Characterization of Two Perkinsus spp. from the Softshell Clam, Mya arenaria Using the Small Subunit Ribosomal RNA Gene

Sequence analysis and riboprinting of the small subunit ribosomal RNA genes were used to characterize two morphologically different Perkinsus species isolates from the gill (G117) and the hemolymph (H49) of the softshell clam, Mya arenaria. Sequence data of the polymerase chain reaction amplified ribosomal RNA loci of G117 and H49 indicated that these genes are 1803 and 1806 base-pair long, respectively. A sequence similarity of > 98.9% was calculated among ribosomal RNA sequences of the two isolates of this study and the published sequences of Perkinsus marinus from the American eastern oyster, Crassostrea virginica, and Perkinsus sp. from the blood cockle of the Australian mollusc, Anadara trapezia. From a phylogenetic tree obtained from Jukes-Cantor distances of the aligned ribosomal RNA gene sequences of 13 eukaryotic taxa using the Neighbor-Joining method, we showed that G117 and H49 clustered within the genus Perkinsus. Guided by the sequence data of Perkinsus marinus (accession # X75762) and Perkinsus sp. (accession # L07375), restriction endonucleases were selected for restriction fragment analysis of polymerase chain reaction products of the small subunit ribosomal RNA genes (riboprinting). Riboprinting was used to distinguish the four members of the genus Perkinsus from each other.     

A riboprinting scheme for identification of unknown Acanthamoeba isolates at species level

We describe a riboprinting scheme for identification of unknown Acanthamoeba isolates at the species level. It involved the use of PCR-RFLP of small subunit ribosomal RNA gene (riboprint) of 24 reference strains by 4 kinds of restriction enzymes. Seven strains in morphological group I and III were identified at species level with their unique sizes of PCR product and riboprint type by Rsa I. Unique RFCP of 17 strains in group II by Dde I, Taq I and Hae III were classified into: (1) four taxa that were identifiable at the species level, (2) a subgroup of 4 taxa and a pair of 2 taxa that were identical with each other, and (3) a species complex of 7 taxa assigned to A. castellanii complex that were closely related. These results were consistent with those obtained by 18s rDNA sequence analysis. This approach provides an alternative to the rDNA sequencing for rapid identification of a new clinical isolate or a large number of environmental isolates of Acanthamoeba.     

Intraspecific Variation and Phylogenetic Relationships in the Genus Entamoeba as Revealed by Riboprinting

ABSTRACT. Eighty-seven isolates of amebae assigned to the genus Entamoeba have been studied by riboprinting (restriction enzyme polymorphism analysis of polymerase chain reaction amplified small subunit ribosomal RNA genes). Twenty-four distinct patterns were obtained, most of which corresponded to previously described species. In three species ( Entamoeba coli, Entamoeba gingivalis and Entamoeba moshkovskii ) intraspecific variation was detected that led to the grouping of isolates into 'ribodemes' (populations of amebae that share the same riboprint pattern). The riboprint data were used to estimate genetic distances among and within species for the construction of phylogenetic trees based on parsimony and distance analyses. The trees obtained with the two methods are largely congruent. In some cases the estimated distances between species were greater than the upper limit recommended for the fragment comigration method of analysis indicating unusually deep branches within this genus. However, it appears that those species producing cysts with eight nuclei, those producing cysts with one nucleus, and those producing cysts with four nuclei form morphologically based groups that are supported by the riboprint data. The oral parasite Entamoeba gingivalis , which does not encyst, clusters with the third group indicating secondary loss of this ability.     

Riboprinting and 16S rRNA Gene Sequencing for Identification of Brewery Pediococcus Isolates

A total of 46 brewery and 15 ATCC Pediococcus isolates were ribotyped using a Qualicon RiboPrinter. Of these, 41 isolates were identified as Pediococcus damnosus using EcoRI digestion. Three ATCC reference strains had patterns similar to each other and matched 17 of the brewery isolates. Six other brewing isolates were similar to ATCC 25249. The other 18 P. damnosus brewery isolates had unique patterns. Of the remaining brewing isolates, one was identified as P. parvulus, two were identified as P. acidilactici, and two were identified as unique Pediococcus species. The use of alternate restriction endonucleases indicated that PstI and PvuII could further differentiate some strains having identical EcoRI profiles. An acid-resistant P. damnosus isolate could be distinguished from non-acid-resistant varieties of the same species using PstI instead of EcoRI. 16S rRNA gene sequence analysis was compared to riboprinting for identifying pediococci. The complete 16S rRNA gene was PCR amplified and sequenced from seven brewery isolates and three ATCC references with distinctive riboprint patterns. The 16S rRNA gene sequences from six different brewery P. damnosus isolates were homologous with a high degree of similarity to the GenBank reference strain but were identical to each other and one ATCC strain with the exception of 1 bp in one strain. A slime-producing, beer spoilage isolate had 16S rRNA gene sequence homology to the P. acidilactici reference strain, in agreement with the riboprint data. Although 16S rRNA gene sequencing correctly identified the genus and species of the test Pediococcus isolates, riboprinting proved to be a better method for subspecies differentiation.     

Phylogenetic Relationships Among Anuran Trypanosomes as Revealed by Riboprinting

Twenty trypanosome isolates from Anura (frogs and toads) assigned to several species were characterized by riboprinting–restriction enzyme digestion of polymerase chain reaction amplified small subunit ribosomal RNA genes. Restriction site polymorphisms allowed distinction of all the recognized species and no intraspecific variation in riboprint patterns was detected. Phylogenetic reconstruction using parsimony and distance estimates based on restriction fragment comigration showed Trypanosoma chattoni to be only distantly related to the other species, white T. ranarum and T. fallisi appear to be sister taxa despite showing non-overlapping host specificities.     

Diversity of endophytic bacterial communities in poplar grown under field conditions

Bacterial endophytes may be important for plant health and other ecologically relevant functions of poplar trees. The composition of endophytic bacteria colonizing the aerial parts of poplar was studied using a multiphasic approach. The terminal restriction fragment length polymorphism analysis of 16S rRNA genes demonstrated the impact of different hybrid poplar clones on the endophytic community structure. Detailed analysis of endophytic bacteria using cultivation methods in combination with cloning of 16S rRNA genes amplified from plant tissue revealed a high phylogenetic diversity of endophytic bacteria with a total of 53 taxa at the genus level that included Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. The community structure displayed clear differences in terms of the presence and relative proportions of bacterial taxa between the four poplar clones studied. The results showed that the genetic background of the hybrid poplar clones corresponded well with the endophytic community structure. Out of the 513 isolates and 209 clones identified, Actinobacteria, in particular the family Microbacteriaceae, made up the largest fraction of the isolates, whereas the clone library was dominated by Alpha- and Betaproteobacteria. The most abundant genera among the isolates were Pseudomonas and Curtobacterium, while Sphingomonas prevailed among the clones.     

Identity of Naegleria strains isolated from organs of freshwater fishes.

Eighteen Naegleria strains were isolated from organs of freshwater fishes belonging to 5 species. Morphometric study allowed the separation of the Naegleria strains from the non-vahlkampfiid amoeboflagellates, but was inadequate for species determination. Six strains, representatives of groups that had a slightly different cyst size, were selected and corresponding derived clones were subjected to sequence analysis and riboprinting restriction fragment length polymorphism (RFLP)-PCR analysis of the small subunit (SSU) rRNA genes. One strain isolated from the brain of a fish with systemic infection was characterised by an intronless 2 kb long SSU rRNA gene and was identified as N. australiensis. Another 5 strains had a 1.3 kb long group I intron in their SSU rRNA gene and, based on the SSU rRNA sequences and riboprints, RFLP-PCR patterns appeared in phylogenetic trees to be closely related to Naegleria clarki.     

Identification and phylogenetic relationships of Vahlkampfia spp. (free-living amoebae) by riboprinting

Abstract The riboprinting technique (restriction fragment length polymorphism analysis of polymerase chain reaction amplified ribosomal DNA) was applied to 8 strains representing 7 species of amoebae from the Vahlkampfia genus (Family Vahlkampfiidae, Class Heterolobosea). The length of the 18S ribosomal gene was found to vary significantly between species. Restriction fragment length polymorphism analysis following digestion of the amplified ribosomal DNA with 18 restriction enzymes confirmed the separate identity of each species, originally based on morphological characteristics, and generated a phylogenetic tree. Two strains assigned to the same species yielded identical riboprints.     

Identifying and Distinguishing Sibling Species in the Tetrahymena pyriformis Complex (Ciliophora, Oligohymenophorea) Using PCR/RFLP Analysis of Nuclear Ribosomal DNA

ABSTRACT We describe a riboprinting strategy for identifying and distinguishing among sibling species in the Tetrahymena pyriformis complex. It involves use of the polymerase chain reaction to amplify a large segment of the nuclear ribosomal DNA and internal transcribed spacers, and digestion of this DNA with restriction enzymes. Unique restriction fragment length patterns or haplotypes were then used to distinguish species into: (1) six taxa that were identifiable to the species level, (2) eight taxa that were separated into four pairs, and (3) a group of eight taxa that were identical to each other. The latter result indicates that a more variable molecule is needed to distinguish the most closely related species in the complex. There was no intraspecific variation between two strains from one species ( Tetrahymena thermophila ) nor among multiple isoiates from another species ( Tetrahymena empidokyrea ). This approach provides an alternative to traditional techniques for identifying T. pyriformis species that require living reference specimens and/or that reveal high levels of intraspecific variation.     

小克银汉霉核糖体DNA PCR扩增产物的限制性片段长度多型性(英文)

应用一对寡核苷酸引物ITS1与ITS4对核DNAG＋C百分数小于30％的小克银汉霉（Cunninghamella）属的核糖体DNA（rDNA）内转录间区（YTS）进行了扩增。测试的属于10个种和变种的22株菌都得到了扩增产物。在同属不同种之间扩增的ITS片段长度有巨大差别。据此可分为三组。这三组所含分类群数及其DNA长度分别为;第一组4种1变种,小于764碱基对（bp）;第二组2种,765～824bp;第三组2种1变种,大于825bp。所研究的许多种中,特别是第三组,单凭其PCR产物的长度就能区分开来。但在第一、二组就需借助限制性内切酶的分析才能予以区分。我们选用了RsaI,TaqI;Tru9I,和HinfI4种限制性内切酶,对所有扩增产物进行了限制性片段长度多型性（RFLPs）的分析。在雅致小克银汉霉（Celegans）、巴西玉蕊小克银汉霉（Cbertholletiae）、和布拉氏霉（Cblakesleeana）各种的限制性酶切图谱,种内非常一致而种与种之间有差别。反之,在某些种其限制性酶切图谱不仅种间互不相同,在种内不同株之间也出现1～3种酶切图型的差异。在这种情况下,只能综合各种资料才能将它们区分开来。本研究肯定了PCR－RFLP在区分小克银汉霉种和变种上的意义,并发现种内个体的差异。这在我们后来所进行的序列分析研究中得到了进一步的证实。     

Molecular evidence indicates that subarctic willow communities in Scotland support a diversity of host-associated Melampsora rust taxa

Rare and threatened subarctic willow scrub communities in the UK are the subject of ongoing conservation programmes, yet little is known about the diversity of fungal taxa that they support. Isolates of the rust genus Melampsora were sampled from 112 leaves of eight subarctic willow (Salix) taxa and their hybrids from twelve sites in the UK. In order to determine the number of Melampsora taxa present in the samples, isolates were sequenced for the Internal Transcribed Spacer (ITS) region of rDNA and data were subject to phylogenetic analysis. Maximum likelihood and Bayesian analysis indicated that the isolates fell into three strongly supported host-associated clades. Clade I contained only isolates from Salix herbacea and was distinguished morphologically by dense urediniospore echinulation and thin cell walls. Clade II contained isolates from Salix arbuscula and Salix reticulata only. These could not be distinguished morphologically from isolates in Clade III which were found on Salix lapponum, Salix myrsinites, Salix myrsinifolia, Salix aurita, Salix lanata, and their hybrids. Clade II was most distinct in ITS sequence, differing by 50 bases from Clades I and III, while the latter clades differed in sequence by only 24 bases on average. Clades I and III are likely to represent the previously recognised taxa Melampsora alpina Juel 1894 and Melampsora epitea Thüm. 1879 respectively, but Clade II has not apparently been described before. Significant differences in the intensity of infection by isolates of Clade III were found among different Salix species at a single site, suggesting either differences in resistance among Salix taxa, or the presence of further cryptic taxa within Clade III. The study illustrates the power of molecular phylogenetic analysis to reveal cryptic biodiversity within Melampsora, and suggests that conserving Salix host diversity within subarctic willow communities will ensure that a diversity of associated Melampsora taxa is maintained.     

Identification and differentiation of Heterotardigrada and Eutardigrada species by riboprinting

In the last decades, the number of known tardigrade species has considerably increased to more than 960 species with new ones being discovered every year. However, the study of tardigrade species presents a general problem which is frequently encountered during the work with invertebrates: small size and remarkable degrees of phenotypic plasticity may sometimes not permit a definite identification of the species. In this investigation we have used riboprinting, a tool to study rDNA sequence variation, in order to distinguish tardigrade species from each other. The method combines a restriction site variation approach of ribotyping with amplified DNAs. In eight investigated species of heterotardigrades and eutardigrades we have amplified the genes for the small subunit ribosomal RNA (SSU; 18S) and subsequently sequenced the genes. Virtual riboprints were used for identification of restriction sites from ten already published 18S rDNA sequences and seven new 18S rDNA sequences. On the basis of the obtained sequences, diagnostic restriction fragment patterns can be predicted with only 11 restriction enzymes. The virtual digestion confirmed the obtained restriction fragment patterns and restriction sites of all amplified and digested tardigrade DNAs. We show that the variation in positions and number of restriction sites obtained by standard restriction fragment analysis on agarose gels can be used successfully for taxonomic identification at different taxonomic levels. The simple restriction fragment analysis provides a fast and convenient method of molecular barcoding for species identification in tardigrades.     

LSU rDNA based RFLP assays for the routine identification of Gambierdiscus species

The Gambierdiscus genus is a group of benthic dinoflagellates commonly associated with ciguatera fish poisoning (CFP), which is generally found in tropical or sub-tropical regions around the world. Morphologically similar species within the genus can vary in toxicity; however, species identifications are difficult or sometimes impossible using light microscopy. DNA sequencing of ribosomal RNA genes (rDNA) is thus often used to identify and describe Gambierdiscus species and ribotypes, but the expense and time can be prohibitive for routine culture screening and/or large-scale monitoring programs. This study describes a restriction fragment length polymorphism (RFLP) typing method based on analysis of the large subunit rDNA that can successfully identify at least nine of the described Gambierdiscus species and two Fukuyoa species. The software programs DNAMAN 6.0 and Restriction Enzyme Picker were used to identify a set of restriction enzymes (SpeI, HpyCH4IV, and TaqαI) capable of distinguishing most of the known Gambierdiscus species for which DNA sequences were available. This assay was tested using in silico analysis and cultured isolates, and species identifications of isolates assigned by RFLP typing were confirmed by DNA sequencing. To verify the assay and assess intra-specific heterogeneity in RFLP patterns, identifications of 63 Gambierdiscus isolates comprising ten Gambierdiscus species, one ribotype, and two Fukuyoa species were confirmed using RFLP typing, and this method was subsequently employed in the routine identification of isolates collected from the Caribbean Sea. The RFLP assay presented here reduces the time and cost associated with morphological identification via scanning electron microscopy and/or DNA sequencing, and provides a phylogenetically sensitive method for routine Gambierdiscus species assignment.     

Molecular characterization of myxozoan parasites from Lake Sasajewun, Algonquin Park, Ontario, by riboprinting

The small subunit-rRNA genes of 18 myxozoans from Lake Sasajewun, Algonquin Park were amplified and digested with restriction endonucleases for riboprinting analysis. Identical riboprints were not found between the myxosporeans and the actinosporeans. The distinct riboprinting patterns observed among these myxozoans indicate considerable genetic diversity within this group. Identical riboprints were found between Myxobolus pendula and Myxobolus pellicides, and between triactinomyxon 'C' and Triactinomyxon ignotum. Parsimony analysis of the riboprints demonstrated that neither the myxosporeans nor the actinosporeans formed a monophyletic group. Some species of Myxobolus are more closely related to forms of triactinomyxon, echinactinomyxon or raabeia than to other Myxobolus species. These results are consistent with the two-host life cycle hypothesis of myxozoans that myxosporeans and actinosporeans are alternating stages of the same organisms.     

Phylogenetic relationships among Acanthamoeba spp. based on PCR-RFLP analyses of mitochondrial small subunit rRNA gene.

We investigated the value of mitochondrial small subunit rRNA gene (mt SSU rDNA) PCR-RFLP as a taxonomic tool for Acanthamoeba isolates with close inter-relationships. Twenty-five isolates representing 20 species were included in the analysis. As in nuclear 18S rDNA analysis, two type strains (A. astronyxis and A. tubiashi) of morphological group 1 diverged earliest from the other strains, but the divergence between them was less than in 18S riboprinting. Acanthamoeba griffini of morphological group 2 branched between pathogenic (A. culbertsoni A-1 and A. healyi OC-3A) and nonpathogenic (A. palestinensis Reich, A. pustulosa GE-3a, A. royreba Oak Ridge, and A lenticulata PD2S) strains of morphological group 3. Among the remaining isolates of morphological group 2, the Chang strain had the identical mitochondrial riboprints as the type strain of A. hatchetti. AA2 and AA1, the type strains of A. divionensis and A. paradivionensis, respectively, had the identical riboprints as A. quina Vil3 and A. castellanii Ma. Although the branching orders of A. castellanii Neff, A. polyphaga P23, A. triangularis SH621, and A. lugdunensis L3a were different from those in 18S riboprinting analysis, the results obtained from this study generally coincided well with those from 18S riboprinting. Mitochondrial riboprinting may have an advantage over nuclear 18S rDNA riboprinting because the mt SSU rDNAs do not seem to have introns that are found in the 18S genes of Acanthamoeba and that distort phylogenetic analyses.     

Ultrastructure and taxonomy of Paraphysomonas (Chrysophyceae) and related genera 2

This is the second of two accounts reporting on the occurrence and systematics of freshwater species of the colourless chrysophycean genus Paraphysomonas in the Cambridge area, using electron microscopy of whole mounts of both cells and scales and sections of scales. Twenty species are included here, eleven of which are new: P. cancellala, P. canistrum, P. homolepis, P. ignivoma, P. limbata, P. morchella, P. runcinifera, P. stelligera, P. stephanolepis, P. subquadrangularis and P. undulata. Five new subspecies are also established: P. circumvallata ssp. mediogranulata, P. poteriophora ssp. manubriata, P. punctata ssp. atrema , ssp. microlepis and ssp. simplicior. P. diademifera (Takahashi) comb. nov. [= Lepidochromonas diademifera (Takahashi) Kristiansen] has been included in Paraphysomonas since it has been found to be colourless and not pigmented as originally described and also to possess scales of a similar structure to those in other species of Paraphysomonas. One previously described species, P. inconspicua Takahashi, is here considered as a synonym of P. butcheri Pennick & Clarke. P. vacuolata Thomsen is removed from Paraphysomonas since it has been found to possess a chloroplast and will be included in a new genus in the third paper of this series. The 37 presently known species of Paraphysomonas have been arranged in 11 different groups according to similarities in scale structure. Consideration of the possible evolution of scale structure in Paraphysomonas and the possible interrelationships within the genus led to the conclusion that all species should be included in a single genus.     

Evaluation of a ribosomal RNA gene probe for the identification of species and subspecies within the genus Staphylococcus.

To evaluate a 16S rRNA gene probe for the identification of staphylococcal species and subspecies, we have augmented previous studies involving 12 staphylococcal species by analysing the remaining 16 species currently classified in the genus Staphylococcus. HindIII- and EcoRI-restricted DNA of isolates from validly described species of Staphylococcus was probed with radiolabelled plasmid pBA2 containing 16S rDNA from Bacillus subtilis. The Dice coefficient was used to assess similarity between the 74 HindIII- and the 81 EcoRI-hybridization patterns obtained from a total of 271 isolates belonging to 31 staphylococcal taxa (28 species, of which three include two subspecies). The use of HindIII yielded a better discrimination of the staphylococci than the use of EcoRI. All of the isolates belonging to the same species or subspecies, except S. hyicus isolates, were recovered as homogeneous clusters using their HindIII hybridization patterns. The phenotypically close taxa were clearly distinguished. Thus, the method presented in this study constitutes a powerful tool for the identification of taxa within the genus Staphylococcus.     

Multiple enzyme restriction fragment length polymorphism analysis for high resolution distinction of Pseudomonas (sensu stricto) 16S rRNA genes

Members of the genus Pseudomonas (sensu stricto) are important phytopathogens and agents of human infections, while other strains and species have beneficial bioremediation and biocontrol activities. Traditionally, these important species have been difficult to differentiate phenotypically; thus, rRNA lineage analyses have often been invoked. In this report, a newly developed approach is described to rapidly detect and distinguish fluorescent Pseudomonas isolates: PCR amplification of a Pseudomonas-specific 990-bp ribosomal RNA gene (rDNA) fragment [Appl. Environ. Microbiol. 64 (1998) 2545.] coupled with multiple enzyme restriction fragment length polymorphism (MERFLP) analysis using a single digestion mixture of AluI, HinfI, RsaI, and Tru9I incubated at 37 degrees C. The method distinguished 116 published sequences and 47 reference strains of authentic Pseudomonas representing 28 nomenspecies. A total of 55% (64/116) of the sequences analyzed by MERFLP were grouped into distinct phylogenetic clusters including Pseudomonas putida, P. syringae, P. aeruginosa, P. stutzeri, and P. fluorescens. The utility of the MERFLPs was confirmed when 100% (33/33) of the above named control reference strains were correctly placed into their phylogenetic clusters. The environmental relevance of the MERFLP method was confirmed when 67% of 28 forest and agricultural soil-derived presumptive Pseudomonas environmental clones and isolates were placed into the five major pseudomonad clusters, one clone fell into the P. agarici cluster, and five clones clustered near related pseudomonads. These data demonstrated that the PCR-MERFLP protocol provides an efficient and powerful tool for distinguishing isolates and rDNA gene libraries of environmental Pseudomonas species.     

Molecular taxonomy and phylogeny of entomopathogenic nematode species (Rhabditida: Steinernematidae) by RFLP analysis of the ITS region of the ribosomal DNA repeat unit

The ITS region of 19 isolates of the genus Steinernema Travassos, 1927 belonging to 17 species was PCR amplified. The resulting products were then digested with 17 different restriction endonucleases and the fragments generated were separated by agarose gel electrophoresis. Some enzymes yielded patterns which were highly diagnostic (e.g. Alu I, Dde I, Hha I and Hinf I) while others showed similar RFLP patterns for the majority of species (e.g. Hpa II). All of the species could be unequivocally identified using this method. A tree was constructed based on band sharing which resulted in clusters of species which exhibited similar morphological features.     

利用核糖体DNA限制性酶切谱(ARDRA)分子技术和小亚基单位核糖体RNA(SSrRNA)基因序列分析对五种双眉虫(原生动物,纤毛门,游仆目)种内及种间关系的探讨

从12种限制性内切酶中筛选出6种可获得种间多样性的内切酶:Alu Ⅰ,Taq Ⅰ,Hae Ⅲ,Hinf Ⅰ,Msp Ⅰ,Xba Ⅰ,对广义双眉虫Diophrys-complex5个种(伪寡毛双眉虫Diophrys apoligothrix、悬游双眉虫D.appendiculata、盾圆双眉虫D.scutum、秀丽拟双眉虫Paradiophrys irmgard、针毛类双眉虫Diophryopsis hystrix)共7个种群的核糖体基因(18S小亚基、部分23S大亚基及其内转录间隔区域)进行多位点酶切。结果显示,种间差异明显大于种内差异。利用RAPDistance1.04软件构建的邻接树表明,盾圆双眉虫的3个种群表现出高度的同源性;3个近缘属(双眉虫属、拟双眉虫属、类双眉虫属)可以被明确区分,并支持类双眉虫属与拟双眉虫属的独立性。从GenBank/EMBL数据库中获得广义双眉虫及相近种的小亚基单位核糖体RNA(SSrRNA)基因序列,利用邻接法(NJ),贝叶斯法(Bayesian)和最大简约法(MP)构建的系统发生树具有基本一致的拓扑结构,结果显示:狭义双眉虫属Diophrys为单源发生系,并与类双眉虫属Diophryopsis组成姐妹群;尽管拟双眉虫属Paradiophrys具有广义双眉虫典型的形态学特征,但与尾刺虫属Uronychia有着较近的亲缘关系。本工作同时表明,核糖体DNA限制性酶切(ARDRA)技术可靠地区分纤毛虫的形态相似种,并在双眉虫属间水平的系统关系推定中存在一定的适用性。