# Resolving Species Limits and Evolutionary Distinctiveness of the Libyan Endemic Arbutus pavarii (Ericaceae) Using Multilocus DNA Barcoding and Phylogenetic Analyses

## TL;DR

This study confirms that Arbutus pavarii is a distinct species, not a variant of A. unedo, using DNA analysis and phylogenetic methods.

## Contribution

The study introduces a multilocus DNA barcoding framework to resolve taxonomic uncertainty in Arbutus species.

## Key findings

- Multilocus DNA barcoding and phylogenetic analyses show Arbutus pavarii is a distinct evolutionary lineage.
- Nuclear and plastid markers together provide strong evidence for species delimitation.
- The study supports conservation efforts by clarifying A. pavarii's taxonomic status.

## Abstract

The taxonomic status of Arbutus pavarii Pamp., a rare and geographically restricted species endemic to northeastern Libya, has long been debated, with some treatments considering it a synonym of A. unedo. To resolve this uncertainty, we applied an integrative molecular framework that combined multilocus DNA barcoding, phylogenetic inference, and multivariate statistical analyses. Five barcode loci—nrITS, matK, rbcL, trnH–psbA, and rps16—were analyzed using barcode-gap diagnostics, TaxonDNA identification tests, and single-locus and concatenated phylogenetic analyses. Barcode-gap analyses based on Kimura 2-parameter distances revealed clear and reproducible separation between intra- and interspecific variation for A. pavarii, particularly for nrITS and the concatenated multilocus dataset, whereas conserved plastid loci showed limited discriminatory power when used individually. Phylogenetic reconstructions consistently recovered A. pavarii as a strongly supported monophyletic lineage, distinct from A. unedo and other Mediterranean congeners, with congruent topologies across the nuclear, plastid, and combined datasets. Multivariate analyses, including principal component analysis and heatmap clustering, further corroborate the genetic cohesion and distinctiveness of A. pavarii samples. Collectively, these results provide robust molecular evidence supporting the recognition of Arbutus pavarii as a distinct evolutionary lineage, rather than an intraspecific variant of A. unedo. This study established a reproducible multilocus framework for species delimitation in Arbutus and highlighted the importance of integrating nuclear and plastid markers to resolve complex taxonomic relationships. The clarified taxonomic status of A. pavarii has important implications for biodiversity assessment and conservation planning in the Mediterranean region, particularly in the Cyrenaican floristic province.