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Better the devil you know? Guidelines for insightful utilization of nrDNA ITS in species-level evolutionary studies in plants
Abstract
The internal transcribed spacers (ITS) of the nuclear ribosomal 18S-5.8S-26S cistron continue to be the most popular non-plastid region for species-level phylogenetic studies of plant groups despite the early warnings about their potential flaws, which may ultimately result in incorrect assumptions of orthology. It has been gradually realized that the alternative target regions in the nuclear genome (low-copy nuclear genes, LCNG) are burdened with similar problems. The consequence is that, to date, developing useful LCNG for non-model organisms requires an investment in time and effort that hinders its use as a real practical alternative for many labs. It is here argued that ITS sequences, despite drawbacks, can still produce insightful results in species-level phylogenetic studies or when non-anonymous nuclear markers are required, provided that a thoughtful use of them is made. To facilitate this, two series of guidelines are proposed. One helps to circumvent problems of ITS amplification from the target organism, including spurious results from contaminants, paralogs and pseudogenes, as well as detection of sequencing artifacts. The other series helps to find out causes for unresolved clades in phylogenetic reconstruction, to integrate gene phylogenies, to distinguish horizontal transfer from lineage sorting, and to reveal if ITS phylogeny is not a good estimate of organism phylogeny.
... exist in a few thousands of copies that are reiterated tandemly at one or more loci which are visible as secondary constrictions or nuclear organizers (NORs) when transcriptionally active 26 . A noteworthy property of 35S rRNA genes is that the sequences of internal (ITS) and external (ETS) transcribed spacers are highly variable between species yet relatively homogeneous within the species genome and often species-specific 47,48 . This makes the comparative study of ITS and ETS sequences in different species a fruitful approach in the molecular phylogeny and DNA barcoding [47][48][49] . ...
... A noteworthy property of 35S rRNA genes is that the sequences of internal (ITS) and external (ETS) transcribed spacers are highly variable between species yet relatively homogeneous within the species genome and often species-specific 47,48 . This makes the comparative study of ITS and ETS sequences in different species a fruitful approach in the molecular phylogeny and DNA barcoding [47][48][49] . It is not well understood how the difference between the repeated 35S rRNA genes of sister species arises in the course of species divergence. ...
... 56 , diploid and polyploid Avena L. 57 , some species of Gossypium L. 58 , and Brassica L. 59 . Generally, the phenomenon of nucleolar dominance is observed in hybrids and allopolyploids 47,60 . Here, the rDNA sequences of the repressed genome rapidly accumulate SNPs and deletions and are gradually lost 34,36,38,40,61 . ...
Sparganium is an emergent aquatic macrophyte widely spread in temperate and subtropical zones. Taxa of this genus feature high phenotypic plasticity and can produce interspecific hybrids. By means of high-throughput sequencing of the internal transcribed spacer (ITS1) of 35S rDNA, the status of 15 Eurasian Sparganium species and subspecies was clarified and the role of hybridization events in the recent evolution of the genus was investigated. It has been shown that a number of species such as S. angustifolium, S. fallax and S. subglobosum have homogenized rDNA represented by one major ribotype. The rDNA of other taxa is represented by two or more major ribotypes. Species with high rDNA heterogeneity are apparently of hybrid origin. Based on the differences in rDNA patterns, intraspecific diversity was identified in S. probatovae and S. emersum. Thus, we have concluded that Sparganium has extensive interspecific hybridization at the subgenus level, and there may also be occasional hybridization between species from different subgenera.
... Among all these units, the internal transcribed spacers (ITS1 and ITS2) are the best-characterized nrDNA sequences 7 partly because ITS sequences show characteristics advantageous for phylogenetic studies, such as biparental inheritance, short length, and high evolution rate 4,8,9 . ITS sequences usually present fast concerted evolution with low levels of intra-genomic sequence variation and very few polymorphic positions 10,11 . However, in some animals (e.g., 12 ) and especially in plants [13][14][15][16][17][18] , sequence homogenization remains incomplete across ITS sequences, resulting in relatively high intra-genomic polymorphism. ...
... These species are closely related and belong to an Iberian clade within this genus 34 . We sequenced ITS1 and ITS2 by NGS to recover all the ITS copies present in the different genomes 11,35 . With these sequences, we then proceeded to quantify the degree of sequence homogenization in both ITS1 and ITS2 within individuals, populations, and species; and the concerted evolution levels in Erysimum spp. ...
... Both ITS and, especially, ITS2 have for a long time been used as phylogenetic and barcoding markers in plants 8,69,[81][82][83] . However, many studies have pointed out that evolutionary inferences based on these markers might lead to misleading or erroneous conclusions in species where sequence homogenization is lacking due to hybridization or other genome rearrangement events 9,11 . In this study, our results indicate that allopolyploidization and hybridization have severely impaired ITS sequence homogenization in Erysimum, implying that ITS-based phylogenies of this genus should be considered with prudence. ...
The internal transcribed spacers (ITS) exhibit concerted evolution by the fast homogenization of these sequences at the intragenomic level. However, the rate and extension of this process are unclear and might be conditioned by the number and divergence of the different ITS copies. In some cases, such as hybrid species and polyploids, ITS sequence homogenization appears incomplete, resulting in multiple haplotypes within the same organism. Here, we studied the dynamics of concerted evolution in 85 individuals of seven plant species of the genus Erysimum (Brassicaceae) with multiple ploidy levels. We estimated the rate of concerted evolution and the degree of sequence homogenization separately for ITS1 and ITS2 and whether these varied with ploidy. Our results showed incomplete sequence homogenization, especially for polyploid samples, indicating a lack of concerted evolution in these taxa. Homogenization was usually higher in ITS2 than in ITS1, suggesting that concerted evolution operates more efficiently on the former. Furthermore, the hybrid origin of several species appears to contribute to the maintenance of high haplotype diversity, regardless of the level of ploidy. These findings indicate that sequence homogenization of ITS is a dynamic and complex process that might result in varying intra- and inter-genomic diversity levels.
... ITS sequences usually present fast concerted evolution with low levels of intra-genomic sequence variation and very few polymorphic positions [9,10]. However, in some animals (e.g., [11]) and especially in plants [12,13,14,15,16,17], sequence homogenization remains incomplete across ITS sequences, resulting in relatively high intra-genomic polymorphism. ...
... For this purpose, we analyzed polymorphisms at the species, population, and individual levels in ITS1 and ITS2 for seven Erysimum species. We sequenced both markers by NGS to recover all the ITS copies present in the different genomes [10,33]. With these sequences, we then proceeded to quantify the degree of sequence homogenization in both ITS1 and ITS2 within individuals, populations, and species; and the concerted evolution levels in polyploid Erysimum spp. ...
... The copyright holder for this preprint (which this version posted May 29, 2022. ; https://doi.org/10.1101/2022.05.29.493735 doi: bioRxiv preprint events [8,10]. In this study, our results indicate that allopolyploidization and hybridization have severely impaired ITS sequence homogenization in Erysimum, implying that ITS-based phylogenies of this genus should be considered with prudence. ...
The internal transcribed spacers (ITS) exhibit concerted evolution by the fast homogenization of these sequences at the intragenomic level. However, the rate and extension of this process are unclear and might be conditioned by the number and divergence of the different ITS copies. In some cases, such as hybrid species and polyploids, ITS sequence homogenization appears incomplete, resulting in multiple haplotypes within the same organism. Here, we studied the dynamics of concerted evolution in 85 individuals of seven plant species of the genus Erysimum (Brassicaceae) with multiple ploidy levels. We estimated the rate of concerted evolution and the degree of sequence homogenization separately for ITS1 and ITS2 and whether these varied with ploidy. Our results showed incomplete sequence homogenization, especially for polyploid samples, indicating a lack of concerted evolution in these taxa. Homogenization was usually higher in ITS2 than in ITS1, suggesting that concerted evolution operates more efficiently on the former. Furthermore, the hybrid origin of several species appears to contribute to the maintenance of high haplotype diversity, regardless of the level of ploidy. These findings indicate that sequence homogenization of ITS is a dynamic and complex process that might result in varying intra- and inter-genomic diversity levels.
... The number of polymorphisms ranged from 0.2 to 7 per 1,000 bp in the nuclear ribosomal DNA assemblies and from 0 to 1.1 per 1,000 bp in the chloroplast assemblies except for Duguetia hadrantha with an average of two polymorphisms per 1,000 bp, concentrated in regions with a coverage lower than 20. Although these polymorphisms may add some noise to the analyses, we interpret the effect to be negligible due to concerted evolution (Feliner & Rosselló 2007, Hillis et al. 1991. Furthermore, most of the polymorphisms were found either in regions with low coverage or near contig breaks, and therefore can be expected to be false positives. ...
... In order to improve resolution of the phylogeny two additional nuclear ribosomal regions, the internal transcribed spacer region (ITS-5.8S-ITS2) and the external transcribed spacer (ETS), which have previously been shown to be highly variable at the species level (reviewed by: Álvarez & Wendel 2003, Feliner & Rosselló 2007, Poczai & Hyvönen 2010, were generated for all accessions, including the newly added species as well as the 42 taxa used in the chloroplast phylogeny by Wang et al. (2012). Voucher information and GenBank accession numbers for all samples included in the dataset are given in the Supplementary information. ...
... 3 Each ribosomal gene cluster consists of a transcribed region (genes 18S, 5.8S, and 26S), internal transcribed spacers located on either side of 5.8S, called ITS1 and ITS2, respectively, and flanking external transcribed spacers ETS1 and ETS2 [40]. ITS markers are widely used as phylogenetic markers to classify plants at different taxonomic levels, including genus, species, and subspecies [41,42]. ...
This article studies the morphological parameters of vegetative and generative organs of different age groups of Crataegus ambigua from four populations in Western Karatau (Mangistau region, Kazakhstan). The study examined four populations Pop 1 Sultan Epe, Pop 2 Karakozaiym, Pop 3 Emdikorgan, and Pop 4 Samal located in various gorges of western Karatau. Some phylogenetic inference methods were applied using six genetic markers atpF-atpH, ITS, matK, psbK-psbI, rbcL, and trnH-psbA. We also used a statistical analysis of the vegetative and generative organs of plants for three age groups of plants (virgin, young generative, and adult generative). According to the age structure, Samal populations have a high concentration of young generative plants - 42.3% and adult generative plants - 30.9%. Morphological analysis showed the significance of the parameters of the generative organs and separated the Samal population into a separate group according to the main PCoA coordinates. The results of the floristic analysis showed that the populations in the Samal have a high concentration of species diversity. The comparative dendrogram of UPGMA shows the informativeness of the use of genetic markers and the psbK-psbI region can be used to judge the difference between the fourth Samal population and the other three.
... Nagy et al., 2012;Schoch et al., 2012). Since the ITSs are present in multiple copies in the genome, potentially both within and among chromosomes, they do not evolve as independent loci but can undergo inter-copy recombination, and individuals can theoretically feature several alleles, in unequal frequencies (Denduangboripant & Cronk, 2000;Feliner & Rosselló, 2007;Göker & Grimm, 2008). Apart from the evolutionary and ensuing technical complications (co-amplification of several copies, of potentially variable length) that would challenge the analysis even of a dichotomous diversification, the scenarios in this study involve the additional level of potential inter-lineage reticulation. ...
Ancient lakes are hotspots of species diversity, posing challenges and opportunities for exploration of the dynamics of endemic diversification. Lake Baikal in Siberia, the oldest lake in the world, hosts a particularly rich crustacean fauna, including the largest known species flock of harpacticoid copepods with some 70 species. Here, we focused on exploring the diversity and evolution within a single nominal species, Harpacticella inopinata Sars, 1908, using molecular markers (mitochondrial COI, nuclear ITS1 and 28S rRNA) and a set of qualitative and quantitative morphological traits. Five major mitochondrial lineages were recognized, with model‐corrected COI distances of 0.20–0.37. A concordant pattern was seen in the nuclear data set, and qualitative morphological traits also distinguish a part of the lineages. All this suggests the presence of several hitherto unrecognized cryptic taxa within the baikalian H. inopinata, with long independent histories. The abundances, distributions and inferred demographic histories were different among taxa. Two taxa, H. inopinata CE and H. inopinata CW, were widespread on the eastern and western coasts, respectively, and were largely allopatric. Patterns in mitochondrial variation, that is, shallow star‐like haplotype networks, suggest these taxa have spread through the lake relatively recently. Three other taxa, H. inopinata RE, RW and RW2, instead were rare and had more localized distributions on either coast, but showed deeper intraspecies genealogies, suggesting older regional presence. The rare taxa were often found in sympatry with the others and occasionally introgressed by mtDNA from the common ones. The mitochondrial divergence between and within the H. inopinata lineages is still unexpectedly deep, suggesting an unusually high molecular rate. The recognition of true systematic diversity in the evaluation and management of ecosystems is important in hotspots, as it is everywhere else, while the translation of the diversity into a formal taxonomy remains a challenge.
... It is well known that nrDNA, unlike chloroplast and mitochondrial genomes, accumulates nucleotide substitutions at approximately the same rate [43]. This feature makes the ITS region genetically diverse, allowing the identification of samples not only at the intragenic level but also, in certain cases, at the intraspecific level of variability [32,44,45]. ...
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.
... Species identification is often complex, mainly among closely related and recently diverged groups (Nieto-Feliner and Rosselló 2007). Determining the limits between species seems to be less changing (Yang and Rannala 2014), especially when applying multiple markers, approaches, and individuals distributed throughout the species range (Binks et al. 2021) and considering the lineage species concept (De Queiroz 2007). ...
Closely related and young species often show high morphological similarity, challenging their identification and correct assignment. Molecular markers and integrative approaches have contributed to solving many taxonomic uncertainties. In this study, we evaluated the genetic variability and ecological features of Petunia guarapuavensis and Petunia scheideana, over which there is a taxonomic debate. Both species are endemic and rare, distributed in the subtropical highland grasslands in southern South America. We based our analyses on nuclear microsatellite and plastid sequences, aiming to disentangle the taxonomic ambiguities that made some consider these entities synonymous despite occupying different clades in the genus phylogenetic tree. Our findings support that there is genetic differentiation between these species, suggesting that they are independent taxonomic entities despite sharing floral traits and a few molecular polymorphisms. The low genetic sharing between the species is likely due to a common ancestor and recent divergence time. In contrast, their morphological similarity can be attributed to the absence of selective pressure, as both grow under similar ecological conditions. This study emphasizes that adding more than one sequence per species, combining data with dissimilar inheritance patterns, and exploring data through different methodologies help to disentangle taxonomic incongruences and reveal diversity that might otherwise remain hidden.
... However, its lower amplification success and sequencing difficulties remain a drawback, particularly in gymnosperms. This finding is in line with previous studies indicating that this nrDNA region evolves rapidly, leading to genetic variations that can differentiate closely related species [32,33]. This finding is consistent with that of Li et al. [34], who found that the Internal Transcribed Spacer (ITS) performed the highest rate of species discrimination of some flowering plants (>95%) among the three markers (matK, rbcL, and ITS) with only a slight improvement in species resolution when combining the three DNA barcodes. ...
... Morphological characterization of the Musa cultivars was carried out focusing on the inflorescence, bract, male flower, and the fruit [21]. Qualitative criteria were used and morphological character traits were matched against the IPB-INIBAP Banana catalog as well as the Musa Germplasm Information System (MGIS) database. ...
Background
Banana production is increasingly under threat due to harsh weather conditions as a result of climate change and different diseases. As such there is a need for the preservation and the characterization of the banana cultivar population for the purposes of crop improvement. The identification of collected banana germplasm in Zimbabwe was conducted based on the Inter-transcribed spacer region as well as morphology. The study was conducted with the aim of distinguishing one cultivar from another towards genetic conservation as well as banana improvement.
Results
ITS 1 and ITS 4 region targeting primers were used to amplify the DNA from twelve cultivars as well as sequence. Blast results identified five Musa groups which are Musa balbisiana (BB), Musa ABB, Musa AB hybrid, Musa acuminata (AAA) , and Musa acuminata subsp. Malaccensis (AA). Phylogenetic analysis was done on the sequences under study and a maximum likelihood tree was generated to determine relationships between the sequences. Further identification was done using the inflorescence, bract, and male bud and fruit characteristics of each cultivar complementing the molecular evaluation.
Conclusion
Genetic and morphological identification of locally grown bananas was therefore successful. An important step towards identifying pure lines suitable for breeding.
... ITS dapat membantu mengidentifikasi nenek moyang atau garis keturunan yang terlibat dalam hibrida dan introgresi dimana evolusi bersama tidak menghomogenkan salinan sekuens (69). Selain itu, ITS sebagai barcode DNA yang telah diaplikasikan secara meluas memiliki kelebihan lainnya yaitu adanya tingkat variasi dan struktur multi-salinan yang memfasilitasi amplifikasi dengan PCR, bahkan dari sampel herbarium (68,70). Alternatif dari permasalahan DNA barcoding menggunakan keseluruhan wilayah ITS dapat berupa penggunaan salah satu spacer, terutama Internal Transcribed Spacer 2 (ITS2) sebagai barcode karena ITS2 memiliki wilayah yang lebih konservatif dibandingkan ITS1 (13,71). ...
Molecular identification using short orthological DNA sequences (DNA barcoding) has been applied for the classification of orchid species which is a major step in biodiversity management, conservation, breeding, authenticating components of herbal products, and tracking the adulteration of orchid species. One of the most widely used loci for phylogenetic inference at the generic and infrageneric levels in plants was ITS located between 18S rDNA, 5.8S rDNA, and 26S rDNA. The ITS or ITS2 region has been suggested as a plant barcode in some previous studies. However, DNA barcoding using the entire ITS genome is considered less effective and efficient in the process of PCR amplification and sequencing. Besides, complete DNA barcodes are difficult to obtain from herbarium samples and herbal products because some DNA sequences have been degraded. DNA mini-barcodes were developed over the past ten years to overcome issues related to DNA barcoding. DNA mini-barcodes use shorter DNA segments for PCR amplification, so they can identify species effectively and efficiently compared to the regular DNA barcoding. Specific primers that encode the ITS1 and ITS2 regions need to be designed for the PCR amplification. DNA mini-barcoding ITS has proven useful in species identification, classification studies, and authentication of specific orchid species. Therefore, a new rapid identification method based on the ITS mini-barcode is expected to be established, especially for orchid species. Keywords : DNA mini-barcode, Internal Transcribed Spacer, Orchid, Primer Design
... Very often, the phylogenetic background is prepared based on the analysis of nrITS (ITS1-5.8S rDNA-ITS2; [7,12,34]). nrITS is one of the most used molecular markers in phylogenetic studies, and it is possible to find the nrITS sequences for many different plant species in GenBank and to use them to construct a phylogenetic tree. ...
Fluorescence in situ hybridization (FISH) with ribosomal DNA (rDNA) sequences provides excellent chromosome markers for comparative cytogenetic analyses, especially in non-model plant species. The tandem repeat nature of a sequence and the presence of a highly conserved genic region make rDNA sequences relatively easy to isolate and clone. In this chapter, we describe the use of rDNA as markers for comparative cytogenetics studies. Traditionally, cloned probes labeled with Nick-translation have been used to detect rDNA loci. Recently, pre-labeled oligonucleotides are also employed quite frequently to detect both 35S and 5S rDNA loci. Ribosomal DNA sequences, together with other DNA probes in FISH/GISH or with fluorochromes such as CMA3 banding or silver staining, are very useful tools in comparative analyses of plant karyotypes.
... Subsequently, three regions of chloroplast genome (trnL-trnF, trnQ-rps16 and rps16 intron) exhibiting high variation were remained and utilized for genotyping all 912 accessions (Supplementary Table S6). In order to ensure the suitability of nuclear ITS fragments for species level phylogeographic analysis, we first checked their characterization with regards to intra-individual ITS polymorphism and pseudogenes, following the guidelines for handling ITS sequences [60] and drawing on our previous molecular study [25]. The PCR conditions were performed to the methods of Chen et al. [25]. ...
The subgenus Cerasus (Rosaceae) contain numerous fruit trees and ornamentals with high economic values. The origin and genetic divergence among various types of fruiting cherries always remain a perplexing issue. We employed three plastom fragments and ITS sequence matrices derived from 912 cherry accessions to elucidate the phylogeographic structure and genetic relationship among fruiting cherries, as well as the origin and domestication of cultivated Chinese cherry. The integration of haplotype genealogies, Approximate Bayesian computation (ABC) approach and estimation of genetic differentiation within and between different groups and lineages has facilitated the resolution of several previously unresolved questions. Firstly, distant phylogenetic relationships between Cerasus and Microcerasus accessions, as indicated by both nuclear and chloroplast data, suggested independent origins and evolution for these two taxa. Moreover, two distinct geographic origin centers (Europe and China) have been confirmed, with significant phylogeographic signals and high genetic differentiation observed between cherries from these regions. This may be attributed to long-term geographic isolation caused by Himalaya-Hengduan Mountains. Our phylogeographic analyses and ABC analysis suggested that cherries inhabiting in China may have undergone multiple hybridization events during the glacial refugia of the eastern edge and southern Himalaya-Hengduan Mountains, followed by rapid radiation throughout their current habitats during interglacial period. The discrepancy between nuclear and chloroplast data may be attributed to hybridization events and incomplete lineage sorting. Furthermore, we speculated that the domesticated Chinese cherries were derived from wild accessions in Longmenshan Fault Zones approximately 2600 years ago. We have also traced the domestication processes and dispersal routes of cultivated Chinese cherries.
... The majority genotypes in O. curvispina and O. martiniana, by contrast, were maternally inherited. Majure (2022) considered that the higher percentage of minority genotypes in O. martiniana could reflect a younger age for that species, and thus reduced time for recombination and gene conversion (Feliner and Roselló 2007). This could also help explain the higher percentage of the minority genotype in G. robertsii. ...
The rare Baja California Sur endemic cactus Grusonia robertsii was originally hypothesized to be an intergeneric hybrid between G. invicta and Cylindropuntia alcahes subsp. alcahes but was described as a Grusonia due to its overall closer resemblance to G. invicta, except in the fruit. A more comprehensive analysis of the morphology and phylogenetic placement of G. robertsii based on plastid and nrDNA sequences has revealed that Rebman's original hypothesis was correct, and this taxon represents the first documented intergeneric hybrid between Cylindropuntia and Grusonia, with G. invicta as the maternal parent and C. alcahes subsp. alcahes as the paternal contributor. We here describe a new nothogenus, Cylindronia, and provide a new combination for the nothospecies Cylindronia robertsii comb. nov.
... An examination of the sequence data used by Schuster et al. (2011b) revealed that the ITS sequence (EF635479) is likely a pseudogene, which inflated sequence divergence and resulted in the artificial separation of M. astonii from the rest of New Zealand Muehlenbeckia. This pseudogene was identified by its relatively low GC content (60.1% versus 65.5%) and the high number of SNPs in the conserved 5.8S region (Buckler et al. 1997;Álvaraez and Wendel 2003;Feliner and Rosselló 2007). We found that pseudogenised ITS copies would readily amplify in this group if 4% DMSO, or some other denaturant, was omitted from the PCR mixture. ...
Japanese knotweed ( Reynoutria japonica ) is native to East Asia, but has been introduced to the West where it is a noxious invasive weed. Taxonomically, Japanese knotweed is placed within subtribe Reynoutriinae (Polygonaceae), which also contains the austral genus Muehlenbeckia (incl. Homalocladium ) and north temperate Fallopia . In the current study, we conducted a phylogenetic analysis using sequence data from six markers, two nuclear ( LEAFYi2 , ITS) and four plastid ( matK , rbcL , rps16-trnK and trnL-trnF ) to further resolve the evolutionary relationships within this group, using the widest sampling of in-group taxa to date. The results of this analysis confirmed that subtribe Reynoutriinae is a monophyletic group, characterised by the presence of extra-floral, nectariferous glands at the base of leaf petioles. Within the subtribe, four main clades were identified: Reynoutria , Fallopia sect. Parogonum, Fallopia s.s. (including Fallopia sects. Fallopia and Sarmentosae ) and Muehlenbeckia . The Fallopia s.s. and Muehlenbeckia clades are sister to one another, while the Fallopia sect. Parogonum clade is immediately basal to them and Reynoutria basal to all three. Fallopia , as currently circumscribed, is paraphyletic as Muehlenbeckia is nested within it. To resolve this, we propose that species of Fallopia sect. Parogonum should be treated as a new genus, Parogonum (Haraldson) Desjardins & J.P.Bailey, gen. et stat. nov. Within Reynoutria , the allied specific and infraspecific taxa that fall under the name Japanese knotweed s.l. form a monophyletic group and their taxonomic status is discussed.
... In angiosperms, the length of the ITS sequences ranges from 500 to 700 bp (Maras 2008;Sultana et al. 2011). The ITS region taxonomic diversity in determining has been found to be frequently used in plants, fungi, lichenicolous fungi and animals (Martín et al. 2003;Cubero et al. 2004;Feliner and Rosselló 2007;Sun et al. 2014;Kaya et al. 2020). ...
In this study, genetic diversity and phylogenetic analysis of Turkish Juglans regia L. populations was conducted based on random-amplified polymorphic DNA (RAPD) and inter-simple sequence repeat polymerase chain reaction ISSR-PCR and nuclear ribosomal DNA internal transcribed spacer (nrDNA ITS) sequences. A total of 14 populations from the Aegean and Marmara regions were included in the research. In all, 12 RAPD and 13 ISSR primers were used for molecular characterization of the J. regia populations. For the amplification of the nrDNA ITS region, ITS4 and ITS5A primers were used in PCR. In RAPD-ISSR-PCR analysis, a total of 55 and 85 bands were obtained, respectively. RAPD and ISSR analyses detected high polymorphism (74.54% and 70.57%, respectively) among J. regia populations. However, ITS sequence analysis was not efficient in revealing the genetic relationship among J. regia samples. In addition, phylogenetic analyses including nrDNA ITS sequences of our populations and that of other Juglans taxa (J. hindsii, J. major, J. nigra, J. microcarpa, J. cathayensis and J. hopeiensis) obtained from the National Center for Biotechnology Information (NCBI) were conducted. Analysis of J. regia populations based on RAPD and ISSR-PCR and ITS sequences revealed that the rate of polymorphism obtained with the ISSR-PCR approach was higher than those obtained using the ITS sequences and RAPD-PCR. Genetic diversity results of these populations will contribute significantly to the identification and breeding programs of walnuts.
... Phylogenetic structures inferred using these different sets of sequences have unique advantages and potential biases (Álvarez and Wendel, 2003;Gitzendanner et al., 2018;Nieto Feliner and Rosselló, 2007). A comprehensive assessment of all phylogenetic trees should allow us to have a relatively accurate understanding of the evolutionary history of Asian ...
Background and aims:
Species of the genus Buddleja in Asia are mainly distributed in the Sino-Himalayan region and forms a challenging taxonomic group with extensive hybridization and polyploidization. A phylogenetic approach to unraveling the history of reticulation in this lineage will deepen our understanding of the speciation in biodiversity hotspots.
Methods:
For this study, we obtained 80 accessions representing all the species in the Asian Buddleja clade, and the ploidy level of each taxon was determined by flow cytometry analyses. Whole plastid genomes, nuclear ribosomal DNA, single nucleotide polymorphisms and a large number of low copy nuclear genes assembled from genome skimming data were used to investigate the reticulate evolutionary history of Asian Buddleja. Complex cytonuclear conflicts were detected through a comparison of plastid and species trees. Gene tree incongruence was further analyzed to detect any reticulate events in the history of this lineage.
Key results:
Six hybridization events were detected, which are able to explain the cytonuclear conflict in Asian Buddleja. Furthermore, PhyloNet analysis combining species ploidy data indicated several allopolyploid speciation events. A strongly supported species tree inferred from a large number of low copy nuclear genes, not only corrected some earlier misinterpretations, but also indicated that there are many Asian Buddleja species that have been mistakenly lumped. Divergent time estimation shows two periods of rapid diversification (8-10 Mya and 0-3 Mya) in the Asian Buddleja clade, which may coincide, respectively, with the final uplift of the Hengduan Mountains and Quaternary climate fluctuations.
Conclusions:
This study presents a well supported phylogenetic backbone for the Asian Buddleja species, elucidates their complex and reticulate evolutionary history, and suggests that tectonic activity, climate fluctuations, polyploidization and hybridization together promoted the diversification of this lineage.
... Polyploidy, namely allopolyploidy, sometimes results in paraphyletic intraspecific relationships resolved by nuclear genes, such as nrITS (Soltis & Soltis 1999;Ruiz-Garcia et al. 2005), because allopolyploid species may possess two or more copies of the gene (or types, in the case of nrITS) from divergent progenitors (Schupp & Feener, 1991;Feliner & Rossello, 2007;Folk et al. 2018). This may explain the intraspecific paraphyly of C. comosum, C. rubicundum, C. klementzii, C. roborowskii, C. caput-medusae and C. arborescens, all of which are polyploid, in the nrITS phylogeny. ...
... Several genomic regions could be utilised for phylogenetic reconstruction and DNA barcoding-based identification (Christin et al. 2012;Wendel & Doyle 1998). In plants, nuclear ribosomal internal transcribed spacers (ITS) which are the internal transcribed spacers of the large subunit of ribosomal DNA, have been utilised as the sequence of choice for phylogenetic markers (Álvarez & Wendel 2003;Feliner & Rosselló 2007). More recently, plastid genes, including those from chloroplast genomes (plastomes), are being extensively used in the reconstruction of phylogenies to identify plant species (Hollingsworth et al. 2009;Li et al. 2015;Sun et al. 2016). ...
Tetrastigma rafflesiae (Miq.) Planch. is a climbing plant species that is known for its unique relationship with holoparasitic plants of Rafflesiaceae. Knowledge on the mitochondrial genes of this species may contribute towards the development of molecular approaches for species identification. This study aims to identify and characterise genes from the T. rafflesiae mitochondrial genome (mitogenome) and assess their potential to discriminate different Tetrastigma species. Mitochondrial-specific sequences were first selected by mapping T. rafflesiae whole-genome sequences to mitogenomes from several reference plant species. De novo assembly of these selected sequences produced a T. rafflesiae mitogenome with a size of 336 kb. Gene annotation revealed that the T. rafflesiae mitogenome contains at least 40 protein coding genes, 20 tRNAs and two rRNAs. Phylogenetic analysis using several mitochondrial genes, namely ccmB, cob, matR, nad6 and rps3 was able to differentiate T. rafflesiae from three other Tetrastigma species, indicating the potential of these genes as species-specific sequence markers. These findings supplement additional genetic information on T. rafflesiae and may aid in the effort of species classification and conservation.
... However, the commonly used nuclear gene fragments have mostly been rDNA sequences (such as ITS and ETS), which are short in length and mutate rapidly, and are only suitable for the reconstruction of phylogenetic relationships of closely related genera and subordinate genera. In addition, rDNA sequences are multicopy, and there incomplete concerted evolution is present, resulting in differences in sequences between different copies, limiting its application in phylogenetic studies (Nieto Feliner & Rosselló, 2007). We used the Skmer method to explore the potential of nuclear genes (after removing the rDNA) obtained by genome skimming for Cymbidium species identification, and the discrimination rate was increased from 68% to 72% ...
Cymbidium is an orchid genus with rapid radiation and high ornamental, economic, ecological and cultural importance, but its classification is controversial based on morphology. The plastid genome (plastome), as an extension of plant standard DNA barcodes, has been widely used as a potential molecular marker for identificating the recently diverged species or complicated plant groups. In this study, we newly generated 237 plastomes of 50 species (at least two individuals per species) by genome skimming, covering 71.4% of the member of the genus Cymbidium. Sequence-based analyses (barcoding gaps and ABGD) and tree-based analyses (ML, BI, and mPTP) methods were conducted for species identification of Cymbidium. Our work provides a comprehensive DNA barcode reference library for Cymbidium species identification. The results show that compared with standard DNA barcodes (rbcL+matK) as well as the plastid trnH-psbA, the species identification rate of the plastome moderately increases from 58% to 68%. At the same time, we propose an optimized identification strategy for Cymbidium species. The plastome cannot completely resolve the species identification of Cymbidium, incomplete lineage sorting, artificial cultivation, nature hybridization and chloroplast capture may be the main reasons. To further explore the potential usage of nuclear data in identifying species, Skmer method was adopted and the identification rate was increased to 72%. It seems that the nuclear genome data acts a vital role in species identification and is expected to be used as next-generation nuclear barcodes.
... Multi-copy nuclear ribosomal (nr)DNA regions are present in numerous copies and undergo intragenomic homogenization (concerted evolution) to different degree [38][39][40]. Single-or low-copy genes are thought not to be prone to concerted evolution, but more susceptible to population genetic processes [41], and their potential for phylogenetic reconstruction has to be assessed with caution as well [42]. Like with classical taxonomy, a single set of characters or one trait (comparable to a single gene region) can be misleading or else come close to the truth. ...
Native American hawkweeds are mainly mountainous species that are distributed all over
the New World. They are severely understudied with respect to their origin, colonization of the vast distribution area, and species relationships. Here, we attempt to reconstruct the evolutionary history of the group by applying seven molecular markers (plastid, nuclear ribosomal and low-copy genes). Phylogenetic analyses revealed that Chionoracium is a subgenus of the mainly Eurasian genus Hieracium, which originated from eastern European hawkweeds about 1.58–2.24 million years ago. Plastid DNA suggested a single origin of all Chionoracium species. They colonized the NewWorld via Beringia and formed several distinct lineages in North America. Via one Central American lineage, the group colonized South America and radiated into more than a hundred species within about 0.8 million years, long after the closure of the Isthmus of Panama and the most recent uplift
of the Andes. Despite some incongruences shown by different markers, most of them revealed the same crown groups of closely related taxa, which were, however, largely in conflict with traditional sectional classifications. We provide a basic framework for further elucidation of speciation patterns. A thorough taxonomic revision of Hieracium subgen. Chionoracium is recommended.
... Aside from lack of sequence variation in the nrDNA regions, the lack of species monophyly in clade C in the nrDNA trees may, in some instances, be due to the presence of multiple rDNA arrays within individuals, incomplete concerted evolution, incomplete lineage sorting, or a combination of these (e.g., Feliner & Rossello, 2007). Indeed, many species that are part of this large clade are polyploids that likely arose from hybridization between closely related B. sect. ...
To investigate phylogenetic relationships among and within major lineages of Bromus , with focus on Bromus sect. Bromus , we analyzed DNA sequences from two nuclear ribosomal (ITS, ETS) and two plastid ( rpl32-trnL UAG , matK ) regions. We sampled 103 ingroup accessions representing 26 taxa of B . section Bromus and 15 species of other Bromus sections. Our analyses confirm the monophyly of Bromus s.l. and identify incongruence between nuclear ribosomal and plastid data partitions for relationships within and among major Bromus lineages. Results support classification of B. pumilio and B. gracillimus within B . sect. Boissiera and B . sect. Nevskiella , respectively. These species are sister groups and are closely related to B. densus ( B . sect. Mexibromus ) in nrDNA trees and Bromus sect. Ceratochloa in plastid trees. Bromus sect. Bromopsis is paraphyletic. In nrDNA trees, species of Bromus sects. Bromopsis , Ceratochloa , Neobromus , and Genea plus B. rechingeri of B . sect. Bromus form a clade, in which B. tomentellus is sister to a B . sect. Genea – B. rechingeri clade. In the plastid trees, by contrast, B . sect. Bromopsis species except B. tomentosus form a clade, and B. tomentosus is sister to a clade comprising B . sect. Bromus and B . sect. Genea species. Affinities of B. gedrosianus , B. pulchellus , and B. rechingeri (members of the B. pectinatus complex), as well as B. oxyodon and B. sewerzowii , are discordant between nrDNA and plastid trees. We infer these species may have obtained their plastomes via chloroplast capture from species of B . sect. Bromus and B . sect. Genea . Within B . sect. Bromus , B. alopecuros subsp. caroli-henrici , a clade comprising B. hordeaceus and B. interruptus , and B. scoparius are successive sister groups to the rest of the section in the nrDNA phylogeny. Most relationships among the remaining species of B . sect. Bromus are unresolved in the nrDNA and plastid trees. Given these results, we infer that most B . sect. Bromus species likely diversified relatively recently. None of the subdivisional taxa proposed for Bromus sect. Bromus over the last century correspond to natural groups identified in our phylogenetic analyses except for a group including B. hordeaceus and B. interruptus .
... The reason for this pattern may be that concerted evolution has operated to maintain only one ITS parental lineage. For several decades, ITS sequences have been the most popular nuclear markers for phylogenetic studies of a wide range of plant taxa ( Alvarez and Wendel, 2003;Feliner and Rossell o, 2007). However, several potential problems have been raised with using ITS data in phylogenetic studies, including paralogs, pseudogenes and concerted evolution (may occur in other multiple-copy gene regions as well) (Arnheim, 1983). ...
Polyploidy after hybridization between species can lead to immediate post-zygotic isolation, causing saltatory origin of new species. Although the incidence of polyploidization in plants is high, it is thought that a new polyploid lineage can succeed only if it establishes a new ecological niche divergent from its progenitor lineages. We tested the hypothesis that Rhodiola integrifolia from North America is an allopolyploid produced by R. rhodantha and R. rosea and determined whether its survival can be explained by the niche divergence hypothesis. To this end, we sequenced two low-copy nuclear genes (ncpGS and rpb2) in a phylogenetic analysis of 42 Rhodiola species and tested for niche equivalency and similarity using Schoener’s D as the index of niche overlap. Our phylogeny-based approach showed that R. integrifolia possesses alleles from both R. rhodantha and R. rosea. Dating analysis showed that the hybridization event that led to R. integrifolia occurred ca. 1.67 Mya and niche modeling analysis showed that at this time, both R. rosea and R. rhodantha may have been present in Beringia, providing the opportunity for the hybridization event. We also found that the niche of R. integrifolia differs from that of its progenitors in both niche breadth and optimum. Taken together, these results confirm the hybrid origin of R. integrifolia and support the niche divergence hypothesis for this tetraploid species. Our results underscore the fact that lineages with no current overlapping distribution could produce hybrid descendants in the past, when climate oscillations made their distributions overlap.
... The minor differences between I. interruptus and I. muliensis are that I. interruptus has shorter habits and petioles and smaller leaves. Despite the constraints of nrDNA sequences (e.g., multiple rDNA arrays, concerted evolution, pseudogenes, and secondary structure) (Álvarez and Wendel, 2003;Feliner and Rossello, 2007), the limited substructures recovered within Clade IV in the nuclear tree are mostly formed by species with similar morphology and from the same distribution area. In contrast, similar patterns of morphology and geographic distribution are hardly found in the plastid tree (Figure 1). ...
As one of the largest genera of Lamiaceae and of great medicinal importance, Isodon is also phylogenetically and taxonomically recalcitrant largely ascribed to its recent rapid radiation in the Hengduan Mountains. Previous molecular phylogenetic studies using limited loci have only successfully resolved the backbone topology of the genus, but the interspecific relationships suffered from low resolution, especially within the largest clade (Clade IV) which comprises over 80% species. In this study, we attempted to further elucidate the phylogenetic relationships within Isodon especially Clade IV using plastome sequences with a broad taxon sampling of ca. 80% species of the genus. To reduce systematic errors, twelve different plastome data sets (coding and non-coding regions with ambiguously aligned regions and saturated loci removed or not) were employed to reconstruct phylogeny using maximum likelihood and Bayesian inference. Our results revealed largely congruent topologies of the 12 data sets and recovered major lineages of Isodon consistent with previous studies, but several incongruences are also found among these data sets and among single plastid loci. Most of the shallow nodes within Clade IV were resolved with high support but extremely short branch lengths in plastid trees, and showed tremendous conflicts with the nrDNA tree, morphology and geographic distribution. These incongruences may largely result from stochasticity (due to insufficient phylogenetic signal) and hybridization and plastid capture. Therefore, the uniparental-inherited plastome sequences are insufficient to disentangle relationships within a genus which has undergone recent rapid diversification. Our findings highlight a need for additional data from nuclear genome to resolve the relationships within Clade IV and more focused studies to assess the influences of multiple processes in the evolutionary history of Isodon. Nevertheless, the morphology of the shape and surface sculpture/indumentum of nutlets is of systematic importance that they can distinguish the four major clades of Isodon.
... The nuclear-encoded 35S ribosomal DNA (rDNA) cistron, comprising the 18S, 5.8S, and 25S rRNA genes, is the most easily accessible nuclear DNA region using genome skimming, i.e. shallow genomic sequencing (Straub et al. 2012). However, interpreting variation in 35S rDNA sequences and their internal transcribed spacers (ITS1 and ITS2) can be challenging due to their mode of evolution (Feliner and Rosselló 2007). In plants, the nuclear-encoded 35S rDNA is located in one or several loci where hundreds of copies follow each other in arrays that tend to be homogeneous at the intra-individual scale due to concerted evolution (Eickbush and Eickbush 2007). ...
Background and aims - Brachystegia is a species-rich tree genus found in tropical Africa and a typical element of Miombo woodlands, a widely distributed subtype of the Zambezian savanna. Plastid DNA was shown to be largely uninformative to assess species phylogenetic relationships due to widespread chloroplast capture among species. Here, we aim to assess the capacity of nuclear ribosomal DNA (rDNA) to clarify the phylogeny of Brachystegia species while accounting for intra-individual site polymorphisms (2ISPs), which are often present in rDNA and potentially phylogenetically informative.
Material and methods - Genome skimming sequencing on 47 samples representing 27 of the 29 currently recognized Brachystegia species, allowed us to retrieve complete nuclear ribosomal cistrons encoding for 18S, 5.8S, and 25S rRNA genes (35S rDNA). We reconstructed the Brachystegia phylogeny using Maximum Likelihood methods based on the standard substitution model or integrating 2ISPs (GENOTYPE implementation in RAxML-NG). We additionally tested the effect of partitioning the data (one partition for rDNA genes and one for the ITS1+ITS2). We also conducted network inferences (Neighbor-Net splits graph), as a strict bifurcative approach might not properly model topological uncertainty at shallow phylogenetic depth.
Key results - 2ISPs-aware and standard phylogenetic reconstructions are largely congruent. We identified several well-supported main clades clarifying the species relationships, including two clades of Miombo woodlands species. Miombo Group A includes species with ovoid to globose axillary dormant buds, while Miombo Group B species have flattened ones. Two morphologically close Brachystegia species (B. kennedyi and B. leonensis) found in Guineo-Congolian rain forests form also a robustly supported clade. 2ISPs coding allowed to identify an additional Guineo-Congolian clade (B. eurycoma and B. nigerica). Ribosomal DNA therefore proves more useful to explore the generic phylogeny than plastid DNA but the species relationships within and among the main clades remain poorly resolved, probably due to recent diversification and/or recurrent hybridization, so that the diversification of Brachystegia remains to be more properly characterised.
Conclusion - Nuclear and plastid phylogenetic reconstructions of Brachystegia species are discordant. Even if not well-resolved, rDNA phylograms and networks are characterized by taxonomic sorting, while we observe a strictly geographic sorting in the plastid dataset. Most of the species' relationships remain to be characterized using additional nuclear markers combined with in-depth morphological investigations.
... The chloroplast marker rbcL, as a conserved gene, is supposed to be a good marker for the resolution of higher taxonomic levels (i.e., families, subfamilies), while trnL-trnF might be more useful for finer scales (i.e., tribe, genus; Gielly & Taberlet, 1994). The nuclear marker ITS (similar to trnL-trnF) also has a high mutation frequency as well (Feliner & Rosselló, 2007). Nuclear markers are characterized by the presence of alleles from both parents, and the phylogeny therefore reflects a joint evolutionary pathway. ...
Juncaceae is a cosmopolitan family belonging to the cyperid clade of Poales together with Cyperaceae and Thurniaceae. These families have global economic and ethnobotanical significance and are often keystone species in wetlands around the world, with a widespread cosmopolitan distribution in temperate and arctic regions in both hemispheres. Currently, Juncaceae comprises more than 474 species in eight genera: Distichia, Juncus, Luzula, Marsippospermum, Oreojuncus, Oxychloë, Patosia and Rostkovia. The phylogeny of cyperids has not been studied before in a complex view based on most sequenced species from all three families. In this study, most sequenced regions from chloroplast (rbcL, trnL, trnL-trnF) and nuclear (ITS1-5.8S-ITS2) genomes were employed from more than a thousand species of cyperids covering all infrageneric groups from their entire distributional range. We analyzed them by maximum parsimony, maximum likelihood, and Bayesian inference to revise the phylogenetic relationships in Juncaceae and Cyperaceae. Our major results include the delimitation of the most problematic paraphyletic genus Juncus, in which six new genera are recognized and proposed to recover monophyly in this group: Juncus, Verojuncus, gen. nov., Juncinella, gen. et stat. nov., Alpinojuncus, gen. nov., Australojuncus, gen. nov., Boreojuncus, gen. nov. and Agathryon, gen. et stat. nov. For these genera, a new category, Juncus supragen. et stat. nov., was established. This new classification places most groups recognized within the formal Juncus clade into natural genera that are supported by morphological characters.
... This specimen conforms morphologically to S. adnatifolius rather than S. cocuccii, so its position in this clade is puzzling. Such position is based mainly on ITS sequence, which is known to have pseudogenes and multiple copies in some groups (Álvarez & Wendel, 2003;Nieto Feliner & Rosselló, 2007), though this is not the case in our study. ITS of S. adnatifolius have neither polymorphic bases nor missing data. ...
The genus Sclerophylax (Atropina clade, Solanaceae) has 14 species recognized to date, all except two endemic to Argentina. To clarify its phylogenetic relationships, molecular sequence data from the trnL‐trnF and the rpl32F‐trnL intergenic spacer regions, the waxy gene and the ITS region were employed in maximum likelihood and Bayesian inference analyses. Divergence times were estimated for the combined dataset using secondary calibrations. Ancestral distribution areas were reconstructed by Bayesian binary analyses and seven morphological traits (one continuous, six discrete) were traced. Sclerophylax is monophyletic and its sister clade is Lycium + Nolana. Sclerophylax is divided into S. sect. Caducifructus (1 sp.) and the monophyletic sect. Sclerophylax (11 spp.). Sclerophylax sect. Sclerophylax includes five well‐supported clades that are also supported by morphological synapomorphies; circumscription of some species remains uncertain, probably as a result of rapid diversifications, but four lineages are recognized by a combination of morphological traits. The ancestor of Sclerophylax was an annual, procumbent/decumbent plant with rhomboidal leaves and sessile, persistent, 2–3‐seeded fruits. The most variable traits were leaf shape and life form, while flower size was shown to be highly informative in diagnosing clades within Sclerophylax. The area of origin for Sclerophylax was the Prepuna biogeographical province, from which it has spread in recent geological times. A total of 12 species are recognized in Sclerophylax, with an updated synonymy, lectotypifications, and re‐circumscriptions provided.
... Therefore, as observed here, more incomplete lineage sorting is expected for ITS than for the chloroplast genes (Naciri and Linder, 2015). Another explanation for conflicting results between ITS and the plastid markers might be the possible incomplete ITS concerted evolution mode after recent hybridization events (Nieto Feliner and Rosselló, 2007) leading to unresolved topologies. In our case, ITS gave almost no resolution at the species level, except for three species of the Eastern Mediterranean basin that have PP values of 1, restricted distribution ranges and no within-species diversity as captured by ThetaS. ...
Recent divergence can obscure species boundaries among closely related taxa. Silene section Italicae (Caryophyllaceae) has been taxonomically controversial, with about 30 species described. We investigate species delimitation within this section using 500 specimens sequenced for one nuclear and two plastid markers. Despite the use of a small number of genes, the large number of sequenced samples allowed confident delimitation of 50% of the species. The delimitation of other species (e.g., Silene nemoralis, S. nodulosa and S. andryalifolia) was more challenging. We confirmed that seven of the ten chasmophyte species in the section are not related to each other but are, instead, genetically closer to geographically nearby species belonging to Italicae yet growing in open habitats. Adaptation to chasmophytic habitats therefore appears to have occurred independently, as a result of convergent evolution within the group. Species from the Western Mediterranean Basin showed more conflicting species boundaries than species from the Eastern Mediterranean Basin, where there are fewer but better-delimited species. Significant positive correlations were found between an estimation of the effective population size of the taxa and their extent of occurrence (EOO) or area of occupancy (AOO), and negative but non-significant correlations between the former and the posterior probability (PP) of the corresponding clades. These correlations might suggest a lower impact of incomplete lineage sorting in species with low effective population sizes and small distributional ranges compared with that in species inhabiting large areas. Finally, we confirmed that S. italica and S. nemoralis are distinct species, that S. nemoralis might furthermore include two different species and that S. velutina from Corsica and S. hicesiae from the Lipari Islands are sister species.
... This plastid gene codes for the large subunit ribulose-1,5-bisphosphate carboxylase/ oxygenase (RUBISCO), an enzyme essential for carbon fixation; thus, genetic variation within this gene could provide insights into local adaptation. The ITS region is also commonly sequenced in plant phylogenetic studies and has been shown to be effective for species discrimination (Feliner and Rosselló, 2007), even within genus Vanilla (Besse et al., 2021). ...
Premise:
Although vanilla is one of the best-known spices, there is only a limited understanding of its biology and genetics within Mexico, where its cultivation originated and where phenotypic variability is high. This study aims to augment our understanding of vanilla's genetic resources by assessing species delimitation and genetic, geographic, and climatic variability within Mexican cultivated vanilla.
Methods:
Using nuclear and plastid DNA sequence data from 58 Mexican samples collected from three regions and 133 ex situ accessions, we assessed species monophyly using phylogenetic analyses and genetic distances. Intraspecific genetic variation was summarized through the identification of haplotypes. Within the primarily cultivated species, Vanilla planifolia, haplotype relationships were further verified using plastome and rRNA gene sequences. Climatic niche and haplotype composition were assessed across the landscape.
Results:
Three species (Vanilla planifolia, V. pompona, and V. insignis) and 13 haplotypes were identified among Mexican vanilla. Within V. planifolia haplotypes, hard phylogenetic incongruences between plastid and nuclear sequences suggest past hybridization events. Eight haplotypes consisted exclusively of Mexican samples. The dominant V. planifolia haplotype occurred throughout all three regions as well as outside of its country of origin. Haplotype richness was found to be highest in regions around Papantla and Chinantla.
Conclusions:
Long histories of regional cultivation support the consideration of endemic haplotypes as landraces shaped by adaptation to local conditions and/or hybridization. Results may aid further genomic investigations of vanilla's genetic resources and ultimately support the preservation of genetic diversity within the economically important crop.
... Despite the fact that nrITS performed quite well (79%) in angiosperms, lower discrimination success was reported for Ranunculales (6.7%) and Laurales (14.3%) (China Plant BOL 2011). The inconsistencies in species ascriptions between nrITS may result from hybridisation and introgression or incomplete lineage sorting (Alvarea and Wendel 2003; Rieseberg et al. 2006;Feliner and Rosselló 2007). As a result, utilising plastid DNA markers alone may not be sufficient to discriminate between closelyrelated species. ...
We describe Polypleurum chinense , a new species of Podostemaceae from Yunxiao County, Fujian Province, China, based on morphological and molecular data and the genus Polypleurum is recorded here for the first time from China. Polypleurum chinense has a gross morphology similar to P. longistylosum , but it can be distinguished from the latter by its narrower roots, more numerous and longer leaves, shorter stigmas and more numerous ovules per locule. To distinguish the new Polypleurum species and study its phylogenetic position, its complete plastome was sequenced and characterised. The plastome is 132,110 bp in length, including a pair of inverted repeat regions (IRs) of 20,389 bp divided by the large single-copy (LSC) and small single-copy (SSC) regions of 79,022 bp and 12,310 bp, respectively. The plastome size of P. chinense is relatively smaller compared to most angiosperms due to the absence of the ycf 1 and ycf 2 genes in the IR regions. The phylogenetic analyses also strongly support the separation of the new species from other taxa.
... In addition, the Internal Transcribed Spacer region (ITS) of nuclear ribosomal DNA can also be used as a reference gene. ITS can be utilized across taxonomic diversity including fungi and lichenicolous fungi, unicellular, and pluricellular algae, non-arthropod invertebrates, arthropods, and vertebrates (Baldwin et al., 1995;Nieto Feliner & Rossell o, 2007). The genes carboxylase (rbcL) and maturase K (matK) from chloroplast are also utilized as a marker in plants. ...
The authentication of food is an urgent concern owing to the increasing population and direct consequences of food on public health. Food authentication using DNA and omics-based methods is gaining ground due to critical advantages notably in the areas of food adulteration in plant and animal-based food and feed products and in determining the quality of food and food spoilage. Besides, there is greater demand for the detection of genetically modified foods (GMOs) and the detection of aller-gens, toxins, and carcinogens like tobacco in the food. The present review summarizes the advanced DNA and omics-based methods (genomics, metabolomics, and proteomics) in the food industry. DNA-based methods rely on specific markers known as reference genes for food authentication. Lactic acid bacteria such as Strep-tococcus thermophiles are being used as probiotics for treating infectious diseases and as an essential culture in the fermentation industry. Novel methods like CRISPR-Cas have been recently introduced for the management of beneficial microorganisms relevant to food like probiotics and starter cultures by enhancing their production and restraining damaging pathogens. These methods can detect the source, presence of allergens, adulteration, and mislabeling in food products.
... Oil Palm Rubber Shrub (20) Oil Palm (19) Rubber (22) Shrub (16) rank), according to the intraspecific level of polymorphism of the barcode locus and availability of reference sequences. The low percentage of identity between the query sequence and the database sequences for the ITS2 region can be probably linked to the intrinsic characteristics of this ribosomal region, such as problems in amplification due to paralogs and pseudogenes [36], or lower universality and sequence quality compared with plastid regions [37]. In our study, the overlap in the plant families identified with the two techniques ranges from 22.2 to 27.8%. ...
Background
Intense conversion of tropical forests into agricultural systems contributes to habitat loss and the decline of ecosystem functions. Plant-pollinator interactions buffer the process of forest fragmentation, ensuring gene flow across isolated patches of forests by pollen transfer. In this study, we identified the composition of pollen grains stored in pot-pollen of stingless bees, Tetragonula laeviceps , via dual-locus DNA metabarcoding (ITS2 and rbcL ) and light microscopy, and compared the taxonomic coverage of pollen sampled in distinct land-use systems categorized in four levels of management intensity (forest, shrub, rubber, and oil palm) for landscape characterization.
Results
Plant composition differed significantly between DNA metabarcoding and light microscopy. The overlap in the plant families identified via light microscopy and DNA metabarcoding techniques was low and ranged from 22.6 to 27.8%. Taxonomic assignments showed a dominance of pollen from bee-pollinated plants, including oil-bearing crops such as the introduced species Elaeis guineensis (Arecaceae) as one of the predominant taxa in the pollen samples across all four land-use types. Native plant families Moraceae, Euphorbiaceae, and Cannabaceae appeared in high proportion in the analyzed pollen material. One-way ANOVA (p > 0.05), PERMANOVA (R² values range from 0.14003 to 0.17684, for all tests p-value > 0.5), and NMDS (stress values ranging from 0.1515 to 0.1859) indicated a lack of differentiation between the species composition and diversity of pollen type in the four distinct land-use types, supporting the influx of pollen from adjacent areas.
Conclusions
Stingless bees collected pollen from a variety of agricultural crops, weeds, and wild plants. Plant composition detected at the family level from the pollen samples likely reflects the plant composition at the landscape level rather than the plot level. In our study, the plant diversity in pollen from colonies installed in land-use systems with distinct levels of forest transformation was highly homogeneous, reflecting a large influx of pollen transported by stingless bees through distinct land-use types. Dual-locus approach applied in metabarcoding studies and visual pollen identification showed great differences in the detection of the plant community, therefore a combination of both methods is recommended for performing biodiversity assessments via pollen identification.
... 5S and 35S rDNAs are most often used as chromosomal markers, as they are highly repetitive, arranged in tandem arrays and highly conserved in the DNA sequence of the coding regions [19][20][21][22][23]. The non-transcribed spacer (NTS) of 5S rDNA and the internal transcribed spacer of 35SrDNA (nrITS 1 and 2) evolve much faster than their coding regions and are thus often used as molecular markers in phylogenetic analyses [24]. In most higher plants, the number and localisation of the 35S and 5S rDNA loci are unlinked [25]. ...
Although Crepis was the first model plant group in which chromosomal changes were considered to play an important role in speciation, their chromosome structure and evolution have been barely investigated using molecular cytogenetic methods. The aim of the study was to provide a better understanding of the patterns and directions of Crepis chromosome evolution, using comparative analyses of rDNA loci number and localisation. The chromosome base number and chromosomal organisation of 5S and 35S rDNA loci were analysed in the phylogenetic background for 39 species of Crepis, which represent the evolutionary lineages of Crepis sensu stricto and Lagoseris, including Lapsana communis. The phylogenetic relationships among all the species were inferred from nrITS and newly obtained 5S rDNA NTS sequences. Despite high variations in rDNA loci chromosomal organisation, most species had a chromosome with both rDNA loci within the same (usually short) chromosomal arm. The comparative analyses revealed several independent rDNA loci number gains and loci repositioning that accompanied diversification and speciation in Crepis. Some of the changes in rDNA loci patterns were reconstructed for the same evolutionary lineages as descending dysploidy.
... As rooted by two Mahonia species, the clades resulting from phylogenetic analyses based on both the plastome (ptDNA) and the nrDNA sequences are depicted in Fig. 2. Given that the low support values of the major clades in the nrDNA phylogram, the topological conflict between the two datasets is most likely due to the influence of concerted evolution of the nrITS sequence (Nieto Feliner and Rossello, 2007), as shown in previous phylogenetic studies of Berberis (Kim et al., 2004b;Yu and Chung, 2017). ...
Though Berberis (Berberidaceae) is widely distributed across the Eurasian landmass it is most diverse in the Himalaya–Hengduan Mountain (HHM) region. There are more than 200 species in China where it is one of the most common mountain shrubs. The study on the taxonomy and evolution of Berberis in this region can thus provide an important insight into the origin and diversification of its flora. A prerequisite to this is mapping and describing the various species of Berberis in the region – a task that despite recent progress is by no means complete. It is clear that in China there may be a significant number of species still to be described and that even with published species much about their distribution remains to be discovered. As a contribution to the first of these tasks seven new species from the northern Hengduan Mountain of N. Sichuan and S. Qinghai: B. chinduensis, B. degexianensis, B. jiajinshanensis, B. jinwu, B. litangensis, B. longquensis and B. riparia, are described here. Differences in overall morphology and especially in floral structures with each other and with similar species of Berberis in the same region are presented. The report is the result of phylogenetic analyses based on plastome and partial nrDNA sequences of both the seven proposed new species and a significant number of similar species already published. Provisional conclusions as to the insights provides on the history of the genetic divergence are discussed.
This article studies the morphological parameters of vegetative and generative organs of different age groups of Crataegus ambigua from four populations in Western Karatau (Mangistau region, Kazakhstan). In this study, we examined four populations: Sultan Epe, Karakozaiym, Emdi-korgan, and Samal, all located in various gorges of Western Karatau. Several phylogenetic inference methods were applied, using six genetic markers to reconstruct the evolutionary relationships between these populations: atpF-atpH, internal transcribed spacer (ITS), matK, psbK-psbI, rbcL, and trnH-psbA. We also used a statistical analysis of plants' vegetative and generative organs for three age groups (virgin, young, and adult generative). According to the age structure, Samal has a high concentration of young generative plants (42.3%) and adult generative plants (30.9%). Morphological analysis showed the significance of the parameters of the generative organs and separated the Samal population into a separate group according to the primary principal component analysis (PCoA) coordinates. The results of the floristic analysis showed that the Samal populations have a high concentration of species diversity. Comparative dendrograms using UPGMA (unweighted pair group method with arithmetic mean) showed that information gleaned from genetic markers and the psbK-psbI region can be used to determine the difference between the fourth Samal population and the other three.
The Dombeya Cav. genus (Malvaceae, ex-Sterculiaceae) comprises around 150 emblematic species from the western Indian Ocean. This genus is found in Africa, the Comoros, the Mascarenes and in many habitats of Madagascar; in each of these areas of distribution, Dombeya has a specific endemism rate of almost 100%. This ecological and specific diversity seems to be linked to major morphological variations and original life-history traits. With an estimated age of less than 10 Ma, this specific diversity and morpho-ecological complexity could be the result of rapid radiation. The taxonomy of the genus is still debated and still requires major taxonomic revision.
Our study is part of this approach and focuses on a particular section of the genus: the autonymic section Dombeya, which occurs in all the genus' distribution areas. The section is characterised by a unique feature within the subfamily: a biparous cyme inflorescence with scorpioid terminal units. Using morphometric, morphological and genetic approaches, we were able to test species hypotheses previously formulated in the literature. The floral organs have proved to be a good object for differentiating taxonomic entities: three major geographical groups, African, Madagascan and Mascarenan, have thus been defined. A molecular approach using genome skimming was then used to extract the largest molecular matrix produced in the genus, which was used for phylogenetic analyses. By combining morphometry, a global morphology approach and phylogeny at the section level, two distinct taxonomic entities emerged within Dombeya sect. Dombeya. The monophyly of the section is not confirmed and it seems that the scorpioid biparous cyme character was acquired twice independently in Africa and Madagascar. The Malagasy species appear to be closely related to the Mascarenan species, which are thought to have derived from them, and form at least 3 distinct taxonomic units, the delimitation of which is debated.
Although both are salient features of genomes, at first glance ribosomal DNAs (rDNAs) and transposable elements (TEs) are genetic elements with not much in common: whereas rDNAs are mainly viewed as housekeeping genes that uphold all prime genome functions, TEs are generally portrayed as selfish and disruptive. These opposing characteristics are also mirrored in other attributes: organization in tandem (rDNAs) versus organization in a dispersed manner (TEs); evolution in a concerted manner (rDNAs) versus evolution by diversification (TEs); and activity that prolongs genomic stability (rDNAs) versus activity that shortens it (TEs). Re-visiting relevant instances in which rDNA-TE interactions have been reported, we note that both repeat types share at least four structural and functional hallmarks: (1) they are repetitive DNAs that shape genomes in evolutionary timescales; (2) they exchange structural motifs and can enter co-evolution processes; (3) they are tightly controlled genomic stress sensors playing key roles in senescence/aging; and (4) they share common epigenetic marks such as DNA methylation and histone modification. Here, we give an overview of the structural, functional and evolutionary characteristics of both rDNAs and TEs, discuss their roles and interactions, and highlight trends and future directions as we move forward in understanding rDNA-TE associations.
We present a first treatment of the Tortella tortuosa complex for Europe. We analysed molecular relationships based on the nuclear ITS and the plastid atpB-rbcL and rps4 in a network context and thereafter characterized the identified entities by their morphology. We found eight morphologically and molecularly distinct entities at the species level, which are also supported in ASAP analyses of the molecular data; one species includes two varieties. In some cases, nuclear and plastid data suggest different relationships and we found a few likely recent hybrid collections. To the main characters of taxonomic importance belong stem anatomy, leaf shape and papillosity. We describe three species as new: T. commutata (a widespread plant; including the new var. valida), T. dolomitica (known only from the Alps) and T. splendida (an Arctic-alpine element), replacing T. arctica auct. For T. angustifolia and T. robusta (both montane) new combinations at the species level are provided. Tortella bambergeri (a submediterranean element), T. fleischeri (an Alpine element, recurring in Scotland) and T. tortuosa s. str. (widespread) complete this informal group of morphologically similar and partly related species. The species differ in ecological requirements and distribution areas, although mixed stands of two or three species are frequent. The area richest in species in Europe is the Alps with all eight species, whereas we found only four from Scandinavia.
The ribosomal RNA genes (rDNA) are universal genome components with a housekeeping function, given the crucial role of ribosomal RNA in the synthesis of ribosomes and thus for life-on-Earth. Therefore, their genomic organization is of considerable interest for biologists, in general. Ribosomal RNA genes have also been largely used to establish phylogenetic relationships, and to identify allopolyploid or homoploid hybridization.Here, we demonstrate how high-throughput sequencing data, through graph clustering implemented in RepeatExplorer2 pipeline ( https://repeatexplorer-elixir.cerit-sc.cz/galaxy/ ), can be helpful to decipher the genomic organization of 5S rRNA genes. We show that the linear shapes of cluster graphs are reminiscent to the linked organization of 5S and 35S rDNA (L-type arrangement) while the circular graphs correspond to their separate arrangement (S-type). We further present a simplified protocol based on the paper by (Garcia et al., Front Plant Sci 11:41, 2020) about the use of graph clustering of 5S rDNA homoeologs (S-type) to identify hybridization events in the species history. We found that the graph complexity (i.e., graph circularity in this case) is related to ploidy and genome complexity, with diploids typically showing circular-shaped graphs while allopolyploids and other interspecific hybrids display more complex graphs, with usually two or more interconnected loops representing intergenic spacers. When a three-genomic comparative clustering analysis from a given hybrid (homoploid/allopolyploid) and its putative progenitor species (diploids) is performed, it is possible to identify the corresponding homoeologous 5S rRNA gene families, and to elucidate the contribution of each putative parental genome to the 5S rDNA pool of the hybrid. Thus, the analysis of 5S rDNA cluster graphs by RepeatExplorer, together with information coming from other sources (e.g., morphology, cytogenetics) is a complementary approach for the determination of allopolyploid or homoploid hybridization and even ancient introgression events.
Barcodes are conserved sequences of genomic, plastid and mitochondrial DNA that can be utilized to uniquely identify an unidentified specimen to its species when conventional identification methods are inapplicable. Among prokaryotic and eukaryotic species, nuclear ribosomal internal transcribed spacer (ITS) sections are one of the most often utilized DNA markers in DNA barcoding and phylogenetic research. In addition to the ribosomal genes, the plastid genes are the most suitable for identifying plant species. The Aksu-Zhabagly Nature Reserve is the oldest nature reserve in Central Asia and is home to 1,312 vascular plant species, 44 of which are categorized as threatened or endangered in Kazakhstan's red data book. In this study, a collection of specimens of uncommon tulip species was compiled, along with their morphological identification and DNA barcoding. The ITS region and parts of the matK and ycf1b genes of tulip plastid DNA were sequenced. The evolutionary link between species of tulips was investigated. Phylogenetic study predicted two Tulipa subclades. Tulipa species have substantially preserved MatK genes. Tulips' ycf1b gene has evolved more slowly than other Liliaceae family members. Nuclear and plastid DNA sequences investigated Tulipa species evolutionary relationships. The findings about the ITS region of nuclear DNA were more definite. Overall, our work shows that genetic data will be important in determining species concepts in this genus, however, even with a molecular perspective pulling apart closely related taxa can be extremely challenging.
Background and aims
Plants have evolved an unrivalled diversity of reproductive strategies, including variation in the degree of sexual versus clonal reproduction. This variation has important effects on the dynamics and genetic structure of populations. We examined the association between large-scale variation in reproductive patterns and intraspecific genetic diversity in two moss species where sex is manifested in the dominant haploid generation and sex expression is irregular. We predicted that in regions with more frequent realised sexual reproduction, populations should display less skewed sex ratios, more often express sex, and have higher genetic diversity than in regions with largely clonal reproduction.
Methods
We assessed reproductive status and phenotypic sex in the dioicous long-lived Drepanocladus trifarius and D. turgescens, in 248 and 438 samples across two regions in Scandinavia with frequent or rare, respectively, realised sexual reproduction. In subsets of the samples, we analysed genetic diversity using nuclear and plastid sequence information and identified sex with a sex-specific molecular marker in non-reproductive samples.
Key results
Contrary to our predictions, sex ratios did not differ between regions, genetic diversity did not differ in D. trifarius and was higher in the region with rare sexual reproduction in D. turgescens. Supporting our predictions, relatively more samples expressed sex in D. trifarius in the region with frequent sexual reproduction. Overall, samples were mostly female. Degree of sex expression and genetic diversity differed between sexes.
Conclusions
Sex expression levels, regional sex ratios and genetic diversity were not directly associated with regional frequency of realised sexual reproduction, and relationships and variation patterns differed between species. We conclude that a combination of species-specific life histories such as longevity, overall degree of successful sexual reproduction and recruitment, and historic factors, are important to explain this variation. Our data on haploid-dominated plants significantly complement plant reproductive biology.
This study aimed to determine the pesticide use in grain maize production in the Yunak district of Konya province and the attitudes and behaviours of producers, as well as their knowledge levels about pesticide use. The model structure designed in the study; was prepared by benefiting from the questionnaire study developed by Günay, (2020). The questionnaire prepared by the researcher was adapted to the regional conditions. Based on demographic variables in the model, it was investigated whether there were statistically significant differences in pesticide use awareness, attitudes and behaviours on pesticide use, and agricultural pesticide supply characteristics scales.
Conservation decision‐making and prioritization of management actions for taxa at high risk of extinction requires a clear understanding of systematic relationships and species boundaries. Taxonomic uncertainty surrounds two threatened species of native olive (genus Notelaea) endemic to Australia. Notelaea ipsviciensis is known from only one small population and is listed as critically endangered under Australian and Queensland legislation, while N. lloydii is listed as vulnerable due to its restricted distribution in south‐east Queensland, Australia. Notelaea ipsviciensis occurs in sympatry with N. lloydii, at the only site where it is found, and exhibits intermediate morphology between N. lloydii and N. ovata raising speculation that it is a natural hybrid of the two. To address this uncertainty, we have reconstructed the molecular phylogeny of the genus Notelaea using both single‐gene sequencing and Single Nucleotide Polymorphism (SNP) data. While the chloroplast DNA markers were not found to be informative, the genome‐wide SNPs used in this study have unequivocally resolved the long‐standing question on the hybrid origin of N. ipsviciensis. Results of the phylogenetics and hybrid analyses of SNP data show N. ipsviciensis is a natural hybrid of N. lloydii and N. ovata, and the taxon's circumscription needs to be re‐evaluated. Our results also revealed unclear species boundaries among numerous other Notelaea species, including the threatened N. lloydii, indicating that further investigation and taxonomic revision may be necessary. This article is protected by copyright. All rights reserved.
The evolution of chromosome number and ribosomal DNA (rDNA) loci number and localisation were studied in Onobrychis Mill. Diploid and tetraploid species, as well as two basic chromosome numbers, x = 7 and x = 8, were observed among analysed taxa. The chromosomal distribution of rDNA loci was presented here for the first time using fluorescence in situ hybridisation (FISH) with 5S and 35S rDNA probes. Onobrychis species showed a high polymorphism in the number and localisation of rDNA loci among diploids, whereas the rDNA loci pattern was very similar in polyploids. Phylogenetic relationships among the species, inferred from nrITS sequences, were used as a framework to reconstruct the patterns of basic chromosome number and rDNA loci evolution. Analysis of the evolution of the basic chromosome numbers allowed the inference of x = 8 as the ancestral number and the descending dysploidy and polyploidisation as the major mechanisms of the chromosome number evolution. Analyses of chromosomal patterns of rRNA gene loci in a phylogenetic context resulted in the reconstruction of one locus of 5S rDNA and one locus of 35S rDNA in the interstitial chromosomal position as the ancestral state in this genus.
The authentication of food is an urgent concern owing to the increasing population and direct consequences of food on public health. Food authentication using DNA and omics‐based methods is gaining ground due to critical advantages notably in the areas of food adulteration in plant and animal‐based food and feed products and in determining the quality of food and food spoilage. Besides, there is greater demand for the detection of genetically modified foods (GMOs) and the detection of allergens, toxins, and carcinogens like tobacco in the food. The present review summarizes the advanced DNA and omics‐based methods (genomics, metabolomics, and proteomics) in the food industry. DNA‐based methods rely on specific markers known as reference genes for food authentication. Lactic acid bacteria such as Streptococcus thermophiles are being used as probiotics for treating infectious diseases and as an essential culture in the fermentation industry. Novel methods like CRISPR‐Cas have been recently introduced for the management of beneficial micro‐organisms relevant to food like probiotics and starter cultures by enhancing their production and restraining damaging pathogens. These methods can detect the source, presence of allergens, adulteration, and mislabeling in food products. Around the world, food diversity and complexity is on steady increase, generating a stable demand for food authentication. Other than chemical methods of testing, biological methods are also gaining ground with the advancement of modern biology. The present review enlists the recent advancement in commercially viable “OMICS”‐based food authentication using small‐molecule‐based, protein‐based, and nucleic acid‐based techniques.
Background: Cercospora leaf spot (CLS) is one of the major diseases causing a major loss in production of mung bean of up to 60%. Although it is cultivated all round the world, its major contribution is attributed to the Asian continent especially the countries of Indian subcontinents which accounts for about 90% of the world’s mungbean production. Due to its high nutritional content, mung bean is a major contributor to the Nation’s economy. The present study was to isolate and characterize the new isolates of Cercospora canescens collected from coastal belts of Odisha, India. Methods: Surveys were conducted in the mung bean growing fields of Bhubaneswar (20.26°N, 85.8°E) and Berhampur (19.36°N, 84.77°E), coastal regions of Odisha during 2018-20. New Cercospora canescens isolates were collected and characterised through morphological, biochemical and molecular basis. Result: New Cercospora isolates were characterized on the basis of morphological, biochemical and molecular analysis to understand the gravity of the disease. Pathogenicity tests were conducted by artificial inoculation to identify the resistant and susceptible mung bean genotypes. On the basis of molecular analysis (internal transcribed spacer (ITS) region), there was a difference between the two isolates and forming different clades. These isolates were sequenced having the NCBI Acc. No. MZ475049 and MZ475050. Both isolates are different from each other because of transitional substitution of thymine and cytosine. The findings will be a complementary contribution for conservation and better management strategies of CLS disease in mung bean.
The geographical distributions of the two cryptic species of the wetland moss Hamatocaulis vernicosus were mapped for western, central and northern Europe, based on identifications of the two by the nuclear ITS1 + 2 and the plastid rpl16 and trnL–trnF. The distributions of the two cryptic species overlap to a large extent. However, in the west and south-west only cryptic species 1 is present, whereas in the boreal north only cryptic species 2 occurs, which agrees with its distribution in Scandinavia. Despite these differences in distribution, no differences between the two cryptic species were revealed in habitat water chemistry, elevation distribution or climatic niches. The difference in distribution therefore suggests that cryptic species 1 could have survived the glacial period in southern Europe and cryptic species 2 in northern or eastern Europe. However, the studied molecular markers did not reveal geographical patterns suggesting origins in different glacial refugia. Although populations of both cryptic species have decreased in large portions of western Europe, a significantly negative Tajima’s D may reflect the long-term expansion south of Scandinavia since the glacial bottleneck, potentially correlated with the expansion of earlier extensive agricultural management of wetlands.
Tragopogon mirus is one of only a few polyploids whose time of origin is known with a high degree of certainty. Previous studies of morphological, karyotypic, and electrophoretic variation suggested multiple origins of allotetraploidy in T. mirus from the Palouse region of eastern Washington and adjacent Idaho. Restriction site analysis of the 17S-25S ribosomal RNA genes (rDNA) was used to test for multiple origins of T. mirus. The rDNA restriction profiles of six populations of T. mirus were compared with those of eight populations of T. dubius and six of T. porrifolius, the diploid progenitors. Populations of T. mirus combine the rDNAs of T. dubius and T. porrifolius, confirming the proposed origin of the tetraploid. Furthermore, the incorporation of restriction site polymorphisms of T. porrifolius into different populations of T. mirus demonstrates at least two independent origins of T. mirus. Although T. mirus often occurs with its diploid progenitors, these mixed populations do not necessarily reflect sites of origin of the tetraploid. In the three mixed populations examined, the diploids could not have produced the tetraploid plants found at the same site. Extensive dispersal of the tetraploid and fluctuating population sizes coupled with local extinctions and subsequent recolonization could explain the discordance between rDNA genotypes and their distributions. Thus, population dynamics in Tragopogon may obscure the sites of independent origin of T. mirus. Finally, although multiple origins of polyploid species have been reported frequently, few studies have demonstrated multiple polyploidization events on a geographic scale of a few kilometers.
The internal transcribed spacer (ITS) region of 18S-26S nuclear ribosomal DNA (nrDNA) has proven to be a useful source of characters for phylogenetic studies in many angiosperm families. The two spacers of this region, ITS-1 and ITS-2 (each <300 bp), can be readily amplified by PCR and sequenced using universal primers, even from DNAs of herbarium specimens. Despite high copy numbers of both spacers, the near uniformity of ITS paralogues, attributed to rapid concerted evolution, allows direct sequencing of pooled PCR products in many species. Divergent paralogues, where detected, require cloning, but may offer a means of obtaining multiple estimates of organismal relationships and of determining placement of the root in a phylogeny independent of outgroup considerations. In reported studies, variation between ITS sequences is mostly attributable to point mutations. A relatively minor proportion of sites is affected by insertions or deletions (indels) among sequences that are similar enough to have retained sufficient signal for phylogenetic analysis. Within these limits, sequence alignment is generally unambiguous except in small regions of apparently lower structural constraint. Phylogenetic analyses of combined data sets from both spacers, where examined, yield trees with greater resolution and internal support than analyses based on either spacer alone. This beneficial effect of simultaneous analysis is not surprising based on the low number of useful characters in each spacer. This effect also suggests high complementarity of spacer data, in accord with similarity in size, sequence variability, and G + C content of ITS-1 and ITS-2 in most investigated groups of closely related angiosperms. Nonindependent evolution of ITS sites involved in intraspacer RNA base-pairing may occur, given possible functional constraints, but preliminary secondary structure analyses of ITS-2 in Calycadenia (Asteraceae) show no definite evidence of compensatory spacer mutations. As expected, levels of ITS sequence variation suitable for phylogenetic analysis are found at various taxonomic levels within families, depending on the lineage. The apparent rates of ITS molecular evolution are roughly correlated with plant life-form, as with chloroplast DNA (cpDNA) data, but reasons for this observation are unclear. ITS characters have improved our understanding of angiosperm phylogeny in several groups by (1) corroborating earlier unexpected findings, (2) resolving conflicts between other data sets, (3) improving resolution of species relationships, or (4) providing direct evidence of reticulate evolution. Hybridization or sorting of ancestral polymorphism in a lineage can complicate interpretation of trees based on any type of evolutionary evidence, including ITS or cpDNA sequences, particularly in the absence of at least one independent phylogenetic data set from the same organisms. The need for phylogenetic markers from the nuclear genome, to complement the rapidly growing body of cpDNA data, makes the ITS region a particularly valuable resource for plant systematists.
Molecular data have had a profound impact on the field of plant systematics, and the application of DNA-sequence data to phylogenetic ,problems ,is now ,routine. The majority of data ,used in plant molecular phylogenetic studies derives from chloroplast DNA and nuclear rDNA, while the use of low-copy nuclear genes has not been widely adopted. This is due, at least in part, to the greater difficulty of isolating and characterising low-copy nuclear genes relative to chloroplast and rDNA sequences ,that are readily amplified with universal primers. The higher level of sequence variation characteristic of low-copy nuclear genes, however, often compensates for the experimental effort required to obtain them. In this review, we briefly discuss the strengths and limitations of chloroplast and rDNA sequences, and then focus our attention on the use of low-copy nuclear sequences. Advantages of low-copy ,nuclear sequences ,include a higher ,rate of evolution ,than for organellar sequences, the potential to accumulate datasets from multiple unlinked loci, and bi-parental inheritance. Challenges intrinsic to the ,use of low-copy ,nuclear sequences ,include distinguishing orthologous ,loci from divergent paralogous loci in the same gene family, being mindful of the complications arising from concerted evolution or recombination among paralogous sequences, and the presence of intraspecific, intrapopulational and intraindividual polymorphism. Finally, we provide a detailed protocol for the isolation, characterisation and use of low-copy nuclear sequences for phylogenetic studies. SB03 015 Us e of n uclear ge nesi n pl antphyl ogeny R. L. S ma ll etal .
Molecular data have had a profound impact on the field of plant systematics, and the application of DNA-sequence data to phylogenetic problems is now routine. The majority of data used in plant molecular phylogenetic studies derives from chloroplast DNA and nuclear rDNA, while the use of low-copy nuclear genes has not been widely adopted. This is due, at least in part, to the greater difficulty of isolating and characterising low-copy nuclear genes relative to chloroplast and rDNA sequences that are readily amplified with universal primers. The higher level of sequence variation characteristic of low-copy nuclear genes, however, often compensates for the experimental effort required to obtain them. In this review, we briefly discuss the strengths and limitations of chloroplast and rDNA sequences, and then focus our attention on the use of low-copy nuclear sequences. Advantages of low-copy nuclear sequences include a higher rate of evolution than for organellar sequences, the potential to accumulate datasets from multiple unlinked loci, and bi-parental inheritance. Challenges intrinsic to the use of low-copy nuclear sequences include distinguishing orthologous loci from divergent paralogous loci in the same gene family, being mindful of the complications arising from concerted evolution or recombination among paralogous sequences, and the presence of intraspecific, intrapopulational and intraindividual polymorphism. Finally, we provide a detailed protocol for the isolation, characterisation and use of low-copy nuclear sequences for phylogenetic studies. SB03 015 Us e of nuclear genes in pl ant phylogeny R. L. S ma ll et al .
Molecular data have had a profound impact on the field of plant systematics, and the application of DNA-sequence data to phylogenetic problems is now routine. The majority of data used in plant molecular phylogenetic studies derives from chloroplast DNA and nuclear rDNA, while the use of low-copy nuclear genes has not been widely adopted. This is due, at least in part, to the greater difficulty of isolating and characterising low-copy nuclear genes relative to chloroplast and rDNA sequences that are readily amplified with universal primers. The higher level of sequence variation characteristic of low-copy nuclear genes, however, often compensates for the experimental effort required to obtain them. In this review, we briefly discuss the strengths and limitations of chloroplast and rDNA sequences, and then focus our attention on the use of low-copy nuclear sequences. Advantages of low-copy nuclear sequences include a higher rate of evolution than for organellar sequences, the potential to accumulate datasets from multiple unlinked loci, and bi-parental inheritance. Challenges intrinsic to the use of low-copy nuclear sequences include distinguishing orthologous loci from divergent paralogous loci in the same gene family, being mindful of the complications arising from concerted evolution or recombination among paralogous sequences, and the presence of intraspecific, intrapopulational and intraindividual polymorphism. Finally, we provide a detailed protocol for the isolation, characterisation and use of low-copy nuclear sequences for phylogenetic studies. S B 0 3 0 1 5 U s e o f n u c l e a r g e n e s i n p l a n t p h y l o g e n y R . L . S ma l l e t a l . Introduction The impact of molecular data on the field of plant systematics can hardly be overstated. In combination with explicit methods for phylogenetic analysis, molecular data have reshaped concepts of relationships and circumscriptions at all levels of the taxonomic hierarchy (Qiu et al. 1999; Soltis et al. 1999; Crawford 2000). As molecular phylogenetic studies have accumulated, it has become apparent that different molecular tools are required for different questions because of varying rates of sequence evolution among genomes, genes and gene regions. The choice of molecular tool is of paramount importance to ensure that an appropriate level of variation is recovered to answer the phylogenetic question at hand. Nonetheless, the plant systematics community is using only a small fraction of the available molecular tools. The preponderance of molecular data applied to plant systematics problems come from two sources: chloroplast DNA (cpDNA) or nuclear ribosomal DNA (rDNA). While the contributions of cpDNA and rDNA to plant systematics are undeniable, reliance on these tools to the exclusion of other, perhaps more appropriate, tools is pervasive (Alvarez and Wendel 2003). Alternatives to cpDNA and rDNA include both mitochondrial (mtDNA) and nuclear (nDNA) sequences other than rDNA. Because of its generally slow rate of sequence evolution and fast rate of structural evolution (Palmer 1992; Palmer et al. 2000), mtDNA generally has been ignored by plant systematists as a potential source of data (but see e.g. Qiu et al. 1998, 1999; Freudenstein and Chase 2001; Anderberg et al. 2002; Sanjur et al. 2002). For this reason, mtDNA will not be considered further in this review. Nuclear sequences other than rDNA represent most of the DNA contained in any given cell, comprising both high-copy repetitive DNA (e.g. transposons, centromeric and telomeric repeats), and low-to moderate-copy DNA elements, including the majority of genes. The evolutionary dynamics of these two classes of DNA can be dramatically different. For example, repetitive DNA may experience non-Mendelian transmission, be subject to concerted evolution, and/or be mobile within a genome (e.g.
The very recent allopolyploid speciation of Cardamine insueta and Cardamine schulzii is well documented. We used this system for a further understanding of the evolution of the internal transcribed spacers
(ITS) of nuclear ribosomal DNA in recently formed hybrids. The ITS sequencing of the two parent species and the alloploid
offspring suggests a synopsis of the types of ITS evolution, reported so far in the literature. We detected homogenization
to one parental ITS type with a very strong bias to the maternal sequence. Nevertheless, maintainance of both parental ITS
sequences in the alloploids was also recorded. Our data suggest: (1) rapid genomic change in newly formed alloploids, and
(2) a multiple origin of C. insueta and C. schulzii.
Chloroplasts and mitochondria originated as bacterial symbionts. The larger, host cells acquired genetic information from their prokaryotic guests by lateral gene transfer. The prokaryotically-derived genes of the eukaryotic cell nucleus now function to encode the great majority of chloroplast and mitochondrial proteins, as well as many proteins of the nucleus and cytosol. Genes are copied and moved between cellular compartments with relative ease, and there is no established obstacle to successful import of any protein precursor from the cytosol. Yet chloroplasts and mitochondria have not abdicated all genes and gene expression to the nucleus and to cytosolic translation. What, then, do chloroplast- and mitochondrially-encoded proteins have in common that confers a selective advantage on the cytoplasmic location of their genes? The proposal advanced here is that co-location of chloroplast and mitochondrial genes with their gene products is required for rapid and direct regulatory coupling. Redox control of gene expression is suggested as the common feature of those chloroplast and mitochondrial proteins that are encoded in situ. Recent evidence is consistent with this hypothesis, and its underlying assumptions and predictions are described.
Previous analyses of species relationships and polyploid origins in the mimosoid legume genus Leucaena have used chloroplast DNA (cpDNA) restriction site data and morphology. Here we present an analysis of a new DNA sequence data set for the nuclear ribosomal DNA (nrDNA) 5.8S subunit and flanking ITS 1 and ITS 2 spacers, a simultaneous analysis of the morphology, ITS and cpDNA data sets for the diploid species, and a detailed comparison of the cpDNA and ITS gene trees, which include multiple accessions of all five tetraploid species. Significant new insights into species relationships and polyploid origins, including that of the economically important tropical forage tree L. leucocephala, are discussed. Heterogeneous ITS copy types, including 26 putative pseudogene sequences, were found within individuals of four of the five tetraploid and one diploid species. Potential pseudogenes were identified using two pairwise comparison approaches as well as a tree-based method that compares observed and expected proportions of total ITS variation contributed by the 5.8S subunit optimized onto branches of one of the ITS gene trees. Inclusion of putative pseudogene sequences in the analysis provided evidence that some pseudogenes in allopolyploid L. leucocephala are not the result of post-allopolyploidization gene silencing, but were inherited from its putative diploid maternal progenitor L. pulverulenta.
Taxonomic consensus is lacking on the Oxytropis arctica and O. campestris species complexes, two polyploid complexes found in the interior and arctic areas of Alaska. One classification has emphasized flower size, whereas flower color is considered a key diagnostic character in another classification. Our analyses of internal transcribed spacer (ITS) sequences and random amplified polymorphic DNA (RAPD) markers provided no support for either classification system. The trees generated from ITS sequences and the phenogram derived from RAPD markers suggest that most recognized taxa in the two complexes are probably polyphyletic, including O. arctica var. barnebyana, which is listed as threatened in Alaska. The only consistent pattern detected by both types of molecular markers was a geographic split dividing the northeastern arctic populations from most other populations (48.60-55.03% in AMOVA analyses). This genetic subdivision probably reflects a Pleistocene barrier formed by the northern coastal ice shield. Our molecular data, in conjunction with the previously reported variation of ploidy levels in these groups, suggest a scenario of recent and multiple origins of polyploidy. It is possible that most Alaskan populations of these two complexes are best referred to as a single taxonomic species despite morphological differentiation within the complexes.
Exploration of the relationship between gene trees and their containing species trees leads to consideration of how to reconstruct species trees from gene trees and of the concept of phylogeny as a cloud of gene histories. When gene copies are sampled from various species, the gene tree relating these copies might disagree with the species phylogeny. This discord can arise from horizontal transfer (including hybridization), lineage sorting, and gene duplication and extinction. Lineage sorting could also be called deep coalescence , the failure of ancestral copies to coalesce (looking backwards in time) into a common ancestral copy until deeper than previous speciation events. These events depend on various factors; for instance, deep coalescence is more likely if the branches of the species tree are short (in generations) and wide (in population size). A similar dependence on process is found in historical biogeography and host-parasite relationships. Each of the processes of discord could yield a different parsimony criterion for reconstructing the species tree from a set of gene trees: with horizontal transfer, choose the species tree that minimizes the number of transfer events; with deep coalescence, choose the tree minimizing the number of extra gene lineages that had to coexist along species lineages; with gene duplication, choose the tree minimizing duplication and/or extinction events. Maximum likelihood methods for reconstructing the species tree are also possible because coalescence theory provides the probability that a particular gene tree would occur given a species tree (with branch lengths and widths specified). In considering these issues, one is provoked to reconsider precisely what is phylogeny. Perhaps it is misleading to view some gene trees as agreeing and other gene trees as disagreeing with the species tree; rather, all of the gene trees are part of the species tree, which can be visualized like a fuzzy statistical distribution, a cloud of gene histories. Alternatively, phylogeny might be (and has been) viewed not as a history of what happened, genetically, but as a history of what could have happened, i.e., a history of changes in the probabilities of inter-breeding.
The nucleolus organizing regions (NORs) on the short arms of chromosomes 1A(m) and 5A(m) of diploid wheat, Triticum monococcum L., are at the most distal loci in the linkage maps of these two chromosome arms. This distal location differs from the interstitial location of the Nor loci on chromosome arms 1BS of tetraploid Triticum turgidum L. and hexaploid T. aestivum L., 5DS of T. aestivum and diploid Ae. tauschii Coss., and 5HS of barley. Moreover, the barley 5HS locus is at a different location than the 5DS locus. However, other markers, including the centromeres, are colinear. These findings showed that the major Nor loci have repeatedly changed position in the chromosome arms during the radiation of species in the tribe Triticeae without rearrangements of the linkage groups. It is suggested that Nor loci may change position via dispersion of minor loci, that are shown here to exist in the T. monococcum genome, magnification of gene copy numbers in these minor loci, and subsequent deletion of the original major loci. Implications of these findings for the use of rRNA nucleotide sequences in phylogenetic reconstructions are pointed out.
I examined three aspects of the cladistic treatment of a set of 17 F1 hybrids of known parental origin: (1) impact of hybrids on consistency index (CI) and number of most parsimonious trees (Trees), (2) placement of hybrids in cladograms, and (3) impact of hybrids on hypotheses of relationship among species. The hybrids were added singly and in randomly selected sets of two to five to a data set composed of Central American species of Aphelandra (including the parents of all hybrids). Compared to analyses with the same number of OTUs all of which were species, the analyses with hybrids yielded results with significantly higher CI. There was no difference in Trees between analyses with hybrids versus species. There was thus no evidence that hybrids would appear to be more problematic for cladistic methods than species. Accordingly, hybrids will not be readily identifiable as taxa that cause marked change in these indices. About % of the hybrids were placed as the cladistically basal members of the lineage that included the most apomorphic parent. Relatively apomorphic hybrids were placed proximate to the most derived parent (ca. 13% of hybrids). Other placements occurred more rarely. The most frequent placements of hybrids thus did not distinguish them from normal intermediate or apomorphic taxa. When analyses with hybrids yielded multiple most parsimonious trees, these were no more different from each other than were the equally parsimonious trees that resulted from analyses with species. Most analyses with one or two hybrids resulted in minor or no change in topology. When hybrids caused topological change, they frequently caused rearrangements of weakly supported portions of the cladogram that did not include their parents. When they disrupted the cladistic placement of their parents, they often caused their parents to change positions, with at least one topology bringing the parental lineages into closer proximity with the hybrid placed between them. Hybrids between parents from the two main lineages of the group caused total cladistic restructuring. In fact, the degree of relationship between a hybrid's parents (measured by both cladistic and patristic distance) was strongly correlated with CI (negatively) and with the degree of disturbance to cladistic relationships (positively). Thus, hybrids between distantly related parents resulted in cladograms with low CI and major topological changes. This study suggests that hybrids are unlikely to cause breakdown of cladistic structure unless they are between distantly related parents. However, these results also indicate that cladistics may not be specially useful in distinguishing hybrids from normal taxa. The applicability of these results to other kinds of hybrids is examined and the likely cladistic treatment of hybrids using other sources of data is discussed.
A population analysis of an unusual NOR-site polymorphism previouslydetected in brown trout (Salmo trutta L.) from North-western Spain wascarried out in 225 individuals from 20 native populations from this area. Theanalysis performed has permitted us to reveal: (i) the ubiquity of thisphenomenon in most river basins from NW Spain, 13 extra-NORs not observed instandard trout being detected; (ii) the rDNA constitution of all extra-NORs, asconfirmed by CMA3-staining and rDNA-FISH, and their capability to constitutetheir own nucleolus; (iii) the constant location of extra-NORs withinindividuals and their stable transmission across generations; and (iv) thetelomeric location of Ag-NORs, which were randomly distributed in the karyotypeof S. trutta, mostly in the heterozygous condition. Repetitivesequences in the IGS or scattered along rDNA units, rather than chromosomerearrangements, could play an important role in the dispersion of NORs. Thepolymorphism described seems to have a single geographical origin, since apositive correlation was demonstrated between the degree of polymorphism andgeographical distance to a central point of the river basin where thephenomenon showed a higher intensity. The distribution of NOR-site variation ascompared with allozyme variation in the populations studied, showed significantstatistical differences. Selection against high copy number, or mutation due tochanges in NOR location, could be acting on this polymorphism to explain theobserved distribution. The high dispersion of NOR-site variants within andamong populations suggests the transposition phenomenon responsible for NORjumping could be still active.
In the early 1960s, researchers noticed that the number of differences between amino acid sequences varied roughly linearly with the time of divergence between the species from which the amino acid sequences were sampled (Margoliash, 1963; Zuckerkandl and Pauling, 1962, 1965). This seminal observation led to the formulation of the “molecular clock” hypothesis (Zuckerkandl and Pauling, 1965), which asserts that change at the molecular level occurs regularly through time. An important corollary prediction of the molecular clock hypothesis is that rates of molecular evolution are equal among diverse evolutionary lineages.
This chapter discusses some practical consequences of disconnections between organizational and hierarchical levels when molecular data are used to reconstruct phylogeny. Crossing from a lower to a higher level—from genes to species or from genotype to phenotype—can simultaneously introduce new kinds of homoplasy and mask homoplasy from the lower level. Sources of homoplasy in reconstructions of “phylogenies” of nucleic acid sequences are presented to illustrate the concept of gene trees. The relationship of homoplasy in single gene trees to homoplasy in multiple gene trees is also discussed. Combining the gene trees help in reconstructing the phylogeny of something other than that of genes themselves, resulting in “species trees” or “organism trees.” The chapter presents species-level problems and deals with the issue of homoplasy in combined data sets involving closely related species. The chapter also provides a discussion on the role of character independence in phylogeny.
The nuclear gene encoding the chloroplast-expressed isozyme of glutamine synthetase (ncpGS) is single copy in diploid angiosperms but is duplicated in species of Glycine, a paleopolyploid genus. The two Glycine paralogues are sister to one another in phylogenetic analyses, a pattern that suggests that this ncpGS duplication occurred subsequent to the divergence of Glycine from extant Glycinnae. This pattern does not support an allopolyploid hypothesis in which genomes from close relatives of extant genera combined to form the polyploid, 2n = 40 genome of Glycine, nor with an andent gene duplication shared with other Glycininae. Rather, it is consistent with autopolyploidy or with a simple gene duplication. Teramnus ncpGS was the closest relative of the two Glycine paralogues, supporting a sister-group relationship between these two genera. In contrast, ncpGS results suggested that Sinodolichos, a genus that has been suggested as a possible congener of Glycine, is more closely related to Pseueminia and Pseudovigna. Both paralogues of ncpGS identify known genome groups among species of Glycine subg. Glycine, but neither strongly resolves relationships among these groups. Incongruence between the two paralogues in the placement of G. falcata mirrors incongruence between the chloroplast genome and other nuclear genes for this species.
The field of systematic biology has been revitalized and transformed during the last few decades by the confluence of phylogenetic thinking with ready access to the tools of molecular biology. Indeed, the title of this volume and the fact that it is already in its second edition offers ample testimony to the impact that molecular approaches have had on efforts to reconstruct the phylogenetic history of plants. Concomitant with the proliferation of molecular tools has been a growing awareness that reliance on a single data set may often result in insufficient phylogenetic resolution or misleading inferences. Accordingly, it is an increasingly widespread practice to apply multiple data sets to a common group of taxa. One of the consequences of analyzing multiple data sets is that the phylogenies inferred may differ from each other in one or more details. This phylogenetic incongruence is not rare; to the contrary, it is almost the rule rather than the exception, being evident to varying degrees.
Numerous DNA regions representing the nuclear and both organellar genomes are now available for comparative sequencing in plants (see Chapter 1); in addition, morphological and chemical data can also be obtained for phylogenetic analyses. With such a diversity of potential data sets available and the relative ease with which DNA sequences can be obtained, the acquisition of multiple data sets for the same suite of taxa is straightforward. As a result, the number of groups for which multiple data sets is available is increasing rapidly. Although it is readily apparent that multiple data sets are needed for estimating phylogenetic relationships reliably, it is also recognized that different genes may, in fact, possess different branching histories (see Chapter 10). Consequently, incorporating multiple data sets into phylogenetic studies is not a casual undertaking. Essential tasks in the analysis of multiple data sets include assessing congruence between different phylogenetic trees and data sets, and ascertaining whether multiple data sets should be combined into a single data matrix prior to phylogenetic reconstruction.
Hierarchy is the main criterion for informativeness in a data set, even if no explicit reference to evolution as a causal process is provided. Sequence data (nuclear ribosomal DNA ITS) from Armeria (Plumbaginaceae) contains a certain amount of hierarchical structure as suggested by data decisiveness and distribution of tree lengths. However, ancillary evidence suggests that extensive gene flow and biased concerted evolution in these multicopy regions have significantly shaped the ITS data set. This argument is discussed using parsimony analysis of four data sets, constructed by combining wild sequences with those from different generations of artificial hybrids (wild + F1, F2, and backcrosses; wild + backcrosses; wild + F1; wild + F2). Compared to the F1 hybrids, F2 show a certain degree of homogenization in polymorphic sites. This effect reduces topological disruption caused by F1 and is considered to be illustrative of how extensive gene flow and biased concerted evolution may have modeled the wild ITS data. The possibility that hierarchy has arisen as a result of—or despite a significant contribution from—those two such potentially perturbing forces raises the question of what kind of signal are we recovering from this molecular data set.
The nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) region was PCR-amplified in 32 genera of non-flowering seed plants. Length of the ITS region was determined by restriction site mapping of PCR products and nucleotide sequences were obtained from the ITS-2 and 5.8S rDNA of selected genera. In contrast to the relatively narrow range of ITS region lengths reported from angiosperms [565-700 base pairs (bp)], substantial length variation (975-3125 bp) is observed in the ITS region of Coniferales, Cycadales, Ginkgoales, and Gnetales. Restriction site analyses indicate that the 5.8S rDNA + ITS-2 ranges from 375-450 bp, while the ITS-1 is responsible for most of the length variation found in gymnosperm ITS regions. The representatives of Pinaceae exhibit the greatest variation in ITS region length (1550-3125 bp), while those of sampled members of Cupressaceae, Taxodiaceae, Cephalotaxaceae, and Taxaceae are relatively stable (975-1125 bp). The observed ITS region lengths in Sciadopityaceae (1250 bp) and Araucariaceae (1325-1350 bp) are somewhat larger than those of Cupressaceae, Taxodiaceae, Cephalotaxaceae, and Taxaceae, while those of sampled Podocarpaceae (2000-2100 bp) fall into the range observed in the Pinaceae. Outside of the Coniferales, ITS region length is similar among Cycadales (1150-1450 bp), Ginkgoales (1200 bp), and two of the three members of Gnetales, Ephedra (1500 bp) and Gnetum (1200 bp). In contrast, the ITS region of Welwitschia is only 750 bp long, ca. 50 bp longer than the longest known angiosperm ITS region. Levels of nucleotide sequence variation as estimated by restriction site mapping suggest that phylogenetic analysis of the ITS region will be informative at the intrageneric level in most non-flowering seed plants. In Cupressaceae, intergeneric comparisons may also be feasible. Because the ITS region is relatively long, it may also permit population- level phylogenetic analysis in many non-flowering seed plants.
Within many lineages diversity of the internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes has proven comparable to that of restriction sites of chloroplast DNA (cpDNA). Although cpDNA polymorphism has proven useful in many phylogenetic studies at the intraspecific level, the utility of ITS sequences in this application has been subjected only to limited testing. The Streptanthus glandulosus complex is an ideal candidate for this evaluation, given the substantial variation in cpDNA restriction site and enzyme electrophoretic markers present among populations. ITS sequences were obtained for 31 populations within the complex; phylogenetic patterns derived from these data were compared to a cpDNA phylogeny based on restriction site data from a previous study. No population exhibited the same ITS sequence, and average divergence among populations was 2.57%. The ITS strict consensus tree bears clades that are geographically distinct, but lacking in internal resolution; we suspect that some combination of gene flow, lineage sorting, and recombination has led to low resolution within these major clades. The ITS tree shares many features with the cpDNA tree, but the two also display substantial incongruities, including one that may have arisen via cytoplasmic introgression. Greater consistency is exhibited by the cpDNA tree, most likely due to the haploid and non-recombining nature of the chloroplast genome. Combining the ITS and cpDNA data results in a more highly resolved tree than those from either of the separate analyses. Despite the pitfalls of using a diploid, recombining data source like ITS at the intraspecific level, potential benefits include tracking of maternal and paternal lineages as well as insight into patterns and levels of gene flow among populations. Furthermore, the joint use of ITS and cpDNA markers facilitates independent assessment of intraspecific phylogenetic hypotheses and can expose cases in which introgression or phylogenetic sorting has caused incongruencies among haplotype trees.
... THE IMPACT OF HYBRIDS ON CLADISTIC ANALYSIS Lucinda A. McDade Department of Botany, Duke University, Durham ... Acanthaceae, Aphelandra, cladistics , hybrids, phylogenetic ... Interspecific hybridization is thought to bridization is thought to be of less evolu- be common ...
This work describes a network server for searching databanks of biopolymer structures and performing other biocomputing procedures; it is available via direct Internet connection. Basic server procedures are dedicated to homology (similarity) search of sequence and D structure of proteins. The homologies found could be used to build multiple alignments, predict protein and RNA secondary structure, and construct phylogenetic trees. In addition to traditional methods of sequence similarity search, the authors propose {open_quotes}non-matrix{close_quotes} (correlational) search. An analogous approach is used to identify regions of similar tertiary structure of proteins. Algorithm concepts and usage examples are presented for new methods. Service logic is based upon interaction of a client program and server procedures. The client program allows the compilation of queries and the processing of results of an analysis.
Reconstruction of phylogenies from molecular data has become an important and increasingly common approach in systematics. The product of such studies is a gene tree, hypothesizing relationships among genes or genomes. This gene tree may be fundamentally incongruent with the true species phylogeny, due to various biological phenomena such as introgression, lineage sorting, or mistaken orthology. In such circumstances all of the gene tree characters defining the relationships of molecular taxa (haplotypes) may be necessarily correlated, and the gene or genome may behave as a single species tree character. In these circumstances robustness of the gene hypothesis is meaningless as a measure of confidence in the species phylogenetic hypothesis. Incongruence between a phylogenetic hypothesis based on numerous, presumably independent, non-molecular characters and a single gene tree should not be assumed to be due to noise in non-molecular data. As with other characters, a character phylogeny, in this case a gene tree, can be tested best by a parsimony analysis in which other characters are included. If independence of molecular characters is assumed, then each is an equivalent phylogenetic hypothesis, as is each non-molecular character, leading to the suggestion that direct combination is appropriate. Swamping becomes an issue when a large molecular data set may be behaving as a single character. To alleviate this problem, a gene tree may be treated as a single multistate character, either ordered or unordered, and included with non-molecular data to obtain a globally parsimonious result. An example is given using published molecular and non-molecular data from the Asteraceae.
Polymerase chain reaction (PCR) amplification of the nuclear internal transcribed spacer (ITS) and 5.8S regions of rDNA from woody bamboos (Bambuseae) led to the recovery of fungal instead of bamboo sequences under a variety of PCR conditions and irrespective of whether the plant DNA was extracted from fresh leaves or silica gel-dried material. Phylogenetic analyses based on the 5.8S sequences indicated that the fungi were most likely basidiomycetes and that none was an ascomycete. A diverse assemblage of nonascomycetous fungi was isolated from different bamboos, and various fungi coexisted in the same host plant. There was no evidence that closely related fungi consistently associate with closely related host bamboos. Phylogenetic analysis based on 5.8S sequences showed that some fungi were in lineages nearVolvariella, Lentinula, Peniophora,andRhizoctonia,but the insufficiency of basidiomycete and zygomycete ITS sequences in sequence data bases precluded more precise fungal identifications. Bamboo ITS regions were amplified only when fresh leaves were surface sterilized before DNA extraction, suggesting that the fungal associates are epiphyllous rather than endophytic. This study highlights the possibility of inadvertent PCR amplification of contaminating DNAs in molecular phylogenetic studies, particularly when using “universal” amplification primers.
▪ Abstract Trans-species polymorphism (TSP) is the occurrence of similar alleles in related species. Excluding instances in which the similarity arose by convergent evolution, TSP is generated by the passage of alleles from ancestral to descendant species. Closely related, recently diverged species, such as those of the Lake Victoria cichlid flock, may share neutral alleles, but long-lasting TSPs occur only in genetic systems evolving under balancing selection. Two such systems have been studied extensively, the major histocompatibility complex (Mhc) of jawed vertebrates and the self-incompatibility (SI) system of flowering plants. Allelic lineages that diverged many millions of years ago and passed through numerous speciation events have been described in both systems. The lineages may differ at up to 50% of their coding sites, both synonymous and nonsynonymous. The differences arise by the process of incorporation of mutations, which is different from the process of fixation. TSP, on the one hand, compl...
As a contribution to establish taxonomic identification methods using DNA sequence data, we evaluate to which extent the present set-up of the nucleotide databases (NCBI, EBI, GenomeNet) allows to use them for reliable routine plant identification applying the implemented sequence similarity and homology search tools. We screened newly determined ITS 1 sequences from Asteraceae (tribes Lactuceae, Anthemideae) against the molecular nucleotide databases where sequences from that exact species, from other species of the same genus, or only from other genera of the same family had already been published. With the present set up of the BLAST® algorithms [MEGABLAST, BLASTN] 100% correct family, 99% correct tribe, and 98% precise genus affiliation could be achieved in Lactuceae representatives and 100% correct family, 100% correct tribe, and 95% precise genus affiliation in Anthemideae. Correct species detection varied between 86% accuracy for Lactuceae and 91% in Anthemideae. Necessary optimization criteria are proposed as well as further requirements for a standardized molecular identification system.
The phylogeny of the genus Diploschistes was investigated using nucleotide sequences of the nuclear ITS rDNA region (ITS1, ITS2 and 5.8S rDNA). Sequences of 22 Diploschistes species were aligned to those of six other species of Thelotremataceae, Graphis scripta and Aspicilia cinerea, with the last used as an outgroup. The alignment was analysed cladistically using maximum parsimony. In the most parsimonious trees, Diploschistes is monophyletic, with D. ocellatus being a sister group to the remaining Diploschistes spp. (= Diploschistes s. str.). A previous cladistic analysis of morphological data suggested an evolutionary trend within the genus from perithecioid to urceolate ascomata. The present ITS data suggest the opposite: perithecioid ascomata are apparently an apomorphic character within the genus, with the actinostomus group forming a derived monophyletic clade. However, the topology within Diploschistes s. str. Lacks strong bootstrap support.
We have designed two taxon-selective primers for the internal transcribed spacer (ITS) region in the nuclear ribosomal repeat unit. These primers, ITS1-F and ITS4-B, were intended to be specific to fungi and basidiomycetes, respectively. We have tested the specificity of these primers against 13 species of ascomycetes, 14 of basidiomycetes, and 15 of plants. Our results showed that ITS4-B, when paired with either a ‘universal’ primer ITS1 or the fungal-specific primer ITS1-F, efficiently amplified DNA from all basidiomycetes and discriminated against ascomycete DNAs. The results with plants were not as clearcut. The ITS1-F/ITS4-B primer pair produced a small amount of PCR product for certain plant species, but the quantity was in most cases less than that produced by the ‘universal’ ITS primers. However, under conditions where both plant and fungal DNAs were present, the fungal DNA was amplified to the apparent exclusion of plant DNA. ITS1-F/ITS4-B preferential amplification was shown to be particularly useful for detection and analysis of the basidiomycete component in ectomycorrhizae and in rust-infected tissues. These primers can be used to study the structure of ectomycorrhizal communities or the distribution of rusts on alternate hosts.
Aim The olive tree is considered one of the best indicators of the Mediterranean climate. The species’ distribution is associated with geographical and bioclimatic factors, as well as being influenced by a long period of cultivation. Despite concerted efforts of different research groups, the origin of the Mediterranean olive tree still remains elusive. In the present study, relationships between taxa and populations covering the entire range of Olea europaea were investigated using both maternal (plastid genome) and biparental (nuclear genome) markers to disclose evolutionary patterns in the olive complex. Phylogenetic and phylogeographical results of the two‐genome analyses were interpreted in a biogeographical context.
Location Mediterranean, temperate and subtropical floristic regions of the Old World.
Methods Phylogeographical reconstructions of plastid DNA polymorphism were performed using microsatellites, restriction sites and indels on a wide sample of 185 representative trees across the Old World, including 28 herbarium specimens from remote areas. Additionally, the potential utility of one ITS‐1 pseudogene for phylogenetic analyses was explored using Bayesian and maximum parsimony approaches on a subsample of 38 olive trees.
Results Forty plastid haplotypes were recognized and split into two lineages and seven sublineages. The analysis of ITS‐1 sequences also allowed the identification of seven well differentiated groups. Distribution of plastid and ribosomal DNA lineages was congruent, but particular cases of phylogenetic incongruence were disclosed (particularly in the Sahara and Madeira). Lastly, two divergent ITS‐1 copies were isolated from the same sample of four individuals of different subspecies.
Main conclusions Phylogenetic congruence of both ITS‐1 and plastid lineages suggested an evolutionary scenario of predominant isolation during the Plio‐Pleistocene in Macaronesia, the Mediterranean, southern Africa, eastern Africa and Asia. The Saharan desert appeared to have played an important role of vicariant barrier between southern and northern African populations in early times. Incongruence of some plastid and nuclear results, as well as intermingled ITS‐1 copies of different lineages in single individuals, was interpreted as a result of recurrent reticulation events in the olive complex. We identified an ancient hybrid zone from the Sahara to north‐eastern African mountains, where divergent plastid and nuclear lineages still co‐exist. Results of this paper, and previous studies, suggest that the cultivated olive originated from a pre‐Quaternary Mediterranean ancestor, with no evidence for a recent hybrid origin. In contrast, a continuous process of olive domestication through local hybridization events of cultivated trees with natural populations may have brought about a remarkably high genomic diversity among cultivated trees across the Mediterranean.
Hierarchy is the main criterion for informativeness in a data set, even if no explicit reference to evolution as a causal process is provided. Sequence data (nuclear ribosomal DNA ITS) from Armeria (Plumbaginaceae) contains a certain amount of hierarchical structure as suggested by data decisiveness and distribution of tree lengths. However, ancillary evidence suggests that extensive gene flow and biased concerted evolution in these multicopy regions have significantly shaped the ITS data set. This argument is discussed using parsimony analysis of four data sets, constructed by combining wild sequences with those from different generations of artificial hybrids (wild + F1, F2, and backcrosses; wild + backcrosses; wild + F1; wild + F2). Compared to the F1 hybrids, F2 show a certain degree of homogenization in polymorphic sites. This effect reduces topological disruption caused by F1 and is considered to be illustrative of how extensive gene flow and biased concerted evolution may have modeled the wild ITS data. The possibility that hierarchy has arisen as a result of—or despite a significant contribution from—those two such potentially perturbing forces raises the question of what kind of signal are we recovering from this molecular data set.
The nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) region has become an important nuclear locus for molecular systematic investigations of angiosperms at the intergenic and interspecific levels. Universal PCR primers are positioned on the conserved rRNA genes (18S, 5.8S, 26S) to amplify the entire ITS spacer region. Recent reports of fungal and algal contaminants, first described as plant ITS sequences, stress the need for diagnostic markers specific for the angiosperm ITS region. This report describes a conserved 14 base pair (bp) motif in the 5.8S rRNA gene that can be used to differentiate between flowering plants, bryophytes, and several orders of algae and fungi, including common plant pathogenic and non-pathogenic fungi. A variant of the motif (found in fungi and algae) contains a convenient EcoRI restriction site that has several applications for eliminating problematic contaminants from plant ITS preparations.
The molecular systematics of Quercus (Fagaceae) was recently assessed by two teams using independently generated ITS sequences. Although the results disagreed in several remarkable features, the phylogenetic trees for either hypothesis were highly supported by bootstrap resampling. We have reanalyzed the ITS sequences used by both teams (eight taxa) to reveal the underlying patterns of this divergence. Within species, conspicuous length and G + C% divergence were evident in most sequence comparisons. In addition, a high rate of substitutions and deletions involving highly conserved motifs in both ITS spacers were present in a set of sequences. This was coupled with a less thermodynamic stability in the RNA structure, lacking conserved hairpins that are putatively involved in the processing of the RNA transcripts. Compelling evidence suggests that the divergent ITS alleles reported by one team are pseudogenes, i.e., nonfunctional paralogous loci. The hypothesis that the contrasting phylogenetic histories drawn from Quercus using ITS data are not strictly related to technical differences between laboratories, but that they have rather been generated from the analysis of paralogous sequences, best reconciles the available data. The risk of incorporating ITS paralogues in plant evolutionary studies which can distortion the phylogenetic signal should caution molecular systematists. Without a detailed inspection of some basic features of the sequence, including the integrity of the conserved motifs and the thermodynamic stability of the secondary structures of the RNA transcripts, errors in evolutionary inferences could be easily overlooked.
Species of Suillus produce fleshy, pored mushrooms. They are important symbiotic (ectomycorrhizal) partners of many coniferous trees. The genus includes several putative eastern Asian and eastern North American disjunct species, i.e., the S. americanus–S. sibiricus and S. decipiens–S. spraguei complexes. Phylogenetic relationships among the groups were determined to further understand the biogeographic pattern. Analyses were based on 40 sequences of the ITS region of the nuclear ribosomal RNA tandem repeats, representing 18 distinct species/populations. Our phylogenetic analyses suggested that: (1) Chinese and United States' (U.S.) S. spraguei plus S. decipiens form a strongly supported monophyletic group, with North American S. decipiens and Chinese S. spraguei being sister taxa; (2) S. americanus, Asian and U.S. S. sibiricus, plus S. umbonatus form a clade supported by a high bootstrap value; and (3) little ITS sequence divergence exists within the latter group compared to the S. decipiens–S. spraguei clade. Phylogenetic patterns revealed by this study imply a close phylogenetic relationship between eastern Asian and eastern North American disjunct population/species of Suillus. These fungi display relatively high host fidelity (at least to the host subgenus level), suggesting potential coevolutionary/comigratory trends.
Discusses a variety of ecological characteristics, summarising their predicted effects on genetic structure within and between populations, then analyses data describing genetic differentiation in plants to determine which ecological variables are consistent predictors of genetic organization. A major section of the review examines ecological and life history traits affecting genetic structure, including such ecological variables as breeding systems, floral morphology, mode of reproduction, pollination mechanisms, seed dispersal, seed dormancy, phenology, life cycle, timing of reproduction, successional stage, geographical range, population size, density, and population spatial structure. -P.J.Jarvis
The reliability of phylogenies reconstructed from data on multigene families is investigated via simulation. The evolutionary scenario used is a character-based model of a twogene family in four species in which clocklike divergence is postulated but neither convergence nor reversal is allowed except as a result of recombination and gene conversion. Thus, any homoplasy emerging from parsimony reconstructions from the simulated data matrices can be attributed to concerted evolution. The probabilities of correctly reconstructing two standard trees are estimated by replicate runs of the simulation. One standard tree (the OP or “orthology/paralogy” tree) reflects the true gene genealogy in the absence of concerted evolution; the other (the CE or “concerted evolution” tree) depicts gene relationships under complete homogenization of the gene family. The probability of correct reconstruction of the OP tree declines quickly as concerted evolution increases, but above an intermediate level of concerted evolution the probability of correctly inferring the CE tree increases rapidly. Trees similar but not identical to the correct trees can be reconstructed above or below the critical intermediate level of concerted evolution. Levels of homoplasy and numbers of equally parsimonious minimal trees are maximized, and bootstrap confidence levels are minimized, near this intermediate level of concerted evolution. When reconstructing the correct gene tree is the goal, both consistency indices and bootstrap levels will show misleadingly high values when concerted evolution is high. However, because the correct species tree can be inferred from either the OP or CE tree (in the absence of homoplasy from sources other than concerted evolution), these same measures correlate well with fidelity of reconstructing the species tree.
The origin of land vertebrates was one of the major transitions in the history of vertebrates. Yet, despite many studies that are based on either morphology or molecules, the phylogenetic relationships among tetrapods and the other two living groups of lobe-finned fishes, the coelacanth and the lungfishes, are still unresolved and debated. Knowledge of the relationships among these lineages, which originated back in the Devonian, has profound implications for the reconstruction of the evolutionary scenario of the conquest of land. We collected the largest molecular data set on this issue so far, about 3,500 base pairs from seven species of the large 28S nuclear ribosomal gene. All phylogenetic analyses (maximum parsimony, neighbor-joining, and maximum likelihood) point toward the hypothesis that lungfishes and coelacanths form a monophyletic group and are equally closely related to land vertebrates. This evolutionary hypothesis complicates the identification of morphological or physiological preadaptations that might have permitted the common ancestor of tetrapods to colonize land. This is because the reconstruction of its ancestral conditions would be hindered by the difficulty to separate uniquely derived characters from shared derived characters in the coelacanthylungfish and tetrapod lineages. This molecular phylogeny aids in the reconstruction of morphological evolutionary steps by providing a framework; however, only paleontological evidence can determine the sequence of morphological acquisitions that allowed lobefinned fishes to colonize land.