Anatomy of mitochondrial DNA from Paramecium aurelia

Summary The linear genome of mitochondrial DNA from four species of Paramecium aurelia was investigated with respect to restriction endonuclease fragments, location and number of ribosomal RNA genes, and interspecies EcoRI and HindIII fragment homologies. One copy of each of the rRNA genes was found in all four species and the 14s and 20s rRNA genes were separated by at least 3,000 bp. R-Loop analysis of the 20s rRNA gene did not reveal the presence of an intervening sequence. Interspecies homology studies showed species 1, 5, and 7 to have a high degree of homology but species 4 was less than 50% homologous to species 1 mt DNA. For all four species, rRNA genes showed good homology indicating that these DNA sequences are highly conserved, even between species having many non-homologous regions. A major region of DNA which displayed little homology between species 1 and 4 was that fragment containing sequences essential for initiation of DNA replication.     

Analysis of NADH dehydrogenase proteins, ATPase subunit 9, cytochrome b, and ribosomal protein L14 encoded in the mitochondrial DNA of Paramecium.

The mitochondrial (mt) encoded ndh1, ndh3, ndh4, ndh5, rpl14, cyt b and atp9 gene products were identified by sequence comparisons with known proteins. Amino acid sequence comparisons between predicted Paramecium mt gene products and proteins in current databases were quantitated approximately by the means of similarity scores for pairs of aligned sequences. The comparisons show that the Paramecium gene products are very divergent from all others with the exception of those from a closely related ciliate, Tetrahymena. The similarity scores of comparisons between a Paramecium mt DNA encoded protein, cytochrome b for example, and the homologous protein from a group of organisms as diverse as other protozoans, vertebrates, fungi, plants, and prokaryotes were all about the same. The Paramecium gene products appear to be equally divergent from proteins representing a number of different kingdoms and organelles.     

Characterization of mitochondrial DNA from Paramecium aurelia with EcoRI and Hae II restriction endonucleases

The mitochondrial DNA from several species of Paramecium aurelia was characterized by its buoyant density, contour length, and cleavage pattern with the restriction endonucleases EcoRI and Hae II. The density and length were the same for all species while the cleavage pattern was unique to each species. In one species the fragments generated by EcoRI were not in equal molar ratios and the amount of an additional fragment appeared to be dependent on the replication stage of the mitochondrial DNA. The uniqueness of each fragmentation pattern was used to identify the mitochondrial DNA in interspecies hybrids.     

Does high intraspecific variability of two genome fragments indicate a recent speciation process of Paramecium dodecaurelia (P. aurelia species complex,Ciliophora, Protozoa)?

Species of the Paramecium aurelia complex show different levels of intraspecific polymorphism, with P. dodecaurelia revealing a high level of intraspecific variation. Paramecium dodecaurelia strains originating from distant localities in the Palaearctic, North America (USA), and Oceania (Hawaii) were studied in terms of intraspecific differentiation and the degree of speciation. Sequences of genes encoding the ITS1–5.8S-ITS2–5’ end of LSU rDNA (1063–1097 bp) and cytochrome c oxidase subunit I mtDNA (638–644 bp) were obtained from 33 strains of P. dodecaurelia, other P. aurelia species, and another species of the genus Paramecium, with Tetrahymena sp. used as an outgroup. In phylograms, the majority of P. dodecaurelia strains from the Palaearctic appear in one cluster, while strains from Japan, Hawaii and the USA are grouped in another cluster, together with some strains from Italy and representatives of the P. aurelia species complex. Our results tend to support the hypothesis that P. dodecaurelia seems to be a polyphyletic species with several haplotypes similar to or even shared with other members of the P. aurelia species complex. However, it is still an open question whether the revealed intraspecific differences within P. dodecaurelia are the result of ongoing speciation, or perhaps they just indicate genetic differentiation within a species that has a wide geographic distribution.     

Crassostrea angulata Bindin Gene and the Divergence of Fucose-Binding Lectin Repeats Among Three Species of Crassostrea

Bindin is a major protein for species-specific recognition between sperm and congenetic egg in many free-spawning marine invertebrates. We cloned a novel bindin gene from the oyster Crassostrea angulata by 3′ and 5′ rapid amplification of cDNA ends. The full-length bindin cDNA was 1,049 bp with a 771-bp open reading frame encoding 257 amino acids. The deduced amino acid sequence contained a putative signal peptide of 24 amino acids. The length of the bindin genomic DNA was 8,508 bp containing four exons and three introns. Three haplotypes of F-lectin repeat were detected from seven sequences of F-lectin repeat of six male oysters. Both neighbor-joining and minimum-evolution phylogenetic trees show that haplotype an1 was close to Crassostrea gigas while an2 and an3 were close to Crassostrea sikamea. Intron-4 in the middle of F-lectin repeat is highly variable in both size and sequence. We classified intron-4 into three types according to their size and the F-lectin repeat they were located in. Intron-4 may play an important role in recombination. We compared the number of nonsynonymous substitutions (Dn) and synonymous substitutions (Ds) per nucleotide site among 19 F-lectin haplotypes of the three species. Dn/Ds ratios suggested that positive selection occurred between C. gigas and C. sikamea and between C. gigas and C. angulata. Nine positive selected positions (p > 90%) are identified among 19 haplotypes of three species. They are located on the F-lectin binding face around the three recognition motif residues. We assume that these nine clustered amino acids are related with species-specific recognition.     

PHYLOGEOGRAPHIC PATTERNS IN MITOCHONDRIAL DNA OF THE DESERT TORTOISE (XEROBATES AGASSIZI), AND EVOLUTIONARY RELATIONSHIPS AMONG THE NORTH AMERICAN GOPHER TORTOISES

Restriction-fragment polymorphisms in mitochondrial DNA (mtDNA) were used to evaluate population-genetic structure in the desert tortoise Xerobates agassizi and to clarify evolutionary affinities among species of the gopher tortoise complex. Fourteen informative endonucleases were employed to assay mtDNAs from 56 X. agassizi representing 22 locations throughout the species' range. The mtDNA genotypes observed were readily partitioned into three major phylogenetic assemblages, each with striking geographic orientation. Overall, the X. agassizi mtDNA genotypes typify a common phylogeographic pattern, in which broad genetic uniformity of populations is interrupted by geographic features that presumably have functioned as dispersal barriers. The geologic history of the Colorado River area, which includes extensive marine incursions, may account for the marked mtDNA divergence between eastern and western X. agassizi assemblages. In mtDNA comparisons among the four species of the gopher tortoise complex, both UPGMA and Wagner parsimony analysis strongly support the recognition of two distinct species groups previously suggested by traditional systematic approaches. Furthermore, the mtDNA data identify the eastern X. agassizi assemblage as the probable inceptive lineage of X. berlandieri. Results from both intra- and interspecific comparisons illustrate how clues to historical events may be present in the geographic structure of mtDNA phylogenies.     

Comparison of the Esterases and Acid Phosphatases in Paramecium multimicronucleatum,Syngens 1–5, P. jenningsi,P. caudatum,and the P. aurelia Complex1

ABSTRACT. Forty-eight stocks in Paramecium jenningsi, syngens 1–5 of P. multimicronucleatum, P. caudatum, P. primaurelia, P. biaurelia, and P. tetraurelia were grown axenically and tested for their esterases and acid phosphatases using starch gel electrophoresis. The five esterases and the acid phosphatases previously characterized in species of the P. aurelia complex were also found in P. jenningsi, and three to four of the esterases and the acid phosphatases were found in the P. multimicronucleatum species complex and in P. caudatum. Additional subtypes were observed for each of the enzyme phenotypes in these new (though here unnamed) species of Paramecium. Two of the new acid phosphatase subtypes, which depart radically in mobility and in pattern, were found in syngen 3 of P. multimicronucleatum and in P. caudatum. Except for syngens 1 and 5 in P. multimicronucleatum, the degree of similarity between syngens 1, 5 and 2, 3, and 4 appears to be very low—perhaps even lower than that seen for species in the aurelia complex. More realistically, the syngens of P. multimicronucleatum should be considered as separate species although they are not here given separate taxonomic names. Limited sharing of subtypes occurred between species in different species complexes. This observation suggests that the molecular distances between species complexes may be even greater than between species within a complex.     

DNA barcoding of nymphalid butterflies (Nymphalidae: Lepidoptera) from Western Ghats of India

We have checked the utility of DNA barcoding for species identification of nymphalid butterflies from Western Ghats of India by using 650 bp sequence of mitochondrial gene cytochrome c oxidase subunit I. Distinct DNA barcoding gap (i.e. difference between intraspecies and interspecies nucleotide divergence), exists between species studied here. When our sequences were compared with the sequences of the conspecifics submitted from different geographic regions, nine cases of deep intraspecies nucleotide divergences were observed. In spite of this, NJ (Neighbour Joining) clustering analysis successfully discriminated all species. Observed cases of deep intraspecies nucleotide divergences certainly warrant further study.     

The D1‐D2 region of the large subunit ribosomal DNA as barcode for ciliates

Ciliates are a major evolutionary lineage within the alveolates, which are distributed in nearly all habitats on our planet and are an essential component for ecosystem function, processes and stability. Accurate identification of these unicellular eukaryotes through, for example, microscopy or mating type reactions is reserved to few specialists. To satisfy the demand for a DNA barcode for ciliates, which meets the standard criteria for DNA barcodes defined by the Consortium for the Barcode of Life (CBOL), we here evaluated the D1‐D2 region of the ribosomal DNA large subunit (LSU‐rDNA). Primer universality for the phylum Ciliophora was tested in silico with available database sequences as well as in the laboratory with 73 ciliate species, which represented nine of 12 ciliate classes. Primers tested in this study were successful for all tested classes. To test the ability of the D1‐D2 region to resolve conspecific and congeneric sequence divergence, 63 Paramecium strains were sampled from 24 mating species. The average conspecific D1‐D2 variation was 0.18%, whereas congeneric sequence divergence averaged 4.83%. In pairwise genetic distance analyses, we identified a D1‐D2 sequence divergence of <0.6% as an ideal threshold to discriminate Paramecium species. Using this definition, only 3.8% of all conspecific and 3.9% of all congeneric sequence comparisons had the potential of false assignments. Neighbour‐joining analyses inferred monophyly for all taxa but for two Paramecium octaurelia strains. Here, we present a protocol for easy DNA amplification of single cells and voucher deposition. In conclusion, the presented data pinpoint the D1‐D2 region as an excellent candidate for an official CBOL barcode for ciliated protists.     

Is the spinule pattern on the leg 4 coxopodite a tactile signal in the specific mate recognition system of Mesocyclops (Copepoda, Cyclopidae)?

Several recent observations on e.g. birds and fruit flies support the hypothesis that when previously geographically separated populations, as neospecies, come into contact, sexual isolation mechanisms become stronger as a result of direct selection against inferior hybrids (reinforcement hypothesis). Many aspects of the reproductive biology of cyclopid copepods are still very poorly known. However, all reports on mating processes in the genus Mesocyclops confirm that males attach to the fourth legs of the females by their geniculate antennulae before transferring spermatophores. Accordingly, it is reasonable to assume that the signals helping recognition of the conspecific mates are located on the leg 4 of the females. The posterior surface of the leg 4 coxopodite in Cyclopidae is ornamented with a complex and diverse spinule pattern which, despite some infraspecific variability, is a reliable species-specific trait in species of Mesocyclops. Depending upon whether the spinule ornamentation has any role in the recognition system, and whether it evolved in geographically separated populations or later when they had begun to overlap, different predictions can be made about the zoogeographical pattern of differences among these character states. In the following study, comparisons are made for 10 allopatric, four sympatric and two partially sympatric species pairs. These comparisons reveal that the degree of divergence in the spinule ornamentation is significantly greater between sympatric than between allopatric species. These results argue in favor of the spinule ornamentation of the leg 4 coxopodite being a signal in specific mate recognition system (SMRS), and that the divergence of this state is initiated when previously separated populations, as neospecies, begin secondary contact.     

Uncovering Cryptic Diversity in Two Amoebozoan Species Using Complete Mitochondrial Genome Sequences

The Amoebozoa are a major eukaryotic lineage that encompasses a wide range of amoeboid organisms. The group is understudied from a systematic perspective: molecular tools have only been applied in the last 15 yr. Hence, there is an undersampling of both genes and taxa in the group especially compared to plants, animals, and fungi. Here, we present the complete mitochondrial genomes of two ubiquitous and abundant morpho‐species (Acanthamoeba castellanii and Vermamoeba vermiformis). Both have mitochondrial genomes of close relatives previously available, enabling insights into recent divergences at a genomic scale, while simultaneously offering comparisons with divergence estimates obtained from traditionally used single genes, SSU rDNA and cox1. The newly sequenced mt genomes are significantly divergent from their previously sequenced conspecifics (A. castellannii 16.4% divergence at nucleotide level and 10.4% amino acid; V. vermiformis 21.6% and 13.1%, respectively), while divergence at the small subunit ribosomal DNA is below 1% within both species. Morphological analyses determined that these lineages are indistinguishable from their previously sequenced counterparts. Phylogenetic reconstructions using 26 mt genes also indicate a level of divergence that is comparable to divergence among species, while reconstructions using the small subunit ribosomal DNA (SSU rDNA) do not. In addition, we demonstrate that between closely related taxa, there are high levels of synteny, which can be explored for primer design to obtain larger fragments than the traditional barcoding genes. We conclude that, although most systematic work has relied on SSU, this gene alone can severely underestimate diversity. Thus, we suggest that the mt genome emerges as an alternative for unraveling the lower level phylogenetic relationships of Amoebozoa.     

Phylogeographic signal variation in mitochondrial DNA among geographically isolated grassland butterflies

Aim We surveyed mitochondrial DNA (mtDNA) sequence variation among regionally isolated populations of 10 grassland‐associated butterfly species to determine: (1) the utility of phylogeographic comparisons among multiple species for assessing recent evolutionary patterns, and (2) the respective roles of isolation attributable to range disjunction versus isolation attributable solely to geographic distance in establishing divergence patterns. Location The Peace River grasslands of northern Alberta and British Columbia, Canada, which are isolated by 300+ km from similar communities to the south. Methods We sequenced mtDNA (1420 bp of cytochrome c oxidase subunit I) from five grassland‐restricted butterfly species that have geographically disjunct populations and from five ecologically broader species that have more continuous distributions across the same regions. Using analysis of molecular variance (AMOVA), Mantel and partial Mantel tests, and haplotype networks, we compared population structure within and between species in order to assess the validity of single‐species phylogeographic characterizations. We then contrasted variance components between disjunct and continuously distributed species to assess whether divergences were correlated more with disjunction or with geographic distance. Results Single‐species analyses varied substantially within both the disjunct and the continuous groups. One species in each of these groups had mtDNA with unusually deep intraspecific mitochondrial lineage divergences. On the whole, however, the five species with disjunct ranges exhibited greater divergence between geographically distant populations than did the five species with continuous distributions. Comparison of variance components between disjunct and continuous species indicated that isolation attributable only to geographic distance was responsible for up to half of the total sequence variation between disjunct populations of grassland butterflies. Main conclusions Our findings show that single‐species phylogeographic analyses of post‐Pleistocene butterfly distributions are inadequate for characterizing regional biogeographic divergence histories. However, comparison of mtDNA sequence divergences between groups of disjunct and continuously distributed species can allow isolation attributable to range interruption to be quantitatively distinguished from isolation attributable solely to gene flow attenuation over the same geographic area.     

Genetic evidence for multiple geographic populations of the giant mottled eel <Emphasis Type="Italic">Anguilla marmorata</Emphasis> in the Pacific and Indian oceans

Anguillid eels have been believed to have a unique catadromous life history completed within a particular ocean current system, and consequently to have extraordinary population structures, with each species comprising a single, virtually panmictic population with a species-specific spawning area far offshore. The giant mottled eel Anguilla marmorata is enigmatic in this respect because it is widely distributed from East Africa to the central South Pacific, where several different current systems exist, and apparently must have multiple spawning areas. To address this issue, the population structure was examined through the mitochondrial (mt) DNA analysis of 162 individuals collected from ten localities representing the overall distributional range. Nucleotide sequencing of a 626-bp segment in the control region of the mtDNA revealed 151 haplotypes, and these were clustered into several major groups supported by high bootstrap probabilities. Sequence differences among geographic samples revealed the existence of five geographic populations around North Pacific, Madagascar, Sumatra, Fiji, and Tahiti. Genetic differentiation among the populations in the Southern Hemisphere was supported by examination of nuclear DNA with amplified fragment length polymorphism (AFLP) analysis performed on 76 individuals from four sampling localities in the Southern Hemisphere. The distribution pattern of five populations was closely associated with the water-mass structure of oceans and major current systems. This observation suggests a hypothesis that present population differentiation in A. marmorata might have resulted from the establishment of new population-specific spawning sites in different oceanic current systems as the species colonized new areas, as its unique catadromous life history of anguillid eels.This revised version was published online in February 2005 with corrections to the repetition of the 1st authors name.     

Interspecies chimeric sperm lysins identify regions mediating species-specific recognition of the abalone egg vitelline envelope.

Abalone sperm lysin is a nonenzymatic, 16-kDa protein that creates a hole in the egg vitelline envelope (VE) through which the sperm swims to fuse with the egg. The dissolution of isolated VE by lysin is species specific. Interspecies comparisons show that the most divergent region of lysin is the N-terminal segment of residues 1-12 which is always species-unique. The C-terminus and three internal segments are moderately variable between species, but not species unique. Analysis of nucleotide substitutions shows that lysin evolves rapidly by positive Darwinian selection, suggesting that there is adaptive value in altering its amino acid sequence. The results reported here, in which segments of lysin were exchanged between two species, prove by direct experimentation that the interspecies variable termini play major roles in the species-specific recognition between sperm lysin and the egg VE.     

Calibration of mitochondrial DNA evolution in geese

Summary Mitochondrial DNA was purified from five American species of geese representing the generaAnser andBranta, which have fossil records. The results of electrophoretic comparisons of about 75 fragments per individual produced by 14 restriction enzymes imply that the mean extent of sequence divergence between species ofAnser andBranta is about 9%. Fossil evidence suggests that these two groups of geese had a common ancestor 4–5 million years ago. Thus, the mean rate of sequence divergence in goose mitochondiral DNA is not far from 2% per million years, the value in mammals.     

The heterochromatin of grasshoppers from the Caledia captiva species complex. I. Sequence evolution and conservation in a highly repeated DNA family

The restriction enzyme TaqI digests 0.2% of the genomic DNA from the grasshopper Caledia captiva to a family of sequences 168 bp in length (length of consensus sequence). The sequence variation of this "Taq family" of repeat units was examined among four races from C. captiva to assay the pattern of evolution within this highly repeated DNA. The Taq-family repeats are located in C-banded heterochromatin on at least one member of each homologous pair of chromosomes; the locations range from centromeric to telomeric. Thirty-nine cloned repeats isolated from two population 1A individuals along with 11 clones from seven populations taken from three of the races demonstrated sequence variation at 72 positions. Pairwise comparisons of the cloned repeats, both within an individual and between different races, indicate that levels of intraspecific divergence, as measured by reproductive incompatibility, do not correlate with sequence divergence among the 168-bp repeats. A number of subsequences within the repeat remain unchanged among all 50 clones; the longest of these is 18 bp. That the same 18-bp subsequence is present in all clones examined is a finding that departs significantly (P less than 0.01) from what would be expected to occur at random. Two other cloned repeats, from a reproductively isolated race of C. captiva, have sequences that show 56% identity with this 18-bp conserved region. An analysis showed that the frequency of occurrence of an RsaI recognition site within the 168- bp repeat in the entire Taq family agreed with that found in the cloned sequences. These data, along with a partial sequence for the entire Taq family obtained by sequencing uncloned repeats, suggest that the consensus sequence from the cloned copies is representative of this highly repeated family and is not a biased sample resulting from the cloning procedure. The 18-bp conserved sequence is part of a 42-bp sequence that possesses dyad symmetry typical of protein-binding sites. We speculate that this may be significant in the evolution of the Taq family of sequences.      

Variation in salmonid mitochondrial DNA: Evolutioinary constraints and mechanisms of substitution

Summary Sequence comparisons were made from 2214 bp of mitochondrial DNA cloned from six Pacific salmonid species. These sequences include the genes for ATPase subunit 6, cytochrome oxidase subunit 3, NADH dehydrogenase subunit 3, NADH dehydrogenase subunit 4L, tRNA^GLY, and tRNA^ARG. Variation is found at 338 silent and 12 nonsilent positions of protein coding genes and 10 positions in the two tRNA sequences. A single 3-bp length difference was also detected. In all pairwise comparisons the sequence divergence observed in the fragment was higher than that previously predicted by restriction enzyme analysis of the entire molecule. The inferred evolutionary relationship of these species is consistent between methods. The distribution of silent variation shows a complex pattern with greatly reduced variation at the junctions of genes. The variation in the tRNA sequences is concentrated in the DHU loop. The close relationship of these species and extensive sequence analyzed allows for an analysis of the spectrum of substitutions that includes the frequencies of all 12 possible substitutions. The observed spectrum of substitutions is related to potential pathways of spontaneous substitution. The salmonid sequences show an extremely high ratio of silent to replacement substitutions. In addition the amino acid sequences of the four proteins coded in this fragment show a consistently high level of identity with theXenopus sequences. Taken together these data are consistent with a slower rate of amino acid substitution among the cold-blooded vertebrates when compared to mammals.     

Studies on Nuclei of Paramecium aurelia. I. Isolation and Purification by Continuous or Discontinuous Sucrose Gradient Centrifugation and Biochemical Composition.*

SYNOPSIS. Nuclei of Paramecium aurelia were isolated and purified by a new method involving the use of continuous or discontinuous sucrose gradient centrifugation. As judged from the DNA levels or nuclear counts in the purified samples, 21–22% of the nuclei were recovered by this method. The single density of the nuclei estimated on the basis of linear sucrose gradients was between 1.35 ± 0.01 and 1.36 ± 0.01. The average picogram quantities of total protein, DNA, and RNA per nucleus were 435, 62.2, and 51.2, respectively.     

Phylogeography and systematics of the Peromyscus eremicus species group and the historical biogeography of North American warm regional deserts

Phylogeographic relationships among 26 populations from throughout the geographic range of the Peromyscus eremicus species group are described based on sequence data for a 699-bp fragment of the mitochondrial DNA COIII gene. Distance, maximum-likelihood, and maximum-parsimony analyses of phylogenetic trees generated under four separate character-weighting strategies and representing five alternative biogeographic hypotheses revealed the existence of a cryptic species (Peromyscus fraterculus, previously included under P. eremicus) on the Baja California Peninsula and adjacent southwestern California and two distinct forms of P. eremicus, one from the Mojave, Sonoran, and northwestern Chihuahuan regional deserts (West) and one from the remainder of the Chihuahuan Desert (East). Distinctiveness of P. fraterculus is supported by previous morphometric and allozyme analyses, including comparisons with neighboring P. eremicus and parapatric P. eva, with which P. fraterculus shares a sister taxon relationship. Divergence of the eva + fraterculus, West + East eremicus, and P. merriami haplotype lineages likely occurred in the late Neogene (3 Ma), in response to northern extension of the Sea of Cortéz and elevation of the Sierra Madre Occidental; divergence of eva from fraterculus is concordant with the existence of a trans-Peninsular seaway during the Pleistocene (1 Ma); and divergence of West from East eremicus occurred during the Pleistocene pluvial-interpluvial cycles, but well before the Wisconsinan glacial interval. The sequence of divergence within the eremicus species group and causal association of geological events of the Neogene and Holocene provide a working hypothesis against which phylogeographic patterns among other arid-adapted species of the warm regional deserts of North America may be compared.