Phylogeny of Allium L. subg. Melanocrommyum (Webb et Berth.) Rouy based on DNA sequence analysis of the internal transcribed spacer region of nrDNA

 Sequence analysis of the ITS region of nuclear ribosomal DNA from subgeneric representatives of Allium L. produced phylogenetic trees which concurred with previous conclusions based on classical taxonomy. Phylogenetic analysis revealed a closer relationship between Nectaroscordum siculum and Allium cernuum (representing Amerallium) than between A. cernuum and the rest of the Allium species employed in this study. The phylogeny of subg. Melanocrommyum based on ITS sequences largely agreed with inferences made by previous researchers based on morphology or a restriction analysis of chloroplast DNA. However, the phylogenetic positions of Allium protensum and Allium macleanii based on ITS sequences did not correspond to their morphological similarity with Allium schubertii and Allium giganteum, respectively. Received: 15 February 1998 / Accepted: 12 March 1998     

Observations on pollination inSarcostemma (Asclepiadaceae)

Field observations on pollination in New World species of the genusSarcostemma R. Br. sensuHolm are reported. Morphological and anatomical comparisons of pollinated flowers ofSarcostemma subg.Ceramanthus Kuntze (New World) andSarcostemma subg.Sarcostemma (Old World) are presented.     

Bulb tunic anatomy and its taxonomic implication in Allium L. (Amaryllidaceae: Allioideae)

Abstract

Allium is taxonomically a difficult genus with blurred taxonomic borders at all taxonomic ranks. In this research, anatomy and morphology of bulb tunics in 42 species of the genus representing its 16 currently recognized sections and 6 subgenera (Allium, Cepa, Reticulatobulbosa, Amerallium, Polyprason and Melanocrommyum) were investigated. Our results indicated the following characters to be most informative at sectional and subgeneric levels: features of calcium oxalate crystals and subepidermal cell layer of the outermost bulb tunic, type of tracheids, and bulbils presence. Three main types of crystals, i.e. prismatic, sand and druses, were fairly common among the investigated species except for the members of A. sect. Acanthoprason (A. subg. Melanocrommyum) that often lack crystals. The subepidermal layers of outer tunics were mostly composed of hexagonal, rectangular, or elongated cells but A. paradoxum (A. subg. Amerallium, sect. Briseis) showed the pentagonal type and A. longisepalum (A. subg. Amerallium, sect. Molium) the elliptic type of subepidermal cells. The members of A. sub. Melanocrommyum showed various types of tunic cells and crystals.     

The 5S RNA genes inPinus radiata and the spacer region as a probe for relationships betweenPinus species

The 5S RNA genes inPinus radiata occur in two size classes of about 850 and 525 bp in length. Representatives from both the long and short size classes were cloned and sequenced. The primary difference in the two size classes was shown to be a 330 bp insertion in the spacer region of the long units. Within an individual breeding clone ofP. radiata there was some sequence heterogeneity between representatives of the short class. The 5S RNA genes in thirty pine species were characterised using either a clone of the short size class or a subclone of the 330 bp insertion characterizing the long size class. Eleven species of subg.Strobus were examined and all lacked the long type of unit of radiata pine. The New World species of subg.Pinus all had both short and long units whereas the Old World species had long units. The implications of these results for the evolution of the 5S DNA sequences and the phylogenetic relationships withinPinus are discussed.     

Karyological studies of Iranian <Emphasis Type="Italic">Allium</Emphasis> L. (Amaryllidaceae) species with focus on sect. <Emphasis Type="Italic">Acanthoprason</Emphasis>. 1. Mitotic chromosomes

Eighteen species and subspecies (34 accessions) of Allium sect. Acanthoprason and 11 species (17 accessions) belonging to other subgenera and sections of Allium were karyologically investigated and include first reports for 12 species. The examined plants of 47 accessions were diploid, three accessions of two species were tetraploid, and in the A. bisotunense accession, we found a mix of di- and triploid individuals. B chromosomes were found in 10 accessions. A basic chromosome number of x = 8 was confirmed for all investigated members of subg. Melanocrommyum and subg. Allium, and x = 9 for Allium tripedale of subg. Nectaroscordum. Idiograms were drawn for each accession, and metaphase images are presented illustrating observed chromosomal variations. Also, karyotype features and asymmetry parameters were calculated for all accessions. Chromosomal aberrations, e.g. aneuploid cells or loss of whole or parts of chromosome arms, were rarely observed. In general, the karyotypes showed low variation in inter- and intrachromosomal asymmetry especially inside of the taxonomic groups, though satellited chromosomes were good markers for subgenera and even specific for two studied sections of subg. Allium. Six different types of satellites were recognized, two of them were newly described: Type P was prevalent in subg. Melanocrommyum, and type O in sect. Codonoprasum. Statistical analyses were performed on five karyological parameters to test correct relationships and also to test previous grouping hypotheses. Although our data confirm distinct karyological characters for the subgenera investigated, the remarkable morphological diversity inside of subg. Melanocrommyum is not mirrored by striking karyological differences.     

Giemsa C-banded karyotypes and taxonomic relationships of some North AmericanAllium species and their relationship to Old World species (Liliaceae)

The somatic karyotypes of six North AmericanAllium species and the EuropeanA. scorzonerifolium have been investigated using a Giemsa C-banding technique. All species have a chromosome number of 2n = 14. InA. scorzonerifolium and the three North American speciesA. dichlamydeum, A. fibrillum andA. unifolium C-bands are restricted to two pairs of nucleolar chromosomes. Each chromosome has a band proximal to the nucleolar constriction and a positively banded satellite. InA. acuminatum, in addition to the bands associated with the nucleolar constrictions, all chromosomes also have pericentromeric bands.A. cernuum exhibits a distinctive banding style: two chromosome pairs with bands adjacent to the nucleolar constrictions and four pairs with telomeric bands on their short arms. In the karyotype ofA. geyeri neither C-bands nor nucleolar chromosomes were found.—A comparison of the banding styles together with other cytological and morphological characters of these species with old world members ofAllium reveals:A. cernuum closely resembles species within subgenusRhizirideum, whereas the other species studies exhibit many similarities with subgenusMolium. Their sectional grouping and their relationships with Old World species are discussed.     

Genetic evidence supports the new anatolian lupine accession,Lupinus anatolicus, as an old world “rough-seeded” lupine (sectionScabrispermae) related toL. pilosus

A noteworthy wild lupine accession was recently discovered in southwestern Turkey and was proposed as a new separate Old World “smooth-seeded” species close toL. micranthus and namedL. anatolicus. Its species status was controversial with respect to cytological and crossability data. In order to examine the position and the evolutionary relationships of this Anatolian accession relative to the Old World lupines, we investigated new data from seed coat micromorphology, and from internal transcribed spacer (ITS) nucleotide sequences of the nuclear ribosomal DNA repeat. The micromorphological seed coat pattern ofL. anatolicus, as revealed by scanning electron microscopy, is characterized by pluricellular tubercles, which represent the typical and unique pattern of the Old World “rough-seeded” lupines (sect.Scabrispermae). In accordance with the micromorphological results, the genetic distances and phylogenetic relationships among the Old World lupines, estimated from ITS data, unambiguously support the new Anatolian lupine accession as part of theL. pilosus-L. palaestinus lineage within the strongly monophyletic group containing all theScabrispermae. The results provided in this study, together with other lines of data available from the literature, are thus hardly compatible with the hypothesis that this new Anatolian lupine accession could be related to Old World “smooth-seeded” lupines (includingL. micranthus); instead, it appears closely related toL. pilosus.     

Pollen morphology and its taxonomic significance of the monotropoideae (Ericaceae)

A palynological survey, including LM, SEM and TEM is presented for eight genera, nine species and 76 samples of the Monotropoideae which is composed of 10 genera and 13 species. On the basis of the aperture number and shape, the following six pollen types are recognized: 1) 3-colp(oroid) ate—Allotropa, 2) 2-colpor(oid)ate—Monotropsis andHemitomes, 3) 4-colpor(oid)ate—Pterospora, Sarcodes andPleuricospora, 4) 2- and 3-colpor(oid)ate—Monotropa hypopitys, 5) 3-colporate and-porate —Monotropa uniflora, 6) 3- to 4- porate—Monotropastrum humile. Relationships among taxa within the Monotropoideae are illustrated on the palynological characters including the aperture type, exine sculpture and structure. Both pollen grains with two and four apertures have evolved independently from pollen grains with three apertures, according to the infraspecific variation of aperture numbers and the usual occurrence of three-aperturate pollen grains in the Ericaceae. 3-colp(oroid)ate pollen ofAllotropa is the most primitive and occupies an isolated position in the subfamily, on the other hand 3- to 4-porate pollen ofMonotropastrum humile is most advanced in the subfamily, probably even in the Ericaceae. A reduced sexine inMonotropastrum humile appears to be a specialized pollen character. Infraspecific geographical difference in palynological characters is revealed in two species ofMonotropa for the first time.M. hypopitys is basically characterized by pollen grains with two apertures in the New World vs. three apertures in the Old World.M. uniflora is basically characterized by 3-porate pollen in the New World vs. 3-colporate in the Old World. In both species pollen grains with more primitive characters usually occur in the Old World.     

Evolution of chromosome Y in primates

We have investigated, by fluorescence in situ hybridization (FISH), the cytogenetic evolution of the Y chromosome in primates using 17 yeast artificial chromosomes, representative of the Y-specific euchromatic region of the human chromosome Y. The FISH experiments were performed on great apes (Homo sapiens, Pan troglodytes, Gorilla gorilla and Pongo pygmaeus pygmaeus), and on two Old World monkeys species as an outgroup (Cercopitecidae Macaca fascicularis and Papio anubis). The results showed that this peculiar chromosome has undergone rapid and unconstrained evolution both in sequence content and organization. Received: 16 January 1998; in revised form: 29 May 1998 / Accepted: 24 June 1998     

A Phylogeny of Euphorbieae Subtribe Euphorbiinae (Euphorbiaceae) Based on Molecular Data

Phylogenetic relationships within Euphorbiinae were inferred from our analysis of the 3′; end of the chloroplast gene ndhF. A sampling of that subtribe covered 88 species; 3 closely related species from the subtribes Anthosteminae and Neoguillauminiinae and the tiribe Hippomaneae were included as outgroups. A phylogenetic assessment was carried out using the parsimony approach. The relationships revealed via these ndhF data supported the monophyly of subg.Esula, subg.Chamaesyce, subg.Euphorbia, and subg.Lacanthis. However, the polyphyly of subg.Agaloma, subg.Lyciopsis, and subg.Eremophyton also was strongly suggested. The African succulent Euphorbiinae can be divided into primarily two independent groups: 1) spiny succulents, which form a strongly supported clade with three subclades (subg.Euphorbia, subg.Lacanthis, andMonadenium+Synadenium); and 2) non-spiny succulents, which consist of sect.Meleuphorbia, sect.Medusae, sect.Anthacantha, sect.Trichadenia, sect.Pseudeuphorbium, sect.Treisia, and sect.Pseudacalypha. In the ndhF tree, the subg.Esula clade is placed as a sister to the rest of the Euphorbiinae. Thus, the origin of theEuphorbia s.I. should be sought within the herbaceous species of subg.Esula. The core North American endemicEuphorbia groups --Agaloma, Chamaesyce, andPoinsettia — are monophyletic and independent of the South American subg.Agaloma. Instead, they are derived from the AfricanEuphorbia subg.Lyciopsis andEremophyton. The Eurasian subg.Esula clade forms two subclades, which are concordant to sect.Esula and sect.Tithymalus.     

Evolution of an intronic microsatellite polymorphism in Toll-like receptor 2 among primates

Nonhuman primates express varying responses to Mycobacterium tuberculosis: New World monkeys appear to be resistant to tuberculosis (TB) while Old World monkeys seem to be particularly susceptible. The aim of this study was to elucidate the presence of the regulatory guanine–thymine (GT) repeat polymorphisms in intron 2 of Toll-like receptor 2 (TLR2) associated with the development of TB in humans and to determine any variations in these microsatellite polymorphisms in primates. We sequenced the region encompassing the regulatory GT repeat microsatellites in intron 2 of TLR2 in 12 different nonhuman primates using polymerase chain reaction amplification, TA cloning, and automatic sequencing. The nonhuman primates included for this study were as follows: chimpanzee (Pan troglodytes), bonobo (Pan paniscus), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), Celebes ape (Macaca nigra), rhesus monkey (Macaca mulatta), pigtail macaque (Macaca nemestrina), patas monkey (Erythrocebus patas), spider monkey (Ateles geoffroyi), Woolly monkey (Lagothrix lagotricha), tamarin (Saguinus labiatus), and ring-tailed lemur (Lemur catta). Nucleotide sequences encompassing the regulatory GT repeat region are similar across species and are completely conserved in great apes. However, Old World monkeys lack GT repeats altogether, while New World monkeys and ring-tailed lemurs have much more complex structures around the position of the repeats. In conclusion, the genetic structures encompassing the regulatory GT repeats in intron 2 of human TLR2 are similar among nonhuman primates. The sequence is most conserved in New World monkeys and less in Old World monkeys.     

Evidence for Gondwanan vicariance in an ancient clade of gecko lizards

Aim Geckos (Reptilia: Squamata), due to their great age and global distribution, are excellent candidates to test hypotheses of Gondwanan vicariance against post‐Gondwanan dispersal. Our aims are: to generate a phylogeny of the sphaerodactyl geckos and their closest relatives; evaluate previous phylogenetic hypotheses of the sphaerodactyl geckos with regard to the other major gecko lineages; and to use divergence date estimates to inform a biogeographical scenario regarding Gondwanan relationships and assess the roles of vicariance and dispersal in shaping the current distributions of the New World sphaerodactyl geckos and their closest Old World relatives. Location Africa, Asia, Europe, South America, Atlantic Ocean. Methods We used parsimony and partitioned Bayesian methods to analyse data from five nuclear genes to generate a phylogeny for the New World sphaerodactyl geckos and their close Old World relatives. We used dispersal–vicariance analysis to determine ancestral area relationships among clades, and divergence times were estimated from the phylogeny using nonparametric rate smoothing. Results We recovered a monophyletic group containing the New World sphaerodactyl genera, Coleodactylus, Gonatodes, Lepidoblepharis, Pseudogonatodes and Sphaerodactylus, and the Old World Gekkotan genera Aristelliger, Euleptes, Quedenfeldtia, Pristurus, Saurodactylus and Teratoscincus. The dispersal–vicariance analysis indicated that the ancestral area for this clade was North Africa and surrounding regions. The divergence between the New World spaherodactyl geckos and their closest Old World relative was estimated to have occurred c. 96 Myr bp . Main conclusions Here we provide the first molecular genetic phylogenetic hypothesis of the New World sphaerodactyl geckos and their closest Old World relatives. A combination of divergence date estimates and dispersal–vicariance analysis informed a biogeographical scenario indicating that the split between the sphaerodactyl geckos and their African relatives coincided with the Africa/South America split and the opening of the Atlantic Ocean. We resurrect the family name Sphaerodactylidae to represent the expanded sphaerodactyl clade.     

Phylogeny of Cleome L. and its close relatives Podandrogyne Ducke and Polanisia Raf. (Cleomoideae, Cleomaceae) based on analysis of nuclear ITS sequences and morphology

A phylogenetic survey of representatives from all New World and several Old World supraspecific groupings of Cleome, as well as from the closely related Podandrogyne and Polanisia, was conducted based on separate analysis of nuclear ribosomal ITS sequences and of morphological characters. Parsimony analysis of the molecular data recognized this group of cleomoid taxa as a strongly supported monophyletic lineage. Podandrogyne was imbedded within a highly supported Andinocleome clade, whereas Polanisia was placed as a member of a North American/Old World Cleome s. l. clade but with low support. The ITS data also indicated the sequential divergence of several basal Old World (sects. Cleome, Gymnogonia, Ranmanissa and Rutidosperma) and North American (subg. Physospermon, sect. Peritoma) lineages followed by more recent splittings of Central and South American lineages (the Andinocleome (sects. Pterosperma and Rimosperma) group, and sects. Melidiscus and Tarenaya). The morphological data showed extensive homoplasy and did not resolve the phylogeny of these cleomoids, although the cladistic analysis distinguished two poorly supported (subg. Eucleome and sect. Tarenaya) clades. Despite this, several sets of morphological and chromosomal secondary synapomorphies served to characterize the main sectional clades recovered in the molecular tree. Based on the strong support of the Cleome + Podandrogyne + Polanisia clade and the insufficient resolution and low support of the basal branches of this tree, and on the inherent homoplasy and unsatisfactory resolution of the morphological diagnostic traits used to characterize these taxa, a reunification of the three cleomoid genera under a large genus Cleome s. l. would be advisable. A biogeographical interpretation of our molecular phylogeny indicated and earlier origin of the ancestral Cleome lineages in the Old World, followed by colonization of North America and then a subsequent expansion towards central and South American with more recent secondary radiations in these subcontinents.     

Phylogeny and Evolution in Cariceae (Cyperaceae): Current Knowledge and Future Directions

The goal of this study was to review the impact of DNA sequence analyses on our understanding of Cariceae phylogeny, classification and evolution. To explore character evolution, 105 taxa from four different studies were included in an nrDNA ITS + ETS 1f analysis of all recognized genera (Carex, Cymophyllus, Kobresia, Schoenoxiphium, Uncinia) and Carex subgenera (Carex, Psyllophora, Vignea, Vigneastra). As in previous analyses, four major Cariceae clades were recovered: (1) a “Core Carex Clade” (subg. Carex, Vigneastra, Psyllophora p.p); (2) A “Vignea Clade” (subg. Vignea, Psyllophora p.p.); (3) a “Schoenoxiphium Clade” (Schoenoxiphium, subg. Psyllophora p.p.), and (4) a “Core Unispicate Clade” (Uncinia, Kobresia, subg. Psyllophora p.p.). All studies provide strong support (86–100% BS) for the Core Carex and Vignea Clades, but only weak to moderate support (<50%–78% BS) for the Core Unispicate and Schoenoxiphium Clades. The relationships of these groups are unresolved. Studies suggest that Carex is either paraphyletic with respect to all Cariceae genera or to all genera except Schoenoxiphium. Kobresia is a grade, but Uncinia and possibly Schoenoxiphium are monophyletic. The monotypic Cymophyllus is indistinct from Carex subg. Psyllophora species. Character analyses indicate that inflorescence proliferation and reduction have occurred in all major clades, and that the Cariceae’s unisexual flowers have evolved from perfect flowers. The ancestor to Cariceae possessed a multispicate inflorescence with cladoprophylls and female spikelets with tristigmatic gynoecia and closed utricles. This morphology is most similar to extant Carex subg. Carex species, which contradicts the nearly unanimous assumption that the highly compound inflorescences of Schoenoxiphium are primitive. Since taxonomic sampling and statistical support for phylogenies have generally been poor, we advocate the temporary maintenance of the four traditional Carex subgenera with androgynous unispicate species placed within subg. Psyllophora and dioecious and gynaecandrous unispicate species distributed amongst subgenera Carex and Vignea. A collective effort focused on developing new nuclear markers, on increasing taxonomic and geographic sampling, and on studying development within the context of phylogeny, is needed to develop a phylogenetic classification of Cariceae.     

Evolution of a new nonclassical MHC class I locus in two Old World primate species

 HLA-G is a nonclassical major histocompatibility complex (MHC) class I molecule that is expressed only in the human placenta, suggesting that it plays an important role at the fetal-maternal interface. In rhesus monkeys, which have similar placentation to humans, the HLA-G orthologue is a pseudogene. However, rhesus monkeys express a novel placental MHC class I molecule, Mamu-AG, which has HLA-G-like characteristics. Phylogenetic analysis of AG alleles in two Old World primate species, the baboon and the rhesus macaque, revealed limited diversity characteristic of a nonclassical MHC class I locus. Gene trees constructed using classical and nonclassical primate MHC class I alleles demonstrated that the AG locus was most closely related to the classical A locus. Interestingly, gene tree analyses suggested that the AG alleles were most closely related to a subset of A alleles which are the products of an ancestral interlocus recombination event between the A and B loci. Calculation of the rates of synonymous and nonsynonymous substitution at the AG locus revealed that positive selection was not acting on the codons encoding the peptide binding region. In exon 4, however, the rate of nonsynonymous substitution was significantly lower than the rate of synonymous substitution, suggesting that negative selection was acting on these codons. Received: 22 April 1998 / Revised: 15 July 1998     

New remains of Egatochoerus jaegeri (Mammalia,Suoidea) from the late Eocene of Peninsular Thailand

Abstract: We report here additional remains referred to Egatochoerus jaegeri from the late Eocene locality of Ban Mark in the Krabi basin, Thailand. The new material described, comprising upper and lower cheek teeth, deciduous premolars and partial cranial remains, makes E. jaegeri the best‐documented Eocene representative of Old World Suoidea at present. Detailed study and comparison of their cheek teeth structure reveal a homogeneity of the molar crest and groove patterns of Old World and New World Palaeogene suoids.     

Plastome sequences of two New World bamboos—Arundinaria gigantea and Cryptochloa strictiflora (Poaceae)—extend phylogenomic understanding of Bambusoideae

• Premise of the study: Two New World species of Bambusoideae, Arundinaria gigantea and Crytpochloa strictiflora, were investigated in a phylogenomic context. Complete plastome sequences have been previously determined and analyzed for nine bambusoid species that exclusively represent Old World lineages. The addition of New World species provides more complete information on relationships within Bambusoideae. • Methods: Plastomes from A. gigantea and C. strictiflora were sequenced using Sanger methods. Phylogenomic and divergence estimate analyses were conducted on both species with 23 other Poaceae. • Key Results: Phylogenomic and divergence analyses suggested that A. gigantea diverged from within Arundinarieae between 1.94–3.92 mya and that C. strictiflora diverged as the sister to tropical woody species between 24.83 and 40.22 mya. These results are correlated with modern relative diversities in the two lineages. • Conclusions: The two New World bamboos show unique plastome features accumulated and maintained in biogeographic isolation from Old World taxa. The overall evidence for A. gigantea is consistent with recent dispersal, and that for C. strictiflora is consistent with vicariance.     

Phylogeny of Osmundaceae Inferred from rbcL Nucleotide Sequences and Comparison to the Fossil Evidences

rbcL sequences of 11 of 15 extant species of Osmundaceae which represent all three genera, Osmunda, Todea and Leptopteris. Our phylogenetic analysis concluded: 1) Osmunda subg. Osmunda and subg. Plenasium are monophyletic groups, but subg. Osmundastrum is not. The genus Osmunda is not monophyletic because Todea and Leptopteris are positioned within Osmunda. 2) Osmunda cinnamomea is the most basally positioned species in Osmundaceae, and it can be called as “a living fossil” because a fossil species (O. claytoniites) with almost the same morphology as this species was recorded from the Triassic. 3) Osmunda japonica and O. regalis are very closely related with only one nucleotide difference in the rbcL gene. 4) Greater nucleotide variation (5–7 nucleotides) was found between conspecific samples of O. cinnamomea and O. claytoniana collected from Japan and United States. Each of these two species may comprise more than two biologically differentiated species. Received 7 July 1999/ Accepted in revised form 25 August 1999     

The T-cell receptor β chain-encoding gene repertoire of a New World primate species,the cotton-top tamarin

 The New World primate, the cotton-top tamarin (Saguinus oedipus), expresses major histocompatibility complex (MHC) class I molecules with limited diversity. The uniqueness of the cotton-top tamarin MHC class I loci may contribute to this species’ unusual susceptibility to viral infections and high incidence of ulcerative colitis. As a prelude to examining the effect of this limited MHC class I diversity on the tamarin CD8⁺ T-cell receptor (TCR) repertoire, we identified expressed tamarin TCR β chain (TCRB) cDNAs by anchored and inverse polymerase chain reaction. Sequence alignments and phylogenetic comparisons with human and rhesus macaque sequences identified homologues of 21 human variable (V) gene families. Only single variable region genes were identified in each of these tamarin VB families, with the exception of the VB 5, 9, and 13 families which were comprised of two or three distinct members. The multiple genes within these three VB families do not appear to have separate human homologues, but rather aligned equally well to a single human gene from their respective VB families. These genes appear to have arisen, therefore, by duplication of certain VB genes in the tamarin ancestors following their divergence from the lineage leading to Old World primates and hominoids. Homologues of 12 of the 13 human joining (J) region genes were also identified in the tamarin. Comparison of the proportion of nonsynonymous (p_N) and synonymous (p_S) substitutions occurring per site within tamarin variable region genes demonstrated a reduction in p_N in the framework regions compared with p_N in the presumed MHC contact regions (CDR1 and CDR2). Taken together, these findings illustrate that the TCR β chain-encoding genes of the cotton-top tamarin are similar in structure and degree of complexity compared with their Old World primate and human counterparts. Received: 19 July 1996 / Revised: 12 August 1996