Reticulate evolutionary history underpins a revised generic circumscription of Paphiopedilum (Orchidaceae): insights from integrative phylogenomics and historical biogeography

Abstract

Background: The taxonomy of Paphiopedilum-a diverse group of slipper orchids-has long posed challenges due to high morphological variability and conflicting phylogenetic signals. Despite intensive research, a clear and stable infrageneric classification remains unresolved.

Results: We present an integrative phylogenetic framework based on multiple nuclear low-copy genes (XDH, PHYC, LFY, RAD51, ACO, DEF4), ITS and plastid (matK) sequences, supported by morphological and biogeographical data. Our analyses consistently recovered monophyletic clades corresponding to both historically recognized and newly proposed lineages. Phylogenetic incongruence observed both between different nuclear loci and between nuclear and plastid phylogenies suggests a reticulate evolutionary history shaped by ancient hybridization events and incomplete lineage sorting. Biogeographic reconstructions place the genus' origin in mainland of Southeast Asia during the Miocene, with subsequent radiation influenced by climatic and geological events. As a result, we propose a revised classification recognizing three genera-Parvisepalum, Brachypetalum, and Paphiopedilum s.str.-with the latter subdivided into six subgenera.

Conclusions: This study provides the most comprehensive phylogenetic and taxonomic treatment of Paphiopedilum to date. Our framework reflects evolutionary relationships more accurately than previous systems and offers a stable basis for future research in orchid systematics, conservation, and evolution.

Keywords: Paphiopedilum; Biogeography; Hybridization; Molecular systematics; Orchidaceae; Phylogenetics; Slipper orchids; Taxonomy.

Fig. 1

Bayesian inference phylogeny of the genus Paphiopedilum based on a concatenated dataset of the low-copy nuclear genes ACO (1-aminocyclopropane-1-carboxylate oxidase), DEF4 (DEFICIENS-like gene 4), LFY (LEAFY), and RAD51. Posterior probability (PP) values are indicated above the branches. Taxon annotations on the phylogenetic tree reflect the new systematic framework proposed in this study. For comparison, the classification system according to Braem & Chiron (2003) is shown in parentheses

Fig. 2

Bayesian inference phylogeny of the genus Paphiopedilum based on a concatenated dataset of the low-copy nuclear genes PHYC (phytochrome C), ACO (1-aminocyclopropane-1-carboxylate oxidase), and DEF4 (DEFICIENS-like gene 4). Posterior probability (PP) values are indicated above the branches. Taxon annotations on the phylogenetic tree reflect the new systematic framework proposed in this study. For comparison, the classification system according to Braem & Chiron (2003) is shown in parentheses

Fig. 3

The Maximum a Posteriori (MAP) network inferred using MCMC_GT, corresponding to the highest posterior probability (log = –38.19071), based on gene tree topologies (nuclear: ACO, DEF4, PHYC, XDH, LFY, RAD51, ITS; and plastid tree), is shown. The blue line indicates a possible hybridization scenario

Fig. 4

Ancestral area reconstruction of Paphiopedilum based on a concatenated dataset of the low-copy nuclear genes PHYC (phytochrome C), ACO (1-aminocyclopropane-1-carboxylate oxidase), and DEF4 (DEFICIENS-like gene 4), along with extant distributions, using (A) the DEC model and (B) BAYAREALIKE + J model in RASP. The map illustrates the coding areas in distinct colors

Fig. 5

Bayesian inference phylogeny of the genus Paphiopedilum based on a concatenated dataset of the low-copy nuclear genes PHYC (phytochrome C), ACO (1-aminocyclopropane-1-carboxylate oxidase), and DEF4 (DEFICIENS-like gene 4), with the overlay of key traits used in the taxonomic circumscription of major clades. Morphological synapomorphies are indicated, as well as the cytogenetic character “multiple duplication of 5S rDNA” (item 8). The tree highlights both genetic relationships and the evolutionary significance of specific morphological features, which are crucial for defining taxonomic boundaries within the genera. Taxon labels reflect the revised systematic framework proposed in this study