Natural hybridizations commonly arise in vascular plants across many different families and floras when infraspecific populations or closely related species come into contact (Ellstrand et al. 1996; Rieseberg 1997; Whitney et al. 2010; Kadereit 2015). These processes play a crucial role in the formation and maintenance of species (Seehausen 2004; Arnold and Martin 2009; Soltis and Soltis 2009; Nolte and Tautz 2010; Abbott et al. 2013). It is estimated that at least a quarter of plant species are involved in hybridization (Mallet 2005).
Whereas there are undoubtedly species of hybrid origin (Barrier et al. 1999; Rieseberg 2006; Meier et al. 2017; Lamichhaney et al. 2018; Wang et al. 2021), it is inappropriate to assign taxonomic rank to each production of hybridization (Marczewski et al. 2016). To date, a large body of endemics spanning a relatively narrow range were proven to be hybrids rather than species (Wiegleb and Kaplan 1998; Zha et al. 2010; Shin et al. 2014; Shi et al. 2016; Zhang et al. 2020 a, b; Lyu et al. 2021; Ao et al. 2022). These hybrids usually have poorer fitness than parental species, and depend on the repeated hybridization between parental species to maintain the populations. As a consequence, they are ordinarily considered as “negative assets” in biodiversity estimation and conservation (Allendorf et al. 2001; Jackiw et al. 2013). For taxonomy, accurate species delimitation is the bedrock and the guarantee. Natural hybrids have long been seen as “troublemakers” by taxonomists as they usually show an intermediate state in a part of characters probably making the morphological divergences between the parents less obvious (Stebbins 1957; Wagner 1969; Dejaco et al. 2016). Thus, it is essential to uncover the hybrid status of potential “disguisers” which should not be attributed with species rank.
Due to the co-occurrence with potential parental species, natural hybrids are often noticed during field investigations at first. Morphological intermediacy further divulges clues of hybridizations for its common application in the identification of natural hybrids (Marczewski et al. 2016). As the development of molecular approaches, there are more available tools to help to unmask hybrids, including but not limited to incomplete ITS (internal transcribed spacer region) concerted evolution (Grimm & Denk 2008; Kou et al. 2017), cytonuclear disequilibrium (Hodkinson et al. 2002; Yu et al. 2014), microsatellite polymorphisms (SSRs; Schroeder & Fladung 2010; Zhang et al. 2020b), heterozygous alleles in single or low-copy nuclear genes (Liao et al. 2015, 2021), and single nucleotide polymorphisms (SNPs; Väli et al. 2010; Zheng et al. 2021). Among these approaches, ITS allied with several plastid makers has a broader application because of the virtues of both high practicality and simplicity, for instance, in the genus Ilex L. (Son et al. 2009; Shi et al. 2016).
Ilex, the sole genus of Aquifoliaceae, consists of at least 14 sections (Yang et al. 2022) and holds more than 600 species as well as a lot of interspecific hybrids both naturally occurring and cultivated (Galle 1997; Powell et al. 2000; Loizeau et al. 2005; Chen et al. 2008). To date, the confirmed inartificial Ilex crosses have only been found to be intra-sectional; however, most natural hybrids are concentrated in the largest section, I. sect. Ilex, which contains over 100 species and has a center of diversity in East Asia (Yang 2020). During the last 40 years (especially the 1980s), I. sect. Ilex had experienced a rapid growth of species number from ca. 50 to over 100. These newly described species commonly are endemics. They usually have narrow native range and are listed in the ICUN red list of endangered species (ICUN 2022), e.g., I. sanqingshanensis W.B.Liao, Q.Fan & S.Shi (Fig. 1) which only occurs in the Sanqing Mountain, eastern China. During the field investigations conducted in 2018 and 2020, we however found that I. sanqingshanensis always co-occurs with the other two members of I. sect. Ilex, i.e., I. ficoidea Hemsl. and I. pernyi Franch. Specifically, I. sanqingshanensis only grows at the elevation of 1300–1600 m where I. ficoidea and I. pernyi have converged (Fig. 1). The discoveries in situ reminds us of the probable hybrid origin of I. sanqingshanensis.
In this study, we analyzed a multi-gene dataset including ITS and two chloroplast DNA (cpDNA) regions (petA-psbJ and psbA-trnH) and a morphological dataset covering eight leaf traits. We aim to: (1) test the hypothesis of the hybrid origin of I. sanqingshanensis, (2) fix the parental species if it is indeed a product of hybridization, and (3) give a reasonable identity to the target, a hybrid species or just a hybrid.