Next Article in Journal
BRACNAC: A BRCA1 and BRCA2 Copy Number Alteration Caller from Next-Generation Sequencing Data
Previous Article in Journal
Dopamine, Norepinephrine and Serotonin Participate Differently in Methylphenidate Action in Concomitant Behavioral and Ventral Tegmental Area, Locus Coeruleus and Dorsal Raphe Neuronal Study in Young Rats
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 24
Issue 23
10.3390/ijms242316629
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

New Insights into Phylogenetic Relationship of Hydrocotyle (Araliaceae) Based on Plastid Genomes

by
Rongrong Yan
1,2,†,
Li Gu
1,2,†,
Lu Qu
3,
Xiaoyu Wang
1,2 and
Guoxiong Hu
1,2,*
1
The Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region, Ministry of Education, Guizhou University, Guiyang 550025, China
2
College of Life Sciences, Guizhou University, Guiyang 550025, China
3
Institute of Medicinal Plant Development Yunnan Branch, Chinese Academy of Medical Sciences, Jinghong 666100, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2023, 24(23), 16629; https://doi.org/10.3390/ijms242316629
Submission received: 11 October 2023 / Revised: 17 November 2023 / Accepted: 21 November 2023 / Published: 22 November 2023
(This article belongs to the Section Molecular Genetics and Genomics)
Browse Figures
Versions Notes

Abstract

:
Hydrocotyle, belonging to the Hydrocotyloideae of Araliaceae, consists of 95 perennial and 35 annual species. Due to the lack of stable diagnostic morphological characteristics and high-resolution molecular markers, the phylogenetic relationships of Hydrocotyle need to be further investigated. In this study, we newly sequenced and assembled 13 whole plastid genomes of Hydrocotyle and performed comparative plastid genomic analyses with four previously published Hydrocotyle plastomes and phylogenomic analyses within Araliaceae. The plastid genomes of Hydrocotyle exhibited typical quadripartite structures with lengths from 152,659 bp to 153,669 bp, comprising a large single-copy (LSC) region (83,958–84,792 bp), a small single-copy (SSC) region (18,585–18,768 bp), and a pair of inverted repeats (IRs) (25,058–25,145 bp). Each plastome encoded 113 unique genes, containing 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. Comparative analyses showed that the IR boundaries of Hydrocotyle plastomes were highly similar, and the coding and IR regions exhibited more conserved than non-coding and single-copy (SC) regions. A total of 2932 simple sequence repeats and 520 long sequence repeats were identified, with specificity in the number and distribution of repeat sequences. Six hypervariable regions were screened from the SC region, including four intergenic spacers (IGS) (ycf3-trnS, trnS-rps4, petA-psbJ, and ndhF-rpl32) and two coding genes (rpl16 and ycf1). Three protein-coding genes (atpE, rpl16, and ycf2) were subjected to positive selection only in a few species, implying that most protein-coding genes were relatively conserved during the plastid evolutionary process. Plastid phylogenomic analyses supported the treatment of Hydrocotyle from Apiaceae to Araliaceae, and topologies with a high resolution indicated that plastome data can be further used in the comprehensive phylogenetic research of Hydrocotyle. The diagnostic characteristics currently used in Hydrocotyle may not accurately reflect the phylogenetic relationships of this genus, and new taxonomic characteristics may need to be evaluated and selected in combination with more comprehensive molecular phylogenetic results.

1. Introduction

Current molecular phylogenetic analyses indicate that Hydrocotyle belongs to the subfamily Hydrocotyloideae of Araliaceae [1,2,3,4]. This genus, comprising approximately 130 species, is widely distributed worldwide but primarily found in the Neotropics, Africa, Southeast Asia, and the warmer regions of China, with a few occurring in Europe [4,5,6]. The majority of the members of Hydrocotyle are perennial, but approximately 35 species endemic to Australia are annual herbs [4]. In China, all taxa, including 18 species, two subspecies, one variety, and two introduced species (Hydrocotyle ranunculoides and H. verticillata), that have been recorded are perennial [7,8]. Hydrocotyle is of economic importance and some species can be used for ornamental, medicinal, and edible purposes. For example, H. verticillata and H. sibthorpioides ‘Variegata’ have been widely used in wetland and aquatic landscape construction [9]. As a traditional Chinese herbal medicine, H. sibthorpioides contains flavonoids, glycerin, and triterpenes that can be used for the treatment of hepatitis and tumors and clearing away heat and toxic materials [10].
Historically, Hydrocotyle is a taxonomically difficult genus in which species differentiation relies heavily on the morphology of the fruit, inflorescence, and leaf [11,12,13]. However, many morphologically similar taxa show continuous variation, which makes accurate identification very difficult between or within species. For example, Hydrocotyle sibthorpioides var. sibthorpioides is morphologically similar to H. sibthorpioides var. batrachium. The main difference is that the leaves of the former are shallowly 5–7 lobed or nearly entire, while the leaves of the latter are deeply 3–5 lobed [7]. In our field investigations, however, there is continuous variation in the degree of leaf division, whereby 3–7 lobed leaves can be seen in the same population. In addition, H. hamelinensis and H. puberula were previously recognized as two putatively distinct species on the basis of morphology in Australia but were finally demonstrated to be conspecific with H. tetragonocarpa and H. scutellifera, respectively [4]. Recently, simple DNA markers, such as ITS, matK, rpl16, trnD-trnT, and psbA-trnH, have been applied to solve the phylogenetic relationships of Hydrocotyle [2,4,14,15]. Although these short DNA fragments can recognize the main clades within Hydrocotyle, topologies among closely related species are still polytomous or have low phylogenetic resolution.
Compared with the complex nuclear genome and unstable mitochondrial genome, the plastid genome shows great advantages, such as uniparental inheritance, a stable genetic structure, and a relatively small size for genetic and evolutionary analyses [16,17,18]. At present, complete plastid genomes are widely being applied to solve phylogenetic relationships at different taxonomical levels within angiosperms [19,20,21,22] and have significantly improved the phylogenetic resolution of Araliaceae compared with Sanger sequencing data [23,24,25,26]. To date, four complete plastid genomes of Hydrocotyle have been reported, including Hydrocotyle nepalensis, H. pseudoconferta, H. sibthorpioides, and H. verticillata. Previous studies have placed a greater focus on the plastome size and gene content for a single species of Hydrocotyle [27,28], but plastid comparative genomes and phylogenomic analyses at the generic level are still lacking. Given the importance of comprehensive genomics to understand plastome evolution and phylogenetic relationships in Hydrocotyle, even Araliaceae, it is necessary to analyze more plastid genomes of Hydrocotyle.
In this study, we newly sequenced and assembled 13 plastid genomes of Hydrocotyle. Comparative genomic analyses were conducted for 17 Hydrocotyle plastomes and phylogenomic analyses were performed within Araliaceae. Our aims were to (1) analyze the structural features and sequence variations of Hydrocotyle plastomes; (2) detect repeat sequences and highly variable regions as potential molecular markers; and (3) infer the phylogenetic relationships among Hydrocotyle species based on the whole plastid data.

2. Results

2.1. Plastome Structure and Features

The complete plastid genomes of the 17 Hydrocotyle taxa ranged from 152,659 bp (Hydrocotyle sibthorpioides ‘Variegata’) to 153,669 bp (H. ranunculoides) in size (Table 1). These plastomes exhibited a typical quadripartite structure, comprising a large single-copy (LSC) region (83,958–84,792 bp), a small single-copy (SSC) region (18,585–18,768 bp), and a pair of inverted repeats (IRs) (25,058–25,145 bp) (Table 1 and Figure 1). The total GC content showed slight variation (37.50–37.67%), with an uneven distribution in the plastid genomes, of which the IR region was highest (43.08–43.30%), followed by the LSC region (35.52–35.70%) and the SSC region (30.95–31.22%).
The gene content was identical among these Hydrocotyle plastomes. A total of 134 genes were annotated, containing 85 protein-coding genes (PCGs), 37 tRNA genes, eight rRNA genes, and four pseudogenes (Table 2). Among these, seven tRNA genes (trnA-UGC, trnI-CAU, trnI-GAU, trnL-CAA, trnN-GUU, trnR-ACG, and trnV-GAC), six protein-coding genes (ndhB, rpl2, rpl23, rps7, rps12, and ycf2), and four rRNA genes (rrn4.5, rrn5, rrn16, and rrn23) were completely duplicated in the IR region. In all Hydrocotyle plastomes, the incompletely duplicated copies of rps19 in the IRa region and ycf1 in the IRb region were identified as pseudogenes. Since the stop codon appeared prematurely, the gene ycf15 in two IR regions was annotated as a pseudogene. There were 17 genes with introns, of which three genes (clpP, rps12, and ycf3) contained two introns and the other 14 genes possessed a single intron.

2.2. Comparative Genomics and Hypervariable Regions

The gene composition for each IR boundary exhibited high similarity, with slight variations in gene arrangement patterns (Supplementary Figure S1). The LSC/IRb junction was located in the coding region of gene rps19, with an incomplete copy (62 bp) in the IRa region, forming a pseudogene (Ψrps19). The SSC/IRb junction was crossed by the partial ycf1 gene, which ranged from 764 bp to 774 bp in the IRb region, and the remaining part of the ycf1 gene expanded into the SSC regions from 4 bp to 76 bp, overlapping with the ndhF gene. Meanwhile, the gene ndhF spanned the SSC/IRb junction, and the length of this gene extending into the IRb region ranged from 28 bp to 70 bp. The SSC/IRa junction was located in the complete ycf1 gene, with 4679 bp to 4759 bp in the SSC region and 764 bp to 774 bp in the IRa region. At the LSC/IRa junction, the rpl2 and trnH genes were entirely distributed within the LSC and IRa regions near the junction.
No gene rearrangements were detected in the plastid genomes of Hydrocotyle, demonstrating that their genome structure was highly conserved (Supplementary Figure S2). The mVISTA analyses showed that the single-copy (SC) and non-coding regions were more divergent than the IR and coding regions (Supplementary Figure S3). To further identify hypervariable regions, we calculated the nucleotide variability (Pi) using sliding window analysis. The Pi values ranged from 0 to 0.02488 in the whole plastid genomes, with an average of 0.005459 (Figure 2). A total of six hypervariable regions were identified (Pi > 0.01700), including four intergenic spacers (IGS) (ycf3-trnS, trnS-rps4, petA-psbJ, and ndhF-rpl32) and two coding genes (rpl16 and ycf1), all of which were from the SC region.

2.3. Repeat Sequence Characteristics

Simple sequence repeats (SSRs) were detected among the 17 Hydrocotyle taxa, with numbers ranging from 171 to 182 (Supplementary Table S1). Mononucleotide repeats accounted for the most part (1876, 63.98%), followed by the dinucleotide (799, 27.25%), the tetranucleotide (184, 6.28%), and the trinucleotide repeats (70, 2.39%). Pentanucleotide repeats were only detected in Hydrocotyle calcicola and H. sibthorpioides ‘Variegata’. Hexanucleotide repeats were present only in H. verticillata (Figure 3A). Two types of mononucleotide repeats and five types of dinucleotide repeats were detected in all species (Figure 3B). Of these, the A/T base was the most abundant, and the dinucleotide repeats were mainly concentrated in the AT/AT base (Supplementary Table S1). The majority of SSRs were located in the LSC region (1843, 62.86%) compared with the SSC (680, 23.19%) and IR (409, 13.95%) regions (Figure 3C). Furthermore, SSRs were distributed mostly in the coding sequence (CDS) (1230, 41.95%) and IGS (1406, 47.95) regions, with a few in the introns (296, 10.10%) (Figure 3D).
Three types of long sequence repeats (LSRs) were recorded, including 251 forward, 252 palindromic, and 17 reverse repeats (Supplementary Table S2). The numbers of LSRs ranged from 26 to 38, of which forward and palindromic repeats were detected in all taxa, but reverse repeats were not detected in Hydrocotyle hookeri, H. hookeri subsp. chinensis, H. hookeri subsp. handelii, H. javanica, H. peltiformis, and H. ranunculoides (Figure 4A). In terms of length, most repeat sequences consisted of 30 bp to 39 bp, followed by 40 bp to 49 bp, with very few sequences greater than 50 bp (Supplementary Table S2 and Figure 4B). These repeat sequences were uneven across different regions, with 261 in the LSC regions, 218 in the IR regions, and 41 in the SSC regions (Figure 4C). Most of the LSRs were scattered in the IGS (164, 31.54%) and CDS (212, 40.77%) regions, with a few in the introns (144, 27.69%) (Figure 4D).

2.4. Codon Usage and Selective Pressure

Similarity in amino acid frequency and codon usage was observed among the 17 Hydrocotyle plastomes (Supplementary Table S3). The number of codons ranged from 25,960 (Hydrocotyle sibthorpioides) to 26,178 (H. ranunculoides and H. salwinica), encoding 20 amino acids. Of these, leucine (Leu) was the most abundant amino acid (10.6% in each taxon), and cysteine (Cys) was the least common (1.1–1.2%). A total of 64 types of codons were detected, of which most amino acids were encoded by more than one codon, except for methionine (Met) and tryptophan (Trp), which only had a single codon. The highest and lowest relative synonymous codon usage (RSCU) values were from UUA (1.85–1.89) and AGC (0.37–0.39), encoding Leu and serine (Ser), respectively. Moreover, 30 codons showed usage bias with RSCU greater than 1, all of which ended with A or U, except for UUG. In contrast, most codons ended with G or C with RSCU values of less than 1, indicating codon usage with less preference. Among the three stop codons (UAA, UAG, and UGA), codon usage showed a bias toward UAA (1.34–1.45).
Non-synonymous (Ka) substitutions, synonymous (Ks) substitutions, and their ratio (Ka/Ks) were calculated based on 79 protein-coding genes (Supplementary Table S4). For the majority of protein-coding genes, the Ka/Ks values were less than 1, meaning that most genes were subjected to purifying selection. The Ka/Ks values of three genes (atpE, rpl16, and ycf2) were greater than 1, implying potentially positive selection. Among these, the rpl16 gene was found with positive selection in Hydrocotyle dielsiana (Ka/Ks = 1.0081), H. himalaica (Ka/Ks = 1.0081), H. hookeri (Ka/Ks = 1.0081), H. hookeri subsp. chinensis (Ka/Ks = 1.0081), H. hookeri subsp. handelii (Ka/Ks = 1.0081), H. javanica (Ka/Ks = 1.0081), H. nepalensis (Ka/Ks = 1.0081), H. peltiformis (Ka/Ks = 1.3440), H. pseudoconferta (Ka/Ks = 1.0081), and H. salwinica (Ka/Ks = 1.0081). The genes atpE (Ka/Ks = 2.3884) and ycf2 (Ka/Ks = 1.0646) with positive selection were detected only in H. ranunculoides.

2.5. Phylogenetic Relationships

To further infer the evolutionary relationships of Hydrocotyle, phylogenetic trees were constructed based on the whole plastid genomes and shared protein-coding genes using the maximum likelihood (ML) and Bayesian inference (BI) methods. The alignment of plastomes yielded a matrix of 150,488 bp, comprising 126,586 constant sites and 11,861 parsimony-informative sites. The aligned protein-coding genes comprised 56,233 bp, of which 49,065 bp were constant sites and 3332 bp were parsimony-informative sites. As the topologies inferred from ML and BI analyses were almost identical, only the phylogram of the ML method was presented with bootstrap support (BS) and posterior probability (PP) values added (Figure 5 and Figure 6). Overall, the supported values based on the whole plastid sequences for each node were much higher than those inferred from protein-coding genes.
Phylogenomic analyses based on the completed plastid genome and protein-coding genes both supported the monophyly of the two subfamilies within the family Araliaceae (BS = 100, PP = 1.00). In subfamily Aralioideae, the sister relationship between the Asian palmate group and the Aralia-panax group was strongly supported (BS = 100, PP = 1.00). In subfamily Hydrocotyloideae, Hydrocotyle was a well-supported monophyletic taxon (BS = 100, PP = 1.00), in which two subclades were recognized. One comprised two species native to the Neotropics (Hydrocotyle verticillata and H. ranunculoides), sister to another subclade consisting of taxa from China. Within the Chinese subclade, H. sibthorpioides, including its variety (H. sibthorpioides var. batrachium) and cultivar (H. sibthorpioides ‘Variegata’), formed a group together with H. wilfordii. Another group included 11 taxa with H. calcicola sister to the remaining 10 taxa.

3. Discussion

3.1. Structural Features and Plastome Evolution

As with most angiosperms [29,30,31,32], the plastid genomes of Hydrocotyle were relatively conserved, with a similar quadripartite structure, gene order, gene content, and GC content (Figure 1 and Table 1). Generally, the expansion and contraction of inverted repeat (IR) regions are common and may lead to size variation, gene loss, and pseudogenization, which play a crucial role in genome evolution [18]. In Hydrocotyle, the plastome size ranged from 152,659 bp to 153,669 bp, with no significant variations observed. The IR boundaries showed high similarity among all species (Supplementary Figure S1), indicating that their plastid genomic structure was highly conserved. Compared with other genera, such as Dendropanax [26], Eleutherococcus [33], and Panax [34] of Araliaceae, the small single-copy (SSC) region of the Hydrocotyle plastomes exhibited a slight expansion due to the ndhF gene extending into the IRb region. The pseudogenization of both shorter copies (rps19 and ycf1) was caused by incomplete duplication, which has also occurred in most Araliaceae plants [23,25,27]. Furthermore, the ycf15 gene containing premature stop codons was annotated as a pseudogene across the 17 Hydrocotyle plastomes. In some Araliaceae plastomes, however, the ycf15 was intact [23,25,33] or completely lost [26,35], demonstrating that a variation in gene content has occurred during the process of evolution. Although pseudogenes have lost their ability to encode proteins, they are still transcribed and have great significance for regulatory processes [36,37]. The ycf15 gene, a hypothetical open reading frame, is distributed in the IR regions among Hydrocotyle plastomes, but its function remains unknown and needs to be further investigated.
Codon usage bias is closely related to gene expression and plants’ adaptation to their environments [38]. Genes with high expression guarantee the efficient recycling of the ribosomes through the use of well-adapted codons to improve cellular fitness [39]. In Hydrocotyle plastomes, the relative synonymous codon usage (RSCU) analyses indicated that most codons showed a strong preference toward A/U-ending synonymous codons (RSCU > 1) (Supplementary Table S3). This is a common phenomenon in the plastid genomes of most land plants and may be correlated with the GC content of codons [40,41,42,43]. Overall, the codon usage pattern presented high similarity among Hydrocotyle plastomes, implying that gene expression is relatively conserved at the plastid level. Moreover, non-synonymous (Ka) substitutions, synonymous (Ks) substitutions, and their ratios (Ka/Ks) are widely used to evaluate selective pressure on protein-coding genes [44]. Previous studies have indicated that most genes are subjected to purifying selection to retain conserved functions throughout plastid evolution [45,46,47]. Similar to previous studies [48,49], most protein-coding genes (76 out of 79 genes) had Ka/Ks values less than 1, of which the Ka/Ks values of photosynthesis-related genes were significantly lower than those of other genes (Supplementary Table S4). In the present study, three genes were likely to undergo positive selection (Ka/Ks > 1), including the subunit of ATP synthase gene atpE, the large subunit of ribosome gene rpl16, and the unknown function gene ycf2. The atpE gene encoding ε subunits of ATP synthase plays an important role in plant photosynthesis, and positive selection on the atpE gene has also been reported in Cucurbitaceae [50], Liliaceae [51], and Zingiberaceae [52]. The rpl16 gene encodes the ribosomal protein L16, which has been proven to be essential in plastid ribosome development [53]. It is worth mentioning that this highly conserved gene exhibiting positive selection needs to be confirmed for future research [54]. As the largest known plastid gene, ycf2 is essential for cell viability but of unknown function [55], presenting positive selection signals in most land plants [22,48,56,57,58]. Given that genes with positive selection may be undergoing adaptive evolution [59], it is necessary to further explore the selective pressure among Hydrocotyle species based on more extensive sampling with phylogenetic clades.

3.2. Sequence Polymorphisms and Hypervariable Regions

The implementation of simple sequence repeat (SSR) molecular markers is characterized by low costs, high polymorphism, reproducibility, and transferability across species [60], and they are widely applied in population genetics [61]. In most land plants, the distribution of SSRs in plastid genomes is uneven, with most of them found in the coding and non-coding regions, with a few in introns [62,63,64], which was confirmed in the current study (Figure 3). The lower number of SSRs in the IR region compared with the single-copy (SC) region may be related to the slower evolutionary rate in the IR region [65]. Consistent with previous studies [66,67], mononucleotide repeats were the most abundant type and primarily dominated by the A/T base. Furthermore, long sequence repeats (LSRs) have great significance for genome recombination and rearrangement [68]. We detected three types of LSRs among Hydrocotyle species, including forward, palindromic, and reverse repeats, of which forward and palindromic repeats were the most common (Figure 4), in line with previous studies [22,46,48,63]. The number and distribution of LSRs presented differences among different taxa, which may be one of the reasons for the diversity of plastid genomes at the generic level [69].
Hypervariable regions screened from the plastid genome can be used as potential DNA barcodes or genetic markers for species identification, biogeographic inference, and phylogenetic analyses [70]. Based on a few universal DNA fragments, however, the phylogenetic topologies of Hydrocotyle among closely related species were polytomous or had a low resolution [2,4,14,15]. Due to the taxonomically challenging and high medicinal and ornamental value, lineage-specific molecular markers are needed to avoid misidentification for Hydrocotyle species. Previous plastome studies have mainly focused on the size and gene content of a single species of Hydrocotyle [27,28], lacking detailed comparative genomics. In this study, six hypervariable regions were identified among 17 Hydrocotyle plastomes, including four intergenic spacers (IGS) (ycf3-trnS, trnS-rps4, petA-psbJ, and ndhF-rpl32) and two coding genes (rpl16 and ycf1), all of which were from the SC region. Of these, ycf3-trnS showed the highest Pi value (0.02488) and was also recognized as a candidate barcode in Dendropanax of Araliaceae [26]. These hypervariable regions provide new insights into the development of DNA barcodes for Hydrocotyle, but further research is needed to determine which of these regions are suitable for species delimitation and identification.

3.3. Phylogenomic Analyses

For a long time, molecular phylogenetic analyses inferred from Sanger sequencing data failed to clearly elucidate the phylogenetic relationships of Hydrocotyle [2,14,15]. In the current study, the phylogenetic trees of Hydrocotyle reconstructed based on whole plastid genomes and protein-coding genes showed a high phylogenetic resolution, in contrast with previous studies using a few molecular markers (Figure 5 and Figure 6). All topologies strongly supported the monophyly of the subfamily Hydrocotyloideae and Aralioideae (BS = 100, PP = 1.00). Historically, Drude [71] placed Hydrocotyloideae in Apiaceae based on the superficial characteristics of the fruit. On the basis of molecular phylogenetic results with more detailed morphological studies, the traditionally defined Hydrocotyloideae was demonstrated to be polyphyletic and some genera (e.g., Hydrocotyle, Trachymene, Neosciadium, and Uldinia) have been transferred from Apiaceae to Araliaceae [1,2,72,73]. In this study, plastid phylogenomic analyses also supported this treatment.
Phylogenomic analyses indicated that the Chinese Hydrocotyle formed two independent lineages (Figure 5 and Figure 6). The result is different from the taxonomic treatment based on morphological evidence. Within Hydrocotyle, the length of the petiole and pedicel/peduncle as well as the size of the leaf blade are regarded as the main diagnostic characteristics [7]. Based on the smaller size of the leaf blade (0.5–1.5 × 0.8–2 cm), Hydrocotyle pseudoconferta, H. sibthorpioides, and H. calcicola are considered to be closely related taxa. However, in our phylogenomic analyses, the three species did not group, with H. pseudoconferta sister to H. himalaica + H. dielsiana, H. sibthorpioides sister to H. wilfordii, and H. calcicola forming an isolated clade. Similarly, on the basis of the synapomorphies of the elongated pedicel (2.5–8 mm) and undensely capitate umbel, H. dielsiana, H. himalaica, and H. hookeri should have gathered in a lineage, but phylogenomic analyses showed that the three species were scattered in two different branches. Traditionally, taxonomic treatment mainly relies on morphological characteristics. However, the selection of taxonomic characteristics is subjective, and different researchers may choose different characteristics, leading to different taxonomic results for the same taxa. When inferring phylogenetic relationships, molecular systematic evidence is often more convincing than morphological evidence. For example, the genera Callicarpa, Clerodendrum, and Vitex were placed in the Verbenaceae because they lack four dry nutlets, but molecular systematic studies have confirmed that these genera with cymose inflorescences should be transferred to the Lamiaceae [21,74]. Therefore, the main diagnostic characteristics applied in the taxonomic treatment of Hydrocotyle may need to be reevaluated, and new diagnostic characteristics should be screened in combination with more comprehensive molecular phylogenetic results.

4. Materials and Methods

4.1. Plant Material, DNA Extraction, and Sequencing

Plant samples were collected from the field and the voucher specimens were deposited in the herbarium of the Natural Museum of Guizhou University (GACP). Details of the sampling information and vouchers are shown in Table 3. Total genomic DNA was extracted from the fresh or silica-gel-dried leaves using the modified cetyltrimethylammonium bromide (CTAB) method [75]. DNA integrity and concentration were monitored with agarose gel electrophoresis and a Qubit Fluorometer, respectively. Total DNA was fragmented randomly to construct shotgun libraries, and then qualified libraries were selected for paired-end (150 bp) sequencing by the Illumina Hiseq 2500 platform at Wuhan BGI Technology Service Co., Ltd. (Wuhan, China). The low-quality reads of raw data were filtered using SOAPnuke v.2.0 [76], generating 3–5 GB of clean data for each taxon.

4.2. Plastome Assembly and Annotation

GetOrganelle v.1.6.2 [77] was applied to assemble plastomes based on clean data with default parameters. The final assembly results were visualized and checked with Bandage v0.8.1 [78]. Plastid Genome Annotator (PGA) [79] was used to annotate the complete plastid genome with Hydrocotyle sibthorpioides (KT589392) as the reference, followed by the validation of the annotation using CPGAVAS2 [80]. Gene start and stop codons were checked and manually inspected in the software Geneious v.10.0.5 [81], and the tRNA genes were further verified using tRNAscan-SE v1.21 [82]. Finally, the plastid genome maps of Hydrocotyle were drawn with OrganellarGenomeDRAW (OGDRAW) https://chlorobox.mpimp-golm.mpg.de/OGDraw.html (accessed on 1 May 2023) [83].

4.3. Comparative Genomic Analyses

In total, 17 Hydrocotyle plastomes were included to perform comparative analyses, of which four plastids’ data (GenBank accession no.: MT561038, OK585058, KT589392, and HM596070) were downloaded from the GenBank database. The annotations of plastomes from GenBank were double-checked according to the above details and manually adjusted when necessary. Gene order and structure rearrangements were detected using Mauve v2.4.0 [84]. Sequence divergence was visualized based on the Shuffle-LAGAN model using mVISTA http://genome.lbl.gov/vista/mvista/submit.shtml (accessed on 10 May 2023) [85]. DnaSPv.6.12.03 [86] was used to calculate nucleotide diversity (Pi), setting the step size and window length to 200 bp and 600 bp, respectively. The expansion and contraction of inverted repeat (IR) regions were detected by the online program IRscope https://irscope.shinyapps.io/irapp/ (accessed on 10 May 2023) [87].

4.4. Repeat Sequence Analyses

Simple sequence repeats (SSRs) were detected using MISA https://webblast.ipk-gatersleben.de/misa/ (accessed on 15 May 2023) [88] by setting the parameters to eight, four, four, three, three, and three for mono-, di-, tri-, tetra-, penta-, and hexa-nucleotides, respectively. Long sequence repeats (LSRs), including forward (F), reverse (R), palindrome (P), and complement (C) repeats, were identified using REPuter https://bibiserv.cebitec.uni-bielefeld.de/reputer (accessed on 15 May 2023) [89], with a minimum repeat size of 30 bp and a Hamming distance of 3.

4.5. Codon Usage Bias and Selective Pressure Analyses

Amino acid frequency and relative synonymous codon usage (RSCU) were calculated using Codon W v.1.4.4 https://sourceforge.net/projects/codonw/ (accessed on 15 May 2023). The RSCU value was greater than 1, equal to 1, and less than 1, indicating preferred, no bias, and less preferred in codon usage, respectively. To assess selective pressure, 79 single-copy protein-coding genes were extracted using Geneious v.10.0.5 [81] after excluding duplicate genes and pseudogenes, and then aligned by MAFFT v.7.3888 [90]. Non-synonymous (Ka) and synonymous (Ks) substitutions were calculated using KaKs_Calculator 2.0 [91] under the Yang–Nielsen (YN) model [92], with Hydrocotyle sibthorpioides (KT589392) as the reference. The Ka/Ks value was greater than 1, equal to 1, and less than 1, demonstrating positive selection, neutral selection, and purifying selection, respectively.

4.6. Phylogenetic Analyses

Phylogenetic analyses including 50 taxa were performed based on the whole plastid genomes and protein-coding genes, of which 47 species from Araliaceae served as the ingroups and the other three species from Apiaceae as the outgroups (Supplementary Table S5). Sequences were aligned using MAFFT v.7.3888 [90] with the auto strategy and then manually adjusted by BioEdit v.7.0.9 [93]. Phylogenetic trees were constructed based on the maximum likelihood (ML) and Bayesian inference (BI) methods in CIPRES Science Gateway https://www.phylo.org/ (accessed on 28 May 2023). ModelFinder [94] was used to select the best-fit model according to the Akaike Information Criterion (AIC) [95]. The ML analyses were performed under the GTRGAMMA model with 1000 bootstrap replicates. The GTR + F + I + G4 model was selected for BI analyses with four parallel Markov chain Monte Carlo (MCMC) chains for 2,000,000 generations and sampling for every 1000 generations. After removing the first 25% of trees as burn-in, the remaining trees were used to generate the consensus tree. Finally, the phylogenetic results were visualized and edited in Figtree v.1.4.4 http://tree.bio.ed.ac.uk/software/figtree/ (accessed on 5 June 2023).

5. Conclusions

In this study, we newly sequenced and assembled 13 whole plastid genomes of Hydrocotyle and performed comparative plastid genome analyses with four previously published data and phylogenomic analyses within Araliaceae. Comparative analyses revealed that the Hydrocotyle plastomes were relatively conserved, with a similar quadripartite structure, gene order, gene content, and GC content. The boundaries of inverted repeats (IRs) and single-copy (SC) regions showed high similarity among all Hydrocotyle plastomes. Six hypervariable regions and 2932 simple sequence repeats were identified as potential molecular markers. Three genes (atpE, rpl16, and ycf2) were observed under positive selection only in a few taxa, implying that most protein-coding genes were relatively conserved and subjected to purifying selection during the plastid evolutionary process. Phylogenomic analyses supported transferring the genus Hydrocotyle from Apiaceae to Araliaceae, and the whole plastid genome was demonstrated to be effective in improving the phylogenetic resolution of Hydrocotyle in contrast with short DNA fragments. The diagnostic characteristics currently used in Hydrocotyle may not accurately reflect the phylogenetic relationships of this genus, and new diagnostic characteristics need to be screened based on more comprehensive molecular phylogenetic results.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms242316629/s1.

Author Contributions

Conceptualization, G.H.; software and formal analysis, L.G. and R.Y.; validation, R.Y. and G.H.; investigation, G.H., L.G., L.Q. and X.W.; writing—original draft preparation, R.Y, L.G. and G.H.; writing—review and editing, G.H. and R.Y.; funding acquisition, G.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (32060048, 32260099) and the Natural Science Foundation of Guizhou Province (Qiankehezhongyindi [2023] 029, Qiankehejichu-ZK [2021] 091).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The Hydrocotyle plastomes generated in this study are available in the NCBI repository https://www.ncbi.nlm.nih.gov (accessed on 1 September 2023) with the accession numbers in Table 3.

Acknowledgments

We are grateful to Jiaxing Yang from the Wuhan Botanical Garden, Chinese Academy of Sciences; Xinxin Zhou from the South China Botanical Garden, Chinese Academy of Sciences; Sirong Yi from Chongqing Three Gorges Medical College; and Chao Ye from the Institute of Botany, Chinese Academy of Sciences for the help in material collection, and Ting Su and Mei Wang from the College of Life Sciences, Guizhou University for the help in data analyses.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Chandler, G.T.; Plunkett, G.M. Evolution in Apiales: Nuclear and chloroplast markers together in (almost) perfect harmony. Bot. J. Linn. Soc. 2004, 144, 123–147. [Google Scholar] [CrossRef]
  2. Nicolas, A.N.; Plunkett, G.M. The demise of subfamily Hydrocotyloideae (Apiaceae) and the realignment of its genera across the entire order Apiales. Mol. Phylogenet. Evol. 2009, 53, 134–151. [Google Scholar] [CrossRef] [PubMed]
  3. Li, R.; Wen, J. Phylogeny and diversification of Chinese Araliaceae based on nuclear and plastid DNA sequence data. J. Syst. Evol. 2016, 54, 453–467. [Google Scholar] [CrossRef]
  4. Perkins, A.J. Molecular phylogenetics and species delimitation in annual species of Hydrocotyle (Araliaceae) from South Western Australia. Mol. Phylogenet. Evol. 2019, 134, 129–141. [Google Scholar] [CrossRef] [PubMed]
  5. Henwood, M.J. Hydrocotyle rivularis: A new trifoliolate species from south-eastern Australia. Telopea 2014, 17, 217–221. [Google Scholar] [CrossRef]
  6. Nicolas, N.A.; Plunkett, M.G. Diversification Times and Biogeographic Patterns in Apiales. Bot. Rev. 2014, 80, 30–58. [Google Scholar] [CrossRef]
  7. She, M.L.; Waston, M.F.A.; Cannon, J.F.M. Hydrocotyle. In Flora of China; Wu, C.Y., Raven, P.H., Hong, D.Y., Eds.; Science Press: Beijing, China, 2005; Volume 14, pp. 14–17. [Google Scholar]
  8. China Check List of Higher Plants. Available online: http://www.sp2000.org.cn/ (accessed on 1 September 2023).
  9. Yu, S. The small grass also becomes a scene. China Flowers Hortic. 2017, 14, 59. [Google Scholar]
  10. Huang, Q.F.; Zhang, S.J.; Huang, R.B.; Wei, L.; Chen, Y.X.; Lv, S.; Liang, C.H.; Tan, S.; Liang, S.; Zhuo, L. Isolation and identification of an anti-hepatitis B virus compound from Hydrocotyle sibthorpioides Lam. J. Ethnopharmacol. 2013, 150, 568–575. [Google Scholar] [CrossRef]
  11. Richard, A. Monographie du genre Hydrocotyle de la famille des Ombellifères. Ann. Gén. Sci. Phys. 1802, 4, 145–224. [Google Scholar]
  12. Candolle, A.P. Umbelliferae. In Prodromus Systematis Naturalis Regni Vegetabilis; Candolle, A.P., Ed.; Sumptibus Victoris Masson: Paris, France, 1830; Volume 4, p. 63. [Google Scholar]
  13. Constance, L.; Dillon, M.O. A new peltate Hydrocotyle (Umbelliferae) from northern Peru. Brittonia 1990, 42, 257–259. [Google Scholar] [CrossRef]
  14. Van De Wiel, C.C.M.; Van Der Schoot, J.; Van Valkenburg, J.; Duistermaat, H.; Smulders, M.J.M. DNA barcoding discriminates the noxious invasive plant species, floating pennywort (Hydrocotyle ranunculoides L.f.), from non-invasive relatives. Mol. Ecol. Resour. 2009, 9, 1086–1091. [Google Scholar] [CrossRef] [PubMed]
  15. Choi, K.S.; Park, S.J. Molecular Phylogenetic Studies of Korean Hydrocotyle L. Korean J. Plant Resour. 2012, 25, 490–497. [Google Scholar] [CrossRef]
  16. Chen, X.; Liu, C. Progress in chloroplast genome analysis. Arch. Biochem. Biophys. 2008, 35, 21–28. [Google Scholar]
  17. Dong, W.; Xu, C.; Cheng, T.; Lin, K.; Zhou, S. Sequencing angiosperm plastid genomes made easy: A complete set of universal primers and a case study on the phylogeny of Saxifragales. Genome Biol. Evol. 2013, 5, 989–997. [Google Scholar] [CrossRef]
  18. Wicke, S.; Schneeweiss, G.M.; Pamphilis, C.W.; Kai, F.M.; Quandt, D. The evolution of the plastid chromosome in land plants: Gene content, gene order, gene function. Plant Mol. Biol. 2011, 51, 273–297. [Google Scholar] [CrossRef]
  19. Parks, M.; Cronn, R.; Liston, A. Increasing phylogenetic resolution at low taxonomic levels using massively parallel sequencing of chloroplast genomes. BMC Biol. 2009, 7, 84. [Google Scholar] [CrossRef]
  20. Li, H.T.; Yi, T.S.; Gao, L.M.; Ma, P.F.; Zhang, T.; Yang, J.B.; Gitzendanner, M.A.; Fritsch, P.W.; Cai, J.; Luo, Y.; et al. Origin of angiosperms and the puzzle of the Jurassic gap. Nat. Plants 2019, 5, 461–470. [Google Scholar] [CrossRef]
  21. Zhao, F.; Chen, Y.P.; Salmaki, Y.; Drew, B.T.; Wilson, T.C.; Scheen, A.C.; Celep, F.; Bräuchler, C.; Bendiksby, M.; Wang, Q.; et al. An updated tribal classification of Lamiaceae based on plastome phylogenomics. BMC Biol. 2021, 19, 2. [Google Scholar] [CrossRef]
  22. Yan, R.R.; Geng, Y.F.; Jia, Y.H.; Xiang, C.L.; Zhou, X.X.; Hu, G.X. Comparative analyses of Linderniaceae plastomes, with implications for its phylogeny and evolution. Front. Plant Sci. 2023, 14, 1265641. [Google Scholar] [CrossRef]
  23. Li, R.; Ma, P.F.; Wen, J.; Yi, T.S. Complete sequencing of five Araliaceae chloroplast genomes and the phylogenetic implications. PLoS ONE 2013, 8, e78568. [Google Scholar] [CrossRef]
  24. Kim, K.; Nguyen, V.B.; Dong, J.; Wang, Y.; Park, J.Y.; Lee, S.C.; Yang, T.J. Evolution of the Araliaceae family inferred from complete chloroplast genomes and 45S nrDNAs of 10 Panax-related species. Sci. Rep. 2017, 7, 4917. [Google Scholar] [CrossRef] [PubMed]
  25. Li, J.; Xie, D.F.; Guo, X.L.; Zheng, Z.Y.; He, X.J.; Zhou, S.D. Comparative Analysis of the Complete Plastid Genome of Five Bupleurum Species and New Insights into DNA Barcoding and Phylogenetic Relationship. Plants 2020, 9, 543. [Google Scholar] [CrossRef] [PubMed]
  26. Wang, Y.; Yu, J.; Chen, Y.K.; Wang, Z.C. Complete Chloroplast Genome Sequence of the Endemic and Endangered Plant Dendropanax oligodontus: Genome Structure, Comparative and Phylogenetic Analysis. Genes 2022, 13, 2028. [Google Scholar] [CrossRef] [PubMed]
  27. Ge, L.; Shen, L.; Chen, Q.; Li, X.; Zhang, L. The complete chloroplast genome sequence of Hydrocotyle sibthorpioides (Apiales: Araliaceae). Mitochondrial DNA B Resour. 2017, 2, 29–30. [Google Scholar] [CrossRef]
  28. Wen, J.; Zhou, W.; Wu, B.; Li, H.; Song, C. The complete chloroplast genome of Hydrocotyle pseudoconferta Masamune 1932 (Araliaceae). Mitochondrial DNA B Resour. 2022, 7, 1199–1200. [Google Scholar] [CrossRef]
  29. Daniell, H.; Lin, C.S.; Yu, M.; Chang, W.J. Chloroplast genomes: Diversity, evolution, and applications in genetic engineering. Genome Biol. 2016, 17, 134. [Google Scholar] [CrossRef]
  30. Huang, Y.; Ma, Q.; Sun, J.; Zhou, L.N.; Lai, C.J.; Li, P.; Jin, X.J.; Zhang, Y.H. Comparative analysis of Diospyros (Ebenaceae) plastomes: Insights into genomic features, mutational hotspots, and adaptive evolution. Ecol. Evol. 2023, 13, e10301. [Google Scholar] [CrossRef]
  31. Liu, Q.; Gao, Y.; Dong, W.; Zhao, L. Plastome evolution and phylogeny of the tribe Ruteae (Rutaceae). Ecol. Evol. 2023, 13, e9821. [Google Scholar] [CrossRef]
  32. Chen, J.; Wang, F.; Zhou, C.; Ahmad, S.; Zhou, Y.; Li, M.; Liu, Z.; Peng, D. Comparative Phylogenetic Analysis for Aerides (Aeridinae, Orchidaceae) Based on Six Complete Plastid Genomes. Int. J. Mol. Sci. 2023, 24, 12473. [Google Scholar] [CrossRef]
  33. Dong, Z.; Zhang, R.; Shi, M.; Song, Y.; Xin, Y.; Li, F.; Ma, J.; Xin, P. The complete plastid genome of the endangered shrub Brassaiopsis angustifolia (Araliaceae): Comparative genetic and phylogenetic analysis. PLoS ONE 2022, 17, e0269819. [Google Scholar] [CrossRef]
  34. Liu, C.; Yang, Z.; Yang, L.; Yang, J.; Ji, Y. The complete plastome of Panax stipuleanatus: Comparative and phylogenetic analyses of the genus Panax (Araliaceae). Plant Divers. 2018, 40, 265–276. [Google Scholar] [CrossRef] [PubMed]
  35. Tian, Z. Characterization of the complete chloroplast genome of Eleutherococcus senticosus (Araliaceae) as an herb in China. Mitochondrial DNA B Resour. 2020, 5, 2133–2134. [Google Scholar] [CrossRef] [PubMed]
  36. Poliseno, L.; Salmena, L.; Zhang, J.; Carver, B.; Haveman, W.J.; Pandolfi, P.P. A coding-independent function of gene and pseudogene mRNAs regulates tumour biology. Nature 2010, 465, 1033–1038. [Google Scholar] [CrossRef] [PubMed]
  37. Prade, V.M.; Gundlach, H.; Twardziok, S.; Chapman, B.; Tan, C.; Langridge, P.; Schulman, A.H.; Stein, N.; Waugh, R.; Zhang, G.; et al. The pseudogenes of barley. Plant J. 2018, 93, 502–514. [Google Scholar] [CrossRef] [PubMed]
  38. Botzman, M.; Margalit, H. Variation in global codon usage bias among prokaryotic organisms is associated with their lifestyles. Genome Biol. 2011, 12, R109. [Google Scholar] [CrossRef]
  39. Kudla, G.; Murray, A.W.; Tollervey, D.; Plotkin, J.B. Coding-sequence determinants of gene expression in Escherichia coli. Science 2009, 324, 255–258. [Google Scholar] [CrossRef] [PubMed]
  40. Li, G.; Pan, Z.; Gao, S.; He, Y.; Xia, Q.; Jin, Y.; Yao, H. Analysis of synonymous codon usage of chloroplast genome in Porphyra umbilicalis. Genes Genom. 2019, 41, 1173–1181. [Google Scholar] [CrossRef]
  41. Xie, D.F.; Yu, H.X.; Price, M.; Xie, C.; Deng, Y.Q.; Chen, J.P.; Yu, Y.; Zhou, S.D.; He, X.J. Phylogeny of Chinese Allium Species in Section Daghestanica and Adaptive Evolution of Allium (Amaryllidaceae, Allioideae) Species Revealed by the Chloroplast Complete Genome. Front. Plant Sci. 2019, 10, 460. [Google Scholar] [CrossRef]
  42. Wang, Y.; Liang, Q.; Zhang, C.; Huang, H.; He, H.; Wang, M.; Li, M.; Huang, Z.; Tang, Y.; Chen, Q.; et al. Sequencing and Analysis of Complete Chloroplast Genomes Provide Insight into the Evolution and Phylogeny of Chinese Kale (Brassica oleracea var. alboglabra). Int. J. Mol. Sci. 2023, 24, 10287. [Google Scholar] [CrossRef]
  43. Chen, M.M.; Zhang, M.; Liang, Z.S.; He, Q.L. Characterization and Comparative Analysis of Chloroplast Genomes in Five Uncaria Species Endemic to China. Int. J. Mol. Sci. 2022, 23, 11617. [Google Scholar] [CrossRef]
  44. Yang, Z.; Nielsen, R. Codon-substitution models for detecting molecular adaptation at individual sites along specific lineages. Mol. Biol. Evol. 2002, 19, 908–917. [Google Scholar] [CrossRef] [PubMed]
  45. Guo, L.; Wang, X.; Wang, R.; Li, P. Characterization and Comparative Analysis of Chloroplast Genomes of Medicinal Herb Scrophularia ningpoensis and Its Common Adulterants (Scrophulariaceae). Int. J. Mol. Sci. 2023, 24, 10034. [Google Scholar] [CrossRef] [PubMed]
  46. Yang, J.X.; Hu, G.X.; Hu, G.W. Comparative genomics and phylogenetic relationships of two endemic and endangered species (Handeliodendron bodinieri and Eurycorymbus cavaleriei) of two monotypic genera within Sapindales. BMC Genom. 2022, 23, 27. [Google Scholar] [CrossRef] [PubMed]
  47. Zhou, S.M.; Wang, F.; Yan, S.Y.; Zhu, Z.M.; Gao, X.F.; Zhao, X.L. Phylogenomics and plastome evolution of Indigofera (Fabaceae). Front. Plant Sci. 2023, 14, 1186598. [Google Scholar] [CrossRef] [PubMed]
  48. Feng, Y.; Gao, X.F.; Zhang, J.Y.; Jiang, L.S.; Li, X.; Deng, H.N.; Liao, M.; Xu, B. Complete Chloroplast Genomes Provide Insights into Evolution and Phylogeny of Campylotropis (Fabaceae). Front. Plant Sci. 2022, 13, 895543. [Google Scholar] [CrossRef]
  49. Zhou, T.; Wang, J.; Jia, Y.; Li, W.; Xu, F.; Wang, X. Comparative Chloroplast Genome Analyses of Species in Gentiana section Cruciata (Gentianaceae) and the Development of Authentication Markers. Int. J. Mol. Sci. 2018, 19, 1962. [Google Scholar] [CrossRef]
  50. Shi, H.; Yang, M.; Mo, C.; Xie, W.; Liu, C.; Wu, B.; Ma, X. Complete chloroplast genomes of two Siraitia Merrill species: Comparative analysis, positive selection and novel molecular marker development. PLoS ONE 2019, 14, e0226865. [Google Scholar] [CrossRef]
  51. Lu, R.S.; Li, P.; Qiu, Y.X. The Complete Chloroplast Genomes of Three Cardiocrinum (Liliaceae) Species: Comparative Genomic and Phylogenetic Analyses. Front. Plant Sci. 2017, 7, 2054. [Google Scholar] [CrossRef]
  52. Gao, B.; Yuan, L.; Tang, T.; Hou, J.; Pan, K.; Wei, N. The complete chloroplast genome sequence of Alpinia oxyphylla Miq. and comparison analysis within the Zingiberaceae family. PLoS ONE 2019, 14, e0218817. [Google Scholar] [CrossRef]
  53. Posno, M.; Vliet, A.V.; Groot, G.S.P. The gene for Spirodela oligorhiza chloroplast ribosomal protein homologous to E. coli ribosomal protein L16 is split by a large intron near its 59 end: Structure and expression. Nucleic Acids Res. 1986, 14, 3181–3195. [Google Scholar] [CrossRef]
  54. Zhao, D.N.; Ren, Y.; Zhang, J.Q. Conservation and innovation: Plastome evolution during rapid radiation of Rhodiola on the Qinghai-Tibetan Plateau. Mol. Phylogenet. Evol. 2020, 144, 106713. [Google Scholar] [CrossRef] [PubMed]
  55. Drescher, A.; Ruf, S.; Calsa, T.; Carrer, H.; Bock, R. The two largest chloroplast genome-encoded open reading frames of higher plants are essential genes. Plant J. 2000, 22, 97–104. [Google Scholar] [CrossRef]
  56. Dong, W.L.; Wang, R.N.; Zhang, N.Y.; Fan, W.B.; Fang, M.F.; Li, Z.H. Molecular Evolution of Chloroplast Genomes of Orchid Species: Insights into Phylogenetic Relationship and Adaptive Evolution. Int. J. Mol. Sci. 2018, 19, 716. [Google Scholar] [CrossRef] [PubMed]
  57. Zhong, Q.; Yang, S.; Sun, X.; Wang, L.; Li, Y. The complete chloroplast genome of the Jerusalem artichoke (Helianthus tuberosus L.) and an adaptive evolutionary analysis of the ycf2 gene. PeerJ 2019, 7, e7596. [Google Scholar] [CrossRef] [PubMed]
  58. Cay, S.B.; Cinar, Y.U.; Kuralay, S.C.; Inal, B.; Zararsiz, G.; Ciftci, A.; Mollman, R.; Obut, O.; Eldem, V.; Bakir, Y.; et al. Genome skimming approach reveals the gene arrangements in the chloroplast genomes of the highly endangered Crocus L. species: Crocus istanbulensis (B.Mathew) Rukšāns. PLoS ONE 2022, 17, e0269747. [Google Scholar] [CrossRef]
  59. Thode, V.A.; Lohmann, L.G. Comparative Chloroplast Genomics at Low Taxonomic Levels: A Case Study Using Amphilophium (Bignonieae, Bignoniaceae). Front. Plant Sci. 2019, 10, 796. [Google Scholar] [CrossRef]
  60. Qi, W.; Lin, F.; Liu, Y.; Huang, B.; Cheng, J.; Zhang, W.; Zhao, H. High-throughput development of simple sequence repeat markers for genetic diversity research in Crambe abyssinica. BMC Plant Biol. 2016, 16, 139. [Google Scholar] [CrossRef]
  61. Yu, J.; Dossa, K.; Wang, L.; Zhang, Y.; Wei, X.; Liao, B.; Zhang, X. PMDBase: A database for studying microsatellite DNA and marker development in plants. Nucleic Acids Res. 2017, 45, D1046–D1053. [Google Scholar] [CrossRef]
  62. Zhu, M.; Feng, P.; Ping, J.; Li, J.; Su, Y.; Wang, T. Phylogenetic significance of the characteristics of simple sequence repeats at the genus level based on the complete chloroplast genome sequences of Cyatheaceae. Ecol. Evol. 2021, 11, 14327–14340. [Google Scholar] [CrossRef]
  63. Su, T.; Geng, Y.F.; Xiang, C.L.; Zhao, F.; Wang, M.; Gu, L.; Hu, G.X. Chloroplast Genome of Salvia Sect. Drymosphace: Comparative and Phylogenetic Analysis. Diversity 2022, 5, 324. [Google Scholar] [CrossRef]
  64. Hong, Z.; Wu, Z.; Zhao, K.; Yang, Z.; Zhang, N.; Guo, J.; Tembrock, L.R.; Xu, D. Comparative Analyses of Five Complete Chloroplast Genomes from the Genus Pterocarpus (Fabacaeae). Int. J. Mol. Sci. 2020, 21, 3758. [Google Scholar] [CrossRef] [PubMed]
  65. Alwadani, K.G.; Janes, J.K.; Andrew, R.L. Chloroplast genome analysis of box-ironbark Eucalyptus. Mol. Phylogenet. Evol. 2019, 136, 76–86. [Google Scholar] [CrossRef] [PubMed]
  66. Ping, J.; Feng, P.; Li, J.; Zhang, R.; Su, Y.; Wang, T. Molecular evolution and SSRs analysis based on the chloroplast genome of Callitropsis funebris. Ecol. Evol. 2021, 11, 4786–4802. [Google Scholar] [CrossRef] [PubMed]
  67. Xie, Y.; Yang, G.; Zhang, C.; Zhang, X.; Jiang, X. Comparative analysis of chloroplast genomes of endangered heterostylous species Primula wilsonii and its closely related species. Ecol. Evol. 2023, 13, e9730. [Google Scholar] [CrossRef] [PubMed]
  68. Pombert, J.F.; Otis, C.; Lemieux, C.; Turmel, M. The chloroplast genome sequence of the green alga Pseudendoclonium akinetum (Ulvophyceae) reveals unusual structural features and new insights into the branching order of chlorophyte lineages. Mol. Biol. Evol. 2005, 22, 1903–1918. [Google Scholar] [CrossRef]
  69. Kawata, M.; Harada, T.; Shimamoto, Y.; Oono, K.; Takaiwa, F. Short inverted repeats function as hotspots of intermolecular recombination giving rise to oligomers of deleted plastid DNAs (ptDNAs). Curr. Genet. 1997, 31, 179–184. [Google Scholar] [CrossRef] [PubMed]
  70. Gregory, T.R. DNA barcoding does not compete with taxonomy. Nature 2005, 434, 1067. [Google Scholar] [CrossRef]
  71. Drude, O. Umbelliferae. In Die Natürlichen Pflanzenfamilien; Engler, A., Prantl, K., Eds.; Verlag von Wilhelm Engelmann: Leipzig, Germany, 1989; Volume 3, pp. 63–250. [Google Scholar]
  72. Plunkett, G.M.; Chandler, G.T.; Lowry, P.P.; Pinney, S.M.; Sprenkle, T.S. Recent advances in understanding Apiales and a revised classification. S. Afr. J. Bot. 2004, 70, 371–381. [Google Scholar] [CrossRef]
  73. Plunkett, G.M.; Soltis, D.E.; Soltis, P.S. Clarification of the relationship between Apiaceae and Araliaceae based on matK and rbcL sequence data. Am. J. Bot. 1997, 84, 565–580. [Google Scholar] [CrossRef]
  74. Li, B.; Cantino, P.D.; Olmstead, R.G.; Bramley, G.L.; Xiang, C.L.; Ma, Z.H.; Tan, Y.H.; Zhang, D.X. A large-scale chloroplast phylogeny of the Lamiaceae sheds new light on its subfamilial classification. Sci Rep. 2016, 6, 34343. [Google Scholar] [CrossRef]
  75. Doyle, J.J.; Doyle, J.L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 1987, 19, 11–15. [Google Scholar] [CrossRef]
  76. Chen, Y.X.; Chen, Y.S.; Shi, C.M.; Huang, Z.B.; Zhang, Y.; Li, S.K.; Li, Y.; Ye, J.; Yu, C.; Li, Z.; et al. SOAPnuke: A MapReduce acceleration supported software for integrated quality control and preprocessing of high-throughput sequencing data. Gigascience 2018, 7, gix120. [Google Scholar] [CrossRef] [PubMed]
  77. Jin, J.J.; Yu, W.B.; Yang, J.B.; Song, Y.; De Pamphilis, C.W.; Yi, T.S.; Li, D.Z. GetOrganelle: A fast and versatile toolkit for accurate de novo assembly of organelle genomes. Genome Biol. 2020, 21, 241. [Google Scholar] [CrossRef] [PubMed]
  78. Wick, R.R.; Schultz, M.B.; Zobel, J.; Holt, K.E. Bandage: Interactive visualization of de novo genome assemblies. Bioinformatics 2015, 31, 3350–3352. [Google Scholar] [CrossRef] [PubMed]
  79. Qu, X.J.; Moore, M.J.; Li, D.Z.; Yi, T.S. PGA: A software package for rapid, accurate, and flexible batch annotation of plastomes. Plant Methods 2019, 15, 50. [Google Scholar] [CrossRef]
  80. Shi, L.; Chen, H.; Jiang, M.; Wang, L.; Wu, X.; Huang, L.; Liu, C. CPGAVAS2, an integrated plastome sequence annotator and analyzer. Nucleic Acids Res. 2019, 47, W65–W73. [Google Scholar] [CrossRef]
  81. Kearse, M.; Moir, R.; Wilson, A.; Stones-Havas, S.; Cheung, M.; Sturrock, S.; Buxton, S.; Cooper, A.; Markowitz, S.; Duran, C.; et al. Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 2012, 28, 1647–1649. [Google Scholar] [CrossRef]
  82. Schattner, P.; Brooks, A.N.; Lowe, T.M. The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Res. 2005, 33, 686–689. [Google Scholar] [CrossRef]
  83. Lohse, M.; Drechsel, O.; Kahlau, S.; Bock, R. OrganellarGenomeDRAW—A suite of tools for generating physical maps of plastid and mitochondrial genomes and visualizing expression data sets. Nucleic Acids Res. 2013, 41, W575–W581. [Google Scholar] [CrossRef]
  84. Darling, A.C.E.; Mau, B.; Blattner, F.R.; Perna, N.T. Mauve: Multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 2004, 14, 1394–1403. [Google Scholar] [CrossRef]
  85. Frazer, K.A.; Lior, P.; Alexander, P.; Rubin, E.M.; Inna, D. Vista:computational tools for comparative genomics. Nucleic Acids Res. 2004, 32, W273–W279. [Google Scholar] [CrossRef]
  86. Rozas, J.; Ferrer-Mata, A.; Sánchez-DelBarrio, J.C.; Guirao-Rico, S.; Librado, P.; Ramos-Onsins, S.E.; Sánchez-Gracia, A. DnaSP 6: DNA Sequence Polymorphism Analysis of Large Data Sets. Mol. Biol. Evol. 2017, 34, 3299–3302. [Google Scholar] [CrossRef] [PubMed]
  87. Amiryousefi, A.; Hyvonen, J.; Poczai, P. IRscope: An online program to visualize the junction sites of chloroplast genomes. Bioinformatics 2018, 34, 3030–3031. [Google Scholar] [CrossRef] [PubMed]
  88. Beier, S.; Thiel, T.; Münch, T.; Scholz, U.; Mascher, M. MISA-web: A web server for microsatellite prediction. Bioinformatics 2017, 33, 2583–2585. [Google Scholar] [CrossRef] [PubMed]
  89. Kurtz, S.; Choudhuri, J.V.; Ohlebusch, E.; Schleiermacher, C.; Stoye, J.; Giegerich, R. REPuter: The manifold applications of repeat analysis on a genomic scale. Nucleic Acids Res. 2001, 29, 4633–4642. [Google Scholar] [CrossRef]
  90. Katoh, K.; Standley, D.M. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol. Biol. Evol. 2013, 30, 772–780. [Google Scholar] [CrossRef]
  91. Wang, D.; Zhang, Y.; Zhang, Z.; Zhu, J.; Yu, J. KaKs_Calculator 2.0: A toolkit incorporating gamma-series methods and sliding window strategies. Genom. Proteom. Bioinform. 2010, 8, 77–80. [Google Scholar] [CrossRef] [PubMed]
  92. Nielsen, R.; Yang, Z. Likelihood models for detecting positively selected amino acid sites and applications to the HIV-1 envelope gene. Genetics 1998, 148, 929–993. [Google Scholar] [CrossRef] [PubMed]
  93. Hall, T.A. BioEdit. A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 1999, 41, 95–98. [Google Scholar] [CrossRef]
  94. Kalyaanamoorthy, S.; Minh, B.Q.; Wong, T.K.F.; von Haeseler, A.; Jermiin, L.S. ModelFinder: Fast model selection for accurate phylogenetic estimates. Nat. Methods 2017, 14, 587–589. [Google Scholar] [CrossRef]
  95. David, P.; Buckley, T.R. Model selection and model averaging in phylogenetics: Advantages of Akaike information criterion and Bayesian approaches over likelihood ratio tests. Syst. Biol. 2004, 53, 793–808. [Google Scholar] [CrossRef]
Ijms 24 16629 g001
Figure 1. Gene map of the plastid genomes of Hydrocotyle. Genes with different functions are coded by color. Genes shown inside the inner circle are transcribed clockwise, and those outside are transcribed counterclockwise. The dark gray color in the inner circle indicates GC content, while the light gray color corresponds to AT content. LSC, large single copy; SSC, small single copy; IR, inverted repeat.
Figure 1. Gene map of the plastid genomes of Hydrocotyle. Genes with different functions are coded by color. Genes shown inside the inner circle are transcribed clockwise, and those outside are transcribed counterclockwise. The dark gray color in the inner circle indicates GC content, while the light gray color corresponds to AT content. LSC, large single copy; SSC, small single copy; IR, inverted repeat.
Ijms 24 16629 g001
Ijms 24 16629 g002
Figure 2. Sliding window analysis of the 17 Hydrocotyle plastomes. The X-axis represents the position of the midpoint of a window, and the Y-axis indicates the nucleotide diversity (Pi) of each window.
Figure 2. Sliding window analysis of the 17 Hydrocotyle plastomes. The X-axis represents the position of the midpoint of a window, and the Y-axis indicates the nucleotide diversity (Pi) of each window.
Ijms 24 16629 g002
Ijms 24 16629 g003
Figure 3. Analyses of simple sequence repeats (SSRs) among 17 Hydrocotyle plastomes. (A), Numbers of different types of SSRs. (B), Types of shared SSRs. (C), Distributions of SSRs in the LSC, SSC, and IR regions. (D), Distributions of SSRs in the CDS, IGS, and intron.
Figure 3. Analyses of simple sequence repeats (SSRs) among 17 Hydrocotyle plastomes. (A), Numbers of different types of SSRs. (B), Types of shared SSRs. (C), Distributions of SSRs in the LSC, SSC, and IR regions. (D), Distributions of SSRs in the CDS, IGS, and intron.
Ijms 24 16629 g003
Ijms 24 16629 g004aIjms 24 16629 g004b
Figure 4. Analyses of long sequence repeats (LSRs) among 17 Hydrocotyle plastomes. (A) Numbers of different types of LSRs. (B) Numbers of different lengths of LSRs. (C) Distributions of LSRs in the LSC, SSC, and IR regions. (D) Distributions of LSRs in the CDS, IGS, and intron.
Figure 4. Analyses of long sequence repeats (LSRs) among 17 Hydrocotyle plastomes. (A) Numbers of different types of LSRs. (B) Numbers of different lengths of LSRs. (C) Distributions of LSRs in the LSC, SSC, and IR regions. (D) Distributions of LSRs in the CDS, IGS, and intron.
Ijms 24 16629 g004aIjms 24 16629 g004b
Ijms 24 16629 g005
Figure 5. Phylogenetic tree of Hydrocotyle and related taxa inferred from the whole plastid genomes using maximum likelihood (ML) and Bayesian inference (BI) methods. The numbers above and below the branches are the ML bootstrap values (BS) and BI posterior probabilities (PP), respectively. BS < 50 and PP < 0.9 are represented by “–”.
Figure 5. Phylogenetic tree of Hydrocotyle and related taxa inferred from the whole plastid genomes using maximum likelihood (ML) and Bayesian inference (BI) methods. The numbers above and below the branches are the ML bootstrap values (BS) and BI posterior probabilities (PP), respectively. BS < 50 and PP < 0.9 are represented by “–”.
Ijms 24 16629 g005
Ijms 24 16629 g006
Figure 6. Phylogenetic tree of Hydrocotyle and related taxa inferred from protein-coding genes using maximum likelihood (ML) and Bayesian inference (BI) methods. The numbers above and below the branches are the ML bootstrap values (BS) and BI posterior probabilities (PP), respectively. BS < 50 and PP < 0.9 are represented by “–”.
Figure 6. Phylogenetic tree of Hydrocotyle and related taxa inferred from protein-coding genes using maximum likelihood (ML) and Bayesian inference (BI) methods. The numbers above and below the branches are the ML bootstrap values (BS) and BI posterior probabilities (PP), respectively. BS < 50 and PP < 0.9 are represented by “–”.
Ijms 24 16629 g006
Table 1. Plastid genome characteristics of the 17 Hydrocotyle taxa.
Table 1. Plastid genome characteristics of the 17 Hydrocotyle taxa.
TaxonLength (bp) GC Content (%)Genes
TotalLSCSSCIRTotalLSCSSCIRPCGstRNArRNAPseudogene
H. calcicola153,30584,29118,72425,14537.60 35.70 30.95 43.25 853784
H. dielsiana153,29784,41218,76725,05937.64 35.74 31.06 43.29 853784
H. himalaica153,29984,41318,76825,05937.64 35.74 31.05 43.29 853784
H. hookeri153,29484,37218,76825,07737.64 35.76 31.07 43.08 853784
H. hookeri subsp. chinensis153,28984,38818,74725,07737.64 35.76 31.08 43.27 853784
H. hookeri subsp. handelii153,32384,40318,76625,07737.64 35.75 31.07 43.27 853784
H. javanica153,32484,40318,76725,07737.63 35.75 31.07 43.27 853784
H. nepalensis *153,35384,43218,76725,07737.63 35.74 31.06 43.27 853784
H. peltiformis153,31184,39018,76725,07737.64 35.75 31.07 43.27 853784
H. pseudoconferta *153,30284,41718,76725,05937.64 35.74 31.07 43.29 853784
H. ranunculoides153,66984,79218,72925,07437.67 35.75 31.22 43.28 853784
H. salwinica153,29484,37218,76825,07737.65 35.76 31.07 43.27 853784
H. sibthorpioides *152,88084,06418,69025,06337.51 35.55 30.95 43.25 853784
H. sibthorpioides var. batrachium152,74084,03018,59425,05837.62 35.69 31.02 43.29 853784
H. sibthorpioides ‘Variegata’152,65983,95818,58525,05837.60 35.56 31.04 43.30 853784
H. verticillata*153,20784,35218,73725,05937.59 35.66 31.08 43.29 853784
H. wilfordii152,95084,13418,69025,06337.50 35.52 30.95 43.25 853784
Note: The sequence from GenBank is indicated by *.
Table 2. Gene content and functional classification of the Hydrocotyle plastomes.
Table 2. Gene content and functional classification of the Hydrocotyle plastomes.
CategoryGene GroupGene Name
PhotosynthesisSubunits of photosystem IpsaA, psaB, psaC, psaI, psaJ
Subunits of photosystem IIpsbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ
Subunits of NADH dehydrogenasendhA a, ndhB a (2), ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
Subunits of cytochrome b/f complexpetA, petB a, petD a, petG, petL, petN
Subunits of ATP synthaseatpA, atpB, atpE, atpF a, atpH, atpI
Large subunit of rubiscorbcL
Self-replicationProteins of large ribosomal subunitrpl14, rpl16 a, rpl2 a (2), rpl20, rpl22, rpl23(2), rpl32, rpl33, rpl36
Proteins of small ribosomal subunitΨrps19, rps11, rps12 b (2), rps14, rps15, rps16 a, rps18, rps19, rps2, rps3, rps4, rps7(2), rps8
Subunits of RNA polymeraserpoA, rpoB, rpoC1 a, rpoC2
Ribosomal RNAsrrn16(2), rrn23(2), rrn4.5(2), rrn5(2)
Transfer RNAstrnA-UGC a (2), trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-UCC, trnG-UCC a, trnH-GUG, trnI-CAU(2), trnI-GAU a (2), trnK-UUU a, trnL-CAA(2), trnL-UAA a, 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 a, trnW-CCA, trnY-GUA, trnfM-CAU
Other genesMaturasematK
ProteaseclpP  b
Envelope membrane proteincemA
Acetyl-CoA carboxylaseaccD
c-type cytochrome synthesis geneccsA
Translation initiation factorinfA
unknown functionConserved hypothetical chloroplast ORFΨycf1, Ψycf15(2), ycf1, ycf2(2), ycf3 b, ycf4
a, gene containing a single intron. b, gene containing two introns. (2), duplicated genes. Ψ, pseudogene.
Table 3. Voucher information and GenBank accession numbers for the 13 newly sequenced Hydrocotyle plastomes.
Table 3. Voucher information and GenBank accession numbers for the 13 newly sequenced Hydrocotyle plastomes.
TaxonVoucher SpecimenLocalityGenBank Accession
H. calcicolaXX Zhou 533 (GACP)Puer, Yunnan, ChinaOM288648
H. dielsianaSR Yi 626 (GACP)Wushan, Chongqing, ChinaOM288647
H. himalaicaL Gu 007 (GACP)Jiangkou, Guizhou, ChinaOM674716
H. hookeriJX Yang 0428 (GACP)Motuo, Tibet, ChinaOM674717
H. hookeri subsp. chinensisisQT 048 (GACP)Kunming, Yunnan, ChinaOM674718
H. hookeri subsp. handeliiL Gu 005 (GACP)Panzhou, Guizhou, ChinaOM674719
H. javanicaGX Hu 746 (GACP)Wangmo, GuiZhou, ChinaOM674725
H. peltiformisGX Hu 637 (GACP)Lushui, Yunnan, ChinaOM674720
H. ranunculoidesL Qu 35 (GACP)Kunming, Yunnan, ChinaOM692359
H. salwinicaGX Hu 638 (GACP)Lushui, Yunnan, ChinaOM674721
H. sibthorpioides var. batrachiumL Gu 004 (GACP)Huaxi, Guizhou, ChinaOM674723
H. sibthorpioides ‘Variegata’C Ye 423 (GACP)Beijing, China (Cultivated)OM674722
H. wilfordiiL Gu 006 (GACP)Dushan, Guizhou, ChinaOM674724
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Yan, R.; Gu, L.; Qu, L.; Wang, X.; Hu, G. New Insights into Phylogenetic Relationship of Hydrocotyle (Araliaceae) Based on Plastid Genomes. Int. J. Mol. Sci. 2023, 24, 16629. https://doi.org/10.3390/ijms242316629

AMA Style

Yan R, Gu L, Qu L, Wang X, Hu G. New Insights into Phylogenetic Relationship of Hydrocotyle (Araliaceae) Based on Plastid Genomes. International Journal of Molecular Sciences. 2023; 24(23):16629. https://doi.org/10.3390/ijms242316629

Chicago/Turabian Style

Yan, Rongrong, Li Gu, Lu Qu, Xiaoyu Wang, and Guoxiong Hu. 2023. "New Insights into Phylogenetic Relationship of Hydrocotyle (Araliaceae) Based on Plastid Genomes" International Journal of Molecular Sciences 24, no. 23: 16629. https://doi.org/10.3390/ijms242316629

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop