Barcode of Life and Tree of Life have attracted much attention in the recent decade and are two hotspots in the realm of biology. The authors reviewed the conceptual initiation, research progress, bottlenecks and resolving strategies for both Barcode of Life and Tree of Life, and discussed perspectives of their development and applications in the future. The concept of  Tree of Life have been developed with a long history, while DNA barcoding has been promoted for a decade, and studies on the two subjects have been greatly accelerated by the advances of DNA sequencing technology, bioinformatics and computational biology. Despite of these, objectives of the two subjects are quite different. DNA barcoding aims to rapidly identify species with a short and standard region of DNA sequences, while Tree of Life focuses on exploring the origin of species and reconstructing phylogenetic relationships among organisms. Therefore, different strategies and overall considerations should be made correspondingly to the distinguishable goals of Barcode of Life and Tree of Life. Uncertainty in species circumscription and sampling strategies is addressed as the major bottleneck deterring the development of Barcode of Life and Tree of Life, and appropriate suggestions are provided. Finally the authors suggest that Chinese scientists should take the advantage of the current resources to push forward theoretical accomplishment of the Tree of Life, as well as the application of DNA barcoding through collaboration with engineers from the fields of bioinformatics and computer sciences.

Clarifying plant species is the base of plant taxonomical research and also the premise of iFlora, for which investigations on plant species provide the most basal and important research materials. However, field investigations traditionally are for the flora of some regions or for collecting some kinds of plant resources, but for the local populations of species. Investigation and research of plant species on population level therefore need to be concerned and implemented in the plan of iFlora, in which “individual (specimens)(local breeding) populationplant species” will be the core clue. Different from the past, the field investigation will be targeted at local breeding population and collects morphological, ecological and genetical information as direct proof applied to taxonomical discussion and foundiation of species′ database. Furthermore we should place great importance on populations of type loaclity. Due to fragmentary type specimens and simple original descriptions, researchers have to actively consider “topotype” and “population of type locality” as the extension of “type”. Populations of type locality, which are like as “Golden Spikes” of geology, are more than just footnotes of type specimens, but provide authoritative and abundant information sources for the taxonomical research of plant species.

The new generation flora, iFlora, is essentially different from other traditional floras, in that it will contain a library of genetic information for use in specimen identification. Based on a combination of recent research and our own laboratory work, we aim in this paper to: 1) discuss the feasibility and irreplaceability of herbarium specimens as materials for use in the iFlora; 2) summarize the major difficulties apparent in the process of genetic information acquisition from herbarium specimens and provide some viable solutions; 3) clarify the importance and significance of herbarium specimens especially the holotype for building the reference library in the iFlora.

 DNA barcoding comprises two distinct aims: specimen identification and species discovery. To achieve these goals, a DNA barcode reference library is indispensable and is currently under construction for Chinese plants. In this paper, we used the existing public database, NCBI, and also our DNA barcode reference library, which is under construction, to identify plant specimens. By combining DNA barcode sequences with scientific names determined by taxonomists, we first attempted to find incorrectly identified specimens and also to check specimens whose DNA sequences were wrongly labeled. Secondly, we attempted the identification of unknown specimens in some groups for which DNA barcodes are available. Thirdly, we make some suggestions for the development of a plant DNA barcode reference library.

 Because of the complexity of plant diversity, we cannot guarantee the accuracy of an iFlora if there is no proper understanding of methods of species identification and classification. The existence of a large number of “difficult” and controversial species, as well as taxonomic variation within species, will further add problems to the construction of such a flora. In this paper, we consider DNA barcoding information and morphological classification with reference to our recent studies of Dioscorea L.(Dioscoreaceae), Polygonum multiflorum Thuna. (Polygonum), Prunella L.(Lamiaceae). We point out that for widely distributed species in these groups we might often ignore continuous variation that links what might be described as different species, but which are better considered as divergent populations of the same species. In such cases, variation in DNA barcodes could exist between populations of one species, which might cause confusion in species identification in an iFlora.

DNA barcoding involves species identification using a short and standardized DNA region. This method has many advantages over traditional morphologybased identification, including greater effectivity, sensitivity, reliability, and standardization. In this study, we used 43 whorled/oppositeleaved species from the genus Pedicularis as a model system to test the utility of DNA barcoding for rapid identification of species in the taxa where traditional identification methods are timeconsuming and inaccurate. Four candidate DNA barcoding loci (three chloroplast: rbcL, matK, trnHpsbA, and one nuclear: ITS) were evaluated by tree and distancebased methods using a total of 164 accessions. Our results showed that species identification rates using the nuclear ITS locus (81.40% and 89.57% for tree and distancebased methods, respectively) surpassed those of the three chloroplast barcodes and all combinations of barcodes. In addition, the nuclear ITS locus was able to help identify some species that had previously been difficult to differentiate using only morphological traits. The demonstrated ability of DNA barcoding to efficiently discriminate among these Pedicularis species highlights the practicality and importance of its utility for the nextgeneration intelligent Flora (iFlora) in fulfilling the rapid and accurate identification of species. This new identification method will provide key information for traditional taxonomy and for investigations of ecology, evolutionary biology, population genetics, and conservation genetics, etc.

Begonia is the biggest genus in the Begoniaceae and exhibits high morphological and ecological diversity, making intrageneric classification and species delimitation difficult, especially in the case of closely related species. In this respect, DNA barcoding has the advantage of achieving rapid species identification without relying on morphological characteristics, once a database containing speciesspecific sequences is established. In this study, we tested four candidate plant barcoding regions, including three chloroplast loci (rbcL, matK, trnHpsbA) and one nuclear locus (ITS), in 136 samples representing 26 species of Begonia. The results demonstrated that species identification based on rbcL, matK and trnHpsbA sequences was poor due to low interspecific variation. Conversely, ITS/ITS2 sequences showed significant intra and interspecific divergence enabling a high level of species discrimination (96%-100%). Consequently, ITS/ITS2 could be considered as a potential barcode for identifying Begonia species, which supports the proposal by the China Plant BOL Group that ITS/ITS2 should be incorporated into the core barcode for seed plants.

iFlora is designed to integrate traditional taxonomy knowledge with DNA sequencing and biological information. Through a series of key technological innovations and integrations, the main objective of iFlora is to construct the nextgeneration Flora or intelligent Flora, which should fulfill the function of accurately and rapidly identifying species and acquiring related digital information. The most important and urgent task for iFlora development is to search for the standard DNA barcode to distinguish most of the plants and economically important mushrooms. To establish a standard DNA barcode for the edible boletes in Yunnan, samples of common boletes in the free markets regarded as four “species” by local mushroom hunters were collected and studied. Our date indicated that these four “species” in fact represent 12 distinct species. Furthermore, four candidate markers, the large subunit nuclear ribosomal RNA (nrLSU), the translation elongation factor 1alpha (tef1α), the RNA polymerase II largest subunit (rpb1) and the RNA polymerase II second largest subunit (rpb2) were tested using the eukaryotic general primers for their feasibility as barcodes of the species of Boletus. This study indicates that the four candidate markers are of very high PCR amplification and sequencing success rate (100% and 100% respectively), and there are no overlapping between intra and inter specific variation. All four candidate markers showed high species resolution. Compared with nrLSU, rpb1, tef1α and rpb2 had much more clearly defined barcoding gaps. This study showed that rpb1 can be used as a primary barcode marker considering that rpb1 showed high sequence variation among species and low variation within species, while tef1α and rpb2 can be proposed as supplementary barcodes for the boletes.

To evaluate how effective DNA barcoding is for the identification of subtropical forest trees, we sampled 525 individuals representing 204 species in 111 genera of 51 plant families that occur in the Ailao Mountains Nature Reserve, and tested the ability of rbcL, matK, trnHpsbA and ITS sequences to discriminate species. PCR success was over 90% for each of these four sequences, while sequencing success rate was highest for rbcL and matK (90.7% and 90.5%, respectively), followed by trnHpsbA (83.6%), and lowest for ITS (73.7%). Thus, all four sequences showed a relatively high level of applicability for subtropical forest trees that occur in the Ailaoshan Mountains Nature Reserve. Using two different “species identification” methods - BLAST and Neighbor Joining (NJ)—the highest rate of success for identification at species (68.4%-81.3%) and genus (99.0%-100%) levels was obtained using ITS when only a single region was used. When two molecular regions were used in combination, rbcL and matK correctly identified 52.8%-60.2% of species and 86.7%-90.5% of genera, while using all four regions in combination correctly discriminated 74.7%-79.6% of species. The relatively low sequencing success rate of ITS was mainly due to failure in certain groups (such as Lauraceae and Fagaceae), which play an important role in subtropical forest, suggesting that the ITS region may not be appropriate for DNA barcoding these particular plant groups.

 Bamboos are a taxonomically difficult group. DNA barcoding provides another supplementary method for the bamboo identification. The core DNA barcodes (rbcL+matK) have quite low discriminatory power in bamboos, therefore, it is crucial to select effective DNA barcodes for bamboos. We scanned the entire chloroplast genomes that were published in Bambusoideae in order to search highly variable regions. Sixteen regions were selected including one coding region and 15 intergenic spacers, and the universality, sequencing success rates, and the variable characters were testified in 30 taxa of 22 species in 10 genera of Arundinarieae and Bambuseae. The results indicated that (1) the amplification success rates were high for each region, but there were some problems in sequencing for some regions due to polystructures; (2) indels should be coded as informative characters; (3) the region trnGtrnT(t) was the most variable one among the 16 regions and it was suggested as the prior candidate DNA barcode in Bambusoideae; (4) the combination “trnGtrnT(t)+X” can be used in DNA barcoding and molecularphylogeny reconstruction study of bamboos.

DNA barcoding is an approach for rapid and accurate species identification using a standard DNA region. Ideal barcodes should have high level of universality in PCR and sequencing. Although the nuclear ribosomal DNA (nrDNA) internal transcribed spacers 2 (ITS2) was proposed as potential DNA barcode for seed plants, including gymnosperms, no suitable ITS2 primer pair for gymnosperms available shows high universality in either aspect. To obtain a highly universal ITS2 primer pair, we newly designed three forward primers for ITS2 based on sequences in the 5.8S gene greatly conserved among 55 genera of gymnosperms. Combined with previously published reverse primers of ITS, seven candidate primer combinations for ITS2 were assessed for their universality. A total of 56 species, representing 40 genera from all the 12 families of the eight orders of gymnosperms, were sampled in this study. Three ITS2 primer combinations, 5.8SR/ITS4, 5.8SRa/ITS4, and 5.8SF2/S3R, were excluded due to their low universality or because of yielding double bands in the PCR in the familylevel assessment. The remaining four primer combinations, GYM_5.8SF1/ITS4, GYM_5.8SF1/S3R, GYM_5.8SF2/ITS4, and S2F/S3R, showed high PCR universality (100%) on all 56 sampled species. Based on our overall assessment of PCR and sequencing universality, brightness of PCR bands, and sequence quality and coverage, we propose the primer combination GYM_5.8SF2/ITS4 as ITS2 barcode for gymnosperms because it was shown to be the most “universal”.

DNA barcoding is a technique used to identify species, which is now widely employed in different biological disciplines. With the development and modification of DNA barcoding, it has become a useful tool for ecological studies. Here, we review the uses of DNA barcoding for quick species identification and/or new and cryptic species discovery, in community phylogenetic reconstruction and ecological forensics, and in interaction networks among species within a community. The techniques of DNA metabarcoding and environmental DNA metabarcoding used in ecological research such as diet analysis and biodiversity assessment are also examined. Finally, we discuss the future prospects of DNA barcoding in ecological research following the development of nextgeneration sequencing technologies, improvement of Largescale Scientific Facilities, and use of new models and software for data analysis.

Botany has entered a postFlora era. Benefited from traditional plant taxonomy and related studies, the success of iFlora requires the integration of DNA barcoding through state of art highthroughputsequencing technologies and portable equipment for rapid taxonomic identification and information platform. Traditional plant identification relies mostly on morphological features, while DNA barcoding has been widely applied as a complementary molecular approach to facilitating fast species identification. However, issues remain in classic DNA barcoding, e.g., specieslevel resolution can only be achieved via multiple barcode markers; taxon identity cannot be revealed for pooled plant samples via Sanger sequencing. In this review, the authors introduce recent developments in metabarcoding, which is based on the nextgenerationsequencing technologies, and its applications in a few key aspects in plant diversity and its potential role in iFlora.

The diversity and conservativeness of seed morphological characteristics are extremely important and useful in identifying and classifying plants, and analyzing their phylogenetic relationships. iFlora is the next generation intelligent flora, its ultimate purpose is to help people identify and understand plants precisely and easily. This paper discusses the assistant roles of seed morphological and anatomical characteristics for the development and application of iFlora, and suggests that these characteristics could be incorporated as one of the essential elements of iFlora, which not only will be referred in identifying and classifying plants, but also may be applied to verify the phylogenetic relationships established through the molecular methods. Meanwhile the information of seed morphology can reenrich the contents of iFlora in the description of plant species.

 Magnetic Bead (MB) has been widely applied in life science for its high surface area, coupling capacity with biomolecules and convenience of being manipulated. With the development of Micro Electro Mechanical systems (MEMS) technologies, MBbased biomicrosystem provide a new approach for separation and detection of biological samples. Integrating modern DNA sequencing technologies and applying highspeed computer digitization, network technology and cloud computing analysis platform for collection and management of species information, the nextgeneration Flora, or iFlora is an intelligent application system for plant species identification and related information acquisition. Efficient genetic information acquiring technology is key in building iFlora. This paper briefly introduces the MBbased biomicrosystem technology and its application in molecular biology. The authors suggest a new MBbased biomicrosystem iFlora genetic information collection system. Combined with MB technology, the total analyses from cell lysis to DNA sequencing will be achieved on microchip, which will realize the quick and efficient acquirement of genetic information.

iFlora Plan aims to raise the public knowledge on plants. As software development for mobile equipments is an important part of iFlora Plan, we designed and developed iFlora beta, a software for iFlora on a mobile platform, based on the China flora database. With Android system and iOS system, iFlora beta can offer services such as basic search for plant species, wizard search, expert interaction and system customization, which makes it possible to quickly search for plant data and identify plant species. We also discussed the way for developing a flora search feature database and an image feature database, which built the bases for the general goal of iFlora Plan.

 There are many differences in how Chinese lycophytes and ferns are classified in Ching’ classification, Fern Families and Genera of China (FFGC), Flora Republicae Popularis Sinicae (FRPS), the new vascular plant linear classification (Christenhusz’s classification), and Flora of China (FOC). In this paper, we compare these major classifications at family and genus levels. We found 63 families and 223 genera of Chinese lycophytes and ferns listed in Ching’s classification, 63 families and 226 genera in FFGC, 61 families and 220 genera in FRPS, 38 families and 160 genera in the linear classification and 38 families and 177 genera listed in FOC. Details of variation in assignment of taxa to families and genera are highlighted, as well as disagreements in presenting Chinese names, Latin names and authorities of species. Some references for data searching and specimen identification are presented. This paper should provide a template for compiling an iFlora of Chinese lycophytes and ferns.