Physalis L., one of the most important genera in the family Solanaceae, contains 75–120 species, which are mainly distributed in tropical, and temperate regions of America, although there are a few species in Eurasia and Southeast Asia (Chinese academy of sciences, 1978; Martinez, 1998; Maggie, 2005; Wei et al., 2012; Zamora-Tavares et al., 2015). There are five Physalis species and two varieties in China and they are mostly found in the east, central, south, and southwest regions of China (Chinese academy of sciences, 1978). They are rich in vitamins, minerals, and antioxidants, and have potential medicinal properties, including anti-bacteria, anti-inflammatory, and anti-cancer actions (Ji et al., 2012; Wei et al., 2012; Hong et al., 2015). Many Physalis species are horticulturally and economically important, and commercial interest has increased in many regions of the world over recent decades (Wu et al., 2006; Wei et al., 2012; Ding et al., 2014). Some Physalis species, including Physalis alkekengi, Physalis pubescens, Physalis peruviana, and Physalis philadelphica have been extensively cultivated for their edible fruit, medicinal properties, or as ornamental plants in many regions of the world, such as China and Mexico (Wei et al., 2012; Zamora-Tavares et al., 2015). Currently, however, most natural Physalis species are rare due to habitat destruction and increased urbanization.

The accurate identification of Physalis species is extremely important in Physalis plant breeding programs and for the conservation of genetic resources. Traditionally, identification of Physalis species has been dependent on morphological characteristics (Menzel, 1951; Axelius, 1996; Martinez, 1998; Vargas et al., 2001). Phenotypic characteristics, however, are often affected by plant variability and growth habitats (Maggie, 2005; Vargas-Ponce et al., 2011; Wei et al., 2012). In addition, plants of the genus Physalis have a similar shape and their morphological characteristics are easily confused (Figure 1). Molecular markers are independent of environmental conditions and have now emerged as important tools for modern taxonomists (Feng et al., 2014). Some DNA marker systems, including simple sequence repeat (SSR), and inter-simple sequence repeats (ISSR), have been used to genetically study Physalis plants (Vargas-Ponce et al., 2011; Wei et al., 2012; Zamora-Tavares et al., 2015). In addition, the DNA sequences of a few genes, including the internal transcribed spacer (ITS) of the nrDNA, the Waxy gene, and chloroplast regions (ndhF and trnLF), have also been used to assess the phylogeny of Physalis and their relationship to other genera in the Solanaceae family (Maggie, 2005; Olmstead et al., 2008).

DNA barcoding is a relatively new taxonomic method that uses short DNA sequences of standard genome regions to make fast, efficient, and reliable species identifications (Hebert et al., 2004; Chen et al., 2010; Gao et al., 2010a; Hajiahmadi et al., 2013; Feng et al., 2015). As an efficient tool for species identification, DNA barcoding has become important in biological systematics, and identification (Chen et al., 2010; Gao et al., 2010a; Liu et al., 2012; Dong et al., 2015; Feng et al., 2015; Wang et al., 2015). Recently, several regions, including matK, rbcL, psbA–trnH, atpF–atpH, ycf 1, and ITS, have been advocated as potential standard DNA barcodes for different taxonomic groups in plants (Chase et al., 2005; Kress et al., 2005; Kress and Erickson, 2007; Lahaye et al., 2008a; Cbol Plant Working Group, 2009; Yao et al., 2009; Parveen et al., 2012; Dong et al., 2015; Larranaga and Hormaza, 2015). ITS2, a sub-region of the nuclear ribosomal ITS, has also been proposed as a novel universal DNA barcode to identify herbs based on 6600 samples of 4800 species (Chen et al., 2010). Compared with whole ITS region, ITS2 was more suitable for species identification because of its short length, and high efficiency for PCR amplification (Chen et al., 2010; Gao et al., 2010a; Han et al., 2013). In addition, the secondary structures of ITS2 sequences could be used as molecular morphological characteristics for species identification (Grajales et al., 2007; Yao et al., 2010). It has been proposed that the ITS2 sub-region should be the standard molecular marker for species authentication and for plant phylogenetic analysis (Schultz and Wolf, 2009; Chen et al., 2010; Gao et al., 2010a,b; Yao et al., 2010; Pang et al., 2011; Liu et al., 2012; Gu et al., 2013; Marghali et al., 2015; Zhao et al., 2015).

In this study, we used ITS2 region to barcode Physalis and then applied it as a phylogenetic marker to infer the genetic relationships among Physalis species.

Physalis species are important medicinal and edible plants that have a significant, economic value. DNA barcoding, using the ribosomal DNA ITS2 region as a tag to identify species, has recently attracted much attention (Chen et al., 2010). Compared with other candidate DNA barcodes, such as psbA-trnH, matK, rbcL, and ITS, ITS2 possesses many advantages, including good universality, small intraspecific variation, but high interspecific divergence, and a small fragment length (~200 bp; Chen et al., 2010; Yao et al., 2010). To our knowledge, this is the first time that the ITS2 regions have been used to identify Physalis species in such a large sample size, an endeavor which has expanded the application of the ITS2 region to the medicinal plant field.

As in some previous studies (Gao et al., 2010a; Liu et al., 2012; Feng et al., 2015), sufficient variation was found in the ITS2 region among Physalis species to allow determination of genetic divergence, and the ITS2 region also demonstrated a higher successful discrimination capability (compared to 82.2% identification success rates for both BLASTA1 and the nearest genetic distance methods). For example, P. angulata and P. minima have extremely similar morphological traits, rendering their differentiation very difficult and sometimes impossible (Figure 1), but they could be accurately discriminated based on their ITS2 regions. In addition, because of sufficient variation in the ITS2 secondary structures, some studies suggested that the secondary structure of ITS2 might be considered as a molecular morphological characteristic (Yao et al., 2010). In this study, we found that the secondary structures of ITS2 perform well in identifying Physalis species (Supplementary Figure 2). For example, some species (P. greenmanii vs. P. hintonii, and P. alkekengi vs. P. alkekengi var. franchetii) from the ambiguous identification cases by the BLAST 1 or nearest distance method could be identified by their ITS2 secondary structures (Supplementary Figure 2).

The ITS2 region cannot solve all the species identification problems in Physalis. For example, P. pubescens vs. P. pruinosa, P. angulate vs. P. angulata var. villosa were found to have identical ITS2 sequences and the same secondary structures,. Therefore, it might be worthwhile using other DNA barcodes as complementary factors for discriminating these species. Certainly, it should be noted that the taxonomic assignment of sequences from GenBank might not be accurate due to the similar morphological characteristics of some Physalis species (for example: P. pubescens and P. pruinosa). Hence, if these factors were taken into account, estimations of in-species discrimination might be lower for Physalis.

Some studies have suggested that although DNA barcoding aims to provide an efficient method for species-level identification, it may also contribute to taxonomic and biodiversity research (Hajibabaei et al., 2007; Wang et al., 2010; Wong et al., 2011; Feng et al., 2015). The ITS2 region could provide taxonomic signatures in systematic evolution (Coleman, 2003; Schultz et al., 2005; Liu et al., 2012; Feng et al., 2015). In our study, ITS2 could be used to barcode Physalis and to serve as a phylogenetic marker for Physalis taxonomy. As in previous studies (Mione et al., 1994; Olmstead et al., 1999; Maggie, 2005), the dendrogram constructed with ITS2 data using the ML method indicated that the genus Physalis was paraphyletic. Maggie (2005) showed that subgenus Rydbergis was morphologically homogeneous and we obtained similar results that showed that most collected species of subgenus Rydbergis (except P. carpenteri from sect. Carpenterianae) were grouped into group I. The species in subgenera Physalodendron, and Physalis were all morphologically atypical, either having multiple flowers per node, corollas which are lobed or odd colors, or unusual fruiting calyx morphology (Martinez, 1999; Maggie, 2005). In our study, these species were distant from the species in subgenus Rydbergis, and were grouped into groups II and III, respectively (Figure 4). P. walteri from subgenus Quincula was included within group I (I-1) together with species from sect. Viscosae (subgenus Rydbergis). In fact, P. walteri was strongly supported as sister species to P. viscosa and P. angustifolia (Waterfall, 1967; Maggie, 2005). In addition, we found that P. microphysa from subgenus Unknown, was an unplaced species in a previous study (Maggie, 2005). However, we were able to group it into group II together with species in subgenus Physalodendron with weak support (BS = 58). Thus, it appears that more sampling and more up-to-date phylogenetic methods are required to understand the taxonomy of P. microphysa.

Although most of the species in subgenus Rydbergis were grouped together within group I, some sections of subgenus Rydbergis were probably polyphyletic, such as Epeteiorhiza (I–1, I–2), and Angulatae (I–1, I–4, I–5) (Figure 4). In addition, the species form sect. Lanceolatae were Clustered together with the species from other sections (such as Viscosae, Angulatae, and Epeteiorhiza). Similar results have been reported by Maggie (2005). As for sect. Carpenterianae, P. carpenteri along with other species from other sections of subgenus Rydbergis, formed group IV with strong support (BS = 99) as previously reported (Maggie, 2005). Our results largely concurred with the view of the previous study that P. carpenteri, P. alkekengi, P. microphysa, and subgenus Physalodendron should be recognized as four small genera (Maggie, 2005).

Some studies have concluded that DNA barcoding sequences do not usually have sufficient phylogenetic signals to resolve evolutionary relationships (Hajibabaei et al., 2006). In addition, multiple copies of ITS2 may suggest that the sequences obtained through PCR are not stable and representative and this might result in misleading phylogenetic inferences (Queiroz Cde et al., 2011). However, after comparison with previous studies, our results have demonstrated that ITS2 is a useful DNA barcode that could be used to identify Physalis species, and build relatively reliable molecular phylogenies for the genus Physalis.

Conceived and designed the study: SF, HW. Collected plant samples: SF, HW, YS, JL, and QY. Performed the experiments: SF, MJ, KJ, and CS. Analyzed the data: SF, MJ, KJ. Wrote the manuscript: SF, HW.

This study was supported in part by the National Natural Science Foundation of China (31470407), the Zhejiang Provincial Public Welfare Technology Applied Research Foundation of China (2014C32090), the Hangzhou Scientific and Technological Program (20150932H04), and the Hangzhou Scientific and Technological Program (20150932H03).