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. 2024 Sep 2;14(9):e70239. doi: 10.1002/ece3.70239

Rampant intraspecific variation of plastid genomes in Gentiana section Chondrophyllae

Shan‐Shan Sun 1,✉, Zhi‐Yong Pan 1, Yu Fu 1, Shen‐Jue Wang 1, Peng‐Cheng Fu 1
PMCID: PMC11368500  PMID: 39224159

Abstract

Exploring the level of intraspecific diversity in taxa experienced radiation is helpful to understanding speciation and biodiversity assembly. Gentiana section Chondrophyllae sensu lato encompasses more than 180 species and occupies more a half of species in the genus. In this study, we collected samples across the range of three species (Gentiana aristata, G. crassuloides and G. haynaldii) in section Chondrophyllae s.l., and recovered the intra‐species variation by comparing with closely related taxon. Using 25 newly sequenced plastid genomes together with previously published data, we compared structural differences, quantified the variations in plastome size, and measured nucleotide diversity in various regions. Our results showed that the plastome size variation in the three Chondrophyllae species ranged from 285 to 628 bp, and the size variation in LSC, IR and SSC ranged from 236 to 898 bp, 52 to 393 bp and 135 to 356 bp, respectively. Nucleotide diversity of plastome or any of the four regions was much higher than the control species. The average nucleotide diversity in plastomes of the three species ranged from 0.0010 to 0.0023 in protein coding genes, and from 0.0023 to 0.0061 in intergenic regions. More repeat sequence variations were detected within the three Chondrophyllae species than the control species. Various plastid sequence matrixes resulted in different backbone topology in two target species, showed uncertainty in phylogenetic relationship based inference. In conclusion, our results recovered that species of G. section Chondrophyllae s.l. has high intraspecific plastome variation, and provided insights into the radiation in this speciose lineage.

Keywords: Gentiana, intraspecific variation, plastome, section Chondrophyllae s.l

Significant high level of intraspecific plastome size variation and genetic diversity are detected in three annual gentians of Gentiana section Chondrophyllae s.l.

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1. INTRODUCTION

The increasing availability of plastid genomes (plastome) represents an excellent opportunity to explore evolutionary processes in plants (Sibbald & Archibald, 2020; Twyford & Ness, 2017). For example, plastid phylogenomics led to a better understanding of evolutionary patterns in taxa experienced radiation or rampant hybridization, as the case of evolutionary radiations in Saussurea (Zhang et al., 2021). Plastome structure is usually conservative in land plants, composing of two inverted repeat (IR) regions that are separated by the large single copy (LSC) region and the small single copy (SSC) region (Jansen & Ruhlman, 2012; Mower & Vickrey, 2018). Comparative analysis among closely related taxa have detected a number of plastome microstructural changes including expansion/contraction or loss of the IR (Lee et al., 2021; Wang et al., 2022) and gene loss (Mower et al., 2021; Wang et al., 2023). Linking the changes with diversification can offer clues to the mechanisms driving their evolution (Fu et al., 2021; Wang et al., 2023; Wicke et al., 2016). Besides phylogeny and molecular evolution, rapid increasing data also offer great benefits to explore intraspecific plastome variation in some diverse groups experienced rapid evolution. Accelerated plastid genome evolution was speculated to contribute to the early stages of speciation (Barnard‐Kubow et al., 2014). Intraspecific variation in plastome sequence and structure has been reported for a few groups of angiosperms, for example the variation in IR extent and boundaries of junction sites in Medicago minima (Choi et al., 2020), length variation in coding region in Medicago truncatula (Gurdon & Maliga, 2014), and gene degradation at individual and population levels in Cymbidium (Kim & Chase, 2017).

Gentiana is a typical alpine genus, with the Qinghai‐Tibet Plateau (QTP) acting as the main center of diversity and the primary source area for dispersal to mountainous areas of the world (Favre et al., 2016; Ho & Liu, 2001). Although Gentiana is distributed in mountain systems around the world, in fact, section Chondrophyllae Bunge sensu lato (s.l.) is the only section globally distributed, whereas another 11 sections of Gentiana are endemic to one or two continents (Ho & Liu, 2001). Section Chondrophyllae s.l composes about 182 species, occupying 51.7% of all Gentiana species. Section Chondrophyllae s.l. is a well‐supported monophyletic group with a long branch in phylogenetic tree (Favre et al., 2020; Fu et al., 2021), hitting rapid evolution in this group (Yuan & Küpfer, 1997). In subtribe Gentianinae, section Chondrophyllae s.l. has experienced the most notable plastome variations including plastome size decreases, gene loss, IR contraction and SSC reduction (Fu et al., 2022), and were supposed to be correlated with the rapid evolution in this section (Fu et al., 2021). In addition, previous studies showed obvious cyto‐nuclear conflict in section Chondrophyllae s.l. (Chen et al., 2021; Fu et al., 2022). Although hybridization is widely accepted to explain cyto‐nuclear conflict, conflicting phylogenetic signals in the plastome which had been observed in various lineages (Walker et al., 2019) are not yet assessed in Gentiana. Since the plastome is rather dynamic in section Chondrophyllae s.l., we wonder what's the level of plastome variation in this lineage, and if the variation has impact on phylogenetic reconstruction.

In this study, we focus on three species, Gentiana aristata Maxim., Gentiana crassuloides Bureau & Franch. and Gentiana haynaldii Kanitz, which came from three series in section Chondrophyllae s.l. By sequencing samples across the range of the three species and comparing with species from the most closely related lineage of section Chondrophyllae s.l., we aim to recover the level of intraspecific plastome diversity and microstructural changes in section Chondrophyllae s.l., and furtherly assess its impact on reconstructing phylogenetic relationship.

2. MATERIALS AND METHODS

2.1. Studied species and sampling

The three target species, Gentiana aristata, G. crassuloides and G. haynaldii, belong to series Humiles Marquand, Orbiculatae Marquand and Dolichocarpa T.N.Ho, respectively (Favre et al., 2020; Ho & Liu, 2001). The three species are annual herbs, and endemic to the QTP. We sampled individuals from different localities to cover the entire range of each species. Because the three species are minute annuals, a whole single plant was collected in the wild for each individual, and conserved in silica gel prior to extraction. In total, individuals from 18, 15 and 10 localities were collected for G. aristata, G. crassuloides and G. haynaldii, respectively (Table 1). No permission to sample was needed because the target species were either in the List of National Key Protected Wild Plants China or sampled from nationally protected regions. Gentiana crassicaulis Duthie ex Burk. from section Cruciata Gaudin which is the sister group of section Chondrophyllae s.l. (Favre et al., 2020; Fu et al., 2021) was served as the control. Species were identified by Dr. Peng‐Cheng Fu, and voucher specimens were deposited either in the herbarium of Luoyang Normal University (no acronym at present).

TABLE 1.

Information of newly sequenced plastid genomes in three species from Gentiana section Chondrophyllae s.l. Gentiana crassicaulis whose voucher no. was Genbank accession number, was served as the control.

Species Voucher no. Latitude Longitude Altitude (m) GenBank no. LSC IR SSC Total
G. aristata fu2016001 35°35′ 102°42′ 3094 PQ154566 73,779 22,336 9394 127,845
fu2016016 34°42′ 102°29′ 3281 PQ154567 74,126 22,333 9398 128,190
fu2017008 35°52′ 99°58′ 3360 PQ154561 73,773 22,347 9386 127,853
fu2017031 33°07′ 97°27′ 4119 PQ154553 73,919 22,506 9206 128,137
fu2017055 32°46′ 97°12′ 4013 PQ154557 74,247 22,404 9128 128,183
fu2017112 31°04′ 96°56′ 4626 PQ154560 73,890 22,506 9208 128,110
fu2017162 31°44′ 100°43′ 4022 PQ154558 73,731 22,335 9384 127,785
fu2017195 32°09′ 102°30′ 3516 PQ154554 73,825 22,327 9407 127,886
fu2017206 33°12′ 101°28′ 3717 PQ154552 73,827 22,327 9407 127,888
fu2017240 33°27′ 100°59′ 3651 PQ154562 73,737 22,329 9388 127,783
fu2017250 33°39′ 99°46′ 4152 PQ154563 73,725 22,217 9403 127,562
fu2017276 34°18′ 100°16′ 3985 PQ154559 73,826 22,327 9407 127,887
fu2017352 36°19′ 101°29′ 3600 PQ154569 73,985 22,113 9390 127,601
fu2018016 34°27′ 102°18 3603 PQ154564 74,075 22,317 9416 128,125
fu2019139 37°07′ 102°27′ 2978 PQ154555 73,706 22,347 9421 127,821
fu2019557 37°47′ 101°19′ 3640 PQ154568 73,706 22,347 9421 127,821
fu2019677 38°00′ 100°21′ 3209 PQ154565 73,706 22,347 9443 127,843
fu2019688 37°35′ 100°45′ 3439 PQ154556 73,705 22,347 9399 127,798
G. crassuloides fu2016024 33°12′ 102°24′ 3545 PQ154539 72,961 22,525 10,232 128,243*
fu2016094 29°51′ 102°01′ 3442 PQ154532 73,206 22,373 10,432 128,384*
fu2017054 32°46′ 97°12′ 4013 PQ154534 72,985 22,562 10,310 128,419*
fu2017086 31°08′ 96°29′ 4187 PQ154537 72,982 22,562 10,310 128,416*
fu2017155 31°42′ 99°37′ 3752 PQ154536 73,068 22,375 10,479 128,297*
fu2017163 31°44′ 100°43′ 4022 PQ154540 72,961 22,662 10,138 128,423*
fu2017181 31°53′ 102°38′ 3343 PQ154528 72,308 22,373 10,468 128,422*
fu2017194 32°09′ 102°30′ 3516 PQ154533 73,116 22,368 10,463 128,315*
fu2017211 33°12′ 101°28′ 3717 PQ154531 72,898 22,383 10,494 128,158*
fu2017241 33°27′ 100°59′ 3926 PQ154529 72,954 22,662 10,138 128,416*
fu2017252 33°39′ 99°46′ 4152 – 72,954 22,662 10,138 128,416*
fu2018053 28°20′ 99°04′ 4326 PQ154538 73,059 22,507 10,370 128,443*
fu2018117 28°48′ 97°42′ 4094 PQ154541 73,071 22,502 10,297 128,372*
fu2019003 34°00′ 107°46′ 3500 PQ154535 73,052 22,510 10,328 128,400*
fu2022005 – – 3600 PQ154530 72,958 22,547 10,346 128,398*
G. haynaldii fu2016063 31°59′ 99°05′ 4021 PQ154542 73,532 22,124 10,117 127,897*
fu2016075 30°59′ 101°08′ 3548 PQ154543 73,296 22,079 10,100 127,554*
fu2017057 32°46′ 97°12′ 4013 PQ154551 73,531 22,124 10,117 127,896*
fu2017067 32°35′ 96°32′ 4133 PQ154545 73,523 22,124 10,117 127,888*
fu2017141 31°19′ 98°04′ 4160 PQ154549 73,531 22,124 10,117 127,896*
fu2017154 31°42′ 99°37′ 3752 PQ154546 73,512 22,124 10,117 127,877*
fu2017180 31°53′ 102°38′ 3343 PQ154544 73,521 22,131 10,136 127,919*
fu2018034 27°48′ 99°38′ 3400 PQ154547 73,307 22,094 10,235 127,730*
fu2020013 30°05′ 91°16′ 4268 PQ154548 73,524 22,124 10,117 127,889*
fu2020024 29°42′ 92°07′ 4552 PQ154550 73,524 22,124 10,117 127,889*
G. crassicaulis – – – KY595457 81,143 25,272 17,070 148,757
– – – KY595458 81,143 25,272 17,070 148,757
– – – KY595459 81,174 25,272 17,070 148,788
– – – KY595460 81,162 25,272 17,071 148,777
– – – KY595461 81,164 25,272 17,070 148,778
– – – KY595462 81,141 25,272 17,070 148,755
– – – KY595463 81,140 25,272 17,070 148,754
– – – KY606171 81,110 25,272 17,070 148,724
– – – KJ676538 81,164 25,271 17,070 148,776
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Note: Columns LSC, IR and SSC report the length (bp) of the large single‐copy, inverted repeat and small single‐copy regions, respectively. Newly sequenced plastid genomes were indicated with asterisks (*) behind the total length.

2.2. Sequencing, assembly and annotation

Total genomic DNA was isolated from dried leaves to perform genome‐skimming sequencing. A 500‐bp DNA Illumina sequencing library was constructed using about 4.0 ng of sonicated DNA as input. The library was multiplexed and sequenced using the Illumina HiSeq 2500 platform, yielding about 2 Gb of 150‐bp paired‐end reads for each sample. The plastid genome was assembled using GetOrganelle v.1.7.1 (Jin et al., 2020) with the default parameters. Each plastid genome was then annotated with PGA (Qu et al., 2019). We converted annotations into graphical maps using OGDRAW (Greiner et al., 2019). After format transfer using GB2sequin (Lehwark & Greiner, 2018), all plastome sequences were deposited in GenBank (accession nos., PQ154528–PQ154569; Table 1). In addition, nine plastomes of Gentiana crassicaulis were retrieved from GenBank for comparison analysis (Table 1).

2.3. Plastome structural changes and nucleotide diversity

Genome comparisons were conducted to identify intra‐species microstructural changes using mVISTA (Frazer et al., 2004). We analyzed genome rearrangement by using the progressive Mauve algorithm in Mauve v2.3.1 (Darling et al., 2010) using the plastid genome sequence with only one IR copy. The genes on the boundaries of the junction sites of the plastome were visualized in IRscope (Amiryousefi et al., 2018). To estimate nucleotide diversity, sequences of all genes, intergenic regions and RNA were extracted in PhyloSuite v.1.2.2 (Zhang, Gao, et al., 2020) and aligned using MAFFT v.7.313 (Katoh et al., 2002). Number of indel and nucleotide diversity (Pi) of different sequences were measured in DnaSP v.5 (Librado & Rozas, 2009). To test whether plastome diversity was correlated with plastome size, Mantel tests were performed in R to clarify the relationship between plastome size and Pi or number of indel.

2.4. Repeat sequence analysis

Microsatellites (SSRs) were identified by MISA (Beier et al., 2017) with the thresholds of 10, 6, 5, 5, 5, 5 repeated units for mono‐, di‐, tri‐, tetra‐, penta‐, and hexa‐nucleotide SSRs, respectively. We used Tandem Repeats Finder (Benson, 1999) to find the tandem repeated sequences with the default settings. We used REPuter (Kurtz et al., 2001) to identify the dispersed repeated sequences, including forward, reverse, complement, and palindromic repeats. The Hamming distance and minimum repeated size were set at three and 30 bp, respectively. To test whether repeats were correlated with plastome size, Mantel tests were performed in R to clarify the relationship between plastome size and number of SSRs, tandem repeated sequences and dispersed repeated sequences.

2.5. Phylogenetic analysis

To reconstruct intra‐species phylogenetic relationship in each of the target species, we built maximum likelihood (ML) trees using three matrices: WP included whole plastome after removing one IR, PCS contained all protein coding genes, IRS comprised intergenic regions. We examined each matrix and removed the most rapidly evolving sites using Gblocks v.0.91b (Talavera & Castresana, 2007) with default setting. The substitution model was chosen using ModelFinder v.2 (Kalyaanamoorthy et al., 2017). Phylogenetic analyses were inferred using IQ‐TREE v.1.6.8 (Nguyen et al., 2015) for 5000 ultrafast (Minh et al., 2013) bootstraps. The trees were visualized with R package ggtree (Yu et al., 2017).

3. RUSULTS

3.1. General plastome characteristics

In this study, we newly sequenced and assembled 25 plastomes of G. crassuloides and G. haynaldii. The 15 plastomes of G. crassuloides varied from 128,158 to 128,443 bp in size, with variation of 285 bp (Figure 1). The size variation in LSC, IR and SSC is 898, 294 and 356 bp, respectively (Table 1; Figure 2). The 10 plastomes of G. haynaldii varied from 127,554 to 127,919 bp in size, with variation of 365 bp (Figure 1). The size variation in LSC, IR and SSC is 236, 52 and 135 bp, respectively (Table 1; Figure 2). The 18 plastomes of G. aristata assembled in Fu et al. (2024) were annotated in this study, and their size vary from 127,562 to 128,190 bp, with size variation of 628 bp (Figure 1). The size variation in LSC, IR and SSC is 542, 393 and 315 bp, respectively (Figure 2). All plastomes of the three species encoded a total of 105 unique genes, of which 18 were duplicated in IR regions. The 105 genes consist of 71 protein‐coding genes, 30 tRNA genes, and 4 rRNA genes. Regarding to the control species, based on 9 plastomes of G. crassicaulis, size variation in total plastome, LSC, IR and SSC was 64, 64, 1 and 1 bp, respectively (Figure 2).

FIGURE 1.

FIGURE 1

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Schematic map of overall features of the chloroplast genome of three annual gentians in Gentiana section Chondrophyllae s.l. Genes drawn inside the circle are transcribed clockwise, and those drawn outside are transcribed counterclockwise. Genes belonging to different functional groups are shown in different colors.

FIGURE 2.

FIGURE 2

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Plastome size variation in three annual gentians in Gentiana section Chondrophyllae s.l. The control is Gentiana crassicaulis that belongs to the sister group of section Chondrophyllae s.l. The median of each boxplot was aligned to keep in a line in each panel. The y‐axle shows the scale of size variation, and the scale for each interval is presented in bracket. IR, inverted repeat; LSC, long single copy; SSC, small single copy.

3.2. Structural changes and nucleotide diversity

Genome comparison showed nucleotide variation within each species, but did not detect either structural changes such as genome rearrangement (Figure S1) or gene loss (Figures S2–S4). We did not detect variation of gene composition in the boundaries of the junction sites in plastomes within each of the three gentians, but gene shift in the boundaries of the junction was observed (Figure 3). For example, the length of rps19 and ycf1 in the IR region was ranged from 56 to 110 bp, and 76 to 318 bp in G. aristata, respectively.

FIGURE 3.

FIGURE 3

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Comparison of LSC, IRs, and SSC junction positions among plastomes of three annual gentians in Gentiana section Chondrophyllae s.l.

In general, the target species had much higher nucleotide diversity than the control. Among the three target species, G. crassicaulis had the highest nucleotide diversity in the total plastome, LSC and SSC region, followed by G. aristata and then G. haynaldii (Table 2; Figure 4). In plastome, SSC region had highest nucleotide diversity in all the three species, and IR had the lowest. G. aristata had the largest number of indels in all regions of the plastome, and G. haynaldii had the lowest (Table 3; Figure 4). Mantel tests did not show significant correlation between plastome size and Pi or number of indels in LSC, SSC and total plastome (p‐value ranged from 0.1619 to 0.6272). The exception occurred in IR region, in which a significant relationship between Pi and IR size was detected (p‐value = 0.0065).

TABLE 2.

Summary of intraspecific length variation in plastid genomes of four Gentiana species.

Species No. Total LSC IR SSC
Min. Max. Variation Min. Max. Variation Min. Max. Variation Min. Max. Variation
G. aristata 18 127,562 128,190 628 73,705 74,247 542 22,113 22,506 393 9128 9443 315
G. crassuloides 15 128,158 128,443 285 72,308 73,206 898 22,368 22,662 294 10,138 10,494 356
G. haynaldii 10 127,554 127,919 365 73,296 73,532 236 22,079 22,131 52 10,100 10,235 135
G. crassicaulis 9 148,724 148,788 64 81,110 81,174 64 25,271 25,272 1 17,070 17,071 1
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Note: Gentiana crassicaulis was served as the control.

Abbreviations: IR, inverted repeat; LSC, large single‐copy; Max., maximum value; Min., minimum value; No., number of plastid genome; SSC, small single‐copy regions.

FIGURE 4.

FIGURE 4

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Nucleotide diversity (Pi) and number of indels in plastid genomes of three annual gentians in Gentiana section Chondrophyllae s.l. The results of total plastome, long single copy (LSC), small single copy (SSC) and inverted repeat (IR) are presented in turn. The control is G. crassicaulis that belongs to the sister group of section Chondrophyllae s.l.

TABLE 3.

Summary of intraspecific nucleotide diversity in plastid genomes of four Gentiana species.

Species Region No. PS No. indel Pi k
G. aristata LSC 971 3795 0.00212 153.731
IR 67 569 0.00051 11.164
SSC 229 997 0.00394 35.474
Total 1334 5927 0.00169 211.532
G. crassuloides LSC 774 2075 0.00309 223.342
IR 40 232 0.00044 9.933
SSC 219 825 0.00649 65.067
Total 1070 3364 0.00242 307.300
G. haynaldii LSC 314 1176 0.00104 75.691
IR 35 235 0.00049 10.727
SSC 81 363 0.00201 19.927
Total 443 1896 0.00087 110.018
G. crassicaulis LSC 27 159 0.00008 6.889
IR 9 5 0.00008 2.000
SSC 7 1 0.00011 1.889
Total 52 170 0.00009 12.778
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Abbreviations: k, average number of nucleotide differences; No. indel, number of indels; No. PS, number of polymorphic sites; Pi., nucleotide diversity.

Comparison of nucleotide diversity of protein coding genes indicated that G. crassicaulis had the highest average nucleotide diversity (Pi = 0.0023), followed by G. aristata (Pi = 0.0013) and then G. haynaldii (Pi = 0.0010). Nucleotide diversity varied among genes, for example, atpE, petG and petN had the highest value in G. crassicaulis, cemA and clpP had the highest value in G. aristata, and psaI had the highest value in G. haynaldii (Figure 5a). Regarding to intergenic regions, G. crassicaulis had the highest average nucleotide diversity (Pi = 0.0061), followed by G. aristata (Pi = 0.0040) and then G. haynaldii (Pi = 0.0023). Among tested intergenic regions, trnH‐psbA had the highest nucleotide diversity in all three target species (Figure 5b). Rpl32‐trnL and psbC‐trnS regions also had high nucleotide diversity within each of the three species. The control had much lower nucleotide diversity than the three target species in both genes and intergenic regions (Figure 5). Nucleotide diversity was very low in most RNAs in all tested species (Figure S5).

FIGURE 5.

FIGURE 5

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Nucleotide diversity (Pi) of protein coding genes (a) and intergenic regions (b) in plastid genomes of three gentians belonging to Gentiana section Chondrophyllae s.l. The control is G. crassicaulis that belongs to the sister group of section Chondrophyllae s.l.

3.3. Repeat sequences

The average number of detected SSRs in plastomes of G. aristata, G. crassuloides, G. haynaldii and control species was 39.3, 36.7, 35.6 and 27.7, respectively. In the three target species, most SSRs occurred in LSC (from 22.4 to 27.1), and rare in IR (from 1.0 to 2.3) (Figure 6a). As expected, the most detected SSR motif was mononucleotide. Regarding to tandem repeated sequences, the average number in G. aristata, G. crassuloides, G. haynaldii and control species was 14.7, 18.1, 19.7 and 28.2, respectively (Figure 6b). The average number of dispersed repeated sequences detected in plastomes of G. aristata, G. crassuloides, G. haynaldii and control species was 10.6, 13.6, 13.8 and 31.9, respectively (Figure 6c). Mantel tests showed a significant negative correlation between total plastome size and number of SSRs, and significant positive correlations between total plastome size and tandem repeated sequences and dispersed repeated sequences (p‐value ranged from 2.20e−16 to 1.02e−11). Among the three plastome regions, only size of LSC showed significant correlation with number of SSRs and tandem repeated sequences (p‐value was 2.27e−10 and 2.20e−16, respectively).

FIGURE 6.

FIGURE 6

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Results of repeat sequence in three annual gentians. (a) SSRs; (b) tandem repeated sequences; (c) dispersed repeated sequences. IR, inverted repeat; LSC, long single copy; SSC, small single copy.

3.4. Phylogenetic analysis

The three matrix in each species resulted in various backbone topologies in the ML trees in which most of deep nodes were highly supported (Figure 7). In G. aristata, the yellow clade being a monophyletic group was weakly supported (bootstrap supporting, BS = 69%) by the WP matrix, but was a polyphyletic group based on PCS and IRS matrixes (Figure 7a). In G. crassuloides, the three matrixes produced the same backbone topology and very similar BS values in deep nodes (Figure 7b). Regarding to G. haynaldii, besides the poor clades existed within the species, the position of the yellow clade was identical based on the WP and PCS matrixes, but differed with the result based on the IRS matrix (Figure 7c).

FIGURE 7.

FIGURE 7

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Phylogenetic relationship in Gentiana aristata (a), G. crassuloides (b) and G. haynaldii (c) based on three matrixes. The outgroup is presented with a gray dot in the base of each tree.

4. DISCUSSION

By sampling and sequencing individuals covering the range of three species belonging to various series in Gentiana section Chondrophyllae s.l., our results indicated high intra‐species plastome diversity in the speciose lineage. The three target species, G. aristata, G. crassuloides and G. haynaldii, had much higher plastome diversity than the control species belonging to the sister group of section Chondrophyllae s.l. For example, the three species had much higher values in plastome size variation, Pi and number of indels than the control species (Figures 2 and 4, 5, 6). The high level of plastome diversity is mainly caused by two reasons. First, large plastome size variation due to a large number of indels covering the whole plastome (Figure 4), rather than structural changes such as expansion/contraction of SSC or IR which commonly detected among species in this section (Fu et al., 2021, 2022). The indels detected in the three target species were much higher (from 1896 to 5927) than in species from G. sect. Monopodiae (e.g., 441 or 581 in plastome; Mao et al., 2023). Second, high nucleotide polymorphism in both genes and intergenic regions. The high nucleotide polymorphism in Chondrophyllae species is much higher than the control species in G. section Cruciata (Figure 5) and two species in G. sect. Monopodiae whose average Pi in genes and intergenic regions were lower than 0.001 and 0.0025, respectively (Mao et al., 2023). Within the three target species, G. crassuloides had the highest plastome diversity, for example large size variation and Pi in SSC and IR, and high Pi in genes and intergenic regions. In particular, as most conserved region in plastome (Guisinger et al., 2011), IR showed the largest size variation in G. crassuloides. The high plastome diversity which could afford more informative sites shall be one of the reasons why the three matrix of G. crassuloides resulted in the same backbone topology in phylogenetic analysis (Figure 7b), although higher diversity did not always mean the better phylogenetic supports due to diverse factors such as the different evolutionary history of genes. In addition, the three annual gentians with high nucleotide diversity had high level of SSRs and repeats, consistent with the observation in Malvaceae that correlations occurred among repeats, SSRs and indels (Abdullah et al., 2021).

Previous study indicated that repeats could yield variable‐length insertions and deletions, and thereby repeat‐mediated genome rearrangement was linked with plastid genome variability (Gurdon & Maliga, 2014). Comparing to the control species, much larger number of SSRs and smaller number of tandem repeated sequences and dispersed repeated sequences were detected in the three Chondrophyllae species (Figure 6). The number of SSRs was variable within species in sect. Chondrophyllae s. l., and most SSRs were mono‐nucleotide repeats, being consistent with other gentians (Mao et al., 2023) and angiosperms (e.g., Li et al., 2023; Mwanzia et al., 2020; Xu et al., 2022). We found that repeat content was significantly correlated with genome size, as plastid with smaller genome size had more SSRs and less repeat elements (dispersed repeat and tandem repeat) in Gentiana. Previous studies also detected that repeat content was positively correlated with genome size and genomic rearrangements, for example in Medicago (Wu et al., 2021) and Alismatidae (Li et al., 2023). Since the repeat elements and plastome size are significantly correlated, we speculate that repeat elements are another factor likely contribute to plastome size variation in G. sect. Chondrophyllae s. l., being consistent with the case in Alismatidae (Li et al., 2023). Variation in repeat elements and plastome degradation such as ndh complex loss (Fu et al., 2022) may be the key factors explaining the genome size variation in sect. Chondrophyllae s. l. In addition, accelerated plastid genome evolution may contribute to the early stages of the speciation process by increasing the likelihood of intraspecific cyto‐nuclear genetic incompatibilities (Barnard‐Kubow et al., 2014). We detected much higher intraspecific diversity in species of sect. Chondrophyllae s. l. than its sister group, hinting accelerated plastid genome evolution. In fact, elevated substitution rate in plastome genes was indeed observed in sect. Chondrophyllae s. l. under broader context (Fu et al., 2021). Therefore, rapid plastid genome evolution maybe one reason explaining the high species diversity in sect. Chondrophyllae s. l.

Plastome was widely used to analyze phylogenetic relationship in both low and high taxonomic units, and could provide robust results in most cases (Lv et al., 2023; Zhou et al., 2022, 2023). Our results based on three matrices showed conflicting phylogenetic signals in two of three species (Figure 7a,c). In fact, conflicting phylogenetic signals in the plastome were observed in various lineages (Walker et al., 2019), from genus level such as Rhododendron (Mo et al., 2022) to higher level such as Leguminosae (Zhang, Wang, et al., 2020), Laureae (Xiao et al., 2020) and Fagales (Yang et al., 2021). It is still uncertain why the conflict in plastome‐inferred phylogenies occurred, and suggested potential reason including heteroplasmic recombination in plastome (Mo et al., 2022; Walker et al., 2019) and complex history of plastome structural evolution (Zhang, Wang, et al., 2020). Currently, no available study detects occurrence of plastid recombination in sect. Chondrophyllae s. l., but heteroplasmy was clarified in an annual gentian in sect. Microsperma T.N. Ho (Sun et al., 2019), suggesting that plastid recombination could not be ruled out in Chondrophyllae species. In addition, hybridization, which could lead to plastid recombination, was proved to be common in sect. Chondrophyllae s. l. (Chen et al., 2021; Fu et al., 2022), including rampant hybridization within G. aristata (Fu et al., 2024). We thereby suggested that heteroplasmic recombination in plastome of sect. Chondrophyllae s. l. shall be possible, but direct evidences are needed. We also found that the species having the highest diversity had consistent phylogenetic topology among the three datasets (Figure 7b) and indicate that conflict in phylogenetic backbone may be easier to be detected in taxon with less plastome sequence diversity. Therefore, heteroplasmic recombination and poor sequence diversity maybe the potential reasons of the conflict in phylogenetic signals, but it is still early to draw a firm conclusion.

AUTHOR CONTRIBUTIONS

Shan‐Shan Sun: Data curation (equal); funding acquisition (equal); project administration (equal); writing – original draft (equal). Zhi‐Yong Pan: Formal analysis (equal); visualization (equal). Yu Fu: Formal analysis (equal); visualization (equal). Shen‐Jue Wang: Formal analysis (equal); visualization (equal). Peng‐Cheng Fu: Formal analysis (equal); investigation (equal); writing – review and editing (equal).

CONFLICT OF INTEREST STATEMENT

None declared.

Supporting information

Figures S1–S5.

ECE3-14-e70239-s001.docx (2.4MB, docx)

ACKNOWLEDGMENTS

We thank West Henan Yellow River Wetland Ecosystem Observation and Research Station for the support in data analysis. This work was financially supported by the National Natural Science Foundation of China (32300190) and Natural Science Foundation of Henan Province (232300420212) to S.S.S.

Sun, S.‐S. , Pan, Z.‐Y. , Fu, Y. , Wang, S.‐J. , & Fu, P.‐C. (2024). Rampant intraspecific variation of plastid genomes in Gentiana section Chondrophyllae . Ecology and Evolution, 14, e70239. 10.1002/ece3.70239

DATA AVAILABILITY STATEMENT

All data are provided within the text, tables, figures and supplements. The raw plastome sequences and annotations are provided in FigShare with the link of https://doi.org/10.6084/m9.figshare.25479385.v1.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figures S1–S5.

ECE3-14-e70239-s001.docx (2.4MB, docx)

Data Availability Statement

All data are provided within the text, tables, figures and supplements. The raw plastome sequences and annotations are provided in FigShare with the link of https://doi.org/10.6084/m9.figshare.25479385.v1.

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