Range extension and re-assessment of Eugenia stirpiflora (O.Berg) Krug & Urb., an endemic Myrtaceae to the Virgin Islands

Biodiversity Data Journal 13: e175549 doi: 10.3897/BDJ.13.e175549 Species Conservation Profiles Range extension and re-assessment of Eugenia stirpiflora (O.Berg) Krug & Urb., an endemic Myrtaceae to the Virgin Islands Sara Bárrios , Clare Weaver , Eleanor Gibney , Thomas Mark Heller , Martin A Hamilton , Marcella Corcoran , Keith Grant , Juan Viruel , Amy Barker , Nancy Woodfield-Pascoe , Colin P. Clubbe ‡ Royal Botanic Gardens Kew, London, United Kingdom § Virgin Islands Rare Plant Initiative (VIRPI), Saint John, Virgin Islands (USA) | Independent Researcher, Saint John, Virgin Islands (USA) ¶ South Carolina Botanical Garden at Clemson University, Clemson, United States of America # National Parks Trust Virgin Islands, Road Town, Virgin Islands (British) Corresponding author: Sara Bárrios ([email protected]) Academic editor: Yuanjun Zhu Received: 20 Oct 2025 | Accepted: 01 Dec 2025 | Published: 16 Dec 2025 Citation: Bárrios S, Weaver C, Gibney E, Heller T, Hamilton M, Corcoran M, Grant K, Viruel J, Barker A, Woodfield-Pascoe N, Clubbe CP (2025) Range extension and re-assessment of Eugenia stirpiflora (O.Berg) Krug & Urb., an endemic Myrtaceae to the Virgin Islands. Biodiversity Data Journal 13: e175549. https://doi.org/10.3897/BDJ.13.e175549 Abstract Background The Caribbean archipelago is a global biodiversity hotspot, with high levels of endemism and a threatened flora. The British Virgin Islands and the US Virgin Islands, two archipelagos situated in the Caribbean Sea are no exception. Despite many decades of floristic investigation, continuing botanical surveys across the region are uncovering previously unrecorded plant populations and improving our understanding of the geographic ranges of locally restricted threatened flora. This study aims to re-evaluate the latest conservation status of Eugenia stirpiflora by examining its current spatial distribution, population numbers, main threats and conservation actions needed to ensure the species' long term survival. ‡ § | ‡ ‡,¶ ‡ # ‡ ‡ # ‡ © Bárrios S et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. New information Thought to be endemic to the island of St. John in the US Virgin Islands (USVI), E. stirpiflora (Myrtaceae), was discovered on the island of Virgin Gorda in the British Virgin Islands (BVI) in 2020, extending its geographic range to a new territory. Despite this wider distribution, the species' extent of occurrence (20 km ) and area of occupancy (12 km ) are still highly restricted. On St. John (USVI), this species is recorded from Europa Bay, including the White Cliffs area, Minna Hill and Upper John's Folly. On Virgin Gorda, the species is only known from Fanny Hill, north of Gorda Peak. The total number of mature individuals ranges between 1,535 and 1,665, with the species' largest population occurring on Europa Bay (St. John) with a number of mature individuals between 1,500 and 1,630. On the island of Virgin Gorda, the species' habitat is declining due to expansion of urban development and uncontrolled grazing by feral ungulates. On St. John, the suitable habitat and the number of individuals are declining due to grazing by free-roaming livestock and feral ungulates, illegal trail clearing and insect pests. Based on these threats, two locations were recognised. Simultaneously, prolonged periods of drought, caused by climate change, are affecting both locations uniformly. This species, previously evaluated on the IUCN Red List as Critically Endangered (CR) under its synonym, Eugenia earhartii, is here re-evaluated as Endangered (EN), based on Criteria B1ab(iii,v)+2ab(iii,v); C2a(ii) according to the IUCN Red List Categories and Criteria. Keywords Caribbean flora, conservation status, distribution, endemism, Myrtaceae, new records, Red List, Virgin Islands Introduction Eugenia stirpiflora (O.Berg) Krug & Urb. grows as a shrub or small tree up to eight metres tall. The bark is grey and thick, becoming gnarled and shedding in irregular patches. The leaves are sessile and coriaceous. Blades are ovate to elliptical, with a rounded apex. The leaf margin is strongly revolute (Fig. 1) similar to the leaves of the close relative E. sessiliflora Vahl. The flowers are cauliflorous, emerging directly from the bark on short pedicels (Fig. 2). Flowers are white with a pink tinge (Fig. 3). The fruit is globose to oblate (Fig. 4) (Acevedo-Rodriguez 1996, Flickinger et al. 2022). This species grows in relatively undisturbed tropical dry forest, in association with other native plant species (Fig. 5) between 20 to 214 metres high (Acevedo-Rodriguez 1996, Flickinger et al. 2022). Thought to be restricted to the island of St. John in the USVI (Acevedo-Rodriguez 1996, Lindsay et al. 2015), this species was discovered on the island of Virgin Gorda in the BVI in February 2020 during a survey of Fanny Hill (Dani Sanchez et al. 2020). Vouchers (T.M. Heller, #1038, K000817252, K!), DNA samples (J. Viruel, #JV22-001, K!) and cuttings for ex-situ collections (G. Gregg, #11, BVI!), collected between 2020 and 2022, 2 2 2Bárrios S et al have now confirmed the identity of this species at this location (Heller 2022, Barrios 2023). Although administered by different countries, the British Virgin Islands and the US Virgin Islands lie in close geographical proximity to each other and, floristically, are part of the Puerto Rican Bank flora (Acevedo-Rodriguez 1996). In situ and ex situ conservation initiatives Trials to asexually propagate this species by cuttings were undertaken at the J.R. O'Neal Botanic Garden on Tortola, BVI in 2022. Cuttings were collected from the recently discovered wild plants at Fanny Hill, on Virgin Gorda, BVI. The cutting trials used semihardwood and hardwood materials and two different media types which were available in Figure 1. Leaves of Eugenia stirpiflora on a tree from Fanny Hill, Virgin Gorda. The leaves are coriaceous with strongly revolute margins (Photo: Thomas Mark Heller, RBG Kew).  Figure 2. Flower buds of Eugenia stirpiflora, emerging from a tree on St. John (USVI) (Photo: Clare Weaver, VIRPI).  Range extension and re-assessment of Eugenia stirpiflora (O.Berg) Krug ... 3 the nursery. All cuttings were dusted with rooting hormone powder (Fig. 6). Pots were placed in a misting unit to maintain a high relative humidity, until the material developed roots. No cuttings survived long-term from these initial propagation trials, suggesting that this species might not be easily propagated by these methods. Further propagation research is needed. In the USVI, the Virgin Islands Rare Plant Initiative (VIRPI) has undertaken regular population monitoring activities since 2012 at Europa Bay, including the White Cliffs area. Figure 3. A cauliflorous flower of Eugenia stirpiflora on Fanny Hill, Virgin Gorda (BVI) (Photo: Sara Bárrios, RBG Kew).  Figure 4. Fruits of Eugenia stirpiflora on a St. John tree (Photo Clare Weaver, VIRPI).  4Bárrios S et al Monitoring includes collecting measurements and photographic data and also observing fruiting cycles (Fig. 7). The VIRPI is working to collect seeds from each individual (maternal lines), to be germinated and conserved at a living gene bank on St. Croix, USVI and shared with other regional institutions. Conservation genetics of Eugenia stirpiflora We assessed the genetic diversity of the BVI population of E. stirpiflora. A total of ten individuals were sequenced and genotyped, using SNPs derived from target capture of 353 nuclear loci. The observed heterozygosity (H) was 0.227 ± 0.003, while the expected heterozygosity (H) was 0.278 ± 0.003, indicating moderate levels of genetic O E Figure 5. Undisturbed tropical dry forest is the preferred habitat for Eugenia stirpiflora (Photo: Thomas Mark Heller, RBG Kew).  Figure 6. Eugenia stirpiflora being propagated by cuttings at J.R. O'Neal Botanic Garden on Tortola, BVI (Photo: Thomas M. Heller, RBG Kew).  Range extension and re-assessment of Eugenia stirpiflora (O.Berg) Krug ... 5 diversity within the population. The inbreeding coefficient (F) was 0.080 ± 0.009, suggesting a slight deficit of heterozygotes relative to Hardy–Weinberg expectations. The moderate levels of genetic diversity observed in Eugenia stirpiflora (H = 0.278) are comparable to those reported in other narrowly endemic plant species studied with target capture SNPs (Liang et al. 2025, Razanamaro et al. 2025). Similarly, Slimp et al. (2021) reported H values between 0.23 and 0.31 in several range-restricted taxa using a comparable Angiosperms353-based pipeline. The inbreeding coefficient for E. stirpiflora (F = 0.080) suggests only mild inbreeding pressure, aligning with values typical for outcrossing species with small census sizes. These preliminary findings suggest that the BVI population of E. stirpiflora retains appreciable genetic variation despite its geographically restricted distribution. The analysis of a single population limits inference on overall species-level diversity and genetic structure; therefore, we recommend the inclusion of additional samples from USVI to cover the species’ distribution range. Such broader sampling will enable an assessment of genetic connectivity, population differentiation and potential local adaptation, all of which are critical for providing information for conservation strategies under ongoing habitat disturbance and climate change. Methods Information on the species' distribution was gathered from digitised herbarium collections held at K, field observations between 2020 and November 2026 and from previous IS E E IS Figure 7. Monitoring plot with tagged Eugenia stirpiflora trees on St. John, USVI (Photo: Clare Weaver, VIRPI).  6Bárrios S et al assessments by Ray (2018), Rivers and Ray (2021). Field surveys have also provided additional information on habitat, main threats and population sizes. The extent of occurrence and the area of occupancy were calculated with GeoCAT (Bachman et al. 2011). The conservation assessment evaluation followed the IUCN Red List Categories and Criteria and guidelines (IUCN Standards and Petitions Committee 2024). For the conservation genetic studies, DNA was extracted from silica gel-dried leaf tissue using a modified Cetyltrimethylammonium bromide (CTAB) protocol following Doyle and Doyle (1987), with all extractions conducted at the Jodrell Laboratory, Royal Botanic Gardens, Kew, UK. Genomic library preparation used 200 ng of input DNA and followed the protocol outlined in Viruel et al. (2019), incorporating half reaction volumes from the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, MA, USA). Indexing was performed using NEBNext® Multiplex Oligos for Illumina® (Dual Index Primer Sets I and II) and purification was achieved using AMPure XP magnetic beads. Enrichment for target loci employed the Angiosperms353 bait set (Johnson et al. 2019, Baker et al. 2022), following the myBaits® manual version 5.03 (Arbor Biosciences). The hybridisation pool consisted of all ten libraries combined in equimolar concentrations. DNA concentrations were assessed with a Quantus™ fluorometer (Promega Corp.) and fragment size distribution was verified using the Agilent 4200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). Libraries were sequenced on an Illumina HiSeq platform at Macrogen (Seoul, South Korea) using 150 bp paired-end reads. Raw genomic data were deposited in the ENA repository prior to publication. Raw reads were assessed for quality using FastQC (Andrews, 2010) and MultiQC (Ewels et al. 2016). Adapter sequences and low-quality bases (LEADING:30, TRAILING:30) were removed using Trimmomatic (Bolger et al. 2014). Cleaned reads were aligned to the Mega353 target reference (McLay et al. 2021) using BWA (Li and Durbin 2009) and assembled de novo with SPAdes (Bankevich et al. 2012) via the HybPiper pipeline (Johnson et al. 2016). Introns were recovered using the intronerate option in HybPiper. To enable SNP discovery, the longest intron per gene was selected to create a reference file. Variant calling followed the pipeline developed by Slimp et al. (2021), modified according to Razanamaro et al. (2025) to integrate Base Quality Score Recalibration (BQSR) using GATK (McKenna et al. 2010). Reads were aligned to the intron reference using BWA, PCR duplicates were removed with GATK and variants were called with GATK’s variant discovery tools (Poplin et al. 2018). SNPs were filtered using the following quality parameters: QD < 5.0, FS > 60.0, MQ < 40.0, MQRankSum < -12.5 and ReadPosRankSum < -8.0. To reduce linkage, SNPs were pruned in PLINK (Chang et al. 2015) using the --indep 50 5 2 setting. Genetic diversity indices, including allelic richness, observed (H) and expected heterozygosity (H) and inbreeding coefficient (F), were calculated in GeneAlEx (Peakall and Smouse 2012). Sequence data generated has been deposited in the European Nucleotide Archive (ENA), with the accession number PRJEB96302. O E IS Range extension and re-assessment of Eugenia stirpiflora (O.Berg) Krug ... 7 Species Conservation Profile Eugenia stirpiflora (O.Berg) Krug & Urb. Species information Synonyms Eugenia earhartii Acev.-Rodr. in Brittonia 45: 133 (1993) Myrciaria stirpiflora O.Berg in Linnaea 30: 702 (1861) Common names Earhart's Stopper Taxonomy Kingdom Phylum Class Order Family Plantae Tracheophyta Magnoliopsida Myrtales Myrtaceae Taxonomic notes According to Flickinger et al. (2022), the review of their type specimens and similar descriptions of Eugenia stirpiflora and Eugenia earhartii, which were both described from St. John, suggests they are the same species and supports their synonymisation. Region for assessment: - Global Reviewers Beech, E.; Wen, L.Y. Editor Barrios, S.; Ray, G.; Rivers, M. Geographic range Biogeographic realm: - Neotropical 8Bárrios S et al Map of records (Google Earth): Basis of EOO and AOO: Min Elevation/Depth (m): Max Elevation/Depth (m): EOO (km2): Trend: Causes ceased?: Causes understood?: Causes reversible?: AOO (km2): Countries: - Virgin Islands, British - Virgin Islands, U.S. Suppl. material 1 Observed Basis (narrative) The extent of occurrence (EOO) was calculated to be 20 km and the area of occupancy to be 12 km , based on a 2 × 2 km cell size, using GeoCAT (Bachman et al. 2011). 30 257 Range description Eugenia stirpiflora is a shrub or small tree, endemic to the British (BVI) and the US Virgin Islands (USVI). Thought to be restricted to the island of St. John in the USVI (AcevedoRodriguez 1996, Lindsay et al. 2015), this species was discovered on the island of Virgin Gorda in the BVI in 2020 (Dani Sanchez et al. 2020). Vouchers and subsequent surveys conducted in 2022, 2023 and 2024 have now confirmed the identity of this species at this location (Heller 2022, Barrios 2023). On St. John in the USVI, the species is known to be extant at three localities: Europa Bay, including the White Cliffs area, Minna Hill and Upper John's Folly. On Virgin Gorda in the BVI, this species is known only from Fanny Hill, north of Gorda Peak (Suppl. material 2). Extent of occurrence 20 Unknown Unknown Unknown Unknown Area of occupancy 12 2 2 Range extension and re-assessment of Eugenia stirpiflora (O.Berg) Krug ... 9 The survey work which took place in the BVI, was funded by the UK Darwin Plus Initiative (DPLUS084 and DPLUS183) and the HSBC 150th Anniversary Fund, which supported the Tropical Important Plant Areas in the British Virgin Islands Project (2016-2019). We also would like to thank the U.S. National Parks Service for issuing the relevant permits to the VIRPI for plant surveys to take place on St. John (Permit numbers: VIIS-00009 & VISS-2025-SCI-0007) and for providing photos from camera traps set in the U.S. National Park (St John). Author contributions SB: Conceptualisation, Methodology, Formal analysis, Investigation, Data curation, Writing-Original Draft, Writing, Review & Editing. CW: Investigation, Data curation, Writing, Review & Editing. EG: Investigation, Writing, Review & Editing. TH: Investigation, Writing, Review & Editing. MAH: Investigation, Writing, Review & Editing. MC: Investigation, Writing, Review & Editing. KG: Investigation. JV: Investigation, Writing, Review & Editing. AB: Data curation, Writing, Review & Editing. NWP: Investigation. CC: Conceptualisation, Investigation, Writing, Review & Editing. All authors have reviewed the final version of the manuscript. 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