Numerical Taxonomic Techniques Research Articles

Numerical taxonomy of Portuguese Tronchuda cabbage and Galega kale landraces using morphological characters

A morphological study was carried out to determine the relationships among Portuguese cole landraces using 58 accessions belonging to: (i) Portuguese Tronchuda cabbage (Brassica oleracea var. tronchuda Bailey syn. var. costata De Candolle, Couve Tronchuda); (ii) Portuguese Galega kale (B. oleracea var. acephala De Candolle, Couve Galega); and (iii) other economically less important coles such as Algarve cabbages (B. oleracea var. capitata L., Couve do Algarve or B. oleracea var. sabauda L., Couve Repolho-lombarda do Algarve). The cole accessions were collected from the growers, studied under field conditions during two consecutive years, and characterized using 46 morphological characters from seedling stage to ripe silique. Morphological data were analysed by numerical taxonomy techniques using UPGMA (Unweighted Pair Group Method Using Arithmetic Averages) and by principal component analysis (PCA). Phenograms based on correlation and distance coefficients showed the existence of 8 main groups (A-H). Five of these groups correspond to Tronchuda cabbage landraces: (A) “Couves de Tras-os-Montes”; (B) “Couves do Minho”; (C) “Couves from Central Portugal”; (D) “Couve Portuguesa”; (E) “Couves from Southern inland Portugal”. The three other groups correspond to: (F) cabbages; (G) miscellaneous coles; and (H) Galega kales. Landrace groups are primarily associated with morphological differences among accessions and secondly with accession geographical origin. The interannual character variation did not affect the clustering patterns of the accessions and therefore the stability of landrace classification. PCA was congruent with the landrace groups defined by the phenograms and gave supplementary information on the usefulness of the characters for the definition of the various groups.

Patterns of acrorenal malformation associations.

Limb and urinary tract defects have frequently been reported to occur together as components of a single acrorenal field defect or in many multiple malformation syndromes. However, the concordance of such anomalies has rarely been studied on a population basis or the relationships between specific limb and renal defects defined. This paper documents the patterns of acrorenal associations seen in over 1,500,000 infants born in Hungary in 1975-1984. In all, 1 in 1,800 infants had a limb deficiency and 9% of these (75 cases) had a urinary tract anomaly. Urinary tract anomalies were most commonly seen with radial ray defects, micromelia and amelia. The commonest recognized patterns were VACTERL association and the cloacal exstrophy and caudal regression sequences. Chromosomal and single gene defects also occurred. Numerical taxonomic techniques delineated six main clusters of patients. Important groupings included micromelia with renal agenesis, split hand/foot with hydronephrosis, and radial ray anomalies with VACTERL defects. The radial ray groups differed in the nature of the VACTERL anomalies seen and with respect to laterality, symmetry, and non-VACTERL anomalies. There was a strong association of bilateral limb defects with bilateral renal anomalies and unilateral with unilateral. Ipsilateral defects tended to occur in typical VACTERL cases, while contralateral defects tended to occur with additional non VACTERL midline anomalies. Although renal and limb anomalies are associated, in almost all cases malformations in other systems are also present. The precise nature of the malformation patterns seen appear to reflect differences in the nature and magnitude of the underlying dysmorphogenetic processes as well as the timing of their effects.

Diversity among Rhizobia Effective with Robinia pseudoacacia L

The diversity of rhizobia that form symbioses with roots of black locust (Robinia pseudoacacia L.), an economically important leguminous tree species, was examined by inoculating seedling root zones with samples of soil collected from the United States, Canada, and China. Bacteria were isolated from nodules, subcultured, and verified to be rhizobia. The 186 isolates varied significantly in their resistance to antibiotics and NaCl, their growth on different carbohydrates, and their effect on the pH of culture media. Most isolates showed intermediate antibiotic resistance, the capacity to use numerous carbohydrates, and a neutral to acid pH response. Isolates had greater similarity within sampling locations than among sampling locations. The isolates were grouped by using numerical taxonomy techniques, and representative strains of 37 groups were selected. The mean generation times of these isolates ranged from 3 to 9 h, and the protein profile of each of the 37 isolates was unique. Nitrogen fixation, total nitrogen accumulation, and plant growth varied significantly among black locust seedlings inoculated with the representative isolates. We conclude that great variation exists among Rhizobium spp. that nodulate black locust, and selection of strains for efficiency of the symbiotic association appears possible.

DNA-DNA hybridization in the systematics of Streptomyces

The DNA relatedness among strains in several different phenotypically defined Streptomyces species clusters was evaluated. It was found that the data from DNA-relatedness studies do not necessarily agree with the clustering generated using numerical taxonomic techniques. A study of the morphologically heterogenous ‘S. cyaneus’ cluster showed that morphological criteria traditionally used to classify and identify Streptomyces species still have value, since strains in DNA-relatedness cluster groups were also similar morphologically (i.e., they had similar spore color, surface properties, and sporophore morphology). An evaluation of DNA relatedness among strains in the S. violaceusniger and S. lavendulae clusters indicated that, if anything, the genus is underspeciated, based on the number of single-member clusters observed. A study of strains of the sweet potato pathogen, S. ipomoea, collected in various locations in the United States and Japan indicated, not surprisingly, that all of the strains belong to the same species.

Numerical classification and identification of Aeromonas genospecies.

A total of 176 Aeromonas strains representing all currently characterized genospecies were tested for 329 biochemical characters. Overall similarities of all strains were determined by numerical taxonomic techniques, the UPGMA algorithm and the SSM and the SJ coefficients as measures of similarity. Sixteen clusters (two or more strains) and seven unclustered strains were recovered at the 93.5% similarity level (SSM). Genospecies 1, 4, 5, 6, 7, 9, 12 and 13 were largely represented by single phena, whereas strains of genospecies 2 and 3 were found in closely-related phena. Strains belonging to genospecies 8 formed two distinct biotypes. Strains belonging to genospecies 11 formed a subcluster within a cluster representing different genospecies. In general, similar groupings were obtained with the Jaccard coefficient at a similarity level of 80.0% (SJ) with minor changes in the definition of clusters. The phenetic data showed good correlation with the taxa defined by DNA/DNA hybridization and those obtained by multilocus enzyme analysis. For all genospecies (independent from cluster assignment) 30 diagnostic characters were selected to construct a matrix for probabilistic identification. The correct identification rate of the matrix was 71.51% taking a Willcox probability greater than 0.99, and 83.7% taking a Willcox probability greater than 0.9 as identification threshold levels.

Physiological Characterization and Identification of Listeria Species

A total of 136 strains of the genus Listeria were physiologically characterized using 329 miniaturized tests. Overall similarities of all strains were determined by numerical taxonomic techniques using the UPGMA algorithm and the SSM and SJ coefficients. A total of 10 clusters (2 or more strains) and 1 single-member cluster were defined at the 95.1 to 95.8% similarity levels (SSM). The same clusters were found at the 76.8% to 77.8% similarity levels (SJ). Strains of L. seeligeri, L. welshimeri, L. ivanovii, L. murrayi and L. grayi were found in separate clusters and formed homogeneous taxospecies. In contrast, strains of L. monocytogenes and L. innocua formed five heterogeneous clusters and one single-member cluster. For differentiation of the phena the following tests are useful: utilization of D-xylose, D-xylitol, D-ribose, D-galactose, alpha-methyl-D-mannoside, methyl-alpha-D-glucopyranoside, inulin and L-rhamnose, acid production from D-glucuronate, alpha-D-galacturonate, alpha-methyl-D-mannoside, methyl-alpha-D-glucopyranoside, D-xylose, L-lyxose, D-sucrose, alpha-D-melizitose, D-tagatose, and D-ribose and hydrolysis of pNP-alpha-D-glucopyranoside, pNP-alpha-D-mannopyranoside and 2-deoxythymidine-5'-pNP-phosphate. Independent of the assignment of some strains of L. monocytogenes and L. innocua to the same cluster, members of these species can be separated by hydrolysis of D-alanine-p-nitroanilide.

Numerical Analysis and DNA Base Compositions of Some Thermophilic Bacillus Species

Morphological, physiological, and biochemical characteristics and the DNA base compositions of 133 thermophilic Bacillus strains were determined. A total of 54 of these strains were received as identified species (mainly Bacillus stearothermophilus, Bacillus coagulans, Bacillus brevis, and Bacillus licheniformis) from international culture collections, and 79 newly isolated strains, which were isolated mainly from sugar diffusion juices of Italian plants, were also examined. Numerical taxonomy techniques (simple matching coefficient and unweight pair grouping using the mathematical average) and DNA G + C values showed that the strains aggregated into nine clusters. Both B. licheniformis and B. brevis were well separated from the other organisms. B. stearothermophilus and B. coagulans were confirmed as separate clusters and exhibited greater heterogeneity than previously shown. The B. stearothermophilus strains clustered into four groups, three of which have been recognized previously by other authors; the members of the fourth group had distinctive characteristics, including considerable biochemical inertness, an inability to grow at temperatures greater than 60 degrees C, and a high G + C content. Within the B. coagulans cluster the strains with characteristics very similar to those of the new species Bacillus smithii clustered together. However, the remaining strains were still clearly separated into two groups; one of these groups was considered B. coagulans sensu stricto, and the other was distinguished by morphological and biochemical criteria, such as spores which do not swell the sporangia, utilization of citrate, a higher proteolytic activity, and acidification of some carbohydrates. Our results were confirmed by comparing them with distinctive characteristics of recently described thermophilic Bacillus species.

A numerical classification of the genera Streptomyces and Streptoverticillium using miniaturized physiological tests

Eight hundred and twenty-one strains of the genera Streptomyces and Streptoverticillium were physiologically characterized using a total of 329 miniaturized tests. Overall similarities of all strains were determined by numerical taxonomic techniques using the UPGMA algorithm and the SSM and the S J coefficients as measures of similarity. Test error was within acceptable limits. Comparison of photometric and visual test reading revealed overall differences of 7·45%. A total of 15 major clusters (six or more strains), 34 minor clusters (less than six strains) and 40 single-member clusters were defined at the 81.5% similarity level (S SM). Two clusters containing physiologically, and in some cases morphologically and genetically, different groups could be further subdivided at the 84·0% similarity level (S SM). Generally, similar groupings were obtained with the Jaccard coefficient at similarity levels ranging from 59·6% to 64·6% similarity (S J), with changes in the definition of clusters and subclusters. The cophenetic correlation coefficients r CS for the UPGMA/S J and the UPGMA/S sm analysis were 0·6929 and 0·8239, respectively. Several phena showed significant overlap with others, indicating the physiological variability within the species. The phenetic data in most cases confirm the major phena of the study of Williams et al. (1983), Journal of General Microbiology 129, 1743-1813 (although the cluster-groups defined in that study could only be detected in part) and the results indicate that the genus Streptomyces is still overspeciated. Some of the major groupings obtained were very much in line with chemotaxonomic and genetical data. However, several clusters containing only a few strains should be regarded as preliminary ‘species’ until further information is available. The majority of Streptoverticillium strains presently assigned to different species formed a homogeneous subcluster defined at the 84·0% similarity level (S SM). Thus, on the basis of numerical phenetic and (published) molecular genetic and chemotaxonomic data, our study supports the suggestion that members of the genus Streptoverticillium be reclassified into the genus Streptomyces.

Numerical taxonomy of soils using spectral information of overlying range vegetation

Unique spectral “signatures” of rangeland vegetation were used to identify and delineate soil map units associated with a hydrosequence. Single-linkage cluster analysis was employed to obtain relatively objective and “natural” groupings of spectral patterns acquired through an airborne-based spectrometer. Similar numerical taxonomic techniques have been used previously for selected physical and chemical properties to classify soils. One hundred and twenty-eight spectral channels between 1.2 and 2.4 μm were used as variables in the cluster analysis. Derived linkages depicted in a dendrogram showed morphological similarities as well as dissimilarities among nine soil map units and two water bodies. Unique clusters of calcareous, non-calcareous, sodic, and marsh soils and open water could be observed in the dendrogram.

Application of miniaturized physiological tests in numerical classification and identification of some bacilli.

Four hundred and sixteen strains of aerobic, endospore-forming bacteria belonging to the genus Bacillus were physiologically characterized using a total of 329 miniaturized tests. Overall similarities of all strains for 295 unit characters were determined by numerical taxonomic techniques using the UPGMA algorithm and the SSM and the SJ coefficients as measures of similarity. The test error and cophenetic correlation were within acceptable limits. Comparison of photometric and visual test reading revealed overall differences of 12.3%. A total of 30 clusters (2 or more strains) and 20 single member clusters were defined at the 86.7 to 89.4% levels (SSM). Generally similar groupings were obtained with the Jaccard coefficient with some changes in the definition of clusters. Heterogeneity was found within the species Bacillus brevis, B. circulans, B. macerans, B. coagulans, B. megaterium, B. sphaericus and B. stearothermophilus, thus confirming the results of previously published data. For the clusters and for some subclusters containing two or more strains the minimum number of differentiating characters was selected by computer programs (CHARSEP, DIACHAR, MOSTTYP) and a frequency matrix for probabilistic identification was constructed consisting of 40 tests versus 36 clusters and subclusters. This matrix was theoretically evaluated using a computer program (MATIDEN), and out of 391 strains, a total of 287 (73.40%) were correctly identified with a Willcox probability p>0.99. Another 34 strains (8.69%) achieved Willcox probabilities above 0.90, for 19 strains (4.85%) p exceeded 0.8, and 27 strains (6.90%) achieved Willcox probabilities>0.5. For 24 strains (6.13%) no correct identification result was obtained.

Numerical classification and identification of some nocardioform bacteria.

A total of 254 strains of the genera Nocardia, Rhodococcus, Amycolata, Amycolatopsis, Gordona and Pseudoamycolata were compared using numerical taxonomic techniques based on 273 physiological characters with the aid of miniaturized tests. Clustering was achieved using the unweighted pair group method with arithmetic averages (UPGMA) and the simple-matching coefficient (SSM) as the measure for similarity. Test error and overlap between the phena were within acceptable limits. Cluster groups were defined at the 87.4 to 91.3% levels (SSM). A total of 25 clusters were obtained in the SSM/UPGMA analysis beside 16 single member clusters, which in most cases were marker strains of different species. For the 25 clusters, containing two or more strains, a matrix comprising frequencies for positive results of 35 tests was constructed. The minimum number of diagnostic characters was selected by computer programs (CHARSEP, DIACHAR, MOSTTYP). The final matrix consisting of 35 tests versus 25 phena was theoretically evaluated using a computer program (MATIDEN) and out of 238 strains, a total of 157 strains (65.96%) were correctly identified with a Willcox probability>0.999, further 18 strains (7.56%) achieved Willcox probabilities above 0.900 and 2 strains (0.84%) were identified correctly with a Willcox probability above 0.800. Correct identification results were obtained for a total of 56 strains (23.52%), but with low Willcox probabilities ranging between 0.500 and 0.800. A total of 5 strains (2.10%) could not be assigned to the correct phenon. In a subsequent practical evaluation of the matrix 32 of 40 tested strains (80.0%) were correctly identified with Willcox probabilities above 0.900.

Ultrametricity, frustration and the graph colouring problem

Using techniques of numerical taxonomy the authors study ultrametricity in the planar graph colouring problem with three and four colours. Evidence of a non-trivial hierarchical organisation is given for the case q=3. For q=4 they find a simpler ultrametricity with only equilateral triangles of side length d approximately=0.77. This example shows that lack of frustration does not rule out ultrametricity completely although it appears in a rather trivial way.

Spatial distribution of pre- and postsynaptic sites of axon terminals in the dorsal horn of the frog spinal cord

Axon terminals which could be interpreted as dorsal root boutons, were photographed from a series of 98 ultrathin sections with a Jeol 100B electron microscope. A total of 13 boutons were recovered for computer reconstruction. Two of them were terminal boutons, eight en passant boutons and three boutons were only partially recovered. All boutons contained multiple synaptic sites (maximum 33 and minimum seven) at which axodendritic and axoaxonic synapses were established. Axodendritic synapses were of the asymmetric type and they were directed toward adjacent dendrites. In axoaxonic synapses, which were of the symmetric type, the boutons were invariably on the postsynaptic side. Among the presynaptic profiles axons with spherical and pleomorphic vesicles and dendrites with flattened vesicles could be discerned. On average, each 2.67-μm 2 bouton surface area contained one presynaptic site at which an axodendritic synapse was established, and each 7-μm 2 surface area contained one postsynaptic site for an axoaxonic (or dendroaxonic) contact. A tendency of grouping of synaptic sites was observed. Distance measurements between the closest neighbours of all synaptic sites were made in four combinations in boutons with the original and with a random distribution of synaptic sites. The arithmetic mean of distances measured between the presynaptic and the closest postsynaptic sites was almost twice as big as that measured in the reverse direction. The difference between these values became greatly reduced in the case of random distribution. The arithmetic mean of distances between the closest neighbours of presynaptic sites was about the same as that between the closest neighbours of postsynaptic sites. This latter value was considerably increased with randomly distributed synaptic sites. The results suggest a non-random distribution of synaptic sites on the surface of boutons. The analysis of cluster formation of synaptic sites performed with a numerical taxonomy technique revealed that the majority of the 153 synaptic sites were comprised in 27 clusters containing both pre- and postsynaptic sites within the 1-μm similarity level. All postsynaptic sites were within 1-μm of one or more presynaptic sites. On the basis of the assumption that the postsynaptic sites are occupied by inhibitory axoaxonic synapses, it is suggested that the transmitter release from the presynaptic sites can be individually controlled in this structural arrangement. A probable mechanism of this function may be the passive invasion of the bouton by the impulse propagating actively along the dorsal root fibre.

Numerical Taxonomic Study of Fast-Growing Soybean Rhizobia and a Proposal that Rhizobium fredii Be Assigned to Sinorhizobium gen. nov.

A total of 33 strains of fast-growing soybean rhizobia isolated from soil and soybean nodules collected in China and 25 strains belonging to the genera Rhizobium, Bradyrhizobium, and Agrobacterium were compared by numerical taxonomic techniques, using 240 different characters. Our results indicated that all of the strains of fast-growing soybean rhizobia which we examined are closely related (guanine-plus-cytosine content, 59.9 to 63.8 mol%) and are separated from Rhizobium and Bradyrhizobium at the generic level. Based on numerical taxonomy, deoxyribonucleic acid (DNA) base ratio determinations, DNA-DNA hybridization data, serological analysis data, the composition of extracellular gum, bacteriophage typing data, and soluble protein patterns, we propose that the fast-growing soybean rhizobia represent members of a new genus rather than a species of Rhizobium (Rhizobium fredii); we propose Sinorhizobium gen. nov. as an appropriate generic name. The type species of the new genus is Sinorhizobium fredii comb. nov. (basonym, Rhizobium fredii Scholla and Elkan 1984), and the type strain is strain ATCC 35423 (= USDA 205). For the other species we propose the name Sinorhizobium xinjiangensis sp. nov.; the type strain of this species is strain CCBAU 110, which has been deposited in the Beijing Agricultural University Culture Collection, Beijing, People’s Republic of China.

Trypsin inhibitor activities as useful taxonomic traits of sweetpotato varieties

Trypsin inhibitor activities (TIAs) of roots of 53 sweetpotato varieties were used as traits for intraspecific grouping. Three numerical taxonomic techniques were evaluated on the basis of breeding records of 42 varieties. When UPGMA was used as the clustering method, phenograms from average Euclidean distance and Manhattan distance performed better than those from correlation, and 8-character matrix gave better phenograms than 4- or 11-character matrix in average, when both node counts and detailed assignments of correct pairs were compared. The best phenogram simultaneously assigned 10 pairs exactly with a probability by chance of 2.84×10−10. For cladistic analysis, mid-point rooting Wagner in the Wagner78 program package was applied to construct 3 cladograms using 4-, 8-, or 11-character matrix as input data. The cladograms from 8- and 11-character matrices were better than that from 4-character matrix, when total relative path length required to search for group members of 11 varieties and assignments of correct pairs were compared. In all, TIAs are useful traits for intraspecific grouping of sweetpotato varieties used. Eleven varieties with unknown breeding records were assigned to appropriate groups accordingly.

Improving data utilization: the case-wise alternative

Both research and practical experience suggest that alternative forms of data analysis and presentation may affect their use in the policy arena. The variable-wise alternative produces estimates of population parameters through conventional statistical techniques. The case-wise alternative defines and identifies qualitatively distinct types of cases through techniques of numerical taxonomy. In the experiment reported here, variable-wise and case-wise presentations of Census data on poverty were used to compare the effects of the alternatives on policy makers in a priority-setting task. The results show that the case-wise alternative can improve utilization, assimilation, recall, and recognition of the data. Under the circumstances examined here, the case-wise alternative is a practical means of increasing the return on the public's investment in data resources to improve policy decisions.

A numerical taxonomic study of Leuconostoc oenos strains from wine

Phenotypic data of 108 tests conducted on 70 malolactic bacteria, including 54 presumptive Leuconostoc oenos strains, five presumptive pediococci isolated from wine and 11 reference strains, were analysed by numerical taxonomic techniques. Using the simple matching coefficient, 58 strains were grouped in six clusters at the 87% S level. Cell wall analysis of the interpeptide bridges and morphology was also used to differentiate between the strains. No L. oenos reference strains were found to group in any cluster. The hypothetical median organism (HMO) of cluster A was related at only the 63% S level to the L. oenos type strain and it is proposed that these strains be regarded as ‘L. gracile’. The majority of the L. oenos strains (35) grouped together at above the 91% S level in cluster B, with the HMO of this cluster related at the 75% S level to the L. oenos type strain. Results indicate that the majority of L. oenos strains included in this study are closely related, but more research is needed to justify the separation of these strains into more than one species.

A survey of endosperm starch granules in the genus Hordeum: a study using image analytic and numerical taxonomic techniques

Image analysis was used to quantify size of endospermic starch granules in 39 species of Hordeum. A set of 279 samples, each with ca. 25 replicates, representing most species of the genus was analyzed by various multivariate techniques. Two groups can be distinguished. One group consisting of 6 species (H. vulgare, H. spontaneum, H. bulbosum, H. glaucum, H. murinum, and H. leporinum) is characterized by possessing granules greater than 35 μm in diameter and a bimodal distribution of all the sizes. The second group, consisting of the remaining 33 species, had granules less than 35 μm in diameter, unimodally size distributed. Univariate statistics for each species are summarized in box plots and bar diagrams. Some species can be uniquely identified. The usefulness of starch in Hordeum systematics is discussed.

Dialogue on systems as clusters: Operationalizing living systems

Numerical taxonomic techniques are shown to be valuable in the operationalization of living systems theory. Living systems theory in turn provides the theoretical foundation for a number of important operational decisions routinely faced by cluster analysts. Among the points of congruence are the choice of operational taxonomic unit, the choice of variables, number of hierarchies, as well as number of clusters. Living systems theory specifies 19 subsystems for seven hierarchical levels, and numerical taxonomy facilitates operationalization of these principles.

Dialogue on systems as clusters: Identifying systems

Numerical taxonomic techniques are advocated for use to help identify the (optimal) hierarchy of certain types of systems in a universe. Such an approach should help to clarify some of the vagueness surrounding operationalizing the concept of a living system. More precise specification of the boundaries and relationships among systems is also possible.