The complete chloroplast genome of Lilium paradoxum Stearn (Liliaceae) from southwestern China

Abstract

Lilium paradoxum, a herb from southeastern Xizang, China, has its first complete chloroplast genome sequenced using next-generation sequencing. The genome is 151,814 bp, consisting of inverted repeats (IRs; 26,323 bp), small single-copy (SSC; 17,524 bp), and large single-copy regions (LSC; 81,644 bp). It encodes 112 unique genes: 78 protein-coding, 30 tRNA, and 4 rRNA genes. Phylogenetic analysis of 22 genomes shows L. paradoxum is closely related to L. gongshanense, L. apertum, and L. souliei. These findings enhance understanding of Lilium's phylogenetic relationships and evolution, particularly the Nomocharis clade in the Hengduan Mountains.

Introduction

Lilium paradoxum Stearn (1956), is an herbaceous plant belonging to the genus Lilium. It thrives in alpine meadows at elevations surpassing 3200 meters in the southeastern Xizang, with purplish-red, bell-shaped flowers nod gracefully, bearing a striking resemblance to those of L. souliei (Franch.) Sealy, distinguished primarily by the exquisite symmetry of its whorled leaves (Figure 1; Stearn 1956; Liang 1980; Liang and Tamura 2000). The genetic relationship based on internal transcribed spacer (ITS) and chloroplast genome fragments reveals that it belongs to Nomocharis clade, which exhibits the contradiction of high divergence in morphology but with low genetic distancing (Liang and Tamura 2000; Gao et al. 2012, 2013, 2015). More genetic information is needed to resolve their phylogenetic relationships. Chloroplasts, vital for photosynthesis, carry genetic information independent of nuclear DNA and encode essential proteins for photosynthesis and biosynthesis (Daniell et al. 2016). With low recombination and nucleotide substitution rates, and typically uniparental inheritance, chloroplast genomes are powerful tools for plant phylogeny studies (Shaw et al. 2005; Tonti-Filippini et al. 2017; Feng et al. 2022).

Figure 1.

Field picture of L. paradoxum, the photo taken by Yun-Dong Gao. L. paradoxum grows in shrubbery, featuring purple bell-shaped flowers and delicately symmetrical whorled leaves that are ovate in shape.

In the present study, we investigate how L. paradoxum contributes to understanding the genetic diversity within the genus Lilium by providing an effective phylogenetic framework based on newly assembled and existing chloroplast genome data. We aim to provide critical insights into the evolutionary mechanisms and the diversification within the genus.

Materials and methods

The fresh leaf samples of Lilium paradoxum (Figure 1) were collected from Xizang Province, China (Bomi County: 29.861603° N, 95.766696°E) and dried in silica gel. Voucher specimens were deposited in the Herbarium of the Chengdu Institute of Biology (CDBI) under voucher specimen number GYD1507 (contact person: Yun-Dong Gao, gaoyd@cib.ac.cn).

DNA was extracted from silica-gel dried leaves using the Doyle and Doyle (1987) protocol. Paired-end sequencing libraries were meticulously constructed with insert sizes of approximately 350 bp, and the sequencing was performed using the DNBseq-4000 platform at the Beijing Genomics Institution (BGI; Shenzhen, China).

About 13 Gb of raw data were filtered by fastp v0.23.2 (Chen et al. 2018) with default parameters and assembled using GetOrganelle v1.7.6.1 (Jin et al. 2020). The chloroplast genome was annotated, and manual corrections were made using Geneious Prime v2023.1.2 (Biomatters Ltd., Auckland, New Zealand) (https://www.geneious.com) and Geseq v.2.03 (Tillich et al. 2017) (https://chlorobox.mpimp-golm.mpg.de/ geseq.html) based on the chloroplast (cp) genome of L. gongshanense (NC_052787) and L. pardanthinum (MG704135). The chloroplast genome map was generated using CPGview (Liu et al. 2023) (http://www.1kmpg.cn/cpgview).

Phylogenetic analysis

To clarify the genetic relationship of Lilium paradoxum, we reconstructed a phylogenetic analysis based on the whole chloroplast genomes of lily species. We selected 18 complete chloroplasts from various clades of the genus Lilium, including the focal species (L. paradoxum), 3–5 representative species each from clades Lophophorum and Nomocharis, and 1–2 species from the rest clades. Additionally, we included 3 outgroups from closely related genera. The selection of sequences is based on our previous work (Gao et al. 2012, 2013, 2015).

The sequences were aligned using MAFFT v7.508 (Katoh and Standley 2019). Ambiguously aligned and low mutation sites were filtered using Gblocks v0.9b (Talavera and Castresana 2007) with default parameters. The best nucleotide substitution model was identified using ModelTest-NG v0.1.7 (Darriba et al. 2020). Bayesian Inference (BI) analyses were performed using MrBayes v3.2 (Ronquist et al. 2012) with the GTR+G + I (lset nst = 6 rates = invgamma) model. Posterior probabilities were estimated using two independent Markov Chain Monte Carlo (MCMC) chains (10 million generations), with the initial 25% of sampled data discarded as burn-in. The phylogenetic tree was enhanced using iTOL v6 (https://itol.embl.de).

Result

From 12.98 GB of raw data, 12.95 GB was retained after filtering. The raw data are available in the China National Center for Bioinformation (CNCB, https://www.cncb.ac.cn/services) under the BioProject PRJCA022586. The average coverage for the assembled cp genome was 4258× (Figure S1). The complete chloroplast genome of Lilium paradoxum was submitted to GenBank under accession number PP073960 and exhibits a typical circular quadripartite structure, composed of a large single-copy (LSC) region of 81,644 bp, a small single-copy (SSC) region of 17,524 bp, and two inverse repeats (IR) regions of 26,323 bp each. The overall GC content stands at 37.0% (Figure 2). A total of 130 genes were annotated (112 unique), including 8 rRNA genes (4 unique), 38 tRNA genes (30 unique), and 84 protein-coding genes (78 unique). Among these genes, the rps12 gene stands out as a notable trans-splicing gene, characterized by its 5′-exon residing in the LSC region and the other end situated in the IR regions. Additionally, 8 tRNA genes (trnA-UGC, trnH-GUG, trnI-CAU, trnI-GAU, trnL-CAA, trnR-ACG, trnV-GAC and trnN-GUU), 6 protein-coding genes (ycf2, ndhB, rpl2, rpl23, rps7 and rps12), and 4 rRNA genes (rrn23S, rrn16, rrn5S and rrn4.5S) were duplicated due to their location within the IR regions (Figure S2 and S3).

Figure 2.

Chloroplast genome map of L. paradoxum. The map was generated by CPGView. Genes located on the inner and outer of the circle are transcribed clockwise and anticlockwise, respectively. The dark grey inner circle indicates GC content. Large single-copy (LSC), small single-copy (SSC), and inverted repeats (IRA and IRB) are indicated in the inner layer. The functional classification of the genes is provided in the bottom left corner.

To clarify the evolutionary and position of L. paradoxum, we utilized three closely related genus species as outgroups and constructed Bayesian Inference (BI) tree based on 18 representative lilies from different clades in the genus Lilium, and the results (Figure 3) mostly consistent with previous studies (Gao et al. 2012, 2013, 2015). The clade Nomocharis and Lophophorum form two well-supported monophyly, and L. paradoxum is in the former, being most closely related to L. gongshanense (Y.D. Gao et X.J. He) Y.D. Gao, L. apertum Franch. 1898 and L. souliei. L. paradoxum and its closest relatives form a distinct clade, with a well-resolved phylogenetic relationship (PP = 1.00).

Figure 3.

The phylogenetic relationships in 22 representative species in Lilium using Bayesian Inference (BI) based on whole plastome sequence data. Bayesian posterior probabilities (PP) on a branch, and PP =1.00 not displayed. L. paradoxum are marked in red. Sequences used for tree construction were as follows: L. amabile, KY940845.1 (Kim et al. 2017); L. apertum, MK493293.1; L. candidum, NC_042399.1 (Kim et al. 2019); L. gongshanense, NC_052787.1; L. hansonii, KM103364.1 (Kim et al. 2016); L. lancifolium, NC_035589.1 (Du et al. 2017); L. longiflorum, KC968977.1 (Du et al. 2017); L. lophophorum, MK493298.1 (Su et al. 2021); L. matangense, MN745201.1 (Su et al. 2021); L. meleagrinum, NC_052788.1; L. nanum, MK493300.1 (Su et al. 2021); L. paradoxum, GYD1507 PP073960; L. philadelphicum, KY940847.1 (Kim et al. 2017); L. regale, MK493302.1; L. sealyi, NC_060548.1 (Li et al. 2021); L. souliei, MW085076.1; L. taliense, KY009938.1 (Zhang et al. 2017); L. tsingtauense, KU230438.1 (Song et al. 2016); L. washingtonianum, MG590100.1 (Kim and Lim 2018); Outgroups: Fritillaria yuminensis, NC_037209.1 (Li et al. 2017); Cardiocrinum giganteum, KX528334.1 (Lu et al. 2016); Notholition macrophyllum, MH011354.1 (Li et al. 2018).

These findings affirm the distinct placement of L. paradoxum within the Nomocharis clade and its close phylogenetic relationship with L. gongshanense, L. apertum, and L. souliei, corroborating previous phylogenetic research.

Discussion and conclusions

The detailed chloroplast genome analysis reveals a typical circular quadripartite structure, with notable gene duplication within the inverse repeat regions. The duplication includes 8 tRNA genes, 6 protein-coding genes, and 4 rRNA genes, which may contribute to the robustness and adaptability of L. paradoxum. The concordance between the chloroplast genome data and the phylogenetic tree underscores the evolutionary significance of L. paradoxum’s placement and enhances our understanding of the genetic underpinnings behind its unique morphological and ecological adaptations (Liao et al. 2021; Feng et al. 2022; Yuan and Gao 2024). The genomic data not only support the placement of L. paradoxum within the Nomocharis clade but also shed light on its close phylogenetic relationships with L. gongshanense, L. apertum, and L. souliei (Figure 3). While the IR regions are more conserved, the LSC and SSC regions show higher nucleotide polymorphism among these species (Yuan and Gao 2024).

This study also provides a genetic basis for parallel evolution within the genus Lilium, particularly in the Nomocharis clade (Sun et al. 2012; Zhang et al. 2014; Gao et al. 2015; Yuan and Gao 2024). Closely related species exhibit similar traits due to environmental pressures. For instance, lily species located at higher altitudes, where they are exposed to heavy rain and strong winds, tend to have bell-shaped flowers that protect their fragile reproductive structures, while species found in shrublands at lower elevations often favor saucer-shaped flowers to attract pollinators (Gao et al. 2015; Lawson and Rands 2019; Yuan and Gao 2024).

Overall, this first reported cp genome of L. paradoxum is a significant addition to the plastid genome for the whole genus Lilium, elucidates the evolutionary position of L. paradoxum and lays the groundwork for subsequent studies on the phylogeny, as well as the unique Nomocharis clade species endemic to the Hengduan mountains.

Funding Statement

This study was supported by the National Natural Science Foundation of China [NSFC Grant No. 32171605] and Natural Science Foundation of Sichuan Province [No. 2023NSFSC0141] to Yun-Dong Gao.

Ethical approval

The collection of leaf samples conformed to the requirement of international ethics, which did not cause damage to the local environment. No endangered or protected species were involved in the study, and the collecting of the samples did not require specific permission from authorities.

Authors’ contributions

LL, YMY and YDG designed the study. YLJ, YMY and YXW performed data analysis. LL, JHM and QLH drafted and YDG revised the manuscript. All authors reviewed and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

The data that support the findings of this study are openly available in GenBank number PP073960 (https://www.ncbi.nlm.nih.gov/nuccore/PP073960) and the related BioProject, raw sequencing files in SRA, and the Bio-Sample number are PRJCA022586, CRR1005869 and SAMC3300506 (https://www.cncb.ac.cn/services), respectively.