L-PEACH has been implemented using the general-purpose plant modeling software. This makes it possible to keep the model code compact by delegating generic issues to the general functionality of. As a result, L-PEACH is easy to maintain and conducive to simulated experimentation. Furthermore, it may serve as a template for constructing other functional–structural models that involve solving complex systems of equations in growing plant structures. In a comprehensive review of carbon-based tree growth models, Le Roux et al. pointed out that three critical issues have not been adequately addressed by most models: adequate representation of the dynamic and feedback aspects of carbon allocation on tree structure and carbon acquisition; explicit treatment of carbon storage reserves and remobilization over multiple years; and integration of below ground processes and tree water and nutrient economies into whole-plant function. Although the work is far from complete, the L-PEACH model provides a platform for addressing all these critical issues. By combining the sink-driven carbon partitioning concepts of the original PEACH model into a distributed network of architecturally explicit sources and sinks, factors such as the proximity of individual sinks to other sinks and sources, as well as the transport resistances between these entities, can be accounted for and become involved in growth and carbon allocation outcomes. However, although there are some experimental data to indicate the functional nature of these relationships , more data will be required before the model is fully calibrated. Considerable conceptual experimental research will be necessary to provide quantitative data required for this calibration. This model also explicitly addresses carbohydrate storage in stems and roots during the growing season,growing blueberries in pots and remobilization of stored carbohydrates during the spring growth flush.

In the process of developing this model, we became increasingly aware of the lack of information about the quantitative dynamics of carbohydrate reserves in trees. As pointed out by Le Roux et al. , the lack of knowledge of the mechanisms driving reserve deposition and remobilization is a major obstacle for evaluating the carbon available at any given time, or for relating reserve dynamics with internal and external variables in tree growth models. Based on preliminary data on root starch concentrations in peach trees we have chosen to treat the starch reserve sinks in stems and root segments as compartments that have sink capacities proportional to their annual growth increment. These reserves then become carbon sources during the spring flush . Current model functions related to carbohydrate reserves are based on preliminary data, and it is our intention to test more fully and quantify these aspects of the model in the near future. The inclusion of the water stress/interaction component in the model is an attempt to demonstrate how root function can be incorporated into a dynamic L-system model of this type. As with carbon storage, the relationships between developing water stress and physiology are based more on published conceptual relationships than on precise quantitative data collected for the purpose of calibrating the model. Nevertheless, the potential of this model to simulate functional interactions between root and shoot processes is readily apparent. Similarly, there is clearly the potential to incorporate additional root processes such as nutrient uptake into the model to more fully capture the functional dynamics of root– shoot interactions. Future developments could also involve the integration of existing architecturally based models of carbon transport and partitioning in roots , in order to model root function more explicitly.The Ranunculaceae is a large and complex plant family, including approximately 59 genera and 2,500 species . Pulsatilla Miller, first described in 1753, consists of about 40 species that are restricted to temperate subarctic and mountainous areas in the Northern Hemisphere .

Plants of Pulsatilla species are often covered with long, soft hairs. Their flowers are solitary and bisexual, with three bracts forming a bell-shaped involucre. The tepal number is always six, and stamens are generally numerous, with the outermost ones resembling degenerated petals . Most authors have treated Pulsatilla as a subgenus or section of the genus Anemone s.l. . However, Miller , Adanson , and Wang et al. have supported a model that separates Pulsatilla from Anemone as an independent genus. Recent phylogenetic studies have shown that all species within Pulsatilla are clustered in a monophyletic group, which is nested within Anemone . Morphologically, Pulsatilla can easily be distinguished from Anemone s.s., since species of the former have a long, plumose beak on the achenes formed by the persistent style and stamens whereas species of the latter do not. Because the primary goal of the present study is to test the use of DNA barcodes for species in the Pulsatilla clade, we here follow the treatment of Wang et al. and Grey-Wilson , regarding Pulsatilla as a distinct genus. There are eleven species of Pulsatilla found in China, most of which are found primarily in the northern part of the country . Some species of Pulsatilla have been used in traditional Chinese medicine for many years for “blood-cooling” or “detoxification” . In particular, the root of Pulsatilla chinensis Regel is a well-known ingredient included in the Chinese Pharmacopoeia . Many species used in folk medicine have been found to contain pharmacologically useful chemical components, including those with anti-cancer and anti-inflammatory activities . The contents of these components differ in various species, resulting in different clinical pharmacological effects. Thus, in cases where target species can be easily confused with their close relatives, undesired species can be inadvertently collected, resulting in negative effects on drug efficacy and patient safety, as has been shown in other plant groups of medicinal importance in China . Pulsatilla is an especially challenging, complex group. In all treatments published to date, the genus has been treated as comprising two to four subgenera: subgenus Miyakea, which contains only one species, P. integrifolia; subgenus Kostyczewianae, which has only one species, located in Central Asia and northwestern China; subgenus Preonanthus, which includes six species; and the largest subgenus Pulsatilla, which comprises 29 species.

However, Pulsatilla shows a frustratingly complicated pattern of intrageneric morphological variability . The recognition and identification of wild Pulsatilla species based on traditional approaches is difficult due to transitional intraspecific morphological characteristics in many Pulsatilla species. For instance, P. turczaninovii and P. tenuiloba were considered to be two separate species that could be told apart by the number of pairs of lateral leaflets . After carefully checking specimens and population investigation, we found that the leaflet numbers of P. turczaninovii and P. tenuiloba are overlapping,drainage gutter and some individuals have both 4 and 5 pairs of lateral leaflets. Flowers nodding before anthesis is recorded as a diagnostic character of P. campanella, but this character was also found in P. ambigua, P. cernua and P. dahurica, and their flower colors show continuous transitional shades of blue . Thus, these characters are not reliable and make Pulsatilla difficult to identify. DNA barcoding aims to achieve rapid and accurate species recognition by sequencing short DNA sequences or a few small DNA regions . This technology was first developed to identify animal species; for example, Hebert et al. argued that “the mitochondrial gene cytochrome c oxidase I , can serve as the core of a global bio-identification system for animals”. Studies have continued to demonstrate that the COI gene fragment efficiently discriminates among animal species, including amphibians , birds , fish , and insects . In plants, however, frequent recombination and low mutation rates restrict the utility of mitochondrial barcode markers . The search for suitable candidates has therefore focused on chloroplast and nuclear DNA markers , although such markers are not always easy to amplify and sequence in all plant taxa using universal primers. Numerous studies have suggested that four standard barcodes — three from the chloroplast genome [the ribulose-bisphosphate/ carboxylase Large-subunit gene , the maturase-K gene , and the trnH-psbA intergenic spacer and the nuclear ribosomal internal transcribed spacers ] — should be used as core barcode markers for the molecular identification of plants . Significant progress has been made in DNA barcoding in plants . However, the discrimination of closely related species using only molecular data is still a major challenge in some genera . Morphological characters, including the shape of nutritive and reproductive organs, remain highly valuable for plant identification and studies of plant evolution . Micromorphological characters have been shown to have great value for species identification and systematics , and these have rarely been considered by previous barcode studies. However, the combination of morphological data and DNA barcodes may be essential for species discrimination, especially in closely related species . Previous molecular phylogenetic studies have included few species from the genus Pulsatilla . In a recent phylogenetic study of Pulsatilla, few species were from Asia and few individuals were collected for one species . Obtaining DNA barcode data from a dataset created by comprehensive sampling of a taxonomically difficult genus such as Pulsatilla should contribute to understanding the discriminatory potential of barcodes in morphologically complex clades. The establishment of an available barcoding system for Pulsatilla may also facilitate further utilization of these taxa, as well as further research into their taxonomy. In this study, four DNA barcode regions were assessed in 19 species of Pulsatilla. Approximately 50% of the accepted species of Pulsatilla found in Europe and the Americas were included, as were 90% of the species found in China.

Our objectives were to: test the effectiveness of common core DNA barcodes in Pulsatilla, evaluate the resolution of these four barcodes, and use 2- to 4-region combinations to correctly identify individuals. We also aimed to develop a protocol that could effectively discriminate among closely related species, primarily for species discrimination of medicinal plants. In addition, we added micro-morphological analyses of leaf tissue obtained using scanning electronic microscopy to reveal the taxonomic relationships among Pulsatilla.In total, 52 accessions representing 19 Pulsatilla species were involved in this study . This sample covered each of the three subgenera from Asia, Europe, and America. Nine samples were sourced from herbarium specimens, while 43 samples were newly collected. All samples were taxonomically identified using published floras, monographs, and references. In total, one to five individuals per species were sampled from different populations in the wild. Fresh leaves were dried in silica gel upon collection and the longitude, latitude, and altitude of each collection site were recorded using a GPS unit . Voucher specimens were stored in the Herbarium of Northwest A&F University and the US National Herbarium . Singleton species were only used as potential causes of failed discrimination, and were not included in the calculation of the identification success rate. Three members of Anemone, two of Clematis, one of Anemoclema, and one of Hepatica were selected as outgroups for tree-based analyses.In this study, the short DNA sequences ITS and trnH–psbA had the best performance in PCR amplification and sequencing among the four barcode markers . Moreover, successful sequencing rates for sequences ITS and trnH–psbA were over 90% for silica-dried samples but lower for herbarium specimens. These findings are consistent with many previous studies . In addition, the varying lengths of insertions/deletions found at the trnH–psbA loci for different species provide important phylogenetic information and species discrimination power . Thus, sequence alignments of this region must be performed with great care to avoid overestimating substitution events. The rbcL and matK genes are approximately 1,428 bp and 1,570 bp in length, respectively . The greatest problem with rbcL and matK was that it was difficult to amplify them from the degraded DNA isolated from old herbarium specimens, since the short lengths of remaining fragments hampered the extension phase of the PCR for these longer genes. Although some problems may be alleviated by using additional pairs of primers, the amplification and sequencing success rate of the old herbarium samples remained poor. Thus, we were not able to obtain all sequences for all herbarium samples.An ideal DNA barcode should be universal, reliable, cost effective, and show considerable discriminatory power. Because none of the proposed single-locus barcodes perfectly meets all these criteria.Multilocus barcodes can often improve the resolution rate of species identification . In the present study, when evaluated alone, the species resolutions based on tree-building for the three chloroplast regions rbcL, matK, and trnH-psbA were 48.78, 14.63, and 9.30%, respectively.