the first strongly supported independent clade (LP=100%, PP=1.0) within Citrus s.l., known as the “Poncirus” clade. “Poncirus” clade further diverges into the “deciduous trifoliate orange” clade (LP=76%, PP=0.81) and the “evergreen trifoliate orange” clade (LP=81%, PP=0.76), which includes C. × polytrifolia and C. × pubinervia. The HYB gene tree is unable to distinguish between the two evergreen trifoliate oranges. C. cavaleriei appears in the HYB gene tree within Citrus s.s., distant from the two species of the evergreen trifoliate orange. The LGT gene tree shows that C. trifoliata and the two evergreen trifoliate oranges also form an independent clade within Citrus s.l. (LP=100%, PP=1.00). However, unlike the HYB gene tree, the LGT gene tree cannot differentiate between the C. trifoliata and the evergreen trifoliate orange. C.cavaleriei remains within Citrus s.s. in the LGT gene tree. There is no significant topological inconsistency of gene trees between the LGT and HYB, but LGT is more conservative and has lower species resolution than HYB. The topology of the P12 gene tree is inconsistent with the previous two. The “Poncirus” clade is embedded within the Citrus s.l., sharing a large clade with Fortunella, which has low support (LP=0, PP=0.66). The P12 gene tree cannot differentiate the three species within the “Poncirus” clade and has limited ability to distinguish other species within Citrus s.l., presenting an overall "comb-like" structure. The topology of the CTV.4 gene tree is significantly different from the first three, with the two evergreen trifoliate oranges separating from C. trifoliata and clustering with C. maxima and C. × aurantium in a large clade with weak support (LP=52%, PP=0.63). This large clade further diverges into two strongly supported smaller clades, namely the “C. maxima” clade (LP=98%, PP=1.0) and the “evergreen trifoliate orange” clade (LP=97%, PP=1.0), showing clear differentiation between them. The CTV.4 gene tree cannot distinguish C. maxima and C. × aurantium as two separate species, nor can it differentiate C. × pubinervia and C. × polytrifolia as two species. The clone sequencing of C. × pubinervia were supplemented to verify this phenomenon. The results show that its three clone sequences are located in the “Poncirus” clade, and two clone sequences are together with C. × polytrifolia in the “C. maxima” clade, exhibiting characteristics of hybrid origin. Direct and cloning sequencing of C. cavaleriei form a strongly supported clade (LP=98%, PP=1.0), adjacent to the “Poncirus” clade and distant from the evergreen trifoliate orange.【Conclusion】The topological structure between CTV.4 and the other three nDNA single-gene trees shows significant inconsistencies, suggesting that C. × polytrifolia and C. × pubinervia may have experienced hybridization events. In the CTV.4 gene tree, as a hybrid related to C. maxima, C. × aurantium is very closely related to C. maxima.Whereas evergreen trifoliate orange has undergone significant genetic differentiation from the " C. maxima" clade, indicating its origin time must be much earlier than C. × aurantium, and evergreen trifoliate orange has experienced an independent evolutionary process. Earlier cpDNA phylogenetic analysis showed that C. trifoliata, C. × polytrifolia, and C. × pubinervia have differentiated at the chloroplast gene level, being three distinguishable species. Therefore, C. × polytrifolia and C. × pubinervia likely originated from natural hybridization between C. trifoliata (♀) and other species of Citrus s.l. (♂), with C. maxima being the most likely maternal species. Since Citrus s.l. has undergone complex reticulate evolution and C. maxima is believed to have participated in the formation of other hybrid species (such as C. × aurantium), so the male parent of evergreen trifoliate orange cannot be determined conclusively through the study of a few gene fragments. Its male parent could be C. maxima or a natural hybrid containing C. maxima lineage. However, this research results does not mean that the two evergreen trifoliate orange are not "good species" in the taxonomic sense, because natural hybridization events are one of the important ways of species formation, especially for many species of Citrus s.l.. This study provides strong evidence of gene exchange existing naturally between the Poncirus and the Citrus s.s., offering new insights into the species origin of C. × pubinervia and C. × polytrifolia. These two species serve as a bridge between the Citrus s.s. and the Poncirus, holding significant value for studying the phylogenetic relationships of the Citrus s.l. and citrus breeding. Therefore, they should receive more attention and protection, suggesting that Citrus × pubinervia be listed as a local protected species or be granted the same level of protection as C. × polytrifolia.
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