A molecular phylogenetic study of Graptopetalum (Crassulaceae) based on ETS, ITS, RPL16, and TRNL-F nucleotide sequences

Nuclear ETS and ITS, as well as plastid rpl16 and trnL-F DNA sequences were used to determine relationships among species of Graptopetalum (Crassulaceae) and closely related genera. Graptopetalum is member of a group of taxa restricted to North America, one of the centers of diversity of Crassulaceae; however, their phylogenetic relationships are not yet understood. Nineteen species of Graptopetalum and 24 species from nine other genera of Crassulaceae were sampled for use in three separate parsimony analyses: ITS alone, ETS alone, and a combined nuclear + plastid DNA analysis using all four gene regions. The ETS data set had the highest number of parsimony-informative sites, about 30% more than in ITS, but the most fully resolved tree resulted when the four DNA regions were combined. Only four subclades of the tree received moderate to strong bootstrap support, one of which includes all species of Graptopetalum having a single whorl of stamens. However, Graptopetalum is not monophyletic. Instead, Tacitus bellus and select species of Cremnophila, Sedum, and Echeveria are interspersed among species of Graptopetalum and show evidence of grouping according to geographical range of distribution more so than habit or floral morphology.     

Evolution of myrmecophytism in western Malesian Macaranga (Euphorbiaceae)

Plants inhabited by ants (myrmecophytes) have evolved in a diversity of tropical plant lineages. Macaranga includes approximately 300 paleotropical tree species; in western Malesia there are 26 myrmecophytic species that vary in morphological specializations for ant association. The origin and diversification of myrmecophytism in Macaranga was investigated using phylogenetic analyses of morphological and nuclear ITS DNA characters and studies of character evolution. Despite low ITS variation, the combined analysis resulted in a well-supported hypothesis of relationships. Mapping myrmecophytism on all most parsimonious trees resulting from the combined analysis indicated that the trait evolved independently between two and four times and was lost between one and three times (five changes). This hypothesis was robust when tested against trees constrained to have three or fewer evolutionary transformations, although increased taxon sampling for the ITS analysis is required to confirm this. Mapping morphological traits on the phylogeny indicated that myrmecophytism was not homologous among lineages; each independent origin involved a suite of different specializations for ant-plant association. There was no evidence that myrmecophytic traits underwent sequential change through evolution; self-hollowing domatia evolved independently from ant-excavated domatia, and different food-body production types evolved in different lineages. The multiple origins of myrmecophytism in Macaranga were restricted to one small, exclusively western Malesian lineage of an otherwise large and nonmyrmecophytic genus. Although the evolution of aggregated food-body production and the formation of domatia coincided with the evolution of myrmecophytism in all cases, several morphological, ecological, and biogeographic factors appear to have facilitated and constrained this radiation of ant-plants.     

Molecular phylogeny of Macaranga, Mallotus, and related genera (Euphorbiaceae s.s.): insights from plastid and nuclear DNA sequence data

Macaranga and Mallotus (Euphorbiaceae s.s.) are two closely related, large paleo(sub)tropical genera. To investigate the phylogenetic relationships between and within them and to determine the position of related genera belonging to the subtribe Rottlerinae, we sequenced one plastid (trnL-F) and three nuclear (ITS, ncpGS, phyC) markers for species representative of these genera. The analyses demonstrated the monophyly of Macaranga and the paraphyly of Mallotus and revealed three highly supported main clades. The genera Cordemoya and Deuteromallotus and the Mallotus sections Hancea and Oliganthae form a basal Cordemoya s.l. clade. The two other clades, the Macaranga clade and the Mallotus s.s. clade (the latter with Coccoceras, Neotrewia, Octospermum, and Trewia), are sister groups. In the Macaranga clade, two basal lineages (comprising mostly sect. Pseudorottlera) and a crown group with three geographically homogenous main clades were identified. The phylogeny of the Mallotus s.s. clade is less clear because of internal conflict in all four data sets. Many of the sections and informal infrageneric groups of Macaranga and Mallotus do not appear to be monophyletic. In both the Macaranga and Mallotus s.s. clades, the African and/or Madagascan taxa are nested in Asian clades, suggesting migrations or dispersals from Asia to Africa and Madagascar.     

Phylogeny and biogeography of the African genus Virectaria Bremek. (Sabiceeae s.l., Ixoroideae, Rubiaceae)

The phylogenetic relationships of the tropical African genus Virectaria with its associated genera within the tribe Sabiceeae s.l. (Ixoroideae and Rubiaceae) were inferred from the combined analysis of nuclear ITS and chloroplast rpoC1 and trnT-F nucleotide sequence data. Phylogenetic relationships within Virectaria were investigated using combined analyses of ETS (nrDNA), ITS, rpoC1 and trnT-F sequence data. The present analyses further show that Hekistocarpa is sister to the Tamridaea–Virectaria–Sabicea clade, Tamridaea and Virectaria are sister genera, and Sabicea s.l. is sister to the Tamridaea–Virectaria clade. Our results strongly support the monophyly of Virectaria and the sister-group relationships between V. multiflora and V. herbacoursi, V. angustifolia and V. procumbens, and V. major and V. belingana. Our analyses indicate a tropical African origin for Sabiceeae s.l., a long isolated evolution for Tamridaea and a wide range of dispersal of Virectaria species in the Lower-Guinean, Upper-Guinean and Congolian regions, without a clearly defined direction of migration.     

樟科黄肉楠属是一个复系类群--基于nrDNA ITS和ETS序列分析

应用nrDNA ITS和ETS序列探讨了樟科Lauraceae黄肉楠属Actinodaphne的系统演化关系。对得到的3个序列矩阵（ITS、ETS和ITS／ETS）,采用MP（maximum parsimony）,ML（maximum likelihood）和Bayesian33分析方法进行了系统发育分析。结果显示,本文选的黄肉楠属Actinodaphne物种与所选的月棒族中的外类群靠近并混和在一起,进一步证实了本属为一个复系类群。结合对传统的形态学性状的重新认识,认为花序类型特征可能是重新界定黄肉楠属的最重要的性状,具有相同化序类型的物种可能具有相同的起源。然而,由于取样数量相对较少以及对矩阵的中．独分析存在一定的差异,还需更详细的研究来验证本文对黄肉楠属系统演化关系的假设,并进一步更精确地重建本属的系统发育关系。     

The complete external transcribed spacer of 18S-26S rDNA: amplification and phylogenetic utility at low taxonomic levels in asteraceae and closely allied families

For molecular phylogenetic reconstruction of some intrageneric groups of plants, a DNA region is needed that evolves more rapidly than the internal transcribed spacer (ITS) of the 18S-26S nuclear ribosomal DNA (nrDNA) repeat. If the region identified is nuclear, it would also be desirable for it to undergo rapid concerted evolution to eliminate problems with coalescence. The external transcribed spacer (ETS) of the nrDNA repeat has shown promise for intrageneric phylogenetic reconstruction, but only the 3' end of the region has been utilized for phylogenetic reconstruction and "universal" primers for PCR amplification have been elusive. We present a method for reliably amplifying and sequencing the entire ETS throughout Asteraceae and some closely allied families. We also show that the ETS is more variable and phylogenetically informative than the ITS in three disparate genera of Asteraceae-Argyranthemum (tribe Anthemideae), Asteriscus (tribe Inuleae), and Helianthus (tribe Heliantheae). The full ETS was amplified using a primer (ETS1f) within the intergenic spacer in combination with a primer (18S-2L) in the 5' end of the highly conserved 18S gene. ETS1f was designed to correspond to a highly conserved region found in Helianthus and Crepis, which are in separate subfamilies of Asteraceae. ETS1f/18S-2L primed in all of the tribes of Asteraceae as well as exemplar taxa from Campanulaceae, Goodeniaceae, and Calyceraceae. For both Argyranthemum and Asteriscus, we were able to directly sequence the ETS PCR products when a single band was produced. When multiple bands were produced, we gel-purified and occasionally cloned the band of interest before sequencing. Although PCR produced single bands for Helianthus species, it was necessary to clone Helianthus amplifications prior to sequencing due to multiple intragenomic ETS repeat types. Alignment of ETS sequences for Argyranthemum and Asteriscus was straightforward and unambiguous despite some subrepeat structure in the 5' end. For Helianthus, different numbers of large tandem subrepeats in different species required analysis of the orthology of the subrepeats prior to alignment. In all three genera, the ETS provided more informative variation for phylogenetic reconstruction and allowed better resolution of relationships than the ITS. Although cloned sequences from Helianthus differed, intragenomic clones consistently formed clades. This result indicated that concerted evolution was proceeding rapidly enough in ETS that species-specific phylogenetic signal was retained. It should be now be possible to use the entire ETS for phylogenetic reconstruction of recently diverged lineages in Asteraceae and at least three other families (approximately 26,000 species or about 8% of all angiosperms).     

Molecular systematics and biogeography of Crawfurdia, Metagentiana and Tripterospermum (Gentianaceae) based on nuclear ribosomal and plastid DNA sequences

BACKGROUND AND AIMS: The systematic position of the genus Metagentiana and its phylogenetic relationships with Crawfurdia, Gentiana and Tripterospermum have not been explicitly addressed. These four genera belong to one of two subtribes (Gentianinae) of Gentianeae. The aim of this paper is to examine the systematic position of Crawfurdia, Metagentiana and Tripterospermum and to clarify their phylogenetic affinities more clearly using ITS and trnL intron sequences. METHODS: Nucleotide sequences from the internal transcribed spacers (ITS) of nuclear ribosomal DNA and the plastid DNA trnL (UAA) intron were analysed phylogenetically. Ten of fourteen Metagentiana species were sampled, together with 40 species of other genera in the subtribe Gentianinae. KEY RESULTS: The data support several previously published conclusions relating to the separation of Metagentiana from Gentiana and its closer relationships to Crawfurdia and Tripterospermum based on studies of gross morphology, floral anatomy, chromosomes, palynology, embryology and previous molecular data. The molecular clock hypothesis for the tested sequences in subtribe Gentianinae was not supported by the data (P < 0.05), so the clock-independent non-parametric rate smoothing method was used to estimate divergence time. This indicates that the separation of Crawfurdia, Metagentiana and Tripterospermum from Gentiana occurred about 11.4-21.4 Mya (million years ago), and the current species of these three genera diverged at times ranging from 0.4 to 6.2 Mya. CONCLUSIONS: The molecular analyses revealed that Crawfurdia, Metagentiana and Tripterospermum do not merit status as three separate genera, because sampled species of Crawfurdia and Tripterospermum are embedded within Metagentiana. The speciation and rapid radiation of these three genera is likely to have occurred in western China as a result of upthrust of the Himalayas during the late Miocene and the Pleistocene.     

Molecular characterization and phylogenetic utility of the rDNA external transcribed spacer region in <Emphasis Type="Italic">Stylosanthes</Emphasis> (Fabaceae)

Supplementary Material The nucleotide sequence of the ribosomal external transcribed spacer (ETS) region of Stylosanthes mexicana was determined and used to evaluate its potential for examination of intra- and inter-specific relationships in Stylosanthes, as compared to the use of the internal transcribed spacer (ITS) region. The entire ETS region comprises 1,145 bp and is composed of a region of non-repetitive sequences consisting of three subregions with organizational and nucleotide-sequence conservation, and a triplicated segment of about 100 bp. A primer designed in the second conserved subregion allowed us to amplify and sequence directly the 3' part (423-431 bp) of the ETS from 22 genotypes of 12 representative Stylosanthes species that were previously used in phylogenetic analysis of the genus. The study revealed that the right-hand part of Stylosanthes ETS contains approximately twice as much variable and informative characters than the ITS. Moreover, pairwise sequence-divergence values are twice as high, on average, when compared to the ITS. The ITS and ETS datasets are consistent in phylogenetic reconstruction of Stylosanthes, and combined parsimony analysis resulted in a strict consensus tree that is better resolved and generally better supported than trees obtained from separate analysis of the spacer regions.     

Molecular phylogeny and adaptive radiation of the endemic Hawaiian Plantago species (Plantaginaceae)

Insular oceanic islands provide excellent opportunities for the study of evolutionary processes and adaptive radiation. The Hawaiian Plantago radiation comprises six endemic taxa showing considerable inter- and intraspecific morphological and ecological diversity. The rDNA internal (ITS) and external (ETS) transcribed spacers and two recently described chloroplast spacers, ndhF-rpl32 and rpl32-trnL, were sequenced to study phylogenetic relationships within this morphologically complex group. Phylogenetic analysis provided strong evidence for the monophyly of Hawaiian Plantago, suggesting that the lineage arose from a single long-distance dispersal event. Inconsistencies between nuclear and chloroplast phylogenies suggest a history of hybridization. The basal, unresolved dichotomy of the combined phylogeny is consistent with rapid phenotypic diversification of the major lineages early in the history of this group. Speciation has largely occurred allopatrically, with divergence a result of intraisland ecological shifts between bog and woodland habitats and interisland dispersal events. Most interisland colonizations were from older to younger islands with initial colonization of Kaua'i. In our analysis, P. pachyphylla is paraphyletic and taxonomic separation of the distinct morphotypes of this species appears justified. Furthermore, the apparent hybrid ancestry and unique morphology and habitat of the endangered P. princeps var. longibracteata support its recognition at the specific rank.     

Species recognition, geographic distribution and host-pathogen relationships: a case study in a group of lignicolous basidiomycetes, Phellinus s.l

Morphological, phylogenetic (sequencing of the ribosomal ITS region) and, if applicable, biological (pairings of single-spore testers) species recognition have been used to resolve relationships among 69 collections belonging to the Hymenochaetales genera Phellinus s.str. and Fomitiporia. The isolates originate from a variety of host plants in Europe, North America and Asia. Separate application of recognition modes led to differing results concerning the number of species, geographic distribution and host range. Sole application of morphological criteria was of limited value, especially in taxa exhibiting a wide distribution, both in terms of geographic origin and ecological niche. Relationships of putatively con-specific collections originating from different continents preferably should be resolved by using an integrative approach. In this study, application of a strict morphological approach led to the recognition of seven species. When using molecular and pairing test data, at least 12 species were detectable. Two of them, F. hesleri and F. polymorpha, are described as new. The number of Phellinus s.str. and Fomitiporia species supposed to have Northern Hemispheric or cosmopolitan distribution, when morphological characters are applied for species recognition, has been reduced significantly. As firm tendencies within morphological species, genetic divergence was more distinct in uniparental than in biparental taxa. In the latter, a strong correlation was observed between phylogenetic and biological species recognition. Overall length of the ribosomal ITS region clearly separated Phellinus s.str. and Fomitiporia but was of limited value as a diagnostic tool at species level. The level of innerspecific morphological plasticity of fruit bodies differs widely between even closely related species, suggesting that morphological transitions occur quite frequently in this fungal group. Considerable instability of the reproduction mode was evident in strains belonging to Phellinus tremulae and among closely related species of Fomitiporia.     

Redefining Phrymaceae: the placement of Mimulus, tribe Mimuleae, and Phryma

Chloroplast trnL/F and nuclear ribosomal ITS and ETS sequence data were used to analyze phylogenetic relationships among members of tribe Mimuleae (Scrophulariaceae) and other closely related families in Lamiales. The results of these analyses led to the following conclusions. (1) The Australian genera Glossostigma and Peplidium and the taxonomically isolated Phryma join four genera of tribe Mimuleae to form a well-supported clade that is distinct from other families in the Lamiales. We refer to that clade as the subfamily Phrymoideae. (2) The genera Mazus and Lancea (tribe Mimuleae) together form a well-supported clade that we recognize as the subfamily Mazoideae. Mazoideae is weakly supported as sister to Phrymoideae. We assign Mazoideae and Phrymoideae to a redefined family Phrymaceae. (3) Mimulus is not monophyletic, because members of at least six other genera have been derived from within it. In light of the molecular evidence, it is clear that species of Phrymaceae (about 190 species) have undergone two geographically distinct radiations; one in western North America (about 130 species) and another in Australia (about 30 species). Phylogenetic interpretations of morphological evolution and biogeographical patterns are discussed.     

Molecular analysis of phylogenetic relationships among Myrmecophytic macaranga species (Euphorbiaceae)

Many species of the paleotropical pioneer tree genus Macaranga Thou. (Euphorbiaceae) live in association with ants. Various types of mutualistic interactions exist, ranging from the attraction of unspecific ant visitors to obligate myrmecophytism. In the latter, nesting space and food bodies are exchanged for protection by highly specific ant partners (mainly species of the myrmicine genus Crematogaster). As a first step toward elucidating the coevolution of ant-plant interactions in the Macaranga-Crematogaster system, we have initiated a molecular investigation of the plant partners' phylogeny. Nuclear ribosomal DNA internal transcribed spacer (ITS) sequences were analyzed for 73 accessions from 47 Macaranga species, representing 17 sections or informally described species groups. Three accessions from the putative sister taxon Mallotus Lour, were included as outgroups. Cladograms of the ITS data revealed Macaranga to be nested within Mallotus. ITS sequences are highly similar within section Pachystemon s.str., suggesting a relatively recent and rapid radiation of obligate myrmecophytes within this section. Forty-three accessions, mainly of ant-inhabited species, were additionally investigated by random amplified polymorphic DNA (RAPD) and microsatellite-primed PCR (MP-PCR) techniques. Phenetic analysis of RAPD and MP-PCR banding profiles generally confirmed the ITS results. Best resolutions for individual clades were obtained when ITS and RAPD/MP-PCR data were combined into a single matrix and analyzed phenetically. The combined analysis suggests multiple (four) rather than a single evolutionary origin of myrmecophytism, at least one reversal from obligate myrmecophytism to nonmyrmecophytism, and one loss of mutualistic specifity.     

Inter-generic relationships of the crows, jays, magpies and allied groups (Aves: Corvidae) based on nucleotide sequence data

Phylogenetic relationships were studied based on DNA sequences obtained from all recognized genera of the family Corvidae sensu stricto . The aligned data set consists 2589 bp obtained from one mitochondrial and two nuclear genes. Maximum parsimony, maximum-likelihood, and Bayesian inference analyses were used to estimate phylogenetic relationships. The analyses were done for each gene separately, as well as for all genes combined. An analysis of a taxonomically expanded data set of cytochrome b sequences was performed in order to infer the phylogenetic positions of six genera for which nuclear genes could not be obtained. Monophyly of the Corvidae is supported by all analyses, as well as by the occurrence of a deletion of 16 bp in the β-fibrinogen intron in all ingroup taxa. Temnurus and Pyrrhocorax are placed as the sister group to all other corvids, while Cissa and Urocissa appear as the next clade inside them. Further up in the tree, two larger and well-supported clades of genera were recovered by the analyses. One has an entirely New World distribution (the New World jays), while the other includes mostly Eurasian (and one African) taxa. Outside these two major clades are Cyanopica and Perisoreus whose phylogenetic positions could not be determined by the present data. A biogeographic analysis of our data suggests that the Corvidae underwent an initial radiation in Southeast Asia. This is consistent with the observation that almost all basal clades in the phylogenetic tree consist of species adapted to tropical and subtropical forest habitats.     

Molecular phylogeny of truffles (Pezizales: Terfeziaceae, Tuberaceae) derived from nuclear rDNA sequence analysis.

Extensive morphological convergence or divergence, a common occurrence in fungi, tends to obscure recognition of phylogenetic relationships among Pezizales, widespread filamentous Ascomycetes with either enclosed underground (hypogeous) or exposed (epigeous) fruit bodies, that often establish mutualistic interactions with arboreous plants. Focusing on hypogeous Pezizales commonly known as truffles, we sequenced the 18S rDNA from nine species belonging to three different families (Tuberaceae, Terfeziaceae, and Balsamiaceae). A data set consisting of 1700 secondary structure-aligned sites, including 24 homologous sequences from the GenBank DNA database and using three reconstruction methods, was employed to infer phylogenies in an interval ranging from the subordinal to the subgeneric level. As revealed by the 18S phylogenetic scheme, Balsamiaceae represent a monophyletic clade, comprising the hypogeous taxa Balsamia and Barssia, nested within Helvellaceae. Similarly, the terfeziacean genera Pachyphloeus and Terfezia constitute together with Cazia a distinct hypogeous clade nested within Pezizaceae. The lack of clustering between Terfezia arenaria and Terfezia terfezioides strongly supports the reassignment of the latter taxon to the original monotypic genus Mattirolomyces. Within Tuberaceae, which are sister to the highly evolved Helvellaceae, the genus Tuber cannot be considered monophyletic if Choiromyces is recognized. The paraphyly of Tuber and other relationships that were not supported by high bootstrap values, nor corroborated by morphological evidence, were supported by a parallel analysis of the faster evolving internal transcribed spacer (ITS) rDNA. Distinct episodes of fruit body morphology shifts are discernable in the 18S rDNA phylogenetic tree. In all cases, the shift from an epigeous to a hypogeous form is the most parsimonious interpretation of character transformation, without any instance of character reversal.     

Nonuniform concerted evolution and chloroplast capture: heterogeneity of observed introgression patterns in three molecular data partition phylogenies of Asian Mitella (saxifragaceae)

Interspecific hybridization is one of the major factors leading to phylogenetic incongruence among loci, but the knowledge is still limited about the potential of each locus to introgress between species. By directly sequencing three DNA regions: chloroplast DNAs (matK gene and trnL-F noncoding region), the nuclear ribosomal external transcribed spacer (ETS) region, and internal transcribed spacer (ITS) regions, we construct three phylogenetic trees of Asian species of Mitella (Saxifragaceae), a genus of perennials in which natural hybrids are commonly observed. Within this genus, there is a significant topological conflict between chloroplast and nuclear phylogenies and also between the ETS and the ITS, which can be attributed to frequent hybridization within the lineage. Chloroplast DNAs show the most extensive introgression pattern, ITS regions show a moderate pattern, and the ETS region shows no evidence of introgression. Nonuniform concerted evolution best explains the difference in the introgression patterns between the ETS region and ITS regions, as the sequence heterogeneity of the ITS region within an individual genome is estimated to be twice that of an ETS in this lineage. Significant gene conversion patterns between two hybridizing taxa were observed in contiguous arrays of cloned ETS-ITS sequences, further confirming that only ITS regions have introgressed bidirectionally. The relatively slow concerted evolution in the ITS regions probably allows the coexistence of multiple alleles within a genome, whereas the strong concerted evolution in the ETS region rapidly eliminates heterogeneous alleles derived from other species, resulting in species delimitations highly concordant with those based on morphology. This finding indicates that the use of multiple molecular tools has the potential to reveal detailed organismal evolution processes involving interspecific hybridization, as an individual locus varies greatly in its potential to introgress between species.     

Molecular Phylogeny and Species Speciation of Adoxaceae. s. s

It remains unclear about the speciation and phylogeny of Adoxaceae s. s ., a small family with 3 genera and 4 species . In this paper , ITS ( nuclear DNA internal transcribed spacer) regions of Adoxa orientalis and Sambucus adnata were firstly sequenced . Phylogenetic trees were constructed for all species of Adoxaceae (four species ) , Sambucus, Viburnum and four genera of Caprifoliaceae . The divergences among four species of this family were further calculated based on the calibration of the fossil records of the Caprifoliaceae and the general evolutionary rate of herbs for ITS . The phylogenetic analyses did not support the previous assumptions on the phylogeny and species divergence of Adoxaceae s. s . based on the morphological evidence : Tetradoxa is not the firstly diverged and it clustered with two species of Adoxa as a monophylogenetic group , paralleling to the other lineage comprising of monotypic Sinadoxa . The allopatric speciation at the diploid level might have contributed to the differentiation among Sinadoxa corydalifolia, Tetradoxa omeiensis and Adoxa moschatellina and the polyploidy to the origin of A. orientalis . The crude timing based on ITS sequence differentiation suggested a recent divergence among all four species probably between the late Miocene and the Tertiary and this speciation process might be closely correlated with habitat fragmentation and change due to the extensive uplifts of the Qinghai-Tibetan Plateau and climatic oscillation during the glacial and interglacial ages occurred at this stage .     

狭义五福花科的分子系统学和物种分化

狭义五福花科(Adoxaceae.s.s)仅含3属4种,但该科的物种分化、系统发育和分类一直存在争议。本文通过测定东方五福花和血满草的ITS(核糖体DNA内转录间隔区)序列,构建了包括狭义五福花科(4种)、广义忍冬科接骨木属、荚属以及其余4属植物在内的系统发育树。研究结果不支持狭义五福花科内根据形态学证据做出的系统发育假设四福花不是该科中最早分化出来的种;该物种与五福花属的两个物种形成一个单系群,与另一分支华福花属相对应。该科中3个物种,四福花、五福花和华福花之间的分化主要是在二倍体水平上的异域分化,而东方五福花则是通过多倍化形成的。粗略的时间估算表明这些物种之间的分化较晚,可能在第三纪末至第四纪早中期,与青池高原近期强烈隆升以及冰期气候反复变化形成的环境变迁密切相关。