What question did this study set out to answer?

To build a comprehensive phylogeny of Diospyros by integrating NGS and Sanger sequencing data.

April 13, 2026Open Access

A global phylogenetic framework for Diospyros L. (Ebenaceae, Ericales): enhanced taxon and sample coverage through NGS, Sanger sequencing, and herbariomics

Puntos clave

To build a comprehensive phylogeny of Diospyros by integrating NGS and Sanger sequencing data.
De-novo sequencing of approximately 324 samples from Diospyros and outgroup species using NGS technology.
Integration of 111 newly collected samples and 213 herbarium samples to generate complete plastome data.
Reconstruction of phylogenies using Maximum Likelihood methods, focusing on single-locus, multi-locus, and whole plastome analyses.
The six-locus tree clarifies genus-wide relationships in Diospyros with enhanced support.
Whole plastome analysis resolves previously ambiguous relationships with robustness.
Identification of ycf1 as the most informative gene for phylogeny building when combined with legacy loci.

Resumen

Abstract Background and Aims Diospyros (∼700 species) is a pan-tropical genus with centres of diversity in poorly studied areas. Fragmented regional classifications have led to confusion in the understanding of this economically important tropical genus. With 20% of species and only six plastid loci previously sampled (atpB, ndhF, rbcL, trnK-matK, trnL-trnF and trnS-trnG), existing phylogenies have been poorly resolved and supported. Next-generation sequencing (NGS) provides the possibility of broad sampling of herbarium specimens. In this study, the three main aims are to: explore methods for integrating legacy Sanger and new NGS sequence data to make more comprehensive datasets; to build the most complete and well-supported phylogenies of Diospyros to date, and to identify the most informative genes for Sanger-based phylogeny building. Methods Approximately 200 Diospyros and outgroup species, totalling 324 samples were de-novo sequenced with NGS technology. One hundred and eleven newly collected samples resulted in 62 whole plastomes and 49 additional partial sequences while 213 herbarium samples generated 48 whole plastomes and 165 additional partial sequences of the six loci. DNA sequences from further 207 samples from 138 taxa were added from public databases. Single-locus, multi-locus and whole plastome phylogenies were reconstructed using a Maximum Likelihood approximation method.. Key results The six-locus tree elucidates the genus-wide relationships and the whole plastome phylogeny resolves multiple previously ambiguous relationships with robust support. There remain only two unresolved clades, possibly results of rapid radiation and/or hybridisation. Whole plastome analysis shows ycf1 to be the best resolving gene when combined with the six legacy loci. Conclusions We provide a well-resolved phylogeny for Diospyros offering a major advance in understanding evolution of the genus. The integration of whole plastome NGS data and legacy chloroplast sequence data enabled us to generate the most comprehensive view of evolutionary relationships for the genus to date (∼250 Diospyros taxa).

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