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Stauroneis (Naviculales, Bacillariaceae) are widespread, mostly in fresh-water habitats, and account for 343 species. They are described mainly on the basis of morphology and morphometric traits. These characteristics vary during life cycles and may overlap between species, making their identification difficult. We isolated two strains of naviculoid diatoms and examined them using an integrative approach (phylogenetic, morphological, ultrastructural data, and life cycle). Phylogenetic analyses based on chloroplast rbcL gene data showed affinity of the new strains to the genus Stauroneis. Our algae share morphological features typical of Stauroneis but differ from similar species in minimal valve length measurements, valve apex shape, and minimal number of striae in 10 µm. Two strains are distinct from each other in maximal valve length and width, partially valve shape, the number of areolae in 10 µm, and cingulum structure. It was revealed that the strains reproduce via isogamy. Three species delimitation methods (ASAP, PTP, and GMYC) also confirmed that the two closely related new strains represent distinct species. Based on molecular data and phenotypic traits examined within the framework of an integrative approach, we describe two new isolates as Stauroneis urbani sp. nov. and Stauroneis edaphica sp. nov.
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Praesagittifera naikaiensis is an acoel flatworm that inhabits the sandy beaches in the intertidal zone of the Seto Inland Sea. This species carries Tetraselmis sp., a green unicellular chlorophyte, as a symbiont in its body, and depends on algal photosynthetic products to survive. However, the eggs of P. naikaiensis contain no symbiotic algae, and juvenile P. naikaiensis acquire symbionts from the surrounding environment through horizontal transfer after hatching, thereby establishing new symbiotic relationships in each generation. Other acoel species, Symsagittifera spp., also inhabit the Seto Inland Sea shores and acquire symbiotic green algae via horizontal transfers. To characterize their symbionts, these acoels were collected from a wide area of the Seto Inland Sea and partial nucleotide sequences of the chloroplast ribulose diphosphate carboxylase large subunit (rbcL) of the symbiotic algae were determined and used for molecular phylogenetic analysis. Symbionts of both P. naikaiensis and Symsagittifera spp. belonged to the genus Tetraselmis but were phylogenetically distant, and both species established symbiotic relationships with different symbionts even when they were sympatric. To test whether each species selects specific algae in the environment for symbiosis, we established algal strains from P. naikaiensis and Symsagittifera sp. symbionts and conducted uptake experiments on aposymbiotic juveniles of P. naikaiensis. The results suggest that symbiotic algae from Symsagittifera could be taken up by P. naikaiensis juveniles, but were unable to establish a normal symbiotic relationship with the juveniles.
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Chlorophyta , Simbiosis , Animales , Chlorophyta/fisiología , Platelmintos/fisiología , Platelmintos/genética , Filogenia , Especificidad de la EspecieRESUMEN
Diatoms are among the most important primary producers in alpine and polar freshwaters. Although temperate diatoms are sensitive to freezing, polar diatoms often exhibit more resistance. This is particularly true for members of the (predominantly terrestrial) Pinnularia borealis species complex. However, it remains unclear to what extent this resistance applies to other representatives of the genus. Here, we compare the freezing-stress tolerance of 11 freshwater, benthic strains representing different species of Pinnularia (including Caloneis) from polar, alpine, and temperate habitats. As vegetative cells, strains were exposed to freezing temperatures of -4, -10, -20, -40, -80, and -196°C. Survival was assessed by light microscopy and photosynthetic measurements. We observed vegetative cells to be sensitive to low freezing temperatures; only "mild" freezing was survived by all tested strains, and most tested strains did not survive treatments ≤-10°C. However, individual strain sensitivities appeared related to their original habitats. For example, polar and alpine strains better withstood "mild" and "moderate" freezing (-4 and -10°C, respectively); although temperate strains were significantly affected by the "mild" freezing treatment, polar and alpine strains were not. The -10°C treatment was survived exclusively by polar strains, and only P. catenaborealis survived all treatments. Interestingly, this species exhibited the lowest survival in the -10°C treatment, potentially implying some metabolic activity even at freezing temperatures. Thus, despite more extensive sampling throughout the genus and finer temperature scaling compared to previous studies, the remarkable freezing-stress tolerance of the P. borealis species complex remains unique within the genus.
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In Kazakhstan, the genus Tulipa is represented by 35 species, 18 of which are listed in the Red Data Book of Kazakhstan and protected by the state. Recent studies of tulip specimens from regions bordering Kazakhstan emphasize the significance of species inventory and report the discovery of several hybrids. In this study, eight tulip species were identified based on morphological characteristics and using DNA barcoding methods. Molecular genetic markers, including nrDNA (ITS) and cpDNA markers (rbcL, matK), of the studied species were sequenced and analyzed using the Bayesian inference and maximum likelihood phylogenetic analysis methods. Our work demonstrates that DNA barcodes based on the ITS, rbcL, and matK marker regions have successful practical applicability, with ITS being the most informative at the intragenic level. However, for distinguishing closely related taxa, the most effective approach would be to use a combined dataset of sequences from multiple DNA markers. The results showed discrepancies in the placement of several taxa (T. kaufmanniana, T. patens), likely due to introgression and natural spontaneous hybridization. The molecular phylogenetic analysis suggests the existence of a previously undescribed hybrid between T. patens and T. alberti. Further detailed population studies are needed to validate this hypothesis.
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While reliance on morphology has been at the expense of clearly distinguishing gracilarioid species, molecular data have proven to be more reliable in discriminating between taxa. Gracilariaphuquocensis was originally described, based on materials collected from Vietnam. Since it was described in 2020, there have been no further reports of this species. Meanwhile, a question has been raised as to whether the identity of a rhodophyte gracilarioid alga collected from the Philippines that has been referred to as an unidentified species of Gracilaria, could be G.phuquocensis. Based on comparative morpho-anatomical features and a molecular phylogeny based on rbcL gene sequences, establishing the identity of the Philippine material has led to the finding of the new record of G.phuquocensis outside its type locality. In addition to the discovery of G.phuquocensis in the Philippines, the species here is also identified as a newly-reported host for the adelphoparasite resembling Gracilariababae.
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The Hildenbrandiales, a typically saxicolous red algal order, is an early diverging florideophycean group with global significance in marine and freshwater ecosystems across diverse temperature zones. To comprehensively elucidate the diversity, phylogeny, biogeography, and evolution of this order, we conducted a thorough re-examination employing molecular data derived from nearly 700 specimens. Employing a species delimitation method, we identified Evolutionary Species Units (ESUs) within the Hildenbrandiales aiming to enhance our understanding of species diversity and generate the first time-calibrated tree and ancestral area reconstruction for this order. Mitochondrial cox1 and chloroplast rbcL markers were used to infer species boundaries, and subsequent phylogenetic reconstructions involved concatenated sequences of cox1, rbcL, and 18S rDNA. Time calibration of the resulting phylogenetic tree used a fossil record from a Triassic purportedly freshwater Hildenbrandia species and three secondary time points from the literature. Our species delimitation analysis revealed an astounding 97 distinct ESUs, quintupling the known diversity within this order. Our time-calibration analysis placed the origin of Hildenbrandiales (crown age) in the Ediacaran period, with freshwater species emerging as a monophyletic group during the later Permian to early Triassic. Phylogenetic reconstructions identified seven major clades, experiencing early diversification during the Silurian to Carboniferous period. Two major evolutionary events-colonization of freshwater habitats and obligate systemic symbiosis with a marine fungus-marked this order, leading to significant morphological alterations without a commensurate increase in species diversification. Despite the remarkable newly discovered diversity, the extant taxon diversity appears relatively constrained when viewed against an evolutionary timeline spanning over 800 million years. This limitation may stem from restricted geographic sampling or the prevalence of asexual reproduction. However, species richness estimation and rarefaction analyses suggest a substantially larger diversity yet to be uncovered-potentially four times greater. These findings drastically reshape our understanding of the deeply diverging florideophycean order Hildenbrandiales species diversity, and contribute valuable insights into this order's evolutionary history and ecological adaptations. Supported by phylogenetic, ecological and morphological evidence, we established the genus Riverina gen. nov. to accommodate freshwater species of Hildenbrandiales, which form a monophyletic clade in our analyses. This marks the first step toward refining the taxonomy of the Hildenbrandiales, an order demanding thorough revisions, notably with the creation of several genera to address the polyphyletic status of Hildenbrandia. However, the limited diagnostic features pose a challenge, necessitating a fresh approach to defining genera. A potential solution lies in embracing a molecular systematic perspective, which can offer precise delineations of taxonomic boundaries.
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Filogenia , Rhodophyta , Simbiosis , Simbiosis/genética , Rhodophyta/genética , Rhodophyta/clasificación , Filogeografía , Ríos , Análisis de Secuencia de ADN , Teorema de Bayes , Biodiversidad , Evolución Molecular , Evolución Biológica , ARN Ribosómico 18S/genéticaRESUMEN
We performed next-generation sequencing of the 18S rDNA-ITS1-5.8S rDNA region along with traditional Sanger sequencing of rbcL, matK, ndhF, and ITS1-5.8S rDNA-ITS2 to clarify the hybridization pattern in the subtribe Alopecurinae and in the genus Alopecurus in particular. Our data support the hybrid origin of Alopecurus × brachystylus from hybridization between A. geniculatus (sect. Alopecurium) and A. pratensis (sect. Alopecurus). Moreover, in the rDNA of hybrid A. × brachystylus, only A. aequalis-like ribotypes from tetraploid A. geniculatus participated. Surprisingly, we found the traces of introgression of A. arundinaceus-like ribotypes not only in hybrid A. × marssonii (A. geniculatus × A. arundinaceus) but in A. aequalis s. str. as well. A high-polyploid group from the section Alopecurus, A. aggr. alpinus has undoubted hybrid origin: e. g., A. brachystachyus has rDNA from the sect. Alopecurium. Alopecurus alpinus, with its allies, is clearly distinct from other members of the sect. Alopecurus (especially by maternal line) and thus we can re-establish a previous opinion about the separate group to which A. alpinus belongs. Species from the section Colobachne (presumably Alpine grasses from Ancient Mediterranean region) probably hybridized with the A. alpinus group. Even A. myosuroides (sect. Pseudophalaris) that could be referred to the separate genus has ribotypes common with the species of the section Alopecurium (A. aequalis, A. geniculatus) in one of the accessions. Additionally, we found that the possible polyphyletic origin of the genus Limnas. Limnas stelleri is very close to Alopecurus magellanicus according to NGS data, while L. malyschevii is more or less distinct from other studied species of the genus Alopecurus.
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Utilising both morphological and molecular analyses, this study unveils Mazusjiangshiensesp. nov., a novel addition to the Mazaceae family, discovered in Shaowu County, Fujian Province, China. The comprehensive description and illustrations provided here are a result of a meticulous exploration of its morphological features. While bearing a resemblance to M.gracilis, this new-found species is distinguished by three distinct characteristics: its stems are delicately soft, its leaves possess a membranous quality and the ovary is notably villous at the apex. Integration of molecular evidence, derived from the nuclear ribosomal DNA (nrITS) and three plastid DNA sequences (rps16, rbcL and trnL-trnF), unequivocally supports the classification of M.jiangshiense as a distinct species. Notably, the molecular analysis positions it as a sister species to M.spicatus, underscoring the phylogenetic relationships within the genus Mazus. Our research not only introduces M.jiangshiense as a novel taxonomic entity, but also provides a nuanced understanding of its morphological differences and molecular affinities, enriching our comprehension of the diversity and evolutionary relationships of Mazaceae.
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Plant DNA barcoding has a multitude of applications ranging from species detection and biomonitoring to investigating ecological networks and checking food quality. The ability to accurately identify species, using DNA barcoding, depends on the quality and comprehensiveness of the reference library that is used. This chapter describes how to create plant reference libraries using the rbcL, matK, and ITS2 DNA barcode regions. It covers the creation of species lists, the collection of specimens from the field and herbarium, DNA extraction, PCR amplification, and DNA sequencing. This methodology gives special attention to using samples from herbaria, as they represent important collections of easily accessible, taxonomically verified plant material.
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Código de Barras del ADN Taxonómico , ADN de Plantas , Plantas , Código de Barras del ADN Taxonómico/métodos , Plantas/genética , ADN de Plantas/genética , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN/métodos , Biblioteca de GenesRESUMEN
This rapid, equipment-free DNA isolation procedure using chromatography paper is a simple method that can be performed in less than 30 min and requires no wet lab experience. With minimal expense, it offers an affordable alternative for anyone wanting to explore biodiversity. It also provides an excellent option for use in classrooms or other activities that are time limited. The method works best for plants or lichens, producing stable DNA on Whatman® chromatography paper at room temperature, which can be eluted as needed.
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Código de Barras del ADN Taxonómico , Código de Barras del ADN Taxonómico/métodos , ADN/aislamiento & purificación , ADN/genética , ADN de Plantas/genética , ADN de Plantas/aislamiento & purificación , Plantas/genética , Cromatografía/métodos , Líquenes/genéticaRESUMEN
While plant species identification in forensics can be useful in cases involving poisonous, psychoactive, or endangered plant species, it can also become quite challenging, especially, when dealing with processed, decaying, colonized or infected material of plant origin. The Animal Plant and Soil Traces expert working group of the European Network of Forensic Science Institutes in their best practice manual has recommended several markers for plant species identification. Current study is a part of implementation of method in a forensic laboratory and its aim is to evaluate four of the recommended markers (ITS, matK, rbcL, and trnH-psbA) for species identification of forensically important plant species including medicinal, poisonous, psychoactive, and other plants. Such parameters as PCR and sequencing success, sequence length, species resolution rate and species cover in GenBank were analysed. Blind testing was performed to evaluate use of the markers for identification of forensically more complicated samples. According to results, a combination of ITS, matK and trnH-psbA is the best choice for plant species identification. The best results with fresh plant material can be achieved with ITS, trnH-psbA, and matK, while ITS and matK are the best choice when working with low quality plant material. rbcL due to its low species discrimination rate can be used only as an indicative marker.
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ADN de Plantas , Plantas , Reacción en Cadena de la Polimerasa , Marcadores Genéticos , ADN de Plantas/genética , Especificidad de la Especie , Análisis de Secuencia de ADN , Ciencias Forenses/métodosRESUMEN
High-sulfur, low-oxygen environments formed by underwater sinkholes and springs create unique habitats populated by microbial mat communities. To explore the diversity and biogeography of these mats, samples were collected from three sites in Alpena, Michigan, one site in Monroe, Michigan, and one site in Palm Coast, Florida. Our study investigated previously undescribed eukaryotic diversity in these habitats and further explored their bacterial communities. Mat samples and water parameters were collected from sulfur spring sites during the spring, summer, and fall of 2022. Cyanobacteria and diatoms were cultured from mat subsamples to create a culture-based DNA reference library. Remaining mat samples were used for metabarcoding of the 16S and rbcL regions to explore bacterial and diatom diversity, respectively. Analyses of water chemistry, alpha diversity, and beta diversity articulated a range of high-sulfur, low-oxygen habitats, each with distinct microbial communities. Conductivity, pH, dissolved oxygen, temperature, sulfate, and chloride had significant influences on community composition but did not describe the differences between communities well. Chloride concentration had the strongest correlation with microbial community structure. Mantel tests revealed that biogeography contributed to differences between communities as well. Our results provide novel information on microbial mat composition and present evidence that both local conditions and biogeography influence these unique communities.
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Invasive plants are known to cause biodiversity loss and pose a major risk to human health and environment. Identification of invasive plants and distinguishing them from native species has been relied on morphological examination. Stringent requirement of floral characters and decreasing number of expert taxonomists are making conventional morphology-based identification system tedious and resource-intensive. DNA barcoding may help in quick identification of invasive species if distinct sequence divergence pattern at various taxonomic levels is observed. The present work evaluates the utility of four molecular markers; rbcL, matK, their combination (rbcL + matK), and psbA-trnH for identification of 37 invasive plant species from India and also in distinguishing them from 97 native species. A psbA-trnH locus was found to be of restricted utility in this work as it was represented by the members of a single family. A hierarchical increase in K2P mean divergence across different taxonomic levels was found to be the maximum for matK alone followed by rbcL + matK and rbcL alone, respectively. NJ clustering analysis, however, confirmed the suitability of combined locus (rbcL + matK) over individual rbcL and matK as the DNA barcode. RbcL showed the lowest resolution power among the three markers studied. MatK exhibited much better performance compared to rbcL alone in identifying most of the species accurately although it failed to show monophyly of genus Dinebra. Two families; Asteraceae and Poaceae, remained polyphyletic in the trees constructed by all three markers. Combined locus (rbcL + matK) was found to be the most suitable marker as it raised the resolution power of both the markers and could identify more than 90% of genera correctly. Phylogenetic tree constructed by Maximum-Parsimony method using combined locus as a molecular marker exhibited the best resolution, thus, supporting the significance of two-locus combination of rbcL + matK for barcoding invasive plant species from India. Present study contributes to the global barcode data of invasive plant species by adding fifty-one new sequences to it. Effective barcoding of additional number of native as well as invasive plant species from India is possible using this dual locus if it is combined with one or more new molecular plastid markers. Expansion of barcode database with a focus on barcode performance optimisation to improve discrimination ability at species level can be undertaken in future.
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An analysis of the phylogeny of Cephaloziellaceae was carried out based on trees constructed for previously and newly obtained sequences of five genes: nuclear ITS1-2 and chloroplast trnL-F, trnG, rbcL, and psbA. Phylogenetic trees inferred from different genes are congruent for the main details; however, the position of several taxa is variable. As a result, a new phylogenetic system of the family was proposed. The narrow genus concept seems to be more appropriate for the family. Cephaloziella spinicaulis is segregated into the new genus Douiniella, the generic status for Prionolobus and Metacephalozia is confirmed, and the dubious generic status of Kymatocalyx is substantiated. The generic independence of Cylindrocolea from Cephaloziella s. str. is confirmed. The small amount of data hinders the description of two more genera from Cephaloziella s.l.
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Pentaclethra macrophylla Benth., commonly referred to as "African oil bean"; is a leguminous tree species that belongs to the subfamily Caesalpinioideae of the family Fabaceae and it is native to the dry tropical rainforest forest of West to Central Africa. It is widely used as a resource for food, medicine, firewood, construction, arts, and craft and particularly of socio-economic and cultural value to indigenous people of southern, Nigeria. Despite its significant potential, it is considered underutilized in the aspect of research attention and global trade. The dataset highlights the distribution pattern, genetic diversity, phylogenetic relationship, and ecological preferences of P. macrophylla accessions collected across the various agro-ecological zones in Nigeria where it is distributed, using the Ribulose 1,5 Bisphosphate Carboxylase/Oxygenase (rbcL) gene.
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Aloina catillum is a variable moss typical of xerophytic environments in the Neotropics, characterized against other closely allied Aloina species with well-differentiated leaf border by its setae twisted to the left throughout. In order to clarify its variability and its relationships with the allied species with differentiated leaf border A. brevirostris, A. obliquifolia, and A. rigida, we performed an integrative study including sequence data from four markers (nuclear ITS, plastid atpB-rbcL, trnG, trnL-F), morphometry, and species assembling by automatic partitioning (ASAP) algorithm. Our data suggest that A. catillum consists of at least three species: A. calceolifolia (an earlier name for A. catillum), and two species described here as a new, A. bracteata sp. nov. and A. limbata sp. nov. This latter species includes the specimens previously identified as A. obliquifolia from South America. Additionally, some morphological and molecular variability was also detected in A. limbata, but was not consistent enough to be recognized taxonomically. The study supports the presence of A. brevirostris in the Neotropics and A. rigida is tentatively excluded from South America. Full descriptions of the A. catillum s.l. species and a diagnostic key to this complex in South America are provided.
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African locust bean (Parkia biglobosa) is a multipurpose leguminous tree species of nutritional and pharmacological value. The plant is widely distributed in Africa and across Nigeria's major agroecological areas (AEAs). Amidst declining cultivation and production, P. biglobosa is genetically threatened in its natural habitats due to overexploitation, deforestation, wildfires and lack of improved tree management practices. Consequently, concerted research efforts directed towards germplasm collection and assessment of genetic relationships are imperative for conserving its genetic resources, sustainable management and selecting promising landraces for breeding programmes. The dataset presents rbcL intraspecific genetic diversity and population structure of 62 P. biglobosa landraces in Nigeria. A relatively high level of diversity and a low degree of nucleotide variability was observed among the landraces. Relatively high values of 642 total allele sites, 601 polymorphic sites, 504 parsimony information sites, 883 total number mutations, 9 haplotypes and 0.55 gene diversity were recorded for the sequence dataset. Low values of 0.35 nucleotide diversity and 5 InDels events were also recorded for the dataset. The gene flow in this dataset demonstrated an extensive exchange of genes between the three populations of P. biglobosa, which influenced the level of genetic differentiation (Gst) between the populations. Significantly low Gst (-0.01) was recorded between the Guinea and Sudan savannah populations, a moderate value (0.03) was recorded between the Sudan savannah and Rainforest populations and a higher Gst value (0.05) was recorded between the Guinea and Rainforest populations. The dataset highlights potential evolutionary dynamics that might influence variations relevant to the breeding and conservation of P. biglobosa in Nigeria and across its range in West and Central Africa.
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As the scope of plant eDNA metabarcoding diversifies, so do the primers, markers and methods. A wealth of primers exists today, but their comparative evaluation is lacking behind. Similarly, multi-marker approaches are recommended but debates persist regarding barcode complementarity and optimal combinations. After a literature compilation of used primers, we compared in silico 102 primer pairs based on amplicon size, coverage and specificity, followed by an experimental evaluation of 15 primer pairs on a mock community sample covering 268 plant species and genera, and about 100 families. The analysis was done for the four most common plant metabarcoding markers, rbcL, trnL, ITS1 and ITS2 and their complementarity was assessed based on retrieved species. By focusing on existing primers, we identify common designs, promote alternatives and enhance prior-supported primers for immediate applications. The ITS2 was the best-performing marker for flowering vascular plants and was congruent to ITS1. However, the combined taxonomic breadth of ITS2 and rbcL surpassed any other combination, highlighting their high complementarity across Streptophyta. Overall, our study underscores the significance of comprehensive primer and barcode evaluations tailored to metabarcoding applications.
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ADN Ambiental , Magnoliopsida , Humanos , Código de Barras del ADN Taxonómico/métodos , ADN Espaciador Ribosómico/genética , Plantas/genética , Magnoliopsida/genéticaRESUMEN
KEY MESSAGE: Cathepsin B plays an important role that degrades the Rubisco large subunit RbcL in freezing stress. Programmed cell death (PCD) has been well documented in both development and in response to environmental stresses in plants, however, PCD induced by freezing stress and its molecular mechanisms remain poorly understood. In the present study, we characterized freezing-induced PCD and explored its mechanisms in Arabidopsis. PCD induced by freezing stress was similar to that induced by other stresses and senescence in Arabidopsis plants with cold acclimation. Inhibitor treatment assays and immunoblotting indicated that cathepsin B mainly contributed to increased caspase-3-like activity during freezing-induced PCD. Cathepsin B was involved in freezing-induced PCD and degraded the large subunit, RbcL, of Rubisco. Our results demonstrate an essential regulatory mechanism of cathepsin B for Rubisco degradation in freezing-induced PCD, improving our understanding of freezing-induced cell death and nitrogen and carbohydrate remobilisation in plants.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Catepsina B/metabolismo , Congelación , Ribulosa-Bifosfato Carboxilasa/metabolismo , Apoptosis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismoRESUMEN
Metabarcoding is a powerful tool, increasingly used in many disciplines of environmental sciences. However, to assign a taxon to a DNA sequence, bioinformaticians need to choose between different strategies or parameter values and these choices sometimes seem rather arbitrary. In this work, we present a case study on ITS2 and rbcL databases used to identify pollen collected by bees in Belgium. We blasted a random sample of sequences from the reference database against the remainder of the database using different strategies and compared the known taxonomy with the predicted one. This in silico cross-validation (CV) approach proved to be an easy yet powerful way to (1) assess the relative accuracy of taxonomic predictions, (2) define rules to discard dubious taxonomic assignments and (3) provide a more objective basis to choose the best strategy. We obtained the best results with the best blast hit (best bit score) rather than by selecting the majority taxon from the top 10 hits. The predictions were further improved by favouring the most frequent taxon among those with tied best bit scores. We obtained better results with databases containing the full sequences available on NCBI rather than restricting the sequences to the region amplified by the primers chosen in our study. Leaked CV showed that when the true sequence is present in the database, blast might still struggle to match the right taxon at the species level, particularly with rbcL. Classical 10-fold CV-where the true sequence is removed from the database-offers a different yet more realistic view of the true error rates. Taxonomic predictions with this approach worked well up to the genus level, particularly for ITS2 (5-7% of errors). Using a database containing only the local flora of Belgium did not improve the predictions up to the genus level for local species and made them worse for foreign species. At the species level, using a database containing exclusively local species improved the predictions for local species by â¼12% but the error rate remained rather high: 25% for ITS2 and 42% for rbcL. Foreign species performed worse even when using a world database (59-79% of errors). We used classification trees and GLMs to model the % of errors vs. identity and consensus scores and determine appropriate thresholds below which the taxonomic assignment should be discarded. This resulted in a significant reduction in prediction errors, but at the cost of a much higher proportion of unassigned sequences. Despite this stringent filtering, at least 1/5 sequences deemed suitable for species-level identification ultimately proved to be misidentified. An examination of the variability in prediction accuracy between plant families showed that rbcL outperformed ITS2 for only two of the 27 families examined, and that the % correct species-level assignments were much better for some families (e.g. 95% for Sapindaceae) than for others (e.g. 35% for Salicaceae).