Phylogenetic relationships of the Santalales and relatives

Summary Determining relationships among parasitic angiosperms has often been difficult owing to frequent morphological reductions in floral and vegetative features. We report 18S (small-subunit) rRNA sequences for representative genera of three families within the Santalales (Olacaceae, Santalaceae, and Viscaceae) and six outgroup dicot families (Celastraceae, Cornaceae, Nyssaceae, Buxaceae, Apiaceae, and Araliaceae). Using Wagner parsimony analysis, one most parsimonius tree resulted that shows the Santalales to be a holophyletic taxon most closely related toEuronymus (Celastraceae). The santalalean taxa showed approximately 13% more transitional mutations than the group of seven other dicot species. This suggests a higher fixation rate for mutations in these organisms, possibly owing to a relaxation of selection pressures at the molecular level in parasitic vs nonparasitic plants. Outgroup relationships are generally in accord with current taxonomic classifications, such as the grouping of Nyssaceae and Cornaceae together (Cornales) and the grouping of Araliaceae with Apiaceae (Apiales). These data provide the first nucleotide sequences for any parasitic flowering plant and support the contention that rRNA sequence analysis can result in robust phylogenetic comparisons at the family level and above.     

Phylogenetic relationships of Cornaceae and close relatives inferred from matK and rbcL sequences

Phylogenetic relationships were inferred using nucleotide sequences of the chloroplast gene matK for members of Cornales, a well-supported monophyletic group comprising Cornaceae and close relatives. The shortest trees resulting from this analysis were highly concordant with those based on previous phylogenetic analysis of rbcL sequences. Analysis of a combined matK and rbcL sequence data set (a total of 2652 bp [base pairs]) provided greater resolution of relationships and higher internal support for clades compared to the individual data sets. Four major clades (most inclusive monophyletic groups) of Cornales are indicated by both sets of genes: (1) Cornus-Alangium, (2) nyssoids (Nyssa-Davidia-Camptotheca)- mastixioids (Mastixia, Diplopanax), (3) Curtisia, and (4) Hydrangeaceae-Loasaceae. The combined evidence indicates that clades 2 and 3 are sisters, with clade 4 sister to the remainder of Cornales. These relationships are also supported by other lines of evidence, including synapomorphies in fruit and pollen morphology and gynoecial vasculature. Comparisons of matK and rbcL sequences based on one of the most parsimonious rbcL-matK trees indicate that matK has a much higher A-T content (66.9% in matK vs. 55.8% in rbcL) and a lower transition:transversion ratio (1.23 in matK vs. 2.21 in rbcL). The total number of nucleotide substitutions per site for matK is 2.1 times that of rbcL in Cornales. These findings are similar to recent comparisons of matK and rbcL in other dicots. Variable sites of matK are almost evenly distributed among the three codon positions (1.0:1.0:1.3), whereas variable sites of rbcL are mostly at the third position (1.8:1.0 :7.5). Among- lineages rates of nucleotide substitutions in rbcL are basically homogeneous throughout Cornales, but are more heterogeneous in matK.     

Fatty acids of some cornaceae,hydrangeaceae, aquifoliaceae,hamamelidaceae and styracaceae

Seed, kernel or fruit oils of the Cornaceae (nine species). Hydrangea, Hamamelis, Ilex (Aquifoliaceae) and the Styracaceae (two species) were analysed for fatty acid composition. Special attention was paid to the occurrence of petroselinic acid (18: 1Δ6c). In the species investigated. C₁₈ acids were always present in greater quantities than C₁₆ fatty acids; C₂₀ and C₂₂ acids were only minor components. The Cornaceae show differing fatty acid patterns which correlate well with anatomical, morphological and other chemical data. In Cornus, Curtisia, Mastixia and Corokia linoleic acid predominates, whereas linoleic and linolenic acid form the major components in Davidia and Nyssa. 18 : 1Δ6c, an aralioid type, occurs in large amounts in Aucuba and Griselinia. Hamamelis, Hydrangea and Ilex show a common fatty acid pattern with linoleic acid as the dominant component in all cases. Classification currently based on morphological and anatomical differences between the two species of Styracaceae which were investigated here should include their different fatty acid compositions: in Halesia linoleic acid predominates over oleic acid, whereas in Styrax equal amounts of these two acids are found.     

从rbcL序列探讨单室茱萸属的系统位置

为了探讨单室茱萸属Mastixia的系统位置,本文对八蕊单室萸（Mastixia euony moides),云南单室茱萸（M.pentandra subsp,chinensis)及近缘物种云南紫树（Nyssa yunnanensis)和毛八角枫（Alangium kurzii)的cpDNA中编码1,5－二磷酸核酸糖羧化酶大亚基的rbcL基因序列进行了双脱氧测定。通过对这些序列与广义山茱萸科Cornaceae和Mastixia曾被归入的科的类群的rbcL序列进行PAUP（Phylogenetic Analysis Using Parsimony)分析,得到24棵最简约的分支树图。PAUP分析表明单室茱萸属与马蹄参属Diplopanax,紫树属Nyssa,喜树属Camptotheca和珙桐属Davidia的亲缘关系最近,并由分支图支持可以将Mastixia提升为山茱萸目Cornales中的一个科－单室萸科Masstixiaceae。     

Metabolite fingerprinting,pathway analyses,and bioactivity correlations for plant species belonging to the Cornaceae,Fabaceae, and Rosaceae families

Key message

A multi-parallel approach gauging the mass spectrometry-based metabolite fingerprinting coupled with bioactivity and pathway evaluations could serve as an efficacious tool for inferring plant taxonomic orders.

Abstract

Thirty-four species from three plant families, namely Cornaceae (7), Fabaceae (9), and Rosaceae (18) were subjected to metabolite profiling using gas chromatography–time-of-flight-mass spectrometry (GC–TOF-MS) and ultrahigh performance liquid chromatography–linear trap quadrupole-ion trap-mass spectrometry (UHPLC–LTQ-IT-MS/MS), followed by multivariate analyses to determine the metabolites characteristic of these families. The partial least squares discriminant analysis (PLS-DA) revealed the distinct clustering pattern of metabolites for each family. The pathway analysis further highlighted the relatively higher proportions of flavonols and ellagitannins in the Cornaceae family than in the other two families. Higher levels of phenolic acids and flavan-3-ols were observed among species from the Rosaceae family, while amino acids, flavones, and isoflavones were more abundant among the Fabaceae family members. The antioxidant activities of plant extracts were measured using ABTS, DPPH, and FRAP assays, and indicated that extracts from the Rosaceae family had the highest activity, followed by those from Cornaceae and Fabaceae. The correlation map analysis positively links the proportional concentration of metabolites with their relative antioxidant activities, particularly in Cornaceae and Rosaceae. This work highlights the pre-eminence of the multi-parallel approach involving metabolite profiling and bioactivity evaluations coupled with metabolic pathways as an efficient methodology for the evaluation of plant phylogenies.

     

Hironoia fusiformis gen. et sp. nov.; a cornalean fruit from the Kamikitaba locality (Upper Cretaceous,Lower Coniacian) in northeastern Japan

The application of sieving techniques to bulk samples from the Ashizawa Formation, Futaba Group (Lower Coniacian) of northeastern Honshu, Japan, has yielded well-preserved mesofossil assemblages comparable with those recently described from eastern North America, Europe, and central Asia. Among the most abundant and distinctive components of these assemblages are fusiform fruits that are assigned here to a new genus and species, Hironoia fusiformis gen. et sp. nov. The fruits developed from an epigynous ovary with three to four locules. Each locule bears one seed and has a distinctive dorsal germination valve. These features of the fruit, along with the adnate calyx, indicate an affinity to extant Cornales and specifically the Cornaceae sensu lato. The recognition of an unequivocal cornalean fruit in the Early Coniacian–Early Santonian of Japan provides the earliest record of this group in the fossil record. It also establishes a minimum age for the early divergence of the asterid clade, a major group of living angiosperms comprising more than a third of all species of extant flowering plants. Electronic Publication     

马蹄参属的解剖学特征及其系统学意义

马蹄参属Diplopanax Hand.-Mazz.被不同的作者放在五加科Araliaceae、山茱萸科Cornaceae或单室茱萸科Mastixiaceae中,本文报道了马蹄参Diplopanax stachyanthus Hand.-Mazz.的解剖学特征,通过比较研究支持将马蹄参属从五加科中移出,改隶于单室山茱萸科。马蹄参属在山茱萸目Cornales中处于比较原始的演化水平。     

INFERIOR OVARY AND GENERIC AFFINITIES OF GARRYA

Characters of the inflorescence and flower distinguish 2 sections in Garrya. Female flowers of sect. Garrya bear 2 small epigynous appendages, usually considered a rudimentary perianth. In sect. Fadyenia, female flowers are nude, but occasionally 2 more or less foliaceous bracts are partially adnate to the ovary, sometimes simulating the appearance of appendages in sect. Garrya. Authors who have observed this infer that the adnate bracts are homologous to epigynous appendages in sect. Garrya and that the ovary, therefore, cannot be considered inferior. This inference, if valid, would weigh against a close relationship between Garrya and Cornaceae. Clearing and sectioning show, however, that the vascular pattern in flowers of sect. Garrya is similar to the pattern in many Cornaceae. Perianth bundles are united with ventral carpellaries for most of their length, whereas bundles of the adnate bracts in sect. Fadyenia are associated with dorsal carpellary strands; therefore, the 2 kinds of appendages are not homologous. In gynoecial characters Garrya most closely resembles Griselinia and, to a lesser extent, A ucuba. These 3 genera probably had a common origin within the Cornaceae.     

Phytoserological systematic survey of the caprifoliaceae

Although several investigators have presented data suggesting that the Caprifoliaceae is polyphyletic in origin, the family has been retained as a taxon in recent classifications. Serological analyses of the taxa included within the family have been undertaken to provide additional taxonomic data. Precipitin techniques of double diffusion (Ouchterlony) and nephelometry (Boyden Procedure) were used to analyse serological similarities of seed proteins from representatives of the family. Three distinct groupings were detected for the taxa included in the Caprifoliaceae. AlthoughViburnum exhibited greatest serological similarity withSambucus, Viburnum was quite distinct from all members of the family investigated.Sambucus also was somewhat distinct from the other genera and exhibited about as much serological correspondence withCornus of the Cornaceae as with any tested member of the Caprifoliaceae. The remaining species examined formed a very close serological grouping. These data can be interpreted as supporting either a monophyletic or polyphyletic origin of the family as presently composed, because of the different arrangements of the families Caprifoliaceae and Cornaceae in the various systems of classification.     

Relationships within Cornales and circumscription of Cornaceae-matK and rbcL sequence data and effects of outgroups and long branches

Phylogenetic relationships in Cornales were assessed using sequences rbcL and matK. Various combinations of outgroups were assessed for their suitability and the effects of long branches and outgroups on tree topology were examined using RASA 2.4 prior to conducting phylogenetic analyses. RASA identified several potentially problematic taxa having long branches in individual data sets that may have obscured phylogenetic signal, but when data sets were combined RASA no longer detected long branch problems. t(RASA) provides a more conservative measurement for phylogenetic signal than the PTP and skewness tests. The separate matK and rbcL sequence data sets were measured as not containing phylogenetic signal by RASA, but PTP and skewness tests suggested the reverse [corrected]. Nonetheless, the matK and rbcL sequence data sets suggested relationships within Cornales largely congruent with those suggested by the combined matK-rbcL sequence data set that contains significant phylogenetic signal as measured by t(RASA), PTP, and skewness tests. Our analyses also showed that a taxon having a long branch on the tree may not be identified as a "long-branched" taxon by RASA. The long branches identified by RASA had little effect on the arrangement of other taxa in the tree, but the placements of the long-branched taxa themselves were often problematic. Removing the long-branched taxa from analyses generally increased bootstrap support, often substantially. Use of non-optimal outgroups (as identified by RASA) decreased phylogenetic resolution in parsimony analyses and suggested different relationships in maximum likelihood analyses, although usually weakly supported clades (less than 50% support) were impacted. Our results do not recommend using t(RASA) as a sole criterion to discard data or taxa in phylogenetic analyses, but t(RASA) and the taxon variance ratio obtained from RASA may be useful as a guide for improved phylogenetic analyses. Results of parsimony and ML analyses of the sequence data using optimal outgroups suggested by RASA revealed four major clades within Cornales: (1) Curtisia-Grubbia, (2) Cornus-Alangium, (3) Nyssa-Camptotheca-Davidia-Mastixia-Diplopanax, and (4) Hydrangeaceae-Loasaceae, with clades (2) and (3) forming a monophyletic group sister to clade (4) and clade (1) sister to the remainder of Cornales. However, there was not strong bootstrap support for relationships among the major clades. The placement of Hydrostachys could not be reliably determined, although most analyses place the genus within Hydrangeaceae; ML analyses, for example, placed the genus as the sister of Hydrangeeae. Our results supported a Cornales including the systematically problematic Hydrostachys, a Cornaceae consisting of Cornus and Alangium, a Nyssaceae consisting of Nyssa and Camptotheca, a monogeneric Davidiaceae, a Mastixiaceae consisting of Mastixia and Diplopanax, and an expanded Grubbiaceae consisting of Grubbia and Curtisia, and two larger families, Hydrangeaceae and Loasaceae.     

SEROLOGICAL INVESTIGATION OF THE SYSTEMATIC POSITION OF THE CAPRIFOLIACEAE. I. CORRESPONDENCE WITH SELECTED RUBIACEAE AND CORNACEAE

Serological techniques of double diffusion (Ouchterlony) and nephelometry (Boyden Procedure) were employed to analyze the serological correspondence of taxa belonging to the Caprifoliaceae with selected taxa belonging to the Rubiaceae and the Cornaceae. Cornus (Cornaceae) exhibited relatively high correspondence with most representatives of the Caprifoliaceae, especially with Sambucus. The correspondence of Viburnum with Cornus was low. The serological correspondence of genera of the Caprifoliaceae with three genera of the Rubiaceae was relatively low indicating that these two families are serologically distinct. The correspondence between the Caprifoliaceae and the Cornaceae suggests a closer relationship between these two families than between the Caprifoliaceae and the tested genera of the Rubiaceae. These limited data suggest that the Rubiaceae and the Caprifoliaceae should not be placed in one taxonomic order. However, the serological variation for additional taxa within the Rubiaceae must be ascertained before more conclusive statements can be made relative to the degree of serological similarity between the two families.     

Some fossil woods from the Palaeogene of Northern Kyushu,II

Four new species of petrified dicotyledonous woods obtained from the Oligocene of Tsuyazaki, fukuoka Prefecture have been described in a continuation of work from the previous paper;Acer palmatoxylum (Aceraceae),Cornus tsuyazakiensis (Cornaceae),Fraxinus oligocenica (Oleaceae) andHovenia palaeodulcis (Rhamnaceae). The fossil woods of those families are new records from the Palaeogene of Japan.     

Aplopsora corni sp. nov. onCornus controversa from Hokkaido,Japan

Aplopsora corni sp. nov. is proposed for a rust fungus whose uredinial and basidial stage occurs onCornus controversa (Cornaceae) in Hokkaido. This new species is separated by its larger urediniospores and probasidia from the morphologically closely relatedA. nyssae onNyssa aquatica andN. sylvatica (Cornaceae) distributed in southern North America.     

Architectural strategies of Cornus sericea, a native but invasive shrub of Southern Quebec, Canada, under an open or a closed canopy

Background and Aims

Qualitative and quantitative studies of the pattern of invasive plant development is considered a key aspect in understanding invasiveness. An architectural analysis was therefore performed in order to understand the relationship between shoot architecture and invasiveness in red-osier dogwood, Cornus sericea (Cornaceae).

Methods

The structural and ontogenic characteristics of individuals in invading and non-invading populations in the native range of the species were compared to test the implication of developmental plasticity on invasiveness.

Key Results and Conclusions

The results show that the shrub has a modular architecture governed by strong developmental rules. Cornus sericea is made up of two levels of organization, each with its own intrinsic sequence of differentiation. These intrinsic mechanisms were used as a framework for comparison and it was found that, in response to the light environment, developmental plasticity was elevated, resulting in two architectural strategies. This developmental plasticity concerns the growth direction and the size of the modules, the speed of their time-course changes, their branching and flowering. Under an open canopy, C. sericea rapidly develops large vertical structures and abundant flowering. This strategy leads the plant to be invasive by excluding competitors and disseminating in the landscape. In the understorey, C. sericea slowly develops long horizontal structures which creep across the soil surface, while assimilating structures are poorly developed. This strategy does not lead to invasiveness but may allow the plant to survive in the understorey and reach sunny patches.     

ComprehendingCornus: Puzzles and progress in the systematics of the dogwoods

Dogwoods evolved in two main lineages, a red-fruited line in which the inflorescences have basal bracts, and a blue-(or white-)fruited line in which the bracts are rudimentary or lacking. The 15 “red-line” species are mostly well marked, whereas “blue-line” species—numbering roughly 50 if some newer treatments be accepted—are mostly hard to tell apart. Red-line ovules are tenuinucellate, blue-line ovules crassinucellate. Dividing the red line separates cornelian cherries from the showy-bracted species; dividing the latter separates dwarf cornels from big-bracted dogwoods. Adding in the blue line there are thus four subgroups, which differ with regard to inflorescences— and with regard to iridoid glucosides, among other things. I downplay further subdivision (into opposite- and alternateleaved blue-line groups, for instance) and focus on few traits because I aim to trace the subgroups backward in the fossil record, to decide the direction of evolutionary changes, and to infer the causes for divergence. Unlike pollen, leaves, and wood, fossil dogwood fruits can often be assigned to one of the four subgroups and sometimes to a species group within a subgroup.Dunstania (based on fruits from England’s early Tertiary and here transferred toCornus’s cornelian cherry subgroup) or something close to the dunstanias gave rise to modernC. volkensii of Africa and four related species found today in California and Eurasia. Fossil fruit-stones of the blue line, like their modern counterparts, can be subglobose, compressed and asymmetric, fluted, or topped by a deep depression. Such a deep depression serves to link a fossil to the extant speciesC. alternifolia andC. controversa, but other features of the blue-line fruitstones, overlapping and less constant, cannot be used to prove a fossil’s tie to groups within the blue line. Evidence of several kinds makes the blue-line dogwoods oldest and connectsCornus to the nyssoids:Nyssa, Camptotheca, andDavidia. Commonly called Nyssaceae after 1910, when Wangerin got their traits wrong, the nyssoids are surer relatives ofCornus than any of Wangerin’s (“Das Pflanzenreich”) cornaceous genera.Mastixia, however, may be the actual sister group because it shares withCornus 1-celled 2-armed hairs that nyssoids lack.Cornus, nyssoids, and mastixioids (modernMastixia and its closest fossil allies) are here regarded as the true Cornaceae. Other genera once thought to be cornaceous are doubtfully to not at all related. As is now widely known, discordant features makeCorokia andKaliphora (for example) out of place in the Cornaceae, butAucuba andGarrya (for example) are neither easily excluded by a listing of their traits nor easily included by construction of branched diagrams. The novelty that brought about the radiation of true cornads was, I think, epigyny, followed fairly quickly by the hard endocarp and singleseeded locules. Mastixioid fruits of the late Cretaceous were dry or leathery and smaller than their successors. This suggests that early members of the family spread abiotically or by means of animals that ate whole plants. Bigger, fleshier fruits came later through interaction with evolving frugivores. The cause of early radiation withinCornus probably was interaction with insect predators and pollinators. One species of the blue line escaped competition for spring pollinators by switching to fall flowering and thereby kept some traits that other dogwoods lost. Though birds are now the main dispersers of the dogwoods, rodents likely play a minor role, and monkeys likely played a major role when compound fruits evolved. Though gynoecial reduction was the rule in dogwoods,C. (Dunstania) multilocularis’s multiple seed chambers reflect an evolutionary increase. I postulate that modernCornus’s 4-merous flower with 2-merous ovary has a strong developmental tie to pairing of the leaves and branches.     

Iridoids in Hydrangeaceae

The content of glycosides in Kirengeshoma palmata and Jamesia americana (Hydrangeaceae) have been investigated. The former contains loganin and secoiridoids, including the alkaloid demethylalangiside. The latter contains no iridoids, but the known glucosides arbutin, picein and prunasin. In order to futher investigate the chemotaxonomy of the family Hydrangeaceae, the distribution of the iridoid and secoiridoid glucosides as well as the known biosynthetic pathways to these compounds have been reviewed. However, only a few genera of the family has been investigated. Loganin, secologanin, and derivatives of these are common. The genus Deutzia is characteristic in containing more structurally simple iridoids in which C-10 has been lost during biosynthesis. Such compounds have so far only been reported from the genus Mentzelia (Loasaceae). The taxonomic relationships between Hydrangeaceae and the closely related Cornaceae and Loasaceae is discussed and found to agree well with recent DNA sequence results.     

Chemical diversity among populations of Mikania micrantha: geographic mosaic structure and herbivory

Populations of the same species vary in their secondary metabolite content. This variation has been attributed to biotic and abiotic environmental conditions as well as to historical factors. Some studies have focused on the geographic variation of chemical diversity in plant populations, but whether this structure conforms to a central–marginal model or a mosaic pattern remains unclear. Furthermore, assessing the chemical diversity of invasive plants in their native distribution facilitates the understanding of their relationships with natural enemies. We examined the geographic variation of chemical diversity in Mexican populations of the bittervine weed Mikania micrantha and its relationship to herbivore damage. The foliar volatile terpenoid blend was analyzed in 165 individuals of 14 populations in the Pacific and Gulf of Mexico tropical watersheds. A cluster analysis grouped individuals with similar terpenoid blends into 56 compositional types. Chemical diversity was measured using the number of compounds and their concentration within the blends for individuals, and the number and frequency of compositional types for populations. A stepwise multiple regression analysis performed with geographic, climatic, and chemical diversity variables explained herbivore damage. However, population-level chemical diversity was the only variable found to be significant (β = ?0.79, P = 0.042) in the model (R ² = 0.89). A Mantel test using Euclidean distances did not indicate any separation by geographic origin; however, four barriers were identified using Monmonier’s algorithm. We conclude that variation in population-level chemical diversity follows a mosaic pattern in which geographic factors (i.e., natural barriers) have some effect and that variation is also associated with the local intensity of herbivore attack.     

A diverse assemblage of moths feeding on aphid honeydew

Adult butterflies and moths (Lepidoptera) use their proboscises to feed on flower nectar and other types of liquid food. Aphids frequently secrete honeydew from their anuses, attracting various types of insects, such as ants. Adult lepidopterans are also known to feed on aphid honeydew. However, very few studies have clarified the species composition and morphology of moths feeding on aphid honeydew. In late June 2017, we found nocturnal moths sipping honeydew secreted by Shivaphis celti and Sitobion cornifoliae (Hemiptera: Aphididae) on leaves of Celtis sinensis (Cannabaceae) and Cornus kousa (Cornaceae), respectively, at the edge of a secondary forest in central Japan. The moths were observed to uncoil their proboscises and feed on honeydew on the leaves. No moths were observed to feed on honeydew directly from aphids. Nocturnal moths of 60 species (11 families) and 16 species (7 families) were collected from honeydew on Ce. sinensis and Co. kousa leaves, respectively. Eleven moth species were shared between the two types of honeydew. The most abundant species, Oncocera semirubella (Lepidoptera: Pyralidae), accounted for 44.7% of all individuals. The sex ratio of O. semirubella was female biased. Of the 65 moth species collected on aphid honeydew, 52.3% (34 species) have been previously reported to visit flowers. Moths visiting honeydew had relatively short proboscises and small body size. These results suggest that aphid honeydew is an important, accessible food resource for moths of small size.     

New flavonoids from the fruits of Cornus mas,Cornaceae

One new β-hydroxychalcone, 4-acetoxy-5,2′,4′,6′,β-pentahydroxy-3-methoxychalcone (1), one new flavanone, 7,3′-dihydroxy-5,4′-dimethoxyflavanone (2) and seven known compounds, 2R, 3R-trans-aromadendrin (3), naringenin-7-O-methylether (4), myricetin (5), quercetin-3-O-rutinoside (6), ursolic acid (7), gallic acid (8) and d-glucose (9) were isolated from the methanolic fruit extract of Cornus mas L. (=Cornus mascula L.), Cornaceae. The structures of the new compounds were elucidated on the basis of extensive spectroscopic methods, including 2D NMR experiments and of known compounds by comparison of physical and spectral data with literature.     

Study on the Main Components Interaction from Flos Lonicerae and Fructus Forsythiae and Their Dissolution In Vitro and Intestinal Absorption in Rats

The Flos Lonicerae-Fructus Forsythiae herb couple is the basic components of Chinese herbal preparations (Shuang-Huang-Lian tablet, Yin-Qiao-Jie-Du tablet and Fufang Qin-Lan oral liquid), and its pharmacological effects were significantly higher than that in Flos Lonicerae or Fructus Forsythiae, but the reasons remained unknown. In the present study, pattern recognition analysis (hierarchical cluster analysis (HCA) and principal component analysis (PCA)) combined with UHPLC-ESI/LTQ-Orbitrap MS system were performed to study the chemical constitution difference between co-decoction and mixed decoction in the term of chemistry. Besides, the pharmacokinetics in vivo and intestinal absorption in vitro combined with pattern recognition analysis were used to reveal the discrepancy between herb couple and single herbs in the view of biology. The observation from the chemical view in vitro showed that there was significant difference in quantity between co-decoction and mixed decoction by HCA, and the exposure level of isoforsythoside and 3, 5-dicaffeoylquinic acid in co-decoction, higher than that in mixed decoction, directly resulted in the discrepancy between co-decoction and mixed decoction using both PCA and HCA. The observation from the pharmacokinetics displayed that the exposure level in vivo of neochlorogenic acid, 3, 4-dicaffeoylquinic acid, isoforsythoside and forsythoside A, higher than that in single herbs, was the main factor contributing to the difference by both PCA and HCA, interestingly consistent with the results obtained from Caco-2 cells in vitro, which indicated that it was because of intestinal absorption improvement of neochlorogenic acid, 3, 4-dicaffeoylquinic acid, isoforsythoside and forsythoside A that resulted in a better efficacy of herb couple than that of single herbs from the perspective of biology. The results above illustrated that caffeic acid derivatives in Flos Lonicerae-Fructus Forsythiae herb couple could be considered as chemical markers for quality control of its preparations.