Plastome phylogenomics of the Diverse Neotropical Orchid Genus Lepanthes with Emphasis on Subgenus Marsipanthes (Pleurothallidinae: Orchidaceae)

Plastome phylogenomics of the Diverse Neotropical Orchid Genus Lepanthes with Emphasis on Subgenus Marsipanthes (Pleurothallidinae: Orchidaceae) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Plastome phylogenomics of the Diverse Neotropical Orchid Genus Lepanthes with Emphasis on Subgenus Marsipanthes (Pleurothallidinae: Orchidaceae) Tatiana Arias, Juan Sebastian Moreno, Sebastian Reyes, Martin Llano Almario, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5738250/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Aug, 2025 Read the published version in BMC Ecology and Evolution → Version 1 posted 15 You are reading this latest preprint version Abstract The first successful resolution of phylogenetic relationships within main lineages in the diverse Neotropical orchid genus Lepanthes Sw. is presented here. Genome skimming produced ten newly sequenced chloroplast genomes, with additional plastome coding genes (17–86) retrieved from GenBank, alongside 26 amplified matK and rITS genes, enabling phylogenetic reconstruction. The Lepanthes plastomes (157,185 − 158,260 bp, 37.15% GC content) contained 136 annotated genes, including 86 protein-coding, 42 tRNA, and 8 rRNA genes. Six hypervariable regions, including parts of the ycf1 gene, were identified as potential DNA barcodes. Phylogenetic analyses revealed that Carl Luer’s subgeneric classifications are non-monophyletic, reflecting significant morphological homoplasy. PCA and correlation analyses confirmed widespread homoplasy in continuous morphological characters. Six major clades were identified, though backbone resolution remains unresolved at two nodes of the phylogeny, requiring the use of nuclear markers or expanded sampling. Subgenus Marsipanthes species are non-monophyletic and constitute an East Andean early divergent clade with species from subgenus Lepanthes , while some derived Biogeographic Choco Marsipanthes clades were recovered, forming a polytomy with species from subgenus Lepanthes . The genus likely originated in southern Ecuador or northern Peru, dispersing across the Andes into the broader Neotropics. Although only a subset of Lepanthes diversity was sampled, the study captures significant taxonomic, geographic, and morphological variation. It provides foundational insights into the genus’s evolution, along with tools and hypotheses that can be expanded upon in future research to further refine our understanding of its evolutionary history. Andes Chloroplast Colombia Eastern Andes Biogeographic Choco Ecuador ycf1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The genus Lepanthes Sw. (Orchidaceae) stands out as one of the largest in the subtribe Pleurothallidinae [1], encompassing around 1,164 accepted species that are widely distributed through the Neotropics from Mexico to Bolivia and the Antilles, and from the Andes to the Amazon Basin [2–4]. The interplay between their specialized habitat requirements and the complex environmental gradients of the Andean region and the cordillera de Talamanca has made Lepanthes one of the most species-rich and endemic-rich genera in the Neotropics. This region is home to numerous endemic Lepanthes species, as highlighted in some studies [5]. The genus displays its highest diversity in Colombia and Ecuador, where around 300 species have been described from each country, 377 species have been recently reported for Colombia [6]. In this region, species cover an impressive altitudinal gradient from sea-level to the snow line of the Andes, close to 4,000 m in elevation [2,7]. These species can thrive in diverse habitats, from lowland areas along the Pacific Ocean and the Amazon foothills to the Sierra Nevada de Santa Marta and the cloud forests of the Andes. Lepanthes species predominantly inhabit ecosystems characterized by high humidity, constant air movement, and horizontal precipitation. They grow attached to litter on the forest floor or on various phorophytes, including shrubs and trees, from the main trunk to branches and even the canopy. This remarkable ecological flexibility, combined with the intersection of three major biodiversity hotspots - Mesoamérica, Chocó/Darién/Western Ecuador, and the tropical Andes [8–9] - and the narrow geographic distributions of many species [5], is proposed to be responsible for the extraordinary diversity observed in the genus. Lepanthes species are characterized by diminutive size, and stems called ramicauls enclosed in funnel-shaped sheaths known as lepanthiform sheaths which often display microscopic cilia or pubescence and variable apical dilation. The flowers typically exhibit transversely elongated, often bilobed petals and a compound lip comprised of a body, connectives, and two blades. The compound lip plays a crucial role in pseudocopulatory pollination [10]. Notably, these orchids possess some of the smallest flowers in the orchid family and within the genus, various morphological groups exhibit specific similarities [11–13]. Lepanthes are notoriously difficult to classify due to the subtle differences between species. Comprehensive molecular studies combine with thorough morphological documentation will provide effective species delimitation and a clearer species concept for this genus. Understanding the genetic diversity and structure within taxa that exhibit subtle morphological differences will be crucial in addressing these challenges. Taxonomy in the genus was developed by Carlyle Luer and collaborators using mostly morphological traits [14–18], and in recent years several molecular studies have been developed for Lepanthes species mostly from central American and the Caribbean [19–20]. It remains a research priority to conduct additional molecular studies across diverse taxa, to be able to critically address proposed taxonomic relationships. Despite hundreds of species distributed throughout diverse ecosystems of the Neotropics, Luer proposed the entire diversity of Lepanthes could be confined within two subgenera: subgenus Lepanthes and subgenus Marsipanthes [21] (Fig. 1 ). The subgenus Lepanthes encompasses the majority of species within the genus and is divided into three subsections, as defined by Luer [14–17]: Breves (including series Breves and Filamentosa ), distinguished by lateral sepals with a single vein; Lepanthes (comprising series Lepanthes , Elongatae , and Mucronatae ), characterized by lateral sepals with two or three veins; and Bilabiatae , in which the lateral sepals are fully fused into a two-veined synsepal. However, the concept of series within these subsections was later abandoned [2]. Subgenus Marsipanthes comprises eleven species distinguished by connate dorsal and lateral sepals with numerous veins forming an inflated tube and thick, fleshy, erect petals with one lobe - saccate flowers [22]. Within Marsipanthes , three sections are recognized: sect. Marsipanthes species have narrowly elongated petals, sect. Caprimulginae have short, transverse petals, and deeply connate sepals, and sect. Felinae have non-inflated flowers and asymmetrical petals with the upper lobe much longer than the lower lobe [7]. Sect. Caprimulginae includes Lepanthes caprimulgus Luer and a recently described species L. attenboroughii Baquero & Monteros [23]. Sect. Marsipanthes includes L. ribes Luer and L. portillae Luer, and sect. Felinae includes L. carunculigera Rchb.f., L. equicalceolata Luer & Escobar, L. felis Luer & Escobar, L. lucifer Luer & Hirtz, L. niessienae Luer and L. quadricornis Luer & Escobar [7, 21]. L. tulcanensis Baquero & Yeager was described in recent years, and based on morphological traits were also considered likely to belong to Marsipanthes sect. Felinae [24]. The current taxonomy of Lepanthes requires reevaluation within an evolutionary framework. Previous studies using few DNA molecular markers have failed to resolve relationships within the studied species [20], limiting our understanding of the genus origin and evolution and hindering the development of effective conservation strategies. Robust phylogenetic analyses based on tens of thousands of nucleotides, can greatly enhance our confidence in the resulting evolutionary hypotheses [25–27], besides adding to the understanding of species delimitation in the group. Plastomes have been widely used in orchid phylogenetics due to their high conservatism and relatively slow evolutionary rates [28]. However, the question remains whether large-scale datasets based on tens to hundreds of thousands of aligned nucleotides, representing both coding and non-coding regions, from the plastome possess enough phylogenetic signal to resolve evolutionary relationships in rapidly diversifying Andean orchids such as Lepanthes [29]. In this study, we employed high-throughput sequencing (HTS) and genome skimming techniques to examine infrageneric taxonomic classifications (e.g. subgenera, sections) and compare the findings with the morphological data. By analyzing the plastome, we aimed to achieve higher levels of sequence divergence to resolve phylogenetic relationships among species in the genus in comparison to what the traditional gene-by-gene approaches have allowed. We present complete plastome sequences of nine representative Lepanthes species to: 1) describe and interpret the plastome structure and evolution of main taxonomic groups within Lepanthes while providing molecular tools useful for the community; 2) reconstruct phylogenetic relationships among selected species of the Lepanthes backbone using plastid genome sequences, further concentrating sequencing efforts in subgenus Marsipanthes ; 3) test preliminary hypothesis about the biogeography and evolution of continuous morphological characters for representatives of the Lepanthes backbone. Methods 1. Taxon sampling, DNA extraction, amplification and sequencing : Fresh leaves from adult plants were collected in Colombia from Colomborquídeas in El Retiro, Antioquia, under a collection permit from the Corporación para Investigaciones Biológicas (resolution ANLA 1263) and CITES (permit number: 42664). We studied ten new plastomes representing valid Lepanthes species, using the circumscription and infrageneric classification of Luer [14, 17, 30] (Fig. 1 ). Additionally, 26 taxa from Ecuador were sampled for this study, 24 species of Lepanthes and two outgroups Pseudolepanthes colombiae Archila and Brachionidium valerioi Ames & C. Schweinf (Table S4 ) under a collection permit for Ecuador (MAATE-DBI-CM-2021-0187). For all species, fresh leaves were stored in silica gel for later DNA extraction using the CTAB method for species sampled in Colombia [31]. Total DNA was purified with silica columns (Epoch Life Science Inc. Missouri, Texas, USA) and eluted in Tris-EDTA. DNA concentration was measured using a fluorometer (Qubit Fluorometer, Invitrogen, USA), and the integrity and purity of the samples were verified through agarose gel electrophoresis. DNA from species sampled in Ecuador was extracted using a rapid extraction procedure [32]. For plastome sequencing, a 300-bp DNA library was constructed using the TruSeq Illumina platform (Illumina Inc., San Diego, CA, USA). A total of 1.5 µg of DNA was fragmented using a Covaris ultrasonicator (Covaris Inc., Woburn, MA), and the fragmented DNA was assessed via gel electrophoresis (Bio-Rad Laboratories, Hercules, CA). The fragmented DNA was treated with End Repair Mix (New England BioLabs, Ipswich, MA) and incubated at 20°C for 30 minutes. The end-repaired DNA was purified with the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany), then treated with A-Tailing Mix (Thermo Fisher Scientific, Waltham, MA) and incubated at 37°C for 30 minutes. The adenylated DNA was ligated with adapters using the Adapter and Ligation Mix, with the ligation reaction incubated at 20°C for 15 minutes. Adapter-ligated DNA was selected by running a 2% agarose gel to recover the target fragments, followed by purification with the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). Multiple rounds of PCR amplification were performed using a PCR Primer Cocktail and PCR Master Mix to enrich the adapter-ligated DNA fragments. The PCR products were again selected via 2% agarose gel and purified with the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). The final library was quantified by determining the average molecule length with an Agilent 2100 Bioanalyzer (Agilent DNA 1000 Reagents, Santa Clara, CA) and by real-time quantitative PCR (qPCR) using TaqMan Probes (Thermo Fisher Scientific, Waltham, MA). Libraries were multiplexed and sequenced on an Illumina HiSeq 4000 platform (San Diego, CA), producing paired end reads of 150 base pairs in length at the Beijing Genomics Institute (BGI), Hong Kong. Polymerase Chain Reaction (PCR) was used for amplification of two regions, nuclear ribosomal internal transcribed spacer (rITS) and plastid Maturase K (matK), with 7.5 µL GoTaq Green Master Mix 2X (Promega), 3 µL of extracted DNA, 7.5 µL ultra-pure water, and 1 µL of each primer (Table S1 ). PCR conditions were as follows: 2 min–95°C, 1 min–95°C, 1 min–55°C, 1 min–72°C, 35 cycles, and 5 min–72°C for final extension. PCR products were purified, and Sanger sequenced (ABI 3500xL Genetic Analyzer, Applied Biosystem). 2. Plastome assembly, annotation, and comparative analyses : In average 4.5 GB of high-quality data was obtained for each sample. To evaluate the quality of the reads and check for the presence of adapters or other contaminants, we used FastQC [33]. Based on this analysis, the reads were trimmed using Trimmomatic v 0.39 [34], removing reads with a Phred score below 33 (parameter “-phred33”) or a length shorter than 100 bp (MINLEN:100). Adapters within the reads (parameter “-ILLUMINACLIP”) and low-quality bases at the start (LEADING) and end (TAILING) of the reads with a Phred score less than 3 were also removed. The remaining paired-end sequences were merged with a maximum overlap of 100 bp (Geneious Prime 2021 v2.2). The quality-filtered reads were then assembled de novo using GetOrganelle v 1.7.5 [35], specifying chloroplasts as the target organelle (parameter “-F embplant_pt”). Gaps between scaffolds were closed through iterative mapping in the Geneious program (Geneious Prime 2021 v2.2) using default settings. Out of the ten sequenced plastomes, only nine were successfully assembled. The sequencing reads for Lepanthes eros were contaminated, and we were unable to assemble its plastome with confidence. The plastome sequences were deposited in GenBank (BioprojectID PRJNA1209138) and the raw sequence data were submitted to GenBank, receiving SRA accessions (Table S4 ). We annotated plastomes assembled using the Geneious annotation tool with default parameters and a 95% similarity threshold. The genomes of Anathallis obovata [36] and Lepanthes caprimulgus [28] were used as reference. Annotations were manually verified to ensure no stop codons within genes. Circular plastome maps for Lepanthes were created using OGDRAW [37]. Several open reading frames (ORFs) that did not match the initial annotations were identified using BLASTn [38] followed by BLASTx [39] searches in NCBI. 3. Plastome Structure and Sequence Divergence Analyses : To assess potential expansions and contractions of the IR boundary, the genes in the boundary regions of LSC/IRb/SSC/IRa were visualized using IRscope v3.1 [40]. Additionally, gene arrangement was analyzed through a collinearity analysis using the Mauve v1.1.3 plugin in Geneious v9.1.4 with default parameters. Sequence divergence of Lepanthes plastomes was examined using the online program mVISTA, with the Anathallis obovata plastome serving as a reference. Nucleotide diversity (Pi) values were calculated using DnaSP6 with a sliding window of 1000 sites and a step size of 25 sites [41]. 4. Plastome Identified Repetitive Sequence and Codon Usage Analyses : Three types of repeat sequences were analyzed in the plastomes: simple sequence repeats (SSRs), tandem repeats, and long repeats. SSRs were detected using MISA v2.1 [42] and visualized with the R package ggplot2. Tandem repeats were identified using Tandem Repeats Finder v0.9 [42]. Forward, reverse, complement, and palindromic long repeats were detected in REPuter [43] using default parameters. The Relative Synonymous Codon Usage (RSCU) ratio for nine Lepanthes plastomes was estimated using CodonW v1.4.2 [44]. An RSCU value greater than 1 indicates positive codon usage bias, while a value less than 1 indicates less frequent usage. The R package "pheatmap" was used to generate a heatmap for the RSCU analysis. 5. Phylogenetic Analysis : Nine Lepanthes species along with Anathallis obovata (Lindl.) Pridgeon and M.W. Chase, Draconanthes aberrans (Schltr.) Luer, Trichosalpinx dirhamphis (Luer) Luer and Zootrophion lappaceum Luer and R. Escobar as outgroups, were included in the phylogenetic analyses. Plastomes were aligned using the MAFFT software [45]. To further explore the phylogenetic relationships within Lepanthes , maximum likelihood (ML) analyses were conducted using RAxML version 7.4.2 [46] under the general time-reversible (GTR) substitution model. Bootstrap support was assessed with 1000 bootstrap replicates. Additionally, Bayesian inference (BI) analysis was performed using MrBayes with 20,000,000 generations and sampled every 1000 generations. The majority rule (> 75%) consensus tree was obtained after removing the first 25% of the sampled trees as “burn-in” [47]. Whole plastid phylogenies were reconstructed based on the following datasets: (1) complete plastid genomes, (2) different outgroup sets, (3) one inverted repeat (IR), (4) coding sequences, and (5) non-coding sequences. The Filamentosa series (Subgen. Lepanthes , Sect. Lepanthes , Subsect. Breves) and the Elongatae series (Subgen. Lepanthes , Sect. Lepanthes , Subsect. Lepanthes ) were not represented in this phylogeny by any species. In addition to constructing complete plastid phylogenies, we downloaded 26 SRA data files for Lepanthes species from Genebank (Table S4 ) and mined all plastid sequences. Chloroplast assemblies were generated using GetOrganelle [35] and Captus [48]. A set of chloroplast coding regions was successfully recovered for multiple Lepanthes species. To perform alignments and phylogenetic reconstructions, we employed two approaches: (1) coding sequences concatenation with an incomplete data matrix for a subset of species, and (2) constructing a matK phylogeny for all available species. For the phylogenetic reconstruction of a combined dataset (rITS + matK) focusing on subgenus Marsipanthes , analyses were conducted using Brachionidium valerioi Ames & C. Schweinf. And Pseudolepanthes colombiae Archila as outgroup species. Maximum Likelihood (ML) analyses were performed using the PhyML plugin [49] in Geneious Prime, employing the GTR + G substitution model with 1000 bootstrap replicates. Bayesian Inference (BI) analyses were conducted in BEAST v.1.10.4 [50], utilizing the GTR + G substitution model and the Yule speciation model. The BI analysis ran for 10 million generations, with tree sampling occurring every 10,000 generations. A single consensus tree was generated using TreeAnnotator v.1.10.4 ( http://beast.community/treeannotator ) 6. Morphological analysis : Patterns of morphological variation and potential correlations between character traits and phylogenetic clades were assessed using Principal Component Analysis (PCA) and correlation matrices to contextualize the evolutionary relationships. Protologs of species included here were used to build a database including continuous measurements of vegetative and floral structures such as the ramicauls, leaves, peduncles, sepals, petals, lip, and column. Both minimum and maximum dimensions were considered for each continuous morphological character. R (version 4.2.1) was the primary programming environment for conducting multivariate analyses of orchid morphological traits. The analysis involved several R packages ggplot2 for data visualization, and ggcorrplot for generating a correlation matrix plot. We pre-processed the data to address missing values using the na.omit function and converted categorical variables to numeric format. After filtering species with missing data only 20 species were used for the analysis. Normalization of the data was performed using z-score scaling to ensure comparability among variables. The correlation matrix was computed to assess the relationships among the traits, and a color-coded visualization was created to facilitate the interpretation of significant correlations. Principal Component Analysis (PCA) was conducted on normalized orchid trait data to capture maximum variance, with results visualized using customized functions for species labeling and heatmaps illustrating trait contributions. The proportion of variance explained by the first two principal components was calculated, and key findings were saved as CSV files for further analysis. RESULTS 1. Plastomes molecular descriptions : The assembled and annotated chloroplasts were uploaded to the NCBI database (BioprojectID PRJNA1209138). Using the Illumina HiSeq 4000 system, we obtained 7,150,658 ( L. eros ) to 11,373,812 bp ( L. narcissus ) paired reads after cleaning, with an average read length of 145 bp and a recovery rate of approximately 82% of the original reads (Table S2 ). The Lepanthes eros genome could not be assembled due to significant contamination in the Illumina reads. The nine Lepanthes plastomes assembled had an average length of 157,710 bp, with L. narcissus having the smallest plastome (157,185 bp) and L. caprimulgus the largest (158,260 bp) (Table 1 ). The G/C content of all plastomes was approximately 37%. Each plastome exhibited the typical quadripartite structure seen in angiosperm plastomes, including a Large Single Copy (LSC), a Small Single Copy (SSC), and two Inverted Repeat (IR) regions (Fig. 2 ). L. ribes had the smallest LSC (89,500 bp) while L. narcissus had the largest (90,156 bp). In terms of SSC, L. nicolasii had the smallest at 19,095 bp, while L. ribes had the largest (23,401 bp). The IR regions ranged from 24,495 bp in L. nicolasii to 21,346 bp in L. ribes . All plastomes contained 136 unique genes, including 42 tRNA genes, 8 rRNA genes, and 86 protein-coding genes. Additionally, 25 genes were duplicated in the IR regions (Table 1 , Fig. 2 ). Table 1 List of the Lepanthes species used in this study and their infrageneric taxonomic classification following Luer [7, 14, 30]. The series Filamentosa (subgen. Lepanthes , sect. Lepanthes , subsect. Breves ) is not represented in this phylogeny by any species (NA nonincluded here). * Lepanthes eros was not included in further analysis due to sequence contamination. Subgenera Section Subsection Series abandoned by Luer and Thoerle [2] Total species number Species sequenced here Species sequenced elsewhere Marsipanthes Caprimulginae - 2 L. caprimulgus L. attenboroughii Felinae - 6 L. felis L. lucifer L. niesseniae L. tulcanensis L. lucifer L. niesseniae Marsipanthes - 3 L. ribes Lepanthes Lepanthes Bilabiatae 7 L. narsissus Breves Breves 85 L. monoptera L. eros * L. elata L. tachirensis Filamentosae 13 NA NA Lepanthes Mucronatae 60 L. mucronata L. hexapus L. ankistra L. heteroloba L. pelix Elongatae 154 NA L. acarina L. clowesii L. katleri L. martineae L. nycteris L. terborchii Lepanthes 795 L. barbelifera L. calodyction L. manabina L. nicolasii L. adrianae L. auriculata L. ballatrix L. calodyction L. ciliisepala L. clareae L. confusa L. crucitasensis L. dactylopetala L. dodsonii L. gargantua L. grandiflora L. imitator L. juninensis L. matamorosii L. meniscophora L. mystax L. orion L. saltatrix L. tentaculata L. terpsichore L. turialvae L. urbaniana L. volador Table 2 Summary of complete chloroplast genomes statistics. Comparison of plastome subunits length and GC content for the samples completely assembled. Species Length (bp) LSC (bp) SSC (bp) IR (bp) GC% L. mucronata 157,848 89,756 19,116 24,488 37.1 L. hexapus 157,629 89,567 21,363 23,363 37.1 L. manabina 157,208 89,953 22,091 22,582 37.1 L. ribes 157,648 89,500 23,401 21,346 37.2 L. felis 157,665 89,735 21,432 23,249 37.2 L. nicolasii 158,213 90,128 19,095 24,495 37.1 L. monoptera 157,920 90,056 21,314 23,275 37.1 L. caprimulgus 158,260 90,089 21,305 23,433 37.2 L. narcissus 157,185 90,156 21,325 22,852 37.1 Several Open Reading Frames (ORFs) were identified across the Lepanthe s genomes. In the LSC region, two hypothetical proteins were detected via BLASTx: one within the trnC-GCA gene in all Lepanthes species except L. nicolasii and L. caprimulgus (408 bp, 136 aa), and another partially overlapping the clpP1 gene only in L. manabina plastome, showing similarity to a Clp protease subunit in Epidendrum ciliare (97.96% identity, e-value: 1e-21). In both inverted repeat regions (IR1 and IR2), a hypothetical protein, M5K25 , previously identified in Dendrobium thyrsiflorum , was detected within the trnI-GAU gene in Lepanthes ribes , L. narcissus , and L. caprimulgus . Another hypothetical protein, conserved across all Lepanthes plastomes, was located within the rr23 rRNA gene (408 bp, 136 amino acids). This protein exhibited high similarity to chloroplast proteins from Acorus calamus (95.56%, e-value: 2e-86), Dendrobium chrysanthum (93.04%), and D. thyrsiflorum (95.6%)." The ycf1 gene located around the boundary of IR1-SSC has different copies and is located in the SSC in most species but L. mucronata , and L. nicolasii. In these species one copy is located in the IR1 and the second one in the SSC (Figure S1 ). L. felis, L. narcissus and L. manabina had two functional copies and L. caprimulgus three. In L. manabina , an additional ORFs has been found embedded within the overlap of the two ycf1 copies, this is like a Dracula erythrocochaete NADH dehydrogenase subunit F (94.51%, e-value: 1e-90) and Masdevallia picturata NADH plastoquinone oxidoreductase subunit 5 (94.51%, e-value: 2e-90). L. narcissus had two copies of the ndhF gene, one of them overlapping with ycf1 and ndhF . Only two species have multiple copies of ycf1 in the boundary of SSC-IR2, L. felis (3 copies) and L. nicolasii (2 copies) (Figure S1 ). 2. Plastome structural variations : An IR boundary map was created by comparing the plastomes of nine Lepanthes species using IRscope (Fig. 3 ). The rpl23 gene was positioned at the end of the LSC, at the junction between the LSC and IR1 (JLB). At the junction between IR1 and SSC (JSB), ycf1 was located within IR1 in L. mucronata and L. nicolasii , while in the rest of species, it was found at the beginning of the SSC. At the junction between SSC and IR2 (JSA) ycf1 were completely located within the SSC, ranging from 89 to 29 bp before the end of IR2 in most species. However, in L. mucronata and L. nicolasii, ycf1 spanned 1072 and 1077 bp, respectively, from the SSC into IR2. At the IR2-LSC junction (JLA), the ycf2 gene was found spanning both regions. Collinearity analysis showed no gene rearrangements or inversions in the Lepanthes plastomes (Figure S2 ). 3. Identification of hypervariable regions : The divergence of complete plastome sequences among nine Lepanthes species was analyzed using mVISTA, with Anathallis obovata as the reference (Figure S3 ). Whole-genome alignment revealed that sequence variation in conserved non-coding regions (highlighted in pink bars) was greater than in protein-coding regions (highlighted in purple bars). The variation rates in both coding and non-coding regions within the two IR regions were lower than those in the LSC and SSC regions. Highly divergent non-coding regions included pskB-psbI , atpB-rbcL , petA-psbJ , psbB-psbN , and rpl32-trnL . In contrast, the rRNA genes were highly conserved compared to other genes. To further investigate DNA polymorphisms within Lepanthes plastomes and to provide molecular tools useful to other researchers working in the group we identified DNA barcodes. Nucleotide diversity (Pi) values were calculated using DnaSP6 (Fig. 4 ). The average Pi value across the nine plastomes was 0.00906, with a total of 4,054 mutations identified. The IR region averaged a Pi value of 0.00229, the LSC region averaged 0.01050, and the SSC region averaged 0.01286. Based on Pi values (ranging from 0.02 to 0.03), eight hypervariable regions of the plastomes were identified: trnKuuu–rps16 , psbK–psbI , psbM–trnEucc , trnTUGU–trnLUAA , accD–psaI , clpP1–psbB , and ycf1 . 4. Repeated sequence analysis – The results of SSR and tandem repeat analysis in Lepanthes plastomes using MISA are presented in Table 3 . The number of identified SSRs ranged from 58 in L. hexapus to 47 in L. monoptera , with a total of 470 SSRs detected across the Lepanthes plastomes. Three types of SSRs (mono-, di-, and complex-nucleotide repeats) were identified, with 436 (92.9%) being mono-nucleotide repeats, predominantly composed of A and T motifs. Additionally, 13 di-nucleotide repeats (2.77%) and 21 complex-nucleotide repeats (4.47%) were identified (Table 3 ). Table 3 Repetitive motif abundance in nine Lepanthes species computed using MISA . Distribution and types of simple sequence repeats. Mononucleotides Dinucleotides Complex repeats SSRs Total L. caprimulgus 50 1 2 53 L. narcissus 47 1 3 51 L. monoptera 45 1 2 47 L. nicolasii 47 1 3 51 L. ribes 46 1 3 50 L. felis 47 1 2 50 L. manabina 48 3 2 53 L. mucronata 52 2 2 56 L. hexapus 54 2 2 58 A total of 215 tandem repeats were detected, with counts ranging from 17 in L. nicolasii to 38 in L. caprimulgus . Notably, all nine Lepanthes plastomes contained 50 long repeats each, comprising palindromic, forward, reverse, or complementary long repeats. Palindromic repeats were the most abundant, ranging from 33 in the L. manabina plastome to 23 in L. narcissus . Forward repeats followed in abundance, with L. felis , L. monoptera , and L. caprimulgus each containing 18 forward repeats, while L. hexapus contained 11. Reverse and complementary repeats accounted for only 59 out of 450 repeats, representing 13.1% of all long repeats (Table S3 ). To assess codon usage bias, the relative synonymous codon usage (RSCU) ratios were calculated for Lepanthes plastomes. Less frequently used codons (RSCU < 1) were generally consistent across species, with most having 32, except L. hexapus and L. narcissus , which had 33. Most preferred codons ended with A or U, except for UGG and AGG. The codons AGA and UCU exhibited the highest RSCU values, averaging 2.11 and 1.58, respectively, while CGC and GAC had the lowest RSCU values, with averages of 0.41 and 0.54 (Figure S4 ). 5. Phylogenomic analysis – Topologies based on whole plastomes, coding regions, and intergenic regions for nine Lepanthes species, representing main taxonomic groups were identical, with similar resolutions using both Bayesian Inference (BI) and Maximum Likelihood (ML) methods (Fig. 5 A). Species of Lepanthes formed a well-supported monophyletic group (PPBI = 1.00, BSML = 100). Within Lepanthes , two sister clades were identified: L. narcissus + L. caprimulgus (PPBI = 1.00, BSML = 100) and the remaining Lepanthes species (PPBI = 1.00, BSML = 100). In the latter clade, L. monoptera was recovered as sister to the rest, followed by L. nicolasii , which is sister to a clade (PPBI = 1.00, BSML = 100) comprising L. manabina sister to L. mucronata + L. hexapus , and L. ribes + L. felis in the Bayesian phylogeny and not resolved in the ML one (Fig. 5 A). A second whole chloroplast phylogeny was reconstructed for 35 Lepanthes species including species sequenced here and 26 more, plus two to five outgroups downloaded from Genebank. For this alignment, one IR was excluded, and only coding regions were considered. The mean sequence length was 37,725 bp, ranging from a maximum of 72,282 bp to a minimum of 7,042 bp. The total length of the alignment was 85,750 bp, with a pairwise identity of 46.6%, and 5.3% of identical sites. Half of the species included here had between 50 and 77 chloroplast genes present in the alignment (Fig. 6 A). Genes represented in this phylogeny included: atpA, atpB, atpE,ndhK, psbB, psbC, psbZ (Fig. 6 B and C). In this phylogeny species from most taxonomic groups described by Luer [14, 17, 30] were included, except for species from the series Filamentosae (Table 1 ). All taxonomic groups below the genus level were found to be non-monophyletic, except for series Mucronatae still to be confirmed since the phylogeny did not fully resolve phylogenetic relationships among species include in this series (Fig. 5 B, Fig. S5 ). Six main clades were recovered, an early-divergent lineage, comprising L. tachirensis and L. juninensis , sister to the remaining species. Clade 1 sister to the rest of Lepanthes and including L. narcissus , sister to L. caprimulgus + L. katleri . Clade 2 consists of L. monoptera , L. rhynchion , and L. speciosa , sister to the remaining species of Lepanthes . A polytomy was observed, including L. acarina , L. clowesii , Clade 3: L. gargantua sister to subclades L. imitator , L. clarae , L. nicolasii + L. adrianae , L. orion and L. auriculata and Clade 4. Clade 4 includes Central America and the Caribbean species L. turialvae , L. urbaniana , and L. grandiflora , sister to the remaining species, Clade 5 including L. tentaculate and L. calodyction , and a second polytomy including several small subclades: L. felis + L. ribes , L. hexapus + L. mucronata , L. lucifer + L. niesseniae , L. heteroloba + L. pelyx , and a subclade consisting of L. dodsonii , L. manabina , L. terpsichore and L. meniscophora (Clade 6) (Fig. 5 B). Further BI and ML analysis focusing on nine of the eleven Marsipanthes species and using a combined rITS + matK dataset recovered several of the clades identified in our main phylogeny but lack of resolution for the backbone, confirming Marsipanthes is non-monophyletic (Figures S6 , S7). Main clades to highlight from this phylogeny include: (1) Species from the Eastern Andes of Ecuador, Perú and Bolivia. L. caprimulgus, L. attenboroughii (both representing subgen. Marsipanthes , sect. Caprimulginae ) and L. martinae, L. nycteris and L. terbochii belonging to subgen. Lepanthes (MLBS = 75, BIPP = 1). (2) Species from a western Andean Marsipanthes clade including L. felis , L. ribes , L. lucifer and L. niesseniae (MLBS = 94, BIPP = 1). L. tulcanesis phylogenetic position is uncertain. Few more clades recovered in this phylogeny include L. calodictyon and closely related species L. barbellifera, L. saltatrix and L. volador (MLBS = 100, BIPP = 1). Lastly, a matK phylogeny for 104 Lepanthes species, including both sequences produced here and those available in GenBank, lacked resolution (Figure S8 ). 6. Morphological continuous characters- PCA analysis indicated that continuous morphological measurements from the species included here did not form distinct groups according to clades (Figs. 7 A, B). PC1 and PC2 account for about 61.96% of the total variance in the dataset, capturing a substantial amount of the variability in the traits we analyzed. PC1 and PC2 account for 35.49% and 26.47% of the total variance respectively. Dorsal sepals (Length Min.-Max., Width Min.-Max.) and Lateral sepals (Length Min.-Max., Width Min.-Max.) have the highest positive loading in this PCA suggesting sepal dimensions are critical for distinguishing among the species in our Lepanthes dataset. Traits like Peduncle Length Min. and Pedicel Length Min. also contribute positively. Traits with negative loadings on PC1 include Ramicauls Length Min. and Number of sheaths Min., indicating they might be less important or show an inverse relationship with the traits positively contributing to PC1. PC2 highlight traits with positive loadings including Dorsal sepal Length Min., Lat sepal Length Min., and Peduncle Length Min. Ramicauls Length Min., Leaf Width Min., Lip Length Min., and Column Length Max. show negative contributions to the data distribution, suggesting they may differentiate the data in an opposite direction compared to the positively loaded traits (Figure S9 ). Continuous morphological characters showed high positive correlations among several, floral and vegetative traits (Fig. 7 C). Strong correlations (above 0.8) were found between Peduncle Length and Floral Bract Length (0.930). Leaf Length and Ovary Length (0.888), Ovary Length and Floral Bract Length (0.849), Dorsal Sepal Length and Dorsal Sepal Width (0.849). None of the negative correlations were strong, for examples Dorsal Sepal Width and Leaf Width were negative correlated (-0.244) (Fig. 7 C). DISCUSSION A genome skimming approach produced nine new chloroplast genomes for Lepanthes species, providing a valuable suite of molecular resources to the scientific community. Additionally, plastome genes sourced from GenBank facilitated the first moderately resolved phylogenetic reconstruction for the genus. Our findings demonstrate that the current taxonomic groups proposed by Luer are not monophyletic, reflecting significant morphological homoplasy. Six main clades were identified; however, the moderate resolution of the phylogenetic backbone highlights the need for nuclear markers and the inclusion of many more species to further refine these relationships. Despite limited species sampling, this study encompasses substantial taxonomic, geographic, and morphological diversity, laying a strong foundation for future evolutionary research on Lepanthes . Plastome evolution of Lepanthes species - A range of plastome lengths, averaging 157,710 base pairs (bp) was observed in Lepanthes . L. narcissus has the smallest plastome 157,185 bp, while L. caprimulgus the largest one 158,260 bp (Table 1 ). The G/C content across all plastomes was consistent at approximately 37%, reflecting typical characteristics for angiosperm and orchid plastomes [51]. The plastome size observed in this study aligns with the broad range reported for Orchidaceae, spanning from the reduced 19,047 bp in the mycoheterotrophic Epipogium roseum [52] to the largest to our knowledge 212,688 bp in the autotrophic Cypripedium subtropicum [53], reflecting the family’s diversity in photosynthetic function and evolutionary adaptation. All sequenced plastomes exhibited the classic quadripartite structure seen in angiosperms, comprising a Large Single Copy (LSC) region, a Small Single Copy (SSC) region, and two Inverted Repeat (IR) regions (Fig. 2 ) [54–55]. Among species, L. ribes had the smallest LSC at 83,758 bp, while Lepanthes narcissus had the largest at 84,423 bp. The SSC size varied, with L. ribes having the smallest at 18,024 bp and L. hexapus the largest at 19,093 bp. Regarding the IR regions, L. narcissus had the smallest at 26,830 bp, while L. ribes had the largest at 27,933 bp. Each plastome contained 136 unique genes, including 111 unique genes, 42 tRNA genes, 8 rRNA genes, and 86 protein-coding genes. Additionally, 25 of these genes were duplicated in the IR regions. Although the IR in many angiosperms is approximately constant in size (~ 25 kb), sequence analysis of its endpoints has shown that small differences exist between species in the extent of the IR [54]. Lepanthes species exhibit consistency in plastome length and structure and aligns with the general characteristics of monocot plastomes [51, 55–56], this stability is important for epiphytes often growing in nutrient-poor soils, maintaining a stable chloroplast genome may be crucial to efficiently capture and use light and other limited resources [57]. Despite the generally conserved nature of chloroplast genomes, the presence of unique open reading frames (ORFs) in Lepanthes plastomes suggests possible lineage-specific evolutionary processes to be further investigated in the future [51, 58]. For example, our analysis identified an ORF within the trnC-GCA gene in all species except L. nicolasii and L. caprimulgus (408 bp, 136 amino acids), indicating potential losses of this ORF at least twice in the genus evolution. Additionally, in the IR regions, we detected a hypothetical protein ( M5K25 ) from Dendrobium thyrsiflorum in several Lepanthes species. A second hypothetical protein was identified in the rr23 rRNA gene in all sequenced species, showing high similarity to proteins in Acorus calamus (95.56%) and Dendrobium thyrsiflorum (95.6%). Furthermore, an ORF in L. manabina , partially overlapping with the clpP1 gene, demonstrated strong similarity to a clp protease subunit in Epidendrum ciliare (97.96% identity, e-value: 1e-21), suggesting either horizontal gene transfer (HGT) or convergent evolution. The ycf1 and ndhF genes exhibit considerable molecular diversity and multiple copies across Lepanthes species sequenced here (Figs. 4 , S1 ). L. felis and L. nicolasii have five and four ycf1 copies respectively, while L. narcissus has two ndhF copies (Figure S1 ). This copy number variation does not seem to have a clear phylogenetic pattern indicating multiple and independent gene gains and losses. Gene duplications and losses pertaining to these regions have been reported in other angiosperms and orchids [59–61]. The ycf1 has been showed to encode products that are essential for cell survival [59]. Multiple copies of this gene observed here may perform slightly different functions. An examination of the IR (Inverted Repeat) boundaries and gene positions reveals slight patterns and variation among different species (Figs. 3 , S1 ). Chloroplast region boundaries are not only fundamental to the structural integrity and function of the chloroplast genome but also provide insights into evolutionary processes, functional genomics, and adaptation strategies. The positions of the LSC-IR junctions vary slightly within groups, but usually this has only negligible effects on plastome size [51]. SSC-IR boundaries were not fixed in Lepanthes ; instead, and like in other plants, they undergo a dynamic and stochastic process that primarily results in conservative expansions and contractions of the IR [54]. Several consistencies were found across all sequenced Lepanthes species: the gene rpl23 is positioned at the end of the LSC, the IR containing a trnH-rps19 cluster as reported in most monocots [55] and an overlap between the genes ycf1 and ndhF near or at the IR1-SSC boundary. In contrast there was some variation at the Inverted repeats and the SSC junctions: the position of ycf1 copies varied among species, both in the SSC and IRb (IR boundary junction JSB) or IRa (IR boundary junction JSA) (Fig. 3 ). For example, the position of ycf1 in L. mucronata and L. nicolasii expands from the SSC into the IRa (IR boundary junction JSA). The IR can terminate within genes, leading to nonfunctional and potentially disruptive 5' or 3' truncated genes, whose translated products may negatively impact essential processes [54]. In Pleurothallidinae, the IR/SSC boundary interrupts the ycf1 gene, resulting in a truncated ycf1 fragment within the IR. Conversely, in Cattleya crispata , the SSC encompasses nearly the entire ycf1 gene [36]. These plastome differences between Pleurothallidinae and Laeliinae may be characteristic of these subtribes. Collinearity analysis further supports the stability of these plastomes, as no gene rearrangements or inversions were detected (Figure S2 ). This lack of major structural changes reinforces the notion that while there are some variations in gene positioning, the overall plastome architecture of Lepanthes remains relatively stable across species. Last, the analysis of relative synonymous codon usage (RSCU) revealed variability in codon preferences across the Lepanthes plastomes. The presence of both preferred (RSCU > 1) and non-preferred codons (RSCU = 1) highlights possible selective pressures influencing plastome evolution. Codons ending in A or U are prevalent, with AGA and UCU exhibiting the highest RSCU values (Figure S4 ). This pattern is consistent with the trend observed in other plant plastomes, where specific codon biases can reflect functional constraints and evolutionary pressures [62]. 2. New molecular resources for Lepanthes - The identification of hypervariable regions in the plastomes of Lepanthes species reveals significant insights into the genetic variability across different regions of the plastome. Using mVISTA for whole-genome alignment with Anathallis obovata as the reference, we observed that sequence divergence was notably higher in the conserved non-coding regions compared to the protein-coding regions. Specifically, non-coding regions such as pskB-psbI, atpB-rbcL, petA-psbJ, psbB-psbN , and rpl32-trnL exhibited higher variability, while rRNA genes remained highly conserved. The analysis of nucleotide diversity (Pi) values further highlights the distribution of genetic variation across different plastome regions. The IR regions showed the lowest nucleotide diversity with an average Pi value of 0.00229, suggesting relative stability in these regions. In contrast, the LSC and SSC regions exhibited higher diversity, with average Pi values of 0.01050 and 0.01286, respectively. This indicates that the LSC regions are more prone to variation, which could be useful for distinguishing between species or for evolutionary studies. The top seven hypervariable regions identified across the plastomes— trnKuuu-rps16 , psbK-psbI , psbM-trnEucc , trnTUGU-trnLUAA , accD-psaI , clpP1-psbB , and ycf1 present promising candidates for developing specific DNA barcodes. Parts of the ycf1 gene are identified as potential good barcode regions for the genus, its utility has also been confirmed for many other angiosperms [60]. These regions, with Pi values between 0.02 and 0.03, offer the highest degree of variability and could serve as effective markers for species identification and phylogenetic studies (Figs. 4 , S3 ). This analysis underscores the utility of non-coding regions and specific hypervariable regions in understanding genetic diversity and developing molecular tools for the study of Lepanthes . The findings not only contribute to the characterization of plastome variability within this genus but also provide a foundation for future research aimed at using these hypervariable regions for practical applications in plant systematics and conservation. SSRs are a type of repeats frequently observed in chloroplast genomes that can be used to discover genome polymorphisms and perform population genetics within and between species [63]. The analysis of repeated sequences in Lepanthes species reveals detailed patterns of sequence variability and bias. Using the MISA tool to identify simple sequence repeats (SSRs) and tandem repeats, we found a total of 469 SSRs across the plastomes. The number of SSRs varied among species, with L.hexapus showing the highest count of 58 and L. monoptera the lowest at 47 (Table 3 ). The structural and evolutionary stability observed in Lepanthes plastomes, particularly in nutrient-poor epiphytic environments, represents an intriguing avenue for future research. Unique features such as open reading frames, gene duplications, and structural variations at IR boundaries suggest lineage-specific evolutionary processes that merit further investigation. 3. Phylogenetic and morphological analyses- A phylogeny of Lepanthes using maternally inherited molecular markers such as those from the plastome was reconstructed here. Our phylogenies have a poor taxonomic representation (9–35/1196 spp.) but a great number of informative molecular characters (≤ 15,834bp). Besides species included in this analysis represented unique morphological groups identified through taxonomic studies. All phylogenies reconstructed here confirmed the monophyly of Lepanthes as proposed by Bogarín et. al [20] (Fig. 5 ); while, both Lepanthes subgenera recognized by Carl Luer [17] are not monophyletic (Figs. 5 , S5 - 7 ). This indicates perianth characters are homoplasic and the fusion of petals and number of veins are not informative at this taxonomic level. Additionally, most of the subclades recovered here do not support further infrageneric classification by Luer [7], except for the subclade comprising L. hexapus and L. mucronata recovered as monophyletic and belonging to series Mucronatae from the section Lepanthes (Fig. 5 A). However, the monophyly of series Mucronatae was not confirmed in the 35-spp. phylogeny, lacking resolution at this scale, further molecular information will have to be analyzed (Fig. 5 B). The series Filamentosa (Subgen. Lepanthes , Sect. Lepanthes , Subsect. Breves ) is not represented in this phylogeny by any species. Those species will remain to be included in future studies to be properly placed in a systematic context for the genus. The current subgeneric circumscription of the genus does not reflect the evolutionary history of the group and a new taxonomic classification using systematics should be proposed. Species L. tachirensis and L. juninensis , are surprisingly recovered as the sister species of the rest of Lepanthes species. L. tachirensis is a widespread taxon in South America originally described from Venezuela, from the Tachira region but further register in Colombia and Ecuador. An interesting aspect of its morphology that sets L. tachirensis apart from the rest of Lepanthes includes—apart from its flowers and open sepals—the tightly sheathed ramicaul, with the ostia, which in other Lepanthes resembles a funnel, closely adhering to the ramicaul. Besides, L. juninensis was originally described from Peru, but its identity is challenging to interpret due to the limited information available—a difficult-to-interpret illustration and description [64]. The species has often been misidentified as L. tachirensis , and this misidentification has been widely propagated among collectors and commercial nurseries. Given that the two species share few similarities, it is possible that the specimen from Genebank identified as L. juninensis is, in fact, misidentified L. tachirensis . Further exploration in the type locality region of Peru will be essential to clarify the identity and morphological characteristics of L. juninensis , if indeed a species. An early divergent clade, The Eastern Andean Marsipanthes clade (Clade 1), is identified in our phylogenies as sister to the rest Lepanthes . This clade comprises two species from the subgenus Marsipanthes section Caprimulginae , specifically L. attenboroughii and L. caprimulgus , which are native to the Eastern Andes of Ecuador and Perú. These species are more closely related to members of Lepanthes section Elongatae ( L. martineae , L. nycteris , L. terbochii , and L. katleri ) and section Bilabiatae ( L. narcissus ), all distributed across the Eastern Andes of Ecuador, Perú, and Bolivia, than to other species within Marsipanthes (Figs. 5 , S5 – 7 , 10). Most species, except for L. narcissus , have shortly pubescent, multi-veined (5–7 at the dorsal sepal and 2–4 in each lateral sepal) sepals, erose to lacerate, carinae adaxially, and long pubescent to fringed at the margins, deeply to shallowly concave, and stripped. The petals are frequently lunate and with the upper and lower lobes of similar shape and size. The lip has long, densely pubescence, narrow to very narrowly triangular lobes which surround the long column (Figs. 1 and S10 ). Based on their floral morphology and distribution on the Eastern Andes, further molecular studies should corroborate whether species from subgen. Lepanthes series Elongatae such as L. echinata Luer & Cloes, L. mulleriana Luer, L. tigrina Luer & Thoerle, besides L. portillae (sect. Marsipanthes ) discovered in El Condor Mountain range of Ecuador, belong to Clade 1. The monophyly of the remaining species in section Bilabiatae (approximately seven species) and their status as the sister group to Clade 1, as suggested by our phylogeny with L. narcissus , remains to be tested. The Monoptera clade (Clade 2), the second early-divergent lineage identified in our study, is resolved as sister to the remainder of Lepanthes and comprises species from subsection Breves , which Dodson and Luer [21] and Luer and Thoerle [18], distinguished by their single-veined lateral sepals. These high-altitude species typically occur above 2500 m and form two distinct groups: (1) the L. monoptera group characterized by a bilaminate lip with oblong to ovate, long-ciliate blades, rounded blade bases, narrowly obtuse apices, short connectives, and a distinctive ovary with a single wing or carina. Other species that could be part of this group include L. cornualis , L. cyrtostele , L. ferax , and L. jucas . And (2) the Rhynchion/Speciosa group, comprising species with large stature, large leaves, and lepanthiform sheaths that are prominent, adnate, or flattened. Intriguingly, L. tachirensis also belongs to subsection Breves and shares morphological traits with L. rhynchion and L. speciosa , yet it is phylogenetically placed at the base of the genus, highlighting the non-monophyly of subsection Breves . Further studies are needed to determine whether other species in this subsection align with one of these two clades or represent additional lineages, based on both molecular and morphological evidence. The Lepanthes phylogeny presented here includes two main polytomies indicating molecular information used here was not enough to resolve parts of the Lepanthes backbone and other molecular markers or more species will have to be included in future studies to improve the resolution. The observed polytomies might also reflect recent divergence or rapid speciation events, in addition to the limitations of molecular data. The first polytomy contains: L. cloesii , L. acarina , the Nicolasii clade (clade 3): L. nicolassi , L. gargantua , L. clareae , L. adrianae , L. auriculata , and L. orion , along with a clade containing the remaining species sampled here of Lepanthes (Fig. 5 B). Lepanthes cloesii and L. acarina did not form a clade, despite their previous classification in the artificial series Elongatae . This finding supports Luer and Thoerle's [2] decision to abandon this grouping. In the ITS/MatK phylogeny, L. cloesii clusters with L. elata and L. ballatrix (Figures S6 –7). Other species with morphological similarities to L. cloesii , such as L. elegantula , L. pastoensis , and L. alexandroi , share features like thick, fleshy leaves and relatively large flowers compared to their smaller leaves. Within this phylogeny, L. cloesii is the sole representative of a larger group that requires further investigation. In contrast, L. acarina —a widely distributed and common species ranging from Colombia to Bolivia—is distinguished by its extremely small size flowers and unique morphology, which complicates its association with other species. These traits suggest that L. acarina may represent a cleistogamous lineage, potentially following an independent evolutionary pathway within the genus. The Nicolasii clade (Clade 3) [7] includes large-sized species with large, erect to suberect leaves, supported by relatively long, robust ramicauls, compact rachis, conspicuous appendix and typically with yellow flowers produced in a raceme on the leaf's abaxial surface. This group might also include species such as L. monitor , L. auriculata , and L. steyermarkii . The remaining Lepanthes species within the first polytomy form a clade with two subclades. A Central American and the Caribbean clade (Clade 4) including species L grandiflora , L. turialvae , L. urbaniana , sister to a clade represented by L calodyction + L. tentaculata and the rest of Lepanthes . Most species from both The Calodictyon clade (Clade 5) and the remaining of Lepanthes have a distribution in the Western Cordillera of Colombia and northern central cordillera of Ecuador known biogeographic Choco [65–66]. The Calodictyon clade (Clade 5) was also recovered in the combined ITS/MatK phylogeny, including species such as L. saltatrix , L. barbelifera , and L. volador (Figures S6 –7). These species are distributed from Central to South America and exhibit notable morphological characteristics. While most species have reticulated leaves, those from Mesoamerica lack this feature. Flowers emerge above the leaf, with sepals that are widely spaced and reflexed, resting on the leaf surface. The petals are unique, with a highly distinct upper and lower lobe. The upper lobe is often elongated, sometimes with a filamentous extension at the apex (though not always), while the lower lobe is typically expanded into a broader segment with a basal filamentous extension (also not always present). The labellum is simple, lacking the typical Lepanthes structure of a body, connectives, and laminae. Instead, it takes on reniform, lunate, ovate, or bilobed shapes and does not include an appendage. The column is elongated, with a conspicuous hood over the anther (clinandrium). Some species have two filaments on their petals, others have one, and some lack filaments entirely. They grow at low to mid elevations 200 to 1800 m. Calodictyon-related species include several from Central America ( L. arachnion , L. pantomima ) and the western Andes ( L. bibarbullata , L. arachnion , L. kayii , L. microcalodictyon , L. pantomima , L. pretiosa , L. tentaculata , L. tortuosa ) [2, 7]. The second polytomy identified within this phylogeny includes L. mucronate + L. hexapus , L. pelix + L. heteroloba, L. ribes + L. felis , L. lucifer + L. niesseniae and the Manabina clade (Clade 6) (Fig. 5 ). The rest of species from subgen. Marsipanthes are recovered within this clade like this: (1) L. ribes (sect. Felinae ) + L. felis . (2) L. niesseniae + L. lucifer (sect. Felinae ). These four species only formed a clade in the combined ITS/Matk , we will refer to them as the western Andes Marsipanthes species (Figure S6 -7, 11). We anticipate species such as L. carunculigera, L. equicalceolata, L. quadricornis will be part of this clade. In our phylogeny, L. hexapus represents a group of species characterized by elliptic to ovate leaves with reticulate veins and a distribution almost exclusively from the biogeographic Choco [65–66]. Based on morphology species like L. satyrica , L. hirsutula , L. acrogenia , L. tetrapus , and L. heptapus among others could be part of this clade. A second group of morphologically similar species are represented here by L. mucronata , these group of species might have a mucron or a small central lobe on the petals. Last, L. pelix + L. heteroloba form part of a group of species with very corrugated leaves. Luer's initial grouping termed these as the series mucronatae [21], however, he later retracted this, possibly due to the complexity and variation in flower and plant morphology. With additional sampled species, this series might be further confirmed or refuted as monophyletic. The phylogenetic position of L. tulcanensis in the combined ITS/MatK phylogenies, along with its morphological similarities to both western Andes Marsipanthes species and the Mucronata group, is intriguing (Figures S6 –7). The plant closely resembles species from the Mucronata group, such as L. rodophylla , L. pelyx , and L. corrugata . However, its flowers bear a stronger resemblance to those of species in the Marsipanthes group (Figure S11 ), particularly L. niesseniae . This is evident in features such as the long peduncle, sepals with more than three veins, a simple lip that folds beneath the column, and three-dimensional petals. Last the Manabina clade (Clade 6) has already been informally recognized by Baquero et al. [67] based on Lepanthes manabina Dodson. All species in this group have centrally concave leaves, ranging from deeply to slightly concave. These leaves have slightly to strongly recurved margins, a microscopically to conspicuously pubescent adaxial surface, and congested inflorescences. The flowers rest on the adaxial side of the leaves and are accompanied by a short to long caudate synsepal and a very small, inconspicuous appendix. Some other species that might belong to this group include L. farallonensis , L. foreroi P.Ortiz, O.Pérez & E.Parra, L. ortiziana O.Pérez, E.Parra & Kolan., L. smaragdina Luer & R.Escobar, L. tomentosa Luer, and L. cincinnata Luer & R.Escobar. Our preliminary morphological analysis, limited to a subset of Lepanthes taxa, confirmed that continuous characters do not exhibit grouping patterns consistent with phylogenetic clades. In only a few cases, such as the artificial subgenus Marsipanthes (Fig. 7 ), do these characters show a taxonomic pattern. However, this analysis revealed intriguing trends using continuous morphological characters, which will be tested further with a broader taxon sampling and additional datasets. The high variability in continuous morphological traits is captured by PC1 and PC2, suggesting that measurements such as sepal size and peduncle length are key in distinguishing among Lepanthes species. Traits like dorsal and lateral sepal dimensions have the highest positive loadings (Figure S9 ), highlighting their importance in species differentiation. This information is particularly valuable for systematics, evolutionary developmental biology (evo-devo), and other areas of study, especially in groups with high morphological similarity. Strong correlations between traits, such as peduncle length and floral bract length, or leaf length and ovary length, suggest functional or developmental linkages. These findings provide insights into how morphological traits may be co-adapted in response to environmental pressures or reproductive strategies. Conversely, weak negative correlations, such as those between dorsal sepal width and leaf width, may indicate independent evolution of certain vegetative and floral traits, pointing to differing selective pressures on these aspects. The hyperdiverse nature of Lepanthes presents significant challenges in resolving its phylogenetic relationships, particularly given the rapid diversification of the genus, which is estimated to have occurred 5–10 million years ago [28, 68]. While this study provides foundational insights, the current dataset reflects the limitations of incomplete taxon sampling and the constrained informativeness of chloroplast markers. Although the sequencing of nine plastomes and the incorporation of additional plastid genes facilitated a moderately resolved phylogenetic reconstruction, the lack of backbone resolution underscores the need for nuclear markers to better elucidate relationships, especially considering morphological homoplasy and the non-monophyly of Luer’s subgeneric classifications. Future efforts must prioritize expanded taxon sampling, both geographically and taxonomically, to capture the genus’s full diversity and refine its taxonomic framework. Biogeographically, the genus is predominantly diverse in Central and South America [20], but smaller clades in other regions highlight the complex evolutionary history of Lepanthes . Understanding these patterns requires phylogenetic reconstructions with greater taxonomic breadth. Current work sequencing whole nuclear genomes of 100 additional taxa aims to address these gaps, incorporating broader sampling and advanced molecular tools to resolve closely spaced speciation events and uncover lineage-specific diversification mechanisms. The discovery of non-monophyletic subgeneric classifications highlights the need for a thorough reassessment of the taxonomic framework for Lepanthes . However, we strongly discourage any taxonomic revisions until a species-rich phylogeny is available, one that includes representatives from all major clades and informal morphological and geographical groups. Taxon sampling and selection are critical factors in phylogenetic reconstructions, as demonstrated by numerous studies [69–71]. Sparse sampling, often constrained by the logistical challenges of acquiring diverse taxa under strict permit regulations can lead to long-branch artifacts and unresolved relationships [25, 72]. Furthermore, the inclusion of distantly related outgroups and taxa with varied evolutionary rates can significantly influence tree topologies [70]. High taxon sampling, even when some data are missing, has consistently been shown to improve branch support, as seen in studies combining whole plastome sequences with datasets containing only a few chloroplast genes [27]. These findings underscore the importance of robust and representative sampling strategies to achieve well-resolved phylogenetic reconstructions for Lepanthes , laying the groundwork for future taxonomic and evolutionary studies. Declarations -Ethics approval and consent to participate: Not applicable -Consent for publication: Not applicable -Availability of data and materials: The datasets generated and/or analysed during the current study are available in the NCBI repository, BioprojectID PRJNA1209138 -Competing interests: We declare that we have no competing interests. None of the authors have financial, personal, or professional conflicts that could be perceived as influencing the work reported in this manuscript. -Funding: Sociedad Colombiana de Orquideología in Colombia for their support with resources. Universidad de las Américas (UDLA) for funding research on Orchidaceae in Ecuador. Idea Wild for their support in supplying equipment to ASS. -Authors' contributions TA: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing – original draft, Writing – review and editing JSM: Conceptualization, Data curation, Investigation, Writing – original draft, Writing – review and editing SR: Formal analysis, Methodology, Writing – review and editing ASS: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Writing – review and editing MLL: Formal analysis, Writing – review and editing GAI: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing – original draft, Writing – review and editing JV: Data curation, Formal analysis, Writing – review and editing LB: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing – original draft, Writing – review and editing AZ: Data curation, Formal analysis, Methodology, Project administration, Resources, Supervision, Visualization, Writing – review and editing -Acknowledgements We extend our gratitude to the Sociedad Colombiana de Orquideología and Colomborquídeas in Colombia for their support with resources and access to plant collections used in this research. We also thank the Universidad de las Américas (UDLA) for funding research on Orchidaceae in Ecuador. Our appreciation goes to the Corporación para Investigaciones Biológicas for providing collection permits for TA and ASS during their affiliation with the institution. We are grateful to Idea Wild for their support in supplying equipment to ASS. Special thanks to Juan Felipe Posada, Luis Eduardo Mejía, Jean Mark Palandre, and Sebastián Vieira for their valuable input, collaboration, and assistance with plant materials. We also acknowledge Juliana Arcila and Jorge Muñoz for their expertise in bioinformatics, which was crucial to the success of this study. Finally, we thank Lourens Grobler and Ron Parsons for kindly allowing the use of their photographs of several Lepanthes species, which greatly enriched this research. We also appreciate Justin Yeager for his help in refining the English for this manuscript. -Authors' information (optional) Declaration of generative AI and AI-assisted technologies in the writing process. During the preparation of this work the author(s) used ChatGPT, OpenAI, December 2024 to improve the readability and language of the manuscript. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the published article. References 1. Karremans AP. 2016 Genera Pleurothallidinarum: An updated phylogenetic overview of Pleurothallidinae Lankesteriana 16 (2):219–241. https://doi.org/10.15517/lank.v16i2.26008. 2. Luer CA and L Thoerle. 2010 Icones Pleurothallidinarum XXXI: Lepanthes of Bolivia Monographs in Systematic Botany from the Missouri Botanical Garden 120: 1–64 3. 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Jansen RK, Cai Z, Raubeson LA, Daniell H, Depamphilis CW, Leebens-Mack J, Muller KF, Guisinger-Bellian M, Haberle RC, Hansen AK, Chumley TW, Lee S-B, Peery R, McNeal JR, Kueh JV, Boore JL. 2007 Analysis of 81 genes from 64 plastid genomes resolves relationships in angiosperms and identifies genome-scale evolutionary patterns Proceedings of the National Academy of Sciences 104(49): 19369–19374 Supplementary Figures Note Supplementary Figures S4 and S9 are not available with this version. Additional Declarations No competing interests reported. Supplementary Files TableS1.xlsx List of primers used for sequence amplification on Marsipanthes and other selected Lepanthes species. TableS2.xlsx Raw and trimmed reads and percentages of reads retain after data filtering for genome skimming readings of ten Lepanthes species representing main taxonomic groups. TableS3.xlsx Repeat sequence analysis found nine Lepanthes species representing main taxonomic groups. TableS4.xlsx List of species, codes, herbaria and accession of orchids used in this study. FigureS1.pdf Number of ycf1 and ndhF gene copies and localization in the chloroplast genome of nine Lepanthes species analyzed here. A. Gene copies located between IR1-SSC. B. Genes copies located between IR2-SSC. FigureS2.pdf Collinearity analysis using Mauve v1.1.3 plugin in Geneious v9.1.4 showed no gene rearrangements or inversions in the Lepanthes plastomes. FigureS3.pdf Sequence divergence of Lepanthes plastomes examined using the online program mVISTA Whole-genome alignment revealed that sequence variation in conserved non-coding regions (highlighted in pink bars) was greater than in protein-coding regions (highlighted in purple bars). FigureS537sppML.pdf Inferred ML phylogeny for the Lepanthes backbone using 35 species with different chloroplast gene sampling size. FigureS6BAYESIANITSMATK.pdf Reconstructed phylogenetic tree of concatenated markers r ITS and matK with BI analysis. FigureS7MLITSMATK.pdf Reconstructed phylogenetic tree of concatenated markers r ITS and matK with ML analysis. FigureS8.pdf MatK Inferred ML phylogeny for all Lepanthes species sequenced here and available in Genebank. FigureS10.tif Marsipanthes species of the Eastern Andes. A. Lepanthes caprimulgus Luer. B. Lepanthes attenboroughii Baquero & Yeager. C. Lepanthes portillae Luer. D. Lepanthes martinae Luer & Cloes. E. Lepanthes mulleriana Luer. F. Lepanthes terborchii Luer & Sijm. G. Lepanthes tigrina Luer & Thoerle. Photos: A & B by Luis E. Baquero, C, E & G by Lourens Grobler and D & F by Ron Parsons FigureS11.png Marsipanthes species of the Western Andes. A. Lepanthes ribes Luer. B. Lepanthes carunguligera Rchb.f. C. Lepanthes quadricornis D. Lepanthes equicalceolata E. Lepanthes felis F. Lepanthes lucifer. G. Lepanthes nisseniae Luer. H. Lepanthes tulcanensis Baquero & Yeager. Photos: A, C & D by Lourens Grobler, B by Ron Parsons and E, F, G & H by Luis E. Baquero. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5738250","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":407948701,"identity":"0d1dbaab-882d-4c57-942d-06899f286a73","order_by":0,"name":"Tatiana Arias","email":"data:image/png;base64,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","orcid":"","institution":"Marie Selby Botanical Gardens","correspondingAuthor":true,"prefix":"","firstName":"Tatiana","middleName":"","lastName":"Arias","suffix":""},{"id":407948702,"identity":"2cb015fc-87e6-42e4-8810-55b07f465a61","order_by":1,"name":"Juan Sebastian Moreno","email":"","orcid":"","institution":"Jardín Botánico de Cali-FZC","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"Sebastian","lastName":"Moreno","suffix":""},{"id":407948703,"identity":"41d27343-4d65-4cbf-bde9-5bf2d0ea62a0","order_by":2,"name":"Sebastian Reyes","email":"","orcid":"","institution":"Universidad de Caldas","correspondingAuthor":false,"prefix":"","firstName":"Sebastian","middleName":"","lastName":"Reyes","suffix":""},{"id":407948704,"identity":"a6da45a7-cbd1-447f-b2a1-19134e667dc5","order_by":3,"name":"Martin Llano Almario","email":"","orcid":"","institution":"Universidad del Valle","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"Llano","lastName":"Almario","suffix":""},{"id":407948705,"identity":"f6511883-2386-4f5c-933a-475a7b0bc124","order_by":4,"name":"Alejandra Serna-Sánchez","email":"","orcid":"","institution":"Universidad de Costa Rica","correspondingAuthor":false,"prefix":"","firstName":"Alejandra","middleName":"","lastName":"Serna-Sánchez","suffix":""},{"id":407948706,"identity":"81695867-e310-440a-a977-2e60eb57bda2","order_by":5,"name":"Gabriel A. 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Genes copies located between IR2-SSC.\u003c/p\u003e","description":"","filename":"FigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5738250/v1/c5f90c5b220ceca34ecad6d3.pdf"},{"id":75753647,"identity":"701eba59-d96e-4544-a374-e6a765a5b0b1","added_by":"auto","created_at":"2025-02-07 21:36:07","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":3676235,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCollinearity analysis using Mauve v1.1.3 plugin in Geneious v9.1.4 showed no gene rearrangements or inversions in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLepanthes \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eplastomes.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"FigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5738250/v1/10b27869c6cb82dbc4e6d3c7.pdf"},{"id":75753649,"identity":"84564ed9-95ae-49d7-a16d-df3d97b34446","added_by":"auto","created_at":"2025-02-07 21:36:07","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1209513,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSequence divergence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLepanthes\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e plastomes examined using the online program mVISTA\u003c/strong\u003e Whole-genome alignment revealed that sequence variation in conserved non-coding regions (highlighted in pink bars) was greater than in protein-coding regions (highlighted in purple bars).\u003c/p\u003e","description":"","filename":"FigureS3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5738250/v1/bdb8059ed60080f701ed335b.pdf"},{"id":75753658,"identity":"645a0321-4e30-4c15-8440-4e7dc8695f9d","added_by":"auto","created_at":"2025-02-07 21:36:08","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":5752,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInferred ML phylogeny for the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLepanthes\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e backbone using 35 species with different chloroplast gene sampling size.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"FigureS537sppML.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5738250/v1/79c76c340cd98903063f192f.pdf"},{"id":75754002,"identity":"7e232a5d-3eae-4910-be11-a8cd4bb0bca5","added_by":"auto","created_at":"2025-02-07 21:44:07","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":2936,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReconstructed phylogenetic tree of concatenated markers r ITS and matK with BI analysis.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"FigureS6BAYESIANITSMATK.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5738250/v1/cfccc48288687697b1429e40.pdf"},{"id":75754006,"identity":"b326e54a-6187-422a-aeac-ec56921eb81b","added_by":"auto","created_at":"2025-02-07 21:44:07","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":3059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReconstructed phylogenetic tree of concatenated markers r ITS and matK with ML analysis.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"FigureS7MLITSMATK.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5738250/v1/3a0e905a9e680f20bc367a79.pdf"},{"id":75753661,"identity":"ea0e2cd6-45d6-4168-bbbb-bdaf5ca4a604","added_by":"auto","created_at":"2025-02-07 21:36:08","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":5757,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMatK Inferred ML phylogeny for all \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLepanthes\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e species sequenced here and available in Genebank.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"FigureS8.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5738250/v1/eca314025114240e9627e02a.pdf"},{"id":75753671,"identity":"11a65ac5-a939-40e0-a548-9cce8666e968","added_by":"auto","created_at":"2025-02-07 21:36:09","extension":"tif","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":34914920,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMarsipanthes \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003especies of the Eastern Andes. \u003c/strong\u003eA. \u003cem\u003eLepanthes caprimulgus \u003c/em\u003eLuer. B. \u003cem\u003eLepanthes attenboroughii \u003c/em\u003eBaquero \u0026amp; Yeager. C. \u003cem\u003eLepanthes portillae \u003c/em\u003eLuer. D. \u003cem\u003eLepanthes martinae \u003c/em\u003eLuer \u0026amp; Cloes. E. \u003cem\u003eLepanthes mulleriana \u003c/em\u003eLuer. F. \u003cem\u003eLepanthes terborchii \u003c/em\u003eLuer \u0026amp; Sijm. G. \u003cem\u003eLepanthes tigrina \u003c/em\u003eLuer \u0026amp; Thoerle. Photos: A \u0026amp; B by Luis E. Baquero, C, E \u0026amp; G by Lourens Grobler and D \u0026amp; F by Ron Parsons\u003c/p\u003e","description":"","filename":"FigureS10.tif","url":"https://assets-eu.researchsquare.com/files/rs-5738250/v1/b600c4f248cfc959c80db9d5.tif"},{"id":75754011,"identity":"3328417f-e45b-4d14-9792-0ab5dcf54892","added_by":"auto","created_at":"2025-02-07 21:44:08","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":8519520,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMarsipanthes \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003especies of the Western Andes. \u003c/strong\u003eA. \u003cem\u003eLepanthes ribes \u003c/em\u003eLuer. B. \u003cem\u003eLepanthes carunguligera \u003c/em\u003eRchb.f. C. \u003cem\u003eLepanthes quadricornis\u003c/em\u003e D. \u003cem\u003eLepanthes equicalceolata\u003c/em\u003e E. \u003cem\u003eLepanthes felis\u003c/em\u003e F. \u003cem\u003eLepanthes lucifer. \u003c/em\u003e\u0026nbsp;G. \u003cem\u003eLepanthes nisseniae \u003c/em\u003eLuer. H. \u003cem\u003eLepanthes tulcanensis \u003c/em\u003eBaquero \u0026amp; Yeager. Photos: A, C \u0026amp; D by Lourens Grobler, B by Ron Parsons and E, F, G \u0026amp; H by Luis E. Baquero.\u003c/p\u003e","description":"","filename":"FigureS11.png","url":"https://assets-eu.researchsquare.com/files/rs-5738250/v1/784d31578549c146e55d9df0.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Plastome phylogenomics of the Diverse Neotropical Orchid Genus Lepanthes with Emphasis on Subgenus Marsipanthes (Pleurothallidinae: Orchidaceae)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe genus \u003cem\u003eLepanthes\u003c/em\u003e Sw. (Orchidaceae) stands out as one of the largest in the subtribe Pleurothallidinae [1], encompassing around 1,164 accepted species that are widely distributed through the Neotropics from Mexico to Bolivia and the Antilles, and from the Andes to the Amazon Basin [2–4]. The interplay between their specialized habitat requirements and the complex environmental gradients of the Andean region and the cordillera de Talamanca has made \u003cem\u003eLepanthes\u003c/em\u003e one of the most species-rich and endemic-rich genera in the Neotropics. This region is home to numerous endemic \u003cem\u003eLepanthes\u003c/em\u003e species, as highlighted in some studies [5].\u003c/p\u003e \u003cp\u003eThe genus displays its highest diversity in Colombia and Ecuador, where around 300 species have been described from each country, 377 species have been recently reported for Colombia [6]. In this region, species cover an impressive altitudinal gradient from sea-level to the snow line of the Andes, close to 4,000 m in elevation [2,7]. These species can thrive in diverse habitats, from lowland areas along the Pacific Ocean and the Amazon foothills to the Sierra Nevada de Santa Marta and the cloud forests of the Andes. \u003cem\u003eLepanthes\u003c/em\u003e species predominantly inhabit ecosystems characterized by high humidity, constant air movement, and horizontal precipitation. They grow attached to litter on the forest floor or on various phorophytes, including shrubs and trees, from the main trunk to branches and even the canopy. This remarkable ecological flexibility, combined with the intersection of three major biodiversity hotspots - Mesoamérica, Chocó/Darién/Western Ecuador, and the tropical Andes [8–9] - and the narrow geographic distributions of many species [5], is proposed to be responsible for the extraordinary diversity observed in the genus.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLepanthes\u003c/em\u003e species are characterized by diminutive size, and stems called ramicauls enclosed in funnel-shaped sheaths known as lepanthiform sheaths which often display microscopic cilia or pubescence and variable apical dilation. The flowers typically exhibit transversely elongated, often bilobed petals and a compound lip comprised of a body, connectives, and two blades. The compound lip plays a crucial role in pseudocopulatory pollination [10]. Notably, these orchids possess some of the smallest flowers in the orchid family and within the genus, various morphological groups exhibit specific similarities [11–13]. \u003cem\u003eLepanthes\u003c/em\u003e are notoriously difficult to classify due to the subtle differences between species. Comprehensive molecular studies combine with thorough morphological documentation will provide effective species delimitation and a clearer species concept for this genus. Understanding the genetic diversity and structure within taxa that exhibit subtle morphological differences will be crucial in addressing these challenges.\u003c/p\u003e \u003cp\u003eTaxonomy in the genus was developed by Carlyle Luer and collaborators using mostly morphological traits [14–18], and in recent years several molecular studies have been developed for \u003cem\u003eLepanthes\u003c/em\u003e species mostly from central American and the Caribbean [19–20]. It remains a research priority to conduct additional molecular studies across diverse taxa, to be able to critically address proposed taxonomic relationships. Despite hundreds of species distributed throughout diverse ecosystems of the Neotropics, Luer proposed the entire diversity of \u003cem\u003eLepanthes\u003c/em\u003e could be confined within two subgenera: subgenus \u003cem\u003eLepanthes\u003c/em\u003e and subgenus \u003cem\u003eMarsipanthes\u003c/em\u003e [21] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The subgenus \u003cem\u003eLepanthes\u003c/em\u003e encompasses the majority of species within the genus and is divided into three subsections, as defined by Luer [14–17]: \u003cem\u003eBreves\u003c/em\u003e (including series \u003cem\u003eBreves\u003c/em\u003e and \u003cem\u003eFilamentosa\u003c/em\u003e), distinguished by lateral sepals with a single vein; \u003cem\u003eLepanthes\u003c/em\u003e (comprising series \u003cem\u003eLepanthes\u003c/em\u003e, \u003cem\u003eElongatae\u003c/em\u003e, and \u003cem\u003eMucronatae\u003c/em\u003e), characterized by lateral sepals with two or three veins; and \u003cem\u003eBilabiatae\u003c/em\u003e, in which the lateral sepals are fully fused into a two-veined synsepal. However, the concept of series within these subsections was later abandoned [2].\u003c/p\u003e \u003cp\u003eSubgenus \u003cem\u003eMarsipanthes\u003c/em\u003e comprises eleven species distinguished by connate dorsal and lateral sepals with numerous veins forming an inflated tube and thick, fleshy, erect petals with one lobe - saccate flowers [22]. Within \u003cem\u003eMarsipanthes\u003c/em\u003e, three sections are recognized: sect. \u003cem\u003eMarsipanthes\u003c/em\u003e species have narrowly elongated petals, sect. \u003cem\u003eCaprimulginae\u003c/em\u003e have short, transverse petals, and deeply connate sepals, and sect. \u003cem\u003eFelinae\u003c/em\u003e have non-inflated flowers and asymmetrical petals with the upper lobe much longer than the lower lobe [7]. Sect. \u003cem\u003eCaprimulginae\u003c/em\u003e includes \u003cem\u003eLepanthes caprimulgus\u003c/em\u003e Luer and a recently described species \u003cem\u003eL. attenboroughii\u003c/em\u003e Baquero \u0026amp; Monteros [23]. Sect. \u003cem\u003eMarsipanthes\u003c/em\u003e includes \u003cem\u003eL. ribes\u003c/em\u003e Luer and \u003cem\u003eL. portillae\u003c/em\u003e Luer, and sect. \u003cem\u003eFelinae\u003c/em\u003e includes \u003cem\u003eL. carunculigera\u003c/em\u003e Rchb.f., \u003cem\u003eL. equicalceolata\u003c/em\u003e Luer \u0026amp; Escobar, \u003cem\u003eL. felis\u003c/em\u003e Luer \u0026amp; Escobar, \u003cem\u003eL. lucifer\u003c/em\u003e Luer \u0026amp; Hirtz, \u003cem\u003eL. niessienae\u003c/em\u003e Luer and \u003cem\u003eL. quadricornis\u003c/em\u003e Luer \u0026amp; Escobar [7, 21]. \u003cem\u003eL. tulcanensis\u003c/em\u003e Baquero \u0026amp; Yeager was described in recent years, and based on morphological traits were also considered likely to belong to \u003cem\u003eMarsipanthes\u003c/em\u003e sect. \u003cem\u003eFelinae\u003c/em\u003e [24].\u003c/p\u003e \u003cp\u003eThe current taxonomy of \u003cem\u003eLepanthes\u003c/em\u003e requires reevaluation within an evolutionary framework. Previous studies using few DNA molecular markers have failed to resolve relationships within the studied species [20], limiting our understanding of the genus origin and evolution and hindering the development of effective conservation strategies. Robust phylogenetic analyses based on tens of thousands of nucleotides, can greatly enhance our confidence in the resulting evolutionary hypotheses [25–27], besides adding to the understanding of species delimitation in the group. Plastomes have been widely used in orchid phylogenetics due to their high conservatism and relatively slow evolutionary rates [28]. However, the question remains whether large-scale datasets based on tens to hundreds of thousands of aligned nucleotides, representing both coding and non-coding regions, from the plastome possess enough phylogenetic signal to resolve evolutionary relationships in rapidly diversifying Andean orchids such as \u003cem\u003eLepanthes\u003c/em\u003e [29].\u003c/p\u003e \u003cp\u003eIn this study, we employed high-throughput sequencing (HTS) and genome skimming techniques to examine infrageneric taxonomic classifications (e.g. subgenera, sections) and compare the findings with the morphological data. By analyzing the plastome, we aimed to achieve higher levels of sequence divergence to resolve phylogenetic relationships among species in the genus in comparison to what the traditional gene-by-gene approaches have allowed. We present complete plastome sequences of nine representative \u003cem\u003eLepanthes\u003c/em\u003e species to: 1) describe and interpret the plastome structure and evolution of main taxonomic groups within \u003cem\u003eLepanthes\u003c/em\u003e while providing molecular tools useful for the community; 2) reconstruct phylogenetic relationships among selected species of the \u003cem\u003eLepanthes\u003c/em\u003e backbone using plastid genome sequences, further concentrating sequencing efforts in subgenus \u003cem\u003eMarsipanthes\u003c/em\u003e; 3) test preliminary hypothesis about the biogeography and evolution of continuous morphological characters for representatives of the \u003cem\u003eLepanthes\u003c/em\u003e backbone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003e1. Taxon sampling, DNA extraction, amplification and sequencing\u003c/b\u003e: Fresh leaves from adult plants were collected in Colombia from Colomborquídeas in El Retiro, Antioquia, under a collection permit from the Corporación para Investigaciones Biológicas (resolution ANLA 1263) and CITES (permit number: 42664). We studied ten new plastomes representing valid \u003cem\u003eLepanthes\u003c/em\u003e species, using the circumscription and infrageneric classification of Luer [14, 17, 30] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Additionally, 26 taxa from Ecuador were sampled for this study, 24 species of \u003cem\u003eLepanthes\u003c/em\u003e and two outgroups \u003cem\u003ePseudolepanthes colombiae\u003c/em\u003e Archila and \u003cem\u003eBrachionidium valerioi\u003c/em\u003e Ames \u0026amp; C. Schweinf (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e) under a collection permit for Ecuador (MAATE-DBI-CM-2021-0187). For all species, fresh leaves were stored in silica gel for later DNA extraction using the CTAB method for species sampled in Colombia [31]. Total DNA was purified with silica columns (Epoch Life Science Inc. Missouri, Texas, USA) and eluted in Tris-EDTA. DNA concentration was measured using a fluorometer (Qubit Fluorometer, Invitrogen, USA), and the integrity and purity of the samples were verified through agarose gel electrophoresis. DNA from species sampled in Ecuador was extracted using a rapid extraction procedure [32].\u003c/p\u003e\u003cp\u003eFor plastome sequencing, a 300-bp DNA library was constructed using the TruSeq Illumina platform (Illumina Inc., San Diego, CA, USA). A total of 1.5 µg of DNA was fragmented using a Covaris ultrasonicator (Covaris Inc., Woburn, MA), and the fragmented DNA was assessed via gel electrophoresis (Bio-Rad Laboratories, Hercules, CA). The fragmented DNA was treated with End Repair Mix (New England BioLabs, Ipswich, MA) and incubated at 20°C for 30 minutes. The end-repaired DNA was purified with the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany), then treated with A-Tailing Mix (Thermo Fisher Scientific, Waltham, MA) and incubated at 37°C for 30 minutes. The adenylated DNA was ligated with adapters using the Adapter and Ligation Mix, with the ligation reaction incubated at 20°C for 15 minutes. Adapter-ligated DNA was selected by running a 2% agarose gel to recover the target fragments, followed by purification with the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). Multiple rounds of PCR amplification were performed using a PCR Primer Cocktail and PCR Master Mix to enrich the adapter-ligated DNA fragments. The PCR products were again selected via 2% agarose gel and purified with the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). The final library was quantified by determining the average molecule length with an Agilent 2100 Bioanalyzer (Agilent DNA 1000 Reagents, Santa Clara, CA) and by real-time quantitative PCR (qPCR) using TaqMan Probes (Thermo Fisher Scientific, Waltham, MA). Libraries were multiplexed and sequenced on an Illumina HiSeq 4000 platform (San Diego, CA), producing paired end reads of 150 base pairs in length at the Beijing Genomics Institute (BGI), Hong Kong.\u003c/p\u003e\u003cp\u003ePolymerase Chain Reaction (PCR) was used for amplification of two regions, nuclear ribosomal internal transcribed spacer (rITS) and plastid Maturase K (matK), with 7.5 µL GoTaq Green Master Mix 2X (Promega), 3 µL of extracted DNA, 7.5 µL ultra-pure water, and 1 µL of each primer (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). PCR conditions were as follows: 2 min–95°C, 1 min–95°C, 1 min–55°C, 1 min–72°C, 35 cycles, and 5 min–72°C for final extension. PCR products were purified, and Sanger sequenced (ABI 3500xL Genetic Analyzer, Applied Biosystem).\u003c/p\u003e\u003cp\u003e \u003cb\u003e2. Plastome assembly, annotation, and comparative analyses\u003c/b\u003e: In average 4.5 GB of high-quality data was obtained for each sample. To evaluate the quality of the reads and check for the presence of adapters or other contaminants, we used FastQC [33]. Based on this analysis, the reads were trimmed using Trimmomatic v 0.39 [34], removing reads with a Phred score below 33 (parameter “-phred33”) or a length shorter than 100 bp (MINLEN:100). Adapters within the reads (parameter “-ILLUMINACLIP”) and low-quality bases at the start (LEADING) and end (TAILING) of the reads with a Phred score less than 3 were also removed. The remaining paired-end sequences were merged with a maximum overlap of 100 bp (Geneious Prime 2021 v2.2).\u003c/p\u003e\u003cp\u003eThe quality-filtered reads were then assembled de novo using GetOrganelle v 1.7.5 [35], specifying chloroplasts as the target organelle (parameter “-F embplant_pt”). Gaps between scaffolds were closed through iterative mapping in the Geneious program (Geneious Prime 2021 v2.2) using default settings. Out of the ten sequenced plastomes, only nine were successfully assembled. The sequencing reads for \u003cem\u003eLepanthes eros\u003c/em\u003e were contaminated, and we were unable to assemble its plastome with confidence. The plastome sequences were deposited in GenBank (BioprojectID PRJNA1209138) and the raw sequence data were submitted to GenBank, receiving SRA accessions (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWe annotated plastomes assembled using the Geneious annotation tool with default parameters and a 95% similarity threshold. The genomes of \u003cem\u003eAnathallis obovata\u003c/em\u003e [36] and \u003cem\u003eLepanthes caprimulgus\u003c/em\u003e [28] were used as reference. Annotations were manually verified to ensure no stop codons within genes. Circular plastome maps for \u003cem\u003eLepanthes\u003c/em\u003e were created using OGDRAW [37]. Several open reading frames (ORFs) that did not match the initial annotations were identified using BLASTn [38] followed by BLASTx [39] searches in NCBI.\u003c/p\u003e\u003cp\u003e \u003cb\u003e3. Plastome Structure and Sequence Divergence Analyses\u003c/b\u003e: To assess potential expansions and contractions of the IR boundary, the genes in the boundary regions of LSC/IRb/SSC/IRa were visualized using IRscope v3.1 [40]. Additionally, gene arrangement was analyzed through a collinearity analysis using the Mauve v1.1.3 plugin in Geneious v9.1.4 with default parameters. Sequence divergence of \u003cem\u003eLepanthes\u003c/em\u003e plastomes was examined using the online program mVISTA, with the \u003cem\u003eAnathallis obovata\u003c/em\u003e plastome serving as a reference. Nucleotide diversity (Pi) values were calculated using DnaSP6 with a sliding window of 1000 sites and a step size of 25 sites [41].\u003c/p\u003e \u003cp\u003e \u003cb\u003e4. Plastome Identified Repetitive Sequence and Codon Usage Analyses\u003c/b\u003e: Three types of repeat sequences were analyzed in the plastomes: simple sequence repeats (SSRs), tandem repeats, and long repeats. SSRs were detected using MISA v2.1 [42] and visualized with the R package ggplot2. Tandem repeats were identified using Tandem Repeats Finder v0.9 [42]. Forward, reverse, complement, and palindromic long repeats were detected in REPuter [43] using default parameters. The Relative Synonymous Codon Usage (RSCU) ratio for nine \u003cem\u003eLepanthes\u003c/em\u003e plastomes was estimated using CodonW v1.4.2 [44]. An RSCU value greater than 1 indicates positive codon usage bias, while a value less than 1 indicates less frequent usage. The R package \"pheatmap\" was used to generate a heatmap for the RSCU analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003e5. Phylogenetic Analysis\u003c/b\u003e: Nine \u003cem\u003eLepanthes\u003c/em\u003e species along with \u003cem\u003eAnathallis obovata\u003c/em\u003e (Lindl.) Pridgeon and M.W. Chase, \u003cem\u003eDraconanthes aberrans\u003c/em\u003e (Schltr.) Luer, \u003cem\u003eTrichosalpinx dirhamphis\u003c/em\u003e (Luer) Luer and \u003cem\u003eZootrophion lappaceum\u003c/em\u003e Luer and R. Escobar as outgroups, were included in the phylogenetic analyses. Plastomes were aligned using the MAFFT software [45]. To further explore the phylogenetic relationships within \u003cem\u003eLepanthes\u003c/em\u003e, maximum likelihood (ML) analyses were conducted using RAxML version 7.4.2 [46] under the general time-reversible (GTR) substitution model. Bootstrap support was assessed with 1000 bootstrap replicates. Additionally, Bayesian inference (BI) analysis was performed using MrBayes with 20,000,000 generations and sampled every 1000 generations. The majority rule (\u0026gt; 75%) consensus tree was obtained after removing the first 25% of the sampled trees as “burn-in” [47].\u003c/p\u003e\u003cp\u003eWhole plastid phylogenies were reconstructed based on the following datasets: (1) complete plastid genomes, (2) different outgroup sets, (3) one inverted repeat (IR), (4) coding sequences, and (5) non-coding sequences. The \u003cem\u003eFilamentosa\u003c/em\u003e series (Subgen. \u003cem\u003eLepanthes\u003c/em\u003e, Sect. \u003cem\u003eLepanthes\u003c/em\u003e, Subsect. Breves) and the \u003cem\u003eElongatae\u003c/em\u003e series (Subgen. \u003cem\u003eLepanthes\u003c/em\u003e, Sect. \u003cem\u003eLepanthes\u003c/em\u003e, Subsect. \u003cem\u003eLepanthes\u003c/em\u003e) were not represented in this phylogeny by any species.\u003c/p\u003e\u003cp\u003eIn addition to constructing complete plastid phylogenies, we downloaded 26 SRA data files for \u003cem\u003eLepanthes\u003c/em\u003e species from Genebank (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e) and mined all plastid sequences. Chloroplast assemblies were generated using GetOrganelle [35] and Captus [48]. A set of chloroplast coding regions was successfully recovered for multiple \u003cem\u003eLepanthes\u003c/em\u003e species. To perform alignments and phylogenetic reconstructions, we employed two approaches: (1) coding sequences concatenation with an incomplete data matrix for a subset of species, and (2) constructing a \u003cem\u003ematK\u003c/em\u003e phylogeny for all available species.\u003c/p\u003e\u003cp\u003eFor the phylogenetic reconstruction of a combined dataset (rITS + matK) focusing on subgenus \u003cem\u003eMarsipanthes\u003c/em\u003e, analyses were conducted using \u003cem\u003eBrachionidium valerioi\u003c/em\u003e Ames \u0026amp; C. Schweinf. And \u003cem\u003ePseudolepanthes colombiae\u003c/em\u003e Archila as outgroup species. Maximum Likelihood (ML) analyses were performed using the PhyML plugin [49] in Geneious Prime, employing the GTR + G substitution model with 1000 bootstrap replicates. Bayesian Inference (BI) analyses were conducted in BEAST v.1.10.4 [50], utilizing the GTR + G substitution model and the Yule speciation model. The BI analysis ran for 10\u0026nbsp;million generations, with tree sampling occurring every 10,000 generations. A single consensus tree was generated using TreeAnnotator v.1.10.4 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://beast.community/treeannotator\u003c/span\u003e\u003cspan address=\"http://beast.community/treeannotator\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e \u003cb\u003e6. Morphological analysis\u003c/b\u003e: Patterns of morphological variation and potential correlations between character traits and phylogenetic clades were assessed using Principal Component Analysis (PCA) and correlation matrices to contextualize the evolutionary relationships. Protologs of species included here were used to build a database including continuous measurements of vegetative and floral structures such as the ramicauls, leaves, peduncles, sepals, petals, lip, and column. Both minimum and maximum dimensions were considered for each continuous morphological character. R (version 4.2.1) was the primary programming environment for conducting multivariate analyses of orchid morphological traits. The analysis involved several R packages \u003cem\u003eggplot2\u003c/em\u003e for data visualization, and \u003cem\u003eggcorrplot\u003c/em\u003e for generating a correlation matrix plot. We pre-processed the data to address missing values using the \u003cem\u003ena.omit\u003c/em\u003e function and converted categorical variables to numeric format. After filtering species with missing data only 20 species were used for the analysis. Normalization of the data was performed using z-score scaling to ensure comparability among variables. The correlation matrix was computed to assess the relationships among the traits, and a color-coded visualization was created to facilitate the interpretation of significant correlations. Principal Component Analysis (PCA) was conducted on normalized orchid trait data to capture maximum variance, with results visualized using customized functions for species labeling and heatmaps illustrating trait contributions. The proportion of variance explained by the first two principal components was calculated, and key findings were saved as CSV files for further analysis.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003e1. Plastomes molecular descriptions\u003c/b\u003e: The assembled and annotated chloroplasts were uploaded to the NCBI database (BioprojectID PRJNA1209138). Using the Illumina HiSeq 4000 system, we obtained 7,150,658 (\u003cem\u003eL. eros\u003c/em\u003e) to 11,373,812 bp (\u003cem\u003eL. narcissus\u003c/em\u003e) paired reads after cleaning, with an average read length of 145 bp and a recovery rate of approximately 82% of the original reads (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The \u003cem\u003eLepanthes eros\u003c/em\u003e genome could not be assembled due to significant contamination in the Illumina reads. The nine \u003cem\u003eLepanthes\u003c/em\u003e plastomes assembled had an average length of 157,710 bp, with \u003cem\u003eL. narcissus\u003c/em\u003e having the smallest plastome (157,185 bp) and \u003cem\u003eL. caprimulgus\u003c/em\u003e the largest (158,260 bp) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The G/C content of all plastomes was approximately 37%. Each plastome exhibited the typical quadripartite structure seen in angiosperm plastomes, including a Large Single Copy (LSC), a Small Single Copy (SSC), and two Inverted Repeat (IR) regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). \u003cem\u003eL. ribes\u003c/em\u003e had the smallest LSC (89,500 bp) while \u003cem\u003eL. narcissus\u003c/em\u003e had the largest (90,156 bp). In terms of SSC, \u003cem\u003eL. nicolasii\u003c/em\u003e had the smallest at 19,095 bp, while \u003cem\u003eL. ribes\u003c/em\u003e had the largest (23,401 bp). The IR regions ranged from 24,495 bp in \u003cem\u003eL. nicolasii\u003c/em\u003e to 21,346 bp in \u003cem\u003eL. ribes\u003c/em\u003e. All plastomes contained 136 unique genes, including 42 tRNA genes, 8 rRNA genes, and 86 protein-coding genes. Additionally, 25 genes were duplicated in the IR regions (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eList of the\u003c/b\u003e \u003cb\u003eLepanthes\u003c/b\u003e \u003cb\u003especies used in this study and their infrageneric taxonomic classification following Luer [7, 14, 30].\u003c/b\u003e The series \u003cem\u003eFilamentosa\u003c/em\u003e (subgen. \u003cem\u003eLepanthes\u003c/em\u003e, sect. \u003cem\u003eLepanthes\u003c/em\u003e, subsect. \u003cem\u003eBreves\u003c/em\u003e) is not represented in this phylogeny by any species (NA nonincluded here). \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eLepanthes eros\u003c/em\u003e was not included in further analysis due to sequence contamination.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubgenera\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSection\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubsection\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSeries abandoned by Luer and Thoerle [2]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal species number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSpecies sequenced here\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSpecies sequenced elsewhere\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eMarsipanthes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCaprimulginae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eL. caprimulgus\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. attenboroughii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eFelinae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eL. felis\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. lucifer\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. niesseniae\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. tulcanensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eL. lucifer\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. niesseniae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eMarsipanthes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eL. ribes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003e\u003cem\u003eLepanthes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003e\u003cem\u003eLepanthes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBilabiatae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eL. narsissus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eBreves\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eBreves\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eL. monoptera\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. eros\u003c/em\u003e\u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eL. elata\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. tachirensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eFilamentosae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eLepanthes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMucronatae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eL. mucronata\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. hexapus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eL. ankistra\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. heteroloba\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. pelix\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eElongatae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e154\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eL. acarina\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. clowesii\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. katleri\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. martineae\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. nycteris\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. terborchii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eLepanthes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e795\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eL. barbelifera\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. calodyction\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. manabina\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. nicolasii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eL. adrianae\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. auriculata\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. ballatrix\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. calodyction\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. ciliisepala\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. clareae\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. confusa\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. crucitasensis\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. dactylopetala\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. dodsonii\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. gargantua\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. grandiflora\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. imitator\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. juninensis\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. matamorosii\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. meniscophora\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. mystax\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. orion\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. saltatrix\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. tentaculata\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. terpsichore\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. turialvae\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. urbaniana\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. volador\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eSummary of complete chloroplast genomes statistics.\u003c/b\u003e Comparison of plastome subunits length and GC content for the samples completely assembled.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLength (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLSC (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSSC (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIR (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGC%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. mucronata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e157,848\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e89,756\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19,116\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e24,488\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e37.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. hexapus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e157,629\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e89,567\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e21,363\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e23,363\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e37.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. manabina\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e157,208\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e89,953\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e22,091\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e22,582\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e37.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. ribes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e157,648\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e89,500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23,401\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e21,346\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e37.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. felis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e157,665\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e89,735\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e21,432\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e23,249\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e37.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. nicolasii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e158,213\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e90,128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19,095\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e24,495\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e37.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. monoptera\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e157,920\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e90,056\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e21,314\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e23,275\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e37.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. caprimulgus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e158,260\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e90,089\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e21,305\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e23,433\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e37.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. narcissus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e157,185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e90,156\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e21,325\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e22,852\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e37.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSeveral Open Reading Frames (ORFs) were identified across the \u003cem\u003eLepanthe\u003c/em\u003es genomes. In the LSC region, two hypothetical proteins were detected via BLASTx: one within the \u003cem\u003etrnC-GCA\u003c/em\u003e gene in all \u003cem\u003eLepanthes\u003c/em\u003e species except \u003cem\u003eL. nicolasii\u003c/em\u003e and \u003cem\u003eL. caprimulgus\u003c/em\u003e (408 bp, 136 aa), and another partially overlapping the \u003cem\u003eclpP1\u003c/em\u003e gene only in \u003cem\u003eL. manabina\u003c/em\u003e plastome, showing similarity to a Clp protease subunit in \u003cem\u003eEpidendrum ciliare\u003c/em\u003e (97.96% identity, e-value: 1e-21). In both inverted repeat regions (IR1 and IR2), a hypothetical protein, \u003cem\u003eM5K25\u003c/em\u003e, previously identified in \u003cem\u003eDendrobium thyrsiflorum\u003c/em\u003e, was detected within the \u003cem\u003etrnI-GAU\u003c/em\u003e gene in \u003cem\u003eLepanthes ribes\u003c/em\u003e, \u003cem\u003eL. narcissus\u003c/em\u003e, and \u003cem\u003eL. caprimulgus\u003c/em\u003e. Another hypothetical protein, conserved across all \u003cem\u003eLepanthes\u003c/em\u003e plastomes, was located within the \u003cem\u003err23\u003c/em\u003e rRNA gene (408 bp, 136 amino acids). This protein exhibited high similarity to chloroplast proteins from \u003cem\u003eAcorus calamus\u003c/em\u003e (95.56%, e-value: 2e-86), \u003cem\u003eDendrobium chrysanthum\u003c/em\u003e (93.04%), and \u003cem\u003eD. thyrsiflorum\u003c/em\u003e (95.6%).\"\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eycf1\u003c/em\u003e gene located around the boundary of IR1-SSC has different copies and is located in the SSC in most species but \u003cem\u003eL. mucronata\u003c/em\u003e, and \u003cem\u003eL. nicolasii.\u003c/em\u003e In these species one copy is located in the IR1 and the second one in the SSC (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). \u003cem\u003eL. felis, L. narcissus\u003c/em\u003e and \u003cem\u003eL. manabina\u003c/em\u003e had two functional copies and \u003cem\u003eL. caprimulgus\u003c/em\u003e three. In \u003cem\u003eL. manabina\u003c/em\u003e, an additional ORFs has been found embedded within the overlap of the two \u003cem\u003eycf1\u003c/em\u003e copies, this is like a \u003cem\u003eDracula erythrocochaete NADH dehydrogenase subunit F\u003c/em\u003e (94.51%, e-value: 1e-90) and \u003cem\u003eMasdevallia picturata NADH plastoquinone oxidoreductase subunit 5\u003c/em\u003e (94.51%, e-value: 2e-90). \u003cem\u003eL. narcissus\u003c/em\u003e had two copies of the \u003cem\u003endhF\u003c/em\u003e gene, one of them overlapping with \u003cem\u003eycf1\u003c/em\u003e and \u003cem\u003endhF\u003c/em\u003e. Only two species have multiple copies of \u003cem\u003eycf1\u003c/em\u003e in the boundary of SSC-IR2, \u003cem\u003eL. felis\u003c/em\u003e (3 copies) and \u003cem\u003eL. nicolasii\u003c/em\u003e (2 copies) (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cp\u003e \u003cb\u003e2. Plastome structural variations\u003c/b\u003e: An IR boundary map was created by comparing the plastomes of nine \u003cem\u003eLepanthes\u003c/em\u003e species using IRscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The \u003cem\u003erpl23\u003c/em\u003e gene was positioned at the end of the LSC, at the junction between the LSC and IR1 (JLB). At the junction between IR1 and SSC (JSB), \u003cem\u003eycf1\u003c/em\u003e was located within IR1 in \u003cem\u003eL. mucronata\u003c/em\u003e and \u003cem\u003eL. nicolasii\u003c/em\u003e, while in the rest of species, it was found at the beginning of the SSC. At the junction between SSC and IR2 (JSA) \u003cem\u003eycf1\u003c/em\u003e were completely located within the SSC, ranging from 89 to 29 bp before the end of IR2 in most species. However, in \u003cem\u003eL. mucronata\u003c/em\u003e and \u003cem\u003eL. nicolasii, ycf1\u003c/em\u003e spanned 1072 and 1077 bp, respectively, from the SSC into IR2. At the IR2-LSC junction (JLA), the \u003cem\u003eycf2\u003c/em\u003e gene was found spanning both regions. Collinearity analysis showed no gene rearrangements or inversions in the \u003cem\u003eLepanthes\u003c/em\u003e plastomes (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cp\u003e \u003cb\u003e3. Identification of hypervariable regions\u003c/b\u003e: The divergence of complete plastome sequences among nine \u003cem\u003eLepanthes\u003c/em\u003e species was analyzed using mVISTA, with \u003cem\u003eAnathallis obovata\u003c/em\u003e as the reference (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Whole-genome alignment revealed that sequence variation in conserved non-coding regions (highlighted in pink bars) was greater than in protein-coding regions (highlighted in purple bars). The variation rates in both coding and non-coding regions within the two IR regions were lower than those in the LSC and SSC regions. Highly divergent non-coding regions included \u003cem\u003epskB-psbI\u003c/em\u003e, \u003cem\u003eatpB-rbcL\u003c/em\u003e, \u003cem\u003epetA-psbJ\u003c/em\u003e, \u003cem\u003epsbB-psbN\u003c/em\u003e, and \u003cem\u003erpl32-trnL\u003c/em\u003e. In contrast, the rRNA genes were highly conserved compared to other genes.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eTo further investigate DNA polymorphisms within \u003cem\u003eLepanthes\u003c/em\u003e plastomes and to provide molecular tools useful to other researchers working in the group we identified DNA barcodes. Nucleotide diversity (Pi) values were calculated using DnaSP6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The average Pi value across the nine plastomes was 0.00906, with a total of 4,054 mutations identified. The IR region averaged a Pi value of 0.00229, the LSC region averaged 0.01050, and the SSC region averaged 0.01286. Based on Pi values (ranging from 0.02 to 0.03), eight hypervariable regions of the plastomes were identified: \u003cem\u003etrnKuuu\u0026ndash;rps16\u003c/em\u003e, \u003cem\u003epsbK\u0026ndash;psbI\u003c/em\u003e, \u003cem\u003epsbM\u0026ndash;trnEucc\u003c/em\u003e, \u003cem\u003etrnTUGU\u0026ndash;trnLUAA\u003c/em\u003e, \u003cem\u003eaccD\u0026ndash;psaI\u003c/em\u003e, \u003cem\u003eclpP1\u0026ndash;psbB\u003c/em\u003e, and \u003cem\u003eycf1\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e4. Repeated sequence analysis\u003c/b\u003e \u0026ndash; The results of SSR and tandem repeat analysis in \u003cem\u003eLepanthes\u003c/em\u003e plastomes using MISA are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The number of identified SSRs ranged from 58 in \u003cem\u003eL. hexapus\u003c/em\u003e to 47 in \u003cem\u003eL. monoptera\u003c/em\u003e, with a total of 470 SSRs detected across the \u003cem\u003eLepanthes\u003c/em\u003e plastomes. Three types of SSRs (mono-, di-, and complex-nucleotide repeats) were identified, with 436 (92.9%) being mono-nucleotide repeats, predominantly composed of A and T motifs. Additionally, 13 di-nucleotide repeats (2.77%) and 21 complex-nucleotide repeats (4.47%) were identified (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eRepetitive motif abundance in nine\u003c/b\u003e \u003cb\u003eLepanthes\u003c/b\u003e \u003cb\u003especies computed using MISA\u003c/b\u003e. Distribution and types of simple sequence repeats.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMononucleotides\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDinucleotides\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eComplex repeats\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSSRs Total\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. caprimulgus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. narcissus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. monoptera\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. nicolasii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. ribes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. felis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. manabina\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. mucronata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. hexapus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eA total of 215 tandem repeats were detected, with counts ranging from 17 in \u003cem\u003eL. nicolasii\u003c/em\u003e to 38 in \u003cem\u003eL. caprimulgus\u003c/em\u003e. Notably, all nine \u003cem\u003eLepanthes\u003c/em\u003e plastomes contained 50 long repeats each, comprising palindromic, forward, reverse, or complementary long repeats. Palindromic repeats were the most abundant, ranging from 33 in the \u003cem\u003eL. manabina\u003c/em\u003e plastome to 23 in \u003cem\u003eL. narcissus\u003c/em\u003e. Forward repeats followed in abundance, with \u003cem\u003eL. felis\u003c/em\u003e, \u003cem\u003eL. monoptera\u003c/em\u003e, and \u003cem\u003eL. caprimulgus\u003c/em\u003e each containing 18 forward repeats, while \u003cem\u003eL. hexapus\u003c/em\u003e contained 11. Reverse and complementary repeats accounted for only 59 out of 450 repeats, representing 13.1% of all long repeats (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo assess codon usage bias, the relative synonymous codon usage (RSCU) ratios were calculated for \u003cem\u003eLepanthes\u003c/em\u003e plastomes. Less frequently used codons (RSCU\u0026thinsp;\u0026lt;\u0026thinsp;1) were generally consistent across species, with most having 32, except \u003cem\u003eL. hexapus\u003c/em\u003e and \u003cem\u003eL. narcissus\u003c/em\u003e, which had 33. Most preferred codons ended with A or U, except for UGG and AGG. The codons AGA and UCU exhibited the highest RSCU values, averaging 2.11 and 1.58, respectively, while CGC and GAC had the lowest RSCU values, with averages of 0.41 and 0.54 (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003e5. Phylogenomic analysis\u003c/b\u003e \u0026ndash; Topologies based on whole plastomes, coding regions, and intergenic regions for nine \u003cem\u003eLepanthes\u003c/em\u003e species, representing main taxonomic groups were identical, with similar resolutions using both Bayesian Inference (BI) and Maximum Likelihood (ML) methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Species of \u003cem\u003eLepanthes\u003c/em\u003e formed a well-supported monophyletic group (PPBI\u0026thinsp;=\u0026thinsp;1.00, BSML\u0026thinsp;=\u0026thinsp;100). Within \u003cem\u003eLepanthes\u003c/em\u003e, two sister clades were identified: \u003cem\u003eL. narcissus\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eL. caprimulgus\u003c/em\u003e (PPBI\u0026thinsp;=\u0026thinsp;1.00, BSML\u0026thinsp;=\u0026thinsp;100) and the remaining \u003cem\u003eLepanthes\u003c/em\u003e species (PPBI\u0026thinsp;=\u0026thinsp;1.00, BSML\u0026thinsp;=\u0026thinsp;100). In the latter clade, \u003cem\u003eL. monoptera\u003c/em\u003e was recovered as sister to the rest, followed by \u003cem\u003eL. nicolasii\u003c/em\u003e, which is sister to a clade (PPBI\u0026thinsp;=\u0026thinsp;1.00, BSML\u0026thinsp;=\u0026thinsp;100) comprising \u003cem\u003eL. manabina\u003c/em\u003e sister to \u003cem\u003eL. mucronata\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eL. hexapus\u003c/em\u003e, and \u003cem\u003eL. ribes\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eL. felis\u003c/em\u003e in the Bayesian phylogeny and not resolved in the ML one (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eA second whole chloroplast phylogeny was reconstructed for 35 \u003cem\u003eLepanthes\u003c/em\u003e species including species sequenced here and 26 more, plus two to five outgroups downloaded from Genebank. For this alignment, one IR was excluded, and only coding regions were considered. The mean sequence length was 37,725 bp, ranging from a maximum of 72,282 bp to a minimum of 7,042 bp. The total length of the alignment was 85,750 bp, with a pairwise identity of 46.6%, and 5.3% of identical sites. Half of the species included here had between 50 and 77 chloroplast genes present in the alignment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Genes represented in this phylogeny included: \u003cem\u003eatpA, atpB, atpE,ndhK, psbB, psbC, psbZ\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and C). In this phylogeny species from most taxonomic groups described by Luer [14, 17, 30] were included, except for species from the series \u003cem\u003eFilamentosae\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All taxonomic groups below the genus level were found to be non-monophyletic, except for series \u003cem\u003eMucronatae\u003c/em\u003e still to be confirmed since the phylogeny did not fully resolve phylogenetic relationships among species include in this series (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). Six main clades were recovered, an early-divergent lineage, comprising \u003cem\u003eL. tachirensis\u003c/em\u003e and \u003cem\u003eL. juninensis\u003c/em\u003e, sister to the remaining species. Clade 1 sister to the rest of \u003cem\u003eLepanthes\u003c/em\u003e and including \u003cem\u003eL. narcissus\u003c/em\u003e, sister to \u003cem\u003eL. caprimulgus\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eL. katleri\u003c/em\u003e. Clade 2 consists of \u003cem\u003eL. monoptera\u003c/em\u003e, \u003cem\u003eL. rhynchion\u003c/em\u003e, and \u003cem\u003eL. speciosa\u003c/em\u003e, sister to the remaining species of \u003cem\u003eLepanthes\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eA polytomy was observed, including \u003cem\u003eL. acarina\u003c/em\u003e, \u003cem\u003eL. clowesii\u003c/em\u003e, Clade 3: \u003cem\u003eL. gargantua\u003c/em\u003e sister to subclades \u003cem\u003eL. imitator\u003c/em\u003e, \u003cem\u003eL. clarae\u003c/em\u003e, \u003cem\u003eL. nicolasii\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eL. adrianae\u003c/em\u003e, \u003cem\u003eL. orion\u003c/em\u003e and \u003cem\u003eL. auriculata\u003c/em\u003e and Clade 4. Clade 4 includes Central America and the Caribbean species \u003cem\u003eL. turialvae\u003c/em\u003e, \u003cem\u003eL. urbaniana\u003c/em\u003e, and \u003cem\u003eL. grandiflora\u003c/em\u003e, sister to the remaining species, Clade 5 including \u003cem\u003eL. tentaculate\u003c/em\u003e and \u003cem\u003eL. calodyction\u003c/em\u003e, and a second polytomy including several small subclades: \u003cem\u003eL. felis\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eL. ribes\u003c/em\u003e, \u003cem\u003eL. hexapus\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eL. mucronata\u003c/em\u003e, \u003cem\u003eL. lucifer\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eL. niesseniae\u003c/em\u003e, \u003cem\u003eL. heteroloba\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eL. pelyx\u003c/em\u003e, and a subclade consisting of \u003cem\u003eL. dodsonii\u003c/em\u003e, \u003cem\u003eL. manabina\u003c/em\u003e, \u003cem\u003eL. terpsichore\u003c/em\u003e and \u003cem\u003eL. meniscophora\u003c/em\u003e (Clade 6) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eFurther BI and ML analysis focusing on nine of the eleven \u003cem\u003eMarsipanthes\u003c/em\u003e species and using a combined \u003cem\u003erITS\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ematK\u003c/em\u003e dataset recovered several of the clades identified in our main phylogeny but lack of resolution for the backbone, confirming \u003cem\u003eMarsipanthes\u003c/em\u003e is non-monophyletic (Figures \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e, S7). Main clades to highlight from this phylogeny include: (1) Species from the Eastern Andes of Ecuador, Per\u0026uacute; and Bolivia. \u003cem\u003eL. caprimulgus, L. attenboroughii\u003c/em\u003e (both representing subgen. \u003cem\u003eMarsipanthes\u003c/em\u003e, sect. \u003cem\u003eCaprimulginae\u003c/em\u003e) and \u003cem\u003eL. martinae, L. nycteris\u003c/em\u003e and \u003cem\u003eL. terbochii\u003c/em\u003e belonging to subgen. \u003cem\u003eLepanthes\u003c/em\u003e (MLBS\u0026thinsp;=\u0026thinsp;75, BIPP\u0026thinsp;=\u0026thinsp;1). (2) Species from a western Andean \u003cem\u003eMarsipanthes\u003c/em\u003e clade including \u003cem\u003eL. felis\u003c/em\u003e, \u003cem\u003eL. ribes\u003c/em\u003e, \u003cem\u003eL. lucifer\u003c/em\u003e and \u003cem\u003eL. niesseniae\u003c/em\u003e (MLBS\u0026thinsp;=\u0026thinsp;94, BIPP\u0026thinsp;=\u0026thinsp;1). \u003cem\u003eL. tulcanesis\u003c/em\u003e phylogenetic position is uncertain. Few more clades recovered in this phylogeny include \u003cem\u003eL. calodictyon\u003c/em\u003e and closely related species \u003cem\u003eL. barbellifera, L. saltatrix\u003c/em\u003e and \u003cem\u003eL. volador\u003c/em\u003e (MLBS\u0026thinsp;=\u0026thinsp;100, BIPP\u0026thinsp;=\u0026thinsp;1).\u003c/p\u003e \u003cp\u003eLastly, a \u003cem\u003ematK\u003c/em\u003e phylogeny for 104 \u003cem\u003eLepanthes\u003c/em\u003e species, including both sequences produced here and those available in GenBank, lacked resolution (Figure \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003e6. Morphological continuous characters-\u003c/b\u003e PCA analysis indicated that continuous morphological measurements from the species included here did not form distinct groups according to clades (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). PC1 and PC2 account for about 61.96% of the total variance in the dataset, capturing a substantial amount of the variability in the traits we analyzed. PC1 and PC2 account for 35.49% and 26.47% of the total variance respectively. Dorsal sepals (Length Min.-Max., Width Min.-Max.) and Lateral sepals (Length Min.-Max., Width Min.-Max.) have the highest positive loading in this PCA suggesting sepal dimensions are critical for distinguishing among the species in our \u003cem\u003eLepanthes\u003c/em\u003e dataset. Traits like Peduncle Length Min. and Pedicel Length Min. also contribute positively. Traits with negative loadings on PC1 include Ramicauls Length Min. and Number of sheaths Min., indicating they might be less important or show an inverse relationship with the traits positively contributing to PC1. PC2 highlight traits with positive loadings including Dorsal sepal Length Min., Lat sepal Length Min., and Peduncle Length Min. Ramicauls Length Min., Leaf Width Min., Lip Length Min., and Column Length Max. show negative contributions to the data distribution, suggesting they may differentiate the data in an opposite direction compared to the positively loaded traits (Figure \u003cspan refid=\"MOESM9\" class=\"InternalRef\"\u003eS9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eContinuous morphological characters showed high positive correlations among several, floral and vegetative traits (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Strong correlations (above 0.8) were found between Peduncle Length and Floral Bract Length (0.930). Leaf Length and Ovary Length (0.888), Ovary Length and Floral Bract Length (0.849), Dorsal Sepal Length and Dorsal Sepal Width (0.849). None of the negative correlations were strong, for examples Dorsal Sepal Width and Leaf Width were negative correlated (-0.244) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eA genome skimming approach produced nine new chloroplast genomes for \u003cem\u003eLepanthes\u003c/em\u003e species, providing a valuable suite of molecular resources to the scientific community. Additionally, plastome genes sourced from GenBank facilitated the first moderately resolved phylogenetic reconstruction for the genus. Our findings demonstrate that the current taxonomic groups proposed by Luer are not monophyletic, reflecting significant morphological homoplasy. Six main clades were identified; however, the moderate resolution of the phylogenetic backbone highlights the need for nuclear markers and the inclusion of many more species to further refine these relationships. Despite limited species sampling, this study encompasses substantial taxonomic, geographic, and morphological diversity, laying a strong foundation for future evolutionary research on \u003cem\u003eLepanthes\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003ePlastome evolution of\u003c/b\u003e \u003cb\u003eLepanthes\u003c/b\u003e \u003cb\u003especies\u003c/b\u003e - A range of plastome lengths, averaging 157,710 base pairs (bp) was observed in \u003cem\u003eLepanthes\u003c/em\u003e. \u003cem\u003eL. narcissus\u003c/em\u003e has the smallest plastome 157,185 bp, while \u003cem\u003eL. caprimulgus\u003c/em\u003e the largest one 158,260 bp (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The G/C content across all plastomes was consistent at approximately 37%, reflecting typical characteristics for angiosperm and orchid plastomes [51]. The plastome size observed in this study aligns with the broad range reported for Orchidaceae, spanning from the reduced 19,047 bp in the mycoheterotrophic \u003cem\u003eEpipogium roseum\u003c/em\u003e [52] to the largest to our knowledge 212,688 bp in the autotrophic \u003cem\u003eCypripedium subtropicum\u003c/em\u003e [53], reflecting the family\u0026rsquo;s diversity in photosynthetic function and evolutionary adaptation.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eAll sequenced plastomes exhibited the classic quadripartite structure seen in angiosperms, comprising a Large Single Copy (LSC) region, a Small Single Copy (SSC) region, and two Inverted Repeat (IR) regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) [54\u0026ndash;55]. Among species, \u003cem\u003eL. ribes\u003c/em\u003e had the smallest LSC at 83,758 bp, while \u003cem\u003eLepanthes narcissus\u003c/em\u003e had the largest at 84,423 bp. The SSC size varied, with \u003cem\u003eL. ribes\u003c/em\u003e having the smallest at 18,024 bp and \u003cem\u003eL. hexapus\u003c/em\u003e the largest at 19,093 bp. Regarding the IR regions, \u003cem\u003eL. narcissus\u003c/em\u003e had the smallest at 26,830 bp, while \u003cem\u003eL. ribes\u003c/em\u003e had the largest at 27,933 bp. Each plastome contained 136 unique genes, including 111 unique genes, 42 tRNA genes, 8 rRNA genes, and 86 protein-coding genes. Additionally, 25 of these genes were duplicated in the IR regions. Although the IR in many angiosperms is approximately constant in size (~\u0026thinsp;25 kb), sequence analysis of its endpoints has shown that small differences exist between species in the extent of the IR [54]. \u003cem\u003eLepanthes\u003c/em\u003e species exhibit consistency in plastome length and structure and aligns with the general characteristics of monocot plastomes [51, 55\u0026ndash;56], this stability is important for epiphytes often growing in nutrient-poor soils, maintaining a stable chloroplast genome may be crucial to efficiently capture and use light and other limited resources [57].\u003c/p\u003e \u003cp\u003eDespite the generally conserved nature of chloroplast genomes, the presence of unique open reading frames (ORFs) in \u003cem\u003eLepanthes\u003c/em\u003e plastomes suggests possible lineage-specific evolutionary processes to be further investigated in the future [51, 58]. For example, our analysis identified an ORF within the \u003cem\u003etrnC-GCA\u003c/em\u003e gene in all species except \u003cem\u003eL. nicolasii\u003c/em\u003e and \u003cem\u003eL. caprimulgus\u003c/em\u003e (408 bp, 136 amino acids), indicating potential losses of this ORF at least twice in the genus evolution. Additionally, in the IR regions, we detected a hypothetical protein (\u003cem\u003eM5K25\u003c/em\u003e) from \u003cem\u003eDendrobium thyrsiflorum\u003c/em\u003e in several \u003cem\u003eLepanthes\u003c/em\u003e species. A second hypothetical protein was identified in the \u003cem\u003err23\u003c/em\u003e rRNA gene in all sequenced species, showing high similarity to proteins in \u003cem\u003eAcorus calamus\u003c/em\u003e (95.56%) and \u003cem\u003eDendrobium thyrsiflorum\u003c/em\u003e (95.6%). Furthermore, an ORF in \u003cem\u003eL. manabina\u003c/em\u003e, partially overlapping with the \u003cem\u003eclpP1\u003c/em\u003e gene, demonstrated strong similarity to a \u003cem\u003eclp\u003c/em\u003e protease subunit in \u003cem\u003eEpidendrum ciliare\u003c/em\u003e (97.96% identity, e-value: 1e-21), suggesting either horizontal gene transfer (HGT) or convergent evolution.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eycf1\u003c/em\u003e and \u003cem\u003endhF\u003c/em\u003e genes exhibit considerable molecular diversity and multiple copies across \u003cem\u003eLepanthes\u003c/em\u003e species sequenced here (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). \u003cem\u003eL. felis\u003c/em\u003e and \u003cem\u003eL. nicolasii\u003c/em\u003e have five and four \u003cem\u003eycf1\u003c/em\u003e copies respectively, while \u003cem\u003eL. narcissus\u003c/em\u003e has two \u003cem\u003endhF\u003c/em\u003e copies (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). This copy number variation does not seem to have a clear phylogenetic pattern indicating multiple and independent gene gains and losses. Gene duplications and losses pertaining to these regions have been reported in other angiosperms and orchids [59\u0026ndash;61]. The \u003cem\u003eycf1\u003c/em\u003e has been showed to encode products that are essential for cell survival [59]. Multiple copies of this gene observed here may perform slightly different functions.\u003c/p\u003e \u003cp\u003eAn examination of the IR (Inverted Repeat) boundaries and gene positions reveals slight patterns and variation among different species (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Chloroplast region boundaries are not only fundamental to the structural integrity and function of the chloroplast genome but also provide insights into evolutionary processes, functional genomics, and adaptation strategies. The positions of the LSC-IR junctions vary slightly within groups, but usually this has only negligible effects on plastome size [51]. SSC-IR boundaries were not fixed in \u003cem\u003eLepanthes\u003c/em\u003e; instead, and like in other plants, they undergo a dynamic and stochastic process that primarily results in conservative expansions and contractions of the IR [54]. Several consistencies were found across all sequenced \u003cem\u003eLepanthes\u003c/em\u003e species: the gene \u003cem\u003erpl23\u003c/em\u003e is positioned at the end of the LSC, the IR containing a \u003cem\u003etrnH-rps19\u003c/em\u003e cluster as reported in most monocots [55] and an overlap between the genes \u003cem\u003eycf1\u003c/em\u003e and \u003cem\u003endhF\u003c/em\u003e near or at the IR1-SSC boundary. In contrast there was some variation at the Inverted repeats and the SSC junctions: the position of \u003cem\u003eycf1\u003c/em\u003e copies varied among species, both in the SSC and IRb (IR boundary junction JSB) or IRa (IR boundary junction JSA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). For example, the position of \u003cem\u003eycf1\u003c/em\u003e in \u003cem\u003eL. mucronata\u003c/em\u003e and \u003cem\u003eL. nicolasii\u003c/em\u003e expands from the SSC into the IRa (IR boundary junction JSA). The IR can terminate within genes, leading to nonfunctional and potentially disruptive 5' or 3' truncated genes, whose translated products may negatively impact essential processes [54]. In Pleurothallidinae, the IR/SSC boundary interrupts the \u003cem\u003eycf1\u003c/em\u003e gene, resulting in a truncated \u003cem\u003eycf1\u003c/em\u003e fragment within the IR. Conversely, in \u003cem\u003eCattleya crispata\u003c/em\u003e, the SSC encompasses nearly the entire \u003cem\u003eycf1\u003c/em\u003e gene [36]. These plastome differences between Pleurothallidinae and Laeliinae may be characteristic of these subtribes. Collinearity analysis further supports the stability of these plastomes, as no gene rearrangements or inversions were detected (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). This lack of major structural changes reinforces the notion that while there are some variations in gene positioning, the overall plastome architecture of \u003cem\u003eLepanthes\u003c/em\u003e remains relatively stable across species.\u003c/p\u003e \u003cp\u003eLast, the analysis of relative synonymous codon usage (RSCU) revealed variability in codon preferences across the \u003cem\u003eLepanthes\u003c/em\u003e plastomes. The presence of both preferred (RSCU\u0026thinsp;\u0026gt;\u0026thinsp;1) and non-preferred codons (RSCU\u0026thinsp;=\u0026thinsp;1) highlights possible selective pressures influencing plastome evolution. Codons ending in A or U are prevalent, with AGA and UCU exhibiting the highest RSCU values (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). This pattern is consistent with the trend observed in other plant plastomes, where specific codon biases can reflect functional constraints and evolutionary pressures [62].\u003c/p\u003e \u003cp\u003e \u003cb\u003e2. New molecular resources for\u003c/b\u003e \u003cb\u003eLepanthes\u003c/b\u003e \u003cem\u003e-\u003c/em\u003e The identification of hypervariable regions in the plastomes of \u003cem\u003eLepanthes\u003c/em\u003e species reveals significant insights into the genetic variability across different regions of the plastome. Using mVISTA for whole-genome alignment with \u003cem\u003eAnathallis obovata\u003c/em\u003e as the reference, we observed that sequence divergence was notably higher in the conserved non-coding regions compared to the protein-coding regions. Specifically, non-coding regions such as \u003cem\u003epskB-psbI, atpB-rbcL, petA-psbJ, psbB-psbN\u003c/em\u003e, and \u003cem\u003erpl32-trnL\u003c/em\u003e exhibited higher variability, while rRNA genes remained highly conserved. The analysis of nucleotide diversity (Pi) values further highlights the distribution of genetic variation across different plastome regions. The IR regions showed the lowest nucleotide diversity with an average Pi value of 0.00229, suggesting relative stability in these regions. In contrast, the LSC and SSC regions exhibited higher diversity, with average Pi values of 0.01050 and 0.01286, respectively. This indicates that the LSC regions are more prone to variation, which could be useful for distinguishing between species or for evolutionary studies.\u003c/p\u003e \u003cp\u003eThe top seven hypervariable regions identified across the plastomes\u0026mdash;\u003cem\u003etrnKuuu-rps16\u003c/em\u003e, \u003cem\u003epsbK-psbI\u003c/em\u003e, \u003cem\u003epsbM-trnEucc\u003c/em\u003e, \u003cem\u003etrnTUGU-trnLUAA\u003c/em\u003e, \u003cem\u003eaccD-psaI\u003c/em\u003e, \u003cem\u003eclpP1-psbB\u003c/em\u003e, and \u003cem\u003eycf1\u003c/em\u003e present promising candidates for developing specific DNA barcodes. Parts of the \u003cem\u003eycf1\u003c/em\u003e gene are identified as potential good barcode regions for the genus, its utility has also been confirmed for many other angiosperms [60]. These regions, with Pi values between 0.02 and 0.03, offer the highest degree of variability and could serve as effective markers for species identification and phylogenetic studies (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). This analysis underscores the utility of non-coding regions and specific hypervariable regions in understanding genetic diversity and developing molecular tools for the study of \u003cem\u003eLepanthes\u003c/em\u003e. The findings not only contribute to the characterization of plastome variability within this genus but also provide a foundation for future research aimed at using these hypervariable regions for practical applications in plant systematics and conservation.\u003c/p\u003e \u003cp\u003eSSRs are a type of repeats frequently observed in chloroplast genomes that can be used to discover genome polymorphisms and perform population genetics within and between species [63]. The analysis of repeated sequences in \u003cem\u003eLepanthes\u003c/em\u003e species reveals detailed patterns of sequence variability and bias. Using the MISA tool to identify simple sequence repeats (SSRs) and tandem repeats, we found a total of 469 SSRs across the plastomes. The number of SSRs varied among species, with \u003cem\u003eL.hexapus\u003c/em\u003e showing the highest count of 58 and \u003cem\u003eL. monoptera\u003c/em\u003e the lowest at 47 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe structural and evolutionary stability observed in \u003cem\u003eLepanthes\u003c/em\u003e plastomes, particularly in nutrient-poor epiphytic environments, represents an intriguing avenue for future research. Unique features such as open reading frames, gene duplications, and structural variations at IR boundaries suggest lineage-specific evolutionary processes that merit further investigation.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3. Phylogenetic and morphological analyses-\u003c/b\u003e A phylogeny of \u003cem\u003eLepanthes\u003c/em\u003e using maternally inherited molecular markers such as those from the plastome was reconstructed here. Our phylogenies have a poor taxonomic representation (9\u0026ndash;35/1196 spp.) but a great number of informative molecular characters (\u0026le;\u0026thinsp;15,834bp). Besides species included in this analysis represented unique morphological groups identified through taxonomic studies.\u003c/p\u003e \u003cp\u003eAll phylogenies reconstructed here confirmed the monophyly of \u003cem\u003eLepanthes\u003c/em\u003e as proposed by Bogar\u0026iacute;n \u003cem\u003eet. al\u003c/em\u003e [20] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e); while, both \u003cem\u003eLepanthes\u003c/em\u003e subgenera recognized by Carl Luer [17] are not monophyletic (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003eS5\u003c/span\u003e-\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This indicates perianth characters are homoplasic and the fusion of petals and number of veins are not informative at this taxonomic level. Additionally, most of the subclades recovered here do not support further infrageneric classification by Luer [7], except for the subclade comprising \u003cem\u003eL. hexapus\u003c/em\u003e and \u003cem\u003eL. mucronata\u003c/em\u003e recovered as monophyletic and belonging to series \u003cem\u003eMucronatae\u003c/em\u003e from the section \u003cem\u003eLepanthes\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). However, the monophyly of series \u003cem\u003eMucronatae\u003c/em\u003e was not confirmed in the 35-spp. phylogeny, lacking resolution at this scale, further molecular information will have to be analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The series \u003cem\u003eFilamentosa\u003c/em\u003e (Subgen. \u003cem\u003eLepanthes\u003c/em\u003e, Sect. \u003cem\u003eLepanthes\u003c/em\u003e, Subsect. \u003cem\u003eBreves\u003c/em\u003e) is not represented in this phylogeny by any species. Those species will remain to be included in future studies to be properly placed in a systematic context for the genus. The current subgeneric circumscription of the genus does not reflect the evolutionary history of the group and a new taxonomic classification using systematics should be proposed.\u003c/p\u003e \u003cp\u003eSpecies \u003cem\u003eL. tachirensis\u003c/em\u003e and \u003cem\u003eL. juninensis\u003c/em\u003e, are surprisingly recovered as the sister species of the rest of \u003cem\u003eLepanthes\u003c/em\u003e species. \u003cem\u003eL. tachirensis\u003c/em\u003e is a widespread taxon in South America originally described from Venezuela, from the Tachira region but further register in Colombia and Ecuador. An interesting aspect of its morphology that sets \u003cem\u003eL. tachirensis\u003c/em\u003e apart from the rest of \u003cem\u003eLepanthes\u003c/em\u003e includes\u0026mdash;apart from its flowers and open sepals\u0026mdash;the tightly sheathed ramicaul, with the ostia, which in other \u003cem\u003eLepanthes\u003c/em\u003e resembles a funnel, closely adhering to the ramicaul. Besides, \u003cem\u003eL. juninensis\u003c/em\u003e was originally described from Peru, but its identity is challenging to interpret due to the limited information available\u0026mdash;a difficult-to-interpret illustration and description [64]. The species has often been misidentified as \u003cem\u003eL. tachirensis\u003c/em\u003e, and this misidentification has been widely propagated among collectors and commercial nurseries. Given that the two species share few similarities, it is possible that the specimen from Genebank identified as \u003cem\u003eL. juninensis\u003c/em\u003e is, in fact, misidentified \u003cem\u003eL. tachirensis\u003c/em\u003e. Further exploration in the type locality region of Peru will be essential to clarify the identity and morphological characteristics of \u003cem\u003eL. juninensis\u003c/em\u003e, if indeed a species.\u003c/p\u003e \u003cp\u003eAn early divergent clade, The Eastern Andean Marsipanthes clade (Clade 1), is identified in our phylogenies as sister to the rest \u003cem\u003eLepanthes\u003c/em\u003e. This clade comprises two species from the subgenus \u003cem\u003eMarsipanthes\u003c/em\u003e section \u003cem\u003eCaprimulginae\u003c/em\u003e, specifically \u003cem\u003eL. attenboroughii\u003c/em\u003e and \u003cem\u003eL. caprimulgus\u003c/em\u003e, which are native to the Eastern Andes of Ecuador and Per\u0026uacute;. These species are more closely related to members of \u003cem\u003eLepanthes\u003c/em\u003e section \u003cem\u003eElongatae\u003c/em\u003e (\u003cem\u003eL. martineae\u003c/em\u003e, \u003cem\u003eL. nycteris\u003c/em\u003e, \u003cem\u003eL. terbochii\u003c/em\u003e, and \u003cem\u003eL. katleri\u003c/em\u003e) and section \u003cem\u003eBilabiatae\u003c/em\u003e (\u003cem\u003eL. narcissus\u003c/em\u003e), all distributed across the Eastern Andes of Ecuador, Per\u0026uacute;, and Bolivia, than to other species within \u003cem\u003eMarsipanthes\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003eS5\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, 10). Most species, except for \u003cem\u003eL. narcissus\u003c/em\u003e, have shortly pubescent, multi-veined (5\u0026ndash;7 at the dorsal sepal and 2\u0026ndash;4 in each lateral sepal) sepals, erose to lacerate, carinae adaxially, and long pubescent to fringed at the margins, deeply to shallowly concave, and stripped. The petals are frequently lunate and with the upper and lower lobes of similar shape and size. The lip has long, densely pubescence, narrow to very narrowly triangular lobes which surround the long column (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003eS10\u003c/span\u003e). Based on their floral morphology and distribution on the Eastern Andes, further molecular studies should corroborate whether species from subgen. \u003cem\u003eLepanthes\u003c/em\u003e series \u003cem\u003eElongatae\u003c/em\u003e such as \u003cem\u003eL. echinata\u003c/em\u003e Luer \u0026amp; Cloes, \u003cem\u003eL. mulleriana\u003c/em\u003e Luer, \u003cem\u003eL. tigrina\u003c/em\u003e Luer \u0026amp; Thoerle, besides \u003cem\u003eL. portillae\u003c/em\u003e (sect. \u003cem\u003eMarsipanthes\u003c/em\u003e) discovered in El Condor Mountain range of Ecuador, belong to Clade 1. The monophyly of the remaining species in section \u003cem\u003eBilabiatae\u003c/em\u003e (approximately seven species) and their status as the sister group to Clade 1, as suggested by our phylogeny with \u003cem\u003eL. narcissus\u003c/em\u003e, remains to be tested.\u003c/p\u003e \u003cp\u003eThe Monoptera clade (Clade 2), the second early-divergent lineage identified in our study, is resolved as sister to the remainder of \u003cem\u003eLepanthes\u003c/em\u003e and comprises species from subsection \u003cem\u003eBreves\u003c/em\u003e, which Dodson and Luer [21] and Luer and Thoerle [18], distinguished by their single-veined lateral sepals. These high-altitude species typically occur above 2500 m and form two distinct groups: (1) the \u003cem\u003eL. monoptera\u003c/em\u003e group characterized by a bilaminate lip with oblong to ovate, long-ciliate blades, rounded blade bases, narrowly obtuse apices, short connectives, and a distinctive ovary with a single wing or carina. Other species that could be part of this group include \u003cem\u003eL. cornualis\u003c/em\u003e, \u003cem\u003eL. cyrtostele\u003c/em\u003e, \u003cem\u003eL. ferax\u003c/em\u003e, and \u003cem\u003eL. jucas\u003c/em\u003e. And (2) the \u003cem\u003eRhynchion/Speciosa\u003c/em\u003e group, comprising species with large stature, large leaves, and lepanthiform sheaths that are prominent, adnate, or flattened. Intriguingly, \u003cem\u003eL. tachirensis\u003c/em\u003e also belongs to subsection \u003cem\u003eBreves\u003c/em\u003e and shares morphological traits with \u003cem\u003eL. rhynchion\u003c/em\u003e and \u003cem\u003eL. speciosa\u003c/em\u003e, yet it is phylogenetically placed at the base of the genus, highlighting the non-monophyly of subsection \u003cem\u003eBreves\u003c/em\u003e. Further studies are needed to determine whether other species in this subsection align with one of these two clades or represent additional lineages, based on both molecular and morphological evidence.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eLepanthes\u003c/em\u003e phylogeny presented here includes two main polytomies indicating molecular information used here was not enough to resolve parts of the \u003cem\u003eLepanthes\u003c/em\u003e backbone and other molecular markers or more species will have to be included in future studies to improve the resolution. The observed polytomies might also reflect recent divergence or rapid speciation events, in addition to the limitations of molecular data. The first polytomy contains: \u003cem\u003eL. cloesii\u003c/em\u003e, \u003cem\u003eL. acarina\u003c/em\u003e, the Nicolasii clade (clade 3): \u003cem\u003eL. nicolassi\u003c/em\u003e, \u003cem\u003eL. gargantua\u003c/em\u003e, \u003cem\u003eL. clareae\u003c/em\u003e, \u003cem\u003eL. adrianae\u003c/em\u003e, \u003cem\u003eL. auriculata\u003c/em\u003e, and \u003cem\u003eL. orion\u003c/em\u003e, along with a clade containing the remaining species sampled here of \u003cem\u003eLepanthes\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cem\u003eLepanthes cloesii\u003c/em\u003e and \u003cem\u003eL. acarina\u003c/em\u003e did not form a clade, despite their previous classification in the artificial series \u003cem\u003eElongatae\u003c/em\u003e. This finding supports Luer and Thoerle's [2] decision to abandon this grouping. In the \u003cem\u003eITS/MatK\u003c/em\u003e phylogeny, \u003cem\u003eL. cloesii\u003c/em\u003e clusters with \u003cem\u003eL. elata\u003c/em\u003e and \u003cem\u003eL. ballatrix\u003c/em\u003e (Figures \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e\u0026ndash;7). Other species with morphological similarities to \u003cem\u003eL. cloesii\u003c/em\u003e, such as \u003cem\u003eL. elegantula\u003c/em\u003e, \u003cem\u003eL. pastoensis\u003c/em\u003e, and \u003cem\u003eL. alexandroi\u003c/em\u003e, share features like thick, fleshy leaves and relatively large flowers compared to their smaller leaves. Within this phylogeny, \u003cem\u003eL. cloesii\u003c/em\u003e is the sole representative of a larger group that requires further investigation. In contrast, \u003cem\u003eL. acarina\u003c/em\u003e\u0026mdash;a widely distributed and common species ranging from Colombia to Bolivia\u0026mdash;is distinguished by its extremely small size flowers and unique morphology, which complicates its association with other species. These traits suggest that \u003cem\u003eL. acarina\u003c/em\u003e may represent a cleistogamous lineage, potentially following an independent evolutionary pathway within the genus.\u003c/p\u003e \u003cp\u003eThe Nicolasii clade (Clade 3) [7] includes large-sized species with large, erect to suberect leaves, supported by relatively long, robust ramicauls, compact rachis, conspicuous appendix and typically with yellow flowers produced in a raceme on the leaf's abaxial surface. This group might also include species such as \u003cem\u003eL. monitor\u003c/em\u003e, \u003cem\u003eL. auriculata\u003c/em\u003e, and \u003cem\u003eL. steyermarkii\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe remaining \u003cem\u003eLepanthes\u003c/em\u003e species within the first polytomy form a clade with two subclades. A Central American and the Caribbean clade (Clade 4) including species \u003cem\u003eL grandiflora\u003c/em\u003e, \u003cem\u003eL. turialvae\u003c/em\u003e, \u003cem\u003eL. urbaniana\u003c/em\u003e, sister to a clade represented by \u003cem\u003eL calodyction\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eL. tentaculata\u003c/em\u003e and the rest of \u003cem\u003eLepanthes\u003c/em\u003e. Most species from both The \u003cem\u003eCalodictyon\u003c/em\u003e clade (Clade 5) and the remaining of \u003cem\u003eLepanthes\u003c/em\u003e have a distribution in the Western Cordillera of Colombia and northern central cordillera of Ecuador known biogeographic Choco [65\u0026ndash;66].\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eCalodictyon\u003c/em\u003e clade (Clade 5) was also recovered in the combined \u003cem\u003eITS/MatK\u003c/em\u003e phylogeny, including species such as \u003cem\u003eL. saltatrix\u003c/em\u003e, \u003cem\u003eL. barbelifera\u003c/em\u003e, and \u003cem\u003eL. volador\u003c/em\u003e (Figures \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e\u0026ndash;7). These species are distributed from Central to South America and exhibit notable morphological characteristics. While most species have reticulated leaves, those from Mesoamerica lack this feature. Flowers emerge above the leaf, with sepals that are widely spaced and reflexed, resting on the leaf surface. The petals are unique, with a highly distinct upper and lower lobe. The upper lobe is often elongated, sometimes with a filamentous extension at the apex (though not always), while the lower lobe is typically expanded into a broader segment with a basal filamentous extension (also not always present). The labellum is simple, lacking the typical \u003cem\u003eLepanthes\u003c/em\u003e structure of a body, connectives, and laminae. Instead, it takes on reniform, lunate, ovate, or bilobed shapes and does not include an appendage. The column is elongated, with a conspicuous hood over the anther (clinandrium). Some species have two filaments on their petals, others have one, and some lack filaments entirely. They grow at low to mid elevations 200 to 1800 m. Calodictyon-related species include several from Central America (\u003cem\u003eL. arachnion\u003c/em\u003e, \u003cem\u003eL. pantomima\u003c/em\u003e) and the western Andes (\u003cem\u003eL. bibarbullata\u003c/em\u003e, \u003cem\u003eL. arachnion\u003c/em\u003e, \u003cem\u003eL. kayii\u003c/em\u003e, \u003cem\u003eL. microcalodictyon\u003c/em\u003e, \u003cem\u003eL. pantomima\u003c/em\u003e, \u003cem\u003eL. pretiosa\u003c/em\u003e, \u003cem\u003eL. tentaculata\u003c/em\u003e, \u003cem\u003eL. tortuosa\u003c/em\u003e) [2, 7].\u003c/p\u003e \u003cp\u003eThe second polytomy identified within this phylogeny includes \u003cem\u003eL. mucronate\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eL. hexapus\u003c/em\u003e, \u003cem\u003eL. pelix\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eL. heteroloba, L. ribes\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eL. felis\u003c/em\u003e, \u003cem\u003eL. lucifer\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eL. niesseniae\u003c/em\u003e and the Manabina clade (Clade 6) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The rest of species from subgen. \u003cem\u003eMarsipanthes\u003c/em\u003e are recovered within this clade like this: (1) \u003cem\u003eL. ribes\u003c/em\u003e (sect. \u003cem\u003eFelinae\u003c/em\u003e)\u0026thinsp;+\u0026thinsp;\u003cem\u003eL. felis\u003c/em\u003e. (2) \u003cem\u003eL. niesseniae\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eL. lucifer\u003c/em\u003e (sect. \u003cem\u003eFelinae\u003c/em\u003e). These four species only formed a clade in the combined \u003cem\u003eITS/Matk\u003c/em\u003e, we will refer to them as the western Andes \u003cem\u003eMarsipanthes\u003c/em\u003e species (Figure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e-7, 11). We anticipate species such as \u003cem\u003eL. carunculigera, L. equicalceolata, L. quadricornis\u003c/em\u003e will be part of this clade.\u003c/p\u003e \u003cp\u003eIn our phylogeny, \u003cem\u003eL. hexapus\u003c/em\u003e represents a group of species characterized by elliptic to ovate leaves with reticulate veins and a distribution almost exclusively from the biogeographic Choco [65\u0026ndash;66]. Based on morphology species like \u003cem\u003eL. satyrica\u003c/em\u003e, \u003cem\u003eL. hirsutula\u003c/em\u003e, \u003cem\u003eL. acrogenia\u003c/em\u003e, \u003cem\u003eL. tetrapus\u003c/em\u003e, and \u003cem\u003eL. heptapus\u003c/em\u003e among others could be part of this clade. A second group of morphologically similar species are represented here by \u003cem\u003eL. mucronata\u003c/em\u003e, these group of species might have a mucron or a small central lobe on the petals. Last, \u003cem\u003eL. pelix\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eL. heteroloba\u003c/em\u003e form part of a group of species with very corrugated leaves. Luer's initial grouping termed these as the series \u003cem\u003emucronatae\u003c/em\u003e [21], however, he later retracted this, possibly due to the complexity and variation in flower and plant morphology. With additional sampled species, this series might be further confirmed or refuted as monophyletic.\u003c/p\u003e \u003cp\u003eThe phylogenetic position of \u003cem\u003eL. tulcanensis\u003c/em\u003e in the combined \u003cem\u003eITS/MatK\u003c/em\u003e phylogenies, along with its morphological similarities to both western Andes \u003cem\u003eMarsipanthes\u003c/em\u003e species and the \u003cem\u003eMucronata\u003c/em\u003e group, is intriguing (Figures \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e\u0026ndash;7). The plant closely resembles species from the \u003cem\u003eMucronata\u003c/em\u003e group, such as \u003cem\u003eL. rodophylla\u003c/em\u003e, \u003cem\u003eL. pelyx\u003c/em\u003e, and \u003cem\u003eL. corrugata\u003c/em\u003e. However, its flowers bear a stronger resemblance to those of species in the \u003cem\u003eMarsipanthes\u003c/em\u003e group (Figure \u003cspan refid=\"MOESM11\" class=\"InternalRef\"\u003eS11\u003c/span\u003e), particularly \u003cem\u003eL. niesseniae\u003c/em\u003e. This is evident in features such as the long peduncle, sepals with more than three veins, a simple lip that folds beneath the column, and three-dimensional petals.\u003c/p\u003e \u003cp\u003eLast the \u003cem\u003eManabina\u003c/em\u003e clade (Clade 6) has already been informally recognized by Baquero et al. [67] based on \u003cem\u003eLepanthes manabina\u003c/em\u003e Dodson. All species in this group have centrally concave leaves, ranging from deeply to slightly concave. These leaves have slightly to strongly recurved margins, a microscopically to conspicuously pubescent adaxial surface, and congested inflorescences. The flowers rest on the adaxial side of the leaves and are accompanied by a short to long caudate synsepal and a very small, inconspicuous appendix. Some other species that might belong to this group include \u003cem\u003eL. farallonensis\u003c/em\u003e, \u003cem\u003eL. foreroi\u003c/em\u003e P.Ortiz, O.P\u0026eacute;rez \u0026amp; E.Parra, \u003cem\u003eL. ortiziana\u003c/em\u003e O.P\u0026eacute;rez, E.Parra \u0026amp; Kolan., \u003cem\u003eL. smaragdina\u003c/em\u003e Luer \u0026amp; R.Escobar, \u003cem\u003eL. tomentosa\u003c/em\u003e Luer, and \u003cem\u003eL. cincinnata\u003c/em\u003e Luer \u0026amp; R.Escobar.\u003c/p\u003e \u003cp\u003eOur preliminary morphological analysis, limited to a subset of \u003cem\u003eLepanthes\u003c/em\u003e taxa, confirmed that continuous characters do not exhibit grouping patterns consistent with phylogenetic clades. In only a few cases, such as the artificial subgenus \u003cem\u003eMarsipanthes\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), do these characters show a taxonomic pattern. However, this analysis revealed intriguing trends using continuous morphological characters, which will be tested further with a broader taxon sampling and additional datasets.\u003c/p\u003e \u003cp\u003eThe high variability in continuous morphological traits is captured by PC1 and PC2, suggesting that measurements such as sepal size and peduncle length are key in distinguishing among \u003cem\u003eLepanthes\u003c/em\u003e species. Traits like dorsal and lateral sepal dimensions have the highest positive loadings (Figure \u003cspan refid=\"MOESM9\" class=\"InternalRef\"\u003eS9\u003c/span\u003e), highlighting their importance in species differentiation. This information is particularly valuable for systematics, evolutionary developmental biology (evo-devo), and other areas of study, especially in groups with high morphological similarity. Strong correlations between traits, such as peduncle length and floral bract length, or leaf length and ovary length, suggest functional or developmental linkages. These findings provide insights into how morphological traits may be co-adapted in response to environmental pressures or reproductive strategies. Conversely, weak negative correlations, such as those between dorsal sepal width and leaf width, may indicate independent evolution of certain vegetative and floral traits, pointing to differing selective pressures on these aspects.\u003c/p\u003e \u003cp\u003eThe hyperdiverse nature of \u003cem\u003eLepanthes\u003c/em\u003e presents significant challenges in resolving its phylogenetic relationships, particularly given the rapid diversification of the genus, which is estimated to have occurred 5\u0026ndash;10\u0026nbsp;million years ago [28, 68]. While this study provides foundational insights, the current dataset reflects the limitations of incomplete taxon sampling and the constrained informativeness of chloroplast markers. Although the sequencing of nine plastomes and the incorporation of additional plastid genes facilitated a moderately resolved phylogenetic reconstruction, the lack of backbone resolution underscores the need for nuclear markers to better elucidate relationships, especially considering morphological homoplasy and the non-monophyly of Luer\u0026rsquo;s subgeneric classifications. Future efforts must prioritize expanded taxon sampling, both geographically and taxonomically, to capture the genus\u0026rsquo;s full diversity and refine its taxonomic framework. Biogeographically, the genus is predominantly diverse in Central and South America [20], but smaller clades in other regions highlight the complex evolutionary history of \u003cem\u003eLepanthes\u003c/em\u003e. Understanding these patterns requires phylogenetic reconstructions with greater taxonomic breadth. Current work sequencing whole nuclear genomes of 100 additional taxa aims to address these gaps, incorporating broader sampling and advanced molecular tools to resolve closely spaced speciation events and uncover lineage-specific diversification mechanisms.\u003c/p\u003e \u003cp\u003eThe discovery of non-monophyletic subgeneric classifications highlights the need for a thorough reassessment of the taxonomic framework for \u003cem\u003eLepanthes\u003c/em\u003e. However, we strongly discourage any taxonomic revisions until a species-rich phylogeny is available, one that includes representatives from all major clades and informal morphological and geographical groups. Taxon sampling and selection are critical factors in phylogenetic reconstructions, as demonstrated by numerous studies [69\u0026ndash;71]. Sparse sampling, often constrained by the logistical challenges of acquiring diverse taxa under strict permit regulations can lead to long-branch artifacts and unresolved relationships [25, 72]. Furthermore, the inclusion of distantly related outgroups and taxa with varied evolutionary rates can significantly influence tree topologies [70]. High taxon sampling, even when some data are missing, has consistently been shown to improve branch support, as seen in studies combining whole plastome sequences with datasets containing only a few chloroplast genes [27]. These findings underscore the importance of robust and representative sampling strategies to achieve well-resolved phylogenetic reconstructions for \u003cem\u003eLepanthes\u003c/em\u003e, laying the groundwork for future taxonomic and evolutionary studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e-Ethics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eNot applicable\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;-Consent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;-Availability of data and materials:\u0026nbsp;\u003c/strong\u003eThe datasets generated and/or analysed during the current study are available in the NCBI repository,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eBioprojectID PRJNA1209138\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;-Competing interests:\u0026nbsp;\u003c/strong\u003eWe declare that we have no competing interests. None of the authors have financial, personal, or professional conflicts that could be perceived as influencing the work reported in this manuscript.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;-Funding:\u0026nbsp;\u003c/strong\u003eSociedad Colombiana de Orquideolog\u0026iacute;a in Colombia for their support with resources.\u0026nbsp;Universidad de las Am\u0026eacute;ricas (UDLA) for funding research on Orchidaceae in Ecuador.\u0026nbsp;Idea Wild for their support in supplying equipment to ASS.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;-Authors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTA:\u0026nbsp;\u003c/strong\u003eConceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review and editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJSM:\u0026nbsp;\u003c/strong\u003eConceptualization, Data curation, Investigation, Writing \u0026ndash; original draft, Writing \u0026ndash; review and editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSR:\u0026nbsp;\u003c/strong\u003eFormal analysis, Methodology, Writing \u0026ndash; review and editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eASS:\u0026nbsp;\u003c/strong\u003eConceptualization, Data curation, Funding acquisition, Investigation, Methodology, Writing \u0026ndash; review and editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMLL:\u0026nbsp;\u003c/strong\u003eFormal analysis, Writing \u0026ndash; review and editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGAI:\u0026nbsp;\u003c/strong\u003eConceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review and editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJV:\u0026nbsp;\u003c/strong\u003eData curation, Formal analysis, Writing \u0026ndash; review and editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLB:\u0026nbsp;\u003c/strong\u003eConceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review and editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAZ:\u0026nbsp;\u003c/strong\u003eData curation, Formal analysis, Methodology, Project administration, Resources, Supervision, Visualization, Writing \u0026ndash; review and editing\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;-Acknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our gratitude to the Sociedad Colombiana de Orquideolog\u0026iacute;a and Colomborqu\u0026iacute;deas in Colombia for their support with resources and access to plant collections used in this research. We also thank the Universidad de las Am\u0026eacute;ricas (UDLA) for funding research on Orchidaceae in Ecuador. Our appreciation goes to the Corporaci\u0026oacute;n para Investigaciones Biol\u0026oacute;gicas for providing collection permits for TA and ASS during their affiliation with the institution. We are grateful to Idea Wild for their support in supplying equipment to ASS. Special thanks to Juan Felipe Posada, Luis Eduardo Mej\u0026iacute;a, Jean Mark Palandre, and Sebasti\u0026aacute;n Vieira for their valuable input, collaboration, and assistance with plant materials. We also acknowledge Juliana Arcila and Jorge Mu\u0026ntilde;oz for their expertise in bioinformatics, which was crucial to the success of this study. Finally, we thank Lourens Grobler and Ron Parsons for kindly allowing the use of their photographs of several \u003cem\u003eLepanthes\u003c/em\u003e species, which greatly enriched this research. We also appreciate Justin Yeager for his help in refining the English for this manuscript.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;-Authors\u0026apos; information (optional)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of generative AI and AI-assisted technologies in the writing process.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the preparation of this work the author(s) used ChatGPT, OpenAI, December 2024 to improve the readability and language of the manuscript. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e1. 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Barrett CF, Specht CD, Leebens-Mack J, Stevenson DW, Zomlefer WB, Davis JI. 2014 Resolving ancient radiations: can complete plastid gene sets elucidate deep relationships among the tropical gingers (Zingiberales)?. \u003cem\u003eAnnals of Botany 113\u003c/em\u003e(1): 119\u0026ndash;133\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e72. Jansen RK, Cai Z, Raubeson LA, Daniell H, Depamphilis CW, Leebens-Mack J, Muller KF, Guisinger-Bellian M, Haberle RC, Hansen AK, Chumley TW, Lee S-B, Peery R, McNeal JR, Kueh JV, Boore JL. 2007 Analysis of 81 genes from 64 plastid genomes resolves relationships in angiosperms and identifies genome-scale evolutionary patterns \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e 104(49): 19369\u0026ndash;19374\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Supplementary Figures Note","content":"Supplementary Figures S4 and S9 are not available with this version. "}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-ecology-and-evolution","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"evob","sideBox":"Learn more about [BMC Ecology and Evolution](http://bmcevolbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/evob/default.aspx","title":"BMC Ecology and Evolution","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Andes, Chloroplast, Colombia, Eastern Andes, Biogeographic Choco, Ecuador, ycf1","lastPublishedDoi":"10.21203/rs.3.rs-5738250/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5738250/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe first successful resolution of phylogenetic relationships within main lineages in the diverse Neotropical orchid genus \u003cem\u003eLepanthes\u003c/em\u003e Sw. is presented here. Genome skimming produced ten newly sequenced chloroplast genomes, with additional plastome coding genes (17\u0026ndash;86) retrieved from GenBank, alongside 26 amplified \u003cem\u003ematK\u003c/em\u003e and rITS genes, enabling phylogenetic reconstruction. The \u003cem\u003eLepanthes\u003c/em\u003e plastomes (157,185\u0026thinsp;\u0026minus;\u0026thinsp;158,260 bp, 37.15% GC content) contained 136 annotated genes, including 86 protein-coding, 42 tRNA, and 8 rRNA genes. Six hypervariable regions, including parts of the \u003cem\u003eycf1\u003c/em\u003e gene, were identified as potential DNA barcodes. Phylogenetic analyses revealed that Carl Luer\u0026rsquo;s subgeneric classifications are non-monophyletic, reflecting significant morphological homoplasy. PCA and correlation analyses confirmed widespread homoplasy in continuous morphological characters. Six major clades were identified, though backbone resolution remains unresolved at two nodes of the phylogeny, requiring the use of nuclear markers or expanded sampling. Subgenus \u003cem\u003eMarsipanthes\u003c/em\u003e species are non-monophyletic and constitute an East Andean early divergent clade with species from subgenus \u003cem\u003eLepanthes\u003c/em\u003e, while some derived Biogeographic Choco \u003cem\u003eMarsipanthes\u003c/em\u003e clades were recovered, forming a polytomy with species from subgenus \u003cem\u003eLepanthes\u003c/em\u003e. The genus likely originated in southern Ecuador or northern Peru, dispersing across the Andes into the broader Neotropics. Although only a subset of \u003cem\u003eLepanthes\u003c/em\u003e diversity was sampled, the study captures significant taxonomic, geographic, and morphological variation. It provides foundational insights into the genus\u0026rsquo;s evolution, along with tools and hypotheses that can be expanded upon in future research to further refine our understanding of its evolutionary history.\u003c/p\u003e","manuscriptTitle":"Plastome phylogenomics of the Diverse Neotropical Orchid Genus Lepanthes with Emphasis on Subgenus Marsipanthes (Pleurothallidinae: Orchidaceae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-07 21:36:01","doi":"10.21203/rs.3.rs-5738250/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-28T23:42:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-28T13:16:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-20T15:18:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-12T05:55:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"67418395633864969426167156039174734090","date":"2025-01-25T15:45:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"246098506987323152774459263909840335045","date":"2025-01-23T21:36:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"158445305941929678416090823927121045495","date":"2025-01-23T14:09:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-21T10:20:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"227670116076676787040182707321367483275","date":"2025-01-19T02:02:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"101483676386503206930013392164145648143","date":"2025-01-18T21:25:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-18T02:43:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-18T02:37:21+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-01-15T20:13:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-01-15T14:42:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Ecology and Evolution","date":"2025-01-15T14:41:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-ecology-and-evolution","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"evob","sideBox":"Learn more about [BMC Ecology and Evolution](http://bmcevolbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/evob/default.aspx","title":"BMC Ecology and Evolution","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f9a824fc-4c4d-48ce-a4d2-1f5a5368c6c8","owner":[],"postedDate":"February 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-11T16:00:28+00:00","versionOfRecord":{"articleIdentity":"rs-5738250","link":"https://doi.org/10.1186/s12862-025-02396-6","journal":{"identity":"bmc-ecology-and-evolution","isVorOnly":false,"title":"BMC Ecology and Evolution"},"publishedOn":"2025-08-07 15:57:18","publishedOnDateReadable":"August 7th, 2025"},"versionCreatedAt":"2025-02-07 21:36:01","video":"","vorDoi":"10.1186/s12862-025-02396-6","vorDoiUrl":"https://doi.org/10.1186/s12862-025-02396-6","workflowStages":[]},"version":"v1","identity":"rs-5738250","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5738250","identity":"rs-5738250","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}