Immunological Distance Research Articles

Ploidy and immunological distance in Swedish water frogs (Rana esculenta complex)

Abstract Frogs of the Rana esculenta complex from some Swedish populations were examined and compared with imported R. ridibunda. Four groups of the frogs were discriminated from morphometrical indices : two correspond to the parental species R. lessonae(LL) and R. ridibunda(RR, not found freeliving in Sweden), one with the triploid hybrids of RRL genotype, and finally one group with LR and LLR hybrids. Albumin patterns revealed by serum electrophoresis confirmed the genotypes, with gene dosage effects being apparent in the triploids. Triploids were furthermore identified by their large erythrocytes in blood smears. Immunological distances measured with antisera to the lessonae albumin and to the slow ridibunda albumin demonstrated the five groups of frogs in the material by distincly resolving all the possible combinations of genomes (LL, LLR, LR, RRL and RR).

Immunological Relationship of Western Palearctic Water Frogs (Salientia: Ranidae)

AbstractReciprocal immunological distances between six albumins of five taxa (Rana lessonae, Italian non-hybrids, R. perezi, the slow and fast albumins of R. ridibunda, and a sample possibly representing R. saharica) were determined with microcomplement fixation. The immunological distances determined by each antiserum were scaled so that the sum of distances to each albumin was identical to the sum of distances from that albumin. The scaled reciprocal values were then averaged and used to generate 39 Fitch-Margoliash phenograms. The three lowest percent standard deviations (% SD) for phenograms were 5.7, 8.3 and 8.6; the next lowest was 12.1; eight phenograms had % SD less than 20. The three phenograms with lowest (% SD agreed in grouping (a) the two albumins of Rana ridibunda; (b) Rana lessonae and the Italian non-hybrid; and (c) Rana perezi and R. "saharica ". In the lowest % SD phenogram, the two ridibunda alleles are linked to the R. lessonae-Italian non-hybrid pair; in the next, the two ridibunda albumins were linked first to the R. perezi-R. "saharica" pair; in the third, the R. lessonae-Italian non-hybrid pair was linked first to the R. perezi-R. "saharica" pair. These latter two phenograms include legs of -5.8 and -4.7, respectively. Five triads of data among the 35 possible in the lowest% SD phenogram fail to conform to the triangle inequality; this is associated with the failure of the antiserum to the rapidly migrating ridibunda albumin to distinguish between the two ridibunda albumins. This posed no problem in grouping the albumins. The negative legs in the second and third ranked phenograms are like those generated by the Fitch-Margoliash algorithm when additive data are forced into a tree other than that specified by the additive data. The three lowest % SD phenograms agree with other data suggesting groupings of the taxa represented. They all place the two ridibunda albumins together, and they all group R. lessonae with the I talian non-hybrid form; these latter two share numerous electrophoretic markers. The data suggest a divergence ofwestern Palearctic water frogs from the eastern North American R. catesbeiana ofabout 43 x 106 years ago. R. lessonae and the Italian non-hybrid may have shared a common ancestor with R. ridibunda some 12 x 106 years ago; R. lessonae and the Italian non-hybrids may have diverged as recently as 4 x 106 years ago. The two ridibunda albumins may differ by as little as one amino acid residue. The Iberian R. perezi and saharica may have diverged from the central European cluster about 16 x 166 years ago, and from each other about 6 x 106 years ago; this latter divergence may reflect the refilling of the Mediterranean Sea following a dry period some 7-5.5 x 106 years ago.

Electrophoretic and Immunological Studies on the Relationship of the Brachyphyllinae and the Glossophaginae

Electrophoretic and albumin immunological data indicate that the Brachyphyllinae as currently conceived is a natural assemblage, with Erophylla sezekorni and Phyllonycteris aphylla being more closely related to each other than either is to Brachyphylla cavernarum . In both data sets, values that distinguish Erophylla from Phyllonycteris are in the general range of values that characterize congeneric species of mammals. Immunological distance values for the species Glossophaga soricina, Monophyllus redmani, Anoura caudifer, Leptonycteris sanborni, Choeroniscus minor , and Hylonycteris underwoodi indicate that these taxa are approximately equi-distant from the Brachyphyllinae. Immunological comparisons of Glossophaga and Monophyllus to Anoura, Leptonycteris, Choeroniscus , and Hylonycteris indicate that the four latter genera are more closely related to Glossophaga and Monophyllus than Glossophaga and Monophyllus are to the genera of the Brachyphyllinae. Values from Lonchophylla thomasi and Lionycteris spurrelli suggest a more distant relationship of these glossophagine genera to the Brachyphyllinae than the other glossophagine genera examined. Our data suggest that a 2N = 32, FN = 60 karyotype like that characteristic of Glossophaga is primitive for a clade including the Brachyphyllinae and the glossophagine genera Anoura, Glossophaga, Monophyllus, Leptonycteris, Hylonycteris , and Choeroniscus . These data suggest that the polyphyletic origin of certain glossophagine genera, as proposed by Baker (1967), is unlikely.

The Relationships of Rana Sylvatica and the Monophyly of the Rana Boylii Group

The Relationships of Rana sylvatica and the Monophyly of the Rana boylii Group. Syst. Zool., 30:170–180.—Reciprocal immunological distances were measured using antisera to the serum albumins of Rana sylvatica and representatives of five groups of Holarctic Rana (R. pipiens, R. catesbeiana, R. temporaria, R. lessonae, and R. muscosa or R. aurora or both). Scaled reciprocal values for three data sets (with R. muscosa, with R. aurora, or with both) were used to generate three sets of Fitch and Margoliash phenograms. The nine lowest percent standard deviations ranged from 6.18 to 8.44, 5.20 to 6.95, and 7.51 to 9.12, respectively. The interspecific distances in both the input and network matrices suggest that R. sylvatica is immunologically most like the eastern North American Rana. Probable cladograms place R. sylvatica either with the eastern North American Rana or in an independent lineage between the brown frogs and the water frogs. Oneway comparisons with antigens representing 12 additional species were made using the first four antisera listed. The R. sylvatica antiserum distinguished two subgroups of eastern North American Rana while the R. temporaria antiserum did not, and the R. temporaria antiserum distinguished two subgroups of both Nearctic (Rana boylii group) and Palearctic brown frogs while the R. sylvatica antiserum did not. Our data support the monophyly of the Rana boylii group, the affinity of the Palearctic brown frogs to the R. boylii group, and a distant association between R. sylvatica and the eastern North American Rana.

Comparative immunological study of albumins of Anolis lizards of the Caribbean islands

1. 1. The immunological diversity of albumins of Anolis shows that a wide variety of structures are capable of carrying out the function of this protein. 2. 2. Groupings of Anolis species based upon albumin divergence are concordant largely with classifications derived from more traditional criteria. 3. 3. The albumin evidence, however, does not support the division of the genus into Alpha- and Beta-sections. 4. 4. Immunological distances between albumins suggest that species of Anolis of at least three evolutionary lines, divergent since about the mid-Oligocene presently occupy the Caribbean Islands.

The Classification of Anolis: Conflict between Genetic and Osteological Interpretation as Exemplified by Anolis cybotes

Anolis cybotes of Hispaniola is a member of the predominantly Puerto Rican Anolis cristatellus series based upon osteological interpretation. Albumin immunological distance data suggest that the closest relatives of A. cybotes are not members of the cristatellus series, but are other Hispaniolan anoles assigned to various, supposedly distantly related series, according to the osteological interpretation. This is one of several examples within Anolis of apparent dis- cordance between the osteologically defined groups and groups that can be defined by genetic criteria. We suggest that the classification of Anolis may need critical re-evaluation.

Anolis Lizards of the Eastern Caribbean: A Case Study in Evolution. III. A Cladistic Analysis of Albumin Immunological Data, and the Definition of Species Groups

Gorman, George C., Donald G. Buth, and Jeff S. Wyles (Department of Biology, University of California, Los Angeles, California 90024) 1980. Anolis lizards of the eastern Caribbean: A case study in evolution. III. A cladistic analysis of albumin immunological data, and the definition of species groups. Syst. Zool., 29:143-158.-Albumin immunological distance data obtained primarily from the literature are interpreted cladistically for representatives of virtually all recognized groups of eastern Caribbean Anolis. A classification is proposed that differs substantially from the classification of Williams (1976). We argue that our classification is highly concordant with the character states used by Williams to generate his genealogy, but his interpretation cannot be forced into compatibility with the albumin evidence. We make no assumptions about regularity of rates of the albumin molecule, but the null hypothesis that rates of albumin evolution are equivalent along each lineage cannot be falsified. [Micro-complement fixation; immunological distance; cladistics; Anolis.] With well over 200 described species, the lizard genus Anolis can be very taxing to the working taxonomist. The current classification of the genus rests primarily on interpretation of data from the detailed comparative osteological study of Etheridge (1960). Williams (1976) used these data as a basis for presenting a formal classification of West Indian Anolis. In the terminology of Williams (1976), the genus is divided into two sections termed a and /8. These sections are distinguished by the absence (a) or pres

The Albumin Immunological and Nei Electrophoretic Distance Correlation: A Calibration for the Saurian Genus Anolis (Iguanidae)

The correlation between Nei electrophoretic and albumin immunological distance is highly significant for the lizard genus Anolis (r = 0.829, P < .001). Deterministic comparisons of the slopes of the regression lines for Anolis and some mammals are significantly different. This observation emphasizes the importance of calibrating this relationship for specific taxonomic groups.

Evolutionary relationships of turtles suggested by immunological cross-reactivity of albumins

1. 1. Plasma albumins of 16 species of turtles representing 9 families and 2 suborders were compared using quantitative microcomplement fixation. 2. 2. Data suggest that Trionyx is related to Carettochelys, but quite distinct from other turtles tested. Dermochelys shows a close affinity to cheloniids, agreeing with the conclusion that it has arisen from chelonioid stock. Chelydrids appear to be derived from a common ancestral stock together with cheloniids, emydids, and dermatemyids. Dermatemys albumin is quite similar to those of other cryptodires which share a common ancestor with it, although it has rather primitive nature morphologically. Terrapene is clearly separated from other emydids studied. Chelodina, a genus of suborder Pleurodira, is quite distinct from those of cryptodires. 3. 3. The estimated divergence time between the genera, calculated from the immunological distances obtained, is fairly concordant with the time derived from fossils.

The Genetic Relationships of the Salamanders of the Genus Plethodon

Highton, Richard and Allan Larson (Department of Zoology, University of Maryland, College Park, Maryland, 20742) 1979. The genetic relationships of the salamanders of the genus Plethodon. Syst. Zool. 28:579-599.-An electrophoretic comparison of proteins is used to evaluate the genetic relationships of the salamanders of the genus Plethodon. The genetic relationships are generally in agreement with the morphological data upon which recent classifications are based but several discrepancies are discussed. In particular, the discovery that the eastern small plethodons are paraphyletic suggests that the ancestral plethodon resembled the small species in size and that the large size of the other eastern species is a derived state. Plethodon neomexicanus, whose relationship to the other plethodons has long been ambiguous, is most closely related to P. larselli; these two species share, in addition to protein similarities, a derived osteological character state not found elsewhere in the genus Plethodon. Electrophoretic genetic distance (D) is highly correlated with albumin irnmunological distance determined for the same set of populations (r = .86) and both measures estimate similar divergence times for Plethodon lineages even when values of D are very large. Average net rate of speciation in Plethodon equals that previously determined for salamanders in general (0.12 speciation events/lineage/million years) but the rates vary considerably. Estimates of genetic distance are used in an analysis of the results of DNA annealing studies. Evolutionary divergence of total single copy and total middle repetitive DNA fractions is highly correlated with genetic distance indicating that both DNA fractions include sequences that diverge in a time dependent manner. Sequence divergence of single copy DNA, which is directly proportional to ATm, is calculated to be 0.17-0.18%/million years as a minimum estimate for Plethodon. The middle repetitive fraction as a whole appears to evolve rapidly in Plethodon as measured by changes in percentages of reassociation and by ATm in interspecific comparisons, but a small subset of the middle repetitive sequences is highly conserved. It is concluded that the eastern and western species of Plethodon diverged slightly over 40 million years ago (in the late Eocene). Compared to other North American salamanders, morphological divergence among plethodons has been slow. [Protein electrophoresis; species phylogeny; rates of speciation; DNA annealing; Amphibia; Plethodontidae; Plethodon.] Electrophoretic analysis of protein variation provides a measure of genetic distance among populations that reflects the evolutionary divergence of structural genes. The largely time dependent or clocklike nature of protein sequence evolution has been established by analyses of the relative amounts of change occurring along the diverging branches of molecular phylogenetic trees (Sarich and Cronin, 1977; Wilson et al., 1977; Carlson et al., 1978). Protein sequence comparisons (Kimura and Ohta, 1974) and immunological protein comparisons (Maxson et al., 1975; Wilson et al., 1977) 1 Present address: Department of Genetics and Museum of Vertebrate Zoology, University of California, Berkeley, California 94720. evaluated in the contexts of fossil evidence and continental drift, relate protein sequence evolution to measures of time. The generally high correlation of electrophoretic genetic distance with albumin immunological distance is used by Sarich (1977) to establish the time dependency of genetic distance and to date divergence of evolutionary lineages from genetic distance estimates. Another estimate of divergence time from electrophoretic data is described by Nei (1975). We again compare the electrophoretic and immunological estimates of genetic divergence because immunological comparisons are available for the same set of populations that we have studied electrophoretically (Maxson et al., 1979). Because of the largely time dependent na-

Production of antibodies to human immunoglobulins in a gibbon.

Immunization of a chimpanzee, a gibbon, a baboon and a rhesus monkey with human globulins gave different results. While chimpanzees did not produce any anti-human antibodies, baboon and rhesus monkey responded by producing anti-human IgG antibodies that did not show subclass or allotype specificity. In the gibbon antiserum, on the other hand, six different antibodies were demonstrated with a specificity against one or more of the four human IgG subclasses, including one allotype. Comparative investigations of non-human primate species sera showed that one of the specificities detected by these antibodies, i.e., the allotype G1m(f), was restricted to man; the other five antibodies reacted with man and with two or more genera of apes. Lower primates did not share any of these antigenic determinants. The extent of immunological distance is parallel to the taxonomic classification. The gibbon shows that there are more similarities between man and chimpanzee or pygmy chimpanzee than between man and gorilla or orangutan.

A study on histones in amoeba nuclei by indirect immunofluorescence method

Nuclear proteins of four species of free-living Amoebidae ( Amoeba proteus, A. discoides, Chaos carolinensis and Polychaos dubia) have been studied by indirect immunofluorescence technique using specific antisera to H1, H2A, H2B, H3 and H4 histone fractions from the calf thymus. It has been shown that the nuclei of the species examined have all these five histone fractions. However, the degree of similarity between homologous fractions from amoebae and the calf thymus varies and can be expressed in terms of immunological distance. Immunological differences between amoebic and calf thymus histones are the most pronounced in H1, being least in H3 and H4. Judged by its immunochemical characteristics, the histone fraction H2A from P. dubia is closer to the corresponding fraction from the calf thymus than is H2A from the other three amoeba species.

Serological analysis of species specificity in the high mobility group chromosomal proteins.

The non-histone chromosomal protein of the high mobility group (HMG-1) present in mouse liver was purified to homogeneity. Antibodies against this protein as well as pure HMG-1 derived from calf thymus and HMG-E purified from duck erythrocytes were elicited in rabbits. The interaction between the antibodies and the immunogens was measured by passive hemoagglutination and by quantitative microcomplement fixation. Quantitative microcomplement fixation assays revealed that the immunological distance between HMG-1 from calf thymus and HMG-1 from mouse liver and duck erythrocytes was 15. This corresponds to 3% sequence differences. It was estimated that amino acid substitution occurred at about seven positions in the polypeptide chain. Thus, HMG-1 proteins display remarkable evolutionary conservation in their primary sequence, similar to that displayed by histones H4 and H3, suggesting that their biological function is dependent on stringent structural requirements. HMG-E protein is significantly different from both HMG-1 and HMG-2 derived from calf thymus. As such, it is a protein unique to avian erythrocytes.

Reciprocal Immunological Distances of Catalase Derived from Strains of Mycobacterium avium, Mycobacterium tuberculosis, and Closely Related Species

The structural divergence of catalase derived from different mycobacteria has previously been studied in terms of immunological distances as determined by titration with antiserum against catalase extracted from Mycobacterium tuberculosis. This study has been extended by addition of a second reference antiserum: One raised against catalase from M. avium. The two systems show a high reciprocality of immunological distance against one another. The catalase from M. intracellulare is serologically very similar to that of M. avium, and the enzyme from M. bovis is very similar to that of M. tuberculosis. Catalase extracted from BCG, a pathovar of M. bovis, appears to exhibit structural modification which diverges from the parent M. bovis and converges on M. avium. Two classes of catalase have been separated from M. scrofulaceum: One is nonreactive with either reference antiserum, and the other exhibits immunological distances that suggest that M. avium is in a position between M. scrofulaceum and M. tuberculosis.

Comparative Study of Molecular Size and Structure of Exo- -Glucanases from Kluyveromyces and Other Yeast Genera: Evolutionary and Taxonomic Implications

A comparative study was made of the molecular weights of the exo-β-glucanases (EC 3.2.1.58) of species belonging to Kluyveromyces or to various other yeast genera. Their immunological distances were determined with the exo-β-glucanases from Kluyveromyces fragilis, K. phaseolosporus, K. aestuarii, and Saccharomyces cerevisiae as references. The molecular sizes of the enzymes were estimated by polyacrylamide gel exclusion chromatography, and the immunological comparison was done by the microcomplement fixation technique. The molecular weights of the enzymes from ascomycetous species ranged from 24 x 103 to 63 x 103, but the majority of the species, including most species of Kluyveromyces, contained exo-β-glucanases with molecular weights between 35 x 103 and 45 x 103. Immunological identity was observed among the exo-β-glucanases from the following species: K. fragilis (reference species), K. marxianus, K. bulgaricus, K. cicerisporus, and K. wikenii; closely related were K. phaseolosporus (reference species), K. delphensis, K. lactis, K. vanudenii, and K. drosophilarum; also closely related were Saccharomyces cerevisiae (reference species), S. italicus, S. uvarum, and S. chevalieri. The exo-β-glucanase from K. aestuarii showed low degrees of homology with those from all tested species of Kluyveromyces. It was concluded from these data that ascospore shape has little significance in the phylogeny of this genus. Other evolutionary implications are discussed.

Preliminary quantification of immunological relationships among the transaldolases of the genus Bifidobacterium.

The immunological relatedness among the transaldolases (dihydroxyacetone transferase, E.C. 2.2.1.2) of twenty species of the genus Bifidobacterum has been tested by the microcomplement fixation method, using B. thermophilum (B. ruminale) RU326 (= ATCC 25866), B. cuniculi RA93 (= ATCC 27916) and B. 'minimum' (DNA homology group) F392 (= ATCC 27916) as references. Based on the serological relationships of the transaldolases, expressed either as indices of dissimilarity or as immunological distances, the twenty species of the genus Bifidobacterium were arranged into clusters. These clusters generally coincided with the immunological groups obtained previously by the immunodiffusion method (Sgorbati and Scardovi, 1979).

Production of Antibodies to Human Immunoglobulins in a Gibbon

Immunization of a chimpanzee, a gibbon, a baboon and a rhesus monkey with human globulins gave different results. While chimpanzees did not produce any anti-human antibodies, baboon and rhesus monkey responded by producing anti-human IgG antibodies that did not show subclass or allotype specificity. In the gibbon antiserum, on the other hand, six different antibodies were demonstrated with a specificity against one or more of the four human IgG subclasses, including one allotype. Comparative investigations of non-human primate species sera showed that one of the specificities detected by these antibodies, i.e., the allotype G1m(f), was restricted to man; the other five antibodies reacted with man and with two or more genera of apes. Lower primates did not share any of these antigenic determinants. The extent of immunological distance is parallel to the taxonomic classification. The gibbon shows that there are more similarities between man and chimpanzee or pygmy chimpanzee than between man and gorilla or orang utan.

A comparative immunological study of catalases from coagulase-positive staphylococci.

Protein homology studies with catalase as a reference point were carried out with coagulase-positive staphylococci belonging to Staphylococcus aureus, S. intermedius and S. hyicus. Antisera against catalases of S. aureus ATCC 12600 and S. aureus ATCC12601 reacted very weakly employing double immunodiffusion and quantitative microcomplement-fixation assay with cell-free extracts or catalase enriched preparations of S. intermedius or S. hyicus. The differences between coagulase-positive staphylococci could be confirmed by using the antiserum against S. intermedius H 11 catalase. Within the strains of the species S. intermedius immunological distances ranging up to 25 indicate a heterogeneity which justify the separation of the biotypes E and F on a subspecies level. Coagulase-positive strains of S. hyicus revealed neither a close relationship to S. aureus nor to S. intermedius.

Comparison of various immunological methods for distinguishing among mammalian pancreatic ribonucleases of known amino acid sequence

Fourteen mammalian pancreatic ribonucleases of known amino acid sequence were compared by 1 or more of 3 different immunological methods: standard quantitative micro-complement fixation, spot-plate micro-complement fixation, and inhibition of phage inactivation. It was found that, while the results obtained by the 3 techniques were correlated with one another, the standard micro-complement fixation procedure was most versatile, economical of materials, and easiest to execute. The standard MC'F technique was more sensitive than the spot-plate technique to differences in amino acid sequence. The inhibition of phage inactivation method was more sensitive than the standard method for measuring differences among closely related RNases but proved impractical for amino acid differences over 15%; the MC'F method could be extended to at least 30% sequence differences. The standard method, moreover, readily detected the single amino acid difference between dromedary and camel RNases. A linear relationship was found between immunological distance (y) in the MC'F test and percent sequence difference (x) which fit the equation y = 7x. The strength of the correlation between immunological distance and percent sequence difference is consistent with the proposal that a large fraction of the evolutionary substitutions of amino acids in ribonuclease are immunologically detectable. This could be explained either by a multideterminant hypothesis or by a pauci-determinant hypothesis which says that substitutions occurring outside determinants produce small conformational changes influencing determinant reactivity.

Evolution of acid phosphatase-1 in the genus Drosophilia. Immunological studies.

The enzyme acid phosphatase-1 was partially purified from 10 Drosophila species. Four antisera were produced and the ten enzymes were reacted against each serum. The method used to quantitate the reactions involved the electrophoretic separation of antigen-antibody complexes from uncomplexed enzyme, followed by densitometry of the free enzyme. Immunological distances were used to obtain correlation coefficients for all pairwise combinations of the 10 species. From these correlation coefficients, a dendrogram was constructed which is very similar to one diagramming the presumed phylogenetic relationships of the ten species. In addition, the data indicate acid phosphatase-1 has evolved at different rates in different lineages within the genus. A preliminary estimate of the unit evolutionary period for this enzyme is 3.25 million years. The method of determining immunological distances which was used in this study is compared to the method of microcomplement fixation in the Discussion.