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Article

Conserved Plastid Genomes of Pourthiaea Trees: Comparative Analyses and Phylogenetic Relationship

by
Ting Ren
1,*,
Chang Peng
2,
Yuan Lu
1,
Yun Jia
1 and
Bin Li
1
1
Institute of Botany of Shaanxi Province, Xi’an Botanical Garden of Shaanxi Province, Xi’an 710061, China
2
Chengdu Park Urban Institute of Plant Science, Chengdu Botanical Garden, Chengdu 610083, China
*
Author to whom correspondence should be addressed.
Forests 2024, 15(10), 1811; https://doi.org/10.3390/f15101811
Submission received: 3 September 2024 / Revised: 7 October 2024 / Accepted: 12 October 2024 / Published: 16 October 2024
(This article belongs to the Special Issue Biodiversity in Forests: Management, Monitoring for Conservation)
Browse Figures
Figure 1
<p>Plastome map of three <span class="html-italic">Pourthiaea</span> species. Genes located inside the circle are transcribed counterclockwise, and genes outside the circle are transcribed clockwise. The colored bars represent different functional groups of genes.</p> "> Figure 2
<p>Comparison of IR boundaries among 13 <span class="html-italic">Pourthiaea</span> plastomes.</p> "> Figure 3
<p>Simple sequence repeats (SSRs) in the 13 <span class="html-italic">Pourthiaea</span> plastomes. (<b>A</b>) Number of SSRs. (<b>B</b>) Number of SSR motifs.</p> "> Figure 4
<p>Long repeats in the 13 <span class="html-italic">Pourthiaea</span> plastomes. (<b>A</b>) Number of different repeat types. (<b>B</b>) Number of different repeat lengths. Forward (F), palindromic (P), reverse (R), and complementary (C) repeats.</p> "> Figure 5
<p>Comparison of the <span class="html-italic">Pourthiaea</span> plastome sequences using mVISTA with <span class="html-italic">P. villosa</span> as the reference.</p> "> Figure 6
<p>Nucleotide diversity (Pi) in whole plastomes. Coding (<b>A</b>) and non-coding (<b>B</b>) regions.</p> "> Figure 7
<p>The phylogenetic tree generated by BI and ML analyses based on 78 shared plastid protein-coding sequences and 29 nrDNA sequences with <span class="html-italic">Photinia glomerata</span> as outgroup. ML replicate values and BI posterior probability values are given at each node. “*” represents the highest support (100%/1). “#” represents that the node does not occur in the ML tree.</p> ">
Versions Notes

Abstract

:
The genus Pourthiaea Decne., a deciduous woody group with high ornamental value, belongs to the family Rosaceae. Here, we reported newly sequenced plastid genome sequences of Pourthiaea beauverdiana (C. K. Schneid.) Hatus., Pourthiaea parvifolia E. Pritz., Pourthiaea villosa (Thunb.) Decne., and Photinia glomerata Rehder & E. H. Wilson. The plastomes of these three Pourthiaea species shared the typical quadripartite structures, ranging in size from 159,903 bp (P. parvifolia) to 160,090 bp (P. beauverdiana). The three Pourthiaea plastomes contained a pair of inverted repeat regions (26,394–26,399 bp), separated by a small single-copy region (19,304–19,322 bp) and a large single-copy region (87,811–87,973 bp). A total of 113 unique genes were predicted for the three Pourthiaea plastomes, including four ribosomal RNA genes, 30 transfer RNA genes, and 79 protein-coding genes. Analyses of inverted repeat/single-copy boundary, mVISTA, nucleotide diversity, and genetic distance showed that the plastomes of 13 Pourthiaea species (including 10 published plastomes) are highly conserved. The number of simple sequence repeats and long repeat sequences is similar among 13 Pourthiaea species. The three non-coding regions (trnT-GGU-psbD, trnR-UCU-atpA, and trnH-GUG-psbA) were the most divergent. Only one plastid protein-coding gene, rbcL, was under positive selection. Phylogenetic analyses based on 78 shared plastid protein-coding sequences and 29 nrDNA sequences strongly supported the monophyly of Pourthiaea. As for the relationship with other genera in our phylogenies, Pourthiaea was sister to Malus in plastome phylogenies, while it was sister to the remaining genera in nrDNA phylogenies. Furthermore, significant cytonuclear discordance likely stems from hybridization events within Pourthiaea, reflecting complex evolutionary dynamics within the genus. Our study provides valuable genetic insights for further phylogenetic, taxonomic, and species delimitation studies in Pourthiaea, as well as essential support for horticultural improvement and conservation of the germplasm resources.

1. Introduction

Rosaceae, one of the most diverse families within angiosperms, comprises 92 genera and 2,805 species [1]. It is currently split into three subfamilies: Amygdaloideae, Rosoideae, and Dryadoideae [2]. The Pourthiaea Decne. is a deciduous woody genus in the subfamily Amygdaloideae. The genus encompasses about 11–22 species and is widely distributed across East Asia to Southeast Asia [3,4,5,6]. Some species of Pourthiaea are important ornamentally and greening trees in China and are frequently utilized in horticulture for their fascinating white flowers in spring followed by red fruits in autumn. Therefore, Pourthiaea plays an indispensable and positive role in ecological construction in the central and southern regions of China.
The genus Pourthiaea was first described by Joseph Decaisne in 1894, yet the taxonomy of Pourthiaea has been controversial. In the early days, Pourthiaea was once considered either as a section of Photinia [2,3,4,7,8], a synonym of Aronia [9], or a segregate genus from Photinia based on morphological characteristics [10]. Until recently, a study first confirmed the monophyly and genus status of Pourthiaea based on two plastid regions (trnL-trnF and psbA-trnH) and the nuclear ribosomal DNA ITS (nrDNA ITS) sequence [11]. Subsequent studies have once again confirmed the monophyly of Pourthiaea based on molecular and morphological evidence [5,6,12]. The monophyly of Pourthiaea shares some distinguishing morphological characteristics, namely deciduous leaves, Kribs’III-I heterogeneous rays in the wood, pedicels and warty peduncles, and clusters of stone cells surrounded by parenchymatous cells in the flesh of pomes [10,13,14]. Additionally, the species delimitation of Pourthiaea remains challenging due to the highly variable morphological characteristics [5,6,15]. Therefore, more efficient and specific DNA barcodes are needed to explore the phylogeny of Pourthiaea and discriminate them from related species.
Typically, the plastid genome (plastome) structure of angiosperms is highly conserved; it is 120 kb–190 kb in size, including a small single-copy (SSC) region, a large single-copy (LSC) region, and two inverted repeat (IR) regions [16]. Variations in plastome size are primarily due to the expansion and contraction of IR regions [17,18]. For example, the plastome of Pelargonium × hortorum is 217 kb in size with the IR region expanding to 75,741 bp [19], while Pinus thunbergii is 119 kb in size, and the IR region is only 495 bp [20]. The IR regions of plastid genomes have even been confirmed to be complete or partial losses in some species [21,22]. Moreover, the plastid genome always encodes 110–130 distinct genes. These include about eighty protein-coding genes (PCGs), about thirty transfer RNA (tRNA) genes, and four ribosomal RNA (rRNA) genes [21]. Nonetheless, gene loss has occurred in the plastid genomes of many plant lineages [16]. Owing to their smaller size compared to the nuclear genome, conserved genomic structure, lack of recombination, low rates of nucleotide substitutions, and usual uniparental inheritance [23], plastid genomes have been more commonly used to infer phylogenetic relationships of green plants [24,25,26]. Recent advances in genome skimming sequencing have facilitated the capture of complete plastomes and nuclear ribosomal DNA (nrDNA) sequences [27,28,29,30]. Therefore, we preferred to use this method to obtain plastomes and nrDNA sequences in order to comprehensively investigate the phylogenetic relationships of the genus Pourthiaea.
Herein, we first analyzed the complete plastid genome sequences of thirteen Pourthiaea species, including ten reported plastomes and three newly sequenced genomes (Pourthiaea beauverdiana, Pourthiaea parvifolia, and Pourthiaea villosa). Our prime objectives were as follows: (1) deduce the plastome structural evolution of 13 Pourthiaea species; (2) screen the most variable regions as specific DNA barcodes for Pourthiaea; and (3) reveal phylogenetic relationships of 13 Pourthiaea species based on our plastome and nrDNA phylogenies. The complete plastome sequence and nrDNA sequence of Photinia glomerata obtained here were used as the outgroup in the phylogenomic analyses of Pourthiaea. As an excellent horticultural tree species, the improvement and cultivation of new varieties in the genus Pourthiaea will not lag behind. This study provides more genetic information for horticultural improvement and conservation of germplasm resources of Pourthiaea. In addition, our study also provides fundamental data that may be useful for future genomics, evolutionary history, and population genetics studies of this genus.

2. Materials and Methods

2.1. Taxon Sampling and DNA Extraction

The plant leaves of Pourthiaea beauverdiana, Pourthiaea parvifolia, and Pourthiaea villosa were collected from Xi’an Botanical Garden of Shaanxi Province (Xi’an, China), and Photinia glomerata were collected from Chengdu Botanical Garden (Chengdu, China) (Table S1). Total genomic DNA was isolated from silica-dried leaves following a modified CTAB protocol [31] by Novogene (Tianjin, China). Voucher specimens of four collected species were deposited in the XBGH (Xi’an, China) (Table S1). The complete plastid genomes of P. amphidoxa (NC_045414) [12], P. arguta (NC_045413) [12], P. blinii (NC_045412) [12], P. hirsute (NC_065652) [6], P. impressivena (NC_065655) [6], P. pilosicalyx (NC_065654) [6], P. pustulata (NC_065653) [6], P. sorbifolia (NC_045416) [12], P. tomentosa (NC_045417) [12], and P. zhejiangensis (NC_062335) [32] were downloaded to conduct subsequent analyses.

2.2. Sequencing, Assembly, and Annotation

High-throughput sequencing was performed using the PE150 sequencing method on the Illumina NovaSeq sequencing platform by Novogene (Tianjin, China). Sequencing yielded 3.40–3.68 Gb of raw data per species. The raw Illumina reads were removed adaptors and low-quality reads using fastP (-q 15 -n 10) [33]. For the de novo plastome assembly, GetOrganelle v1.7.2 [34] (-k 21,45,65,85,105 -R 15 -F embplant_pt) was used to assemble the clean reads into circular plastid genomes. The annotation of the four plastomes was conducted using GeSeq [35] and further manually adjusted in Geneious v9.0.2 (https://www.geneious.com/) (accessed on 7 December 2023). The gene map of the plastome was generated with the program OrganellarGenomeDRAW (OGDRAW) [36]. Additionally, the nrDNA sequences of the four species were also assembled using GetOrganelle v1.7.2 [34] (-k 35,85,115 -R 10 -F embplant_nr). The four newly obtained complete plastid genomes (PQ197388-PQ197391) and nrDNA sequences (PQ117736-PQ117739) were deposited in GenBank (Table 1 and Table S9). The inverted repeat/single-copy (IR/SC) boundaries of the 13 Pourthiaea plastomes (including 10 published plastomes) were compared to visualize the contraction and expansion of IR regions.

2.3. SSRs and Long Repeat Sequences

Simple sequence repeats (SSRs) in the 13 Pourthiaea plastomes were excavated using MISA [37] with the following parameters: ≥10 repeat units for mononucleotide SSRs, ≥5 for dinucleotide SSRs, ≥4 for trinucleotide SSRs, and ≥3 for tetranucleotide, pentanucleotide, and hexanucleotide SSRs. The four repeats, including forward (F), reverse (R), palindromic (P), and complementary (C) repeats, were excavated using the REPuter program [38] based on the following parameters: hamming distance = 3, and minimum repeat size = 30 bp. The redundant repeat sequences were removed.

2.4. Comparative Plastid Genomic Analyses

The alignment of the 13 Pourthiaea plastomes was performed using the mVISTA program [39] with P. villosa as a reference. The non-coding and coding regions were extracted and aligned. Then, the nucleotide diversity (Pi) of the regions was evaluated with an aligned size of more than 200 bp using DnaSP v5.1 [40]. The genetic distances among the 13 Pourthiaea plastomes were computed with MEGA v6 [41].

2.5. Evolutionary and Phylogenomic Analyses

To investigate the selection patterns of the 79 plastid PCGs, the synonymous (dS) and nonsynonymous (dN) nucleotide substitution rates, and their ratio (ω = dN/dS) were computed using a site-specific model executed in the Codeml program (NSsites = 0, 1, 2, 3, 7, 8, model = 0, seqtype = 1) [42] of PAML4.9 [43] with F3 × 4 codon frequency and cleandata = 1. To run the Codeml program, two files are required, namely seqfile and treefile. After extracting using PhyloSuite v1.2.2 [44], aligning using MAFFT v7 [45], and removing the stop codons manually, 79 plastid protein-coding sequences were used as the input seqfiles. The ML tree constructed using RAxML v8.2.8 [46] based on 79 plastid protein-coding sequences was used as the input treefile. A likelihood ratio test (LRT) was used to calculate a chi-square approximation to determine the model fit. The Bayes Empirical Bayes (BEB) analysis was used to recognize the sites under positive selection with posterior probabilities ≥ 0.95.
Phylogenetic trees were constructed using 29 complete plastome sequences and corresponding 29 nrDNA sequences to explore the phylogenetic relationship of Pourthiaea, with Photinia glomerata as the outgroup. Bayesian inference (BI) and maximum likelihood (ML) methods were used for phylogenomic analyses. The PhyloSuite v1.2.2 [44] was used to extract the 78 plastid protein-coding sequences shared by 29 species. The 78 shared plastid protein-coding sequences were aligned using MAFFT v7 [45] with the codon mode, trimmed using trimAl v1.2 [47] with automated1, and then concatenated using PhyloSuite v1.2.2 [44]. The nrDNA sequences were aligned using MAFFT v7 [45] with the Auto algorithm. Then, the alignment was manually adjusted to minimize nucleotide mismatches. The ML analysis was performed using RAxML v8.2.8 [46] with 1,000 bootstrap replicates. Other parameters were set to “raxmlHPC -p 12,345 -f a -m GTRGAMMA -x 12,345 -s input -n output”. The BI analysis was performed using MrBayes v3.1.2 [48] with the best-fit substitution model (TVM + I + G for 78 shared plastid protein-coding sequences and GTR + I + G for nrDNA sequences) determined by Modeltest v3.7 [49] with the Akaike Information Criterion [50]. The two independent Markov chain Monte Carlo algorithms were run for 5,000,000 generations with one cold chain and three heated chains. The trees were sampled every 100 generations and the first 25% were discarded. Stationarity was achieved when the average standard deviation of the split frequencies < 0.01.

3. Results

3.1. Plastome Structure

We obtained at least 3.0 Gb of raw reads for Pourthiaea beauverdiana (24,239,328 reads), Pourthiaea parvifolia (22,662,706 reads), Pourthiaea villosa (24,516,204 reads), and Photinia glomera (23,435,210 reads) through Illumina NovaSeq sequencing (Table 1). We used 24,062,356 clean reads for P. beauverdiana, 22,404,606 clean reads for P. parvifolia, 24,214,064 clean reads for P. villosa, and 23,321,484 clean reads for Ph. glomera to assemble the complete plastid genomes with the mean coverage depth ranging from 61.31× (P. parvifolia) to 214.50× (Ph. glomera) (Table 1).
The plastomes of three Pourthiaea species shared typical quadripartite structures, ranging from 159,903 bp (P. parvifolia) to 160,090 bp (P. beauverdiana) (Table 2). The three plastomes contained a pair of IRs (26,394–26,399 bp), separated by a SSC region (19,304–19,322 bp) and a LSC region (87,811–87,973 bp) (Table 2). A total of 113 unique genes were predicted for the plastomes of the three Pourthiaea species, including four rRNA genes, 30 tRNA genes, and 79 PCGs (Table 2). The three plastomes had eighteen genes replicated in both IR regions, comprising four rRNA genes (rrn5, rrn4.5, rrn16, and rrn23), seven tRNA genes (trnI-CAU, trnR-ACG, trnA-UGC, trnI-GAU, trnL-CAA, trnV-GAC, and trnN-GUU), and seven PCGs (rps12, rpl23, rpl2, rps7, ycf1, ycf2, and ndhB) (Figure 1; Table 2 and Table S2). The GC content of P. beauverdiana and P. villosa was 36.5%, while the GC content of P. parvifolia was 36.6% (Table 2). The plastome of Ph. glomera was similar to the other three Pourthiaea species, being 160,261 bp in length and containing 113 unique genes. The overall GC content of the Ph. glomera plastome was 36.4% (Figure S1; Table 2 and Table S2).
The IR/SC boundary regions were highly conserved among 13 Pourthiaea plastomes (Figure 2). The LSC/IRb borders were situated in the rps19 genes and extended 120 bp into the rps19 genes. The ycf1 genes and ndhF genes were overlapped in the IRb/SSC regions, overlapping by 7 bp and 14 bp, respectively. The ycf1 genes crossed the SSC/IRa borders, with 4564 bp in the SSC regions and 1076 bp in the IRa regions. The IRa/LSC borders extended into the rpl2-trnH and were 190 bp and 5–145 bp away from the rpl2 genes and trnH genes, respectively.

3.2. SSRs and Long Repeats Analyses

A total of 1345 SSRs were excavated in the 13 Pourthiaea plastomes, with distributions ranging from 100 to 107 SSRs per genome (Figure 3A; Table S3). The greatest number of the SSRs were mononucleotide repeats (1016), followed by dinucleotide (234), tetranucleotide (80), pentanucleotide (11), trinucleotide (3) and hexanucleotide (1) repeats. The only hexanucleotide motif is AAATAT/ATATTT in P. pustulata (Figure 3B). The greatest number of the mononucleotide SSRs were A/T (979, 96.36%) motifs, the dinucleotide SSRs were AT/AT (221, 94.44%) motifs, the trinucleotide SSRs were AAT/ATT (2, 66.67%) motifs, and all the tetranucleotide SSRs were AAAT/ATTT (80) motifs (Figure 3B). The AAAAT/ATTTT motif accounts for 9.09% (1) of the pentanucleotide SSRs (Figure 3B). Therefore, our results indicated that SSRs in plastomes usually consisted of polythymine (polyT) or polyadenine (polyA) repeats. Further analysis showed that 78.44% of the SSRs were located in the LSC region, 15.17% in the SSC region, and 6.39% in the IR region.
For long repeats, the repeat number of each species ranged from 60 (P. amphidoxa, P. arguta, and P. blinii) to 132 (P. pilosicalyx) (Figure 4A; Table S4). Forward repeats are the richest types (516), followed by palindromic (278), reverse (116), and complementary (82) repeats (Figure 4A). The long repeats were recorded mostly in sequence lengths of 30–40 bp (933) and 40–50 bp (61) (Figure 4B). P. amphidoxa and P. pustulata plastomes had one long repeat with a sequence length over 60 bp, while long repeats ranging from 50 to 60 bp were found in P. beauverdiana (1), P. parvifolia (1), P. villosa (1), P. amphidoxa (1), P. hirsute (1), P. impressivena (1), and P. pilosicalyx (1) plastomes (Figure 4B).

3.3. Comparative Analyses of Plastome Sequences

The complete plastomes of 13 Pourthiaea species were plotted using mVISTA [39] to reveal the levels of sequence divergence, and the annotation of P. villosa was a reference (Figure 5). The results indicated that the plastome sequences of these 13 species are highly conserved (Figure 5). Obviously, the IR regions were more conserved than the two SC regions. Furthermore, the coding regions were more conserved, and most of the sequence divergence was focused on the non-coding regions. The Pi value also indicated that the divergence in the non-coding regions (0.0013 on average) is higher than that in the coding regions (0.0003 on average), and the LSC/SSC regions (0.001 on average) are more variable than the IR regions (0.00026 on average) (Table S5). Moreover, some highly variable hotspots were found in the non-coding regions. The most divergent non-coding regions among these plastomes were trnT-GGU-psbD (0.007713), trnR-UCU-atpA (0.007555), and trnH-GUG-psbA (0.008712) (Figure 6, Table S5). The pairwise genetic distance of 13 Pourthiaea plastomes ranged from 0 to 0.003 with an overall average of 0.001, indicating that these plastomes are highly conserved (Table S6).

3.4. Positive Selection Analysis

To identify the selective pressure within the 79 PCGs of the 13 Pourthiaea plastomes, the ratio dN/dS (ω) of non-synonymous to synonymous nucleotide substitution rates was computed. In all genes, the dN/dS ranged between 0.0001 and 1.2729 (Table S7). The great mass of genes were under strong purifying selection (dN/dS values < 0.5) (Table S7). Three genes, including rpoC2, rps18, and rbcL, showed dN/dS values > 1 (Table S7). However, only the rbcL gene with three sites was detected under positive selection (BEB probability > 0.95) after the likelihood ratio test (Table S8).

3.5. Phylogeny Inference

In this study, 78 shared plastid protein-coding sequences and 29 nrDNA sequences were used to investigate the phylogenetic relationships of 13 Pourthiaea species, with Photinia glomerata as the outgroup (Table S9). The aligned matrix of 78 shared plastid protein-coding sequences and nrDNA sequences were 68,085 bp and 6250 bp long, with 451 and 301 parsimony informative sites, and proportions of 0.66% and 4.82%, respectively. Two methods (ML and BI) produced the same topologies with generally high support values using the 78 shared plastid protein-coding sequences, while two methods (ML and BI) yielded slightly inconsistent topologies using the 29 nrDNA sequences (Figure 7). The monophyly of Pourthiaea was strongly supported based on our analyses (ML, BS = 100; BI, PP = 1) in both plastome and nrDNA phylogenies (Figure 7). Pourthiaea was sister to Malus (ML, BS = 100; BI, PP = 1) in plastome phylogenies, while it was sister to the remaining genera in nrDNA phylogenies (Figure 7). Significant cytonuclear discordance was observed between our plastome phylogenies and nrDNA phylogenies within Pourthiaea (Figure 7). The monophyly of the three sections (sect. Amphidoxae, sect. Impressivenae, and sect. Pourthiaea) was supported in nrDNA phylogenies, whereas none of the three sections formed a monophyletic group in plastome phylogenies (Figure 7).

4. Discussion

4.1. Characteristics and Comparison of the Pourthiaea Plastomes

The conserved plastomes in numerous plant lineages have been reported, namely Araceae [51], Asteraceae [52], Betulaceae [53], Fagaceae [54], etc. Here, we newly sequenced three plastomes of Pourthiaea species, which are similar to formerly published Rosaceae plastomes in total length, gene number, and GC content [55]. While the total lengths of the three Pourthiaea plastomes, ranging from 159,903 bp to 160,090 bp, were larger than some species within Rosaceae, such as Alchemilla (152,427–152,438 bp) [56], Rosa (156,405–156,749 bp) [57], and Rubus (155,566–156,236 bp) [58]. The gene number in the three Pourthiaea species (131) was fewer than Alchemilla (133) [56] and Rosa (134) [57], but was identical to Rubus (131) [58]. The GC content of three Pourthiaea species (36.5%–36.6%) was lower than Alchemilla (37.0%) [56], Rosa (37.2%) [57], and Rubus (37.0%–37.3%) [58]. Moreover, the total length of the three Pourthiaea plastomes differs by only 187 bp. Perhaps this proves the conservatism of the Pourthiaea plastomes from one aspect, which may be attributed to the conservatism of the IR region. Overall, the newly sequenced plastomes contribute to understanding the characteristics of plastomes of the members within Pourthiaea and Rosaceae.
The expansion and contraction of the IR regions are significant factors contributing to plastome length variations and evolutionary dynamics [59,60,61]. Our comparison of IR/SC boundary regions among the 13 Pourthiaea plastomes revealed that the gene numbers and orders in these borders were identical, with only slight extensions of the IRa/LSC borders into rpl2-trnH, while other IR/SC borders have not shifted. This indicates that the IR regions are highly conserved with minimal structural variation in the 13 Pourthiaea plastomes. There are differences in the IR/SC boundary regions between Pourthiaea and other members of the Rosaceae, such as Alchemilla [56], Fragaria [56], Rubus [62], etc. Moreover, highly conserved genomic structures have been observed in other tree species, such as Quercus [63], Tilia [64], and Ehretia [65].
SSRs, often containing one to six nucleotides, are employed as molecular markers for biogeographic and population genetics studies [66,67]. Consistent with previous studies on Rosaceae or other plant groups, our results found that SSRs in the 13 Pourthiaea plastomes predominantly consist of polythymine (polyT) or polyadenine (polyA) repeats [62,68,69]. Additionally, the high polyT or polyA SSRs likely contribute to the overall AT richness in the plastomes [70,71]. The uneven distribution of SSRs observed in the 13 Pourthiaea plastomes is similar to findings in Cnidium monnieri [71] and Oryza minuta [72]. Our study provides essential molecular resources for the conservation of Pourthiaea germplasm, offering SSRs that can be used in breeding programs and population genetic studies aimed at preserving the genetic diversity of this genus. It is well known that repeat sequences play a major role in mutation, rearrangement, and recombination events in plastomes [73,74,75]. Among the identified 992 long repeat sequences, those with 30–40 bp and 40–50 bp were the most common (99.1%), consistent with many plant plastomes [64,76]. The palindromic and forward repeats were the most universal repeat types, while the reverse and complementary repeats were the less universal, as reported in prior studies [64,76,77].
The plastome sequences of the 13 Pourthiaea species showed low divergence in general, as indicated by mVISTA, Pi, and genetic distance analyses, similar to other woody species genera [63,65,78]. The three most divergent non-coding regions (trnH-GUG-psbA, psbZ-trnG-GCC, and trnR-UCU-atpA) were identified as the mutational hotspots. Among them, trnH-GUG-psbA is the universal DNA barcode of land plants [79]. The psbZ-trnG-GCC and trnR-UCU-atpA were identified as the mutational hotspots in the genera of Quercus [63], Sanicula [77], Rehmannia [80], Argentina [81], and Castanea [82]. Therefore, these regions could be good candidate DNA barcodes to discriminate Pourthiaea species. A study based on 101 complete plastomes showed that the large single-copy (LSC) region can serve as a highly effective DNA marker for accurately identifying medicinal Dendrobium species [83]. We may be able to collect more plastomes of Pourthiaea species in the future to determine the efficiency of LSC regions in the species delimitation of this genus. In short, our findings offered a precious reference for developing DNA barcodes for Pourthiaea.
The pattern of dN/dS is a very crucial marker in gene evolution studies. The dN/dS < 1 represents purifying selection (especially less than 0.5), dN/dS = 1 represents neutral evolution, and dN/dS > 1 represents positive selection [84]. The great mass of protein-coding genes in our study held low dN/dS values, suggesting a strong purifying selection and the conservatism of plastid protein-coding genes in Pourthiaea. We identified that the rbcL gene was under positive selection in Pourthiaea. The rbcL gene is essential for photosynthesis as it encodes the large subunit of the RuBisCO [23,85]. Positive selection on this gene has been observed in other plant groups, for instance Lamiales [86], Amphilophium [87], and tribe Selineae [71]. In fact, compared with aquatic plants, bacteria, and algae, positive selection on the rbcL gene is often observed in terrestrial plants [88]. This may be because the thermal and water regime of the aquatic habitat is more stable, while the habitat of terrestrial plants is more variable, and therefore requires positive selection to regulate the activity of the RuBisCO [89]. The identification of positive selection on the rbcL gene suggests that Pourthiaea may have undergone adaptive changes, which warrants further investigation to understand their relation to environmental pressures.

4.2. Phylogenetic Relationship of Pourthiaea

To explore the phylogenetic relationships of Pourthiaea, we used 78 shared plastid protein-coding sequences and 29 nrDNA sequences, and two methods to conduct phylogenomic analyses. As expected, all phylogenetic analysis results support the monophyly of the 13 Pourthiaea species. Previous studies divided Pourthiaea into three sections: sect. Amphidoxae, sect. Impressivenae, and sect. Pourthiaea [6]. However, the monophyly of the three sections was supported in nrDNA phylogenies, but not in plastome phylogenies. For the discordances, the nrDNA phylogeny was usually better supported by morphological evidence than the plastome phylogeny [6]. Morphological characteristics distinct to each section include shrubs, occasionally small trees, shorter petioles, and umbellate inflorescences or corymbose in sect. Pourthiaea; tomentose branchlets, adaxial leaf blade, petioles, pedicels, peduncles, and sepals in sect. Amphidoxae; and ferruginous villous pubescence on the leaf blade, petiole, branchlet, hypanthium, and inflorescences in sect. Impressivenae. The cytonuclear discordance, common in angiosperms, is mostly explained as caused by incomplete lineage sorting (ILS) and/or hybridization [26,90,91]. In the case of Pourthiaea, our study implies that the conflicting nuclear and plastome phylogenies are likely the result of hybridization [6]. Pourthiaea belongs to Maleae, and the hybridization event within this tribe is widespread [7]. The origin or expansion of some genera is related to hybridization such as Micromeles, Pseudocydonia, Phippsiomeles, and Rhaphiolepis [12,92,93]. Therefore, the observed cytonuclear discordance likely stems from hybridization events within Pourthiaea, reflecting complex evolutionary dynamics within the genus. This finding highlights the necessity of using nuclear and plastid markers for phylogenetic studies to fully unravel the evolutionary history of these species. Future research could focus on resolving the cytonuclear discordance of Pourthiaea through whole-genome approaches.

5. Conclusions

Here, we sequenced three plastomes of P. beauverdiana, P. parvifolia, and P. villosa using high-throughput technology, providing new genetic insights into this genus. The three newly obtained Pourthiaea species had a typical tetrad structure and contained one hundred and thirteen unique genes, including four rRNA, thirty tRNA, and seventy-nine PCGs. We first revealed the conservatism of the 13 Pourthiaea plastomes (including 10 published plastomes) through IR/SC boundary, mVISTA, Pi value, and genetic distance analyses. We identified 100–107 SSRs, 60–132 long repeat sequences, and three mutational hotspots among the 13 Pourthiaea plastomes. The selection pressure analysis revealed positively selected sites on the rbcL gene. Phylogenetic analyses based on 78 shared plastid protein-coding sequences and 29 nrDNA sequences support the monophyletic origin of 13 Pourthiaea species, providing a reliable phylogenetic framework for understanding the evolution of the Pourthiaea species. The cytonuclear discordance observed may result from hybridization among Pourthiaea species. This study not only contributes to coming population genetics, species delimitation, and evolutionary history studies of Pourthiaea species, but also provides essential support for horticultural improvement and the conservation of the germplasm resources.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f15101811/s1, Figure S1. Gene map of Photinia glomerata plastome. Table S1. Voucher information of four newly sequenced species. Table S2. Gene list of four newly sequenced plastomes. Table S3. SSRs comparison of 13 Pourthiaea species. Table S4. Long repeat sequences comparison of 13 Pourthiaea species. Table S5. Pi values of non-coding and coding regions of 13 Pourthiaea plastomes. Table S6. Genetic distance among 13 Pourthiaea species. Table S7. The ω (dN/dS) of 79 protein-coding genes among 13 Pourthiaea species. Table S8. The LRT of the ω under different models. Table S9. Species list used for constructing the phylogenetic tree.

Author Contributions

Formal analysis, T.R. and C.P.; funding acquisition, T.R., Y.J. and B.L.; methodology, C.P. and Y.J.; project administration, T.R.; resources, T.R., C.P. and Y.L.; Software, Y.L. and Y.J.; validation, Y.L. and B.L.; writing—original draft, T.R.; writing—review and editing, T.R., C.P., Y.L., Y.J. and B.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Project of the Science and Technology Program of Shaanxi Academy of Science (2023K-48, 2023K-25, 2023K-14), National Natural Science Foundation of China (32300314).

Data Availability Statement

Four annotated nrDNA sequences and plastomes have been submitted into NCBI with accession numbers: PQ117736-PQ117739 and PQ197388-PQ197391.

Acknowledgments

We thank Novogene company for sequencing.

Conflicts of Interest

We declare that we have no conflicts of interest.

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Figure 1. Plastome map of three Pourthiaea species. Genes located inside the circle are transcribed counterclockwise, and genes outside the circle are transcribed clockwise. The colored bars represent different functional groups of genes.
Figure 1. Plastome map of three Pourthiaea species. Genes located inside the circle are transcribed counterclockwise, and genes outside the circle are transcribed clockwise. The colored bars represent different functional groups of genes.
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Figure 2. Comparison of IR boundaries among 13 Pourthiaea plastomes.
Figure 2. Comparison of IR boundaries among 13 Pourthiaea plastomes.
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Figure 3. Simple sequence repeats (SSRs) in the 13 Pourthiaea plastomes. (A) Number of SSRs. (B) Number of SSR motifs.
Figure 3. Simple sequence repeats (SSRs) in the 13 Pourthiaea plastomes. (A) Number of SSRs. (B) Number of SSR motifs.
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Figure 4. Long repeats in the 13 Pourthiaea plastomes. (A) Number of different repeat types. (B) Number of different repeat lengths. Forward (F), palindromic (P), reverse (R), and complementary (C) repeats.
Figure 4. Long repeats in the 13 Pourthiaea plastomes. (A) Number of different repeat types. (B) Number of different repeat lengths. Forward (F), palindromic (P), reverse (R), and complementary (C) repeats.
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Figure 5. Comparison of the Pourthiaea plastome sequences using mVISTA with P. villosa as the reference.
Figure 5. Comparison of the Pourthiaea plastome sequences using mVISTA with P. villosa as the reference.
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Figure 6. Nucleotide diversity (Pi) in whole plastomes. Coding (A) and non-coding (B) regions.
Figure 6. Nucleotide diversity (Pi) in whole plastomes. Coding (A) and non-coding (B) regions.
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Figure 7. The phylogenetic tree generated by BI and ML analyses based on 78 shared plastid protein-coding sequences and 29 nrDNA sequences with Photinia glomerata as outgroup. ML replicate values and BI posterior probability values are given at each node. “*” represents the highest support (100%/1). “#” represents that the node does not occur in the ML tree.
Figure 7. The phylogenetic tree generated by BI and ML analyses based on 78 shared plastid protein-coding sequences and 29 nrDNA sequences with Photinia glomerata as outgroup. ML replicate values and BI posterior probability values are given at each node. “*” represents the highest support (100%/1). “#” represents that the node does not occur in the ML tree.
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Table 1. Assembly information for three Pourthiaea species and Photinia glomera.
Table 1. Assembly information for three Pourthiaea species and Photinia glomera.
SpeciesGenBank NumbersRaw ReadsClean ReadsMean Coverage
Pourthiaea beauverdianaPQ19738924,239,32824,062,356114.48×
Pourthiaea parvifoliaPQ19739022,662,70622,404,60661.31×
Pourthiaea villosaPQ19739124,516,20424,214,064109.37×
Photinia glomeraPQ19738823,435,21023,321,484214.50×
Table 2. Summary of the complete plastomes of three Pourthiaea species and Photinia glomera.
Table 2. Summary of the complete plastomes of three Pourthiaea species and Photinia glomera.
SpeciesTotal SizeLSCSSCIRUnique GenePCGstRNArRNAGC%
(bp)(bp)(bp)(bp)Numbers
P. beauverdiana160,09087,97319,31926,39911379 (7)30 (7)4 (4)36.5
P. parvifolia159,90387,81119,30426,39411379 (7)30 (7)4 (4)36.6
P. villosa160,03087,92019,32226,39411379 (7)30 (7)4 (4)36.5
Ph. glomera160,26188,22219,27326,38311379 (7)30 (7)4 (4)36.4
Numbers in parentheses represent the number of genes duplicated in IRs.
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Ren, T.; Peng, C.; Lu, Y.; Jia, Y.; Li, B. Conserved Plastid Genomes of Pourthiaea Trees: Comparative Analyses and Phylogenetic Relationship. Forests 2024, 15, 1811. https://doi.org/10.3390/f15101811

AMA Style

Ren T, Peng C, Lu Y, Jia Y, Li B. Conserved Plastid Genomes of Pourthiaea Trees: Comparative Analyses and Phylogenetic Relationship. Forests. 2024; 15(10):1811. https://doi.org/10.3390/f15101811

Chicago/Turabian Style

Ren, Ting, Chang Peng, Yuan Lu, Yun Jia, and Bin Li. 2024. "Conserved Plastid Genomes of Pourthiaea Trees: Comparative Analyses and Phylogenetic Relationship" Forests 15, no. 10: 1811. https://doi.org/10.3390/f15101811

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