# Comparative Analysis of the Complete Chloroplast Genomes inAllium Subgenus Cyathophora (Amaryllidaceae): Phylogenetic Relationship and Adaptive Evolution

Authors: Xin Yang; Deng-Feng Xie; Jun-Pei Chen; Song-Dong Zhou; Yan Yu; Xing-Jin He
Journal: BioMed Research International
 (2020)

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## Abstract

Recent advances in molecular phylogenetics provide us with information ofAllium L. taxonomy and evolution, such as the subgenus Cyathophora, which is monophyletic and contains five species. However, previous studies detected distinct incongruence between the nrDNA and cpDNA phylogenies, and the interspecies relationships of this subgenus need to be furtherly resolved. In our study, we newly assembled the whole chloroplast genome of four species in subgenus Cyathophora and two allied Allium species. The complete cp genomes were found to possess a quadripartite structure, and the genome size ranged from 152,913 to 154,174 bp. Among these cp genomes, there were subtle differences in the gene order, gene content, and GC content. Seven hotspot regions (infA, rps16, rps15, ndhF, trnG-UCC, trnC-GCA, and trnK-UUU) with nucleotide diversity greater than 0.02 were discovered. The selection analysis showed that some genes have elevated Ka/Ks ratios. Phylogenetic analysis depended on the complete chloroplast genome (CCG), and the intergenic spacer regions (IGS) and coding DNA sequences (CDS) showed same topologies with high support, which revealed that subgenus Cyathophora was a monophyletic group, containing four species, and A. cyathophorum var. farreri was sister to A. spicatum with 100% bootstrap value. Our study revealed selective pressure may exert effect on several genes of the six Allium species, which may be useful for them to adapt to their specific living environment. We have well resolved the phylogenetic relationship of species in the subgenus Cyathophora, which will contribute to future evolutionary studies or phylogeographic analysis of Allium.


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## Body

## 1. Introduction

SubgenusCyathophora (R. M. Fritsch) R. M. Fritsch is a small group of Allium that has been put forward lately [1]. The special subgenus Cyathophora contains about six species and one variety according to Li et al. [2]; besides, A. spicatum (Prain) N. Friesen has a wild distribution range, extending from China to Nepal, while the rest of them are endemic species in China and mainly distributed in the southeastern margin of the Qinghai-Tibet Plateau (QTP): A. mairei Lév, A. kingdonii Stearn, A. rhynchogynum Diels, A. trifurcatum (F. T. Wang and Tang) J. M. Xu, and A. cyathophorum Bur. and Franch and its variety A. cyathophorum var. farreri (Stearn) Stearn. Although it contains a small number of species, the boundary of subgenus Cyathophora and the involved species have experienced some alterations with the development of molecular biology. In previous study, A. spicatum was classified at different taxonomic levels because of its idiographic spicate inflorescence based on morphological and molecular evidences [3]. Five species have been proposed by Huang et al. about subgenus Cyathophora [4]: A. mairei, A. rhynchogynum, A. cyathophorum, A. cyathophorum var. farreri, and A. spicatum, while A. kingdonii and A. trifurcatum did not belong to this group. Micromorphological and cytological features supported that the subgenus Cyathophora is a monophyly and contains five species [5, 6]. Among species of the subgenus Cyathophora, A. spicatum grows in the droughty western QTP with the extremely abnormal spicate inflorescence [3], while A. cyathophorum and A. cyathophorum var. farreri with the umbel inflorescence stretch to the moist HMR [7] (Figure 1). Furthermore, Li et al. [6] suggested that A. cyathophorum and A. farreri were independent species based on molecular phylogeny and the striking distinctiveness in micromorphology. A. rhynchogynum has never been sampled since it was published in 1912 [8]. We also performed a lot of field work to collect it but failed. The Flora of China recorded that A. rhynchogynum only distributed in northwest of Yunnan province in China. Therefore, we speculate that A. rhynchogynum might become extinct or there is an identification error in previous research studies. Li et al. [6] performed phylogenetic and biogeographic analyses for A. cyathophorum and A. spicatum based on chloroplast and nuclear ribosomal DNA and detected distinct different topologies between these two molecular methods, in which A. cyathophorum showed close relationship with A. spicatum in nuclear DNA tree but was sister to A. cyathophorum var. farreri in cpDNA tree [4, 6]. Other than this, the relationship between these species is not exactly determined, and phylogenetic analysis using single or several combined chloroplast fragments does not solve the problem effectively, and the complete cp genome can well resolve the relationship of subgenus Cyathophora. Hence, it is imperative to reconstruct the relationship of subgenus Cyathophora and clarify the contained species depending on the complete chloroplast genomes. To evaluate the subgenus Cyathophora resources comprehensively, we also need more efficient molecular markers.Figure 1
The morphological characters of flowers of subgenusCyathophora species. (a) A. cyathophorum. (b) A. cyathophorum var. farreri. (c) A. mairei. (d) A. spicatum.
(a)
(b)
(c)
(d)Chloroplast is one of the basic organelles in plant cells, which is in charge of photosynthesis of green plants [9]. The chloroplast genomes have a highly conserved structure and gene content, which have a quadripartite structure composed by large single-copy (LSC) and small single-copy (SSC) regions separated by two parts of inverted repeat (IR) [10, 11]. Previous studies suggested that genome size of angiosperms ranged from 120 kb to 170 kb with gene number changed from 120 to 130 [12]. Complete chloroplast genome has long been a core issue in plant molecular evolution and systematic studies because of its oversimplified structure, highly conservative sequence, and maternal hereditary traits [13]. Since the complete cp genome analysis can provide more genetic information contrasted with just single or few cpDNA fragments [14], by using cp genome sequences, many long existing phylogenetic problems of different angiosperms at various taxonomic levels have been successfully resolved [15–20].In addition to exploring phylogenetic studies, the whole cp genome has important significance to reveal the photosynthesis mechanism, metabolic regulation, and adaptive evolution of plants. Research has shown that adaptive evolution is mainly promoted by evolutionary processes like natural selection, which affects genetic changes caused by genetic recombination and mutations [21]. Many recent studies have analyzed the selection pressures that undergo by species in the evolutionary processes based on complete chloroplast genome, for example, a positive selection for the atpF gene may suggest that it has made an important impact on the divergence in deciduous and evergreen oak tree [9], and there also existed positive selection on ycf2 in watercress chloroplasts [22]. With the development of sequencing technology, the number of cp genomic sequences has increased dramatically in recent years. However, a few plastid genomes of Allium were reported until now, and it is necessary to develop more complete chloroplast genome in Allium for future phylogenetic and evolutionary research studies.In our report, we assembled and characterized the complete cp genome sequence of the sixAllium species using next-generation sequencing technologies to (1) reveal common structural patterns and hotspot regions, (2) gain a better understanding of the relationship about subgenus Cyathophora based on complete chloroplast genome, and (3) investigate adaptive evolution in the cp genomes of the six Allium species. We hope our study will provide valuable genetic resources for further evolutionary studies about subgenus Cyathophora.


## 2. Materials and Methods

### 2.1. Plant Materials, DNA Extraction, and Sequencing

Fresh leaves ofA. cyathophorum, A. cyathophorum var. farreri, A. spicatum, A. mairei, A. trifurcatum, and A. kingdonii were collected from different places (Table 1). Morphological characters were measured using karyotype [23]. The healthy leaves were immediately dried with silica gel to use for DNA extraction. The voucher specimens were stored in the Herbarium of Sichuan University (SZ Herbarium). Their total genomic DNA was extracted from the sampled leaves according to the manufacturer's instructions for the Plant Genomic DNA Kit (Tiangen Biotech, Beijing, China). Genomic DNA was indexed by tags and pooled together in one lane of Illumina HiSeq platform for sequencing (paired-end, 350 bp) at Novogene (Beijing, China).Table 1
Samples information.
Species
Location
Geographical coordinate
A. cyathophorum
Mangkang, Tibet
29°43′24″N, 98°31′51″E
A. cyathophorum var. farreri
Zhouqu, Gansu
33°47′10″N, 104°22′7″E
A. spicatum
NaiDong, Tibet
29°13′40″N, 91°45′36″E
A. mairei
Shangri-La, Yunnan
27°25′19″N, 100°09′22″E
A. trifurcatum
Shangri-La, Yunnan
27°52′22″N, 99°43′10″E
A. kingdonii
Nyingchi, Tibet
29°37′27″N, 94°39′2″E


### 2.2. Chloroplast Genome Sequence Assembly and Annotation

We firstly used FastQC v0.11.7 to assess the quality of all reads [24]. To select the best reference, we filtrated the chloroplast genome related reads by mapping all reads to the published chloroplast genome sequences in Allium. SOAPdenovo2 was used to assemble all relevant reads into contigs [25]. The clean reads were assembled using the program NOVOPlasty [26] with the complete chloroplast genome of its close relative A. cepa as the reference (GenBank accession no. KM088014). Geneious 11.0.4 was used to finish the annotation of the assembled chloroplast genome, and it was corrected manually after comparison with references [27]. The circular plastid genome maps were generated utilizing the OGDRAW program [28]. The GenBank accession numbers of A. cyathophorum, A. cyathophorum var. farreri, A. spicatum, A. mairei, A. trifurcatum, and A. kingdonii are MK820611, MK931245, MK931246, MK820615, MK931247, and MK294559, respectively.


### 2.3. Repeat Sequences and Simple Sequence Repeat (SSR) Analysis

REPuter [29] was selected to investigate the location and size of repeat sequences, which included four types of repeats in the chloroplast genomes about the six Allium species. The sequence identity and minimum length of repeat size was set to >90% and 30 bp, with the hamming distance of 3. We used IMEx, ImperfectMicrosatelliteExtractor (http://43.227.129.132:8008/IMEX/imex_advanced.html) [30], to find chloroplast SSRs in six chloroplast genome sequences of Allium. Its specifications were set up as follows: the minimum number of repeats for mononucleotide, dinucleotides, trinucleotides, tetranucleotides, pentanucleotide and hexanucleotides was 10, 5, 5, 4, 3, and 3, respectively, the repeat type was imperfect, the imperfection % was Mono: 10%, Di: 10%, Tri: 15%, Tetra: 20%, Penta: 5%, and Hexa: 5%, mismatches allowed in pattern were Mono: 1, Di: 1, Tri: 2, Tetra: 4, Penta: 0, and Hexa: 3, and the level of standardization was level 1 standardization.


### 2.4. Codon Usage Analysis

Codon usage of the species in subgenusCyathophora was analyzed by the software of CodonW [31]. Protein-coding genes (CDS) were selected with the following filter requirements: (1) each CDS was longer than 300 nucleotides [18, 32]; (2) repeat sequences were deleted. Totally, 53 CDS of each species in Allium were selected for further study.


### 2.5. Genome Comparison (IR Contraction and Expansion)

The mVISTA program was chosen to analyze the whole sequence similarity of all sixAllium species with Shuffle-LAGAN model [33], using the chloroplast genomes to compare their difference in sequences at the chloroplast genome level and A. cyathophorum as the reference. The boundaries between single copy regions (LSC and SSC) and inverted repeats (IR) regions among the six chloroplast genome sequences were compared by using Geneious v11.0.4 software [27].


### 2.6. Hotspot Regions Identification in SubgenusCyathophora

To analyze nucleotide diversity (Pi), we extracted the shared 112 genes of the six species inAllium after alignment. DnaSP 5.10 was employed to calculate the nucleotide variability [34].


### 2.7. Gene Selective Pressure Analysis of SixAllium Plastomes

To investigate selection pressures, nonsynonymous (Ka) and synonymous (Ks) substitution rates of 65 selected protein-coding genes between the cp genomes of subgenusCyathophora and the other two Allium species were calculated by KaKs Calculator version 2.0 [35].


### 2.8. SubgenusCyathophora Phylogenomic Analysis Based on Chloroplast Genome

Phylogenetic analysis of subgenusCyathophora was totally depended on twenty-nine complete chloroplast genome sequences, which were twenty-one species of Allium (including 6 newly assembled species; 15 other species of Allium were collected from NCBI), six species of Lilium, and two species of Asparagus as the out groups (Table S1). Three different databases were used to build the phylogenetic tree, which include the complete genome sequences, the IGS sequences, and all CDS sequences, and three different methods, Bayesian-inference (MrBayes v3.2), maximum parsimony (PAUP-version4.0), and maximum likelihood (RAxmL8.0), were used to build the tree. The sequences were aligned using MAFFT [36] in Geneious 11.0.4 with the set parameters and manually trimmed. GTR + I + G was selected as the best model using software ModelTest v3.7 [37]. Maximum likelihood (ML) analyses were performed using RAxmL8.0 with 1000 bootstrap replications [38]. PAUP was used to conduct maximum parsimony (MP) analyses [39]. MP was run using a heuristic search with 1000 random addition sequence replicates with the tree-bisection-reconnection (TBR) branch-swapping tree search criterion. Bayesian inference (BI) was executed with Mrbayes v3.2 [40], and the Markov chain Monte Carlo (MCMC) analysis was run 1 × 108 generations. The trees were sampled every 1000 generations: the first 25% were discarded as burn in and the remaining trees were used to establish a 50% majority rule consensus tree. When the average standard deviation of the splitting frequency was kept below 0.001, it was considered that the stationarity is achieved.


## 2.1. Plant Materials, DNA Extraction, and Sequencing

Fresh leaves ofA. cyathophorum, A. cyathophorum var. farreri, A. spicatum, A. mairei, A. trifurcatum, and A. kingdonii were collected from different places (Table 1). Morphological characters were measured using karyotype [23]. The healthy leaves were immediately dried with silica gel to use for DNA extraction. The voucher specimens were stored in the Herbarium of Sichuan University (SZ Herbarium). Their total genomic DNA was extracted from the sampled leaves according to the manufacturer's instructions for the Plant Genomic DNA Kit (Tiangen Biotech, Beijing, China). Genomic DNA was indexed by tags and pooled together in one lane of Illumina HiSeq platform for sequencing (paired-end, 350 bp) at Novogene (Beijing, China).Table 1
Samples information.
Species
Location
Geographical coordinate
A. cyathophorum
Mangkang, Tibet
29°43′24″N, 98°31′51″E
A. cyathophorum var. farreri
Zhouqu, Gansu
33°47′10″N, 104°22′7″E
A. spicatum
NaiDong, Tibet
29°13′40″N, 91°45′36″E
A. mairei
Shangri-La, Yunnan
27°25′19″N, 100°09′22″E
A. trifurcatum
Shangri-La, Yunnan
27°52′22″N, 99°43′10″E
A. kingdonii
Nyingchi, Tibet
29°37′27″N, 94°39′2″E


## 2.2. Chloroplast Genome Sequence Assembly and Annotation

We firstly used FastQC v0.11.7 to assess the quality of all reads [24]. To select the best reference, we filtrated the chloroplast genome related reads by mapping all reads to the published chloroplast genome sequences in Allium. SOAPdenovo2 was used to assemble all relevant reads into contigs [25]. The clean reads were assembled using the program NOVOPlasty [26] with the complete chloroplast genome of its close relative A. cepa as the reference (GenBank accession no. KM088014). Geneious 11.0.4 was used to finish the annotation of the assembled chloroplast genome, and it was corrected manually after comparison with references [27]. The circular plastid genome maps were generated utilizing the OGDRAW program [28]. The GenBank accession numbers of A. cyathophorum, A. cyathophorum var. farreri, A. spicatum, A. mairei, A. trifurcatum, and A. kingdonii are MK820611, MK931245, MK931246, MK820615, MK931247, and MK294559, respectively.


## 2.3. Repeat Sequences and Simple Sequence Repeat (SSR) Analysis

REPuter [29] was selected to investigate the location and size of repeat sequences, which included four types of repeats in the chloroplast genomes about the six Allium species. The sequence identity and minimum length of repeat size was set to >90% and 30 bp, with the hamming distance of 3. We used IMEx, ImperfectMicrosatelliteExtractor (http://43.227.129.132:8008/IMEX/imex_advanced.html) [30], to find chloroplast SSRs in six chloroplast genome sequences of Allium. Its specifications were set up as follows: the minimum number of repeats for mononucleotide, dinucleotides, trinucleotides, tetranucleotides, pentanucleotide and hexanucleotides was 10, 5, 5, 4, 3, and 3, respectively, the repeat type was imperfect, the imperfection % was Mono: 10%, Di: 10%, Tri: 15%, Tetra: 20%, Penta: 5%, and Hexa: 5%, mismatches allowed in pattern were Mono: 1, Di: 1, Tri: 2, Tetra: 4, Penta: 0, and Hexa: 3, and the level of standardization was level 1 standardization.


## 2.4. Codon Usage Analysis

Codon usage of the species in subgenusCyathophora was analyzed by the software of CodonW [31]. Protein-coding genes (CDS) were selected with the following filter requirements: (1) each CDS was longer than 300 nucleotides [18, 32]; (2) repeat sequences were deleted. Totally, 53 CDS of each species in Allium were selected for further study.


## 2.5. Genome Comparison (IR Contraction and Expansion)

The mVISTA program was chosen to analyze the whole sequence similarity of all sixAllium species with Shuffle-LAGAN model [33], using the chloroplast genomes to compare their difference in sequences at the chloroplast genome level and A. cyathophorum as the reference. The boundaries between single copy regions (LSC and SSC) and inverted repeats (IR) regions among the six chloroplast genome sequences were compared by using Geneious v11.0.4 software [27].


## 2.6. Hotspot Regions Identification in SubgenusCyathophora

To analyze nucleotide diversity (Pi), we extracted the shared 112 genes of the six species inAllium after alignment. DnaSP 5.10 was employed to calculate the nucleotide variability [34].


## 2.7. Gene Selective Pressure Analysis of SixAllium Plastomes

To investigate selection pressures, nonsynonymous (Ka) and synonymous (Ks) substitution rates of 65 selected protein-coding genes between the cp genomes of subgenusCyathophora and the other two Allium species were calculated by KaKs Calculator version 2.0 [35].


## 2.8. SubgenusCyathophora Phylogenomic Analysis Based on Chloroplast Genome

Phylogenetic analysis of subgenusCyathophora was totally depended on twenty-nine complete chloroplast genome sequences, which were twenty-one species of Allium (including 6 newly assembled species; 15 other species of Allium were collected from NCBI), six species of Lilium, and two species of Asparagus as the out groups (Table S1). Three different databases were used to build the phylogenetic tree, which include the complete genome sequences, the IGS sequences, and all CDS sequences, and three different methods, Bayesian-inference (MrBayes v3.2), maximum parsimony (PAUP-version4.0), and maximum likelihood (RAxmL8.0), were used to build the tree. The sequences were aligned using MAFFT [36] in Geneious 11.0.4 with the set parameters and manually trimmed. GTR + I + G was selected as the best model using software ModelTest v3.7 [37]. Maximum likelihood (ML) analyses were performed using RAxmL8.0 with 1000 bootstrap replications [38]. PAUP was used to conduct maximum parsimony (MP) analyses [39]. MP was run using a heuristic search with 1000 random addition sequence replicates with the tree-bisection-reconnection (TBR) branch-swapping tree search criterion. Bayesian inference (BI) was executed with Mrbayes v3.2 [40], and the Markov chain Monte Carlo (MCMC) analysis was run 1 × 108 generations. The trees were sampled every 1000 generations: the first 25% were discarded as burn in and the remaining trees were used to establish a 50% majority rule consensus tree. When the average standard deviation of the splitting frequency was kept below 0.001, it was considered that the stationarity is achieved.


## 3. Results

### 3.1. Chloroplast Genome Organization and Gene Content in Six Species

These six acquiredAllium cp genomes were detected to have a circular DNA structure of angiosperm cp genomes that comprises LSC, SSC, and two IR regions (Figure 2). The sizes of the six CP genomes ranged from 152,913 bp for A. mairei to 154,174 bp for A. cyathophorum, which were similar with other Allium CP genomes [41]. The size varied from 82,493 bp (A. mairei) to 83,423 bp (A. kingdonii) in the LSC region, from 17,811 bp (A. kingdonii) to 21,706 bp (A. trifurcatum) in the SSC region, and from 24,561 bp (A. trifurcatum) to 26,467 bp (A. cyathophorum) in the IR region (Table 2). The entire GC content of the cp genome sequences was 36.8–36.9%, and the GC contents of the LSC, SSC, and IR regions were 34.6–34.8%, 29.5–31.2%, and 42.7–43.1%, respectively. A total of 132 genes were discovered from the complete cp genome: 8 ribosomal RNA (rRNA) genes, 86 protein-coding genes, and 38 transfer RNA (tRNA) genes (Table 3).Figure 2
Gene map ofA. cyathophorum, A. cyathophorum var. farreri, A. spicatum, A. mairei, A. trifurcatum, and A. kingdonii complete chloroplast genomes. The circle inside genes is transcribed clockwise, and the outside genes are transcribed counterclockwise. Genes belonging to different functional groups are represented by distinct colors.Table 2
Details comparison of the complete chloroplast genomes of six species ofAllium.
A. cyathophorum
A. cyathophorum var. farreri
A. spicatum
A. mairei
A. trifurcatum
A. kingdonii
Genome size (bp)
154,174
153,111
153,187
152,913
153,456
153,559
LSC length (bp)
83,359
82,544
82,625
82,493
82,628
83,423
SSC length (bp)
17,881
17,811
17,920
18,762
21,706
17,810
IR length (bp)
26,467
26,378
26,321
25,829
24,561
26,163
LSC GC content (%)
34.6
34.7
34.7
34.8
34.8
34.8
SSC GC content (%)
29.5
29.7
29.7
29.9
31.2
29.9
IR GC content (%)
42.7
42.7
42.7
42.9
43.1
42.7
Total GC content (%)
36.8
36.9
36.9
36.9
36.9
36.9
Total number of genes
132
132
132
132
132
132
Protein coding genes
86
86
86
86
86
86
rRNA
8
8
8
8
8
8
tRNA
38
38
38
38
38
38Table 3
Genes existing in the chloroplast genome ofAllium.
Category
Gene name
Number
Photosystem I
psaA, psaB, psaC, psaI, psaJ
5
Photosystem II
psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ
15
Cytochrome b6/f
petA, petB, petD, petG, petL, petN
6
ATP synthase
atpA, atpB, atpE, atpF, atpH, atpI
6
Rubisco
rbcL
1
NADH oxido reductase
ndhA, ndhB(×2), ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
12
Large subunit ribosomal proteins
rpl2(×2), rpl14, rpl16, rpl20, rpl22, rpl23(×2), rpl32, rpl33, rpl36
11
Small subunit ribosomal proteins
rps3, rps4, rps7(×2), rps8, rps11, rps12(×2), rps14, rps15, rps16, rps18, rps19(×2)
14
RNAP
rpoA, rpoB, rpoC1, rpoC2
4
Other proteins
accD, ccsA, matK, cemA, clpP, infA
6
Proteins of unknown function
ycf1(×2), ycf2(×2), ycf3, ycf4
6
Ribosomal RNAs
rrn23(×2), rrn16(×2), rrn5(×2), rrn4.5(×2)
8
Transfer RNA
trnA-UGC(×2), trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnH-GUG(×2), trnG-GCC, trnG-UCC, trnI-CAU(×2), trnI-GAU(×2), trnK-UUU, trnL-CAA(×2), trnL-UAA, trnL-UAG, trnM-CAU, trnN-GUU(×2), trnP-UGG, trnQ-UUG, trnR-ACG(×2), trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC(×2), trnV-UAC, trnW-CCA, trnY-GUA
38
Total
132


### 3.2. Repeat and Simple Sequence Repeat (SSR) Analysis

Many research studies of cp genomes revealed that repeat sequences have been widely used in phylogeny, population genetics, and other studies [42]. Four types of repeats (forward repeats, reverse repeats, complement repeats, and palindromic repeats) were detected in the six Allium species. There were only 3 complement repeats in A. cyathophorum, while the other species did not have. The number of repeats varied from 37 to 77 in the six species; the A. cyathophorum showed the most abundant number of repeats, including 29, 40, 5 and 3 palindromic forward reverse and complement repeats, respectively. The number of forward repeats ranged from 15 to 40, the number of palindromic repeats ranged from 17 to 29, and the number of reverse repeats ranged from 1 to 5 (Figure 3). The lengths of forward, palindromic, and reverse repeats ranged from 30 to 267 bp, and most of them were concentrated in 30–50 bp (81.48%), while those of 50–70 bp (9.09%), >100 bp (6.40%), and 70–90 bp (3.03%) were less common (Figure S1). Earlier reports recommend that the appearance of the repeats indicates that this locus is a staple hots-pot for reconfiguration of the genome [43–45]. Nevertheless, these repeats are valuable for developing genetic markers in population genetics studies [46, 47].Figure 3
Investigation of repeated sequences inA. cyathophorum, A. cyathophorum var. farreri, A. spicatum, A. mairei, A. trifurcatum, and A. kingdonii chloroplast genomes. (a) Four repeat types. (b) Number of the forward repeat by length. (c) Number of the palindromic repeat by length. (d) Number of the reverse repeat by length.
(a)
(b)
(c)
(d)SSRs, also called as microsatellites, are 1- to 6-bp repeating sequences that are extensively distributed in the chloroplast genome. SSRs are highly polymorphic and codominant, which are valuable markers for study involving gene flow, population genetics, and gene mapping [48]. In this study, six classes of SSRs (mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats) were found in the cp genome of the six species, whereas the number of hexanucleotide repeats ranged from 1 to 3 in the six species, and the pentanucleotide repeats just existed in A. kingdonii and A. spicatum. The total number of SSRs in the genome of the six Allium species was 185 in A. cyathophorum, 158 in A. cyathophorum var. farreri, 159 in A. spicatum, 171 in A. mairei, 201 in A. trifurcatum, and 165 in A. kingdonii (Figure 4(a)). The highest number was mononucleotide repeat, which accounted for about 30.41% of the total SSRs (Figure 4(c)); the number ranged from 36 in A. kingdonii to 71 in A. trifurcatum, and all mononucleotide repeats are composed of A or T bases; these conclusions were unanimous in previous studies that SSRs in cp genomes usually contained short polyA or polyT repeats [49], while those of dinucleotide repeats (28.20%), trinucleotide repeats (20.79%), tetranucleotide repeats (19.15%), pentanucleotide repeats (0.38%), and hexanucleotide repeats were the least abundant (1.06%). In the whole SSR locus, the SSRs located in the LSC area are much more than those in the SSC and IR areas (Figure 4(b)), which is identical with previous research studies that SSRs are unevenly distributed in cp genomes [50].Figure 4
Analysis of simple sequence repeats (SSRs) in sixAllium chloroplast genome sequences. (a) Number of six SSR types discovered in six Allium chloroplast genome sequences. (b) Number of SSRs in the LSC, IR, and SSC regions in six Allium chloroplast genome sequences. (c) Presence of different SSR types in total SSRs of six Allium chloroplast genome sequences.
(a)
(b)
(c)


### 3.3. Codon Usage Analysis

Codon usage bias is a phenomenon that the synonymous codons usually have different frequencies of use in plant genomes, which was caused by evolutionary factors that affect gene mutations and selections [51, 52]. The relative synonymous codon usage (RSCU) is a method that estimates nonuniform synonymous codon usage in coding sequences, in which RSCU less than 1 demonstrates lack of bias, whereas RSCU value greater than 1 stands for more frequent use of a codon. In view of the sequences of 53 protein-coding genes (CDS), the codon usage frequency was calculated for the six Allium cp genomes (Table 4). Altogether, the number of codons ranged from 33058 in A. kingdonii to 23791 in A. mairei. In addition, the result indicated that a total of 13218 codons encoding leucine in the cp genomes of the six species and 1453 codons encoding cysteine as the most common and least common universal amino acids, respectively. As recently discovered in other cp genomes of plants, our study revealed that except tryptophan and methionine, there was preference in the use of synonymous codons, and the RSCU value of 30 codons exceeded 1 for each species, and they were A or T-ending codons. The result is in accordance with other researches, which the codon usage preference for A/T ending in plants [53–55].Table 4
Codon usage in sixAllium chloroplast genomes.
AA
Codon
Number
RSCU
Acy
Afa
Asp
Ama
Aki
Atr
Acy
Afa
Asp
Ama
Aki
Atr
Phe
UUU
826
825
827
822
809
827
1.33
1.33
1.33
1.33
1.32
1.33
UUC
420
415
418
414
421
419
0.67
0.67
0.67
0.67
0.68
0.67
Leu
UUA
758
758
756
751
744
746
2.05
2.05
2.05
2.05
2.04
2.04
UUG
447
450
447
441
446
444
1.21
1.22
1.21
1.21
1.22
1.21
CUU
455
453
453
448
448
452
1.23
1.23
1.23
1.22
1.23
1.24
CUC
138
137
138
139
136
138
0.37
0.37
0.37
0.38
0.37
0.38
CUA
295
295
298
294
292
288
0.8
0.8
0.81
0.8
0.8
0.79
CUG
122
121
124
122
119
125
0.33
0.33
0.34
0.33
0.33
0.34
Ile
AUU
943
946
945
955
938
931
1.49
1.49
1.49
1.5
1.48
1.48
AUC
350
345
346
339
350
344
0.55
0.54
0.55
0.53
0.55
0.55
AUA
611
610
613
615
616
616
0.96
0.96
0.97
0.97
0.97
0.98
Met
AUG
497
500
498
495
497
495
1
1
1
1
1
1
Val
GUU
431
432
432
429
440
438
1.48
1.48
1.49
1.48
1.51
1.5
GUC
129
130
129
128
131
131
0.44
0.45
0.44
0.44
0.45
0.45
GUA
432
435
434
434
430
433
1.49
1.49
1.5
1.5
1.47
1.48
GUG
171
167
165
165
167
165
0.59
0.57
0.57
0.57
0.57
0.57
Ser
UCU
467
468
473
461
473
476
1.73
1.73
1.75
1.72
1.76
1.76
UCC
246
254
249
252
249
244
0.91
0.94
0.92
0.94
0.93
0.9
UCA
321
320
320
318
311
325
1.19
1.18
1.18
1.19
1.16
1.2
UCG
151
151
150
151
159
154
0.56
0.56
0.55
0.56
0.59
0.57
Pro
CCU
338
339
338
337
338
338
1.56
1.57
1.56
1.56
1.55
1.56
CCC
191
188
189
187
199
194
0.88
0.87
0.87
0.86
0.91
0.9
CCA
247
244
247
243
246
238
1.14
1.13
1.14
1.12
1.13
1.1
CCG
92
94
93
98
89
94
0.42
0.43
0.43
0.45
0.41
0.44
Thr
ACU
448
449
449
446
438
440
1.65
1.67
1.67
1.65
1.63
1.63
ACC
186
184
184
185
191
193
0.69
0.68
0.68
0.69
0.71
0.72
ACA
335
331
331
334
335
329
1.24
1.23
1.23
1.24
1.25
1.22
ACG
116
114
114
114
111
115
0.43
0.42
0.42
0.42
0.41
0.43
Ala
GCU
533
536
538
537
528
542
1.85
1.85
1.85
1.85
1.85
1.86
GCC
159
163
161
162
161
164
0.55
0.56
0.55
0.56
0.56
0.56
GCA
343
345
348
341
335
348
1.19
1.19
1.2
1.18
1.18
1.19
GCG
117
118
115
119
116
111
0.41
0.41
0.4
0.41
0.41
0.38
Tyr
UAU
683
669
668
674
679
666
1.63
1.61
1.62
1.62
1.64
1.61
UAC
155
160
156
157
150
160
0.37
0.39
0.38
0.38
0.36
0.39
TER
UAA
29
29
29
29
30
26
1.47
1.47
1.5
1.47
1.73
1.47
UAG
18
18
17
18
12
15
0.92
0.92
0.88
0.92
0.69
0.85
His
CAU
415
416
415
412
419
420
1.56
1.57
1.56
1.57
1.56
1.56
CAC
118
113
116
113
118
119
0.44
0.43
0.44
0.43
0.44
0.44
Gln
CAA
583
585
585
587
587
591
1.53
1.54
1.54
1.54
1.54
1.54
CAG
178
176
177
177
175
177
0.47
0.46
0.46
0.46
0.46
0.46
Asn
AAU
803
799
797
803
798
784
1.56
1.56
1.56
1.56
1.55
1.55
AAC
229
226
227
226
231
227
0.44
0.44
0.44
0.44
0.45
0.45
Lys
AAA
876
878
884
866
838
863
1.55
1.55
1.55
1.53
1.51
1.52
AAG
255
258
254
264
269
269
0.45
0.45
0.45
0.47
0.49
0.48
Asp
GAU
692
692
691
681
682
691
1.64
1.64
1.64
1.63
1.62
1.62
GAC
154
152
154
156
158
160
0.36
0.36
0.36
0.37
0.38
0.38
Glu
GAA
861
864
857
861
869
852
1.49
1.49
1.48
1.49
1.5
1.49
GAG
296
299
299
291
291
289
0.51
0.51
0.52
0.51
0.5
0.51
Cys
UGU
187
185
185
186
184
186
1.52
1.54
1.54
1.54
1.52
1.54
UGC
59
56
56
55
58
56
0.48
0.46
0.46
0.46
0.48
0.46
TER
UGA
12
12
12
12
10
12
0.61
0.61
0.62
0.61
0.58
0.68
Trp
UGG
386
389
391
392
391
391
1
1
1
1
1
1
Arg
CGU
279
280
280
280
281
278
1.37
1.37
1.37
1.38
1.37
1.35
CGC
74
78
78
76
71
80
0.36
0.38
0.38
0.37
0.34
0.39
CGA
269
270
269
269
272
274
1.32
1.32
1.31
1.32
1.32
1.33
CGG
88
89
87
88
89
89
0.43
0.43
0.43
0.43
0.43
0.43
Ser
AGU
339
342
342
342
331
335
1.26
1.26
1.27
1.27
1.23
1.24
AGC
91
88
88
86
87
92
0.34
0.33
0.33
0.32
0.32
0.34
Arg
AGA
390
392
395
388
398
400
1.91
1.91
1.93
1.91
1.93
1.94
AGG
125
121
119
119
124
119
0.61
0.59
0.58
0.59
0.6
0.58
Gly
GGU
489
487
487
493
483
489
1.33
1.33
1.33
1.34
1.31
1.33
GGC
133
130
131
131
137
132
0.36
0.36
0.36
0.36
0.37
0.36
GGA
620
621
625
622
623
616
1.69
1.7
1.7
1.69
1.69
1.67
GGG
228
226
226
225
229
236
0.62
0.62
0.62
0.61
0.62
0.64
RSCU represents relative synonymous codon usage. RSCU more than one is highlighted in bold. Acy, Afa, Asp, Ama, Aki, and Atr stand forA. cyathophorum, A. cyathophorum var. farreri, A. spicatum, A. mairei, A. kingdonii, and A. trifurcatum, respectively.


### 3.4. Comparative Analysis of the Chloroplast Genomes among Six Species inAllium

mVISTA online software in the Shuffle-LAGAN mode was employed to analyze the comprehensive sequence discrepancy of the six chloroplast genomes ofAllium with the annotation of A. cyathophorum as a reference. In this study, the whole chloroplast genome alignment showed great sequence consistency of the six cp genomes, indicating that Allium cp genomes are very conservative (Figure 5). We found that among the six cp genomes, their IR region is more conserved compared to the LSC and SSC regions, which is similar with other plants [56, 57]. Furthermore, as we have found in other angiosperms, the coding areas were more conserved than the noncoding areas, and there were more variations in the intergenic spacers of the LSC and SSC areas, whereas the IR areas presented a lower sequence divergence [58, 59]. A. cyathophorum var. farreri had the highest sequence similarity to A. cyathophorum in sequence identity analysis. Noncoding regions displayed varying degrees of sequence differences in these six Allium cp genomes, including trnK-rps16, trnS-trnG, atpH-atpI, petN-psbM, trnT-psbD, trnF-ndhJ, accD-psaI, and petA-psbL. The coding areas with significant diversity contain matK, rps16, rpoC2, infA, ycf1, ndhF, and rps15 genes. The highly diverse regions found in this study may be used to develop molecular markers that can improve efficiency to study phylogenetic relationships within the Allium species.Figure 5
Sequence alignment of sixAllium chloroplast genomes (A. cyathophorum as the reference). The y-axis represents the percent similarity between 50% and 100%. Different colors represent different genetic regions.Though the cp genome is usually well conserved, having typical quadripartite structure, gene number, and order, a phenomenon recognized as ebb and flow exists, and this is where the IR area often expands or contracts [60]. Expansion and contraction of IR region is related to the size variations in the cp genome and has great differences in its evolution [61, 62]. We compared the IR/SC boundary areas of the six Allium cp genomes, and we found that there are obvious differences in the IR/LSC and IR/SSC connections (Figure 6). At the boundary of LSC/IRa junction, rps19 gene of different species distance the boundary were from 1 to 81 bp, while the rpl22 genes distance the border were from 29 to 273 bp. At the boundary of LSC/IRb connections, the psbA genes distance the border were reached from 108 to 605 bp. The inverted repeat b (IRb)/SSC border located in the coding region, and the ycf1 genes of the six species with a region ranged from 4193 to 5223 bp located in the SSC regions, which the ycf1 gene of A. trifurcatum all located in the SSC region. The shorter ycf1 gene crossed the inverted repeat (IRa)/SSC boundary, with 56–919 bp locating in the SSC regions. And the ndhF genes were situated in the SSC regions, which distance from the IRa/SSC boundary ranged from 1 to 1962 bp. Undoubtedly, the full-length differences in the sequence of the six cp genomes are caused by changes in the IR/SC boundaries.Figure 6
Comparison of the boundaries of the LSC, SSC, and IR areas of the whole chloroplast genomes of the six species.


### 3.5. Hotspot Regions Identification in SubgenusCyathophora

We totally extracted the shared 112 genes of the six species in chloroplast genomes; the nucleotide variability (Pi) ranged from 0.00041 (rrn16) to 0.08125 (infA) among these shared genes (Figure 7; Table S2). Seven genes (infA, rps16, rps15, ndhF, trnG-UCC, trnC-GCA, and trnK-UUU) were considered to be hotspot regions with a nucleotide diversity greater than 0.02. These regions can be used to develop useful markers for phylogenetic analysis and distinguish the species in Allium.Figure 7
The nucleotide diversity of the shared 112 genes of the six species in chloroplast genomes.
(a)
(b)


### 3.6. Synonymous (Ks) and Nonsynonymous (Ka) Substitution Rate Analysis

The Ka/Ks ratio is a significant index for understanding the evolution of protein-coding genes to assess gene differentiation rates and to determine whether positive, purified, or neutral selections have been performed; a Ka/Ks ratio >1 illustrates positive selection and Ka/Ks < 1 illustrates purifying selection, while the ratio of Ka/Ks close to 1 illustrates neutral selection [63]. In our study, the Ka/Ks ratio was calculated for 65 shared protein-coding genes in all six chloroplast genomes (Table S3), and the results are shown in Figure 8. The conservative genes with Ka/Ks ratio of 0.01, indicating powerful purifying selection pressure, were rpl2, rpl32, psaC, psbA, rpoC2, petN, psbZ, psaB, psaJ, and psbT, when the averaging Ka/Ks method showed ycf1 and ycf2 genes with Ka/Ks > 1, which shows that they may undergo some selective pressure among the six Allium species. The Ka/Ks ratios ranging from 0.5 to 1 were found for matK, rps16, psaI, cemA, petA, and rpl20, representing relaxed selection. The majority (56 of 65 genes) had an average Ka/Ks ratio ranging from 0 to 0.49 for the six compared groups, indicating that most genes were under purifying selection. Other than this, four genes (matK, rpoB, petA, and rpoA) with Ka/Ks > 1 in one or more pairwise comparisons (Figure 8) suggest that these genes may undergo selective pressure which is unknown, which is very important for researching the evolution of species.Figure 8
KA/KS analysis of 65 common protein coding genes in sixAllium species. Acy, Afa Asp, Ama, Aki, and Atr stand for A. cyathophorum, A. cyathophorum var. farreri, A. spicatum, A. mairei, A. kingdonii, and A. trifurcatum, respectively. KA: nonsynonymous; KS: synonymous.
(a)
(b)
(c)


### 3.7. Phylogenetic Analysis of Subgenus Cyathophora Depends on Chloroplast Genome

The cp genome of sequence is significant and helpful to construct phylogenetic relationships and explore the evolutionary history in many previous reports [64, 65]. To explore the phylogenetic relationship of the six Allium species, we constructed the phylogenetic tree using three different methods and databases containing twenty-one Allium species, six Lilium species, and two Asparagus species as the out groups (Figure 9). Three databases of the complete genome sequences, the IGS sequences, and all CDS sequences using MP, BI, and ML methods all showed the same topologies with high support (Figures S2 and S3). The results strongly supported that subgenus Cyathophora is a monophyletic group, comprising A. cyathophorum, A. cyathophorum var. farreri, A. spicatum, and A. mairei in this study with 100% bootstrap value; subgenus Cyathophora does not contain A. kingdonii and A. trifurcatum, and the phylogenetic tree indicates that A. cyathophorum var. farreri is a direct sister to A. spicatum, which is in accordance with the results of previous molecular research studies [4, 6]. The sister relationship of A. cyathophorum var. farreri and A. spicatum strongly suggests that A. spicatum is closely related to subgenus Cyathophora though it is a special species with the significant abnormal spicate inflorescence compared to other species with capitate or umbellate inflorescence. Furthermore, Allium kingdonii was the closest relative of Allium paradoxum and Allium ursinum.Figure 9
Phylogenetic relationship of subgenusCyathophora with relational species. The whole chloroplast genomes dataset was analyzed using three different methods: maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI). Numbers on the branches stand for bootstrap values in the ML, MP, and posterior probabilities in the BI trees. Six species of Lilium and two species of Asparagus were considered as the out groups. Purple shows the branch of the subgenus Cyathophora.


## 3.1. Chloroplast Genome Organization and Gene Content in Six Species

These six acquiredAllium cp genomes were detected to have a circular DNA structure of angiosperm cp genomes that comprises LSC, SSC, and two IR regions (Figure 2). The sizes of the six CP genomes ranged from 152,913 bp for A. mairei to 154,174 bp for A. cyathophorum, which were similar with other Allium CP genomes [41]. The size varied from 82,493 bp (A. mairei) to 83,423 bp (A. kingdonii) in the LSC region, from 17,811 bp (A. kingdonii) to 21,706 bp (A. trifurcatum) in the SSC region, and from 24,561 bp (A. trifurcatum) to 26,467 bp (A. cyathophorum) in the IR region (Table 2). The entire GC content of the cp genome sequences was 36.8–36.9%, and the GC contents of the LSC, SSC, and IR regions were 34.6–34.8%, 29.5–31.2%, and 42.7–43.1%, respectively. A total of 132 genes were discovered from the complete cp genome: 8 ribosomal RNA (rRNA) genes, 86 protein-coding genes, and 38 transfer RNA (tRNA) genes (Table 3).Figure 2
Gene map ofA. cyathophorum, A. cyathophorum var. farreri, A. spicatum, A. mairei, A. trifurcatum, and A. kingdonii complete chloroplast genomes. The circle inside genes is transcribed clockwise, and the outside genes are transcribed counterclockwise. Genes belonging to different functional groups are represented by distinct colors.Table 2
Details comparison of the complete chloroplast genomes of six species ofAllium.
A. cyathophorum
A. cyathophorum var. farreri
A. spicatum
A. mairei
A. trifurcatum
A. kingdonii
Genome size (bp)
154,174
153,111
153,187
152,913
153,456
153,559
LSC length (bp)
83,359
82,544
82,625
82,493
82,628
83,423
SSC length (bp)
17,881
17,811
17,920
18,762
21,706
17,810
IR length (bp)
26,467
26,378
26,321
25,829
24,561
26,163
LSC GC content (%)
34.6
34.7
34.7
34.8
34.8
34.8
SSC GC content (%)
29.5
29.7
29.7
29.9
31.2
29.9
IR GC content (%)
42.7
42.7
42.7
42.9
43.1
42.7
Total GC content (%)
36.8
36.9
36.9
36.9
36.9
36.9
Total number of genes
132
132
132
132
132
132
Protein coding genes
86
86
86
86
86
86
rRNA
8
8
8
8
8
8
tRNA
38
38
38
38
38
38Table 3
Genes existing in the chloroplast genome ofAllium.
Category
Gene name
Number
Photosystem I
psaA, psaB, psaC, psaI, psaJ
5
Photosystem II
psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ
15
Cytochrome b6/f
petA, petB, petD, petG, petL, petN
6
ATP synthase
atpA, atpB, atpE, atpF, atpH, atpI
6
Rubisco
rbcL
1
NADH oxido reductase
ndhA, ndhB(×2), ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
12
Large subunit ribosomal proteins
rpl2(×2), rpl14, rpl16, rpl20, rpl22, rpl23(×2), rpl32, rpl33, rpl36
11
Small subunit ribosomal proteins
rps3, rps4, rps7(×2), rps8, rps11, rps12(×2), rps14, rps15, rps16, rps18, rps19(×2)
14
RNAP
rpoA, rpoB, rpoC1, rpoC2
4
Other proteins
accD, ccsA, matK, cemA, clpP, infA
6
Proteins of unknown function
ycf1(×2), ycf2(×2), ycf3, ycf4
6
Ribosomal RNAs
rrn23(×2), rrn16(×2), rrn5(×2), rrn4.5(×2)
8
Transfer RNA
trnA-UGC(×2), trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnH-GUG(×2), trnG-GCC, trnG-UCC, trnI-CAU(×2), trnI-GAU(×2), trnK-UUU, trnL-CAA(×2), trnL-UAA, trnL-UAG, trnM-CAU, trnN-GUU(×2), trnP-UGG, trnQ-UUG, trnR-ACG(×2), trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC(×2), trnV-UAC, trnW-CCA, trnY-GUA
38
Total
132


## 3.2. Repeat and Simple Sequence Repeat (SSR) Analysis

Many research studies of cp genomes revealed that repeat sequences have been widely used in phylogeny, population genetics, and other studies [42]. Four types of repeats (forward repeats, reverse repeats, complement repeats, and palindromic repeats) were detected in the six Allium species. There were only 3 complement repeats in A. cyathophorum, while the other species did not have. The number of repeats varied from 37 to 77 in the six species; the A. cyathophorum showed the most abundant number of repeats, including 29, 40, 5 and 3 palindromic forward reverse and complement repeats, respectively. The number of forward repeats ranged from 15 to 40, the number of palindromic repeats ranged from 17 to 29, and the number of reverse repeats ranged from 1 to 5 (Figure 3). The lengths of forward, palindromic, and reverse repeats ranged from 30 to 267 bp, and most of them were concentrated in 30–50 bp (81.48%), while those of 50–70 bp (9.09%), >100 bp (6.40%), and 70–90 bp (3.03%) were less common (Figure S1). Earlier reports recommend that the appearance of the repeats indicates that this locus is a staple hots-pot for reconfiguration of the genome [43–45]. Nevertheless, these repeats are valuable for developing genetic markers in population genetics studies [46, 47].Figure 3
Investigation of repeated sequences inA. cyathophorum, A. cyathophorum var. farreri, A. spicatum, A. mairei, A. trifurcatum, and A. kingdonii chloroplast genomes. (a) Four repeat types. (b) Number of the forward repeat by length. (c) Number of the palindromic repeat by length. (d) Number of the reverse repeat by length.
(a)
(b)
(c)
(d)SSRs, also called as microsatellites, are 1- to 6-bp repeating sequences that are extensively distributed in the chloroplast genome. SSRs are highly polymorphic and codominant, which are valuable markers for study involving gene flow, population genetics, and gene mapping [48]. In this study, six classes of SSRs (mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats) were found in the cp genome of the six species, whereas the number of hexanucleotide repeats ranged from 1 to 3 in the six species, and the pentanucleotide repeats just existed in A. kingdonii and A. spicatum. The total number of SSRs in the genome of the six Allium species was 185 in A. cyathophorum, 158 in A. cyathophorum var. farreri, 159 in A. spicatum, 171 in A. mairei, 201 in A. trifurcatum, and 165 in A. kingdonii (Figure 4(a)). The highest number was mononucleotide repeat, which accounted for about 30.41% of the total SSRs (Figure 4(c)); the number ranged from 36 in A. kingdonii to 71 in A. trifurcatum, and all mononucleotide repeats are composed of A or T bases; these conclusions were unanimous in previous studies that SSRs in cp genomes usually contained short polyA or polyT repeats [49], while those of dinucleotide repeats (28.20%), trinucleotide repeats (20.79%), tetranucleotide repeats (19.15%), pentanucleotide repeats (0.38%), and hexanucleotide repeats were the least abundant (1.06%). In the whole SSR locus, the SSRs located in the LSC area are much more than those in the SSC and IR areas (Figure 4(b)), which is identical with previous research studies that SSRs are unevenly distributed in cp genomes [50].Figure 4
Analysis of simple sequence repeats (SSRs) in sixAllium chloroplast genome sequences. (a) Number of six SSR types discovered in six Allium chloroplast genome sequences. (b) Number of SSRs in the LSC, IR, and SSC regions in six Allium chloroplast genome sequences. (c) Presence of different SSR types in total SSRs of six Allium chloroplast genome sequences.
(a)
(b)
(c)


## 3.3. Codon Usage Analysis

Codon usage bias is a phenomenon that the synonymous codons usually have different frequencies of use in plant genomes, which was caused by evolutionary factors that affect gene mutations and selections [51, 52]. The relative synonymous codon usage (RSCU) is a method that estimates nonuniform synonymous codon usage in coding sequences, in which RSCU less than 1 demonstrates lack of bias, whereas RSCU value greater than 1 stands for more frequent use of a codon. In view of the sequences of 53 protein-coding genes (CDS), the codon usage frequency was calculated for the six Allium cp genomes (Table 4). Altogether, the number of codons ranged from 33058 in A. kingdonii to 23791 in A. mairei. In addition, the result indicated that a total of 13218 codons encoding leucine in the cp genomes of the six species and 1453 codons encoding cysteine as the most common and least common universal amino acids, respectively. As recently discovered in other cp genomes of plants, our study revealed that except tryptophan and methionine, there was preference in the use of synonymous codons, and the RSCU value of 30 codons exceeded 1 for each species, and they were A or T-ending codons. The result is in accordance with other researches, which the codon usage preference for A/T ending in plants [53–55].Table 4
Codon usage in sixAllium chloroplast genomes.
AA
Codon
Number
RSCU
Acy
Afa
Asp
Ama
Aki
Atr
Acy
Afa
Asp
Ama
Aki
Atr
Phe
UUU
826
825
827
822
809
827
1.33
1.33
1.33
1.33
1.32
1.33
UUC
420
415
418
414
421
419
0.67
0.67
0.67
0.67
0.68
0.67
Leu
UUA
758
758
756
751
744
746
2.05
2.05
2.05
2.05
2.04
2.04
UUG
447
450
447
441
446
444
1.21
1.22
1.21
1.21
1.22
1.21
CUU
455
453
453
448
448
452
1.23
1.23
1.23
1.22
1.23
1.24
CUC
138
137
138
139
136
138
0.37
0.37
0.37
0.38
0.37
0.38
CUA
295
295
298
294
292
288
0.8
0.8
0.81
0.8
0.8
0.79
CUG
122
121
124
122
119
125
0.33
0.33
0.34
0.33
0.33
0.34
Ile
AUU
943
946
945
955
938
931
1.49
1.49
1.49
1.5
1.48
1.48
AUC
350
345
346
339
350
344
0.55
0.54
0.55
0.53
0.55
0.55
AUA
611
610
613
615
616
616
0.96
0.96
0.97
0.97
0.97
0.98
Met
AUG
497
500
498
495
497
495
1
1
1
1
1
1
Val
GUU
431
432
432
429
440
438
1.48
1.48
1.49
1.48
1.51
1.5
GUC
129
130
129
128
131
131
0.44
0.45
0.44
0.44
0.45
0.45
GUA
432
435
434
434
430
433
1.49
1.49
1.5
1.5
1.47
1.48
GUG
171
167
165
165
167
165
0.59
0.57
0.57
0.57
0.57
0.57
Ser
UCU
467
468
473
461
473
476
1.73
1.73
1.75
1.72
1.76
1.76
UCC
246
254
249
252
249
244
0.91
0.94
0.92
0.94
0.93
0.9
UCA
321
320
320
318
311
325
1.19
1.18
1.18
1.19
1.16
1.2
UCG
151
151
150
151
159
154
0.56
0.56
0.55
0.56
0.59
0.57
Pro
CCU
338
339
338
337
338
338
1.56
1.57
1.56
1.56
1.55
1.56
CCC
191
188
189
187
199
194
0.88
0.87
0.87
0.86
0.91
0.9
CCA
247
244
247
243
246
238
1.14
1.13
1.14
1.12
1.13
1.1
CCG
92
94
93
98
89
94
0.42
0.43
0.43
0.45
0.41
0.44
Thr
ACU
448
449
449
446
438
440
1.65
1.67
1.67
1.65
1.63
1.63
ACC
186
184
184
185
191
193
0.69
0.68
0.68
0.69
0.71
0.72
ACA
335
331
331
334
335
329
1.24
1.23
1.23
1.24
1.25
1.22
ACG
116
114
114
114
111
115
0.43
0.42
0.42
0.42
0.41
0.43
Ala
GCU
533
536
538
537
528
542
1.85
1.85
1.85
1.85
1.85
1.86
GCC
159
163
161
162
161
164
0.55
0.56
0.55
0.56
0.56
0.56
GCA
343
345
348
341
335
348
1.19
1.19
1.2
1.18
1.18
1.19
GCG
117
118
115
119
116
111
0.41
0.41
0.4
0.41
0.41
0.38
Tyr
UAU
683
669
668
674
679
666
1.63
1.61
1.62
1.62
1.64
1.61
UAC
155
160
156
157
150
160
0.37
0.39
0.38
0.38
0.36
0.39
TER
UAA
29
29
29
29
30
26
1.47
1.47
1.5
1.47
1.73
1.47
UAG
18
18
17
18
12
15
0.92
0.92
0.88
0.92
0.69
0.85
His
CAU
415
416
415
412
419
420
1.56
1.57
1.56
1.57
1.56
1.56
CAC
118
113
116
113
118
119
0.44
0.43
0.44
0.43
0.44
0.44
Gln
CAA
583
585
585
587
587
591
1.53
1.54
1.54
1.54
1.54
1.54
CAG
178
176
177
177
175
177
0.47
0.46
0.46
0.46
0.46
0.46
Asn
AAU
803
799
797
803
798
784
1.56
1.56
1.56
1.56
1.55
1.55
AAC
229
226
227
226
231
227
0.44
0.44
0.44
0.44
0.45
0.45
Lys
AAA
876
878
884
866
838
863
1.55
1.55
1.55
1.53
1.51
1.52
AAG
255
258
254
264
269
269
0.45
0.45
0.45
0.47
0.49
0.48
Asp
GAU
692
692
691
681
682
691
1.64
1.64
1.64
1.63
1.62
1.62
GAC
154
152
154
156
158
160
0.36
0.36
0.36
0.37
0.38
0.38
Glu
GAA
861
864
857
861
869
852
1.49
1.49
1.48
1.49
1.5
1.49
GAG
296
299
299
291
291
289
0.51
0.51
0.52
0.51
0.5
0.51
Cys
UGU
187
185
185
186
184
186
1.52
1.54
1.54
1.54
1.52
1.54
UGC
59
56
56
55
58
56
0.48
0.46
0.46
0.46
0.48
0.46
TER
UGA
12
12
12
12
10
12
0.61
0.61
0.62
0.61
0.58
0.68
Trp
UGG
386
389
391
392
391
391
1
1
1
1
1
1
Arg
CGU
279
280
280
280
281
278
1.37
1.37
1.37
1.38
1.37
1.35
CGC
74
78
78
76
71
80
0.36
0.38
0.38
0.37
0.34
0.39
CGA
269
270
269
269
272
274
1.32
1.32
1.31
1.32
1.32
1.33
CGG
88
89
87
88
89
89
0.43
0.43
0.43
0.43
0.43
0.43
Ser
AGU
339
342
342
342
331
335
1.26
1.26
1.27
1.27
1.23
1.24
AGC
91
88
88
86
87
92
0.34
0.33
0.33
0.32
0.32
0.34
Arg
AGA
390
392
395
388
398
400
1.91
1.91
1.93
1.91
1.93
1.94
AGG
125
121
119
119
124
119
0.61
0.59
0.58
0.59
0.6
0.58
Gly
GGU
489
487
487
493
483
489
1.33
1.33
1.33
1.34
1.31
1.33
GGC
133
130
131
131
137
132
0.36
0.36
0.36
0.36
0.37
0.36
GGA
620
621
625
622
623
616
1.69
1.7
1.7
1.69
1.69
1.67
GGG
228
226
226
225
229
236
0.62
0.62
0.62
0.61
0.62
0.64
RSCU represents relative synonymous codon usage. RSCU more than one is highlighted in bold. Acy, Afa, Asp, Ama, Aki, and Atr stand forA. cyathophorum, A. cyathophorum var. farreri, A. spicatum, A. mairei, A. kingdonii, and A. trifurcatum, respectively.


## 3.4. Comparative Analysis of the Chloroplast Genomes among Six Species inAllium

mVISTA online software in the Shuffle-LAGAN mode was employed to analyze the comprehensive sequence discrepancy of the six chloroplast genomes ofAllium with the annotation of A. cyathophorum as a reference. In this study, the whole chloroplast genome alignment showed great sequence consistency of the six cp genomes, indicating that Allium cp genomes are very conservative (Figure 5). We found that among the six cp genomes, their IR region is more conserved compared to the LSC and SSC regions, which is similar with other plants [56, 57]. Furthermore, as we have found in other angiosperms, the coding areas were more conserved than the noncoding areas, and there were more variations in the intergenic spacers of the LSC and SSC areas, whereas the IR areas presented a lower sequence divergence [58, 59]. A. cyathophorum var. farreri had the highest sequence similarity to A. cyathophorum in sequence identity analysis. Noncoding regions displayed varying degrees of sequence differences in these six Allium cp genomes, including trnK-rps16, trnS-trnG, atpH-atpI, petN-psbM, trnT-psbD, trnF-ndhJ, accD-psaI, and petA-psbL. The coding areas with significant diversity contain matK, rps16, rpoC2, infA, ycf1, ndhF, and rps15 genes. The highly diverse regions found in this study may be used to develop molecular markers that can improve efficiency to study phylogenetic relationships within the Allium species.Figure 5
Sequence alignment of sixAllium chloroplast genomes (A. cyathophorum as the reference). The y-axis represents the percent similarity between 50% and 100%. Different colors represent different genetic regions.Though the cp genome is usually well conserved, having typical quadripartite structure, gene number, and order, a phenomenon recognized as ebb and flow exists, and this is where the IR area often expands or contracts [60]. Expansion and contraction of IR region is related to the size variations in the cp genome and has great differences in its evolution [61, 62]. We compared the IR/SC boundary areas of the six Allium cp genomes, and we found that there are obvious differences in the IR/LSC and IR/SSC connections (Figure 6). At the boundary of LSC/IRa junction, rps19 gene of different species distance the boundary were from 1 to 81 bp, while the rpl22 genes distance the border were from 29 to 273 bp. At the boundary of LSC/IRb connections, the psbA genes distance the border were reached from 108 to 605 bp. The inverted repeat b (IRb)/SSC border located in the coding region, and the ycf1 genes of the six species with a region ranged from 4193 to 5223 bp located in the SSC regions, which the ycf1 gene of A. trifurcatum all located in the SSC region. The shorter ycf1 gene crossed the inverted repeat (IRa)/SSC boundary, with 56–919 bp locating in the SSC regions. And the ndhF genes were situated in the SSC regions, which distance from the IRa/SSC boundary ranged from 1 to 1962 bp. Undoubtedly, the full-length differences in the sequence of the six cp genomes are caused by changes in the IR/SC boundaries.Figure 6
Comparison of the boundaries of the LSC, SSC, and IR areas of the whole chloroplast genomes of the six species.


## 3.5. Hotspot Regions Identification in SubgenusCyathophora

We totally extracted the shared 112 genes of the six species in chloroplast genomes; the nucleotide variability (Pi) ranged from 0.00041 (rrn16) to 0.08125 (infA) among these shared genes (Figure 7; Table S2). Seven genes (infA, rps16, rps15, ndhF, trnG-UCC, trnC-GCA, and trnK-UUU) were considered to be hotspot regions with a nucleotide diversity greater than 0.02. These regions can be used to develop useful markers for phylogenetic analysis and distinguish the species in Allium.Figure 7
The nucleotide diversity of the shared 112 genes of the six species in chloroplast genomes.
(a)
(b)


## 3.6. Synonymous (Ks) and Nonsynonymous (Ka) Substitution Rate Analysis

The Ka/Ks ratio is a significant index for understanding the evolution of protein-coding genes to assess gene differentiation rates and to determine whether positive, purified, or neutral selections have been performed; a Ka/Ks ratio >1 illustrates positive selection and Ka/Ks < 1 illustrates purifying selection, while the ratio of Ka/Ks close to 1 illustrates neutral selection [63]. In our study, the Ka/Ks ratio was calculated for 65 shared protein-coding genes in all six chloroplast genomes (Table S3), and the results are shown in Figure 8. The conservative genes with Ka/Ks ratio of 0.01, indicating powerful purifying selection pressure, were rpl2, rpl32, psaC, psbA, rpoC2, petN, psbZ, psaB, psaJ, and psbT, when the averaging Ka/Ks method showed ycf1 and ycf2 genes with Ka/Ks > 1, which shows that they may undergo some selective pressure among the six Allium species. The Ka/Ks ratios ranging from 0.5 to 1 were found for matK, rps16, psaI, cemA, petA, and rpl20, representing relaxed selection. The majority (56 of 65 genes) had an average Ka/Ks ratio ranging from 0 to 0.49 for the six compared groups, indicating that most genes were under purifying selection. Other than this, four genes (matK, rpoB, petA, and rpoA) with Ka/Ks > 1 in one or more pairwise comparisons (Figure 8) suggest that these genes may undergo selective pressure which is unknown, which is very important for researching the evolution of species.Figure 8
KA/KS analysis of 65 common protein coding genes in sixAllium species. Acy, Afa Asp, Ama, Aki, and Atr stand for A. cyathophorum, A. cyathophorum var. farreri, A. spicatum, A. mairei, A. kingdonii, and A. trifurcatum, respectively. KA: nonsynonymous; KS: synonymous.
(a)
(b)
(c)


## 3.7. Phylogenetic Analysis of Subgenus Cyathophora Depends on Chloroplast Genome

The cp genome of sequence is significant and helpful to construct phylogenetic relationships and explore the evolutionary history in many previous reports [64, 65]. To explore the phylogenetic relationship of the six Allium species, we constructed the phylogenetic tree using three different methods and databases containing twenty-one Allium species, six Lilium species, and two Asparagus species as the out groups (Figure 9). Three databases of the complete genome sequences, the IGS sequences, and all CDS sequences using MP, BI, and ML methods all showed the same topologies with high support (Figures S2 and S3). The results strongly supported that subgenus Cyathophora is a monophyletic group, comprising A. cyathophorum, A. cyathophorum var. farreri, A. spicatum, and A. mairei in this study with 100% bootstrap value; subgenus Cyathophora does not contain A. kingdonii and A. trifurcatum, and the phylogenetic tree indicates that A. cyathophorum var. farreri is a direct sister to A. spicatum, which is in accordance with the results of previous molecular research studies [4, 6]. The sister relationship of A. cyathophorum var. farreri and A. spicatum strongly suggests that A. spicatum is closely related to subgenus Cyathophora though it is a special species with the significant abnormal spicate inflorescence compared to other species with capitate or umbellate inflorescence. Furthermore, Allium kingdonii was the closest relative of Allium paradoxum and Allium ursinum.Figure 9
Phylogenetic relationship of subgenusCyathophora with relational species. The whole chloroplast genomes dataset was analyzed using three different methods: maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI). Numbers on the branches stand for bootstrap values in the ML, MP, and posterior probabilities in the BI trees. Six species of Lilium and two species of Asparagus were considered as the out groups. Purple shows the branch of the subgenus Cyathophora.


## 4. Discussion

### 4.1. Variations among the SixAllium Species

In this research, we assembled the complete cp genome of the six species inAllium. They were very conservative in genome structure and size; it showed a typical circular DNA structure and similar cp genome sequence length, ranging from 152,913 bp in A. mairei to 154,174 bp in A. cyathophorum. The six species had the identical numbers of protein-coding, tRNA, and rRNA genes. There were some expansion or contraction of IRs among these species (Figure 6); the expansion and contraction of IR regions are related to the divergences in chloroplast genome size [66]. To some extent, it is contributed to the cp genome variation and evolution. Other than this, variations in the IR/SC boundaries in the six cp genomes lead to the distinction in the whole length of sequence [61]. Previous research studies showed that SSRs have been widely known as important resources of molecular markers and have been broadly applied in phylogenetic and biogeographic studies [67, 68]. We surveyed and analyzed the quantities and distributions of SSRs with the six species in Allium, the largest number of SSR type was mononucleotide repeats, and the SSRs in the LSC area are much higher than those in the SSC and IR areas (Figure 4), showing that SSRs have a unevenly distribution in cp genome [50]. Additionally, we also explored seven common genes (infA, rps16, rps15, ndhF, trnG-UCC, trnC-GCA, and trnK-UUU) with nucleotide diversity more than 0.02 in the six cp genome sequences of Allium; among them, trnK-UUU, trnG-UCC, ndhF, and rps15 have been previously known as hypervariable regions in Allium [17], and we consider that these SSRs and genes with greater nucleotide diversity can be used as helpful DNA barcodes to identify the species in Allium.


### 4.2. Phylogenetic Relationships

The results of phylogenetic analysis clearly show thatAllium subgenus Cyathophora is a monophyletic group, and comprise four species (A. cyathophorum, A. farreri, A. spicatum and A. mairei), A. cyathophorum var. farreri has been upgraded to the level of the species as A. farreri in a recent study [69]. Besides A. farreri is a direct sister to A. spicatum with 100% strong bootstrap value, while the previous study showed low bootstrap value by the combined plastid dataset (trnL-F + rpl32-trnL) [4]. Currently, most phylogenetic relationships are obtained with chloroplast fragments, while single ITS, chloroplast fragment, or chloroplast combined fragment does not have a better effect in phylogenetic analysis compared to the whole cp genome. We convinced that the complete chloroplast genomes have more advantages to solve the phylogenetic issues about the subgenus Cyathophora. In previous studies, many phylogenetic problems in many plants have been successfully resolved by using complete cp genome sequences [18, 19, 70]; the lately published article about Allium also well resolved the phylogenetic relationship [17, 71]. Although the morphological characteristics of the A. farreri and A. spicatum are obviously different, in which A. spicatum has distinctive spicate inflorescence compared to A. farreri with umbel hemispheric inflorescence, our results undoubtedly showed A. farreri is a direct sister to A. spicatum, which is in accordance with Li et al. [6]. According to previous study, different inflorescence may imply that the umbel inflorescence was replaced by spicate inflorescence to adapt the harsh environment [6]. The phylogenetic tree revealed A. cyathophorum had a closer relationship with A. spicatum and A. farreri compared to A. mairei. Furthermore, the members of subgenus Cyathophora do not contain A. kingdonii and A. trifurcatum; A. kingdonii was the closest relative of A. paradoxum and A. ursinum, which is consistence with previous studies [4, 6]. Certainly, our study persuasively constructed reliable phylogeny relationship of subgenus Cyathophora by using the complete cp genome data.


### 4.3. Selection Events in Protein Coding Genes

DNA base mutations can be divided into two categories based on their effects to the encoded amino acids: synonymous mutations (Ks) and nonsynonymous mutations (Ka). Synonymous mutations do not result in amino acid changes, the frequency of which is represented by Ks; nonsynonymous mutations resulted in a change of amino acid, the frequency of which is indicated by Ka [72]. The ratio (Ka/Ks) is an important indicator to reveal evolutionary rate and natural selection pressure [73]. Interestingly, the synonymous nucleotide substitutions occurred at a higher frequency than nonsynonymous substitutions, and thus Ka/Ks ratios are constantly <1 in most genes [9, 74], and our study is similar with this. Between different regions and genes, the Ka/Ks ratios were usually specific (Figure 8). Most conserved genes (56 of 65 genes) had an average Ka/Ks value ranging from 0 to 0.49 for the fifteen comparison groups, indicating that most genes were under purifying selection. On the contrary, the average Ka/Ks values of the ycf1 and ycf2 genes were >1 in the fifteen comparison groups, revealing that some selective pressure may execute on them in six Allium species. Previous studies have shown that ycf1 and ycf2 genes were two large open reading frames; they were important to tobacco, and the gene knockout experiments showed that ycf1 and ycf2 played important role in a healthy cell [75]. Hu et al. [76] suggested that plants have a variety of adaptation strategies in response to unforeseen environmental conditions. Recent studies about Allium species also suggested that the selective pressure in chloroplast genomes play an important role in Allium species adaptation and evolution [17, 71]. In our field investigations, the species of subgenus Cyathophora grows in slopes or grasslands with altitude ranging from 2700 m to 4800 m. The elevated Ka/Ks ratios observed about some genes in the six Allium species may suggest that it is relate to their specific living environment. What is more, there were four genes (matK, rpoB, petA, and rpoA) with Ka/Ks > 1 in at one or more pairwise comparisons (Figure 8, Table S3), and among these genes, rpoA was also undergone positive selection in species of Annonaceae [77]. Previous study demonstrated that rpoA encodes the α subunit of plastid RNA polymerase (PEP), which is in charge of the expression of most photosynthesis-related genes [78]. It is generally believed that low temperature and strong ultraviolet radiation are not conducive to effective photosynthesis of plants; therefore, plants that survive and reproduce at high altitudes need a special photosynthetic protection strategy [79, 80]. In this study, the population of subgenus Cyathophora is mainly distributed in the Qinghai-Tibet Plateau and its adjacent high-altitude regions [2]. Therefore, we speculated that the positive selection of these genes may be related to the difference between their optimal growth environment.


## 4.1. Variations among the SixAllium Species

In this research, we assembled the complete cp genome of the six species inAllium. They were very conservative in genome structure and size; it showed a typical circular DNA structure and similar cp genome sequence length, ranging from 152,913 bp in A. mairei to 154,174 bp in A. cyathophorum. The six species had the identical numbers of protein-coding, tRNA, and rRNA genes. There were some expansion or contraction of IRs among these species (Figure 6); the expansion and contraction of IR regions are related to the divergences in chloroplast genome size [66]. To some extent, it is contributed to the cp genome variation and evolution. Other than this, variations in the IR/SC boundaries in the six cp genomes lead to the distinction in the whole length of sequence [61]. Previous research studies showed that SSRs have been widely known as important resources of molecular markers and have been broadly applied in phylogenetic and biogeographic studies [67, 68]. We surveyed and analyzed the quantities and distributions of SSRs with the six species in Allium, the largest number of SSR type was mononucleotide repeats, and the SSRs in the LSC area are much higher than those in the SSC and IR areas (Figure 4), showing that SSRs have a unevenly distribution in cp genome [50]. Additionally, we also explored seven common genes (infA, rps16, rps15, ndhF, trnG-UCC, trnC-GCA, and trnK-UUU) with nucleotide diversity more than 0.02 in the six cp genome sequences of Allium; among them, trnK-UUU, trnG-UCC, ndhF, and rps15 have been previously known as hypervariable regions in Allium [17], and we consider that these SSRs and genes with greater nucleotide diversity can be used as helpful DNA barcodes to identify the species in Allium.


## 4.2. Phylogenetic Relationships

The results of phylogenetic analysis clearly show thatAllium subgenus Cyathophora is a monophyletic group, and comprise four species (A. cyathophorum, A. farreri, A. spicatum and A. mairei), A. cyathophorum var. farreri has been upgraded to the level of the species as A. farreri in a recent study [69]. Besides A. farreri is a direct sister to A. spicatum with 100% strong bootstrap value, while the previous study showed low bootstrap value by the combined plastid dataset (trnL-F + rpl32-trnL) [4]. Currently, most phylogenetic relationships are obtained with chloroplast fragments, while single ITS, chloroplast fragment, or chloroplast combined fragment does not have a better effect in phylogenetic analysis compared to the whole cp genome. We convinced that the complete chloroplast genomes have more advantages to solve the phylogenetic issues about the subgenus Cyathophora. In previous studies, many phylogenetic problems in many plants have been successfully resolved by using complete cp genome sequences [18, 19, 70]; the lately published article about Allium also well resolved the phylogenetic relationship [17, 71]. Although the morphological characteristics of the A. farreri and A. spicatum are obviously different, in which A. spicatum has distinctive spicate inflorescence compared to A. farreri with umbel hemispheric inflorescence, our results undoubtedly showed A. farreri is a direct sister to A. spicatum, which is in accordance with Li et al. [6]. According to previous study, different inflorescence may imply that the umbel inflorescence was replaced by spicate inflorescence to adapt the harsh environment [6]. The phylogenetic tree revealed A. cyathophorum had a closer relationship with A. spicatum and A. farreri compared to A. mairei. Furthermore, the members of subgenus Cyathophora do not contain A. kingdonii and A. trifurcatum; A. kingdonii was the closest relative of A. paradoxum and A. ursinum, which is consistence with previous studies [4, 6]. Certainly, our study persuasively constructed reliable phylogeny relationship of subgenus Cyathophora by using the complete cp genome data.


## 4.3. Selection Events in Protein Coding Genes

DNA base mutations can be divided into two categories based on their effects to the encoded amino acids: synonymous mutations (Ks) and nonsynonymous mutations (Ka). Synonymous mutations do not result in amino acid changes, the frequency of which is represented by Ks; nonsynonymous mutations resulted in a change of amino acid, the frequency of which is indicated by Ka [72]. The ratio (Ka/Ks) is an important indicator to reveal evolutionary rate and natural selection pressure [73]. Interestingly, the synonymous nucleotide substitutions occurred at a higher frequency than nonsynonymous substitutions, and thus Ka/Ks ratios are constantly <1 in most genes [9, 74], and our study is similar with this. Between different regions and genes, the Ka/Ks ratios were usually specific (Figure 8). Most conserved genes (56 of 65 genes) had an average Ka/Ks value ranging from 0 to 0.49 for the fifteen comparison groups, indicating that most genes were under purifying selection. On the contrary, the average Ka/Ks values of the ycf1 and ycf2 genes were >1 in the fifteen comparison groups, revealing that some selective pressure may execute on them in six Allium species. Previous studies have shown that ycf1 and ycf2 genes were two large open reading frames; they were important to tobacco, and the gene knockout experiments showed that ycf1 and ycf2 played important role in a healthy cell [75]. Hu et al. [76] suggested that plants have a variety of adaptation strategies in response to unforeseen environmental conditions. Recent studies about Allium species also suggested that the selective pressure in chloroplast genomes play an important role in Allium species adaptation and evolution [17, 71]. In our field investigations, the species of subgenus Cyathophora grows in slopes or grasslands with altitude ranging from 2700 m to 4800 m. The elevated Ka/Ks ratios observed about some genes in the six Allium species may suggest that it is relate to their specific living environment. What is more, there were four genes (matK, rpoB, petA, and rpoA) with Ka/Ks > 1 in at one or more pairwise comparisons (Figure 8, Table S3), and among these genes, rpoA was also undergone positive selection in species of Annonaceae [77]. Previous study demonstrated that rpoA encodes the α subunit of plastid RNA polymerase (PEP), which is in charge of the expression of most photosynthesis-related genes [78]. It is generally believed that low temperature and strong ultraviolet radiation are not conducive to effective photosynthesis of plants; therefore, plants that survive and reproduce at high altitudes need a special photosynthetic protection strategy [79, 80]. In this study, the population of subgenus Cyathophora is mainly distributed in the Qinghai-Tibet Plateau and its adjacent high-altitude regions [2]. Therefore, we speculated that the positive selection of these genes may be related to the difference between their optimal growth environment.


## 5. Conclusions

Here, we sequenced, assembled, and annotated six chloroplast genomes ofAllium with high-throughput sequencing technology. The gene contents and orders of the cp genomes were extremely conservative, and their cp genomes are also quadripartite structure. Repeated sequence and SSRs are helpful sources for developing new molecular markers. Codon usage analyses detected that some amino acids of the six species showed distinct codon usage preferences, and we should comprehend codon usage bias to learn evolution process. We also discovered seven highly variable common genes which can be used to develop useful markers for phylogenetic analysis and distinguish species in Allium. The Ka/Ks analysis indicated that some selective pressure may exert on several genes in the chloroplast genomes of six Allium L. species. The maximum likelihood (ML), BI, and MP phylogenetic results clearly showed that subgenus Cyathophora comprised the four assembled species: A. cyathophorum, A. cyathophorum var. farreri, A. spicatum, and A. mairei, and A. cyathophorum var. farreri has a closer relationship with A. spicatum. This study will not only provide insights into the cp genome characteristics of species in subgenus Cyathophora but also supply useful genetic resources for phylogenetic analysis of genus Allium.



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*Source: 1732586-2020-01-17.xml*