To examine structural variation among the plastomes of Dipodium, whole plastome alignments were generated using MAFFT (v7.388; Katoh et al., 2002; Katoh and Standley, 2013) implemented in Geneious Prime with full annotations. Alignments were manually checked, in cases of divergent regions, e.g., the operon region of ndhC, ndhK, and ndhJ genes or junctions between the large single copy (LSC)/inverted repeat B (IRB)/SSC/inverted repeat A (IRA) regions; respective regions (including annotations) were extracted in Geneious Prime, separately aligned, proofread, and subsequently visualised using OGDRAW (ver. 1.3.1, Greiner et al., 2019).

To classify the level of degradation of plastid genes in Dipodium, we used the following categories. 1) Functional: The reading frame was intact, and less than 10% of the open reading frame was disrupted by small indels. 2) Moderately pseudogenised: Less than 10% of the open reading frame was disrupted by internal stop codons or indels, causing non-triplet frameshifts. 3) Severely pseudogenised: More than 10% of the open reading frame was disrupted by internal stop codons, large deletions (>10%), or non-triplet frameshifts (based on Barrett et al., 2019). 4) Lost: The gene was not identified in the annotation process of the de novo assembly (e.g., Joyce et al., 2018) and/or was not detectable within the reference-guided assembly. A gene was considered as not detectable within the reference-guided mapping process if at least 70% of the gene sequence could not be identified for calculation of the consensus sequence within the Geneious mapping process. The coded matrix of gene degradation was plotted against the maximum likelihood phylogenetic tree of Dipodium.

Plastome sizes of Dipodium ranged from 142,949 bp (D. variegatum) to 152,956 bp (D. aff. roseum 3) ( Table 2 , Figure 5 ; Supplementary Material 7 ). The largest average plastome size (150,578 bp) was found in the D. roseum complex, closely followed by the leafy D. ensifolium (150,084 bp) and the D. punctatum complex (149,512 bp). Plastome sizes within the D. stenocheilum complex were markedly lower with an average size of 146,305 bp. Similarly, small plastomes were also found in D. hamiltonianum (145,902 bp), D. interaneum (146,497 bp), and the leafy climber D. pandanum 1 (sect. Leopardanthus) (146,204 bp) ( Table 2 ). Dipodium plastomes possess the typical quadripartite structure of angiosperms, with the SSC region ranging from 12,039 bp (D. variegatum) to 16,756 bp (D. ensifolium), the LSC region ranging from 81,514 bp (D. stenocheilum 2) to 83,172 bp (D. punctatum), and the pair of IRs ranging from 24,436 bp (D. variegatum) to 26,817 bp (D. aff. roseum 3) ( Table 2 ).

Total mean GC content of Dipodium plastomes was 36.9%, ranging between 36.8% (D. roseum 2 and D. pardalinum 1) and 37.1% (D. hamiltonianum) ( Table 2 ). Within the D. roseum complex, the GC content was 36.8%–36.9%, followed by the D. punctatum complex (36.9%) and D. stenocheilum complex (37.0%), and the highest GC content was 37.1% and 37.0% (D. hamiltonianum and D. interaneum, respectively) ( Table 2 ).

The plastid genes of each plastome were rated as functional, moderately to severely pseudogenised, or physically lost. The total number of functional genes in Dipodium plastomes ranged slightly from 119 to 121, including a total of 73 or 74 functional protein-coding sequence regions [protein-coding sequence (CDS)] (68 or 69 unique CDS), 37 to 39 functional tRNA genes (30 or 31 unique tRNA genes), and eight rRNA genes (four unique rRNA genes) ( Table 2 ).

The IR region was largely conserved among all examined Dipodium plastomes. All species showed six duplicated coding regions in the IRs (i.e., rpl2, rpl23, rps7, rps12, rps19, and ycf2) and all four rRNA genes ( Table 3 ). Most plastomes showed eight duplicated tRNA genes in the IR regions with the exception of the plastomes of D. interaneum and D. elegantulum, which comprised a duplicated trnV-GAC within the IRB, and the plastomes of D. ammolithum, D. hamiltonianum, and D. stenocheilum 2, which contained a duplicated trnV-GAC within the IRA ( Table 3 , Figures 5B, C ). All plastomes contained 16 functional intron genes (i.e., atpF, clpP, petB, petD, rpl2, rpl16, rpoC1, rps12, rps16, trnA-UGC, trnG-UCC, trnI-GAU, trnK-UUU, trnL-UAA, trnV-UAC, and ycf3), except for D. pandanum 1, which possessed two pseudogenes with introns (i.e., ndhA and ndhB) ( Table 3 , Figure 5 ). The rps12 gene was trans-spliced with the 5′ end located in the LSC region, and the 3′ end was duplicated in the IRs in all studied plastomes ( Figure 5 ; Supplementary Material 7 ).

The SSC region was found to vary the most among the examined samples. All plastomes showed a contraction of the SSC with a reduction of 20%–40% compared to the average size of the angiosperm SSC regions (ca. 20 kb) (Ruhlman and Jansen, 2014).

Three plastomes (D. pandanum 1, D. stenocheilum 1, and D. variegatum) lost the ndhF gene. This complete loss of the ndhF gene resulted in the ycf1 fragment being located in the vicinity of the rpl32 ( Figures 5B–D ) and caused a boundary shift of the IRB/SSC region located at the 3′ end of the ycf1 fragment and spacer region of rpl32 ( Figure 5 ). All other plastomes exhibited a severely truncated ndhF gene but did not exhibit an IRB/SSC boundary shift ( Figure 5 ; Supplementary Material 7 ). The IRA/SSC junction in all examined plastomes was located within the 5′ portion of the functional ycf1 gene, ranging from 97 bp (D. pandanum 1) to 1,072 bp (D. aff. roseum 3) ( Figure 5 ).

In contrast to the instability of the IR/SSC boundaries, IR/LSC boundaries were found to be relatively stable. For all studied plastomes, the LSC/IRA boundaries were located near the 3′ end of psbA ( Figure 5 ). Variations within the LSC regions were limited to the operon, which contained ndhC, ndhJ, ndhK ( Figure 5A ), and the independent pseudogenisation of cemA in the plastome of D. aff. roseum 4 and trnD-GUC in the plastome of D. campanulatum ( Table 3 ; Supplementary Material 7 ).

All ndh genes exhibited varying degrees of putative loss or pseudogenisation; not a single ndh gene remained functional in the examined Dipodium plastomes ( Table 3 , Figures 5 , 6 ).

The most severe ndh gene loss occurred in the plastome of D. variegatum, with ndhA, ndhB, ndhD, and ndhK severely pseudogenised and ndhJ moderately pseudogenised.

The greatest degradation processes within Dipodium occurred for the ndhG gene, which was putatively lost in almost all plastomes, except D. stenocheilum 1, which retained a severely pseudogenised ndhG gene ( Figure 6 ). This was followed by ndhK, which was lost in 19 out of 24 plastomes (D. ensifolium, D. hamiltonianum, D. interaneum, the D. roseum complex, and the D. stenocheilum complex) ( Figure 6 ). In the six remaining plastomes, ndhK was conserved to different degrees. D. punctatum, D. pulchellum, and D. campanulatum 2 retained more than 90% of the homologous bases compared to the functional ndhK gene of M. coccinea (KP205432, Kim et al., 2015) but showed severe frameshift mutations and indels, which caused several internal stop codons. The plastomes with severely pseudogenised ndhK genes exhibited large truncations.

Nine Dipodium samples (D. ammolithum, D. atropurpureum 1, D. elegantulum, D. pardalinum 1, D. roseum 1 and 2, D. aff. roseum 2, D. stenocheilum 2, and D. variegatum) putatively lost the ndhC gene. In D. punctatum, ndhC was moderately pseudogenised, and in the remaining plastomes, ndhC was severely pseudogenised ( Figure 6 ). Only D. ensifolium showed an intact start codon for the ndhC gene but suffered a severe truncation with the loss of ca. 50% of homologous bases compared to the functional ndhC gene of M. coccinea (KP205432, Kim et al., 2015).

In D. pandanum 1, D. stenocheilum 1, and D. variegatum, the ndhF gene was putatively lost ( Figure 6 ). All other samples possessed severely truncated ndhF genes with absent start codons and multiple internal stop codons. The ndhH gene was present in D. pandanum 1, possessing a length of 1,176 bp (99.2% of homologous length compared to M. coccinea; KP205432, Kim et al., 2015). In most other samples, the ndhH gene was degraded, possessing several stop codons. D. stenocheilum 1 and D. variegatum lost the ndhH gene ( Figures 5 , 6 ).

Moreover, ndhE, ndhI, and ndhA were found to be putatively lost in the plastome of D. variegatum and D. pandanum 1 ( Figures 5 , 6 ). No gene loss occurred for ndhB, ndhD, and ndhJ; however, all three genes exhibited various degrees of degradation within all examined Dipodium plastomes and were either moderately or severely pseudogenised due to internal stop codons or frameshift mutations.

The ndhD gene was found to have undergone the fewest degradation processes in regard to gene length, which was largely conserved ranging from 1,122 bp (D. campanulatum 1) to 1,521 bp (D. pulchellum), and in most plastomes, ndhD possessed the alternative start codon ACG (Threonine). Furthermore, almost all Dipodium plastomes showed the canonical AUG (Methionine) start codon for ndhA, ndhB, ndhE, and ndhI. The intron-containing ndhA and ndhB genes exhibited the strongest degradation (i.e., large deletions) within the intron regions and the downstream exon in all Dipodium samples. Exon1 of ndhB was almost complete and in-frame for most plastomes and showed only one point mutation (from A to C), which resulted in a stop codon at amino acid position 68 (after 201 bp from the beginning of the first exon in ndhB).

Within different Dipodium complexes, the patterns for putative ndh gene losses and severe or moderate pseudogenisations were similar for examined plastomes of D. hamiltonianum and D. interaneum. Both plastomes putatively lost ndhG and ndhK and showed severe pseudogenisations of ndhA, ndhB, ndhC, ndhF, ndhH, and ndhJ and a moderately pseudogenised ndhE gene, but differed in the level of putative pseudogenisation of ndhD and ndhI ( Figure 6 ).

Other similarities were found in the D. roseum complex, in which the ndhD gene was moderately pseudogenised in all samples. Almost all samples of the D. roseum complex, except for D. roseum 2, harboured moderately pseudogenised ndhE and ndhI genes.

Within the same species, only D. aff. roseum 3 and D. aff. roseum 4 showed the same pattern of ndh gene loss and level of degradation, which was also present in the plastome of D. pardalinum 2. Within the D. stenocheilum complex, D. stenocheilum 1 putatively lost ndhI and ndhF.

Across other samples, only two plastome pairs (D. ensifolium and D. aff. roseum 1; D. basalticum and D. atropurpureum 2) shared the same pattern of ndh gene loss and degradation. In comparison to all other species of examined Dipodium plastomes, D. variegatum independently lost ndhE and ndhI, and D. pandanum 1 lost the ndhA gene ( Figure 6 ).

In this study, whole plastome assemblies were generated for 24 Dipodium samples, including representatives of all leafless, putatively full mycoheterotrophs of sect. Dipodium found in Australia, one leafy photosynthetic species of sect. Dipodium (D. ensifolium), and one leafy photosynthetic species of sect. Leopardanthus (D. pandanum). The overall organisation and the plastid gene content are generally conserved in most examined Dipodium plastomes ( Figure 5 , Tables 2 , 3 ). All examined plastomes showed the typical quadripartite structure of angiosperms (Ruhlman and Jansen, 2014). However, some genomic features among several Dipodium plastomes were not conserved, including 1) differences in total genome length; 2) independent boundary shifts IRB/SSC/IRA within the plastome of D. pandanum 1, D. stenocheilum 1, and D. variegatum; 3) triplication of the trnV-GAC in the plastomes of D. ammolithum, D. elegantulum, D. hamiltonianum, D. stenocheilum 2, and D. interaneum; 4) the independent pseudogenisation of cemA in the plastome of D. aff. roseum 4 and trnD-GUC in the plastome of D. campanulatum 1; and 5) the pseudogenisation or loss to varying degrees of ndh genes ( Figure 5 , Table 3 ; Supplementary Material 7 ).

The total genome length of Dipodium plastomes displayed differences of approximately 10,000 bp between the smallest (142,949 bp; D. variegatum) and largest plastomes (152,956 bp, D. aff. roseum 3), which correlated with the level of ndh gene degradation. Some Dipodium plastomes were similar to the average size of orchid plastomes (152,442 bp) published on the NCBI database (286 Orchidaceae chloroplast genome, accessed on June 13, 2022; NCBI, 2022) however, most plastomes were smaller (average size Dipodium plastomes: 148,703 bp; Table 2 ). Average GC contents in Dipodium were very similar to the average GC content of published orchid plastomes on the NCBI database (NCBI, 2022) (ca. 36.8%; 286 Orchidaceae chloroplast genome, accessed on June 13, 2022), and all fell into the range of typical angiosperm plastomes (ca. 30%–40%) ( Table 2 ).

In orchids, ndh gene losses and pseudogenisations, which occurred in both autotrophic and heterotrophic species, have been documented in various genera (e.g., Kim et al., 2015; Feng et al., 2016; Niu et al., 2017; Barrett et al., 2018; Roma et al., 2018; Barrett et al., 2019; Lallemand et al., 2019; Kim et al., 2020; Peng et al., 2022; Kim et al., 2023). This study is in line with these general findings in that ndh gene degradation was also observed within the orchid genus Dipodium. All chloroplast ndh genes in Dipodium plastomes exhibited varying degrees of putative pseudogenisation and loss, and not a single ndh gene remained functional among the examined chloroplast genomes ( Table 3 , Figures 5 , 6 ). These findings include all plastomes of leafless putatively fully mycoheterotrophic species and two autotrophic leafy species (D. pandanum and D. ensifolium) and thus suggest that all examined species, independently of their nutritional status, have lost the functionality of the plastid NADH dehydrogenase complex. Hence, the last common ancestor of extant Dipodium is likely to have lacked a functional NDH complex. Previous studies in Cymbidiinae, the first diverging lineage in Cymbideae, found that all species studied so far exhibited at least one degraded ndh gene (e.g., Yang et al., 2013; Kim and Chase, 2017). As the next diverging lineage in Cymbidieae is Dipodium, this suggests that the degradation of ndh genes in Cymbidieae was likely a dynamic process from functional to non-functional. However, further research is needed, e.g., ancestral state reconstructions of gene degradation with increased taxonomic sampling. The inclusion of more species among sect. Leopardanthus is warranted to clarify if some ndh genes have remained functional in some autotrophic species of sect. Leopardanthus.

Previous studies examined ndh gene loss at the genus level and revealed an independent loss of function of the NADH dehydrogenase complex for several genera (e.g., Kim et al., 2015; Lin et al., 2015). However, comparative whole plastome studies examining gene degradation and loss among closely related mycoheterotrophic species are still scarce. For a better understanding of ndh gene degradation patterns, this study investigated the degree of ndh gene degradation among closely related orchid species ( Figure 6 ). The greatest degradation within Dipodium was found for ndhG, which is putatively lost in almost all examined plastomes, except D. stenocheilum 1, which retained a putative severely pseudogenised ndhG ( Figure 6 ). The ndhG gene is located within the SSC region. In general, it is well established that genes in the SSC region experience higher substitution rates compared to genes located within IR regions (Ruhlman and Jansen, 2014). The latter is the case for ndhB, which is located in the IRs and structurally more conserved in Dipodium compared to most ndh genes located in the SSC. The greatest degree of ndh gene degradation occurred in D. variegatum, which putatively lost ndhC and ndhE–ndhI. All other plastomes putatively lost at least one to three ndh genes and showed different levels of degradation ( Figure 6 ).

Interestingly, the level of ndh gene degradation varied even among closely related species within species complexes. For example, D. stenocheilum 1 independently lost ndhI and ndhF, whereas all other studied samples of the D. stenocheilum complex retained those two genes as moderately or severely pseudogenised ( Figure 6 ). Different levels of gene degradation and loss were even found within the same species. For example, D. atropurpureum 1 lost ndhC, whereas D. atropurpureum 2 retained a severely pseudogenised ndhC ( Figure 6 ). Moreover, the study of Kim et al. (2020) included one individual of D. roseum, which showed a different pattern of ndh gene loss and degradation to those found among the D. roseum samples of this study. D. roseum (MN200386) experienced complete loss of ndhA, ndhC–ndhI, and ndhK but retained pseudogenised ndhB and ndhJ genes (Kim et al., 2020). These findings also agree with the recent comparative plastome study on D. roseum and D. ensifolium: D. roseum (OQ885084) has retained truncated ndhB, ndhD, and ndhJ genes but completely lost ndhA, ndhC, ndhE–ndhI, and ndhK (McLay et al., 2023).

Overall, some patterns of ndh gene degradation found in this study in Dipodium are similar; however, many were unique for each individual examined. Hence, this suggests that sect. Dipodium has undergone a recent and active ndh gene degradation, which strongly implies a relaxed evolutionary selective pressure for the retention of the NDH complex.

Orchidaceae plastomes frequently show an expansion/shift of the IR towards the SSC region (e.g., Kim et al., 2020). This instability of the IR/SSC junction is assumed to correlate with the deletion of ndhF and has resulted in a reduction of the SSC, as observed in several Orchidaceae plastomes (e.g., Kim et al., 2015; Niu et al., 2017; Dong et al., 2018; Roma et al., 2018) and other land plant plastomes (e.g., Amaryllidaceae, Bignoniaceae, and Orobanchaceae) (Thode and Lohmann, 2019; Könyves et al., 2021; Li et al., 2021). This study revealed reduced SSC regions for most examined plastomes, which correlated with the degradation of the ndh gene suite located in the SSC. Compared to typical SSC regions found in angiosperms (ca. 20 kb; Ruhlman and Jansen, 2014), the smallest SSC region was reduced by ca. 7,900 bp (D. variegatum), and the largest SSC region was reduced by ca. 4,700 bp (D. ensifolium) ( Table 2 , Figure 5 ). However, a large expansion of the IR such as found in Vanilla and Paphiopedilum plastomes (Kim et al., 2015) was not found in Dipodium (IR sizes ranging between 24,436 and 26,817 bp, Table 2 ).

In angiosperms, the ycf1 gene usually occupies ca. 1,000 bp in the IR (Kim et al., 2015; Sun et al., 2017). Dipodium plastomes in this study displayed varying positions of ycf1 within the IR. In plastomes in which the ndhF gene was completely lost or severely truncated, the portion of ycf1 within the IRA was mostly shorter compared to plastomes, which contained moderately truncated ndhF genes ( Figure 5 ). These results are similar to the findings of Kim et al. (2015), who compared the locations of the IR/single-copy region junctions among 37 orchid plastomes and closely related taxa in Asparagales. In at least three plastomes (D. pandanum 1, D. stenocheilum 1, and D. variegatum), ndhF was independently lost, and the SSC/IRB junction was shifted into the spacer region near the rpl32 gene in direct adjacency to the partially duplicated ycf1 fragment ( Figures 5B–D ). These findings suggest the deletion of ndhF correlated with the shift of the SSC/IRB junction. Interestingly, the boundaries between SSC and IR regions were found to be variable even among closely related species, e.g., in Cymbidium. Some species in Cymbidium showed similar patterns of IR/SSC shifts (Kim and Chase, 2017) as found in Dipodium.

In at least five plastomes (D. ammolithum, D. elegantulum, D. hamiltonianum, D. interaneum, and D. stenocheilum 2), the trnV-GAC gene was triplicated (i.e., duplicated trnV-GAC version in close proximity to each other in either IRA or IRB) ( Figures 5B, C , Table 3 ). To the best of our knowledge, similar tRNA duplication patterns within the IR regions have not yet been found in any other Orchidaceae plastomes. However, a recent study on plastomes of the angiosperm genus Medicago (Wu et al., 2021) yielded similar patterns. Wu et al. (2021) found three copies of the trnV-GAC gene in the plastomes of two closely related species within the IR (Medicago archiducis-nicolai and Medicago ruthenica), which were linked to forward and tandem repeats. Interestingly, the findings of Wu et al. (2021) support the hypothesis that repetitive sequences lead to genomic rearrangements and thus affect plastome stability. This may also apply to some Dipodium plastomes. However, to rule out any technical issues throughout the next-generation sequencing (NGS) process and to validate findings of duplicated tRNAs (and the above-mentioned boundaries of IR/SC regions), PCR amplification of affected regions should be carried out in future studies. However, in strong support of tRNA duplication is their independent presence within the IR of five plastomes among individuals of the same species complexes (D. stenocheilum complex and D. hamiltonianum complex). However, an increased sampling is necessary to better understand the impacts of genomic rearrangements due to repetitive sequences and thus plastome instability in Dipodium.

Heterotrophic plants are remarkable survivors, exhibiting often curious morphological, physical, or genomic modifications. Multiple heterotrophs were found to have suffered plastid genome degradations due to relaxed pressure on photosynthetic function. In recent years, evidence has accumulated that plastid genomes have undergone gene degradation in the evolutionary transition from autotrophy to heterotrophy (e.g., Wicke et al., 2016; Graham et al., 2017; Barrett et al., 2019). Among these, the first stage is the loss and pseudogenisation of genes involved in encoding the NDH complex. Interestingly, all examined plastomes of Dipodium have lost or pseudogenised all 11 ndh genes regardless of their nutritional status ( Figure 6 ). Two photosynthetic species with green leaves were included in this study, D. pandanum (sect. Leopardanthus) and D. ensifolium (sect. Dipodium). Degradation in ndh genes among photosynthetic species is not surprising and was frequently reported in previous plastome studies in land plants. The large-scale study on Orchidaceae plastomes of Kim et al. (2020) observed ndh gene pseudogenisation and losses among species in many epiphytes and several terrestrials that have retained their photosynthetic capacity. The NDH complex is thought to mediate the Photosystem I cyclic electron transport, fine-tune photosynthetic processes, and alleviate photooxidative stress (e.g., Yamori et al., 2015; Peltier et al., 2016; Sabater, 2021). D. pandanum is a terrestrial or climbing epiphytic orchid and highly localised in rainforest habitats, whereas the terrestrial D. ensifolium grows in open forests and woodlands (Jones, 2021); thus, both species seem to prefer shaded understory habitats. For epiphytic or terrestrial plants living in low-light habitats, it has been proposed that the NDH complex may not be essential anymore (e.g., Barrett et al., 2019). One reason for this may be that they are less exposed to photooxidative stress (e.g., Feng et al., 2016; Barrett et al., 2019). However, the NDH complex is composed of 11 chloroplast-encoded subunits and additional subunits encoded by the nucleus (e.g., Peltier et al., 2016). It has been established that genomic material was repeatedly exchanged between the nucleus, mitochondrion, and chloroplast in the evolutionary course of endosymbiosis. Thus, previous studies examined whether genes were transferred from the chloroplast to the nucleus and/or mitochondrion genome or whether nuclear genes for the NDH complex suffered under degradation. Indeed, Lin et al. (2015) reported ndh fragments within the mitochondrial genomes of orchids; however, no copies were found in the nuclear orchid genomes. Similar findings were reported from the orchid genus Cymbidium (Kim and Chase, 2017). However, further studies are needed to determine whether ndh gene transfer into the nucleus or mitochondrion may play a role within Dipodium.

The proposed subsequent next steps towards (myco-) heterotrophy is the functional loss of photosynthetic genes (e.g., psa, psb, pet, rbcL, or rpo) followed by genes for the chloroplast ATP synthase and genes with other functions such as housekeeping genes (e.g., matK, rpl, and rnn; e.g., Graham et al., 2017; Barrett et al., 2019). Most examined Dipodium plastomes displayed no additional plastid gene degradation in addition to ndh gene degradation, except in D. aff. roseum 4, where cemA was pseudogenised, and in D. campanulatum 1, where the trnD-GUC gene was pseudogenised ( Table 3 ). The cemA gene encodes the chloroplast envelope membrane protein and was found to be non-essential for photosynthesis; however, cemA-lacking mutants of the green alga Chlamydomonas were found to have a severely affected carbon uptake (Rolland et al., 1997) and may therefore be classified as directly involved in photosynthesis. Transfer RNA genes (trn) are involved in the translation process and categorised as “housekeeping” genes (e.g., Graham et al., 2017; Wicke and Naumann, 2018; Barrett et al., 2019). Moreover, similar gene degradation patterns were found in the plastomes of D. roseum (MN200386, Kim et al., 2020 and OQ885084, McLay et al., 2023) and D. ensifolium (OQ885084, McLay et al., 2023), which functionally lost all ndh genes. However, most photosynthesis-related genes in the plastomes of Dipodium were found to be functional. Thus, mycoheterotrophic species of Dipodium display evidence of being at the beginning of plastid gene degradation, in contrast with the majority of fully mycoheterotrophic orchids, which are in more advanced stages of degradation, e.g., Cyrtosia septentrionalis (Kim et al., 2019), Epipogium (Schelkunov et al., 2015), and Rhizanthella (Delannoy et al., 2011). In contrast, mycoheterotrophs such as Corallorhiza trifida (Barrett et al., 2018), Cymbidium macrorhizon (Kim et al., 2017), Hexalectris grandiflora (Barrett et al., 2019), and Limodorum abortivum (Lallemand et al., 2019) display functional losses within the plastid ndh genes only, and some species among them additionally lost one or two other genes, similar to findings in Dipodium. Interestingly, most of these species are leafless but considered putatively partially mycoheterotrophic. Suetsugu et al. (2018) demonstrated that the leafless green orchid C. macrorhizon contains chlorophyll and can fix significant quantities of carbon during the fruit and seed production phase and, thus, is photosynthetically active. Chlorophyll is present in C. trifida also, but this green, leafless coralroot is an inefficient photosynthesiser (Barrett et al., 2014). Some species among leafless orchids within sect. Dipodium (e.g., D. elegantulum, D. stenocheilum, and D. variegatum) appear green on stems ( Figure 1 , Jones, 2021), which suggests that they may contain some chlorophyll and be able to photosynthesise. Coupled with relatively mild plastid gene degradation compared to other fully mycohetrotrophic orchids, this suggests that some leafless species among sect. Dipodium may be partially mycoheterotrophic rather than fully mycoheterotrophic as has been hypothesised for D. roseum (Kim et al., 2020; McLay et al., 2023). However, no studies so far have examined whether leafless species among sect. Dipodium contain chlorophyll and whether they are capable of carrying out photosynthesis at sufficient rates. Therefore, more research is needed to assess the trophic status, including analysis of chlorophyll quantities and the ratio of photosynthetic carbon to fungal carbon for Dipodium.

Compared with recently published studies on mycoheterotrophic orchids such as Corallorhiza and Hexalectris (Barrett et al., 2018, 2019), which incorporated divergence-time estimations, plastomes of Dipodium showed the least degradation. Hexalectris crown age was estimated to be ca. 24 Ma, and plastomes of mycoheterotophs were more degraded compared to mycoheterotrophic plastomes of Corallorhiza, which diversified ca. 9 Ma onwards (Barrett et al., 2018, 2019). Dipodium diversified in the late Miocene ca. 11 Ma and the mycoheterotrophic lineage divergent from the autotrophic lineage ca. 8.1 Ma, which is slightly younger compared to Corallorhiza. Hence, time of divergence may play a role in the degree of degradation of Dipodium plastomes, which show an early stage of plastome degradation compared to older diverging mycoheterotrophic lineages that are in more advanced stages of plastome degradation.