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. 2024 Nov 5;24:1046. doi: 10.1186/s12870-024-05766-2

Comparison of the chloroplast genomics of nine endangered Habenaria species and phylogenetic analysis

Jinkui Zhang 1,2, Dangwei Zhou 2,4,5,✉, Weidong Chen 1, Pengcheng Lin 2, Suqin Zhao 3, Min Wang 2, Huan Wang 4, Shengbo Shi 4, Furrukh mehmood 6, Xing Ye 2, Jing Meng 2, Wenyuan Zhuang 2
PMCID: PMC11536600  PMID: 39497089

Abstract

Background

Habenaria, a genus in the family Orchidaceae, are the nearly cosmopolitan orchids, and most species have significant medicinal and ornamental values. Despite the morphological and molecular data that have been studied in recent years, the phylogenetic relationship is still unclear.

Results

We sequenced, assembled, and annotated the chloroplast (cp) genomes of two species (Habenaria aitchisonii Rchb.f. and Habenaria tibetica Schltr.ex Limpricht) of Habenaria grown on the Qinghai-Tibetan Plateau (QTP), and compared them with seven previously published cp genomes which may aid in the genomic profiling of these species. The two genomes ranged from 155,259–155,269 bp in length and both included 132 genes, encoding 86 proteins, 38 tRNAs and 8 rRNAs. In the cp genomes, the tandem repeats (797), SSRs (2195) and diverse loci (3214) were identified. Comparative analyses of codon usage, amino frequency, microsatellite, oligo repeats and transition and transversion substitutions revealed similarities between the species. Moreover, we identified 16 highly polymorphic regions with a nucleotide diversity above 0.02, which may be suitable for robust authentic barcoding and inferring in the phylogeny of Habenaria species. Among the polymorphic regions, positive selection was significantly exerted on several genes, such as cemA, petA, and ycf1. This finding may suggest an important adaptation strategy for the two Habenaria species on the QTP. The phylogenetic relationship revealed that H. aitchisonii and H. tibetica were more closely related to each other than to the other species, and the other seven species were clustered in three groups. In addition, the estimated divergence time suggested that the two species separated from the others approximately 0.39 Mya in the Neogene period. Our findings also suggest that Habenaria can be divided into different sections.

Conclusions

The results of this study enriched the genomics resources of Habenaria, and SSR marker may aid in the conservation management of two endangered species.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-024-05766-2.

Keywords: Habenaria, Chloroplast genome, Molecular marker, Positive selection, Orchidaceae, Phylogenomic

Background

Habenaria (Habenariinae, Orchidoidea) is a large genus including more than 891 species [1, 2], that are widely dispersed across the tropical, subtropical, temperate, and alpine regions worldwide [3, 4]. The species in this genus not only have great ornamental values, but their tubers also have significant therapeutic properties and could be used as diuretics to treat swelling, waist strength and kidney conditions, lumbago, and hernia [5–7]. Additionally, people frequently cook the tubers for use in meals to cure HIV/AIDs in several regions of Africa [8]. However, owing to variations in morphology, such as tubers, spurred lip, long columns, wide U-shaped anthers, long caudicles, naked viscidia and free stigmas [3, 9], new descriptions of Habenaria species are still being reported [10–13], and the phylogenetics relationships within the genus are still controversial [14–16].

The plastome has a quadripartite structure, which consisted of two inverted repeats (IRa and IRb), a large single-copy (LSC) and small single-copy (SSC) regions [17, 18]. The size of the chloroplast genomes ranges from 107 to 218 kb [17]. Like the nuclear genome, the chloroplast genome also contains variations such as deletions, insertions, losses, and single-nucleotide mutations in specific regions [19, 20]. The polymorphisms of the chloroplast genome reveal the evolutionary profiling of the paternal inheritance of the species and could be used in population genetics, phylogenetics and barcoding for plants [21]. Owing to the polymorphisms in the plastome, several studies have been performed to resolve taxonomic discrepancies and identify the phylogenetic relationships in Orchidaceae with high resolution [20, 22, 23]. These studies can provide not only information to resolve the taxonomic discrepancies of plant lineages, but also in-depth insight into the evolution of the plastomes [19, 20, 24].

Adaptive evolution reflects the adaptability of the species during the evolution process in which natural selection is achieved through genetic variation, genetic recombination, and gene flow [25, 26]. Therefore, exploring the selection characteristics that species have undergone during their evolutionary process is another topic of interest in the chloroplast genome analyses. Yang et al. (2002) reported that heterogeneity in the plastid matK and rbcL genes in different species of the family of mangrove genus [27]. In recent years, many studies have identified positively selected chloroplast genes through the assessment of Ka (non-synonymous substitution) and Ks (synonymous substitutions) [24, 28, 29].

Habenaria aitchisonii Rchb.f. and Habenaria tibetica Schltr.ex Limpricht of genus Habenaria grow on the QTP or adjacent regions [4, 12], especially H. tibetica, which is an endemic endangered species that is found only in shrub meadows or alpine meadows. Ecological and biogeographical forces have a substantial impact on the heterogeneity rate of the chloroplast genome [27]. In general, the substitution rates of cp genomes have undergone minimal evolution [29]. Previous studies revealed that the positive selection could increase the Ka value but does not affect the Ks value [30]. However, there is little known about the evolution and adaptation profiling of Habenaria species.

In this study, two Habenaria species (H. aitchisonii and H. tibetica) grown on the QTP were sequenced using the next-generation Illumina platform. Combined with the cp genomes of 40 species of Orchidaceae, we aimed to (1) compare the cp genome structure of these two species with those of the seven species in the Habenaria genus; (2) reconstruct the phylogeny of Habenaria species according to cp genome data and the species relationships in Orchidaceae at the plastid level; (3) investigate selective or adaptive evolution cues in the cp genomes of known Habenaria species.

Results

Chloroplast genomes of Habenaria aitchisonii and Habenaria tibetica

The Habenaria aitchisonii and Habenaria tibetica chloroplast genomes were sequenced with average coverage depths ranging from 646 × to 1588 × . The two genomes were 155, 259 bp (GenBank accession number: OQ701055) and 155, 269 bp (GenBank accession number: OQ701056) in length, respectively (Table S1). Both species presented typical quadripartite structures (Fig. 1a and b), in which the IRs were separated by the SSC and LSC regions. The LSC regions had lengths of 84,234 bp and 84,143 bp in H. aitchisonii and H. tibetica, respectively, with a 91 bp constriction in H. tibetica, the SSC regions were 17,643 bp and 17,646 bp in H. aitchisonii and H. tibetica respectively, with an expansion of 3 bp in H.tibetica. Similarly, the IR regions also exhibited a 49 bp expansion in H. tibetica. The cp genomes of both species included 132 genes, encoding 86 proteins, 38 tRNAs and 8 rRNAs. Among them, 20 genes were duplicated in the IR regions, including 4RNA genes (rrn16, rrn23, rrn4.5 and rrn5), 7 tRNA genes (trnA-UGC, trnH-GUG, trnI-CAU, trnL-CAA, trnN-GUU, trnR-ACG and trnV-GAC) and 8 protein-coding genes (ndhB, rpl2, rpl23, rps12, rps19, rps7, ycf1 and ycf2). In addition, 17 genes had one intron, and 1 had two introns (Clp P) (Table S1 and Table 1). The ycf1 gene was also found to be truncated at the junction of the IR/SSC with a functional copy (Fig. 1). The GC content was similar between the two species (36.83% and 36.84%); the IR regions had the highest GC content (42.91% to 42.86%), and the SSC regions had the lowest GC content (29.38% to 29.40%).

Fig. 1.

Fig. 1

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Chloroplast genome maps of Habenaria aitchisonii Rchb.f. a and Habenaria tibetica Schltr.ex Limpricht. b Genes of different functional groups are displayed in different colours. The darker gray color corresponds to the GC content, and large single-copy (LSC), small single copy (SSC) and inverted repeat (IRA and IRB) regions are indicated

Table 1.

General characteristics of the chloroplast genomes of the nine Habenaria species

Genome feature H. aitchisonii H.tibetica H.dentata H.cruciformis H.ciliolaris H.radiata H.flagellifera H.checiformis H.pantlingiana
Genome Size(bp) 155,259 155,269 153,682 155,708 154,544 155, 353 155,298 153,896 153,951
LSC (bp) 84,234 84,143 83,963 85,131 84,032 84, 833 85,749 83,732 83,641
SSC (bp) 17,643 17,646 17,041 17,659 19,602 17, 718 18,373 17,026 17,370
IR (bp) 26,691 26,740 26,339 26,459 25,455 26,401 25,595 26,569 26,470
GC content (%) 36.83 36.84 36.62 36.60 37.90 36.60 37.90 36.70 36.60
Total number of genes 132 132 133 131 132 113 130 131 133
Protein-coding gene 86 86 87 79 86 79 81 85 87
tRNA 38 38 37 30 38 30 37 38 38
rRNA 8 8 8 4 8 4 8 8 8
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Comparation of plastome features of the Habenaria genus

To determine the cp genome features of the Habenaria genus, we compared the H. aitchisonii and H. tibetica with the other seven species. The plastome structure of all the species was highly conserved (Fig. 2; Table S2). The complete plastome sizes ranged from 151,210 bp (H. flagellifera) to 155,708 bp (H. cruciformis), the LSC length ranged from 81,072 bp (H. flagellifera) to 85,131 bp (H. cruciformis), the IR region ranged from 26,399 bp (H. dentata) to 26,856 bp (H. ciliolaris), and the SSC length ranged from 17,026 bp (H. chejuensis) to 17,718 bp (H. radiata) (Fig. 2, Table 1). Moreover, the GC content of all plastomes ranged from 36.60% to 37.90%.

Fig. 2.

Fig. 2

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Mauve alignment of organization of the plastomes of nine Habenaria species using collinear blocks. The green colour represents the inversion of single copy and the small colour blocks represent different types of genes (black represents tRNA, red reprents rRNA, white represents protein-coding, and green represents intron-containing tRNA)

Mauve-based analysis revealed similar gene arrangements contents in the Habenaria genus (Fig. 2). Gene rearrangement did not occur in the nine plastomes, and the differences appeared in the intron-contained RNA regions. All plastomes displayed similarity at the LSC/IRb and SSC/IRa junctions (Fig. 3). rpl22 and ycf1 are located in these junction regions, respectively. However, at the IRb and SSC junctions, some differences were detected. In H. pantlingiana plastome, ycf1 clearly covered the junction’s regions, whereas in other plastomes, the gene was close to the junction regions. Interestingly, the ycf1 gene was inverted in the plastome of H. chejuensis (Fig. 3).

Fig. 3.

Fig. 3

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Analyses of the expansion and contraction of inverted repeats in the nine Habenaria chloroplast genomes

Relative synonymous codon usage and amino acid frequency

The RSCU frequency plays an important role in reflecting mutation bias during evolution. To determine the codon usage and amino acid frequency, RSCU and amino acid frequency were analysed in the plastomes, protein-coding genes contained 79,767 bp and 79,749 bp in H. aitchisonii and H. tibetica, respectively. The two plastomes exhibited similar RSCU frequencies (Figure S1 and Table S3). Among the protein-coding genes, leucine was the most abundant amino acid (10.35% and 10.34%), followed by serine (7.91% and 7.09%) and arginine (5.96% and 5.95%). However, only 461 (1.73%) encoded tryptophan, which is the least common amino acid (Figure S1 and Table S3). There were 29 and 28 codons that displayed clearly biased usage (RSCU > 1) in H. aitchisonii and H. tibetica, respectively. However, the tryptophan usages were not biased in either species (RSCU = 1) (Table S3). Furthermore, codon usage appeared biased in H. aitchisonii and H. tibetica compared with other species (Fig. 4; Table S4). The results revealed that the four parameters involved in codon usage bias were slightly greater than those of the other seven Habenaria species, Goodyera, Anoetochilus and Vanilla, while the CAI and CBI appeared to be the lower (Fig. 4).

Fig. 4.

Fig. 4

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The comparative analysis of codon usage bias in Habenaria and other species. (Habenaria 1: seven other Habenaria species; Habenaria 2: H.tibetica and H.aitchisonii). A ENC, effective number of codons; B GC3s, GC content of synonymous codons in the 3rd position; C G3s, GC content of synonymous codons in 3rd position; D CBI, codon bias index; E CAI, codon adaptation index; F Fop, Frequency of optimal codons index

Analysis of microsatellites and oligonucleotide repeats

A total of 233 SSRs and 232 SSRs were identified in the plastomes of H. aitchisonii and H. tibetica, respectively. Among them, 136 SSRs (58.40%) occurred in the LSC region, 52 SSRs (22.30%) in the IR region, and 45 SSRs (19.30%) in the SSC region in H. aitchisonii. Similarly, 134 SSRs (57.80%), 46 SSRs (19.80%) and 52 SSRs (22.40%) occurred in LSC, IR and SSC regions, respectively (Table S5a). In both species, 99 SSRs appeared in the intergenic region.

To obtain more information about the SSRs in the Habenaria genus, we compared all 2195 SSRs in the nine species. Mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide SSRs accounted for 67.26, 5.65, 27.43, 1.82, 0.68 and 0.23%, respectively, of all SSRs. Among all 27 types of SSRs, A/T was the most common, followed by the AAG/CTT and AAT/ATT types (Fig. 5a). Among the nine species, H. pantlingiana had a greater number of unique SSR types of Pentra (4), H. flagellifera had 3, and H. chejenensis and H. radiata had one Pentra type. The other species exhibited no unique SSR types of Pentra.

Fig. 5.

Fig. 5

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Comparison of SSR profiling and the oligonucleotide repeats in the nine Habenaria species. (a. SSR profiling; b. oligonucleotide repeats)

To compare the oligonucleotide repeats in all nine species, we used REPuter software. As shown in Fig. 5b, the total number of repeats clearly differed among the nine plastomes (Table S5b). H. ciliolaris contained the greatest number of repeats (211) whereas H. radiata contained the fewest number of repeats (26) (Fig. 5b-a). Moreover, H. ciliolaris contained the most forward repeats (77) whereas H. radiata contained the fewest forward repeats (9) (Fig. 5b-b). H. ciliolaris contained the greatest number of reverse repeats (35) whereas H. flagellifera contained no reverse repeats (5b-c). H. aitchisonii contained the most palindromic repeats, followed by H. tibetica. In contrast, H. cruciformis and H. radiata presented the fewest palindromic repeats (Fig. 5b-d). Four species (H. cruciformis, H. flagellifera, H. radiata and H. aitchisonii) contained one complementary repeat, and H. ciliolaris contained the greatest number of complementary repeats (38) (Fig. 5b-e).

Identification of polymorphic loci

To understand the nucleotide polymorphic profiling, the cp genomes of nine Habenaria species were analysed with DnaSP software. As shown in Fig. 6, LSC, SSC and IR regions exhibited clearly differences in polymorphic loci. In the IR region, the Pi value slightly changed, and the value ranged from 0–0.0074, with an average 0.0001431. The greatest number of polymorphisms appeared in SSC regions with an average value 0.021857. The highest Pi value was 0.04527(ycf1). The LSC region had the highest Pi value among those greater than 0.0001431, despite the average value of 0.010343(Fig. 6; Table S6).

Fig. 6.

Fig. 6

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Nucleotide variability values compared between the nine chloroplast genomes of Habenaria species using sliding window analysis

Molecular evolution analysis

The pairwise Ka/Ks were analyzed in nine species of Habenaria (Fig. 7), which reflected the selected pressure on the sequences. H. aitchisonii and H. tibetica species displayed no positive sites (Ka/Ks > 1) or neutral sites (Ka/Ks = 1). Only six genes exhibited purified pressure, and most genes presented no changes in Ka or Ks (Table S7). However, for H. aitchisonii, H. tibetica and other Habenaria species, 3, 4 or 5 genes, respectively, may be subjected to positive selection. These genes included cemA, petA, rps11, rpl14, ycf1, psbK, rpl22, ycf2, ycf2-2, psbH, and ndhI (Table S7). To determine the positive selection genes of Habenaria, cemA, petA, rps11, rpl14, ycf1, psbK, rpl22, ycf2, ycf2-2, psbH, and ndhI were analysed in the H. aitchisonii and H.tibetica branch and other closely related species (Table S8), and all the genes had no significant posterior probabilities on the H. aitchisonii and H. tibetica branch (Table S4). However, six genes petA, rps11, ndhI, rpl22, ycf1 and ycf2 contained the sites with positive selection in the BEB test. Among them petA, ndhI, rpl22, ycf1 and ycf2 had more than one positive selection site. Moreover, two sites were detected in the ndhI, rpl22, ycf1 and ycf2 genes on the H. aitchisonii and H. tibetica branch (Table 2; Table S8). These results suggested that the two species experienced adaptation evolution on the QTP.

Fig. 7.

Fig. 7

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Pairwise Ka/Ks for nine species of Habenaria

Table 2.

The potential positive selection test based on the branch-site model

Gene name Null hypothesis Alternative hypothesis Significant test
InL df ω InL df ω BEB p-value
cemA -1967.69 1 1 -1971.76 3 0.547 104 K p < 0.05
petA -2147.03 1 1 -2147.03 3 1.000 none p > 0.05
rps11 -1126.26 1 1 -1124.08 3 999.0 38 V,82A,88 T p < 0.05
ndhi -1371.40 1 1 -1369.59 3 147.6 38I,95F p < 0.05
psbH -493.70 1 1 -492.37 3 999.0 10S p < 0.05
psbK -462.73 1 1 -462.73 3 2.140 none p > 0.05
rpl14 -851.21 1 1 -850.77 3 52.192 17Q,119P p > 0.05
rpl22 -1130.19 1 1 -1130.11 3 1.643 120 V p > 0.05
ycf1 -23,972.75 1 1 -23,972.75 3 1.000 none p < 0.05
ycf2 -16,608.99 1 1 -16,608.99 3 1.000 None p < 0.05
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Phylogenetic relationship and divergence time analysis

To understand the phylogenetic relationships between H. aitchisonii and H. tibetica and other species of orchid, we constructed the phylogenetic trees using 50 cp genomes (Table S9). The phylogenetic trees revealed that H. aitchisonii and H. tibetica were clustered together with 100% bootstrap support (Fig. 8). The nine Habenaria species were clustered in one clade and were closely related to Goodyeras. Among the Habenaria clade, H. pantingiana and H. flagellifera were grouped together, and H. dentata, H. ciliolaris, and H. chejuensis were clustered in other branches. H. crucicifomis and H. radiata appeared as two parallel branches. Using the fossil records for calibration with the FBD model, we estimated that the nine Habenaria species appeared approximately 15.36 Mya and diverged. Moreover, the two species separated into ancestors of H. cruciformis and H. radiata approximately 0.39 Mya in the Neogene period (Fig. 8).

Fig. 8.

Fig. 8

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The Phylogenetic relationships Habenaria species and related Orchidaceae species using the ML method. Bootstrap values were shown at the nodes

Discussion

Here, we sequenced and assembled cp genomes of H. aitchisonii and H. tibetica grown on the QTP (Fig. 1). Our results revealed that the cp genomes of the two species were very similar in structure and genome size. Combined with the published cp genomes [23, 31], our result also revealed that the genome size of the Habenaria species was 153,682–155,708 bp, which is smaller than that of Cypripedium [31] and larger than that of Vanilla, Cyrtosia, Gastrodia and Epipogium species [23]. Moreover, the GC content of the nine Habenaria species ranged from 36.60% to 37.90% (Table 1), with an average GC content of 36.95%, which was slightly greater than the average GC content of orchid plastomes (36.40%) [31] and Cypripedium species (31.8%) [21].The GC content might be related to the ancestral feature of monocot genomes [32], in addition, selection and mating system could also drive GC content and GC3 usage [33].We suggest that high GC content of Habenaria species may be related to the mutations resulting from these processes.

Gene number and gene order are other evolutionary characteristics of cp genomes. A previous study reported that the average number of genes in 124 orchid species was 113 [22]. However, our results revealed that most of the species contained more than 130 coding genes, except H. radiata (113 genes) (Table 1), which is also a slightly greater number than that in Cypripedium (128–130 genes). This difference may be caused by the greater number of duplicated genes. In these two species, more than 20 genes were duplicated. Ndh genes have been lost or deleted in Cypripedium and other orchid species [23, 31, 34], or pseudogenes [34, 35]. Interestingly, our results revealed that the Habenaria species contained all 11 ndh genes (Table S1 and S7). Ndh genes are involved in photosynthesis or plastome stability [22, 36]. The NDH complex plays a crucial role as a safeguard of the photosynthetic machinery against environmental stress and absent of ndh displayed poor to adapt to photooxidative protection [36, 37]. Therefore, all ndh genes keep may play important roles in Habenaria adaptation to adverse environment.

Highly variable regions could be used to design the markers for phylogenetic and biogeographic analysis [38]. In this study, the SSC regions contained the greatest number of polymorphisms and had the highest average Pi value of 0.021857, and ycf1 presented a high Pi value (Fig. 6). This result was consistent with that reported for Blumea species [19]. Excluding the conserved IR regions, the LSC regions also presented high variations (matK-rps16, psbK-psbI, atpA-atpF, rps2-rpoC2, ycf4-cemA, rpl33-rps18, and rps11-rpl36) (Fig. 6; Table S4). These findings may suggest that this region has potential for use as markers. Moreover, 16 genes (atpF, matK, ycf4, cemA, psbK, rps18, psbI, rps11, rpl22, rps15, ycf1, ndhF, rpl32, ccsA, ndhD and ndhE) with nucleotide diversity greater than 0.02 were detected (Fig. 7; Table S5). Among these loci, ndhF, rps15, ccsA, and rpl32 have been found to be highly variable regions in different species [19, 22]. Therefore, the high variation information identified in this study has the potential to be exploited as candidate barcode sequences in the phylogenetic analysis of Habenaria. Moreover, 233 SSRs and 232 SSRs were identified in the plastomes of the two species, and more SSRs occured in IGS regions (Fig. 5a; Table S5a). Of the six SSR types, more than sixty SSRs were mononucleotides, and the results were consistent with those of other species [19, 22]. Moreover, our results revealed 27 types of SSRs in the nine Habenaria species, which is lower than that in Cypripedium [22]. This difference may be due to the Cyripedium having larger cp genomes and enlarged IR regions. In Cypripedium, cp genome expansion is associated with the proliferation of IGS regions [22]. Therefore, the repeat regions identified in this study may aid in population genetics studies of Habenaria. On the QTP, the two Habenaria displayed plaque-like distribution and would more vulnerable to threats. Chloroplast SSRs have a faster rate of evolution than other types of polymorphism [39] and are particular sensitive markers for assessing population size changes and genetic diversity [40]. Using the SSRs marker to analysis genetic diversity would help to prioritize allocation of conservation resources [41] and to identify seed source zones for reforestation and translocation [42]. Hence, our results also provide the data for current and future conservation measures of two Habenaria species on the QTP.

Habenaria is a large genus, and most Habenaria species are terrestrial orchids that are nearly cosmopolitan, and occur in the tropical, subtropical, temperate, and alpine regions [3, 4]. In the ML tree, our results revealed that Habenaria is not the monophyletic and that the nine species could be divided into five different groups (Fig. 8). Among them, the two species in this study (H. aitchisonii and H. tibetica) clustered together with 100% bootstrap support. Interestingly, both species were grown on the QTP and exhibited similar morphological characteristics [4]. Both species have two basal subopposite leaves, slightly 2-lobed petals, racemes with flowers, deeply 3-lobed lips and slightly clavate spurs [4]. Our cp genomes result also revealed that the two species are closely related. These results are also supported by the previous studies [10, 11]. Using the rbcL + matK + ITS, Habeniara can be divided into eleven clades and the species from tropical and alpine regions can be grouped into different subclades [11]. Subclade I included mostly species from the tropical region, and Subclade II included the alpine species. Our results revealed that H. chejuensis, H. ciliolaris and H. dentata were grouped into other branches (Fig. 8), which may originate in tropical regions [11]. Although the cp genomes data presented here are still quite limited and could not clarify the phylogenetic relationships in the Habenaria, our results still suggest that the cp genomes could be used for phylogenetic inference when additional cp genome information is obtained in the future. Moreover, the divergence time revealed that the nine Habenaria species have originated 15.36 Mya, which was in accordance with the previous reports [43, 44]. Interestingly, our results also suggested that the two species diverged at only 0.39 Mya in Neogene period. The orchid subfamily appeared in the Eocene and cooler temperatures in the ecosystem promoted their adaptive radiation [43]. The growth of the Qinghai-Tibetan Plateau led to a cold and dry climate [45], which increased the separation of the from the other Habenaria species.

In the previous study, some chloroplast genes were shown to be targets of natural selections [24, 27–29, 46]. Our results revealed that there were no genes subjected to natural selection between the two alpine species (Ka/Ks < 1). However, more than 3 genes had a Ka/Ks > 1 in both species including cemA, petA, rps11, rpl14, ycf1, psbK, rpl22, ycf2, ycf2-2, psbH, and ndhI (Fig. 7). To further understand the positive genes in Habenaria genus, codon model was used for ten genes [25, 26]. Codon sites with higher posterior values are another indicator of divergent selective pressure [25, 26]. The results also revealed that six genes may be under significant positive selection (Table 2; Table S8), suggesting that the positive selections occurred in the two alpine species. In addition, the genes associated with photosynthesis (petA, psbH), NADH-dehydrogenase subunits(ndhI), self-replication process gene (rps11, rpl22) and ycf1, ycf2 also exhibited positive selection. Photosynthesis system and NADH-dehydrogenase contribute to light harvesting and electron transport to produce ATP under positive selection in Allium [46]. Here, our results were consistent with their conclusion. Moreover, our results also revealed that genes related to the cp ribosome (rpl22 and rpl11) exhibited significant positive selection. This finding may suggest that protein synthesis plays an important role in stress adaptation of two alpine species.

The QTP is the largest and highest plateau in the world and an important hotspot of biodiversity [47]. Orchid species are extremely sensitive to environmental change [48]. Acharya et al. (2011) reported that the precipitation and temperature could affect the abundance and distribution of the orchid species [49]. Similarly, Hu et al. (2022) reported that annual precipitation, elevation, and topsoil pH (H2O) strongly influence the distribution of the orchid species in the QTP [50]. Ka/Ks ration have been widely used to infer evolutionary dynamics and identify adaptive signatures among species. The Ka/Ks ratios suggested that positive selection existed in Allioideae species [33] and Solanaceae species [20, 24]. Our results also showed that positive selection occurred in two alpine Habenaria species. The divergence time revealed that the two species separated from the other species in the Neogene period and climate was believed to have become dry and cold [39, 46], Therefore, positive selection of genes in cp genome in two species may help their adaptation to adverse environmental factors on the QTP.

Conclusions

In this study, the two chloroplast genomes of Habenaria species on the QTP were sequenced and compared via genomic profiling with those of seven other published species. We revealed similarities in gene arrangement and gene content in the Habenaria genus. The rearrangement of genes did not occur in the nine plastomes. Comparisons of tandem of codon usage, amino frequency, microsatellites, oligo repeats and transition and transversion substitutions revealed similarity between the two species. Moreover, we identified 16 highly polymorphic regions with a nucleotide diversity above 0.02, which may be suitable for robust authentic barcoding and inference in the phylogeny of Habenaria species. Among the polymorphic regions, positive selection was significantly exerted on cemA, petA, ndhI, rpl22, rps11and psbH. The phylogenetic relationships revealed that H. aitchisonii and H. tibetica are more closely related to each other than to the other species and that the two species diverged from the other species approximately 0.39 Mya. The data obtained in this study also enriched the genomic resources of Habenaria in Orchidaceae, which may be helpful for the conservation efforts of these endangered species with the new barcoding and SSRs and checking the positive sites for chloroplast biotechnology in the future.

Methods

Sample collection and DNA extraction

In the flowering season of 2021, the fresh leave of H. aitchisonii and H. tibetica were collected from Maixiu National Forestry Park (35. 2619°N, 101.8861°E, Alt. 3200 m) and Sanjiang Source National Park (32.9385°N, 100.7436°E, Alt. 3300 m). The voucher specimens (ZDW-2021–024 and ZDW-2021–030) were identified by Prof. Pengcheng Lin and deposited in the Herbarium of Northwest Institute of Plateau Biology, Chinese Academy of Sciences. The total DNA was extracted using the CTAB method [51].

Plastome genome sequencing, assembly and annotation

The high-quality DNA was sequenced with the Illumina NovaSeq 6000 sequencing platform (Nanjing Genepioneer Biotechnologies, Inc.). fastp (version 0.20.0, https://github.com/OpenGene/fastp) was used to filter the raw reads, and the clean reads were mapped to the chloroplast genomes in the GenBank. The contigs were obtained with SOAPdenovo2 v3.10.1 (http://cab.spbu.ru/software/spades/) under kmer = 55, 87 and 121 [52].The scaffold was constructed by SSPACE v2.0 [53], and GapCloser was used to fill the gaps [52]. The annotation of the complete chloroplast genomes was executed using DOGMA (http://dogma.ccbb.utexas.edu/) [54] and the circular chloroplast genome map was generated by OGDRAW [55].

SSRs, codon usage and nucleotide diversity analysis

The Web-based tool REPuter (https://bibiserv.cebitec.uni-bielefeld.de/reputer/) was used to detect repeats including forward, palindrome, reverse and complement repeats. The minimal repeat size was set to 30 bp, and the sequence identity was > 90%. Simple sequence repeats (SSRs) were identified using the Micro Satellite identification tool (MISA) [56] with the minimum repeats of mono-, di-, tri-, tetra-, penta- and hexanucleotides set to 8, 5, 4, 3, 3 and 3, respectively.

In the codon usage analysis, only 53 protein-coding genes with lengths > 300 bp were chosen for synonymous codon using the tool CodonW1.4.2 to avoid sampling errors (http://codonw.sourceforge.net). The overall codon usage and the relative synonymous codon usage (RSCU) were analysed. The number of polymorphic sites and nucleotide variability (Pi) were evaluated using a sliding window with a 200 bp step size and a 600 bp window length implemented in DnaSP v.5.10.1 [57].

Comparative analysis of cp genomes

The plastomes of H.aitchisonii and H. tibetica were compared with the cp genomes of seven other Habenaria species from GenBank using Mauve 2.4.0. to identify evolutionary events such as gene loss, duplication, and rearrangement [58]. The junctions of the cp genomes were analysed with IRscope [59].

Molecular evolution analysis

To test the evolution imprinting, the protein-coding genes of nine species of Habenaria genus were analysed. The corresponding functional protein-coding genes were separately aligned using MAFFT [60], and the synonymous (Ks) and non-synonymous (Ka) substitution rates, as well as the Ka/Ks value were subsequently calculated using the Ka/Ks_calculator 2.0 [61] with the settings of genetic code Table 11 (bacterial and plant plastid code) and Nei and Gojobori method of calculation. Genes with Ka/Ks values of “NA” in the results, were defined as not applicable, the results occurred when Ks = 0, this happened (in cases with no substitutions in the alignment, or 100% match). To further test the positively selected genes, genes (Ka/Ks > 1) of nine Habenaria genus and corresponding genes from Anoectochilus emeiensis, Anoectochilus zhejiangensis, Goodyera foliosa, Goodyera fumata, Goodyera procera, Goodyera rosulacea, Goodyera velutina, Ludisia discolor, and Vanilla planifolia were employed and genes in Ludisia discolor, and Vanilla planifolia as outgroup, the branch-site model was used in PAML software (http://abacus.gene.ucl.ac.uk/software/PAML.html).

Phylogenetic analysis

The cp genomes and coding-proteins of 50 Orchidaceae species were retrieved from the GenBank and used to construct a phylogenetic tree. The cp genomes of Iris dichotoma (Iridaceae) and Lycorris sanguinea (Amaryllidaceae) were used as the outgroup [22]. The two sets of sequences were aligned by MAFFT [60], and the alignments were subsequently adjusted by the Gblocks program [62]. The maximum likelihood (ML) method was employed to construct phylogenetic trees with RAxMLversion 8.0 software using the GTRGAMMA model [63]. Bootstrap analysis for each branch was performed with 1000 replications. We used the Markov Chain Monte Carlo(MCMC)tree program from the software package (burnin = 20,000, sampfreq = 10, nssample = 100,000), phylogenetic analysis by maximum likelihood (PAML) version 4.9j [64] to estimate divergence times on a fixed topology. Age calibration was constrained to the phylogeny of Habenaria and its close relatives and fossil selection was performed according to previous methods [45].

Supplementary Information

12870_2024_5766_MOESM1_ESM.pdf (287.2KB, pdf)

Supplementary Material 1: Figure S1. Codon content of 20 amino acid and the stop codon of 81 coding genes of Habenaria aitchisonii (a)and Habenaria tibetica(b).

12870_2024_5766_MOESM2_ESM.docx (16.8KB, docx)

Supplementary Material 2: Table S1. Accession numbers, quantity of raw data, and coverage depth of de novo assembled plastomes of Habenaria aitchisonii andHabenaria tibetica.

12870_2024_5766_MOESM3_ESM.xls (43KB, xls)

Supplementary Material 3: Table S2. Comparison of genes inHabenaria aitchisonii and Habenaria tibetica with genes in other Habenaria species.

12870_2024_5766_MOESM4_ESM.xls (3.6KB, xls)

Supplementary Material 4: Table S3. Codon usage and codon-anoticodon recognition patterns in Habenaria aitchisonii and Habenaria tibetica cp genomes.

12870_2024_5766_MOESM5_ESM.zip (15.3KB, zip)

Supplementary Material 5: Table S4 a Comparison of repeat sequences in Habenaria aitchisonii, and Habenaria tibetica. Table S4 b Distribution of SSRs in the Habenaria aitchisonii and Habenaria tibetica cp genomes.

12870_2024_5766_MOESM6_ESM.zip (16.3KB, zip)

Supplementary Material 6: Table S5. a Comparative of codon usage bias of selected 18 species of Orchidaceae. Table S5b Oligonucleotide analysis of the Habenaria cp genomes.

12870_2024_5766_MOESM7_ESM.txt (4KB, txt)

Supplementary Material 7: Table S6. Comparison of nucleotide variability (Pi) among Habenaria aitchisonii, Habenaria tibetica and related species.

12870_2024_5766_MOESM8_ESM.xlsx (36.2KB, xlsx)

Supplementary Material 8: Table S7. The Ka/Ks ratios of protein-coding genes of Habenaria aitchisonii, Habenaria tibetica and related species.

12870_2024_5766_MOESM9_ESM.docx (40.3KB, docx)

Supplementary Material 9: Table S8. Codon model analysis of ten cp genome genes.

12870_2024_5766_MOESM10_ESM.docx (20.2KB, docx)

Supplementary Material 10: Table S9. List of chloroplast genomes belonging to Orchidaceae used for the phylogenetic analysis

Acknowledgements

We acknowledge Qinghai Forestry and Grassland Administration for providing material collected for public interest research and Nanjing Genepioneer Biotechnologies, Inc. for sequencing. We thank Chuanbei Jiang, Yue Wang, and Mingxing Yang for software help. We also thanks for preprint organization for providing help with the manuscript (https://sciety.org/articles/activity/10.20944/preprints202406.0585.v1.) In addition, We thank Prof.Guo-mei Li, Mr.A-Qiong Gongsong, and Mr. Pengguo Lin for their help with the photograph and location guide. We also thank Lei Zhang, Yong-qing Li, Fu-hao Zhang, Peng-cuo Gama, Dong-peng He, and Cheng-qiang Qiao for their help in the materials collection.

Authors' contributions

Z-DW, L-PC, and C-WD conceived and designed the experiments. Z-JK, Z-SQ, W-M, W-H and S-SB analyzed the data. Y-X, M-J, and Z-YW participated in the material collection. Z-DW, F-M, Z-JK, S-SB and W-H prepared the manuscript and revised the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the foundation of Qinghai Forestry and Grassland Administration (QHBY-2021–004-01), the foundation of the Qinghai Science and Technology Department (2021-ZJ-734), Construction Project for Innovation Platform of Qinghai Province (2024-ZJ-T02) and the fund of the Institute of Biotechnology of Medicine Herb of Qinghai Nationalities University.

Data availability

The chloroplast genome sequences of H. aitchisonii and H. tibetica were submitted to the GenBank (SRA accession number: SRX25202832 and SRX25202831) and the accession numbers were: OQ701055 and OQ701056, respectively.

Sequence data that support the findings of this study have been deposited in NCBI. Habenaria aitchisonii Cp genome accession number:OQ701055 and raw data numer: SRX25202832; Habenaria tibetica Cp genome accession number: OQ701056 and raw data number: SRX25202831.

Declarations

Ethics approval and consent to participate

All the samples collected were permitted by the Qinghai Forestry and Grassland Administration.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

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

Supplementary Materials

12870_2024_5766_MOESM1_ESM.pdf (287.2KB, pdf)

Supplementary Material 1: Figure S1. Codon content of 20 amino acid and the stop codon of 81 coding genes of Habenaria aitchisonii (a)and Habenaria tibetica(b).

12870_2024_5766_MOESM2_ESM.docx (16.8KB, docx)

Supplementary Material 2: Table S1. Accession numbers, quantity of raw data, and coverage depth of de novo assembled plastomes of Habenaria aitchisonii andHabenaria tibetica.

12870_2024_5766_MOESM3_ESM.xls (43KB, xls)

Supplementary Material 3: Table S2. Comparison of genes inHabenaria aitchisonii and Habenaria tibetica with genes in other Habenaria species.

12870_2024_5766_MOESM4_ESM.xls (3.6KB, xls)

Supplementary Material 4: Table S3. Codon usage and codon-anoticodon recognition patterns in Habenaria aitchisonii and Habenaria tibetica cp genomes.

12870_2024_5766_MOESM5_ESM.zip (15.3KB, zip)

Supplementary Material 5: Table S4 a Comparison of repeat sequences in Habenaria aitchisonii, and Habenaria tibetica. Table S4 b Distribution of SSRs in the Habenaria aitchisonii and Habenaria tibetica cp genomes.

12870_2024_5766_MOESM6_ESM.zip (16.3KB, zip)

Supplementary Material 6: Table S5. a Comparative of codon usage bias of selected 18 species of Orchidaceae. Table S5b Oligonucleotide analysis of the Habenaria cp genomes.

12870_2024_5766_MOESM7_ESM.txt (4KB, txt)

Supplementary Material 7: Table S6. Comparison of nucleotide variability (Pi) among Habenaria aitchisonii, Habenaria tibetica and related species.

12870_2024_5766_MOESM8_ESM.xlsx (36.2KB, xlsx)

Supplementary Material 8: Table S7. The Ka/Ks ratios of protein-coding genes of Habenaria aitchisonii, Habenaria tibetica and related species.

12870_2024_5766_MOESM9_ESM.docx (40.3KB, docx)

Supplementary Material 9: Table S8. Codon model analysis of ten cp genome genes.

12870_2024_5766_MOESM10_ESM.docx (20.2KB, docx)

Supplementary Material 10: Table S9. List of chloroplast genomes belonging to Orchidaceae used for the phylogenetic analysis

Data Availability Statement

The chloroplast genome sequences of H. aitchisonii and H. tibetica were submitted to the GenBank (SRA accession number: SRX25202832 and SRX25202831) and the accession numbers were: OQ701055 and OQ701056, respectively.

Sequence data that support the findings of this study have been deposited in NCBI. Habenaria aitchisonii Cp genome accession number:OQ701055 and raw data numer: SRX25202832; Habenaria tibetica Cp genome accession number: OQ701056 and raw data number: SRX25202831.

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