Organellar phylogenomics at the epidendroid orchid base, with a focus on the mycoheterotrophic Wullschlaegelia

Abstract

Background and aims: Heterotrophic plants have long been a challenge for systematists, exemplified by the base of the orchid subfamily Epidendroideae, which contains numerous mycoheterotrophic species.

Methods: Here we address the utility of organellar genomes in resolving relationships at the epidendroid base, specifically employing models of heterotachy, or lineage-specific rate variation over time. We further conduct comparative analyses of plastid genome evolution in heterotrophs and structural variation in matK.

Key results: We present the first complete plastid genomes (plastomes) of Wullschlaegelia, the sole genus of the tribe Wullschlaegelieae, revealing a highly reduced genome of 37 kb, which retains a fraction of the genes present in related autotrophs. Plastid phylogenomic analyses recovered a strongly supported clade composed exclusively of mycoheterotrophic species with long branches. We further analysed mitochondrial gene sets, which recovered similar relationships to those in other studies using nuclear data, but the placement of Wullschlaegelia remains uncertain. We conducted comparative plastome analyses among Wullschlaegelia and other heterotrophic orchids, revealing a suite of correlated substitutional and structural changes relative to autotrophic species. Lastly, we investigated evolutionary and structural variation in matK, which is retained in Wullschlaegelia and a few other 'late stage' heterotrophs and found evidence for structural conservation despite rapid substitution rates in both Wullschlaegelia and the leafless Gastrodia.

Conclusions: Our analyses reveal the limits of what the plastid genome can tell us on orchid relationships in this part of the tree, even when applying parameter-rich heterotachy models. Our study underscores the need for increased taxon sampling across all three genomes at the epidendroid base, and illustrates the need for further research on addressing heterotachy in phylogenomic analyses.

Keywords: Triphora aff. wagneri; Triphora trianthophoros; Uleiorchis ulei; Wullschlaegelia calcarata; Epidendroideae; Orchidaceae; comparative genomics; herbarium; heterotachy; mitochondrial; mycoheterotrophy; plastid.

Fig. 1.

Plastome map of Wullschlaegelia calcarata accession NYBG Vincent #15 387 (Rio Grande, Puerto Rico, USA). *Gene contains one intron; **gene contains two introns (i.e. clpP); ***gene contains two cis-spliced exons and one trans-spliced exon (i.e. rps12).

Fig. 2.

Structural comparison among representative early-diverging epidendroid plastomes from Mauve analysis. Colours indicate locally collinear blocks (LCBs) 1–9, listed above the plastome of Nervilia. Black bars above plastome maps for Nervilia and Triphora represent the inverted repeat (IR; with one copy removed); both Gastrodia and Wullschlaegelia lack an inverted repeat. Asterisks indicate the location of the matK gene.

Fig. 3.

(A) Plastid gene relationships based on the best-scoring GHOST heterotachy model (GTR+FO*H6). Epidendroid tribes (and subtribes) are listed to the right of the tree. Numbers adjacent to branches are bootstrap values based on 2000 ultrafast replicates in IQ-TREE 2 (no value indicates 100 % support). (B) Phylogram of the same tree as that in (A) showing relative estimates for branch lengths under the GHOST model. Taxa in red are fully mycoheterotrophic, while purple indicates W. calcarata. Reference taxa were acquired from NCBI GenBank or from Serna-Sánchez et al. (2021), with the latter indicated by an asterisk (*); **, matK, psaB and rbcL genes from Xerorchis amazonica (GenBank numbers AF074148, AF263688 and AF074244, respectively). (C) Unrooted phylogram among six accessions of W. calcarata based on whole aligned plastomes.

Fig. 4.

(A) Mitochondrial gene relationships based on the best-scoring GHOST heterotachy model (GTR+FO*H4). Epidendroid tribes are listed to the right of the tree. Numbers adjacent to branches are bootstrap values based on 2000 ultrafast pseudoreplicates in IQ-TREE 2. (B) Phylogram of the same tree as that in (A) showing relative estimates for branch lengths under the GHOST model. Taxa in red are fully mycoheterotrophic, while purple indicates W. calcarata. Reference taxa, excluding newly sequenced accessions of Triphora, Wullschlaegelia and Uleiorchis, were acquired from Li et al. (2019). Newly sequenced accessions are indicated by an asterisk (*).

Fig. 5.

Retained, putatively functional genes in representative orchid plastomes of different trophic modes. Gene colours indicate different functional complexes, with the five ‘core non-bioenergetic’ genes (sensuGraham et al., 2017) in yellow. Coloured bar on left indicates the trophic mode of each species: dark green, leafy autotrophic; light green, leafless partially mycoheterotrophic; magenta, leafless fully mycoheterotrophic. Wullschlaegelia calcarata is in purple text.

Fig. 6.

Phylogenetic principal components analysis (PPCA) of nine log-transformed plastome features. Red font indicates leafless, fully mycoheterotrophic species, blue font indicates leafy, autotrophic species. Inset shows the PPCA biplot, with feature names defined in the caption of Table 7.

Fig. 7.

Structural analysis of Dali z-scores for predicted matK gene products among representative leafy, autotrophic (blue) and leafless, fully heterotrophic (red) epidendroid orchids. (A, B) Correspondence analysis of z-scores for axes 1 vs 2 and 1 vs 3, respectively. (C) Average linkage dendrogram based on the pairwise z-score matrix showing divergence in structural similarity. (D) Heat map of pairwise z-scores, with lower structural similarity shown in in blue and higher similarity in orange-red.

Fig. 8.

Representative topology summaries for early-diverging Epidendroideae from previous studies and the current study. Analyses including Wullschlaegelia are indicated in purple. The solid square indicates the node constrained in topology tests (Wullschlaegelieae, Gastrodieae). Support values adjacent to branches are as follows: Freudenstein and Chase (2015), maximum likelihood (ML) bootstrap; Serna-Sanchez et al. (2021), maximum likelihood bootstrap; Pérez-Escobar et al. (2024), number of gene trees concordant (top) and discordant (bottom) with the inferred species tree; Zhang et al. (2023), maximum likelihood bootstrap support for all gene trees for a particular node (e.g. >70 indicates that all gene trees received at least 70 % support); current study (plastome and mitochondrial genes), maximum likelihood bootstrap. Asterisk and square in the summary tree from Pérez-Escobar et al. (2024) indicate a non-monophyletic Neottieae and the node used for topology tests in the current study, respectively.