Reprint of: Using nuclear gene data for plant phylogenetics: Progress and prospects

The paper reviews the current state of low and single copy nuclear markers that have been applied successfully in plant phylogenetics to date, and discusses case studies highlighting the potential of massively parallel high throughput or next-generation sequencing (NGS) approaches for molecular phylogenetic and evolutionary investigations. The current state, prospects and challenges of specific single- or low-copy plant nuclear markers as well as phylogenomic case studies are presented and evaluated.     

Utility of Low-Copy Nuclear Gene Sequences in Plant Phylogenetics

Low-copy nuclear genes in plants are a rich source of phylogenetic information. They hold a great potential to improve the robustness of phylogenetic reconstruction at all taxonomic levels, especially where universal markers such as cpDNA and nrDNA are unable to generate strong phylogenetic hypotheses. Low-copy nuclear genes, however, remain underused in plant phylogenetic studies due to practical and theoretical complications in unraveling the evolutionary dynamics of nuclear gene families. The lack of the universal markers or universal PCR primers of low-copy nuclear genes has also hampered their phylogenetic utility. It has recently become clear that low-copy nuclear genes are particularly helpful in resolving close interspecific relationships and in reconstructing allopolyploidization in plants. Gene markers that are widely, if not universally, useful have begun to emerge. Although utilizing low-copy nuclear genes usually requires extra lab work such as designing PCR primers, PCR-cloning, and/or Southern blotting, rapid accumulation of gene sequences in the databases and advances in cloning techniques have continued to make such studies more feasible. With the growing number of theoretical studies devoted to the gene tree and species tree problem, a solid foundation for reconstructing complex plant phylogenies based on multiple gene trees began to build. It is also realized increasingly that fast evolving introns of the low-copy nuclear genes will provide much needed phylogenetic information around the species boundary and allow us to address fundamental questions concerning processes of plant speciation. Phylogenetic and molecular evolutionary analyses of developmentally important genes will add a new dimension to systematic and evolutionary studies of plant diversity.     

Utility of low-copy nuclear gene sequences in plant phylogenetics

Low-copy nuclear genes in plants are a rich source of phylogenetic information. They hold a great potential to improve the robustness of phylogenetic reconstruction at all taxonomic levels, especially where universal markers such as cpDNA and nrDNA are unable to generate strong phylogenetic hypotheses. Low-copy nuclear genes, however, remain underused in plant phylogenetic studies due to practical and theoretical complications in unraveling the evolutionary dynamics of nuclear gene families. The lack of the universal markers or universal PCR primers of low-copy nuclear genes has also hampered their phylogenetic utility. It has recently become clear that low-copy nuclear genes are particularly helpful in resolving close interspecific relationships and in reconstructing allopolyploidization in plants. Gene markers that are widely, if not universally, useful have begun to emerge. Although utilizing low-copy nuclear genes usually requires extra lab work such as designing PCR primers, PCR-cloning, and/or Southern blotting, rapid accumulation of gene sequences in the databases and advances in cloning techniques have continued to make such studies more feasible. With the growing number of theoretical studies devoted to the gene tree and species tree problem, a solid foundation for reconstructing complex plant phylogenies based on multiple gene trees began to build. It is also realized increasingly that fast evolving introns of the low-copy nuclear genes will provide much needed phylogenetic information around the species boundary and allow us to address fundamental questions concerning processes of plant speciation. Phylogenetic and molecular evolutionary analyses of developmentally important genes will add a new dimension to systematic and evolutionary studies of plant diversity.     

Molecular markers applied to plant tissue culture

Summary The last decade has witnessed successful applications of plant tissue culture techniques in several crops. During that same period, studies in plant molecular genetics have also grown exponentially. Molecular markers (isozymes, RFLPs, and PCR-based markers such as RAPDs) are now used to study many of the current limitations of tissue culture. They have been used to investigate mechanisms that underlie somaclonal variation in the nuclear, mitochondrial, and chloroplast genomes. One recurrent problem with several tissue culture systems has been the difficulty of determining the origin of regenerants. Molecular markers represent powerful tools to determine precisely the origin of plants derived from microspore or anther culture, protoplast fusion, and other tissue culture studies where this information is important. With improvements in tissue culture techniques, populations of doubled haploid lines have been produced in several major crop species. Doubled haploid populations have proven useful in the production of molecular maps and in tagging important agronomic traits. This review describes the use of molecular markers to address fundamental and practical questions of plant tissue culture, and discusses the potential of improvements in molecular techniques and new molecular markers such as SCAR and STS along with high-resolution mapping strategies.     

Secondary Structure Analyses of the Nuclear rRNA Internal Transcribed Spacers and Assessment of Its Phylogenetic Utility across the Brassicaceae (Mustards)

The internal transcribed spacers of the nuclear ribosomal RNA gene cluster, termed ITS1 and ITS2, are the most frequently used nuclear markers for phylogenetic analyses across many eukaryotic groups including most plant families. The reasons for the popularity of these markers include: 1.) Ease of amplification due to high copy number of the gene clusters, 2.) Available cost-effective methods and highly conserved primers, 3.) Rapidly evolving markers (i.e. variable between closely related species), and 4.) The assumption (and/or treatment) that these sequences are non-functional, neutrally evolving phylogenetic markers. Here, our analyses of ITS1 and ITS2 for 50 species suggest that both sequences are instead under selective constraints to preserve proper secondary structure, likely to maintain complete self-splicing functions, and thus are not neutrally-evolving phylogenetic markers. Our results indicate the majority of sequence sites are co-evolving with other positions to form proper secondary structure, which has implications for phylogenetic inference. We also found that the lowest energy state and total number of possible alternate secondary structures are highly significantly different between ITS regions and random sequences with an identical overall length and Guanine-Cytosine (GC) content. Lastly, we review recent evidence highlighting some additional problematic issues with using these regions as the sole markers for phylogenetic studies, and thus strongly recommend additional markers and cost-effective approaches for future studies to estimate phylogenetic relationships.     

Beyond complementation. Map-based cloning in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii is an excellent model system for plant biologists because of its ease of manipulation, facile genetics, and the ability to transform the nuclear, chloroplast, and mitochondrial genomes. Numerous forward genetics studies have been performed in Chlamydomonas, in many cases to elucidate the regulation of photosynthesis. One of the resultant challenges is moving from mutant phenotype to the gene mutation causing that phenotype. To date, complementation has been the primary method for gene cloning, but this is impractical in several situations, for example, when the complemented strain cannot be readily selected or in the case of recessive suppressors that restore photosynthesis. New tools, including a molecular map consisting of 506 markers and an 8X-draft nuclear genome sequence, are now available, making map-based cloning increasingly feasible. Here we discuss advances in map-based cloning developed using the strains mcd4 and mcd5, which carry recessive nuclear suppressors restoring photosynthesis to chloroplast mutants. Tools that have not been previously applied to Chlamydomonas, such as bulked segregant analysis and marker duplexing, are being implemented to increase the speed at which one can go from mutant phenotype to gene. In addition to assessing and applying current resources, we outline anticipated future developments in map-based cloning in the context of the newly extended Chlamydomonas genome initiative.     

General functions to transform associate data to host data,and their use in phylogenetic inference from sequences with intra-individual variability

Background  

Amongst the most commonly used molecular markers for plant phylogenetic studies are the nuclear ribosomal internal transcribed spacers (ITS). Intra-individual variability of these multicopy regions is a very common phenomenon in plants, the causes of which are debated in literature. Phylogenetic reconstruction under these conditions is inherently difficult. Our approach is to consider this problem as a special case of the general biological question of how to infer the characteristics of hosts (represented here by plant individuals) from features of their associates (represented by cloned sequences here).     

Land plants and DNA barcodes: short-term and long-term goals

Land plants have had the reputation of being problematic for DNA barcoding for two general reasons: (i) the standard DNA regions used in algae, animals and fungi have exceedingly low levels of variability and (ii) the typically used land plant plastid phylogenetic markers (e.g. rbcL, trnL-F, etc.) appear to have too little variation. However, no one has assessed how well current phylogenetic resources might work in the context of identification (versus phylogeny reconstruction). In this paper, we make such an assessment, particularly with two of the markers commonly sequenced in land plant phylogenetic studies, plastid rbcL and internal transcribed spacers of the large subunits of nuclear ribosomal DNA (ITS), and find that both of these DNA regions perform well even though the data currently available in GenBank/EBI were not produced to be used as barcodes and BLAST searches are not an ideal tool for this purpose. These results bode well for the use of even more variable regions of plastid DNA (such as, for example, psbA-trnH) as barcodes, once they have been widely sequenced. In the short term, efforts to bring land plant barcoding up to the standards being used now in other organisms should make swift progress. There are two categories of DNA barcode users, scientists in fields other than taxonomy and taxonomists. For the former, the use of mitochondrial and plastid DNA, the two most easily assessed genomes, is at least in the short term a useful tool that permits them to get on with their studies, which depend on knowing roughly which species or species groups they are dealing with, but these same DNA regions have important drawbacks for use in taxonomic studies (i.e. studies designed to elucidate species limits). For these purposes, DNA markers from uniparentally (usually maternally) inherited genomes can only provide half of the story required to improve taxonomic standards being used in DNA barcoding. In the long term, we will need to develop more sophisticated barcoding tools, which would be multiple, low-copy nuclear markers with sufficient genetic variability and PCR-reliability; these would permit the detection of hybrids and permit researchers to identify the 'genetic gaps' that are useful in assessing species limits.     

Development of genetic markers for Ochradenus arabicus (Resedaceae), an endemic medicinal plant of Saudi Arabia

Some species of the genus Ochradenus are difficult to identify based on morphological markers. Similar limitations are found for biochemical markers. We developed genetic markers based on DNA sequences for Ochradenus arabicus, which is an endemic plant to Saudi Arabia, locally utilized as a medicinal shrub. The internal transcribed spacer sequence of nuclear ribosomal DNA and chloroplast (rpoB and rpoC1) markers were more informative than other chloroplast DNA markers. Based on these markers, we were able to discriminate this species from another species of the same genus (O. baccatus) that is widely distributed in Saudi Arabia, despite a high degree of morphological similarity. These genetic markers facilitate its identification, even when acquired in a dried state from local markets.     

Tracking ancient polyploids: a retroposon insertion reveals an extinct diploid ancestor in the polyploid origin of belladonna

Polyploidy is a prominent process in plant evolution and adaptation, but molecular phylogenetic studies of polyploids based on DNA sequences have often been confounded by their complex gene and genome histories. We report here a retroposon insertion in the nuclear gene granule-bound starch synthase I (GBSSI or "waxy") that clearly reveals the ancient hybrid history of the medically important polyploid species belladonna (Atropa belladonna) and resolves the controversy over the taxonomic group to which it belongs, the tribe Hyoscyameae (Solanaceae). Our inferences based on the pattern of presence or absence of the retroposon insertion are corroborated by phylogenetic analyses of the GBSSI gene sequences. This case may suggest that retroposons are promising molecular markers to study polyploid evolution.     

Characterisation of insect and plant origins using DNA extracted from small volumes of bee honey

A DNA-based tool was validated that potentially enables the characterisation of both plant and insect of origin of small (approximately 1 ml) samples of bee honey. Using this method, mitochondrial, nuclear and chloroplast DNA (mtDNA, nuDNA, cpDNA) markers were successfully extracted, PCR amplified, and sequenced from a range of honeys, and the relative amount of plant nuDNA and cpDNA, and bee mtDNA in the samples was quantified using quantitative real-time PCR. Short, but taxonomically informative lengths of insect and plant organelle DNA could be routinely recovered from all honey samples tested, and longer organelle, and nuclear DNA sequences can be recovered from many. The data also enabled preliminary characterisation of the quality of these different DNA sources in honey. Although the absolute quantity of the different genetic markers varied considerably between sample, a general trend was observed of insect mtDNA dominating over plant organelle DNA, and with plant nuclear DNA at the lowest levels. Furthermore there was a clear correlation between the plant DNA content and the success of the PCR assays. To maximise successful characterisation of samples, future studies are recommended to focus on the use of organelle markers, and limit the size of PCR amplicons targeted, although with appropriate sample selection and assay optimisation, other approaches may be possible.     

Reprogramming of human cancer cells to pluripotency for models of cancer progression

The ability to study live cells as they progress through the stages of cancer provides the opportunity to discover dynamic networks underlying pathology, markers of early stages, and ways to assess therapeutics. Genetically engineered animal models of cancer, where it is possible to study the consequences of temporal‐specific induction of oncogenes or deletion of tumor suppressors, have yielded major insights into cancer progression. Yet differences exist between animal and human cancers, such as in markers of progression and response to therapeutics. Thus, there is a need for human cell models of cancer progression. Most human cell models of cancer are based on tumor cell lines and xenografts of primary tumor cells that resemble the advanced tumor state, from which the cells were derived, and thus do not recapitulate disease progression. Yet a subset of cancer types have been reprogrammed to pluripotency or near‐pluripotency by blastocyst injection, by somatic cell nuclear transfer and by induced pluripotent stem cell (iPS) technology. The reprogrammed cancer cells show that pluripotency can transiently dominate over the cancer phenotype. Diverse studies show that reprogrammed cancer cells can, in some cases, exhibit early‐stage phenotypes reflective of only partial expression of the cancer genome. In one case, reprogrammed human pancreatic cancer cells have been shown to recapitulate stages of cancer progression, from early to late stages, thus providing a model for studying pancreatic cancer development in human cells where previously such could only be discerned from mouse models. We discuss these findings, the challenges in developing such models and their current limitations, and ways that iPS reprogramming may be enhanced to develop human cell models of cancer progression.     

Navigating the tip of the genomic iceberg: Next-generation sequencing for plant systematics

? Premise of the study: Just as Sanger sequencing did more than 20 years ago, next-generation sequencing (NGS) is poised to revolutionize plant systematics. By combining multiplexing approaches with NGS throughput, systematists may no longer need to choose between more taxa or more characters. Here we describe a genome skimming (shallow sequencing) approach for plant systematics. ? Methods: Through simulations, we evaluated optimal sequencing depth and performance of single-end and paired-end short read sequences for assembly of nuclear ribosomal DNA (rDNA) and plastomes and addressed the effect of divergence on reference-guided plastome assembly. We also used simulations to identify potential phylogenetic markers from low-copy nuclear loci at different sequencing depths. We demonstrated the utility of genome skimming through phylogenetic analysis of the Sonoran Desert clade (SDC) of Asclepias (Apocynaceae). ? Key results: Paired-end reads performed better than single-end reads. Minimum sequencing depths for high quality rDNA and plastome assemblies were 40× and 30×, respectively. Divergence from the reference significantly affected plastome assembly, but relatively similar references are available for most seed plants. Deeper rDNA sequencing is necessary to characterize intragenomic polymorphism. The low-copy fraction of the nuclear genome was readily surveyed, even at low sequencing depths. Nearly 160000 bp of sequence from three organelles provided evidence of phylogenetic incongruence in the SDC. ? Conclusions: Adoption of NGS will facilitate progress in plant systematics, as whole plastome and rDNA cistrons, partial mitochondrial genomes, and low-copy nuclear markers can now be efficiently obtained for molecular phylogenetics studies.     

Impact assessment of nuclear and thermal power plants on zooplankton in cooling ponds

The current state of assessment of nuclear and thermal power plant toxic effects on zooplankton entrained in the water supply hardware is reviewed. Common research approaches and their inherent problems are considered and particular emphasis is given to specific features of cooling reservoirs and their ecosystems. Complex analysis of zooplankton mortality in various conditions and areas of a cooling reservoir is presented. Our data from field and laboratory studies allow major factors resulting in damage to and mortality of zooplankton to be determined and suggest that, among all plankton species in the cooling reservoir, zooplankton is the most sensitive group to entrainment-induced stress. Our results clearly demonstrate that both field observations and laboratory studies are required to reliably estimate power plant impact on zooplankton and to predict mortality of organisms.     

Postglacial intra-lacustrine divergence of Icelandic threespine stickleback morphs in three neovolcanic lakes

The geographical context of divergence and local adaptation of lacustrine fish is controversial. Despite recent theoretical support for sympatric and parapatric divergence, empirical studies providing unequivocal support for this remain scant. An important component of such a case would be where multiple lakes have different morphs and a range of markers, both mitochondrial and nuclear, show monophyly within lakes. Here we describe such a situation for threespine sticklebacks in three lakes in Iceland. By analysing the variation at nuclear and mitochondrial markers in several freshwater and marine populations as well as three pairs of intra-lacustrine morphs we infer their phylogenetic relationships and colonization pattern. There were high levels of microsatellite variation in all populations and no evidence was found for either repeated colonization of marine fish or colonization from distinct glacial refugia. Intra-lacustrine threespine stickleback morphs in all three lakes show significant genetic divergence probably indicating restricted gene flow.     

Protocol for the analysis of n-alkanes and other plant-wax compounds and for their use as markers for quantifying the nutrient supply of large mammalian herbivores

Plant-wax markers can be used for estimating forage intake, diet composition and supplement intake in grazing livestock, wild ruminants and other mammals. We describe protocols for using the saturated hydrocarbons (alkanes) of plant wax as markers for estimating fecal output, intake and digestibility. Procedures for investigating digestion kinetics are also discussed. Alkanes can also be used to estimate diet composition and the procedures required to do this are also described, including the special case where supplementary feed is treated as a component of the diet composition estimate. The long-chain alcohols (LCOHs) and very long-chain fatty acids (VLCFAs) of plant wax show particular promise for discriminating a greater number of species in the diet. The use of all these plant-wax markers in nutrition studies depends on having quantitative, repeatable and mutually compatible assay procedures for alkanes, LCOHs and VLCFAs; we present protocols for these assays in detail. Analysis of a single sample of feces or plant material for all these plant-wax markers can be completed within 2 days; however, it is possible to process up to 50 samples (analyzed in duplicate) per week.     

Better the devil you know? Guidelines for insightful utilization of nrDNA ITS in species-level evolutionary studies in plants

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.     

Markers in time and space: A review of the last decade of plant phylogeographic approaches

Plant studies comprise a relatively small proportion of the phylogeographic literature, likely as a consequence of the fundamental challenges posed by the complex genomic structures and life history strategies of these organisms. Comparative plastomics (i.e., comparisons of mutation rates within and among regions of the chloroplast genome) across plant lineages has led to an increased understanding of which markers are likely to provide the most information at low taxonomic levels. However, the extent to which the results of such work have influenced the literature has not been fully assessed, nor has the extent to which plant phylogeographers explicitly analyse markers in time and space, both of which are integral components of the field. Here, we reviewed more than 400 publications from the last decade of plant phylogeography to specifically address the following questions: (i) What is the phylogenetic breadth of studies to date? (ii) What molecular markers have been used, and why were they chosen? (iii) What kinds of markers are most frequently used and in what combinations? (iv) How frequently are divergence time estimation and ecological niche modelling used in plant phylogeography? Our results indicate that chloroplast DNA sequence data remain the primary tool of choice, followed distantly by nuclear DNA sequences and microsatellites. Less than half (42%) of all studies use divergence time estimation, while even fewer use ecological niche modelling (14%). We discuss the implications of our findings, as well as the need for community standards on data reporting.