Microcomplement Fixation Research Articles

Seroprevalence of antibodies to Chlamydophila abortus in ovine in the State of Alagoas, Brazil

The goal of this study was to perform a seroepidemiological investigation and to identify risk factors associated with infection of Chlamydophila abortus of sheep herds in the Brazilian state of Alagoas. The study was conducted with samples of 274 ewes with ages equal to or higher than 24 months in 25 herds and in 23 towns located in three regions of the state (Sertão, Agreste and Eastern Alagoas). Anti-C. abortus antibodies were detected using the microcomplement fixation test. The risk factors, were determined based on questionnaires consisting of objective questions, about the farmer and general characteristics of the herd like size, sanitary situation and reproductive management. Among 274 sera samples analyzed for C. abortus, 59 (21.5%) were positive with titers ≥32, 187 (68.3%) negative and 28 (10.2%) suspect with titers ≥16. In the 23 towns studied, 20 had positive animals. Among herds 21 (77.7%) of had positive animals. The only variable which appeared to be significant in the multivariate analysis was the region, and Sertão was the most significant (p<0.001; OR=3.48; T.I. 1.79 - 6.76). Results indicate that infection by Chlamydophila abortus is widespread on sheep farms in the State of Alagoas. Others studies, however, have to be conducted to isolate the agent in order to confirm the role of the bacteria is reproductive disturbances in sheeps. In addition to that, control and prophylactic measures along with health promoting programs have to be encouraged on the studied farms so that infection reates are reduced.

Seroprevalence of antibodies to Chlamydophila abortus in Ovine in the State of Alagoas, Brazil

The goal of this study was to perform a seroepidemiological investigation and to identify risk factors associated with infection of Chlamydophila abortus of sheep herds in the Brazilian state of Alagoas. The study was conducted with samples of 274 ewes with ages equal to or higher than 24 months in 25 herds and in 23 towns located in three regions of the state (Sertão, Agreste and Eastern Alagoas). Anti-C. abortus antibodies were detected using the microcomplement fixation test. The risk factors, were determined based on questionnaires consisting of objective questions, about the farmer and general characteristics of the herd like size, sanitary situation and reproductive management. Among 274 sera samples analyzed for C. abortus, 59 (21.5%) were positive with titers ≥32, 187 (68.3%) negative and 28 (10.2%) suspect with titers ≥16. In the 23 towns studied, 20 had positive animals. Among herds 21 (77.7%) of had positive animals. The only variable which appeared to be significant in the multivariate analysis was the region, and Sertão was the most significant (p<0.001; OR=3.48; T.I. 1.79 – 6.76). Results indicate that infection by Chlamydophila abortus is widespread on sheep farms in the State of Alagoas. Others studies, however, have to be conducted to isolate the agent in order to confirm the role of the bacteria is reproductive disturbances in sheeps. In addition to that, control and prophylactic measures along with health promoting programs have to be encouraged on the studied farms so that infection reates are reduced.

Perspective: conservation genetics enters the genomics era

Throughout most of my professional career (which began in the late 1960s), a long-sought Holy Grail in molecular ecology and evolution was to obtain extensive nucleic acid sequences from large numbers of loci and organisms. In lieu of efficient DNA-sequencing technologies, researchers adopted a succession of less direct approaches for estimating various genomic parameters such as heterozygosities, kinship coefficients, or genetic distances. These laboratory techniques included allozyme electrophoresis (mid-1960s), the immunological approach of micro-complement fixation (1960s), gel-sieving and other methods to reveal hidden protein variation (early 1970s), restriction-enzyme assays especially of mitochondrial DNA (late 1970s), DNA/DNA hybridization (1970s), DNA fingerprinting by minisatellites (1980s), PCR-based sequencing of particular target genes for which conservative primers were developed (late 1980s), RAPD (randomly amplified polymorphic DNA) assays (1990s), microsatellite analyses (1990s), DNA barcoding based on a mitochondrial gene (2000s), and several other molecular approaches for revealing genetic variation in particular proteins or classes of nucleic acids (see Hillis et al. 1996; Avise 2004; Freeland 2005). Different laboratory methods yielded genetic markers well-suited for addressing different sections along a phylogenetic spectrum from the microto the macro-evolutionary: detection of clonal identity or non-identity (e.g., via DNA fingerprinting or multi-locus allozymes), population demography and mating systems (allozymes, microsatellites), intraspecific population structure and phylogeography (allozymes, mtDNA), speciational processes and species differences (barcoding, allozymes, mtDNA), hybridization and introgression (allozymes, mtDNA), and supra-specific phylogenetics at many temporal scales (via microcomplement fixation, DNA-DNA hybridization, and DNA sequencing of particular nuclear or cytoplasmic loci). In many cases, the data also served to improve our mechanistic understanding of a wide range of molecular-level phenomena such as mutation rates and patterns, gene duplications, the phenomenon of concerted evolution, and the operation of natural selection on particular loci. The primary limitation of most methods (with the possible exception of DNA hybridization) was that only a tiny fraction of the genome was accessible from which to make estimates of the genome-wide parameters that ultimately were of interest. In recent years, later-generation molecular technologies have made mass-scale nucleic acid sequencing almost routine. For example, by the spring of 2009, at least one entire genome had been sequenced from each of about 1,000 species (including 100 eukaryotes), with another 1,000 species in various stages of sequence completion. Modern molecular methods such as 454 pyrosequencing also make it possible to sequence thousands of proteincoding genes using expressed sequence tags (ESTs) from the transcriptomes (messenger RNA pools) of multiple individuals, even in non-model organisms (Papanicolaou et al. 2005; Hudson 2007). Furthermore, in this ‘‘genomics revolution,’’ dramatic advancements in microchip arrays and related technologies have made gene-expression profiling (transcriptomics, proteomics, and metabolomics) practicable at unprecedented genomic scales (Gibson and Muse 2009). Indeed, molecular technologies are no longer the limiting factor in genetic analysis, often having been J. C. Avise (&) Department of Ecology and Evolutionary Biology, University of California, Irvine, Irvine, CA 92697, USA e-mail: javise@uci.edu

Phylogenetic relationships in the genus Aethomys (Rodentia: Muridae)

Phylogenetic relationships in the genus Aethomys were examined based on qualitative cranial data for all currently recognized species. A cladistic analysis suggested the presence of three clades: 1) A. bocagei, A. thomasi, A. silindensis, A. kaiseri, and A. nyikae;2) A. chrysophilus, A. ineptus, and A. hindei;3) A. granti, A. namaquensis, and A. stannarius. These phylogenetic groupings are largely inconsistent with previously postulated supraspecific relationships for the genus. Nonetheless, relationships within each clade broadly correspond with some previously suggested evolutionary hypotheses among some members of the genus, the only notable exception being a close affinity between two southern African taxa (A. namaquensis and A. granti) and the geographically disjunct West African endemic, A. stannarius. A phylogram derived from an analysis of anagenetic divergence among taxa supported the widely-held view of a close affinity between A. namaquensis and A. granti that has traditionally led to their inclusion in the subgenus Micaelamys. These results are relatively concordant with recent molecular studies based on microcomplement fixation of albumin, mitochondrial DNA cytochrome b and 16S rRNA data, which collectively suggest that the genus Aethomys is paraphyletic. Possible paraphyly is also reflected by a relatively low bootstrap value at the node uniting all ingroup taxa in this study, as well as previous studies on dental morphology, modes of karyotypic change, and gross sperm and bacular morphology, and strongly supports the taxo-nomic elevation of the currently recognized subgenera Micaelamys and Aethomys to full generic rank. Despite the broad correspondence between the morphological and molecular data, homoplasy and low bootstrap support in the present investigation point to the need for a careful consideration of the power and choice of morphological characters in future studies on this and related groups of murine rodents.

A short history of the initial application of anti-5-BrdU to the detection and measurement of S phase.

Because this article is a bit of history rather than ascientific paper, we will depart from the formality of ajournalarticle.Thisarticlehastwopurposes.Thefirstistogive credit to two deceased individuals, Albert Castro,M.D., and Howard Gratzner, Ph.D. (1). Castro was the keyperson who was able to produce a polyclonal antibodythat was specific for bromodeoxyuridine (BrdU). For 10years, Gratzner took the lead in converting an idea into afunctional assay that affected DNA analysis and led to thedevelopment of many other assays with similar method-ologies. The second is to inform young scientists that (a)inventions or creations have utility in fields other than thepurpose for which they were created; (b) it is possible todo useful work in an institution that certainly would nothave been classified as one of the best; and (c) the pres-ence of talented people who are willing to collaborate isinvaluable.Scientifically, Gratzner is best known for the develop-ment of the first monoclonal antibody (mAb) to BrdU andiododeoxyuridine. This work was the culmination of pre-vious efforts with Robert Leif to develop polyclonal anti-bodies, which detected BrdU with variable specificity.Collaborations with scientists at the Lawrence LivermoreLaboratory encouraged the development of a widely usedflow cytometric test for the simultaneous measurementsof DNA and BrdU. The use of mAbs to halogenated pyri-midines was a significant scientific advance and currentlyis an integral tool for the analysis by flow and digitalmicroscopy of the cell cycle.On a personal level, Gratzner’s enthusiasm for scienceand his intelligence, curiosity, loyal interactions with fel-low collaborators, and friendship are sorely missed by allwhoknewhim.Hewasgenuinelyawarmandnicepersonwho was liked by almost everyone with whom he came incontact (1).Castro was a jovial man who interacted well with hiscollaborators. He was generous and very knowledgeable.He ran an immunodiagnostics laboratory at the universi-ties of Oregon and Miami and was an expert in the pro-duction of antibodies in animals. His warm smile, gener-ous giving of his knowledge, common sense, enthusiasmfor scientific research, and ability to get the job done wereessential to this project.Because this is a story of events that happened approx-imately 30 years ago and the existing records are limited,the accuracy of this article is limited by our memory andthose of our colleagues who have kindly helped with thisproject. Of course, this is our version of the events.Cell division and DNA replication are fundamental tobiology and specifically to the biology of cancer. Thisarticledescribeshowasimpleprocedureforthedetectionand quantitation of DNA synthesis was developed. Theprecise determination of when S phase occurs in the cellcycle is of use to maximize the selective killing of tumorcells with cycle-specific chemotherapeutic drugs. Ofgreater significance, studies of the control of the cellcycle, including its detour into apoptosis, can provideextremely useful insights into the creation of new thera-peutic regimens.The original process to detect S-phase cells included theuse of tritiated thymidine with autoradiography to mea-sure the labeling index (percentage of S-phase cells)and/or the fraction of labeled mitotic cells. Before and atthe infancy of flow cytometry, it was believed that thequantitative DNA analysis of normal and neoplastic cellsmight provide an objective marker for the diagnosis ofneoplasia. These measurements were performed with amicroscope on Feulgen-stained cells. In a normal popula-tion, it was established that there were two predominantpeaks in the DNA distribution, with a ratio of 1:2 in DNAcontent, and that cells that were synthesizing DNA (Sphase) were scattered in between, with a relative DNAcontent between 1 and 2. The detection of tritiated thy-

Molecular Phylogenetics of Australo–Papuan Possums and Gliders (Family Petauridae)

Phylogenetic relationships within the possums of the family Petauridae, including their affinities with the family Pseudocheiridae, were inferred from DNA sequences obtained for the mitochondrial ND2 gene (1040 bp) combined with previously published partial 12S rDNA sequences. Short, deep internodes characterize some of the divergences obtained. The robustness of these nodes was assessed by several methods such as exclusion of taxa and partitioning of characters. In all analyses a monophyletic Pseudocheiridae was evident, whereas a monophyletic Petauridae was not as well supported. Within the Petauridae, Gymnobelideus was more closely related to Dactylopsila–Dactylonax than to Petaurus. This supports the results obtained from microcomplement fixation of albumin and DNA–DNA hybridization studies but conflicts with morphological data.

A Molecular Phylogeny for the Frog Genus Limnodynastes (Anura: Myobatrachidae)

Members of the genus Limnodynastes are a prominent and widespread feature of the Australian frog fauna. Yet despite their potential to be informative about biogeographic history and mechanisms of speciation, the relationships among these taxa are not well known. We investigated phylogenetic relationships within the genus Limnodynastes via sequencing of mitochondrial (mt)DNA from current members of the genus Limnodynastes and the monotypic genus Megistolotis. a 450-bp fragment of the 16S rRNA gene and a 370-bp fragment of the protein-coding gene ND4 were used to infer a molecular phylogeny. We revise traditional species groupings and now recognize four species groups within Limnodynastes: the L. ornatus group (L. ornatus and L. spenceri), the L. peronii group (L. peronii, L. tasmaniensis, L. fletcheri, the L. depressus), the L. salmini group (L. salmini, L. convexiusculus, and L. lignarius), and the L. dorsalis group (L. dorsalis, L. terraereginae, L. dumerilii and L. interioris). The L. ornatus species group forms a highly distinctive clade that is a sister group to the other Limnodynastes groups. Pending broader phylogenetic studies it could be removed from the genus Limnodynastes. Our results concur with previous suggestions that Megistolotis lignarius is nested within Limnodynastes, and we therefore reclassify this species as Limnodynastes lignarius. Furthermore, specimens identified as L. depressus form a mtDNA lineage distinct from other species in the genus, confirming the validity of the species. Specimens of species from the L. dorsalis group (L. dorsalis, L. dumerilii, L. interioris, and L. terraereginae) are closely related such that L. dumerilii is paraphyletic with two other species. Finally, our study provides broad support for previous phylogenies based on microcomplement fixation.

Ocorrência e distribuição de esporos de Clostridium botulinum tipos C e D em áreas de criação de búfalos na Baixada Maranhense

Botulismo é enzoótico na criação de búfalos da Baixada Maranhense, Estado do Maranhão. No presente trabalho foram realizados estudos para verificar a ocorrência e distribuição de esporos de Clostridium botulinum tipos C e D em amostras de solo, limo e fezes de búfalos, colhidas aleatoriamente em áreas inundáveis da criação de búfalos nessa Baixada. A evidenciação de esporos foi realizada em 40 amostras de fezes, 65 de limo e 35 de solo, provenientes de quatro municípios, pelo cultivo em meio de cultura com carne cozida e posterior inoculação do sobrenadante filtrado em camundongo, na tentativa de verificação da presença de toxina botulínica. A tipificação de amostras positivas foi realizada pela microfixação de complemento. Os resultados revelaram que 104 (74,28%) das 140 amostras examinadas foram positivas para a presença de esporos de C. botulinum pelo teste indireto. Não houve diferença significativa (P&gt;0,05) entre os valores obtidos quando das análises das amostras de solo (77,1%), limo (60,0%) e fezes (95,0%). Das 28 amostras de solo, limo e fezes positivas, que foram utilizadas para a tipificação, quatro (14,29%) foram classificadas como tipo C, 23 (82,14%) como tipo D e uma (3,5%) como pertencente ao complexo CD. Os resultados revelaram uma alta contaminação ambiental por C. botulinum em áreas de criação de búfalos da Baixada Maranhen-se. A identificação de outros tipos e de subtipos de C. botulinum não foi realizada.

Sensibilidade toxicológica e especificidade do teste de microfixação de complemento na detecção de toxinas botulínicas C e D em meio de cultura e fígado de camundongos

No presente estudo pretendeu-se verificar a sensibilidade toxicológica e especificidade do Teste de Microfixação de Complemento (MCF) na detecção de toxinas botulínicas C e D no sobrenadante de cultivos bacterianos e em fígados de camundongos inoculados com doses letais e subletais. As toxinas foram produzidas em meio de cultura Hemoline, tituladas através da determinação da DL50 pelo Bioensaio em Camundongo e diluídas nas concentrações de 10, 1, 0,1, 0,01 e 0,001 DL50. Desta forma, foram utilizadas em dois modelos experimentais, onde foi determinada a sensibilidade toxicológica do MCF no sobrenadante do meio de cultura com as diluições descritas acima e ainda em extratos hepáticos de camundongos com peso corporal de 20g, inoculados com as mesmas diluições. A tentativa de evidenciação das toxinas botulínicas nos extratos hepáticos de camundongos foi realizada através da sua extração após a morte pela administração das doses letais e ainda pelo sacrifício dos animais inoculados com doses subletais, em intervalos de 5 dias. Os resultados evidenciaram uma sensibilidade toxicológica para o MCF de 100% para os dois tipos de toxinas ao nível de 0,01 DL50, quando testados os sobrenadantes de meio de cultura, portanto 100 vezes superior ao Bioensaio em Camundongo. A sensibilidade toxicológica do MCF, quando examinados extratos hepáticos de camundongos inoculados com 1 e 10 DL50 de toxinas botulínicas C e D, foi inferior, com valores de 100, 80, 89 e 72%, respectivamente. Pelo teste foi possível detectar toxinas botulínicas tipos C e D nos extratos hepáticos de camundongos inoculados com doses subletais até 15 dias após a sua inoculação. A especificidade do MCF foi de 88 e 92%, quando testados extratos hepáticos de camundongos sadios, e confrontados com as antitoxinas C e D; e 100% no sobrenadante do meio de cultura. Os resultados apontam para uma possível utilização do teste como importante instrumento de pesquisa e ainda na eventual substituição dos testes in vivo pelas suas implicações éticas e limitações práticas.

Cell specific nuclear antigens of boar spermatozoa.

Demembranated boar sperm heads were differentially extracted at conditions involving high salt-urea, proteolysis and DNase I cleavage that mimic the conditions promoting the in vivo decondensation of the fertilizing sperm nucleus in the egg ooplasm. The sperm-unique subset of proteins was studied which remained bound in the residual salt-resistant nuclear structure operationally defined as sperm nuclear matrix. By means of polyvalent antisera the immune specificity of the sperm nucleoprotein complex was estimated using ELISA and microcomplement fixation test as compared to somatic type dehistonized chromatin of boar liver. To define immunologically specific sperm DNA-associated proteins hybridomas were generated by fusing lymphocytes immunized with boar sperm protein/DNA complex. Monoclonal antibodies were selected (Mab 1A8, 1B3, 2B5, 2H5 and 3A4) which identified protein moieties in the sperm DNA-tight binding proteins complex resistant to cleavage with DNase I and sensitive upon digestion with high concentration of proteases. No appreciable reactivity was recorded of the antibodies to somatic chromatin and no significant binding to ssDNA. A polypeptide in the residual sperm nuclear structure of apparent Mr 27 kDa was recognized by Mab 3A4 as detected by Western blotting. The enhanced reactivity to the DNase I digested sperm nuclear fraction (except for Mab 2H5) suggests that DNA protected from nuclease digestion by a protein might be essential for immune reactivity and full antigenic integrity as well as the dependence of the cognate proteins on the binding to DNA for antigenicity and immune specificity. The functioning of the identified putative sperm specific proteins is anticipated in the structural rearrangement of chromatin in the zygote.

Divergent Lineages Within the Bufo margaritifera Complex (Amphibia: Anura; Bufonidae) Revealed by Albumin Immunology

The toads belonging to the Bufo margaritifera complex (Amphibia: Anura; Bufonidae) are widely distributed in the Neotropics. A molecular analysis, using the quantitative immunological technique of micro-complement fixation, assessed the degree of divergence in a plasma protein, serum albumin, among representative Central and South American toads currently placed in this complex. This analysis revealed a surprisingly large amount of genetic diversity. Comparisons of estimates of albumin sequence evolution in representatives of the Bufo margaritifera complex from 32 localities in one central and seven South American countries indicate that this complex indudes more than three lineages that are very distinct genetically but show relatively little morphological variation. The degree of albumin divergence implies that some of these lineages have been isolated for at least 30 million years. This study also suggests that the amount of genetic diversity within other Amazonian species or species groups could be substantial.

Relationships among palearctic Hyla: Insights from immunology

The European or green tree frog, Hyla arborea (family Hylidae; subfamily Hylinae), was once believed to be a single widespread species ranging throughout Europe and Asia. Based on data from more detailed studies of morphology and mating call, and information from molecular techniques, H. arborea is now recognized as a superspecies group. To examine relationships among these frogs, we used the quantitative immunological technique of micro-complement fixation to estimate the degree of amino acid sequence divergence in the proetin serum albumin. We estimate the divergence time between the European H. arborea and the Middle Eastern H. savignity to be about 6 MYA, while some Asian species appear to have diverged much earlier, approximately 24 MYA. Several distinct patterns of albumin cross-reactivity to the two antisera used in this study were observed. The groups distinguished by these patterns are consistent with patterns of speciation that can be supported by biogeographical history.

Geographic Variation of Serum Albumin in the Monotypic Snake Genus Diadophis (Colubridae: Xenodontinae)

-The ringneck snake, Diadophis punctatus (Colubridae [s.l.l Xenodontinae), is one of the most widespread snake species in North America. Thirteen subspecies currently are recognized based upon variation in several morphological characters. An antiserum prepared against the serum albumin of the eastern subspecies, D. p. edwardsii, revealed very high levels of variation in albumin across the range of this species. The immunological distances (direct estimates of amino acid differences) range from 0 to 25; this degree of albumin divergence typically is found between congeneric species in other vertebrates, including snakes, and indicates substantial genetic differentiation within D. punctatus. The primary division within this genus appears to separate eastern and western (including midwestern) subspecies and dates to approximately the Miocene. These data indicate that Diadophis may not be a monotypic genus, but instead contains at least two genetically distinct species. Molecular techniques have proven to be invaluable in detecting genetic variation between taxa having no obvious or consistent morphological differentiation (Highton, 1979; Donnellan and Aplin, 1989; Hedges and Thomas, 1991). Some workers have suggested that investigations of genetic variation at the species level is the province of isozyme, cytogenetic, and, most recently, DNA sequence data (Hillis and Moritz, 1990). However, estimates of amino acid sequence divergence between proteins obtained using the immunological technique of micro-complement fixation (MC'F) have helped to reveal a number of cryptic species (Maxson, 1978; Scanlan et al., 1980; Maha et al., 1983). Although MC'F primarily has been applied to problems of inter-specific and inter-generic lecular techniques have proven to be inluable in det cting genetic variation between a having no bvious or consistent morphoical dif erentiation (Highton, 1979; Donneland Aplin, 198 ; Hedges and Thomas, 1991). e workers have suggested that investigai ns of genetic vari t on at the species level is e province of isozyme, cytogenetic, and most cently, DNA sequence data (Hillis and Movariation, this technique has proven useful in intra-specific studies of geographic variation within widespread species (i.e., Plethodon glutinosus; Highton et al., 1989). Sequence divergence in serum albumin has been shown to increase in an approximately linear manner with time across a wide diversity of vertebrate taxa; an average of 10 amino acid substitutions between taxa accumulate every 6 million years (Maxson, 1992). This relationship indicates that data generated through MC'F can provide insights on divergence times between lineages. The xenodontine snake genus Diadophis (Baird and Girard, 1853), currently recognized as a monotypic species (punctatus), is one of the most widely distributed North American snakes, ranging from southern Canada into Mexico, and 105 This content downloaded from 157.55.39.177 on Fri, 18 Nov 2016 04:12:50 UTC All use subject to http://about.jstor.org/terms

Evolution in the Murinae (Rodentia) Assessed by Microcomplement Fixation of Albumin

The inter-relationship of 17 genera of Murinae rodents were studied using microcomplement fixation of albumin to measure immunological distances among taxa. The genera chosen represented six major clades and four monogeneric groups identified in earlier companion studies (an African group, a South-east Asian group, a New Guinean group, an Australasian group, an Acomys group, a Micromys group and Apodemus, Millardia, Mus and Phloeomys). When these companion studies are included 136 species and 67 genera were studied. Ten major clades were consistently supported by the data but apart from linking the South-east Asian and Australasian clades, the data do not resolve their inter-relationships. The Acomys-Uranomys-Lophouromys clade may be the sister-group to the rest of the Murinae. Otomys (Otomyinae) is considered to be a murine related to the African group of genera. Of the ten major clades identified, only one (Mus) occurs significantly in more than one primary biogeographic region. It is suggested that following an early period of relatively rapid dispersal from the place of origin, little further dispersal took place and subsequent evolution occurred within relatively confined geographic areas.

Generic level relationships of the Papionini (Cercopithecoidea).

Phylogenetic hypotheses for the Old World monkey tribe Papionini based on molecular data are incongruent with those inferred from previous morphological analyses. Morphologists have often inferred a close relationship between Mandrillus and Papio based on their overall similarity. Theropithecus has been variously proposed to be either quite distantly related to these two genera, their sister taxon, or anywhere in between. Molecular and chromosomal analyses on the other hand unambiguously group Theropithecus and Papio together to the exclusion of Mandrillus. Additionally, molecular and chromosomal analyses reveal that mangabeys (Cerocebus) are paraphyletic. Morphologists have acknowledged this possibility resurrecting the genus name Lophocebus for one group of mangabeys. A review and reanalysis of the morphological characters put forth by various researchers find little to contradict the consensus phylogeny derived from analysis of chromosomal banding, nuclear RNA restriction mapping, alpha and beta hemoglobin sequences, albumin and transferrin microcomplement fixation, DNA-DNA hybridization, repetitive DNA patterns, immunodiffusion, hemoglobin and adenylate kinase isozymes, and mitochondrial cytochrome oxidase subunit II DNA sequences.

The colubrid radiation in Africa (Serpentes: Colubridae): phylogenetic relationships and evolutionary patterns based on immunological data

Phylogenetic relationships among genera of African colubrids were evaluated using estimates of divergence among serum albumins compared by microcomplement fixation. Representatives of about half of the extant genera of African colubrids, as well as the Elapidae, Atractaspis and the Madagascan colubrid Leioheterodon, were analysed. The tree of best fit to the data has an unresolved basal polychotomy comprising at least five lineages of colubrids, as well as Elapidae and Atractaspis; thus, colubrids were not demonstrably monophyletic with these data. Two cosmopolitan clades, colubrines and natricines, are represented in Africa by series of closely related genera, but divergence among other genera is relatively great. Rate tests show that this is apparently not due to higher rates of albumin evolution in these, relative to other colubrids. Among the other associations supported by the immunological data are: (1) Psammophis -( Rhamphiophis-Dipsina)- Malpolon-Psammophylax; (2) Amblyodipsas-Macrelaps; (3) ( Lycodonomorphus-Lamprophis)- Mehelya; and (4) Colubrinae-Natricinae. Grayia is questionably associated with the colubrine-natricine lineage. Prosymna and Lycodon are clearly members of the colubrine clade, and Amplorhinus possibly associates with Leioheterodon . Gonionotophis, Duberria, Lycophidion and Pseudaspis show no strong association with any other genera, and represent other basal or near-basal clades within the colubrid/elapid radiation. The immunological data do not support a clade comprising the Elapidae, Atractaspis and some 'aparallactines' relative to Viperidae and other colubrids. The basal colubrid-elapid- Atractaspis divergence occurred more than 30 Myr ago, and the fossil record of colubrids in Africa greatly underestimates both the age and clade diversity of this group. In contrast to the pattern of radiation in the neotropics, where most colubrids belong to one of three major clades, in Africa only the colubrine lineage comprises a substantial portion of the extant generic diversity; most other genera stem from relatively ancient cladogenetic events and have few living representatives.

The colubrid radiation in Africa (Serpentes: Colubridae): phylogenetic relationships and evolutionary patterns based on immunological data

Abstract Phylogenetic relationships among genera of African colubrids were evaluated using estimates of divergence among serum albumins compared by microcomplement fixation. Representatives of about half of the extant genera of African colubrids, as well as the Elapidae, Atractaspis and the Madagascan colubrid Leioheterodon , were analysed. The tree of best fit to the data has an unresolved basal polychotomy comprising at least five lineages of colubrids, as well as Elapidae and Atractaspis ; thus, colubrids were not demonstrably monophyletic with these data. Two cosmopolitan clades, colubrines and natricines, are represented in Africa by series of closely related genera, but divergence among other genera is relatively great. Rate tests show that this is apparently not due to higher rates of albumin evolution in these, relative to other colubrids. Among the other associations supported by the immunological data are: (1) Psammophis -( Rhamphiophis-Dipsina )- Malpolon-Psammophylax ; (2) Amblyodipsas-Macrelaps ; (3) ( Lycodonomorphus-Lamprophis )- Mehelya ; and (4) Colubrinae-Natricinae. Grayia is questionably associated with the colubrine-natricine lineage. Prosymna and Lycodon are clearly members of the colubrine clade, and Amplorhinus possibly associates with Leioheterodon . Gonionotophis, Duberria, Lycophidion and Pseudaspis show no strong association with any other genera, and represent other basal or near-basal clades within the colubrid/elapid radiation. The immunological data do not support a clade comprising the Elapidae, Atractaspis and some 'aparallactines' relative to Viperidae and other colubrids. The basal colubrid-elapid- Atractaspis divergence occurred more than 30 Myr ago, and the fossil record of colubrids in Africa greatly underestimates both the age and clade diversity of this group. In contrast to the pattern of radiation in the neotropics, where most colubrids belong to one of three major clades, in Africa only the colubrine lineage comprises a substantial portion of the extant generic diversity; most other genera stem from relatively ancient cladogenetic events and have few living representatives.

Evolution in Some South-East Asian Murinae (Rodentia), as Assessed by Microcomplement Fixation of Albumin, and Their Relationship to Australian Murines

The interrelationships of 16 genera and 49 species of predominantly South-east Asian murine rodents were studied by means of microcomplement fixation of albumin to measure immunological distances among taxa. The results are viewed as a hypothesis of the phylogenetic relationship of these taxa that can be tested by other data sets. Three main groupings are suggested: (1) Maxomys; (2) Leopoldomys, Niviventer and Tokudaia; and (3) Bandicota, Berylmys, Bullimus, Bunomys, Komodomys, Nesokia, Papagomys, Paruromys, Rattus, Stenomys, Sundamys and Taeromys. Within this latter group, Bunomys chrsogasta, Komodomys and Rattus timorensis group together, as do Bullimus, Rattus and Stenomys, and Bandicota with Nesokia. The Australian murines, represented by Mesembriomys, may be part of this South-east Asian radiation but, if so, arose early in its history. Biogeographically, the results support South-east Asia as being a centre of murine evolution with secondary foci in Sulawesi, New Guinea and Australia. There is some evidence to suggest that a relatively recent land bridge between Sulawesi, Flores and Timor may have existed.

Evolution in New-Guinean Muridae (Rodentia) Assessed by Microcomplement Fixation of Albumin

The interrelationships of 19 genera and 28 species of New Guinean rodents representing 80% of the currently recognised New Guinean genera, were studied using microcomplement fixation of albumin to measure immunological distances among genera. The phylogenetic distinctiveness of Rattus and the closely related Stenomys from all other genera was confirmed. The remaining genera fall into three groups: (i) Lorentzimys; (ii) Anisomys, Coccymys, Chiruromys, Hyomys, Macruromys, Mallomys and Pogonomys; and (iii) Crossomys, Hydromys, Leptomys, Mayermys, Melomys, Neohydromys, Parahydromys, Pseudohydromys and Uromys. Solomys, from the Solomon islands, and Leggadina, Mesembriomys and Xeromys, from Australia, were shown to belong to this latter group. Groups (i) and (ii) are essentially endemic to New Guinea, while the third shares genera and species with Australia.

The sequence-immunology correlation revisited: Data for cetacean myoglobins and mammalian lysozymes

Quantitative microcomplement fixation tests employing rabbit antisera were done to compare immunologically 13 cetacean myoglobins and 15 mammalian lysozymes c of known amino acid sequence. In both cases there was a strong correlation between immunological distance (y) and percent sequence difference (x), as had been found for several other globular proteins. For myoglobin the relationship could be described by y = 10.5x and for lysozyme by y = 8.5x. The coefficients in both of these equations are appreciably higher than the values of 5.1-6.9 reported for three other vertebrate globular proteins (bird lysozyme c, mammalian ribonuclease, and mammalian serum albumin), and they imply that rabbit antisera to mammalian myoglobins and lysozymes are more sensitive to evolutionary substitutions. A strong inverse correlation (r = -0.95) was found when the slope of the line relating y to x for these five data sets was plotted against the percent sequence difference between the rabbit's own protein and the proteins immunized with. Specifically, the cetacean myoglobins on average differ in amino acid sequence from rabbit myoglobin by less than 13% and exhibit the steepest slope (10.5), while bird lysozyme sequences differ by nearly 40% from rabbit lysozyme and exhibit the shallowest slope (5.1).