A Comparative Analysis of the Chloroplast Genomes of Three Lonicera Medicinal Plants

Both Lonicerae japonicae flos and Lonicerae similis flos are important components in traditional Chinese medicine (TCM) with precious medicinal value. However, the absence of studies on their chloroplast genomes and chromatography has considerably hindered the study of their evolutionary and phylogenetic relationships. In this study, the complete chloroplast (cp) genomes of Lonicera acuminata Wall. and Lonicera similis Hemsl. were sequenced using the Illumina sequencing platform and compared with that of Lonicera japonica Thunb., which has been previously reported. Furthermore, the chromatographic fingerprints of the three plants were constructed using HPLC and the content of quality marker (Q-Marker) was calculated. The annotation results showed that the two chloroplast genomes were typical quadripartite structures with lengths of 155,330 bp (L. acuminata) and 155,207 bp (L. similis). A total of 126 different genes were annotated, containing 82 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. The expansion and contraction of the inverted repeat (IR) regions suggested that the boundary regions of IR/SC were comparatively conserved in the three species, and six regions (trnH-GUG-psbA, rps2-rpoC2, rbcL-psaI, trnN-GUU-ndhF, rps15-ycf1, and infA) with nucleotide diversity values (Pi) of variable sites higher than 1% were identified. Phylogenetic relation indicated that L. similis had a closer genetic relationship with L. japonica than L. acuminata. Additionally, the chromatographic fingerprints showed that the characteristic peaks of the three medicinal plants were similar, including Neochlorogenic acid, Chlorogenic acid, 4-Dicaffeoylquinic acid, Sweroside, Secoxyloganin, Luteoloside, Isochlorogenic acid A, Isochlorogenic acid B, and Isochlorogenic acid C. The content of chlorogenic acid and total phenolic acid in L. acuminata (7.4633 ± 0.4461%, 14.8953 ± 0.0728%) and L. similis (14.1055 ± 0.2566%, 21.9782 ± 0.1331%) was much higher than that of L. japonica (3.9729 ± 0.0928%, 6.0964 ± 0.1228%), respectively. This study provides appropriate information for species identification, phylogeny, quality assessment, and rational use of three medicinal plants of the genus Lonicera.


Introduction
Lonicerae japonicae flos, a traditional Chinese medicine (TCM), has been extensively used for several thousand years in China [1][2][3][4]. In the 2020 edition of the Pharmacopoeia of the People's Republic of China (Chinese Pharmacopeia), Lonicerae japonicae flos is defined as the dried flower buds or initial flowers of L. japonica Thunb., which belongs to Caprifoliaceae [5]. As a famous TCM, Lonicerae japonicae flos has antiviral, anti-inflammatory, and antibacterial activity, and pharmacological studies have shown it is effective in the treatment of inflammation and infectious diseases [3,[6][7][8][9]. Significantly, Lianhua Qingwen Capsule/Granule, Jinhua Qinggan Granule, and Shuanghuanglian Oral Liquid, three

Cp Genome Organizations
We used high-throughput Illumina paired-end sequencing data to acquire cp DNA sequences of L. acuminata and L. similis, and the annotated, complete chloroplast genomes of the two species were then deposited in GenBank under accession numbers MZ901373 and MZ241297, respectively. Three complete cp genome sequences, including the two Lonicera species sequenced in this study and the L. japonica accession from GenBank, were combined for integrated analysis. The genomes ranged in size from 155,207 (L. similis) to 155,330 bp (L. acuminata) (Figure 1). The genomes of these species have a typical quadripartite structure, with two IRs (23,777) separated by the LSC (88,859-89,149) and SSC (18,672) regions. The GC content of the L. japonica, L. similis, and L. acuminata cp genomes is 38.58%, 38.59%, and 38.55%, respectively (Table 1); these three species showed similar GC levels (~39%). Lonicera cp genomes displayed analogous gene content and order, containing 126 genes, 82 protein-coding genes, 36 tRNA genes, and 8 rRNA genes.
Genes 2023, 14, x FOR PEER REVIEW 3 of 16 lated using HPLC. This study provides a valuable resource for species identification, quality evaluation, and genetic improvement of the genus Lonicera.

Cp Genome Organizations
We used high-throughput Illumina paired-end sequencing data to acquire cp DNA sequences of L. acuminata and L. similis, and the annotated, complete chloroplast genomes of the two species were then deposited in GenBank under accession numbers MZ901373 and MZ241297, respectively. Three complete cp genome sequences, including the two Lonicera species sequenced in this study and the L. japonica accession from GenBank, were combined for integrated analysis. The genomes ranged in size from 155,207 (L. similis) to 155,330 bp (L. acuminata) (Figure 1). The genomes of these species have a typical quadripartite structure, with two IRs (23,777) separated by the LSC (88,859-89,149) and SSC (18,672) regions. The GC content of the L. japonica, L. similis, and L. acuminata cp genomes is 38.58%, 38.59%, and 38.55%, respectively (Table 1); these three species showed similar GC levels (~39%). Lonicera cp genomes displayed analogous gene content and order, containing 126 genes, 82 protein-coding genes, 36 tRNA genes, and 8 rRNA genes.

Repeat and Simple Sequence Repeat (SSR) Analyses
A comparative analysis of the repeats in the three Lonicera cp genomes suggested that the three types of repeats (tandem, forward, and palindromic repeats) were similar in number and distribution (Figure 2a). L. japonica presented the highest frequency of tandem repeats (72) and forward repeats (37), while L. acuminata had the lowest with (53) and (33), respectively. Palindromic repeats did not differ much between L. japonica (12), L. similis (14), and L. acuminata (13). The tandem repeats in the three Lonicera cp genomes ranged from 10 to 30 bp in length (Figure 2b), while the forward and palindromic repeats were mostly between 36 and 65 bp in length (Figure 2c,d).  The cp genome is uniparentally inherited and SSRs share a high level of variation within the same species [27]. As such, they are used as molecular markers in developmental research and also help to identify species [37]. MISA detected 155 SSRs in L. japonica, comprising 109 mono-, 32 di-, 2 tri-, 9 tetra-, and 3 hexanucleotide repeats. Here, a grand total of 155 SSRs were detected in the L. similis cp genome; among these SSRs, there were 109, 32, 2, 8, and 4 for mono-, di-, tri-, tetra-, and hexa-nucleotide repeats, respectively. No pentanucleotide repeats were discovered. A total of 155 SSRs, comprising 112 mono-, 31 di-, 4 tri-, and 8 tetra-nucleotide repeats were observed in the L. acuminata cp genome, while no penta-and hexa-nucleotide repeats were found ( Figure 3). SSRs of the three cp genomes were composed of mono-and di-repeat motifs. In the L. japonica and L. similis cp genomes, there are the same number of mono-repeats and di-repeats: 109 and 32, respectively. In the L. acuminata cp genome, the mono-repeats and di-repeats number 112 and 31, respectively. However, the number of three or more oligonucleotide repeats is comparatively low.

IR Expansion and Contraction
It is reported that the chloroplast genome of angiosperms is conserved in structure and size [37]; the differences in IRs may reflect phylogenetic history [38]. Here, we selected three species of the family Caprifoliaceae and compared the sizes and junctions of their LSC, SSC, and IR regions. Although the lengths of the IR regions, ranging from 23,760 bp to 23,777 bp, varied little among the three species, some differences in the IR expansions and contractions were observed. As shown in Figure 4, the rpl2 gene of all three species was located in the LSC region, the rpl23 gene was located at the LSC/IRb border expanded 121 bp into the IRb region, and the ndhF gene of the three species extended 8 bp into the IRb region. For the SSC/IRa boundaries in L. japonica, L. similis, and L. acuminata, the distances were 220 bp, 208 bp, and 208 bp from the ycf1 gene to the boundary, respectively ( Figure 4). 23,760 bp to 23,777 bp, varied little among the three species, some differences in the IR expansions and contractions were observed. As shown in Figure 4, the rpl2 gene of all three species was located in the LSC region, the rpl23 gene was located at the LSC/IRb border expanded 121 bp into the IRb region, and the ndhF gene of the three species extended 8 bp into the IRb region. For the SSC/IRa boundaries in L. japonica, L. similis, and L. acuminata, the distances were 220 bp, 208 bp, and 208 bp from the ycf1 gene to the boundary, respectively ( Figure 4).

Sequence Divergence Analysis
The three complete cp genomes were compared with mVISTA using L. japonica as a reference. As shown in Figure 5, high sequence conservation across the three Lonicera cp genomes was observed, especially in gene regions. Variant loci in intergenic regions were distinguished higher than those in the gene regions. Most of the highly variable regions were in the conserved non-coding sequences (CNS) region, included rps16-trnQ, ycf1-trnN, psaA-ycf3, rbcL-accD, psaJ-clpP, and petB-petD, etc. In order to clarify the variation in the higher regions, the nucleotide diversity values (Pi) were calculated using the DnaSP v.6.10 software ( Figure 6). The variation in intergenic regions ranged from 0 to 2.23%, with an average of 0.36%, which was twofold higher than that in the CDS regions (0.16% on average). Five divergent loci in intergenic regions (trnH-GUG-psbA, rps2-rpoC2, rbcL-psaI, trnN-GUU-ndhF, and rps15-ycf1) and one in the CDS regions (infA) had a Pi exceeding 1%. These six divergence hotspot regions should be applied to the development of molecular markers for phylogenetic and phylogeographic analyses, as well as plant identification of Lonicera species.

Sequence Divergence Analysis
The three complete cp genomes were compared with mVISTA using L. japonica as a reference. As shown in Figure 5, high sequence conservation across the three Lonicera cp genomes was observed, especially in gene regions. Variant loci in intergenic regions were distinguished higher than those in the gene regions. Most of the highly variable regions were in the conserved non-coding sequences (CNS) region, included rps16-trnQ, ycf1-trnN, psaA-ycf3, rbcL-accD, psaJ-clpP, and petB-petD, etc. In order to clarify the variation in the higher regions, the nucleotide diversity values (Pi) were calculated using the DnaSP v.6.10 software ( Figure 6). The variation in intergenic regions ranged from 0 to 2.23%, with an average of 0.36%, which was twofold higher than that in the CDS regions (0.16% on average). Five divergent loci in intergenic regions (trnH-GUG-psbA, rps2-rpoC2, rbcL-psaI, trnN-GUU-ndhF, and rps15-ycf1) and one in the CDS regions (infA) had a Pi exceeding 1%. These six divergence hotspot regions should be applied to the development of molecular markers for phylogenetic and phylogeographic analyses, as well as plant identification of Lonicera species.

Phylogenetic Analysis
To further analyze the relationships between L. acuminata, L. similis, and L. japonica, 22 chloroplast genome sequences of the family Caprifoliaceae and one outgroup (Antirhea chinensis) were selected and downloaded from GenBank to construct phylogenetic trees with L. acuminata and L. similis, adopting the maximum-likelihood (ML) method. The ML tree based on the GTR + G + I optimal model was constructed through IQ-tree (Version 2.04) with 1000 bootstrap replicates [39]. In our study, the selected species only came from six genera of the family Caprifoliaceae. Figure 7 clearly displays that all species generated six major clades, which correspond to the six selected genera. All Lonicera were clustered together in one monophyletic group, between L. acuminata, L. similis, and L. japonica of the genus Lonicera and family Caprifoliaceae. L. japonica was clustered together with L. confusa and L. similis, which implicates that L. similis may have a closer genetic relationship with L. japonica than L. acuminata. s 2023, 14, x FOR PEER REVIEW 9 of

Phylogenetic Analysis
To further analyze the relationships between L. acuminata, L. similis, and L. japoni 22 chloroplast genome sequences of the family Caprifoliaceae and one outgroup (An hea chinensis) were selected and downloaded from GenBank to construct phylogene trees with L. acuminata and L. similis, adopting the maximum-likelihood (ML) metho The ML tree based on the GTR + G + I optimal model was constructed through IQ-tr (Version 2.04) with 1000 bootstrap replicates [39]. In our study, the selected species on came from six genera of the family Caprifoliaceae. Figure 7 clearly displays that all sp cies generated six major clades, which correspond to the six selected genera. All Lonic were clustered together in one monophyletic group, between L. acuminata, L. similis, a L. japonica of the genus Lonicera and family Caprifoliaceae. L. japonica was clustered gether with L. confusa and L. similis, which implicates that L. similis may have a clo genetic relationship with L. japonica than L. acuminata.

Plant Morphology
It can be seen in Table 2 that the main medicinal parts (flower buds and flowers) Lonicerae japonicae flos and Lonicerae similis flos have a fresh scent and a light a slightly bitter taste, but they differ more markedly in color, length, and upper swoll parts. In our study, L. japonica was yellow-white in color, L. similis was yellow-brow and L. acuminata was yellow-green. For the medicinal parts of the three plants (flow buds and flowers), L. similis was the longest but shortest in upper diameter, L. acumin was the longest in upper diameter, and L. japonica was in the middle.

Plant Morphology
It can be seen in Table 2 that the main medicinal parts (flower buds and flowers) of Lonicerae japonicae flos and Lonicerae similis flos have a fresh scent and a light and slightly bitter taste, but they differ more markedly in color, length, and upper swollen parts. In our study, L. japonica was yellow-white in color, L. similis was yellow-brown, and L. acuminata was yellow-green. For the medicinal parts of the three plants (flower buds and flowers), L. similis was the longest but shortest in upper diameter, L. acuminata was the longest in upper diameter, and L. japonica was in the middle.

HPLC Fingerprints and the Content of Main Q-Marker
In terms of chemical composition, HPLC fingerprints of the three species were tained by high performance liquid chromatography (HPLC) analysis at 240 nm with erence to the Chinese Pharmacopoeia [5] and superimposed on the control (Figure 8). Fr the fingerprints, we can clearly see that the main active ingredients of Lonicerae sim flos and Lonicerae japonicae flos are basically the same, including Neochlorogenic ac Chlorogenic acid, 4-Dicaffeoylquinic acid, Sweroside, Secoxyloganin, Luteoloside, I chlorogenic acid A, Isochlorogenic acid B, Isochlorogenic acid C, etc., but the conten main Q-Marker differs ( Table 3). The evaluation standards for L. japonica in the Chin Pharmacopoeia [5] require it to contain not less than 1.5% chlorogenic acid, not less th 3.8% total phenolic acid, and not less than 0.050% Luteoloside. Here, the content chlorogenic acid and total phenolic acid in L. acuminata (7.4633 ± 0.4461%, 14.895 0.0728%) and L. similis (14.1055 ± 0.2566%, 21.9782 ± 0.1331%) was much higher than t of L. japonica (3.9729 ± 0.0928%, 6.0964 ± 0.1228%), respectively. The content of Luteo side in L. similis (0.0291 ± 0.0044%) was lower than that specified in the pharmacopo but it is noteworthy that the content of chlorogenic acid, its main purgative and deto fying component, was higher than that of all other species at 14.8953 ± 0.0728%.

HPLC Fingerprints and the Content of Main Q-Marker
In terms of chemical composition, HPLC fingerprints of the three species were obtained by high performance liquid chromatography (HPLC) analysis at 240 nm with reference to the Chinese Pharmacopoeia [5] and superimposed on the control (Figure 8). From the fingerprints, we can clearly see that the main active ingredients of Lonicerae similis flos and Lonicerae japonicae flos are basically the same, including Neochlorogenic acid, Chlorogenic acid, 4-Dicaffeoylquinic acid, Sweroside, Secoxyloganin, Luteoloside, Isochlorogenic acid A, Isochlorogenic acid B, Isochlorogenic acid C, etc., but the content of main Q-Marker differs ( Table 3). The evaluation standards for L. japonica in the Chinese Pharmacopoeia [5] require it to contain not less than 1.5% chlorogenic acid, not less than 3.8% total phenolic acid, and not less than 0.050% Luteoloside. Here, the content of chlorogenic acid and total phenolic acid in L. acuminata (7.4633 ± 0.4461%, 14.8953 ± 0.0728%) and L. similis (14.1055 ± 0.2566%, 21.9782 ± 0.1331%) was much higher than that of L. japonica (3.9729 ± 0.0928%, 6.0964 ± 0.1228%), respectively. The content of Luteoloside in L. similis (0.0291 ± 0.0044%) was lower than that specified in the pharmacopoeia, but it is noteworthy that the content of chlorogenic acid, its main purgative and detoxifying component, was higher than that of all other species at 14.8953 ± 0.0728%.

HPLC Fingerprints and the Content of Main Q-Marker
In terms of chemical composition, HPLC fingerprints of the three species were obtained by high performance liquid chromatography (HPLC) analysis at 240 nm with reference to the Chinese Pharmacopoeia [5] and superimposed on the control (Figure 8). From the fingerprints, we can clearly see that the main active ingredients of Lonicerae similis flos and Lonicerae japonicae flos are basically the same, including Neochlorogenic acid, Chlorogenic acid, 4-Dicaffeoylquinic acid, Sweroside, Secoxyloganin, Luteoloside, Isochlorogenic acid A, Isochlorogenic acid B, Isochlorogenic acid C, etc., but the content of main Q-Marker differs ( Table 3). The evaluation standards for L. japonica in the Chinese Pharmacopoeia [5] require it to contain not less than 1.5% chlorogenic acid, not less than 3.8% total phenolic acid, and not less than 0.050% Luteoloside. Here, the content of chlorogenic acid and total phenolic acid in L. acuminata (7.4633 ± 0.4461%, 14.8953 ± 0.0728%) and L. similis (14.1055 ± 0.2566%, 21.9782 ± 0.1331%) was much higher than that of L. japonica (3.9729 ± 0.0928%, 6.0964 ± 0.1228%), respectively. The content of Luteoloside in L. similis (0.0291 ± 0.0044%) was lower than that specified in the pharmacopoeia, but it is noteworthy that the content of chlorogenic acid, its main purgative and detoxifying component, was higher than that of all other species at 14.8953 ± 0.0728%.

HPLC Fingerprints and the Content of Main Q-Marker
In terms of chemical composition, HPLC fingerprints of the three species were obtained by high performance liquid chromatography (HPLC) analysis at 240 nm with reference to the Chinese Pharmacopoeia [5] and superimposed on the control (Figure 8). From the fingerprints, we can clearly see that the main active ingredients of Lonicerae similis flos and Lonicerae japonicae flos are basically the same, including Neochlorogenic acid, Chlorogenic acid, 4-Dicaffeoylquinic acid, Sweroside, Secoxyloganin, Luteoloside, Isochlorogenic acid A, Isochlorogenic acid B, Isochlorogenic acid C, etc., but the content of main Q-Marker differs ( Table 3). The evaluation standards for L. japonica in the Chinese Pharmacopoeia [5] require it to contain not less than 1.5% chlorogenic acid, not less than 3.8% total phenolic acid, and not less than 0.050% Luteoloside. Here, the content of chlorogenic acid and total phenolic acid in L. acuminata (7.4633 ± 0.4461%, 14.8953 ± 0.0728%) and L. similis (14.1055 ± 0.2566%, 21.9782 ± 0.1331%) was much higher than that of L. japonica (3.9729 ± 0.0928%, 6.0964 ± 0.1228%), respectively. The content of Luteoloside in L. similis (0.0291 ± 0.0044%) was lower than that specified in the pharmacopoeia, but it is noteworthy that the content of chlorogenic acid, its main purgative and detoxifying component, was higher than that of all other species at 14.8953 ± 0.0728%.

Plant Material and DNA Extraction
L. similis was collected in the town of Dangwu, Huaxi District, Guiyang City (26°23′34.49″ N, 106°35′56.89″ E, 1158 m above sea level), Guizhou Province, China, and L. acuminata was harvested from the village of Hekou, the town of Muxi, Muchuan County, Sichuan Province, China (28°51′7.40″ N, 103°50′55.46″ E, 1114 m altitude) by Chenju Yang. According to the macroscopic morphological characteristics of the specimen, the species was determined [40]. Total genomic DNA of two species was extracted using an E.Z.N.A ® plant DNA kit (FEIYANG, Guangzhou, China). For each sample a combined total of 1000 ng of DNA served as input to the DNA sample preparation. The DNA library was established utilizing the TruseqTM RNA Sample Prep Kit. Whole DNA was utilized to generate libraries of an average insert size of 400 bp. An Illumina platform was used to sequence the library preparation and obtained approximately 3 GB of 150 bp paired-end reads, which were saved in fastq format.

Assembly and Annotation
FastQC was used to examine the acquired raw data for analysis of quality, including single base content quality, base content mapping, GC content distributions, and quality of sequence bases. After removal of the adapter sequences, program GetOrganelle was used to assemble the filtered reads [41] using L. japonica (GenBank accession number: NC_026839) for the original genome reference, and the genome of the assembled chloroplast was annotated by the GeSeq online package [42]. Finally, the accurate, annotated, complete chloroplast genomes of two Lonicera species were deposited to GenBank with accession numbers MZ241297 and MZ901373. Here, the document we use was further annotated based on the accession number.

Characterization of Repeat Sequences and SSRs
Repeat sequences, comprising the forward and palindromic, were identified by REPuter [43] with a Hamming distance set at 3 bp and a minimum repeat size set at 30    According to the macroscopic morphological characteristics of the specimen, the species was determined [40]. Total genomic DNA of two species was extracted using an E.Z.N.A ® plant DNA kit (FEIYANG, Guangzhou, China). For each sample a combined total of 1000 ng of DNA served as input to the DNA sample preparation. The DNA library was established utilizing the TruseqTM RNA Sample Prep Kit. Whole DNA was utilized to generate libraries of an average insert size of 400 bp. An Illumina platform was used to sequence the library preparation and obtained approximately 3 GB of 150 bp paired-end reads, which were saved in fastq format.

Assembly and Annotation
FastQC was used to examine the acquired raw data for analysis of quality, including single base content quality, base content mapping, GC content distributions, and quality of sequence bases. After removal of the adapter sequences, program GetOrganelle was used to assemble the filtered reads [41] using L. japonica (GenBank accession number: NC_026839) for the original genome reference, and the genome of the assembled chloroplast was annotated by the GeSeq online package [42]. Finally, the accurate, annotated, complete chloroplast genomes of two Lonicera species were deposited to GenBank with accession numbers MZ241297 and MZ901373. Here, the document we use was further annotated based on the accession number.

Characterization of Repeat Sequences and SSRs
Repeat sequences, comprising the forward and palindromic, were identified by RE-Puter [43] with a Hamming distance set at 3 bp and a minimum repeat size set at 30 bp. Tandem repeats were analyzed using the Tandem Repeats Finder with default parameters [44]. SSRs were detected using MISA with the following thresholds [45]: eight repeat units for mono SSRs, four repeat units for di-and trinucleotide repeat SSRs, and three repeat units for tetra-, penta-, and hexanucleotide repeat SSRs.

Comparative Genome Analysis and Sequence Variation
The program mVISTA in the Shuffle-LAGAN mode was applied to compare the cp genomes of Lonicerae japonicae flos (L. japonica) and Lonicerae similis flos (L. acuminata and L. similis) [46]. The variable character for coding and non-coding region with an aligned length of more than 200 bp was obtained based on the method of Zhang et al. [47]. The nucleotide variability was calculated with DnaSP 6.10 [48]. The online software IRSCOPE (https://irscope.shinyapps.io/irapp/, accessed on 31 October 2022) was adopted to carry out boundary analysis on the IR regions of the three species [49].

Phylogenetic Analysis
The chloroplast genome sequences of 22 Caprifoliaceae family and one outgroup (A. chinensis) were downloaded from GenBank to build phylogenetic trees by maximumlikelihood (ML) with L. acuminata and L. similis. The ML tree was constructed using IQtree (Version 2.04, Trifinopoulos J., Vienna ，Austria) based on the GTR + G + I optimal model with 1000 bootstrap replicates [39,50].

Discussion
In this study, we conducted a comprehensive comparative analysis of Lonicerae similis flos and Lonicerae japonicae flos. The cp genomes of the three species ranged in length from 155,079 (L. japonica) to 155,330 bp (L. acuminata); they had a typical quadripartite structure, usually consisting of an LSC, an SSC, and two IR regions, which is consistent with highly conserved features of the cp genome of most angiosperms [25]. Compared with previous studies [33,51,52], chloroplast genomes of Lonicera plants differ little in terms of genome organization, number of genes, and types. In a comparative analysis of repeat sequences in the cp genomes, L. japonica (a total of 121 repeats, 72 tandem, 37 forward, 12 palindromic repeats) and L. similis (a total of 115 repeats, 66 tandem, 35 forward, 14 palindromic repeats) repeats were similar, while L. acuminata (a total of 99 repeats, 53 tandem, 33 forward, 13 palindromic repeats) had shorter fragment repeats. The three cp genomes had similar repeat unit lengths, with tandem repeats ranging from 10 to 30 bp and forward and palindromic repeats ranging from 36 to 65 bp. The cp genome is uniparentally inherited and SSRs have a high level of variation within the same species [27]. As such, they are used as the molecular indicators in developmental studies and also help to identify species [37]. SSRs are commonly used as genetic markers in community genetics and evolutionary studies [31]. The most enriched were mononucleotide repeats, next to dinucleotide repeats. As a whole, tetranucleotide repeats were slightly more abundant than trinucleotide and hexanucleotide repeats, and in all three cp genomes pentanucleotide repeats were rare. The three species shared similar IR expansion and contraction, and in sequence alignments had higher variance genes in the CNS region, including rps16-trnQ, ycf1-trnN, psaA-ycf3, rbcL-accD, psaJ-clpP, and petB-petD, etc. The nucleotide diversity values (Pi) showed that six regions (trnH-GUG-psbA, rps2-rpoC2, rbcL-psaI, trnN-GUU-ndhF, rps15-ycf1, and infA) had a Pi exceeding 1%, and these six divergence hotspot regions should be useful for developing molecular markers. In the phylogenetic tree, L. japonica may be more closely related to L. similis than L. acuminata. Our evolutionary tree formed three main branches, which was consistent with previous research [33]. At the same time, plants in the same growing environment may be more closely related.
Despite their different origins, they have close proximity of plant species, appearances, and functions, together with traditional applications [5,19]. Studies have shown that chromatographic fingerprint analysis provides a more rational method for the identification and quality assessment of traditional Chinese herbal medicines [35,36], which can effectively identify the authenticity, merit, and demerit of traditional Chinese medicines and can increase the accuracy of species identification. In this study, we used high performance liquid chromatography (HPLC) to obtain HPLC fingerprints of three species and calculated their contents with reference to the Chinese Pharmacopoeia [5]. HPLC fingerprints showed that Lonicerae similis flos and Lonicerae japonicae flos shared the same characteristic peaks, except for Lonicerae similis flos where Caffeic acid was not detected (peak 4), but the contents of quality marker (Q-Marker) were different. The content of Luteoloside (0.0291 ± 0.0044%) in L. similis was lower than that specified in the pharmacopoeia for Lonicerae japonicae flos, but it is noteworthy that the content of chlorogenic acid and total phenolic acid in L. acuminata (7.4633 ± 0.4461%, 14.8953 ± 0.0728%) and L. similis (14.1055 ± 0.2566%, 21.9782 ± 0.1331%) was much higher than that of L. japonica (3.9729 ± 0.0928%, 6.0964 ± 0.1228%), respectively. Chlorogenic acid is known as "plant gold", which has hypotensive effects, antioxidant, and antibacterial activities [53][54][55][56][57]. It is may be one of the potential active ingredients of Lonicerae japonicae flos in the treatment of COVID-19 [58]. With such high chlorogenic acid content, L. similis may be selected as a better genetic line for superior breeding and a better source species for chlorogenic acid. These results also provide a basis for future in-depth studies on the synthesis mechanisms of high chlorogenic acid in L. similis.

Conclusions
In this study, the cp genomes of Lonicerae similis flos (L. acuminata and L. similis) were sequenced and annotated through high-throughput sequencing technology. We compared the cp genomes of these three species, and bioinformatics analysis revealed that the structure and gene content of the cp genomes of these three Lonicerae were highly similar and conserved, suggesting a close relationship between them. We identified 155 SSR loci that could potentially be used as molecular markers to study the diversity of the genus Lonicera. Six different mutation hot spots (trnH-GUG-psbA, rps2-rpoC2, rbcL-psaI, trnN-