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Comprehensive characterization and phylogenetic analysis of the complete plastomes of two ant–orchids, Caularthron bicornutum and Myrmecophila thomsoniana

BMC Plant Biology volume 24, Article number: 1146 (2024) Cite this article

Abstract

Background

Myrmecophytes, characterized by specialized structures like hollow stems that facilitate mutualistic relationships with ants, serve as an important system for studying ant-plant interactions and the adaptation mechanisms. Caularthron and Myrmecophila are exemplary myrmecophytes within Orchidaceae. Previous studies suggested a genetic relationship between these two genera, placing them within Laeliinae (Epidendreae), yet the precise phylogenetic positioning remained uncertain. The absence of available plastome resources has hindered investigations into plastome evolution and phylogeny.

Results

In this study, we sequenced and assembled the complete plastomes of Caularthron bicornutum and Myrmecophila thomsoniana to elucidate their plastome characteristics and phylogenetic relationships. The determined plastome sizes were 150,557 bp for C. bicornutum and 156,905 bp for M. thomsoniana, with GC contents of 37.3% and 37.1%, respectively. Notably, M. thomsoniana exhibited a distinctive IR expansion and SSC contraction, with the SSC region measuring only 4532 bp and containing five genes (ccsA, ndhD, rpl32, psaC, and trnL-UAG), a unique feature observed for the first time in Epidendreae. Comparative analyses with species from the related genus Epidendrum revealed that C. bicornutum plastome exhibited conserved genome size, GC content, gene content, and gene order. A total of 32 and 33 long sequence repeats, 50 and 40 tandem repeats, and 99 and 109 SSRs were identified in the plastomes of C. bicornutum and M. thomsoniana, respectively. The RSCU analysis demonstrated a consistent pattern in both plastomes, with 29 out of 30 codons with RSCU values greater than 1 featuring A/U at the third codon position. Leucine was the most prevalent amino acid, while Cysteine was the least common. Four potential DNA barcoding regions with Pi values exceeding 0.07, namely ycf1, ccsA–psaC, petN–psbM, and accD–psaI, were identified for subsequent phylogenetic reconstructions within Laeliinae. Phylogenetic analysis underscored the close relationships among Caularthron, Epidendrum, and Myrmecophila.

Conclusions

This study represents the first comprehensive analysis of the plastome characteristics of Caularthron bicornutum and Myrmecophila thomsoniana. Through our characterization and phylogenetic analyses, we unveiled the unique IR expansion/SSC contraction and further elucidated their phylogenetic positions. Our research contributes significant data and insights into the dynamic evolution of ant–orchid plastomes and the phylogeny of the Laeliinae.

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Background

Myrmecophytes (ant–plants) exhibit specialized structures such as enlarged stems or leaf sheaths that provide nesting spaces for symbiotic relationships with ants [1, 2]. These plants are believed to offer domatia to house ants in exchange for protection, nutrients, and the elimination of competitors [3]. Ant–plants are predominantly found in families such as Asclepiadaceae, Bromeliaceae, Orchidaceae, Rubiaceae, and ant-associated ferns [4]. Among these myrmecophytes are epiphytic species like Dischidia (Apocynaceae) with domatia formed by folded leaves [5], Lecanopteri (Polypodiaceae) with hollow rhizomes [6], and Myrmecodia (Rubiaceae) characterized by prominent caudex and natural cavities [7]. Caularthron Raf. (1837) and Myrmecophila Rolfe (1917) are classic examples of myrmecophytes within the Orchidaceae [8]. These two myrmecophytic genera forego part of water storage tissues to develop hollow pseudobulbs suitable for ant nesting [9]. Moreover, ant waste serves as a significant nutrient source for epiphytic Caularthron bilamellatum, facilitating nitrogen transfer from ant waste to orchid tissues through fungal hyphae [10].

Ant–orchids (myrmecophytic orchids) may derive some nutrients from ant waste, but photosynthesis plays a central role in biomass accumulation. The plastid, with its distinct genomic DNA known as the plastid genome (plastome), plays a crucial role in the photosynthetic process [11]. Plant plastomes typically exhibit a highly conserved quadripartite structure consisting of a large single copy (LSC) region, a small single copy (SSC) region, and a pair of inverted repeat (IR) regions [12, 13]. The plastome contains approximately 120 conserved genes that are vital for plant viability as they encode components for photosynthesis and plastid gene expression [14]. To date, nearly 13,000 complete plastome sequences have been reported and utilized in various studies [11, 12]. Phylogenetic reconstructions within the Orchidaceae using plastome sequences have showcased the utility of plastomes and offered insights into the evolutionary history of the Orchidaceae [15,16,17]. Nevertheless, no plastome sequence of ant–orchids have yet been reported.

Laeliinae, a subtribe within the Epidendreae, is renowned for its species diversity and horticultural value, encompassing well-known genera such as Cattleya, Laelia, Epidendrum, and other popular flowers. It is mainly found across subtropical regions of the Americas and the Caribbean [18]. The majority of Laeliinae genera are epiphytic or rupicolous, including two myrmecophytic genera, Caularthron and Myrmecophila [19]. Caularthron comprises four species indigenous to tropical America. Its growth habit resembles that of reed-stem Epidendrum species, with only the uppermost leaves remaining on the stem, which swells into hollow pseudobulbs that serve as habitats for ants. Unlike other Epidendrum-related genera, the lip of Caularthron is not distinctly fused to the column, and the peculiar calli on the lip differ from those of other Laeliinae [18]. Myrmecophila consists of nine species found from Mexico southwards to northern Costa Rica, with disjunct species in coastal Venezuela and the Cayman Islands. Initially grouped within the broadly defined Schomburgkia Lindl. due to characteristics such as elongated inflorescence peduncles, eight pollinia, and twisted sepals and petals. Myrmecophila was later proposed by Rolfe to encompass four species that deviated from the typical Schomburgkia morphology. Despite this proposal, the classification was not widely accepted, leading to various taxonomic revisions over time [20].

Phylogenetic reconstructions utilizing combined internal transcribed spacer (ITS) and plastid data (matK, trnK, and trnL-F) had provided a fundamental phylogenetic framework of Laeliinae [21, 22]. These analyses indicate that Caularthron belongs to the Epidendrum alliance. However, the placement of Myrmecophila remains ambiguous. ITS data suggest its affinity with the Cattleya alliance, while combined analyses position it within the Epidendrum alliance and closest to Caularthron [21, 22]. Further investigations are warranted to resolve its taxonomic placement and elucidate the evolution of myrmecophily within Myrmecophila and Caularthron.

Caularthron and Myrmecophila, serving as representative examples of ant–orchids, are two related genera within Laeliinae. Their phylogenetic positions warrant further exploration, and plastome sequences have demonstrated their effectiveness in phylogenetic reconstructions within Orchidaceae. However, representative plastome sequences of these genera have been lacking. In this study, we have assembled and analyzed the complete plastome sequences of Caularthron bicornutum and Myrmecophila thomsoniana with the aim of: (1) revealing the plastome characteristics and evolution of Caularthron and Myrmecophila; (2) identifying potential mutation hotspots within Laeliinae using plastome data; and (3) investigating the phylogenetic positions of Caularthron and Myrmecophila to gain insights into the phylogeny of Laeliinae.

Results

Basic plastome characteristics

The complete plastomes of Caularthron bicornutum and Myrmecophila thomsoniana were successfully assembled with average depth of 7865.09× and 7926.55×, respectively (Fig. S1). The genome sizes of C. bicornutum and M. thomsoniana were determined to be 150,557 bp and 156,905 bp, with GC contents of 37.3% and 37.1%, respectively. Both plastomes exhibited a typical quadripartite structure, consisting of a large single copy (LSC) region, a small single copy (SSC) region, and two inverted repeat (IR) regions (Fig. 1). In the plastome of C. bicornutum, the LSC, SSC, and IR regions measured 86,166 bp, 12,297 bp, and 26,047 bp, with GC contents of 34.9%, 28.7%, and 43.3%, respectively. For M. thomsoniana, the corresponding lengths were 85,855 bp (LSC), 4,537 bp (SSC), and 33,259 bp (IR), with GC contents of 34.9%, 31.3%, and 40.3%, respectively. A total of 130 genes were identified in the C. bicornutum plastome, comprising 84 protein-coding genes (PCGs), 30 transfer RNA genes (tRNAs), and 8 ribosomal RNA genes (rRNAs) (Table 1). Among these, 20 genes, including 8 PCGs, 8 tRNAs, and 4 rRNAs, were found to be duplicated. Furthermore, 17 genes contained one intron, and the clpP and ycf3 genes harbored two introns. The plastome of M. thomsoniana contained 134 genes, with an additional four duplicated genes compared to C. bicornutum, namely ndhE, ndhH, rps15, and ycf1.

Fig. 1
figure 1

Annotation maps of C. bicornutum (A) and M. thomsoniana (B) plastomes. The darker gray in the inner circle corresponds to the GC content. The IRA and IRB (two inverted repeating regions); LSC (large single-copy region); and SSC (Small single-copy region) are indicated outside of the GC content

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Table 1 Annotated genes in C. bicornutum plastome. (×2) indicates gene was duplicated, * indicates gene with one intron and ** indicates gene with two introns
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To determine the gene order and structural organization of these two plastomes, comparison analyses were conducted with five Epidendrum species (including E. avicula, E. ciliare, E. diffusum, E. eburneum, and E. porpax), which represent other complete plastomes of the subtribe Laeliinae. The mauve visualization results revealed IR expansion and SSC contraction in the M. thomsoniana plastome, with no gene rearrangements detected among these plastomes (Fig. 2). Additionally, the results of the IR boundary map also indicated the IR expansion and SSC contraction patterns (Fig. 3). The IR boundaries of C. bicornutum were generally consistent with those of the Epidendrum species, while the IR boundaries of the M. thomsoniana plastome were notably different due to IR expansion. The JLB (LSC/IRb) boundaries were located at rpl22 gene among testing plastomes, no significant difference was observed. The JLA (LSC/IRa) boundary was identified between the rps19 and psbA genes. The JSA (SSC/IRa) boundary was situated to the right of the ndhE gene with a distance of 328 bp, while the JSB (SSC/IRb) boundary was located between the rpl32 and ndhE genes (Fig. 3).

Fig. 2
figure 2

Mauve alignment of seven Laeliinae plastomes, where blocks of the same color connected by lines indicate local collinear blocks within each alignment

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Fig. 3
figure 3

Comparison of junctions between the LSC, SSC, and IR regions among seven Laeliinae plastomes

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Sequence repeats

Long sequence repeats, including complementary (C), forward (F), palindrome (P), and reverse (R) types, were detected in both plastomes. A total of 32 and 33 long sequence repeats ranged from 30 to 57 bp were identified in the plastomes of C. bicornutum and M. thomsoniana, respectively (Fig. 4A and B, Table S1). For C. bicornutum, the plastome contained F and P types of long sequence repeats, and for M. thomsoniana, F, P, and R types were observed. No C type sequence repeats were found in either plastome. Long repeat sequences ranged from 30 to 39 bp were most common, and no sequence repeats in the range of 40–49 bp were observed in the M. thomsoniana plastome (Fig. 4A and B, Table S1). A total of 50 and 40 tandem sequence repeats were identified in the plastomes of C. bicornutum and M. thomsoniana, respectively (Table S2). The distribution and length range of these repeats are summarized in Fig. 4D and E. Furthermore, 99 and 119 simple sequence repeats (SSRs) were identified in the plastomes of C. bicornutum and M. thomsoniana, respectively (Fig. 4F and G, Table S3). The LSC regions were found to contain the highest number of SSRs, with 69 and 73 occurrences. The IR region of C. bicornutum and the SSC region of M. thomsoniana exhibited the least occurrences of SSRs. Mononucleotide SSRs were the most abundant type in both plastomes. The pentanucleotide SSRs were absent in C. bicornutum, while hexanucleotide SSRs were lacking in M. thomsoniana (Fig. 4F and G, Table S3).

Fig. 4
figure 4

Summary of sequence repeats across the two newly assembled plastomes. (A) The number of each of four long sequence repeat types (C, complement; F, forward; P, palindrome; and R, reverse). (B) The number of long sequence repeats of different lengths. (C) The distribution of tandem sequence repeats in LSC/SSC/IR region. (D) The number of tandem sequence repeats of different lengths. (E) The distribution of SSRs in LSC/SSC/IR region. (F) The number of SSRs containing one- to six-nucleotide motifs

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Relative synonymous codon usage

The relative synonymous codon usage (RSCU) of 70 unique protein-coding genes (PCGs) excluding the ndh genes in the two plastomes was calculated and illustrated in Fig. 5 and Table S4. These genes were encoded by 19,659 and 19,627 codons and displayed a highly conserved codon usage bias (CUB). Leucine (Leu) emerged as the most abundant amino acid, occurring 1977 and 1974 times, and Cysteine (Cys) was the least common, appearing 217 and 218 times. The RSCU analysis revealed that AGA (encoding arginine, Arg) had the highest RSCU values, at 1.9219 and 1.9008, whereas AGC (encoding serine, Ser) displayed the lowest RSCU values, at 0.3221 and 0.3320. Among these codons, 30 exhibited RSCU values above 1, while 33 had values below 1. Additionally, the RSCU values for AUG (encoding methionine, Met) and UGG (encoding tryptophan, Trp) were both determined to be 1.

Fig. 5
figure 5

Codon usage biases of C. bicornutum (left) and M. thomsoniana (right) plastomes

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Plastome variation hotspot

The mVISTA online tool was utilized to investigate sequence variation hotspot regions, using Epidendrum eburneum (OR460025) as the reference sequence (Fig. S3). In the plastome of M. thomsoniana, one duplicated region (ndhE–ycf1) was removed due to IR expansion and SSC contraction. The results indicated a high level of conservation across the plastomes, and the IR regions showed higher conservation compared to the LSC and SSC regions. Subsequently, nucleotide diversity (Pi) was calculated using the plastomes of nine Laeliinae species, which included two Cattleya species and five Epidendrum species. The Pi values ranged from 0 to 0.08194 (Fig. 6A, Table S5). Four highly variable regions were identified, namely ycf1 (Pi = 0.08194), ccsA–psaC (Pi = 0.08056), petN–psbM (Pi = 0.07417), and accD–psaI (Pi = 0.07111) (Fig. 6A). The alignment lengths and Pi values of the 68 common PCGs among the nine plastomes were summarized in Supplementary Table 6. The genes ycf2 (6957 bp), ycf1 (5699 bp), and rpoC2 (4176 bp) exhibited the longest alignments. Among these, the most variable PCGs were ycf1, rps15, rpl20, rpl16, and ccsA, with Pi values of 0.02471, 0.01720, 0.01713, 0.01688, and 0.01588, respectively (Fig. 6B, Table S6).

Fig. 6
figure 6

The nucleotide diversity (Pi) of Laeliinae plastomes and 68 protein-coding sequences. (A) For the nucleotide diversity of the complete plastome using a sliding window test, four mutation hotspot regions were annotated. The window size was set to 100 bp and the sliding windows size was 25 bp. X-axis, the position of the midpoint of a window; Y-axis, Pi values of each window. (B) The nucleotide diversity of 68 protein-coding sequences. The bar indicates gene lengths, the line indicates Pi values

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Phylogenetic analysis

A total of 60 complete plastome sequences, comprising 53 Epidendreae species and 7 outgroup species, were utilized to conduct phylogenetic analyses and elucidate the phylogenetic positions of C. bicornutum and M. thomsoniana (Fig. 7, Table S7). To improve the quality and accuracy of alignment matrix, the matrix was trimmed after initial aligned. The trimmed alignment matrix consisted of 99,473 bp, with 12,795 variable sites and 5,954 parsimony-informative sites. Phylogenetic trees were constructed using maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI) methods, yielding mostly consistent topologies. The analysis resolved five subtribes with high support, including Agrostophyllinae, Calypsinae, Bletiinae, Laeliinae, and Pleurothallidinae (BSML = 94, BSMP = 97, PP = 1.00). It was determined that both C. bicornutum and M. thomsoniana belonged to the subtribe Laeliinae (Fig. 7). The phylogenetic relationships within the subtribe Laeliinae were deciphered as (Cattleya (Myrmecophila (Caularthron, Epidendrum))) with high support (BSML ≥ 94, BSMP ≥ 97, PP = 1.00).

Fig. 7
figure 7

The phylogenetic tree of 53 Epidendreae species and 7 outgroup species obtained by maximum-likelihood analysis based on complete plastome sequences. The numbers near the nodes are bootstrap percentages and Bayesian posterior probabilities (BPML, BPMP, PP); * the node is the 100 bootstrap percentage or 1.00 posterior probability

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Discussion

Plastome characteristics and structure

In this study, we determined the complete plastome sequences of two epiphytic ant–orchids, Caularthron bicornutum and Myrmecophila thomsoniana, with lengths of 150,557 bp and 156,905 bp, respectively, and GC contents of 37.3% and 37.1%. These findings are consistent with the range of plastome sizes observed in other members of tribe Epidendreae. The diversity of lifestyles (terrestrial, epiphytic, and heterotrophic) among Epidendreae species has been a contributing factor to the variation in plastome sizes [23]. Plastome sizes in this tribe have been reported to range from 43,091 bp (Yoania prainii, heterotrophic) to 159,493 bp (Cremastra appendiculata, terrestrial), while GC contents have shown variation from 28.7% (Yoania squamipes, heterotrophic) to 37.4% (Tipularia josephi, terrestrial) [24].

Compared to closely related Epidendrum plastomes, which range in size from 147,902 bp to 150,986 bp [25], M. thomsoniana (156,905 bp) shows notable expansion due to structural variations within its plastome. This expansion is primarily attributed to a unique IR expansion and SSC contraction, resulting in an SSC region measuring only 4532 bp and containing five genes (ccsA, ndhD, rpl32, psaC, and trnL-UAG) (Fig. 1). This phenomenon was initially identified in tribe Epidendreae and has since been documented in other lineages within the Orchidaceae, including Hetaeria (Orchidoideae), Paphiopedilum (Cypripedioideae), Pogonia (Vanilloideae), and Vanilla (Vanilloideae) [26]. While the plant plastomes are generally conserved, similar plastome variations have been observed across diverse groups, such as in the subfamily Apioideae (Apiaceae) [27, 28], Cistanche (Orobanchaceae) [29], and Euphorbia (Euphorbiaceae) [30]. However, the biological drivers underlying this SSC contraction, as well as the possibility of similar contractions in other Myrmecophila species, warrant further investigation. Moreover, despite the unique SSC contraction leading to different IR boundaries in the M. thomsoniana plastome, comparative analyses revealed a conserved gene order (Figs. 2 and 3). The plastome of C. bicornutum has lost the ndh A/D/F/G/I/K genes, while the plastome of M. thomsoniana lacks the ndh A/F/G/I genes. This diversity in ndh gene loss aligns with previous findings in Epidendrum [25]. The loss of ndh genes is common in orchids, as observed in Angraecum [31] and Chiloschista [32], and it may be linked to their epiphytic lifestyle, though ndh gene loss may vary among different species.

Plastome evolution and candidate DNA barcodes

The identification of sequence repeats plays a significant role in the recognition of plant germplasm resources [33]. Despite variations in plastome lengths and gene numbers between the two species, the sequence repeats exhibited a generally similar pattern, with differences in distribution within the SSC/IR regions (Fig. 4). Our results indicated that this distributional difference is related to the size of the SSC and IR regions. Codon usage patterns serve as indicators of molecular evolution, with codon usage bias (CUB) playing a crucial role in providing insights into molecular evolution processes [34]. In this study, the relative synonymous codon usage (RSCU) values were mostly consistent for a single or two codons encoding the same amino acid, while displaying variations in most other codons (Fig. 5). Among the 30 codons with RSCU values greater than 1 in the two plastomes, 29 codons featured A/U at the third codon position, a trend commonly observed in the plastomes of many angiosperms due to their high AT contents [35]. Leucine (Leu) was identified as the most prevalent amino acid, while cysteine (Cys) was the least common, a finding consistent with observations in the related genus Epidendrum [25]. The high frequency of Leu may be related to its critical function in photosynthesis-related metabolism [36], and this similar frequency pattern has also been observed in other groups [37,38,39].

Consistent with findings in other orchid studies, plastome mutational hotspots have proven to be practical regions for DNA barcoding development and exploring plastome evolution [33, 34]. Following this rationale, we conducted nucleotide diversity analyses of the complete plastome and coding genes to identify potential DNA barcodes for phylogenetic analysis within the subtribe Laeliinae. Our results indicated that the IR regions exhibited higher conservation compared to the LSC and SSC regions, aligning with previous research [32, 40, 41]. Comparative with previous studies on Epidendrum plastomes revealed similar levels of nucleotide diversity in coding genes [25]. We identified four highly variable regions (ycf1, ccsA–psaC, petN–psbM, and accD–psaI; Pi > 0.07) as promising candidate DNA barcodes. Interestingly, our findings differed from the barcode identification in Epidendrum species, suggesting that the candidate DNA barcodes selected for Epidendrum may not be suitable for the broader Laeliinae group [25]. The Laeliinae subtribe comprises approximately 38 genera [19, 42], yet to date, plastome sequences have only been published for Epidendrum and Cattleya. While our study analyzed the plastomes of two additional genera, further plastome data from additional genera are needed to more accurately identify the most suitable DNA barcodes.

Phylogenetic inference and taxonomic implication

Since Bentham established the subtribe Laeliinae in 1881 [43], various taxonomists have proposed different classifications based on diverse morphological characteristics, such as the presence of a column foot, column structure, and the number of pollinia [44,45,46]. However, these classifications have been criticized as highly artificial in some aspects, emphasizing specific morphological features and being found to be unnatural [22]. A comprehensive phylogenetic analysis within Laeliinae, utilizing combined internal transcribed spacer (ITS) and plastid (matK, trnK, and trnL-F) data, strongly indicated a close relationship between Caularthron and Epidendrum [22]. The phylogenetic positions of Myrmecophila showed conflicting results when inferred from separate ITS and plastid regions; however, the combined data placed Myrmecophila at the base of the Epidendrum alliance and closely related to Laelia. This placement was considered reasonable by Van den Berg et al., as the hollow pseudobulbs that host ant nests in Myrmecophila are related to Caularthron, while the long stems and similar flower morphology are reminiscent of Laelia [47]. In the present study, we reconstructed the phylogenetic relationships of Epidendreae using complete plastome sequences and employing ML, MP, and BI methods (Fig. 7). Our analysis revealed close relationships among Caularthron, Epidendrum, and Myrmecophila. However, the accurate phylogenetic position of Myrmecophila could not be confidently confirmed due to limitations in sampling, particularly the lack of Laelia. We propose Myrmecophila belongs to the Laelia alliance, suggesting that previous phylogenetic reconstructions using various molecular markers lacked sufficient genetic information [22]. Species within the Laeliinae are of exceptional horticultural value and possess important breeding capacities. However, there are limited plastome sequences available, with only a few including two Cattleya species (incomplete) and five Epidendrum species. Here, we present newly sequenced plastomes of two genera, which will serve as valuable resources for further research.

Materials and methods

Sample sampling and sequencing

Plant materials from two ant–orchid species were collected from the Forest Orchid Garden greenhouse at Fujian Agriculture and Forestry University, Fuzhou, Fujian Province, China. The species were formally identified by Dr. Jun-Wen Zhai and Prof. Zhong-Jian Liu. Voucher specimens have been deposited in the Herbarium of Fujian Agriculture and Forestry University, with deposition numbers MHLi or159 (C. bicornutum) and MHLi or143 (M. thomsoniana). The corresponding GenBank accession numbers for these species are PQ279222 and PQ279223. In total, 60 species representing 35 genera were analyzed alongside publicly available plastome data. Seven species from seven genera (Calanthe, Cymbidium, Dendrobium, Eria, Neottia, Thunia, and Vanda) were selected as outgroup taxa (Table S7).

Total genomic DNA was extracted from fresh leaves using the Plant Mini Kit (Qiagen, CA, USA) following the manufacturer’s protocol. DNA quality and integrity were assessed via 1% agarose gel electrophoresis to check for degradation and contamination. Library preparation was carried out using the HiSeq 3000 PE Cluster Kit (Illumina) and clustered on a cBot Cluster Generation System in accordance with the manufacturer’s instructions. Sequencing was performed on the Illumina HiSeq 4000 platform, generating 150-bp paired-end reads. Custom scripts were employed to filter the Illumina data during clustering (default parameters: -L 5, -p 0.5, -N 0.1). Paired-end reads were discarded if more than 50% of the bases had a quality score of Q ≤ 5 or if the N content exceeded 10% of the total base number.

Plastome assembly and annotation

Paired-end reads were processed using the GetOrganelle pipeline (https://github.com/Kinggerm/GetOrganelle) [48] for filtering. The filtered reads were subsequently assembled using SPAdes v3.10 [49]. Plastid contigs were refined from the final ‘fastg’ files using the GetOrganelle script, and De Bruijn graphs were analyzed and corrected with Bandage [50] to produce circular sequences. Sequencing depth and coverage were calculated and visualized following the ‘Generating Sequencing Depth and Coverage Map for Organelle Genomes’ protocol (https://doi.org/10.17504/protocols.io.4r3l27jkxg1y/v1) [51].

The newly assembled plastomes were annotated using GeSeq [52], and tRNA gene annotations were validated with tRNAscan-SE v2.0.3 [53]. Protein-coding gene annotations from GeSeq were manually verified and corrected for start and stop codons in Geneious R11.1.5 [54]. The translation of each protein-coding gene was also cross-checked using Geneious R11.1.5 [54]. The plastome annotation file was prepared using GB2Sequin [55] and submitted to GenBank at the National Center for Biotechnology Information (NCBI) under unique accession numbers. Annotated plastomes were illustrated with circle diagrams generated by OGDRAW [56].

Characterization and comparative analysis

To identify rearrangements in Laeliinae plastomes, Mauve software (https://gel.ahabs.wisc.edu/mauve) [57] was used. Visual analysis and evaluation of LSC/IR/SSC boundary differences across five Epidendrum plastomes were performed using the CPJSdraw 0.0.1 online R Shiny application [58]. Tandem repeats were identified with Tandem Repeats Finder v4.09 [59] using default parameters. Four types of long repeats—forward (F), palindrome (P), reverse (R), and complement (C)—were detected with REPuter software [60] under default settings. Simple sequence repeats (SSRs) were identified using the Perl script MISA [61], with the minimum repeat thresholds set to 10 for mono-, 5 for di-, 4 for tri-, and 3 for tetra-, penta-, and hexa-nucleotide motifs. The relative synonymous codon usage (RSCU) was analyzed using DAMBE 7 [62]. The results were visualized using ChiPlot Online Tools (https://www.chiplot.online) [63].

Sequence variation hotspots and phylogenetic analysis

Plastome diversity was analyzed using the mVISTA online program, employing the Shuffle-LAGAN alignment algorithm [64]. Protein-coding genes (PCGs) were extracted with PhyloSuite v1.2.2 [65] and aligned using MAFFT7 [66]. Nucleotide diversity (Pi) across the eight plastomes was calculated using DnaSP 6 [67], with a window length of 100 bp and a step size of 25 bp.

For phylogenetic analysis, whole plastome sequences were aligned using MAFFT [64] and trimmed with trimAl v1.2 [68] using the heuristic (-automated1) approach to reduce errors from poor-quality alignments. Phylogenetic analyses were conducted on the CIPRES Science Gateway platform (RaxML-HPC2 on XSEDE 8.2.12, PAUP on XSEDE 4.a168, and MrBayes on XSEDE 3.2.7) [69], utilizing maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI) methods.

For MP analysis, 1000 tree-bisection-reconnection (TBR) searches were performed with unlimited MAXTREES using PAUP [70], employing a heuristic search with 1000 random sequence additions and TBR branch swapping, treating all characters as equally weighted and unordered. ML analysis was conducted with 1000 bootstrap replicates using the GTRCAT model [71]. Bayesian inference was performed with the GTR + I + Γ substitution model in MrBayes v3.2.7 [72], running the Markov chain Monte Carlo (MCMC) algorithm for 10,000,000 generations, sampling trees every 100 generations. The first 25% of trees were discarded as burn-in to generate a majority-rule consensus tree and estimate posterior probabilities (PP).

Conclusions

In this study, we successfully obtained the complete plastomes of two ant–orchids belonging to Laeliinae, namely Caularthron bicornutum and Myrmecophila thomsoniana, and conducted comprehensive characterization and phylogenetic analysis. The sizes and GC contents of the plastomes were found to be consistent with the ranges observed in Epidendreae species. Notably, the ndh genes had undergone independent loss in the two plastomes. Our results revealed a unique IR expansion and SSC contraction in the plastome of M. thomsoniana. We have identified potential DNA barcode regions, namely ycf1, ccsA–psaC, petN–psbM, and accD–psaI, which can serve as valuable resources to facilitate research on Laeliinae. Through phylogenetic analysis based on the available data, we further explored the phylogenetic positions of the two ant–orchids and confirmed the close relationship among Caularthron, Epidendrum, and Myrmecophila. These discoveries enhance our understanding of the characteristics and evolution of Laeliinae plastomes. Future research endeavors can leverage these findings to delve deeper into the evolutionary relationships of plastomes within the Laeliinae and related taxa.

Data availability

The two plastomes generated in this study are available in NCBI (https:// www.ncbi.nlm.nih.gov) with accession numbers PQ279222 and PQ279223. The raw data has been submitted to the Genome Sequence Archive (https://ngdc.cncb.ac.cn/gsa/) of the National Genomics Data Center database (BioSample: SAMC4158175 and SAMC4158176; BioProject: PRJCA030575; SRA: CRA019286). Voucher specimens were identified by Dr. Jun-Wen Zhai and Prof. Zhong-Jian Liu (Fujian Agriculture and Forestry University), deposited in the Herbarium of Fujian Agriculture and Forestry University with deposition numbers (MHLi or159 and MHLi or143).

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Funding

This work was supported by the National Key Research and Development Program of China (2023YFD1600504), the Construction and Management Program of the Research Center for the Protection and Utilization of Orchids in Motuo County, Xizang Autonomous Region, China (KH230350A), and the Research and Innovation Team for the Development and Application of Landscaping Plant Resources at Zhangzhou Institute of Technology (zzyt23008).

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  1. Jin-Wei Li and Ru-Yi Li contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of National Forestry and Grassland Administration for Orchid Conservation and Utilization, College of Landscape Architecture, Fujian Agriculture and Forestry University, Fuzhou, 350002, China

    Jin-Wei Li, Ru-Yi Li, Yu-Ming Chen, Yu-Han Wu, Shu-Ling Tang & Jun-Wen Zhai

  2. Zhangzhou Institute of Technology, Zhangzhou, 363000, China

    Shu-Ling Tang

  3. State Key Laboratory of Subtropical Silviculture, Bamboo Industry Institute, Zhejiang Agriculture and Forestry University, Hangzhou, 311300, China

    Long-Hai Zou

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  1. Jin-Wei Li
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  6. Shu-Ling Tang
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Contributions

Conceptualization, J-W L., S-L T., J-W Z.; validation, R-Y L., Y-M C., Y-H W., L-H Z.; data curation, R-Y L., J-W L., Y-M C.; writing—original draft preparation, J-W L. and R-Y L.; writing—review and editing, S-L T. and J-W Z.; funding acquisition, S-L T. and J-W Z. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Shu-Ling Tang or Jun-Wen Zhai.

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The plant materials used in the study were collected under permission. The collection of plant materials and use comply with relevant institutional, national, and international guidelines and legislation. This article does not contain any studies with human participants or animals and does not involve any endangered or protected species.

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12870_2024_5827_MOESM1_ESM.jpg

Supplementary Material 1: Overall coverage depth of the plastome assembly of C. bicornutum (A) and M. thomsoniana (B). The horizontal coordinate is the position of the chloroplast genome, and the vertical coordinate is the sequencing depth.

12870_2024_5827_MOESM2_ESM.jpg

Supplementary Material 2: Global alignment of the two newly assembled and other two Epidendrum plastomes using mVISTA with Epidendrum eburneum (OR460025) as reference. The y-axis shows the coordinates between the plastomes. The red boxes mean high variation regions in plastome sequence

12870_2024_5827_MOESM3_ESM.xls

Supplementary Material 3: Table S1 The statistics of identified long repeat sequences. Table S2 The statistics of identified tandem sequence repeats. Table S3 The statistics of identified SSRs. Table S4 The RSCU values of all 64 codons for the two newly assembled plastomes. Table S5 Nucleotide diversity among seven Laeliinae plastomes. Table S6 Nucleotide diversity of the PCGs of seven Laeliinae plastomes. Table S7 The organism and accession number obtained from NCBI in the phylogenetic analysis.

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Li, JW., Li, RY., Chen, YM. et al. Comprehensive characterization and phylogenetic analysis of the complete plastomes of two ant–orchids, Caularthron bicornutum and Myrmecophila thomsoniana. BMC Plant Biol 24, 1146 (2024). https://doi.org/10.1186/s12870-024-05827-6

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