Journal of Systematics and Evolution

This investigation aimed at compiling all phylogenetic lineages within and around genus Cyanoboletus. The evolutionary inference obtained from the nuclear ribosomal genes internal transcribed spacer region (ITS) suggests that part of the species currently classified in Cyanoboletus belong in lineages separate from the genus, thus suggesting a narrower boundary that includes only the species that develop a strong staining reaction to touch and to air exposure of the context. The separate lineages are the monotypic Cupreoboletus genus and a few species that do not develop such reaction, which are part of a clade together with genera Cacaoporus and Acyanoboletus, thus broadening the concept of Cacaoporus to encompass all of them. The emerging 3C perspective of Cupreoboletus, Cacaoporus and Cyanoboletus offers a remarkably consistent morphological diagnosis, overcoming the problems of a too broad concept for Cyanoboletus. This work reveals that Boletus neotropicus, B. novae-zelandiae and B. sensibilis belong respectively in Cyanoboletus, Cacaoporus and Lanmaoa, and by studying multigene alignment concatenates it identifies lineages that probably represent undescribed species: at least four in Cacaoporus and at least five in Cyanoboletus. Diagnostic tables and dichotomic keys are presented by geographic region. The present work also includes a study of the phylogenetic position of Neoboletus flavosanguineus, a species once classified in Cyanoboletus. The complexity of assigning species epithets in some lineages is addressed, namely for the boundaries between Cacaoporus instabilis and Ca. fagaceophilus as well as the diversity under the names Cyanoboletus sinopulverulentus and Cy. pulverulentus. The overall picture of evolutionary lineages sets a framework for the choice of reference data that can provide, in future phylogenetic studies that involve the 3C, a balanced and efficient coverage. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/724631v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@7f618corg.highwire.dtl.DTLVardef@dd6a14org.highwire.dtl.DTLVardef@5f7399org.highwire.dtl.DTLVardef@9e7443_HPS_FORMAT_FIGEXP M_FIG C_FIG

The diversity of the brown frog genus Rana may be underestimated as the high similarity of morphological characters. A new species belonging to the genus Rana is delineated based on eight specimens obtained from the Tianma National Nature Reserve, Jinzhai County, Luan City, Anhui Province, China. The phylogenetic analysis based on three mitochondrial genes (12S, ND2, and Cyt b) and one nuclear gene (BDNF) showed that the new species formed an independent clade closely related to R. culainensis and received strong support. In addition, morphological differentiation confirmed the phylogenetic results, and both supported the validity of a new species (Rana tianmaensis sp. nov.) in the R. japonica species group. The discovery of this new species enhances peoples understanding of the biodiversity of Rana and can provide important foundational data for scientific decision-making on protected area construction, ecological conservation, and species diversity. With the inclusion of newly described species in this study, the distribution of Rana genus in China now includes 31 recognized species.

O_LIPlants and fungi are major sources of natural products beneficial to society, making the study of distinct species essential for discovering new drugs and bioactive compounds. The medicinal mushroom "Lingzhi" or "Reishi" (Ganoderma lingzhi) is widely used in traditional medicine and extensively studied for its bioactive triterpenoids, yet it is commonly identified as Ganoderma lucidum, the type species of the genus, which lacks a type specimen. C_LIO_LIWe sequenced a G. lucidum specimen preserved in the Kew fungarium, which agreed with the original description and was collected from wood of Corylus avellana in southern England. Using this reference specimen, we compiled genomic and ITS barcoding datasets to explore the genetic and geographic variation within this species. C_LIO_LIWe showed that G. lingzhi and G. lucidum diverged more than 12 million years ago and that all seven "G. lucidum" genomes deposited in public databases belong to other species. More than 1000 barcoding sequences showed that the widely used homology-based ITS barcoding is not working in this group, which can be mitigated by a phylogenetic placement approach. The 149 sequences assigned to G. lucidum with high confidence showed a Eurasian distribution and introductions to North and South America and Africa. C_LIO_LIOur study underscores the importance of accurate species identification and provides guidance for a group of pharmaceutical and socially significant species. To further support future studies and the wider public in differentiating between G. lingzhi and G. lucidum, we propose using "False Lingzhi" as the English name for G. lucidum. C_LI Societal Impact StatementTraditional Chinese Medicine has expanded far beyond Asia, with growing markets in North America and Europe for supplements and functional foods. Lingzhi or Reishi (Ganoderma lingzhi), a well-known medicinal mushroom, is valued for its anti-inflammatory and anticancer properties. However, it is often misidentified with species that may not provide the same health benefits. This confusion poses risks to consumer safety, product regulation, and research. Here, we establish a reference using morphological and molecular tools for the most commonly misidentified species (Ganoderma lucidum) and propose the name "False Lingzhi" to distinguish it, supporting accurate identification, safer product development, and reliable research.

Understanding evolutionary relationships in hyperdiverse plant groups remains a major challenge in systematics. The orchid genus Bulbophyllum, the second largest genus of flowering plants, represents an exceptional example of phylogenetic and morphological complexity. Relationships, particularly within the species-rich Asian clade, have remained poorly resolved due to extensive morphological variation and limited resolution in previous phylogenetic studies. Here, we reconstructed phylogenetic relationships using 63 plastid genes from 355 specimens representing 322 species and 65 of the 97 recognised sections of Bulbophyllum. Our analyses confirmed that the genus comprises five major evolutionary lineages comprised of species predominantly from Australasia, Madagascar, Continental Africa, Neotropics, and Asia. We provide the first robust phylogenetic evidence for a dichotomous split within the Asian clade into two well-supported lineages: the Asian-Malesian clade and the Malesian-Papuasian clade, with the latter containing a strongly supported Papuasian subclade. Additionally, this study supports the monophyly of several currently recognised sections while clarifying relationships in previously problematic groups. This study provides the most comprehensive plastid-based phylogenomic framework for Bulbophyllum to date and establishes a foundation for future taxonomic revision and integrative analyses of diversification and trait evolution within this hyperdiverse genus.

Forest trees pose numerous potential challenges to phylogenomic inference. Their large effective population sizes and relatively long generation times lead to deep allele coalescence and consequently incomplete lineage sorting (ILS), which biases inferences of divergence times toward older ages and introduces gene tree discordance. Deep phylogenetic divergences, reaching back into the Paleocene, introduce reference-mapping biases. Introgression--the movement of genes between lineages--may result in different phylogenies being inferred depending on which individuals are included in analysis, even if the plurality of the genome favors the divergence history unaffected by introgression. These factors influence phylogenetic inference across the Tree of Life but are particularly prevalent in forest trees. Oaks (Quercus) are notable for all three influences. In addition, our knowledge of the oak phylogeny is currently based strongly on restriction site associated DNA sequencing (RADseq) datasets published over the past decade, which may introduce additional sources of uncertainty. In this chapter, we analyze a 322-species RADseq dataset and genome resequencing data from across the genus to address sources of uncertainty in our understanding of the global oak phylogeny, which we hope will serve as a model for other research groups working on comparable woody plant groups.

Although genetic diversity is a fundamental component of biodiversity, we lack data for a majority of species, particularly in biodiversity hotspots such as tropical forests. We present a comparative genetic dataset of 19 tropical tree species (including one palm) from the Caribbean island of Puerto Rico and neighboring islands (Hispanola and the US Virgin Islands). Using a reduced-representation sequencing technique (SLAF-seq), we identified species-specific single-nucleotide polymorphism (SNP) datasets with 24,413 to 433,637 high-quality SNPs per species. The focal species represent a range of life-history and climate associations, which may be relevant to their genetic structure. Therefore, we also include complementary information on species functional traits (wood density, leaf thickness, specific leaf area, maximum height, and seed dry mass), as well as geographic distributions and climatic associations from species distribution models.

The remarkable diversity of life on Earth results from evolutionary processes functioning across different spatial and temporal scales. Species diversification occurs through various mechanisms and at widely varying rates, but identifying the conditions that trigger bursts of diversification over short timescales remains a central challenge in evolutionary biology. This difficulty is more pronounced when incomplete lineage sorting (ILS), hybridization, and ongoing gene flow obscure evolutionary relationships and complicate species delimitation. In this study, we investigated the evolutionary history and species boundaries within a group of recently diverged Petunia lineages shaped by pervasive gene flow. We integrated phylogenomic, population genetic, and species delimitation approaches to reconstruct lineage relationships and assess whether these lineages represent distinct species or stages along a speciation continuum. By applying methods that account for both ILS and gene flow, we revealed that most lineages are not fully independent evolutionary units but rather occupy intermediate positions along this continuum. Gene flow played a crucial role during diversification, blurring species boundaries and generating reticulate evolutionary patterns. Our findings demonstrate that traditional phylogenetic trees may oversimplify relationships in such systems, while phylogenetic networks offer a more accurate representation of evolutionary history. Comprehensive and integrative analyses, such as those employed here, are essential for capturing these complex dynamics. Ultimately, only four lineages could be confidently recognized as distinct species, whereas the remaining represent cases of ongoing divergence. These results emphasize the need to refine species delimitation frameworks for systems characterized by recent divergence and extensive reticulation.

AimThe commercial interest of grapevines (Vitis vinifera L.) has prompted numerous studies on their origin and genetic resources in the context of global change. However, genomic-scale information on diversity patterns and genetic structure in southwestern Europe remains scarce. This study infers the genetic structure, gene flow events between genetic groups, and genetic refugia of Vitis vinifera ssp. sylvestris in the Iberian Peninsula. LocationThe Iberian Peninsula. TaxonThe wild grapevine, Vitis vinifera L. ssp. sylvestris MethodsWe reanalyzed a set of 137 complete genomes of V. vinifera ssp. sylvestris. After variant calling, validation and annotation, we obtained a high-quality SNP dataset. Using these markers, we performed phylogenetic and population structure analyses to determine the number and spatial distribution of genetic groups and their contact zones. Next, we inferred the timing and directionality of gene flow events between groups. Finally, heterozygosity and allele rarity were estimated to identify populations with high conservation value. ResultsWe detected three major ancestral populations and four putative genetic refugia in the south of the Iberian Peninsula. Demographic analyses indicate sustained gene flow between [~]21,000 and [~]7,000 years ago from a North African ancestral group into Iberian wild populations in the south. Heterozygosity and allele rarity analyses identified populations of high conservation value in a variety of areas within the Iberian Peninsula. Main ConclusionsWe identify the biogeographical factors behind the long-known singularity of wild Iberian grapevines. The southern Iberian Peninsula is a hotspot of genetic diversity for wild grapevines, hosting three ancestral populations and multiple contact zones that acted as micro-refugia. The current genetic variability of Iberian wild grapevines is best explained by natural, climate-driven gene flow between African lineages with Middle Eastern origin and Iberian groups. These contacts were favored by climatic conditions during the late Pleistocene ([~]21,000 years) and early Holocene ([~]8,300 years). Our results dismiss a significant anthropogenic influence during Neolithic domestication for explaining the genetic composition of Iberian wild grapevine genotypes.

We present a genome assembly from a specimen of Quercus canariensis (Fagaceae; Fagales; Magnoliopsida). The assembly was generated using PacBio HiFi long reads with an approximate sequencing depth of 39X and scaffolded using a reference-guided approach. The genome sequence has a total length of 816.0 megabases for haplotype 1 and 804.8 megabases for haplotype 2. The two haplotypes are each resolved into 12 chromosomal pseudomolecules, with only 3.48% and 1.36% of sequences remaining unplaced in haplotypes 1 and 2, respectively. Assembly completeness is supported by BUSCO scores of 98.3% and 98.2% complete genes for haplotypes 1 and 2, respectively. Structural annotation identified 51,882 and 46,482 protein-coding genes in haplotypes 1 and 2, respectively. This genome assembly provides the first chromosome-scale reference genome for Q. canariensis, laying the base for future genomic and evolutionary studies in this understudied species of the hybridizing white oak species complex. TaxonomyLineage cellular organisms; Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae; rosids; fabids; Fagales; Fagaceae; Quercus EBI:txid568684 Quercus canariensis Willd. 1809 (Willdenow)

Phylogenetic trees represent the evolutionary histories of taxa and support tasks such as clustering and Tree of Life reconstruction. Many established comparison methods, including the Robinson-Foulds (RF) distance, assume identical taxon sets. A methodological gap remains for trees with distinct but overlapping taxa. Existing approaches either prune non-common leaves, which can discard information, or complete both trees such that they share the same taxa. Completion is more comprehensive, but current methods typically ignore branch lengths, which are essential for identifying evolutionary patterns. This paper introduces k-Nearest Common Leaves (k-NCL), an algorithm for completing rooted phylogenetic trees defined on different but overlapping taxa. The method uses branch lengths and topological characteristics and does not rely on a specific distance measure. The k-NCL algorithm is designed to preserve evolutionary relationships in the trees under comparison. The running time is O(n2), where n is the size of the union of the two leaf sets. Additional properties include preservation of original distances and topology, symmetry, and uniqueness of the completion. Implemented in Python, k-NCL is evaluated on biological datasets of amphibians, birds, mammals, and sharks. Experimental results show that RF combined with k-NCL improves phylogenetic tree clustering performance compared to the RF(+) tree completion approach. Availability and implementationAn open-source implementation of k-NCL in Python and the datasets used in this study are available at https://github.com/tahiri-lab/KNCL.

The combination of target capture sequencing (TCS) with low-coverage whole genome sequencing (lcWGS), an approach known as Hyb-Seq, has allowed the integration of natural history collections into the genomics revolution, transforming biodiversity research. To implement Hyb-Seq, a collection of genomic targets, often nuclear orthologs, is needed to design probes for TCS. In flowering plants, the universal Angiosperms353 probe set has been proven resolutive at multiple evolutionary scales, with caveats. Malpighiales is known to be one of the most challenging flowering plant orders to resolve. Within this order, the clusioid clade ([~]2.2K species, 94 genera, five families) is no exception. To resolve phylogenetic relationships in this recalcitrant clade, we design a custom probe set, the Clusioids626 kit, composed of 39,936 120-mer probes targeting 626 nuclear orthologs ([~]6.6M nucleotides). This probe set includes all Angiosperms353 targets and 273 clusioid-specific ones, carefully chosen taking copy-number, length evenness, and phylo-informativeness into account. We test our probe set on 70 accessions representing all families and tribes in the clusioid clade. On average, 50.4% of TCS reads mapped to our targets, recovering a median of [~]600 orthologs. Relationships for all clusioid families are fully resolved for our nuclear targets. Additionally, 105 plastid coding DNA sequences were retrieved from the lcWGS fraction. A strong cyto-nuclear conflict was detected. The Clusioids626 kit performs better than the universal Angiosperms353 enrichment panel alone. Our kit design workflow can be extended into other lineages for which a universal probe set exists but more resolution is needed.

Phylogenetic network inference methods are increasingly used to detect hybridization and gene flow from genomic data, but their robustness to common sources of model violation remains poorly characterized. We conducted a simulation study to evaluate the effects of hidden paralogy and substitution rate variation on two widely used network inference methods: find_graphs from ADMIXTOOLS 2 and SNaQ. Using an eight-taxon species tree calibrated from an empirical reptile phylogeny, we simulated data under various levels of hidden paralogy (from none to strong) and three levels of rate variation (none, gene-specific, and lineage-specific). We found that hidden paralogy had limited impact on network inference under the conditions examined: both network methods correctly favored a tree without reticulation, and ASTRAL recovered the correct species tree every time. In contrast, lineage-specific rates severely biased find_graphs, inflating worst f-statistic residuals well beyond the standard acceptance threshold. SNaQ correctly selected a tree model almost always across all conditions, though its network with h = 1 reticulation displayed the true species tree with a lower probability under lineage-specific rates. We also show that the standard worst residuals threshold of 3 for find_graphs produces inflated type I error even without rate variation, and we recommend empirical calibration of this threshold within each study system.

Renowned for its aromatic fruits and economic importance, the genus Vanilla poses longstanding taxonomic and phylogenetic challenges. Despite recent molecular studies, a comprehensive species tree is lacking, and the evolutionary processes and historical patterns shaping the genus remain poorly understood. We present a new, comprehensive phylogenomic framework for Vanilla, based on 349 low-copy nuclear genes and 76 plastid loci from the Angiosperms353 probe set, which we used to infer evolutionary relationships, assess cyto-nuclear and gene-species tree discordance, and thoroughly investigate its historical distribution and diversification. Sampling 43% of the genus, our framework resolves phylogenetic uncertainties, clarifies major clades, confirms prior hypotheses, and reveals novel placements, including V. planifolia and Vanilla subg. Gondwana. Discordances are primarily driven by incomplete lineage sorting, particularly in the vanillin-producing clade, with evidence of both ancient and recent hybridization, including a natural hybrid from the Yucatan Peninsula. Biogeographic analyses indicate a Guiana Shield origin ([~]30 Mya), Amazonia as a major diversification source, the Andes as a permeable barrier, and Central America as the main diversification sink. This study provides a robust evolutionary framework for Vanilla, supporting taxonomic revisions, comparative trait analyses, and a deeper understanding of the processes shaping this economically and biologically important orchid genus.

If youve ever complained about a species name thats a mouthful--say, the soldier fly Parastratiosphecomyia stratiosphecomyioides or the myxobacterium Myxococcus llanfairpwllgwyngyllgogerychwyrndrobwllllantysiliogogogochensis--youre in very good company. But could the readability of binomial scientific names cause more than complaints? Could it influence how much species are studied and talked about? We examined a random sample of 3,019 species names spanning 29 phyla/divisions. We tested whether name length and reading difficulty are associated with species representation in the scientific literature (measured via literature mentions) and their visibility to the public (measured via Wikipedia pageviews). Both species name traits showed significant negative relationships with literature mentions and Wikipedia reads. Increasing name length from 10 to 30 characters is associated with a 66% decrease in expected mentions and a 65% decrease in Wikipedia reads, while shifting from the most to the least readable name in the dataset corresponds to 53% and 76% decreases. These patterns are consistent with something familiar: the fickleness of human attention, responding to features of the world that are far from rational. While creativity in naming is a cherished part of taxonomy, a touch of orthographic restraint may ultimately benefit both science and the species themselves--especially among understudied uncharismatic taxa.

Wild bees face declines, and forests may serve as critical habitats for pollinators. However, how forest composition and the associated floral environment shape pollen provisioning and resource partitioning among cavity-nesting bees remains poorly understood. Here, we leveraged BEF-China, a large-scale subtropical forest biodiversity experiment with experimentally controlled plant (tree and shrub) communities, to investigate how forest composition and spatial context shape pollen provisioning, resource partitioning, and reproductive success of cavity-nesting bees. We used DNA metabarcoding to analyze floral composition of pollen provisioned by five cavity-nesting bee species, with samples collected from BEF-China across three years (2022- 2024). By comparing pollen taxonomic composition from whole-nest pooled samples and individual brood-cell samples with the experimentally planted species pool, we characterized dietary patterns and temporal dynamics of five bee species. Bees primarily relied on floral resources from the surrounding landscape, with planted trees providing essential but temporally restricted pollen supplements during specific phenological stages. Co-occurring bee species exhibited staggered nesting phenology and distinct dietary preferences for different plant families, with fine-scale resource differentiation even during periods of phenological overlap. Our results suggest that managed forests support cavity-nesting bees by providing critical woody floral resources during specific phenological gaps and offering stable nesting environments. To mitigate pollinator declines, forest management should prioritize maintaining diverse, phenologically complementary flowering vegetation within and surrounding forest stands. This ensures temporal continuity of pollen availability throughout the nesting season, which is particularly crucial for restoring pollinator services in simplified forest landscapes.

Characterized by the earliest use of pottery, the Jomon culture was a unique Neolithic culture that spread throughout the Japanese Archipelago. Previous archaeological evidence suggests that Jomon hunter-gatherers colonized the southernmost islands, the Ryukyu Archipelago, by approximately 7,000 years before present (YBP). However, genetic characteristics of the Ryukyu Jomon population and its contribution to the modern population have not been elucidated yet. In this study, we newly sequenced 273 modern and 25 ancient (6,700-900 YBP) whole genomes collected across the Ryukyu Archipelago. Our analysis demonstrated a genetic differentiation between the Hondo (Japanese mainland) and Ryukyu Jomon, dating back to [~]6,900 YBP. After the divergence from the Hondo Jomon, the Ryukyu Jomon experienced severe bottlenecks, with an effective population size of [~]2,000. Admixture between the Ryukyu Jomon and migrants from the historic Hondo population occurred [~]1,000 YBP, which corresponds to the widespread adoption of iron tools and agriculture in the Central Ryukyus. Different demographic histories between modern Hondo and Ryukyu populations resulted in different rates of Jomon ancestry in these populations. By providing a new perspective on the peopling of the Ryukyu Archipelago, this study significantly enhances our understanding of cultural transitions in the region.

Background and AimsNon-native species are now ubiquitous members of regional floras. The factors that lead to establishment and dominance of non-native species are continuously debated. Fundamental hypotheses about drivers of invasion success include the role of phylogeny, polyploidy, genome size, and rapid niche evolution. These hypotheses have been tested in the seed plants, but ferns, the second largest group of vascular plants, have rarely been considered in these analyses, despite making up a non-trivial portion of non-native floras. MethodsWe compiled a dataset of global non-native ferns and categorized them along the invasion spectrum using descriptions from the literature and natural history collections. Using this dataset, we assessed I) the taxonomic diversity and phylogenetic clustering of non-native ferns, II) the geographic distribution of fern introductions, testing for shifts in climatic niches, and III) test for the association of invader traits across the invasion continuum, including smaller genome sizes and higher ploidal levels. Key ResultsWe generated a dataset that includes 83 taxa; of these, we classified 18 as casual, 35 as naturalized (but not invasive), and 30 as invasive. Using this dataset, we found I) weak or no phylogenetic clustering of non-native ferns, II) some regions are overrepresented as sources and recipients of introductions, III) climatic niches are often conserved between native and introduced ranges, but can differ between introductions, IV) naturalized ferns have smaller genomes, and V) invaders have higher ploidal levels. ConclusionsWe integrated regional floras, occurrence and climate data, phylogeny, and cytology to test fundamental hypotheses regarding the colonization success of ferns. This study provides insights into the ecological, genomic, and phylogenetic features associated with the colonization of new habitats by non-native ferns, a largely overlooked portion of non-native plant taxa.

Invasive species pose a major threat to biodiversity, yet our understanding of failed invasions/translocations, instances where alien/introduced species fail to establish, remains limited. Investigating the factors behind failed invasions is critical for improving prevention and management strategies for modern biological invasions. Here, we propose a novel framework that utilizes archaeological archives to uncover evidence of failed invasions. We estimated the biological and ecological factors contributing to the failed invasion of pigs from later prehistory to recent times (299 cal BC to 1900 AD) on Rebun Island in far northern Japan by synthesizing the evidence obtained from stable isotopes, zooarchaeology, and historical documents. Despite the anthropogenic introductions of pigs into Rebun Island, pigs did not establish a feral population and disappeared after ca. 1200 AD. We identified reduced propagule pressure, abiotic resistance due to the cold climate, and decreased resources as the three key factors that contributed to the disappearance of pigs. Pigs are one of the most widespread invasive species globally, and this study represents a novel approach to studying failed invasions using archaeological data, which aligns with the framework of conservation paleobiology.

For over four decades, the bivalve Anomalocardia flexuosa has been recorded in Hong Kong coastal waters. However, the known native distribution of this heavily exploited commercial species is restricted to the Atlantic coast of South America, raising questions about the biogeographical validity of the Hong Kong populations. By employing an integrative taxonomic approach combining morphological re-evaluations and molecular phylogenetic analysis of the COI gene, we confirm that the species in Shui Hau, Hong Kong, China, has been historically misidentified. The population belongs to Cryptonema producta (syn. Anomalocardia producta).

Background and aimsThis study combines morphological and environmental data to better understand a Brunfelsia (Solanaceae) species complex, aiming to clarify patterns of variation and identify ecological factors that shape morphotype boundaries. Such an approach provides a broader perspective on how organisms respond to environmental gradients and contributes to a more comprehensive understanding of biodiversity. MethodsWe analyzed 273 herbarium specimens for 13 morphological traits using univariate and ordination analyses, namely PCA and CVA. Climatic and edaphic variables were extracted for 147 specimens with georeferenced records. To assess habitat suitability and the ecological niche of each predefined morphotype, niche models under present conditions and niche overlap tests were conducted. A redundancy analysis (RDA) was applied to evaluate how environmental predictors explain variation in vegetative and floral traits. Finally, DAPC was used to estimate membership probabilities based on morphological and environmental data. Key ResultsTwo well-differentiated groups were recovered: the capitata-hydrangeiformis morphotype, allegedly composing a cline, and the ecologically and morphologically distinct "bahia" morphotype. Variation in floral traits was better explained by environmental predictors than variation in vegetative traits; moreover, floral traits were able to delineate morphotypes more robustly when plotted in isolation. However, when analyzing the results of ecological niche overlap, a significant ecological separation of the "bahia" morphotype from the others was observed. Therefore, key morphological characters for the taxonomy of Brunfelsia covary in part with environmental variables. ConclusionsThese findings support the recognition of "bahia" morphotype as a distinct species to be formally described. This integrative approach contributes to understanding diversification processes in biodiversity hotspots and highlights hidden taxonomic diversity within Brunfelsia, where many rare and narrow-endemic taxa lie.