The Phylogenetic Relationships of the Gobioninae (Teleostei: Cyprinidae) Inferred from Mitochondrial Cytochrome b Gene Sequences

The Gobioninae are a group of morphologically and ecologically diverse Eurasian freshwater cyprinid fishes. The intergeneric relationships of this group are unresolved and the possible monophyly of this subfamily remains to be established. We used complete mitochondrial cytochrome b gene sequences from most genera within the gobionine group, in addition to a selection of cyprinid outgroups, to investigate the possible monophyly of this group and resolve the interrelationships within the group. Our results support the monophyly of the Gobioninae and identify four monophyletic groups within the subfamily; the Hemibarbus group, the Sarcocheilichthys group, the Gobio group, and the Pseudogobio group. The morphologically aberrant genera Gobiobotia, Xenophysogobio and Gobiocypris are included in the Gobioninae, with the latter a sister group of Gnathopogon.     

Complete mitochondrial genome of the grass carp (Ctenopharyngodon idella, Teleostei): insight into its phylogenic position within Cyprinidae

Genome comparison has shed light on many fields of both basic and applied research, including the study of species phylogeny. Grass carp (Ctenopharyngodon idella) belongs to Cyprinidae, the largest freshwater fish family; but which subfamily it belongs to remains a controversial issue. In this study, the complete mitochondrial genome (mitogenome) sequence of grass carp was determined and phylogenetic analyses of all mitochondrial protein-coding genes and a nuclear gene (RAG 2) were conducted to explore the evolutionary relationship of grass carp with other cyprinid species. The mitogenome of grass carp is 16,609 bp in length. As with most other vertebrates, it contains the same gene order and an identical number of genes or regions, including 13 protein-coding genes, two rRNA genes, 22 tRNA genes and one putative control region. Phylogenetic analyses using two different datasets (mitochondrial and nuclear) and three different computational algorithms (Bayesian, MP and ML) all revealed two distinct groups with high statistical support, indicating that Cyprininae and Leuciscinae are two separate, valid subfamilies. Importantly, our phylogenetic result provides strong molecular evidence in support of the placement of Ctenopharyngodon in Leuciscinae rather than in Cyprininae.     

PERMANENT GENETIC RESOURCES: Identification of microsatellite loci for parentage analysis in roach Rutilus rutilus and eight other cyprinid fish by cross-species amplification, and a novel test for detecting hybrids between roach and other cyprinids

In order to identify microsatellite loci for parentage analysis in roach Rutilus rutilus, 59 published primer sets were tested on roach and eight other cyprinid fish. Twenty polymorphic loci were identified for roach, of which the polymerase chain reaction products of seven could be pooled for sequencer analysis. Together, these seven loci have an exclusion probability of 0.997 for parentage, when no parents are known. We also describe a novel test for hybrids between roach and four other cyprinids, based on intraspecies length differences of internal transcribed spacer region 1.     

Cyprinid phylogeny based on Bayesian and maximum likelihood analyses of partitioned data: implications for Cyprinidae systematics

Cyprinidae is the biggest family of freshwater fish, but the phylogenetic relationships among its higher-level taxa are not yet fully resolved. In this study, we used the nuclear recombination activating gene 2 and the mitochondrial 16S ribosomal RNA and cytochrome b genes to reconstruct cyprinid phylogeny. Our aims were to (i) demonstrate the effects of partitioned phylogenetic analyses on phylogeny reconstruction of cyprinid fishes; (ii) provide new insights into the phylogeny of cyprinids. Our study indicated that unpartitioned strategy was optimal for our analyses; partitioned analyses did not provide better-resolved or -supported estimates of cyprinid phylogeny. Bayesian analyses support the following relationships among the major monophyletic groups within Cyprinidae: (Cyprininae, Labeoninae), ((Acheilognathinae, ((Leuciscinae, Tincinae), Gobioninae)), Xenocyprininae). The placement of Danioninae was poorly resolved. Estimates of divergence dates within the family showed that radiation of the major cyprinid groups occurred during the Late Oligocene through the Late Miocene. Our phylogenetic analyses improved our understanding of the evolutionary history of this important fish family.     

Utility and evolution of cytochrome b in insects

Cytochrome b (cyt-b) is widely used in molecular phylogenetic studies of vertebrate, but not invertebrate, taxa. To determine whether this situation is an historical accident or reflects the utility of cyt-b, we compared the abilities of cyt-b, COI, and one nuclear ribosomal gene region (D1 of 28S) to recover intergeneric relationships within the tiger moth tribes Ctenuchini and Euchromiini. Additionally, we compared the rate of sequence and amino acid evolution of cyt-b across insects. Cytochrome b had the same level of sequence variation and A/T bias as COI, but was less useful for recovering intergeneric relationships. The total evidence tree casts doubt on the traditional taxonomy of the group. For the class Insecta, we found that functional conservation of amino acids occurs for the same regions as those found in vertebrates with the exception of Mallophaga (lice). Lice have an accelerated rate of nonsynonymous substitutions. Accelerated rate of cyt-b nucleotide and amino acid evolution in Apidae (bees) may be correlated with increased metabolic rates associated with facultative endothermy (= heterothermy).     

A comparison of complete mitochondrial genomes of silver carp Hypophthalmichthys molitrix and bighead carp Hypophthalmichthys nobilis: implications for their taxonomic relationship and phylogeny

Based upon morphological characters, Silver carp Hypophthalmichthys molitrix and bighead carp Hypophthalmichthys nobilis (or Aristichthys nobilis ) have been classified into either the same genus or two distinct genera. Consequently, the taxonomic relationship of the two species at the generic level remains equivocal. This issue is addressed by sequencing complete mitochondrial genomes of H. molitrix and H. nobilis , comparing their mitogenome organization, structure and sequence similarity, and conducting a comprehensive phylogenetic analysis of cyprinid species. As with other cyprinid fishes, the mitogenomes of the two species were structurally conserved, containing 37 genes including 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA (tRNAs) genes and a putative control region (D-loop). Sequence similarity between the two mitogenomes varied in different genes or regions, being highest in the tRNA genes (98·8%), lowest in the control region (89·4%) and intermediate in the protein-coding genes (94·2%). Analyses of the sequence comparison and phylogeny using concatenated protein sequences support the view that the two species belong to the genus Hypophthalmichthys . Further studies using nuclear markers and involving more closely related species, and the systematic combination of traditional biology and molecular biology are needed in order to confirm this conclusion.     

Characterization of Genomic Inheritance of Intergeneric Hybrids between Ascocenda and Phalaenopsis Cultivars by GISH,PCR-RFLP and RFLP

BackgroundThe intergeneric hybrids between Ascocenda John De Biase ‘Blue’ and Phalaenopsis Chih Shang''s Stripes have been generated to introduce the blue color into the Phalaenopsis germplasm in prior study. In order to confirm the inheritance in hybrid progenies, genomic in situ hybridization (GISH) and restriction fragment length polymorphism (RFLP) analysis were conducted to confirm the intergeneric hybridization status.Methods/ResultsGISH analysis showed the presence of both maternal and paternal chromosomes in the cells of the putative hybrids indicating that the putative hybrid seedlings were intergeneric hybrids of the two parents. Furthermore, twenty-seven putative hybrids were randomly selected for DNA analysis, and the external transcribed spacer (ETS) regions of nrDNA were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and RFLP analyses to identify the putative hybrids. RFLP analysis showed that the examined seedlings were intergeneric hybrids of the two parents. However, PCR-RFLP analysis showed bias to maternal genotype.ConclusionsBoth GISH and RFLP analyses are effective detection technology to identify the intergeneric hybridization status of putative hybrids. Furthermore, the use of PCR-RFLP analysis to identify the inheritance of putative hybrids should be carefully evaluated.     

Inheritance of organelle DNA sequences in a citrus-poncirus intergeneric cross

Many land plants deviate from the maternal pattern of organelle inheritance. In this study, heterologous mitochondrial and chloroplast probes were used to investigate the inheritance of organelle genomes in the progeny of an intergeneric cross. The seed parent was LB 1-18 (a hybrid of Citrus reticulata Blanco cv. Clementine x C. paradisi Macf. cv. Duncan) and the pollen parent was the cross-compatible species Poncirus trifoliata (L.) Raf. All 26 progeny examined exhibited maternal inheritance of plastid petA and petD loci. However, 17 of the 26 progeny exhibited an apparent biparental inheritance of mitochondrial atpA, cob, coxII, and coxIII restriction fragment length polymorphisms (RFLPs) and maternal inheritance of mitochondrial rrn26 and coxI RFLPs. The remaining nine progeny inherited only maternal mitochondrial DNA (mtDNA) configurations. Investigations of plant mitochondrial genome inheritance are complicated by the multipartite structure of this genome, nuclear gene control over mitochondrial genome organization, and transfer of mitochondrial sequences to the nucleus. In this study, paternal mtDNA configurations were not detected in purified mtDNA of progeny plants, but were present in progeny DNA preparations enriched for nuclear genome sequences. MtDNA sequences in the nuclear genome therefore produced an inheritance pattern that mimics biparental inheritance of mtDNA.     

Mapping unexplored genomes: a genetic linkage map of the woody sonchus alliance (Asteraceae: Sonchinae) in the Macaronesian Islands

As an initial step to mapping quantitative trait loci for species differences and adaptive radiation of insular endemics in Macaronesia, a genetic linkage map was constructed from an intergeneric backcross between Lactucosonchus webbii and Sonchus radicatus, core members of the tree lettuces in the Macaronesian Islands. A total of 152 amplified fragment length polymorphism markers were mapped into 10 major and 3 minor linkage groups for a total map length of 644 cM with an average distance of 4.53 cM for the 10 major groups. The genetic linkage map length is considerably less than the estimated, and this may reflect incomplete genomic coverage in the current study or reduced recombination, which is a common feature of maps for hybrids of divergent taxa. Segregation distortion occurred in 34% of the mapped markers, and they were located primarily in 4 linkage groups. Segregation distortion in the current BC(1) intergeneric population is slightly lower than average (40%) for BC(1) interspecific populations. This level of segregation distortion implies that unlike what we normally assume no to few reproductive barriers, oceanic island plant taxa do exhibit some degree of postmating reproductive isolation.     

Chloroplast DNA restriction site study of Verbesina (Asteraceae: Heliantheae)

Chloroplast DNA variation was examined for 79 species of Verbesina and 24 outgroups. Two independent analyses of the data were performed. An intergeneric study to elucidate the phylogenetic relationships of Verbesina used 22 genera, most of which are regarded in the literature as closely related to Verbesina. Coreopsis and Hymenoxys served as outgroups for this analysis. The 16 6-bp (base pair) restriction endonucleases used in the intergeneric study revealed 263 phylogenetically informative sites. Wagner analyses of these characters resulted in four equally parsimonious trees with a length of 857 steps and a Consistency Index of 0.492. Results from this study indicate that Verbesina is monophyletic, a member of the tribe Heliantheae, and that its sister taxa are the mostly Mexican genera Podachaenium, Squamopappus, and Tetrachyron. The infrageneric study of Verbesina included species belonging to all of its infrageneric taxa, except the monotypic sect. Stenocarpha. The 17 6-bp restriction endonucleases used in the infrageneric study revealed 137 sites 77 of which were phylogenetically informative. Wagner analyses of these characters generated 180 equally parsimonious trees with a length of 158 steps and a Consistency Index of 0.786. The genera Podachaenium, Squamopappus, and Tetrachyron served as outgroups. Two major clades, which correspond to traditional divisions of the genus based on leaf arrangement, are supported by the study. No support was found for the monophyly of seven of the 11 sections examined; further sampling of sections Lipactinia, Ochractinia, Verbesina, and Ximenesia is needed. Results support a major reassessment of the traditional infrageneric classification of the genus and provide the basis for the reevaluation of the sectional taxonomy of Verbesina. These studies support a North American origin for the genus with several independent introductions into South America producing significant diversity, especially in the Andean region.     

A comparative ultrastructural study of spermatozoa of the teiid lizards Cnemidophorus gularis gularis,Cnemidophorus ocellifer,and Kentropyx altamazonica (Reptilia,Squamata, Teiidae)

The ultrastructure of the spermatozoa of Cnemidophorus gularis gularis, Cnemidophorus ocellifer, and Kentropyx altamazonica is described for the first time. Mature spermatozoa of Cnemidophorus spp. and K. altamazonica differ in the occurrence of a perforatorial base plate, the enlargement of axonemal fibers 3 and 8, and shape of mitochondria. The comparisons of the ultrastructure sperm of Cnemidophorus spp. and K. altamazonica with Ameiva ameiva [J. Morphol. (2002) in press] suggest that Ameiva and Cnemidophorus are more similar to each other than either is to Kentropyx. Statistical analyses reveal that sperm of all three species studied are significantly different in the following dimensions: head, acrosome, distal centriole length, and nuclear shoulders width. There was no variable statistically different between the Cnemidophorus spp. only. The length of the tail, midpiece, entire sperm, and nuclear rostrum are significantly different between K. altamazonica and Cnemidophorus spp. Our results indicate that sperm ultrastructure presents intra and intergeneric variability.     

How objective are genera in euascomycetes?

Intra- and intergeneric distances derived from maximum-likelihood phylogenetic trees inferred from 254 nuclear ITS rDNA sequences were examined for seven families of euascomycetes, representing five classes. The intra- and intergeneric distances were well separated in most cases, but the distances varied between families. The analysis of the distance distributions provides a powerful tool for identifying certain taxa with highly deviating distances and thus cases of excessive lumping or splitting. Some cases of lumping and splitting found in different families are briefly discussed. The results of the analysis show that the generic concepts differ between the families. The consequences for nomenclature are discussed and a method abandoning binomial nomenclature while keeping the style of species names is recommended to ensure nomenclatural stability.     

Molecular evidence for the monophyly of East Asian groups of Cyprinidae (Teleostei: Cypriniformes) derived from the nuclear recombination activating gene 2 sequences

The family Cyprinidae is one of the largest families of fishes in the world and a well-known component of the East Asian freshwater fish fauna. However, the phylogenetic relationships among cyprinids are still poorly understood despite much effort paid on the cyprinid molecular phylogenetics. Original nucleotide sequence data of the nuclear recombination activating gene 2 were collected from 109 cyprinid species and four non-cyprinid cypriniform outgroup taxa and used to infer the cyprinid phylogenetic relationships and to estimate node divergence times. Phylogenetic reconstructions using maximum parsimony, maximum likelihood, and Bayesian analysis retrieved the same clades, only branching order within these clades varied slightly between trees. Although the morphological diversity is remarkable, the endemic cyprinid taxa in East Asia emerged as a monophyletic clade referred to as Xenocypridini. The monophyly for the subfamilies including Cyprininae and Leuciscinae, as well as the tribes including Labeonini, Gobionini, Acheilognathini, and Leuciscini, was also well resolved with high nodal support. Analysis of the RAG2 gene supported the following cyprinid molecular phylogeny: the Danioninae is the most basal subfamily within the family Cyprinidae and the Cyprininae is the sister group of the Leuciscinae. The divergence times were estimated for the nodes corresponding to the principal clades within the Cyprinidae. The family Cyprinidae appears to have originated in the mid-Eocene in Asia, with the cladogenic event of the key basal group Danioninae occurring in the early Oligocene (about 31-30 MYA), and the origins of the two subfamilies, Cyprininae and Leuciscinae, occurring in the mid-Oligocene (around 26 MYA).     

Phenotypic shift of an alien piscivorous chub following translocation from a large lake to small irrigation ditches

The piscivorous chub (Opsariichthys uncirostris) is a Japanese predatory cyprinid with a native distribution restricted to a few large lakes. It has now established in a large region of Japan following accidental translocation. Although large water bodies with abundant food resources were long considered essential for establishing this species, the chub has settled in small irrigation ditches in Kyushu. In this study, we explored the phenotypic responses of the chub in these small water bodies by comparing life history traits and morphology with those of chub inhabiting a native lake. Growth rate, fecundity-related traits, and trunk length shifted markedly following translocation to the new habitat. These phenotypic shifts were typical reactions to characteristic conditions of irrigation ditches, such as habitat instability, lotic conditions, and limited food. Adaptability via a rapid phenotypic shift by the chub may have facilitated establishment of populations in small irrigation ditches.     

Genome size variation in North American minnows (Cyprinidae). II. Variation among 20 species

Genome sizes (nuclear DNA contents) from 200 individuals representing 20 species of North American cyprinid fishes (minnows) were examined spectrophotometrically. The distributions of DNA values of individuals within populations of the 20 species were essentially continuous and normal; the distribution of DNA values among species was continuous and overlapping. These observations suggest that changes in DNA quantity in cyprinids are small in amount, involve both gains and losses of DNA, and are cumulative and independent in effect. Significant heterogeneity in mean genome size occurs both between individuals within populations of species and among species. The former averages maximally around 6% of the cyprinid genome and is nearly the same as the amount of DNA theoretically needed for the entire cyprinid structural gene component. The majority of the DNA content variation among the 20 species is distributed above the level of individuals within populations. Comparisons of average genome size difference or distance between individuals drawn from different levels of taxonomic organization indicate that considerably greater divergence in genome size has occurred in the extremely speciose cyprinid genus Notropis as compared with other North American cyprinid genera. This may suggest that genome size change is concentrated in speciation episodes. Finally, no associations were found between interspecific variation in genome size and five life-history characters. This suggests that much of the variation in genome size within and among the 20 species may be phenotypically inconsequential.     

Phylogeny and Divergence Times of Gymnosperms Inferred from Single-Copy Nuclear Genes

Phylogenetic reconstruction is fundamental to study evolutionary biology and historical biogeography. However, there was not a molecular phylogeny of gymnosperms represented by extensive sampling at the genus level, and most published phylogenies of this group were constructed based on cytoplasmic DNA markers and/or the multi-copy nuclear ribosomal DNA. In this study, we use LFY and NLY, two single-copy nuclear genes that originated from an ancient gene duplication in the ancestor of seed plants, to reconstruct the phylogeny and estimate divergence times of gymnosperms based on a complete sampling of extant genera. The results indicate that the combined LFY and NLY coding sequences can resolve interfamilial relationships of gymnosperms and intergeneric relationships of most families. Moreover, the addition of intron sequences can improve the resolution in Podocarpaceae but not in cycads, although divergence times of the cycad genera are similar to or longer than those of the Podocarpaceae genera. Our study strongly supports cycads as the basal-most lineage of gymnosperms rather than sister to Ginkgoaceae, and a sister relationship between Podocarpaceae and Araucariaceae and between Cephalotaxaceae-Taxaceae and Cupressaceae. In addition, intergeneric relationships of some families that were controversial, and the relationships between Taxaceae and Cephalotaxaceae and between conifers and Gnetales are discussed based on the nuclear gene evidence. The molecular dating analysis suggests that drastic extinctions occurred in the early evolution of gymnosperms, and extant coniferous genera in the Northern Hemisphere are older than those in the Southern Hemisphere on average. This study provides an evolutionary framework for future studies on gymnosperms.     

Merodiploidy in ESCHERICHIA COLI-SALMONELLA TYPHIMURIUM Crosses: The Role of Unequal Recombination between Ribosomal RNA Genes

Previous workers have shown that intergeneric crosses between Salmonella typhimurium and Escherichia coli produce a high proportion of merodiploid recombinants among the viable progeny. We have examined the unequal cross-over event that was responsible for a number of intergeneric merodiploids. The merodiploids that we studied were all heterozygous for the metB-argH interval and were the products of intergeneric conjugal crosses. We found that when the S. typhimurium donor had its transfer origin closely linked to metB and argH, all recombinants examined were merodiploid, and they generally arose as F-prime factors. Many of these F-prime factors had been created by recombination between flanking rrn genes in the donor. When the S. typhimurium Hfr transfer origin was more distant from the selected markers, quite different results were obtained. Depending on the donor, 19-47% of the recombinants that acquired the donor argH+ or metB+ genes were merodiploid for these loci, but none of the recombinants were F-prime. A majority of the merodiploids had a novel (nonparental) rrn gene, indicating that unequal recombination between nonidentical rrn genes was a prevalent mechanism for establishing the merodiploidy. Both tandem and nontandem duplications were found. Some of the merodiploids duplicated E. coli genes in addition to acquiring S. typhimurium genes. Some merodiploids contained the oriC region from each parent. Of a total of 118 intergeneric merodiploids characterized from all donors, 48 different genotypes were observed, and 38 of the 48 had one or more nonparental rrn operons.     

Partial combination applied to phylogeny of European cyprinids using the mitochondrial control region

Previous molecular phylogenies of European cyprinids led to some solid facts and some uncertainties. This study is based on a stretch of more than 1 kb in the mitochondrial control region newly sequenced for 35 European cyprinids and on previous cytochrome b and 16S rDNA data. The trees based on the control region are more accurate and robust than those obtained from the two other genes. Character incongruence among the three genes was tested using the incongruence length difference (ILD) test. Iterative removals of individual sequences followed by new ILD tests identified two sequences responsible for statistically significant incongruence. A partial combination was conducted, that is, a combination of the three data sets, removing the two sequences previously identified. The phylogenetic analysis of this partial combination gives a more robust and resolved picture of subfamilial interrelationships. The Rasborinae are the sister group of all other cyprinids. The monophyletic Cyprininae emerges next. Tinca tinca first and then Rhodeus are the sister groups of all the remaining nonrasborine and noncyprinine species. Gobio is the sister group of the Leuciscinae, in which the Phoxinini are the sister group of the Leuciscini. Within the Leuciscini, the genus Leuciscus and the subfamily Alburninae are both paraphyletic. The Rasborinae are the most basal cyprinid subfamily and the Tincinae are not the sister group of the Cyprininae. These two results challenge only two anatomical characters, which need to be reinterpreted or considered as homoplastic in cyprinid evolution: the modification of the first pleural rib and its parapophysis and the bony composition of the interorbital septum.     

Intergeneric nuclear gene transfer between somatically and sexually incompatible plants through asymmetric protoplast fusion

Summary A stable intergeneric transfer of nuclear genes from Physalis minima into the genome of Datura innoxia has been achieved through asymmetric protoplast fusion. No hybrid plants could be obtained from these species either by traditional methods of sexual breeding or by somatic hybridization via fusion of protoplasts containing complete nuclear genomes. The incompatibility barriers were bypassed by the fusion of highly X-irradiated (LD₁₀₀) wild-type Physalis with nuclear albino mutant Datura protoplasts. In this intergeneric reconstruction, 1.15% of the total heterokaryons restored the chlorophyll synthesis in their regenerants. Two representative transformed lines, TRL-A and TRL-D, were further characterized, showing 0.43–0.78 pg. additional nuclear DNA (4.45–8.07% nuclear DNA of P. minima). Since chromosomes of the species of Datura and Physalis were distinguishable, the mitotic complement of the transformed regenerants showed only 3 and 1 chromosomes of the donor in tetraploid (2n=48) and octoploid (2n=96) genomes of the recipient, respectively. The introduction and expression of limited genes of Physalis in Datura have also been confirmed by the allelic expression of various isoenzymes. Such stable gene transfer via asymmetric fusion of protoplasts has been discussed in relation to its application in the genetic manipulation of plants.