Complete mitochondrial genomes of three vulnerable cave bat species and their phylogenetic relationships within the order Chiroptera.

The IUCN Red List of Threatened Species contains 175 Brazilian bat species that are threatened by extinction in some degree. From this perspective, it is essential to expand the knowledge about the genetic diversity of vulnerable bats. Genomic sequencing can be useful to generate robust and informative genetic references, increasing resolution when analyzing relationships among populations, species, or higher taxonomic levels. In this study, we sequenced and characterized in detail the first complete mitochondrial genomes of Furipterus horrens, Lonchorhina aurita, and Natalus macrourus, and investigated their phylogenetic position based on amino acid sequences of protein-coding genes (PCGs). The mitogenomes of these species are 16,516, 16,697, and 16,668 bp in length, respectively, and each comprises 13 PCGs, 22 tRNA genes, two rRNA genes, and a putative control region (CR). In the three species, genes were arranged similarly to all other previously described bat mitogenomes, and nucleotide composition was also consistent with the reported range. The length and arrangement of rrnS and rrnL were also consistent with those of other bat species, showing a positive AT-skew and a negative GC-skew. Except for trnS1, for which we did not observe the DHU arm, all other tRNAs showed the cloverleaf secondary structure in the three species. In addition, the mitogenomes showed minor differences in start and stop codons, and in all PCGs, codons ending in adenine were more common compared to those ending in guanine. We found that PCGs of the three species use multiple codons to encode each amino acid, following the previously documented pattern. Furthermore, all PCGs are under purifying selection, with atp8 experiencing the most relaxed purifying selection. Considering the phylogenetic reconstruction, F. horrens was recovered as sister to Noctilio leporinus, L. aurita and Tonatia bidens shared a node within Phyllostomidae, and N. macrourus appeared as sister to Molossidae and Vespertilionidae.

New taxonomic insights for Brazilian Syrbatus Reitter (Coleoptera: Staphylinidae: Pselaphinae), including three new species and their mitochondrial genomes.

Here we present a taxonomic treatment for the Brazilian species of Syrbatus (Reitter, 1882), including the description of three new species (Syrbatus moustache Asenjo & Valois sp. nov., Syrbatus obsidian Asenjo & Valois sp. nov. and Syrbatus superciliata Asenjo & Valois sp. nov.) from the Quadrilátero Ferrífero (Minas Gerais, Brazil). In addition, we designated lectotypes for the Brazilian species of species-group 2, Syrbatus centralis (Raffray, 1898), Syrbatus hetschkoi (Reitter, 1888), Syrbatus hiatusus (Reitter, 1888), Syrbatus transversalis (Raffray, 1898), and Syrbatus trinodulus (Schaufuss, 1887), besides recognizing the holotype for Syrbatus brevispinus (Reitter, 1882), Syrbatus bubalus (Raffray, 1898), and Syrbatus grouvellei (Raffray, 1898). The mitochondrial genomes (mitogenomes) of the three new species are presented, for which we present the phylogenetic placement among Staphylinidae with previously published data.

Cytomolecular diversity among Vigna Savi (Leguminosae) subgenera.

The genus Vigna (Leguminosae) comprises about 150 species grouped into five subgenera. The present study aimed to improve the understanding of karyotype diversity and evolution in Vigna, using new and previously published data through different cytogenetic and DNA content approaches. In the Vigna subgenera, we observed a random distribution of rDNA patterns. The 35S rDNA varied in position, from terminal to proximal, and in number, ranging from one (V. aconitifolia, V. subg. Ceratotropis) to seven pairs (V. unguiculata subsp. unguiculata, V. subg. Vigna). On the other hand, the number of 5S rDNA was conserved (one or two pairs), except for V. radiata (V. subg. Ceratotropis), which had three pairs. Genome size was relatively conserved within the genus, ranging from 1C = 0.43 to 0.70 pg in V. oblongifolia and V. unguiculata subsp. unguiculata, respectively, both belonging to V. subg. Vigna. However, we observed a positive correlation between DNA content and the number of 35S rDNA sites. In addition, data from chromosome-specific BAC-FISH suggest that the ancestral 35S rDNA locus is conserved on chromosome 6 within Vigna. Considering the rapid diversification in the number and position of rDNA sites, such conservation is surprising and suggests that additional sites may have spread out from this ancestral locus.

Organellar-genome analyses from the lycophyte genus Isoetes L. show one of the highest frequencies of RNA editing in land plants.

RNA editing is a post-transcriptional process that challenges the central dogma of molecular biology by modifying RNA sequences, introducing nucleotide changes at specific sites, and generating functional diversity beyond the genomic code, especially when it concerns organellar transcripts. In plants, this phenomenon is widespread, but its extent varies significantly among species and organellar genomes. Among land plants, the heterosporous lycophytes (i.e., Isoetes and Selaginella) stand out for their exceptionally high numbers of RNA-editing sites, despite their morphological stasis and ancient lineage. In this study, we explore the complete set of organellar protein-coding genes in the aquatic plant group Isoetes, providing a detailed analysis of RNA editing in both the mitochondrial and plastid genomes. Our findings reveal a remarkable abundance of RNA editing, particularly in the mitochondrial genome, with thousands of editing sites identified. Interestingly, the majority of these edits result in non-silent substitutions, suggesting a role in fine-tuning protein structure and function. Furthermore, we observe a consistent trend of increased hydrophobicity in membrane-bound proteins, supporting the notion that RNA editing may confer a selective advantage by preserving gene functionality in Isoetes. The conservation of highly edited RNA sequences over millions of years underscores the evolutionary significance of RNA editing. Additionally, the study sheds light on the dynamic nature of RNA editing, with shared editing sites reflecting common ancestry whereas exclusive edits matching more recent radiation events within the genus. This work advances our understanding of the intricate interplay between RNA editing, adaptation, and evolution in land plants and highlights the unique genomic features of Isoetes, providing a foundation for further investigations into the functional consequences of RNA editing in this enigmatic plant lineage.

The complete mitochondrial genome and description of a new cryptic Brazilian species of Metopiellus Raffray (Coleoptera: Staphylinidae: Pselaphinae).

Metopiellus Raffray, 1908 is a genus of South American rove beetles typically found in tropical humid forests. Here we describe a new cryptic species from Eastern Amazon, in northern Brazil, Metopiellus crypticus Asenjo sp. nov., and its major morphologic diagnostic features, which were photographed and illustrated. In addition, we bring the complete mitochondrial genome sequence of M. crypticus sp. nov., and its position within the phylogenetic context of the family, including previously available mitogenomes of Staphylinidae species.

Relationship between fruit phenotypes and domestication in hexaploid populations of biribá (Annona mucosa) in Brazilian Amazonia.

Background: Biribá (Annona mucosa Jacq.) is a fruit tree domesticated in Amazonia and has polyploid populations. The species presents ample phenotypic variation in fruit characteristics, including weight (100-4,000 g) and differences in carpel protrusions. Two cytotypes are recorded in the literature (2n = 28, 42) and genome size records are divergent (2C = 4.77, 5.42 and 6.00 pg). To decipher the role of polyploidy in the domestication of A. mucosa, we examined the relationships among phenotypic variation, chromosome number and genome size, and which came first, polyploidization or domestication. Methodology: We performed chromosome counts of A. mucosa from central and western Brazilian Amazonia, and estimated genome size by flow cytometry. We performed phylogenetic reconstruction with publicly available data using a Bayesian framework, time divergence analysis and reconstructed the ancestral chromosome number for the genus Annona and for A. mucosa. Results: We observed that variation in fruit phenotypes is not associated with variation in chromosome number and genome size. The most recent common ancestor of A. mucosa is inferred to be polyploid and diverged before domestication. Conclusions: We conclude that, when domesticated, A. mucosa was already polyploid and we suggest that human selection is the main evolutionary force behind fruit size and fruit morphological variation in Annona mucosa.

SPLACE: A tool to automatically SPLit, Align, and ConcatenatE genes for phylogenomic inference of several organisms.

The reconstruction of phylogenomic trees containing multiple genes is best achieved by using a supermatrix. The advent of NGS technology made it easier and cheaper to obtain multiple gene data in one sequencing run. When numerous genes and organisms are used in the phylogenomic analysis, it is difficult to organize all information and manually align the gene sequences to further concatenate them. This study describes SPLACE, a tool to automatically SPLit, Align, and ConcatenatE the genes of all species of interest to generate a supermatrix file, and consequently, a phylogenetic tree, while handling possible missing data. In our findings, SPLACE was the only tool that could automatically align gene sequences and also handle missing data; and, it required only a few minutes to produce a supermatrix FASTA file containing 83 aligned and concatenated genes from the chloroplast genomes of 270 plant species. It is an open-source tool and is publicly available at https://github.com/reinator/splace.

New plastomes of eight Ipomoea species and four putative hybrids from Eastern Amazon.

Ipomoea is a large pantropical genus globally distributed, which importance goes beyond the economic value as food resources or ornamental crops. This highly diverse genus has been the focus of a great number of studies, enriching the plant genomics knowledge, and challenging the plant evolution models. In the Carajás mountain range, located in Eastern Amazon, the savannah-like ferruginous ecosystem known as canga harbors highly specialized plant and animal populations, and Ipomoea is substantially representative in such restrictive habitat. Thus, to provide genetic data and insights into whole plastome phylogenetic relationships among key Ipomoea species from Eastern Amazon with little to none previously available data, we present the complete plastome sequences of twelve lineages of the genus, including the canga microendemic I. cavalcantei, the closely related I. marabaensis, and their putative hybrids. The twelve plastomes presented similar gene content as most publicly available Ipomoea plastomes, although the putative hybrids were correctly placed as closely related to the two parental species. The cavalcantei-marabaensis group was consistently grouped between phylogenetic methods. The closer relationship of the I. carnea plastome with the cavalcantei-marabaensis group, as well as the branch formed by I. quamoclit, I. asarifolia and I. maurandioides, were probably a consequence of insufficient taxonomic representativity, instead of true genetic closeness, reinforcing the importance of new plastome assemblies to resolve inconsistencies and boost statistical confidence, especially the case for South American clades of Ipomoea. The search for k-mers presenting high dispersion among the frequency distributions pointed to highly variable coding and intergenic regions, which may potentially contribute to the genetic diversity observed at species level. Our results contribute to the resolution of uncertain clades within Ipomoea and future phylogenomic studies, bringing unprecedented results to Ipomoea species with restricted distribution, such as I. cavalcantei.

Complete mitochondrial genome of the greater round-eared bat, Tonatia bidens (Chiroptera: Phyllostomidae) from Brazil and phylogenetic relationships within the family.

The greater round-eared bat, Tonatia bidens, is a locally rare species belonging to the highly diverse family Phyllostomidae. In this study, the complete mitogenome of T. bidens was sequenced using optimized protocols of DNA extraction from fixed cells originally prepared for cytogenetic studies. Here we present the complete mitogenome and place our results in a phylogenetic context with other data generated for the family Phyllostomidae. The circular genome had 16,717 bp in size, comprising 37 genes and GC content of 42.24%. Furthermore, the phylogenetic tree indicated a well-supported relationship between the representatives of Tonatia into the subfamily Phyllostominae.

Preliminary placement and new records of an overlooked Amazonian tree, Christiana mennegae (Malvaceae).

Christiana mennegae is a phylogenetically enigmatic taxon and represents a case in point of a species whose presence escaped the radar of the Amazon lists and the Brazilian Flora project. Here we expand its distribution by adding new records from Peru and overlooked ones from Brazil. To investigate its phylogenetic placement in the Brownlowioideae, part of the rbcL gene of the plastid and the intergenic ITS2 region were sequenced. Macro- and micro-morphological investigation of features of C. mennegae using SEM of foliar, flower, fruit and seed structures are presented. A lectotype for the name is designated here. The morphology of trichomes revealed five types of trichomes ranging from glandular to branched and unbranched and we also report stomata on the seed surface for the first time in Brownlowioideae. Christiana mennegae and C. africana were recovered as sister species in the phylogenetic analysis, albeit with low to moderate support, and more species of this and closely related genera must be sampled and analyzed in order to obtain a clearer picture of the group's affinities and relationships. We provide an update of its conservation status from Vulnerable to Least Concern. We also highlight the need for investment in the digitization of biological collections, botanical capacity building at the local level and the importance of the availability of online literature to speed the study of Amazonian plant diversity.

Going out for dinner-The consumption of agriculture pests by bats in urban areas.

Insectivorous bats provide ecosystem services in agricultural and urban landscapes by consuming arthropods that are considered pests. Bat species inhabiting cities are expected to consume insects associated with urban areas, such as mosquitoes, flying termites, moths, and beetles. We captured insectivorous bats in the Federal District of Brazil and used fecal DNA metabarcoding to investigate the arthropod consumed by five bat species living in colonies in city buildings, and ascertained whether their predation was related to ecosystem services. These insectivorous bat species were found to consume 83 morphospecies of arthropods and among these 41 were identified to species, most of which were agricultural pests. We propose that bats may roost in the city areas and forage in the nearby agricultural fields using their ability to fly over long distances. We also calculated the value of the pest suppression ecosystem service by the bats. By a conservative estimation, bats save US$ 94 per hectare of cornfields, accounting for an annual savings of US$ 390.6 million per harvest in Brazil. Our study confirms that, regardless of their roosting location, bats are essential for providing ecosystem services in the cities, with extensive impacts on crops and elsewhere, in addition to significant savings in the use of pesticides.

Plastome-based phylogenomics elucidate relationships in rare Isoëtes species groups from the Neotropics.

The genus Isoëtes is globally distributed. Within the Neotropics, Isoëtes occurs in various habitats and ecosystems, making it an interesting case study to address phylogenetic and biogeographic questions. We sequenced and assembled plastomes and ribosomal DNA (rDNA) sequences to reconstruct phylogenetic relationships in Isoëtes from tropical regions in the Neotropics. The ploidy level of nine taxa was established to address the potential source of phylogenetic incongruence in the genus. Node ages were estimated using MCMCTree. The ancestral range estimates were conducted in BioGeoBEARS. Plastome-based phylogenies were congruent throughout distinct matrices and partition schemes, exhibiting high support for almost all nodes. Whereas, we found incongruences between the rDNA and plastome datasets. Chromosome counts identified three diploids, five tetraploids and one likely hexaploid among Neotropical species. Plastome-based node age estimates showed that the radiation of the crown Isoëtes group occurred at 20 Ma, with the diversification of the tropical American (TAA) clade taking place in the Pleistocene at 1.7 Ma. Ancestral range estimates showed that the ancestor of the TAA clade may have evolved first in the dry diagonal area in South America before reaching more humid regions. In addition, the colonization of the Brazilian semiarid region occurred three times, while the occupation of the Cerrado and Amazon regions occurred twice and once, respectively. Our study showed a large unobserved diversity within the genus in warm-dry regions in the Neotropics. Plastomes provided sufficient genomic information to establish a robust phylogenetic framework to answer evolutionary questions in Isoëtes from the Neotropics.

Complete mitochondrial genome of a cave dwelling Desmopachria (Insecta: Coleoptera: Dytiscidae) from the Eastern Amazon.

Coleoptera presents most of the cave fauna biodiversity, with several troglobite species belonging to the aquatic family Dytiscidae. However, very little is known on both genetic and genomic diversity traits of Neotropical cave beetles. Thus, here we present the complete mitochondrial genome sequence of five specimens of Desmopachria collected in a ferruginous cave from Serra dos Carajás in Parauapebas (Pará, Brazil, Eastern Amazon). Besides the general characteristics of the mitogenome of the analyzed specimens, we present their phylogenetic position within the family, considering the available genome sequences of different subfamilies within Dytiscidae.

Complete mitochondrial genome of Glomeridesmus spelaeus (Diplopoda, Glomeridesmida), a troglobitic species from iron-ore caves in Eastern Amazon.

We report the complete mitochondrial genome sequence of Glomeridesmus spelaeus, the first sequenced genome of the order Gomeridesmida. The genome is 14,825 pb in length and encodes 37 mitochondrial (13 PCGs, 2 rRNA genes, 22 tRNA) genes and contains a typical AT-rich region. The base composition of the mitogenome was A (40.1%), T (36.4%), C (15.8%), and G (7.6%), with an GC content of 23.5%. Our results indicated that G. spelaeus is only distantly related to the other Diplopoda species with available mitochondrial genomes in the public databases. As the broadest genetic characterization of a Glomeridesmida species available to date, the mitogenome of G. spelaeus will help understanding the evolution of such a little-known millipede group. Also, our data will be important for the characterization and conservation of the diverse invertebrate troglofauna of the Amazonian caves.

Geography is essential for reproductive isolation between florally diversified morning glory species from Amazon canga savannahs.

The variety, relative importance and eco-evolutionary stability of reproductive barriers are critical to understanding the processes of speciation and species persistence. Here we evaluated the strength of the biotic prezygotic and postzygotic isolation barriers between closely related morning glory species from Amazon canga savannahs. The flower geometry and flower visitor assemblage analyses supported pollination by the bees in lavender-flowered Ipomoea marabaensis and recruitment of hummingbirds as pollinators in red-flowered Ipomoea cavalcantei. Nevertheless, native bee species and alien honeybees foraged on flowers of both species. Real-time interspecific hybridization underscored functionality of the overlap in flower visitor assemblages, questioning the strength of prezygotic isolation underpinned by diversification in flower colour and geometry. Interspecific hybrids were fertile and produced offspring in nature. No significant asymmetry in interspecific hybridization and hybrid incompatibilities among offspring were found, indicating weak postmating and postzygotic isolation. The results suggested that despite floral diversification, the insular-type geographic isolation remains a major barrier to gene flow. Findings set a framework for the future analysis of contemporary evolution of plant-pollinator networks at the population, community, and ecosystem levels in tropical ecosystems that are known to be distinct from the more familiar temperate climate models.

Diversity of yeasts during fermentation of cocoa from two sites in the Brazilian Amazon / Diversidade de leveduras presentes na fermentação de cacau de duas localidades na Amazônia brasileira

The purpose of this study was to identify the yeasts involved in spontaneous fermentation of cocoa from the Brazilian Amazon region. The fermentation process was carried out experimentally with cocoa seeds from two sites (Medicilândia and Tucumã), State of Pará, northern Brazil, during a six-day period. Totals of 44 yeasts were isolated from Medicilândia and 29 from Tucumã. Molecular identification was carried out by sequencing the D1/D2 region fragment of the rRNA 26S gene, expanded with universal primers for the NL1GC and LS2 eukaryotes. Pichia manshurica and Saccharomyces cerevisiae were identified in Medicilândia and five yeast species (Pichia fermentans, P. kudriavzevii, P. manshurica, S. cerevisiae and Zygosaccharomyces bailii) were identified in Tucumã. The results showed that P. manshurica and S. cerevisiae may have potential for use as starter cultures in future studies to improve the quality of cocoa seeds fermented in the Brazilian Amazon region.(AU)

A proposta deste estudo foi identificar as leveduras envolvidas na fermentação espontânea de cacau da Amazônia brasileira. A fermentação foi realizada em Medicilândia e Tucumã, Pará, Brasil, durante 6 dias. Em total foram obtidos 44 isolados de leveduras de Medicilândia e 29 de Tucumã. A identificação molecular foi realizada por sequenciamento do fragmento da região D1/D2 do gene rRNA 26S, amplificado com primers universais para eucariotos NL1GC e LS2. Em Medicilândia, foram identificadas Pichia manshurica e Saccharomyces cerevisiae. Em Tucumã foram identificadas cinco espécies (Pichia fermentans, P. kudriavzevii, P. manshurica, S. cerevisiae e Zygosaccharomyces bailii). Os resultados sugerem que P. manshurica e S. cerevisiae podem ter potencial para uso como culturas starter em estudos futuros, para melhorar a qualidade das sementes de cacau fermentadas na Amazônia brasileira.(AU)

Diversity of yeasts during fermentation of cocoa from two sites in the Brazilian Amazon

The purpose of this study was to identify the yeasts involved in spontaneous fermentation of cocoa from the Brazilian Amazon region. The fermentation process was carried out experimentally with cocoa seeds from two sites (Medicilândia and Tucumã), State of Pará, northern Brazil, during a six-day period. Totals of 44 yeasts were isolated from Medicilândia and 29 from Tucumã. Molecular identification was carried out by sequencing the D1/D2 region fragment of the rRNA 26S gene, expanded with universal primers for the NL1GC and LS2 eukaryotes. Pichia manshurica and Saccharomyces cerevisiae were identified in Medicilândia and five yeast species (Pichia fermentans, P. kudriavzevii, P. manshurica, S. cerevisiae and Zygosaccharomyces bailii) were identified in Tucumã. The results showed that P. manshurica and S. cerevisiae may have potential for use as starter cultures in future studies to improve the quality of cocoa seeds fermented in the Brazilian Amazon region. (AU)

A proposta deste estudo foi identificar as leveduras envolvidas na fermentação espontânea de cacau da Amazônia brasileira. A fermentação foi realizada em Medicilândia e Tucumã, Pará, Brasil, durante 6 dias. Em total foram obtidos 44 isolados de leveduras de Medicilândia e 29 de Tucumã. A identificação molecular foi realizada por sequenciamento do fragmento da região D1/D2 do gene rRNA 26S, amplificado com primers universais para eucariotos NL1GC e LS2. Em Medicilândia, foram identificadas Pichia manshurica e Saccharomyces cerevisiae. Em Tucumã foram identificadas cinco espécies (Pichia fermentans, P. kudriavzevii, P. manshurica, S. cerevisiae e Zygosaccharomyces bailii). Os resultados sugerem que P. manshurica e S. cerevisiae podem ter potencial para uso como culturas starter em estudos futuros, para melhorar a qualidade das sementes de cacau fermentadas na Amazônia brasileira.(AU)

Karyotype heterogeneity in Philodendron s.l. (Araceae) revealed by chromosome mapping of rDNA loci.

Philodendron s.l. (Araceae) has been recently focus of taxonomic and phylogenetic studies, but karyotypic data are limited to chromosome numbers and a few published genome sizes. In this work, karyotypes of 34 species of Philodendron s.l. (29 species of Philodendron and five of Thaumatophyllum), ranging from 2n = 28 to 36 chromosomes, were analyzed by fluorescence in situ hybridization (FISH) with rDNA and telomeric probes, aiming to understand the evolution of the karyotype diversity of the group. Philodendron presented a high number variation of 35S rDNA, ranging from two to 16 sites, which were mostly in the terminal region of the short arms, with nine species presenting heteromorphisms. In the case of Thaumatophyllum species, we observed a considerably lower variation, which ranged from two to four terminal sites. The distribution of the 5S rDNA clusters was more conserved, with two sites for most species, being preferably located interstitially in the long chromosome arms. For the telomeric probe, while exclusively terminal sites were observed for P. giganteum (2n = 30) chromosomes, P. callosum (2n = 28) presented an interstitial distribution associated with satellite DNA. rDNA sites of the analyzed species of Philodendron s.l. species were randomly distributed considering the phylogenetic context, probably due to rapid evolution and great diversity of these genomes. The observed heteromorphisms suggest the accumulation of repetitive DNA in the genomes of some species and the occurrence of chromosomal rearrangements along the karyotype evolution of the group.

Quillworts from the Amazon: A multidisciplinary populational study on Isoetes serracarajensis and Isoetes cangae.

Isoetes are ancient quillworts members of the only genus of the order Isoetales. The genus is slow evolving but is resilient, and widespread worldwide. Two recently described species occur in the Eastern Brazilian Amazon, Isoetes serracarajensis and Isoetes cangae. They are found in the ironstone grasslands known as Canga. While I. serracarajensis is present mostly in seasonal water bodies, I. cangae is known to occur in a single permanent lake at the South mountain range. In this work, we undertake an extensive morphological, physiological and genetic characterization of both species to establish species boundaries and better understand the morphological and genetic features of these two species. Our results indicate that the morphological differentiation of the species is subtle and requires a quantitative assessment of morphological elements of the megaspore for diagnosis. We did not detect differences in microspore output, but morphological peculiarities may establish a reproductive barrier. Additionally, genetic analysis using DNA barcodes and whole chloroplast genomes indicate that although the plants are genetically very similar both approaches provide diagnostic characters. There was no indication of population structuring I. serracarajensis. These results set the basis for a deeper understanding of the evolution of the Isoetes genus.

Blind Testing: DNA Barcoding Sheds Light Upon the Identity of Plant Fragments as a Subsidy for Cave Conservation.

Plants living above and around caves represent an important, albeit poorly studied, resource within cave ecosystems. The presence of plant material (root-like structures or rhizothemes, saplings, seeds, and seedlings) correlates positively with the biodiversity of the cave dwelling animals as shown for iron-ore caves in Carajás, Pará, Brazil. Plant material collected in caves has proven to be difficult to identify by traditional botanical methods, thus this research aims to provide a qualitative insight into the taxonomy and morphology of rhizothemes and other plant fragments found in the caves. The identification process used a combination of different molecular markers (ITS2, rbcL, and trnH-psbA) followed by a comparison of the sequences obtained against publicly available databases. The rhizothemes were submitted to micromorphological analysis to ascertain their putative root or stem origin and to compare their anatomy with known patterns found in the plant families or genera recovered through molecular matches. All studied samples were Angiosperms, mostly belonging to subclass Rosideae, within four orders: Malpighiales (Euphorbiaceae, Hypericaceae), Sapindales (Anacardiaceae and Sapindaceae), Myrtales (Myrtaceae), Fabales (Fabaceae), and only two belonging to subclass Asteridae, order Gentianales (Apocynaceae). Some of the samples were matched to generic level, with ITS2 being the best marker to identify the fragments because it shows high degree of sequence variation even at specific level and result reliability. All rhizothemes turned out to be roots, and correspondence was found between the existing literature and the individual anatomical patterns for the families and genera retrieved. DNA barcode has proved to be a useful tool to identify plant fragments found in this challenging environment. However, the existence of well curated, authoritatively named collections with ample biological information has proven to be essential to achieve a reliable identification.