Distinct mycorrhizal communities in sympatric Lepanthes orchids revealed by long-read sequencing

ABSTRACT Mycorrhizal fungi form essential mutualisms with the majority of land plants, yet their role on plant community composition and diversification remains poorly understood. Orchids provide a model system for studying these interactions because the orchid life cycle obligatorily depends on mycorrhizae. Historically, studies have emphasized the role of niche partitioning and competition avoidance resulting in distinct mycobiome compositions among coexisting orchids. Since closely related orchid species have been found to associate with similar groups of fungi, it has been speculated that different fungal species are needed for coexistence but not for speciation. However, fungi have often been examined at lower resolution levels (i.e., class, order, family, or genus). Using third-generation long-read sequencing (PacBio) of the full ITS region, we characterized fungal communities at fine scale with high accuracy. We evaluated alpha- and beta-diversity of fungal communities in four closely related, narrowly endemic epiphytic orchid species from the rapidly diversifying genus Lepanthes in one of the world’s richest biodiversity hotspots. Our analyses reveal that orchid species have distinct mycobiont assemblages, with differences in composition unevenly distributed across species. These results suggest that shifts in fungal partners may contribute to speciation and rapid diversification in Lepanthes. This study highlights the potential evolutionary role of mycorrhizal fungi in orchid diversification and demonstrates the value of high-resolution sequencing in uncovering cryptic fungal diversity. Competing Interest Statement The authors have declared no competing interest. Footnotes Data availability statement: All sequence data generated in this study have been deposited in publicly accessible repositories. PacBio reads are available in the NCBI Short Read Archive (SRA) under BioProject PRJNA1345165, accession numbers: SRX31179501 - SRX31179909. Scripts, along with input metadata, feature tables and ASV FASTA files necessary to conduct analyses are available in UGA Open Scholar repository (DOI: https://doi.org/10.71927/uga.27592). Processed fungal ITS sequence data and metadata used for diversity analyses will be made publicly available in Dryad Digital Repository following acceptance of the manuscript (DOI: [insert DOI]). All analysis scripts and R code used for data processing and statistical analyses will be also available in Dryad upon publication (DOI: [insert DOI]). The precision of geographic coordinates in metadata has been reduced to prevent misuse and for species protection. Funding statement: This research was supported by the American Orchid Society, Botanical Society of America, Tinker Foundation, and the UGA Plant Biology Department. Additional logistical and field support was provided by Centro Científico Tropical, Reserva Biológica del Bosque Nuboso de Monteverde, and Jardín Botánico Lankester, Costa Rica. The funders had no role in study design, data collection, data analysis, or manuscript preparation. Conflict of interest disclosure: The authors declare that they have no competing interests or conflicts of interest related to the research, authorship, or publication of this work. Ethics approval statement: All field and collection activities were conducted under the research permits issued by the Costa Rican Ministry of Environment and Energy (MINAE) through Centro Científico Tropical (permit numbers M-P-SINAC-PNI-ACAT-027-2019, M-P-SINAC-PNI-ACAT-033-2021; CITES export permits: 2019-CR4603/SJ, 2021-CR52292/SJ). Import permit was issued by USDA (permit number P37-19-00225). No protected species were harmed, and all procedures complied with local and institutional ethical guidelines for the collection of plant material.