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Comparison of plastid genomes and ITS of two sister species in Gentiana and a discussion on potential threats for the endangered species from hybridization

BMC Plant Biology volume 23, Article number: 101 (2023) Cite this article

Abstract

Background

Gentiana rigescens Franchet is an endangered medicinal herb from the family Gentianaceae with medicinal values. Gentiana cephalantha Franchet is a sister species to G. rigescens possessing similar morphology and wider distribution. To explore the phylogeny of the two species and reveal potential hybridization, we adopted next-generation sequencing technology to acquire their complete chloroplast genomes from sympatric and allopatric distributions, as along with Sanger sequencing to produce the nrDNA ITS sequences.

Results

The plastid genomes were highly similar between G. rigescens and G. cephalantha. The lengths of the genomes ranged from 146,795 to 147,001 bp in G. rigescens and from 146,856 to 147,016 bp in G. cephalantha. All genomes consisted of 116 genes, including 78 protein-coding genes, 30 tRNA genes, four rRNA genes and four pseudogenes. The total length of the ITS sequence was 626 bp, including six informative sites. Heterozygotes occurred intensively in individuals from sympatric distribution. Phylogenetic analysis was performed based on chloroplast genomes, coding sequences (CDS), hypervariable sequences (HVR), and nrDNA ITS. Analysis based on all the datasets showed that G. rigescens and G. cephalantha formed a monophyly. The two species were well separated in phylogenetic trees using ITS, except for potential hybrids, but were mixed based on plastid genomes. This study supports that G. rigescens and G. cephalantha are closely related, but independent species. However, hybridization was confirmed to occur frequently between G. rigescens and G. cephalantha in sympatric distribution owing to the lack of stable reproductive barriers. Asymmetric introgression, along with hybridization and backcrossing, may probably lead to genetic swamping and even extinction of G. rigescens.

Conclusion

G. rigescens and G. cephalantha are recently diverged species which might not have undergone stable post-zygotic isolation. Though plastid genome shows obvious advantage in exploring phylogenetic relationships of some complicated genera, the intrinsic phylogeny was not revealed because of matrilineal inheritance here; nuclear genomes or regions are hence crucial for uncovering the truth. As an endangered species, G. rigescens faces serious threats from both natural hybridization and human activities; therefore, a balance between conservation and utilization of the species is extremely critical in formulating conservation strategies.

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Background

Gentiana is a famous but extremely complex genus in the family Gentianaceae, comprising approximately 362 species worldwide. Alpine and subalpine regions in southwestern China are the centre of the highest concentration of species in Gentiana [1]. Gentiana rigescens Franchet is a perennial herb with beautiful flowers and is mainly distributed in Yunnan, Sichuan, and Guizhou [1, 2]. According to Chinese Pharmacopoeia (2020), the species is one of the original species of famous Gentianae Radix et Rhizoma, the so-called “Jianlongdan” [3]. The root and rhizome of the species are traditionally used to clear damp-heat and quench the fire of liver and gall bladder, and have recently been shown to possess neuroprotective and anti-Alzheimer effects[4, 5]. The medicine contains medicinal components, such as iridoids and flavonoids [6, 7]. Owing to over-harvesting and habitat destruction, wild resources of G. rigescens have decreased sharply over the last few decades. In addition, the species is narrowly distributed in altitude gradients, probably due to its rigid requirements for biotope, according to our field investigation and a previous study [8]. As a result, G. rigescens has been listed in the third class of National Key Protected Wild Medicinal Materials in China [9,10,11].

Gentiana cephalantha Franchet is a sister species to G. rigescens with similar morphology, but with better adaptation capacity in diverse habitats (Fig. 1). In Yunnan and adjacent regions, G. cephalantha is often used as a medicinal substitute for G. rigescens because of its rich resources and confusing morphology [12]. According to Flora of China (1995), both G. rigescens and G. cephalantha belong to the sect. Kudoa [2], but since have been adjusted to sect. Monopodiae in the latest taxonomic classification [1]. In terms of morphology, G. cephalantha has well-developed rosette leaves and violet corolla, whereas G. rigescens does not possess well-developed rosette leaves and carries blue corolla (Fig. 1). Although the two species are widely distributed in SW China with an obvious overlap in geographic ranges, they dominate different altitude gradients. According to a previous field investigation, G. rigescens narrowly grows in low elevation areas, but G. cephalantha occupies a wide range and diverse habitats in middle and high elevation regions (Table 1). They are generally separated by geography, except for some sympatric distributions. Under such conditions, are G. rigescens and G. cephalantha conspecific or independent ones in phylogeny? Does nature hybridization occur between the two species (Fig. S1), and will the process pose a threat to the endangered species?

Fig. 1
figure 1

Morphology of Gentiana rigescens and G. cephalantha, as well as their habitats. (A) Habitats of sympatric and allopatric distributions of G. rigescens and G. cephalantha; (B) whole plant of typical G. rigescens without rosette leaves; (C) whole plant of typical G. cephalantha with rosette leaves; (D) comparison of floral and leaf morphology between G. rigescens (upper) and G. cephalantha (under) in the sympatric distribution

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Table 1 Information of sample collection for Gentiana rigescens and G. cephalantha
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Over the last few decades, molecular techniques have been widely adopted to study species evolution and phylogeny [13, 14], but phylogenetic relationships among Gentiana species are not well understood because of the complexity of this genus, insufficient sampling, and low resolution of traditional molecular markers [15,16,17,18,19,20]. The complete chloroplast genome could provide more useful genetic information than the cpDNA fragments for exploring the phylogeny of complicated genera and discriminating closely related species [21,22,23]. Dong et al. [24] used plastid genomes to clarify the phylogenetic relationships between 20 complex species in Lagerstroemia. Chen et al. [25] also adopted plastid genomes to perform analysis of species discrimination and phylogeny for 21 Fritillaria species in China and revealed that the chloroplast genome could efficiently identify closely related species and resolve complicated phylogeny. Ji et al. [26] used complete chloroplast genomes to clarify the phylogenetic relationships among 29 Paris species, providing important molecular evidence for clarifying long-standing taxonomic problems in this genus. However, plastid genomes or regions might not really reveal the phylogeny of certain groups due to maternal inheritance; therefore, genetic information from other genomes, such as nrDNA ITS and nuclear genes, could play a vital role here, as well as explore potential hybridization and its threats to endangered species [27, 28].

In the present study, both the plastid genome and ITS were analysed to: (1) compare the basic characteristics of plastid genomes of G. rigescens and G. cephalantha from sympatric and allopatric distributions; (2) reveal phylogenetic relationships between the two species in Gentiana; and (3) explore natural hybridization and genetic introgression between the two species, as well as potential threats to G. rigescens. Undoubtedly, this study would be beneficial to understanding the phylogeny of Gentiana, as well as the conservation of G. rigescens and the pharmacophylogeny of Gentianae Radix et Rhizoma.

Results

Characteristics of plastid genomes for G. rigescens and G. cephalantha

The plastid genomes of G. rigescens and G. cephalantha were highly similar in their lengths, ranging from 146,795 to 147,001 bp and 146,856 to 147,016 bp, respectively. The intergenic spacer regions (IGS) in G. cephalantha were significantly longer than those in G. rigescens. Moreover, all chloroplast genomes of the two species had typical circular quadripartite structures, consisting of a large single-copy region (LSC), a small single-copy region (SSC) and a pair of inverted repeat regions (IR) with similar lengths (Table S1, Fig. 2). In total, 116 genes were annotated in these plastid genomes, including 78 protein-coding genes, 30 tRNA genes, four rRNA genes and four pseudogenes (ψinfA, ψycf1, ψrps16, ψrps19). Most genes were located in the LSC and SSC regions. Nineteen genes were duplicated in the IR regions, including eight protein-coding genes, seven tRNA genes and four rRNA genes. In addition, 11 protein-coding genes and six tRNA genes were found to contain introns (Table 2).

Fig. 2
figure 2

Plastid genome maps of Gentiana rigescens and G. cephalantha. Genes drawn inside the circle are transcribed clockwise, while those outside the circle are transcribed counter-clockwise. The inner dark gray circle corresponds to GC content and the inner light gray circle corresponds to the AT content. Different colors represent of distinctive genes within separate functional groups

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Table 2 Information of the complete chloroplast genomes of Gentiana rigescens and G. cephalantha
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Comparative analysis of re-sequencing revealed that the types and numbers of SSRs were highly similar between G. rigescens and G. cephalantha. This study identified five types of SSRs (mono-, di-, tri-, tetra-, and penta-nucleotides). Among them, mono-nucleotides occurred more frequently in the genomes of the two species (63.6%), followed by tetra-nucleotide repeats (19.6%) and tri-nucleotide repeats (11.3%) (Fig. 3A and B). Most of these SSRs were distributed in the LSC region (60.0%), and a few were distributed in the IR regions (5.6%) (Fig. 3C). In addition, the distribution of SSRs in the intergenic spacer regions (51.8%) was more abundant than that in coding genes (38.6%) or in intronic regions (9.6%) (Fig. 3D).

Fig. 3
figure 3

Analysis of simple repeat sequences (SSRs) in complete chloroplast genomes of Gentiana rigescens and G. cephalantha. (A) Numbers of SSRs of five types; (B) type of shared SSRs among the eighteen plastid genomes; (C) numbers of SSRs in LSC, SSC, and IR regions (IGS), and intronic regions; (D) numbers of SSRs in the coding regions (CDS), intergenic spacer regions

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Comparison of the plastid genome structures of G. rigescens and G. cephalantha

To compare the plastid genome structures of G. rigescens and G. cephalantha, IR region expansion and contraction were analyzed using IRscope. The results showed that the boundaries of the genomes were highly consistent between the two species. The rps19 gene had a 145 bp expansion in the IRb region, and the SSC/IRb junction was located in the overlapping region of ycf1 and ndhF genes. In addition, because ycf1 gene crossed IRa/SSC, most of its sequences were located in the SSC region (4,404 bp), and the remaining sequences were located in IRa (912 bp ), with the same length repetition in IRb (Fig. 4).

Fig. 4
figure 4

Comparison of LSC, SSC, and IR border regions among the plastid genomes of Gentiana rigescens and G. cephalantha. Colored boxes for genes represent the gene position

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For the plastid genomes, nucleotide diversity (Pi) of G. cephalantha was higher than that of G. rigescens. Most of the highly variable sites were concentrated in the LSC and SSC regions. In contrast, the Pi value was relatively low in the IR regions (Table 3). Furthermore, the Pi values were calculated for the coding genes and intergenic spacer regions of the two species. A total of 28 coding genes and 24 intergenic regions in the plastid genomes of G. rigescens showed genetic variability within species, as well as 32 coding genes and 30 intergenic regions in those of G. cephalantha. Among these, five coding genes and seven intergenic regions possessed high nucleotide diversity (Pi > 0.001; Fig. 5).

Table 3 Variable sites in the chloroplast genomes of Gentiana rigescens and G. cephalantha
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Fig. 5
figure 5

Comparative analysis of nucleotide diversity (Pi) values among the plastid genomes of Gentiana rigescens and G. cephalantha. (A) Nucleotide diversity (Pi) values of coding genes; (B) nucleotide diversity (Pi) values of intergenic regions

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The mVISTA analysis showed that the alignments of plastid genomes were highly similar between G. rigescens and G. cephalantha. Hypervariable regions (HVR) were mainly distributed in the LSC region, but few were distributed in the IR regions. Moreover, the genetic variation of the non-coding regions was greater than that of the coding regions. Therefore, most HVR, including trnH-GUG-psbA, atpH-atpI, trnY-GUA-trnT-GGU, psbL-psbF, psbB-psbT, rpl32-trnL-UAG, and rps7-ndhB, were located in the non-coding regions, in addition to psbT, ndhB, rpoC2, and ycf1 present in the coding regions (Fig. 6).

Fig. 6
figure 6

Visualization alignment of the plastid genomes of Gentiana rigescens and G. cephalantha

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Analysis of heterozygotes in ITS sequences

In this study, 18 individuals from G. rigescens and G. cephalantha were used to acquire ITS sequences by the Sanger sequencing. Subsequently, DNA sequences of good quality from 17 individuals were used for final analysis, except that of GcS23, because of confused overlapping peaks. The total length of the alignment of the ITS sequences was 626 bp, including six informative sites (Table 4). Heterozygotes occurred intensively in individuals from the sympatric distribution, especially in those belonging to G. rigescens without rosette leaves. Furthermore, GcS21, GcS24, GrS27, and GrS33 possessed complete heterozygosity across all the variable sites.

Table 4 Variable sites of ITS in Gentiana cephalantha and G. rigescens
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Phylogenetic analysis of the two species

To explore the phylogenetic relationship between G. rigescens and G. cephalantha, maximum likelihood (ML) and Bayesian inference (BI) were mainly performed based on chloroplast genomes, coding sequences (CDS), and ITS sequences; and then phylogenetic analysis using representative HVR was also carried out. The results showed that topological structures between the ML and BI trees were highly identical for plastid datasets and showed little inconformity for the ITS samples (Fig. 7), but they were obviously inconsistent for the HVR sequences (Fig. S2). However, there were significant differences among the trees based on plastid genomes, CDS, and ITS sequences. For all the three datasets, both G. rigescens and G. cephalantha were clustered into a monophyletic branch; however, the phylogeny between the chloroplast genomes and ITS sequences of the two species were completely different (Fig. 7). Individuals of G. rigescens and G. cephalantha were intermingled in the trees of plastid genomes, as well as those of HVR sequences, but could be weakly distinguished by the CDS dataset. On the contrary, the phylogenetic trees based on ITS sequences showed that the two species were well discriminated, except for the potential hybrids, namely GcS21, GrS27, and GrS33 in BI, as well as GcS21 and GcS24 in the ML tree. Moreover, the ML and BI analysis using ITS sequences covering nine sections of Gentiana (14 sect.) supported the independence of sect. Monopodiae separated from sect. Kudoa.

Fig. 7
figure 7

Phylogenetic relationship of Gentiana rigescens and G. cephalantha. (A) complete chloroplast genomes; (B) CDS regions; (C) BI (left) and ML (right) phylogeny based on the ITS sequences. Number above nodes are support values with BI posterior probabilities (PP) values on the left and ML bootstrap (BS) values on the right. Orange fonts represent G. rigescens, dark blue fonts represent G. cephalantha, and grey shades indicate the individuals located in the sympatric distribution

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Discussion

Comparison on the plastid genomes between G. rigescens and G. cephalantha

In this study, the structures of the plastid genomes between G. rigescens and G. cephalantha were highly similar. The total lengths of the genomes were from 146,759 bp to 147,001 bp in G. rigescens and from 146,856 bp to 147,016 bp in G. cephalantha, which were shorter than those of most of the other species in Gentiana, as well as other genera in Gentianaceae [29, 30]. Four common pseudogenes were found in the plastid genomes of G. rigescens and G. cephalantha. Among them, the existence of ψycf1 and ψrps19 could be attributed to boundary effects, whereas ψinfA and ψrps16 are probably caused by gene transfer and loss during evolution, which are also common in species of Gentianaceae and other family [29, 31,32,33,34,35]. In addition, deletion of ndh gene has been frequently reported in Gentiana, but we could not detect it in either of the two species [29].

Simple sequence repeats (SSRs) are useful tools for evaluating population genetic diversity and structure of species and are thus widely adopted in studies on the conservation of endangered species and evolution of complicated groups [36, 37]. The number of SSRs was 35 – 37 in the two species, and mono-nucleotide repeats were the most abundant, especially A/T repeat units. These results were consistent with those of our previous study [30]. In addition, most of the SSRs were distributed in the intergenic spacer region, followed by the coding genes (rpoC1, psaB, atpB, ndhF, ycf1), but the least in the intronic region, which was also similar to those reported for other Gentiana species [35].

Hypervariable regions (HVR) not only resolve phylogeny and identify species at the species level but also provide critical information for exploring species differentiation and genetic structure at the population level [38]. The results of mVISTA and the sliding window have been used for screening the HVR [39, 40]. Herein, we detected eight HVR from non-coding (trnH-GUG-psbA, atpH-atpI, petG-trnW-CCA, rpl32-trnL-UAG, rps7-ndhB) and coding regions (ndhB, rpoC1, psbH), which have been widely adopted for studies on phylogenetic analysis and DNA barcodes in angiosperms [25, 41,42,43,44,45]. In contrast, matK and rbcL as core DNA barcodes showed no genetic variation in G. rigescens and G. cephalantha, further supporting the results of our previous study on DNA barcodes in Gentiana [19]. It should be noted that the trnH-psbA, as an efficient barcode for identifying species, showed the highest Pi value in the sliding window. A possible reason is that Gentiana species are prone to base inversion in this region, leading to overestimation of genetic variation within the species [46]. Therefore, this region was deemed unsuitable to identify Gentiana species, as it has been reported before [19, 20]. Expansion and contraction of the IR regions are important factors leading to changes in plastid genome size and play an important role in the stability and evolution of the genome structure [47,48,49]. The results showed that the genetic compositions of the four junctions in the chloroplast genomes were highly identical between the two species, similar to the findings from studies on other species belonging to the Gentianaceae [29, 31]. The expansion and contraction of rps19, ndhF, and ycf1 located at the boundaries of plastid genomes were consistent between G. rigescens and G. cephalantha, probably due to conservatism during plastid evolution of Gentiana [30].

Phylogenetic analysis and species definition of G. rigescens and G. cephalantha

Currently, plastid genomes have been widely used to reveal the phylogeny of complicated genera or closely related species and developed as field known as phylogenomics [24, 26, 50]. Gentiana is an extremely complicated genus in Gentianaceae with approximately 362 species worldwide, including a series of confusing species such as G. rigescens and G. cephalantha [2]. Although the two species can be discriminated by the basal rosettes of G. cephalantha and its blue corolla, they are still easily confused because of their similar but variable morphology, especially in sympatric distributions [51]. In our study, the two species were clustered into a monophyletic group using plastid genomes, CDS, and ITS datasets, which showed that the two species were closely related in terms of phylogeny. According to the ML and BI analysis, complete chloroplast genomes, coding sequences, and HVR regions could not correctly discriminate individuals from G. rigescens and G. cephalantha; on the contrary, ITS could gather the two species into distinct independent clades after removal of potential hybrids (Fig. 7). First, in the phylogenetic tree based on the plastid genomes, all individuals were heavily mixed which was also supported by comparative analysis of the chloroplast genomes of the two Gentiana species. Comparatively, the tree constructed using CDS showed better resolution, in which most individuals of G. cephalantha (except GcS20) formed a monophyletic group. The present result also supports that from the previous report in which protein-coding sequences were adopted to reveal phylogenetic relationships of species in Gentiana sect. Kudoa and evaluate divergence times [45]. On the contrary, G. rigescens and G. cephalantha were clustered into two independent clades based on ITS sequences, regardless of the possible hybrids (Fig. 7). Therefore, both G. cephalantha and G. rigescens are closely related but independent species, according to their morphological and molecular evidences. The present results further verified the value of ITS as shown in previous phylogenetic analyses of Gentiana species [52]. Moreover, molecular evidence from the current study also supported the adjustment of the two species into sect. Monopodiae from sect. Kudoa [1, 2, 16].

It is well known that the chloroplast genome could provide much richer genetic information and a better solution than DNA regions for revealing the phylogeny of complicated genera and discriminating closely related species [53, 54]. In our previous study, the plastid genome was confirmed to be a DNA super barcode that could efficiently discriminate most species in Fritillaria in China, but the results from ITS and other universal DNA barcodes analyses were disappointing [25, 53]. Zhang et al. [55] adopted plastid phylogenomics to reveal the deep phylogenetic relationships and diversification history of Rosaceae. Li et al. [56] also reported that phylogenomic analyses of Fagopyrum supported the division of the cymosum and urophyllum groups, and resolved the systematic position of subclades within the urophyllum group. However, it should be noted that plastid genomes could enhance species discrimination and reveal phylogeny, but they are still not powerful enough to resolve all species in complicated genera and recently diverged lineages, such as Paris, Berberis, and Rhododendron [26, 57, 58]. This research provides a case study in which only the plastid genome resulted in incorrect assessment of phylogenetic relationships and species discrimination; therefore, nuclear genes should be adopted alongside plastid genomes to reveal intrinsic phylogeny and trace species boundaries.

Species evolution in Gentiana and potential hybridization between G. rigescens and G. cephalantha

According to previous studies, both G. rigescens and G. cephalantha are widely distributed in Yunnan, Sichuan, Guizhou and adjacent regions with obvious geographical overlap [1, 2]. Field investigations revealed that the two species are generally distributed in allopatric regions. G. rigescens narrowly grows at lower altitudes, but G. cephalantha can occur in a much wider range at higher altitude gradients (Fig1). It is well known that the Qinghai-Tibet Plateau (QTP) and its adjacent mountains are key regions for evolution of alpine plants, and Gentiana was revealed to originate from QTP in the Eocene and then spread to other distributions from the late Miocene onwards, a phenomenon named as the "out of Tibet" hypothesis [59]. According to molecular clock dating using pollen and seed fossils, divergence time of Gentiana was deduced to be 29 million years, and the divergence of G. rigescens and G. cephalantha happened about 0.51 Ma and supposedly is the youngest node of differentiation. Obviously, G. cephalantha is more adaptable to the cooling climate and diverse habitats, so it colonizes a much broader territory than G. rigescens in altitude gradients (Fig. 1). The two species generally grow in allopatric areas at altitudes, except for a few sympatric distributions that are generally located in the border zones between G. rigescens and G. cephalantha. In the present study, potential hybridization was observed in sympatric distributions in the Diancang Mountains, which showed a visible intermediate corolla color compared to typical G. rigescens and G. cephalantha (Fig. S1); meanwhile, possible hybrids were preliminarily identified based on the ITS sequences (Table 4). Natural hybrids between Gentiana straminea and G. siphonantha have been confirmed based on molecular evidence [60]. Considering the overlapping of flowering, geographic isolation might be the main factor for the two species, but possible physiological factors should be explored using pollination biology and other evidences [61].

Threats to G. rigescens from hybridization and the balance between conservation and utilization of the endangered species

Natural hybridization occurs widely among species and evolutionary lineages in plants and plays critical role in species evolution [62, 63]. Hybridization probably leads to phenotypic novelty and results in the formation of new species [64]. Meanwhile, the same process may cause the breakdown of species integrity and ultimately drive rare species to extinction through genetic swamping, where the rare form is replaced by hybrids or demographic swamping [63, 65]. G. rigescens, a rare species, is threatened by hybridization with its common congeneric species. According to the ITS sequences of the 17 individuals of the two species (Table 4 and Fig. 7), hybridization was massively detected in the sympatric distribution and sporadically in the territory of G. rigescens, but seemingly not in the allopatric distribution of G. cephalantha. Therefore, asymmetric introgression had a significantly more serious impact on G. rigescens than G. cephalantha. Hybridization, as well as subsequent backcrossing between hybrids and the common species, constantly dilutes the genetic loci of the rare one [65,66,67], which finally results in genetic erosion of G. rigescens. As two sister species, no steady reproductive barrier exists between G. rigescens and G. cephalantha, in addition to geographic isolation along altitude gradients. Habitat change along with human activities provides more possibilities for hybridization between the newly divergent species and affords new niches to hybrids [63]. As a result, the rare species may be replaced by hybrids, even faced with extinction due to genetic swamping or demographic swamping from hybridization [63, 66,67,68].

As an endangered species possessing medicinal value, balance between efficient conservation and rational utilization of G. rigescens is an urgent problem to be resolved. First, strict conservation of the species should be adopted for basic measurements, so as to avoid serious harvesting and destruction. Then, exploring new original species of Gentianae Radix et Rhizoma by pharmacophylogeny could be an efficient method to satisfy medicinal demands, such as G. cephalantha [69]. Although both species are used as traditional Chinese medicine “Jianlongdan” in Yunnan and adjacent regions, further evaluation of the medicinal quality and efficacy of medicines between the two species is necessary. Moreover, developing artificial cultivation for G. rigescens would be beneficial for balancing conservation and utilization. It should be noted that screening pure individuals without genetic pollution is critically important to ensure the quality of the medicine in consideration of hybridization and genetic introgression. Finally, protecting habitat diversity could provide different niches for G. rigescens and G. cephalantha, and geographic isolation might be an efficient reproductive barrier to maintaining the independence of the species [66, 68].

Conclusion

In this study, the next-generation sequencing and Sanger sequencing were used to investigate plastid genomes and ITS sequences of G. rigescens and G. cephalantha from sympatric and allopatric distributions. Comparative analysis of the plastid genomes showed that the two Gentiana species possess highly similar genome structures. G. rigescens and G. cephalantha were clustered into a monophyletic group during phylogenetic analyses based on the plastid genome, CDS, and ITS regions, supporting their close relationship. Moreover, the two species were mixed in the trees constructed using plastid genomes, CDS, and HVR, but they could form obvious monophyly in phylogenetic analysis based on ITS, excluding the potential hybrids. The present study supports the current treatment of G. rigescens and G. cephalantha as independent but closely related species in Gentiana based on the present morphological and molecular evidences. Natural hybridization occurs intensively between the two species, especially in sympatric distribution. Asymmetric introgression has a significantly more serious impact on G. rigescens than G. cephalantha, and this process probably leads to constant dilution of genetic loci and even extinction of the rare species. Conserving wild resources, exploring alternative species, developing artificial cultivation, and maintaining habitat diversity are efficient measurements for ensuring a balance between conservation and utilization. In summary, the complete chloroplast genome possesses rich genetic information; thus, it can be used to explore the phylogenetic relationships of closely related species or complicated genera. However, phylogenetic trees might not reveal real phylogeny when we only use plastid genomes or genes because of matrilineal inheritance, and datasets from nuclear genomes or regions are crucial for exploring the truth.

Methods

Materials collection

Dali (Tali) in China is the origin of the type specimens of G. rigescens and G. cephalantha. In the present study, fresh and clean leaves of adult individuals were collected from the Diancang and Luoping Mountians in Dali, China (Fig. 1 and Table 1). The leaves were directly dried using allochroic silica gel during fieldwork and were used as molecular materials. Herein, we sampled six accessions of each species from their sympatric distribution in the Diancang Mountians (2,422 m). Considering the morphological similarity between the two species, rosette leaves and blue corolla were regarded as distinctive features for G. cephalantha that was discriminated from G. rigescens (Fig. 1). Three individuals of G. cephalantha were also sampled from an allopatric distribution of alpine shrubbery in the alpine region of the same mountain (3,402m), and three individuals representing G. rigescens were collected from one allopatric distribution in the Luoping Mountains (Fig. 1). Eight individuals representing the two species were used for DNA sequencing. To avoid sampling individuals from the same female parent, geographic distances among individuals were above 30 m. Meanwhile, 2-3 mature individuals with flowers from each site were excavated and used as the voucher specimens (Table 1). The collection of molecular materials and specimens was approved by the Forestry and Grassland Administration in Dali Prefecture, China (Grant no. 2021-137). All specimens were identified by Professor Dequan Zhang according to Ho’s classification system (1995) and Flora of China (2001) [1, 2] and then deposited at the Herbarium of Medicinal Plants and Crude Drugs of the College of Pharmacy, Dali University.

Molecular experiments

Total genomic DNA was extracted from the dried leaves using a modified CTAB method [70]. DNA quality and concentration were detected using 1.2% agarose gel electrophoresis and spectrophotometer (Bio-Rad, Hercules, CA, USA). The DNA was then sheared to yield approximately 500 bp long fragments for library construction. The library was sequenced on an Illumina Hiseq 2500 platform according to the standard protocol of manufacturer’s instructions. About 2-4 Gb raw paired-end reads (2×150 bp) were obtained for each individual of the two species. Next-generation sequencing were performed by Novogene Bioinformatics Technology Co. Ltd., Beijing, China.

The amplification reaction system of ITS sequence was: 2×Taq PCR MasterMix (10 μl), two ITS primers (0.3 μl), template DNA (1 μl) and ddH2O (8.4 μl). The PCR amplification procedure was as follows: pre-denaturation at 94°C for 2 min; 94°C for 40 s, 55°C for 45 s, and 72°C for 55 s, after 35 cycles; 72°C extension for 7 min primer sequence: ITS4: 5'-TCC TCC GCT TAT TGA TAT GC-3'; ITS5: 5'-GGA AGT AAA AGT CGT AAC AAG G-3'. The entire ITS sequence was sequenced by the Kunming Institute of Botany, Chinese Academy of Sciences.

Assembly, annotation, and submission of plastid genomes and ITS sequence

Raw data were filtered using Trimmomatic v.0.32 with default settings [71], and paired-end reads in the clean data were assembled into contigs using GetOrganelle.py [72]. After assembly, the de novo assembly graphs were visualized and edited using Bandage, and then a whole or nearly whole circular plastid genome was generated [73]. Using G. rigescens (MT062862) downloaded from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/) as the reference sequence, MAFFT was performed in the Geneious v.11.1.4 [74, 75], and annotation, modification and manual correction were performed. Circular genome visualization was generated using the online program OGDRAW (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html).

Original ITS DNA sequences were assembled using the SeqMan program (DNASTAR, Lasergene) [76] and then aligned in MEGA v.7.0.26 [77]. The boundaries of ITS1, 5.8S, and ITS2 were determined using the reference sequence of G. cephalantha (KT907627) from NCBI. All annotated plastid genomes and ITS sequences were confirmed, edited using Sequin software, and submitted to the GenBank database (Table 1).

Comparative analysis of the plastid genomes

Using the website IRscope (https://irscope.shinyapps.io/irapp/), the IR boundaries of 18 plastid genomes of G. rigescens and G. cephalantha were analyzed [78], followed by manual editing. The lengths of large single copy (LSC), small single copy (SSC), inverted repeats (IRs), protein-coding genes, and intergenic spacer regions for each of genomes were counted by Geneious v.11.1.4. The online program mVISTA (https://genome.lbl.gov/vista/mvista/instructions.shtml) was used for sequence alignment and variation analysis of plastid genomes in Shuffle-LAGAN mode [79, 80], with the annotation of G. cephalantha (MN199135) as a reference. In addition, parsimony informative sites and variable sites were analyzed, and a sliding window was used to analyze the nucleotide diversity (Pi) of complete chloroplast genomes and protein-coding genes in G. rigescens and G. cephalantha by DnaSP v.6.11 (with a window length of 600 bp and a step size of 200 bp) [81]. MISA was adopted to evaluate SSRs in plastid genomes, among which the SSRs parameters of mono-, di-, tri-, tetra-, penta-, and hexa-nucleotide motifs were set as 10, 5, 4, 3, 3, and 3 [82].

Phylogenetic analysis for G. rigescens and G. cephalantha

A total of 18 new plastid genomes from G. rigescens and G. cephalantha, and 23 published plastid genomes for other Gentiana species from NCBI were used for phylogenetic analysis (Table S2). In addition, phylogenetic trees were constructed based on the 17 new ITS sequences (excluding GcS23) and 32 reported ITS sequences from other Gentiana species (Table S3). Two Gentianopsis species were selected as outgroups for constructing phylogenetic trees based on the plastid genomes, CDS, and ITS datasets; moreover, phylogenetic analysis using HVR was additionally performed for the two species. The most appropriated model of sequence substitution for plastid genomes (GTR + G + I), CDS (GTR + G + I), HVR (GTR or GTR+G), and ITS (GTR + G + I) were screened by MEGA v.7.0.26 [78]. Phylogenetic analysis was performed using Maximum likelihood (ML) and Bayesian inference (BI). ML analysis was performed using the RAxML v.8.2.10. The local bootstrap (BS) probability of each branch was calculated with 1,000 repetitions [83]. BI analysis was performed using MrBayes v.3.2.6 [84]. The Markov Chain Monte Carlo (MCMC) algorithm was calculated for 1000,000 generations with a sampling of trees every 1,000 generations. The first 25% of the generations were discarded as burn-in, and posterior probability (PP) values were determined from the remaining trees to evaluate the support rate of each branch. The state was considered to have been reached when the average standard deviation of the split frequency was < 0.01. Finally, all the methods were performed in accordance with relevant guidelines and regulations.

Availability of data and materials

All sequences (plastid genomes and ITS sequences) used in this study have been submitted to the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/) with accession numbers (OM961144, OM961148, OM961150, OM9652-OM961154, OM961160, OM961164, OM961165, and OM961167-OM961175 for plastid genomes; ON820192-ON820203 for ITS sequences) (Table 1). All the sequences will be available after publication of this manuscript.

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Acknowledgements

We thanks Miss Haisu Hu and Qian Zhang in Dali University for their assistance in field work for sample collection and data analysis of plastid genomes.

Funding

This research was supported by National Natural Science Foundation of China (32060091, 31660081), Reserve Talents Project for Young and Middle-Aged Academic and Technical Leaders of Yunnan Province (202105AC160063).

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  1. College of Pharmacy, Dali University, Dali, 671000, Yunnan, China

    Jiuyang Mao, Yuze Liang, Xue Wang & Dequan Zhang

  2. Yunnan Key Laboratory of Screening and Research on Anti-pathogenic Plant Resources from Western Yunnan (Cultivation), Dali, 671000, Yunnan, China

    Dequan Zhang

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Contributions

JM performed molecular experiments, analyzed the data, and drafted the manuscript. YL and XW participated in field work and data analysis. DZ conceived this study, collected molecular materials and specimens, and revised the manuscript finally. All authors have read and approved the manuscript.

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Correspondence to Dequan Zhang.

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The collection of molecular materials and specimens had been approved by the Forestry and Grassland Administration in Dali Prefecture, Yunnan, China (Grant no. 2021-137).

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Supplementary Information

Additional file 1: Figure S1.

Phenotype of potential hybrids between Gentiana rigescens and G. cephalantha.

Additional file 2: Figure S2.

Phylogenetic relationships of Gentiana rigescens and G. cephalantha based on each HVR regions.

Additional file 3: Table S1.

Gene contents of the plastid genomes of Gentiana rigescens and G. cephalantha.

Additional file 4: Table S2.

The complete chloroplast genomes of Gentiana species downloaded from NCBI.

Additional file 5: Table S3.

The nrITS sequences of Gentiana species downloaded from NCBI.

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Mao, J., Liang, Y., Wang, X. et al. Comparison of plastid genomes and ITS of two sister species in Gentiana and a discussion on potential threats for the endangered species from hybridization. BMC Plant Biol 23, 101 (2023). https://doi.org/10.1186/s12870-023-04088-z

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BMC Plant Biology

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