Plastome phylogenomics of the diverse neotropical orchid genus Lepanthes with emphasis on subgenus Marsipanthes (Pleurothallidinae: Orchidaceae)

Abstract

Well-resolved phylogenetic relationships within the diverse Neotropical orchid genus Lepanthes are presented based on a genome skimming approach that yielded nine newly sequenced chloroplast genomes. We complemented this with 17-86 plastome coding genes for 26 species retrieved from GenBank, alongside amplified matK and rITS regions. The Lepanthes plastomes (157,185-158,260 bp, 37.15% GC content) contained 136 annotated genes, including 86 protein-coding, 42 tRNA, and 8 rRNA genes. We identified six hypervariable regions, including parts of the ycf1 gene, as potential DNA barcodes. Phylogenetic analyses revealed that Carl Luer's subgeneric classifications are non-monophyletic, a finding confirmed by PCA of continuous morphological traits, reflecting significant morphological homoplasy. Six major clades were identified, though resolution for the phylogenetic backbone remains unresolved at two nodes. Subgenus Marsipanthes is not monophyletic as currently circumscribed, with two subclades recovered in distinct positions within the phylogeny. An early-diverging lineage, comprising species restricted to the eastern Andean slopes from southern Colombia to Peru, includes members of both Marsipanthes and Lepanthes. A derived clade, consisting of species from both subgenera, confined to the Chocó biogeographic region, forms an unresolved polytomy. Although only a subset of Lepanthes diversity was sampled, this study captures significant taxonomic, geographic, and morphological variation. It provides foundational insights into the genu's evolutionary history, along with tools and hypotheses that can be expanded upon in future research to further refine our understanding of its biogeographic history.

Keywords: Ycf1; Andes; Chloroplast; Chocó Biogeographic Region; Colombia; Eastern Andes; Ecuador.

Fig. 1

Taxonomic representatives and distribution of Lepanthes. Left panel: Representatives of each taxonomic category proposed by Luer [–8] and their morphological diversity. A. L. caprimulgus B. L. eros C. L. felis D. L. hexapus E. L. manabina F. L. monoptera G. L. mucronata H. L. nicolasii I. L. narcissus J. L. ribes. Photographs by Ron Parsons (A), J. S. Moreno (B, C, D, E, F and G), SCO (H), J. Riksen (I) and S. Vieira-Uribe (J). Scale bar = 5 mm. Right Panel: Spatial distribution of Lepanthes species richness across the Neotropics. Species richness was calculated within a 1-degree grid based on occurrence of 4582 records obtained from GBIF, iNaturalist, Species link and Moreno [9].

Fig. 2

Plastome structure of nine Lepanthes species. The darker gray in the inner circle corresponds to the GC content. Bars of different colors indicate different functional groups. Genes on the inside circle are transcribed clockwise, while genes on the outside circle are transcribed counterclockwise. LSC: large single copy region; SSC: small single copy region IR: inverted repeat regions. Lepanthes nicolasii chloroplast

Fig. 3

Comparison of the boundaries between the LSC, SSC and IR regions in the nine Lepanthes plastomes. Tree topology from the ML phylogeny of complete plastomes. JLB: LSC/IRb junctions; JSB: SSC/IRb junctions; JSA: SSC/IRa junctions; JLA: LSC/IRa junctions

Fig. 4

Nucleotide variability (Pi) of whole plastomes from nine Lepanthes species including a single Inverted Repeat

Fig. 5

Inferred phylogenies for the Lepanthes backbone using Bayesian Inference based on 17-86 coding plastid regions. A. Molecular phylogeny of 35 Lepanthes species and two outgroups. B. Molecular phylogeny of Lepanthes highlighting the main clades and subclades. Colors represent equivalent clades and subclades in both phylogenies. Red asterisk shows support value below 0.8 PP

Fig. 6

Plastid coding genes representation in Lepanthes and outgroups. A. Number of plastid coding genes per species included in the alignment. B. Overall completeness of the alignment for each gene across species. Yellow color indicates the absence of a coding gene. C. Percentage coverage of each gene in the total number of species within the alignment

Fig. 7

Morphological analysis of continuous vegetative and reproductive characters in Lepanthes. A. Recovered phylogenetic relationships and main Lepanthes clades, this panel highlights species included in the PCA, with colors representing different clades. B. PCA, colored dots represents the clades where the species belong in the phylogeny. C. Heat map of a correlation matrix showing either positive or negative correlations among all continuous morphological characters studied here