MBE Advance Access originally published online on April 2, 2007
Molecular Biology and Evolution 2007 24(7):1492-1505; doi:10.1093/molbev/msm068
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Research Articles |
Analysis of Nuclear Copies of Mitochondrial Sequences in Honeybee (Apis mellifera) Genome
Department of Biochemistry, Purdue University
E-mail: sbehura{at}purdue.edu.
 |
Abstract |
|---|
 TOP Abstract IntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
|---|
At least 0.08% of the Apis mellifera nuclear genome contains sequences that originated from mitochondria. These nuclear copies of mitochondrial sequences (numts) are scattered all over the honeybee chromosomes and have originated by multiple independent insertions of mitochondrial DNA (mtDNA) as evident by phylogenetic analysis. Apart from original insertions, moderate duplications of numts also contributed to the present pattern and distribution of mitochondrial sequences in honeybee chromosomes. Assimilation of mitochondrial genes in the nuclear genome is mediated by extensive fragmentations of the original inserts. Replication slippage seems to be a major mechanism by which small sequences are inserted or deleted from mtDNA destined to nucleus. Most of the honeybee numts (84%) are located in the nongenic regions. The majority (94%) of the numts that are located in predicted nuclear genes have originated from mitochondrial genes coding for cytochrome oxidase and NADH dehydrogenase subunits. On the other hand, the mitochondrial rRNA or tRNA gene sequences are predominantly (88%) located in nongenic regions of the genome. Evidences also support for exertion of purifying selection on numts located in specific genes. Comparative analysis of numts of European, African, and Africanized honeybees suggests that numt evolution in A. mellifera is probably not demarked by speciation time frame but may be a continuous and dynamic process.
Key Words: Apis mellifera • numt • mitochondrial • insertion • duplication • molecular marker
|
Introduction |
|---|
|
TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
|---|
According to the endosymbiotic theory, the ancestors of animal mitochondria and plant plastids were once free-living prokaryotes, resembling
-proteobacteria (in the case of mitochondria) and cyanobacteria (in the case of plastids). Present mitochondrial genomes in animals are about 100- to 300-fold smaller than those of free-living bacteria (4000–6000 kb) (Selosse et al. 2001
). Since the origin of eukaryotes roughly 2 billion years ago, the eubacterial autonomy of organellar genomes was gradually lost. This is supported from evidences of continuous transfer of mitochondrial genes to the nuclear genome resulting in reduction of organellar genome size (Thorsness and Weber 1996; Timmis et al. 2004). Evidences of transfer of mitochondrial genes to the nuclear genome have been supported from genome sequences of eukaryotes (Richly and Leister 2004). These copies of mitochondrial sequences present in nuclear genomes are commonly referred to as "numts". Phylogenetic analysis of numts paralogous to mitochondrial DNA (mtDNA) suggests that migration of genes from mitochondria to nucleus is a continuous and dynamic evolutionary process (Ricchetti et al. 2004; Timmis et al. 2004). Theoretical models suggest that the rate of transfer of mtDNA to nucleus depends on the intensity of intracellular competition (compactness of organelle genomes is advantageous in intracellular competition) and also on probability of paternal organelle transmission and the effective population size of the species (Yamauchi 2005).
Transfer of mitochondrial sequences to the nuclear genome is a ubiquitous phenomenon. More than 80 eukaryotes including 20 insect species have been implicated to have numts in their genome (Bensasson et al. 2001). In insects, mtDNA is extensively used as molecular markers in ecological and evolutionary studies (Behura 2006). In most of these works, common methodologies are practiced where mtDNA fragments are amplified by polymerase chain reactions (PCRs), sequenced, and the sequence data are used for population genetic and evolutionary analysis. However, PCR amplification of mitochondrial gene fragments from total genomic DNA is vulnerable for amplification of the corresponding nuclear paralogous sequences that may mislead the phylogenetic and diagnostic inferences (Arctander 1995; Zischler et al. 1995; Zhang and Hewitt 1996). Hence, presence of numts can be problematic in these studies of insects. Not much has been done to address these issues although the presence of numts has already been implicated in all orders of insects (Bensasson et al. 2001) with more detailed analysis in tiger beetles (Pons and Vogler 2005), grasshopper (Bensasson et al. 2000), and Sitobion aphids (Sunnucks and Hales 1996). However, the nuclear genome sequences of Anopheles gambiae showed no detectable mitochondrial sequences, whereas that of Drosophila melanogaster showed only a few (6 to 8) numts in the sequenced genome (Richly and Leister 2004). The recently completed genome sequencing of Apis mellifera (HGSC 2006) provides unique opportunities in this direction to study the transposed copies of mtDNA in the nuclear genome of this important insect (A. mellifera).
The Western honeybee A. mellifera is regarded as the premier pollinator of major fruit crops accounting for more than 10 billion dollars a year in the United States alone (Morse and Calderone 2000). It has a long history of association with mankind because of their cavity-nesting lifestyle and use in beekeeping for producing honey, wax, and royal jelly. Honeybee mtDNA sequences have been used to infer geographic origin, genetic relatedness, phylogeny, and population structure (Smith and Brown 1988; Hall and Muralidharan 1989; Garnery et al. 1992; Franck et al. 2000, 2001; Clarke et al. 2001). Apart from the Western honeybees, 4 Eastern honeybee species, Apis cerana, Apis nigrocincta, Apis koschevnikovi, and Apis nuluensis, are known that are native to eastern Asia as far north as Korea and Japan. Apis species (at least 10) have been identified by using both genotypic and phenotypic differences between them (Cameron 1993; Engel and Schultz 1997; Arias and Sheppard 2005). Within A. mellifera, at least 24 subspecies have been identified that are distributed in Europe, Africa, and Asia (Garnery et al. 1992; Arias and Sheppard 1996; Franck et al. 2000). In spite of the extensive utility of mtDNA markers in phylogeography of honeybees, doubts still prevail about the ancestral relation among the honeybee species (Arias and Sheppard 2005). A recent investigation using genome-wide analysis of single nucleotide polymorphisms among New World and Old World honeybees suggested that A. mellifera originated from Africa (Whitfield et al. 2006) as opposed to earlier belief that they might have originated from Eastern honeybees (Arias and Sheppard 2005). Within A. mellifera, hybridization between selected subspecies has been reported (Smith et al. 1989; Franck et al. 2000; Jensen et al. 2005), and one of the consequences of such hybridization of African subspecies Apis mellifera scutellata with the native Western honeybees has resulted in rapid spread of Africanized bees (killer bees) that have largely replaced European bees throughout its range in the New World (Pinto et al. 2004, 2005; Schneider et al. 2004; Collet et al. 2006).
Characterization of honeybee populations can be benefited from identification of numts in the honeybee genome. Identification and characterization of numts in the honeybee are important not only to avoid their interference in generating mtDNA as genetic markers but also for using them as nuclear markers to infer ancestral history of the species and subspecies. Once a mtDNA fragment is transposed to the nuclear genome, it remains in the genome as a "molecular fossil" (Bensasson et al. 2001). Hence, numts collectively form a "natural library" of all ancestral mitotypes migrated to the nucleus providing invaluable information on population and speciation history of the mtDNA of the organism. Moreover, comparison of orthologous numts with paralogous copies is important to understand duplication and differentiation of nuclear mtDNA in relation to speciation time of the organism. In principle, duplication of numts gives rise to paralogous copies in the same genome. But, the time of duplication of a numt with respect to that of speciation event marks the specificity of paralogous copies to the descendent species. However, not much research has been done to characterize numts in a comparative manner in closely related species or subspecies (Sunnucks and Hales 1996; Hazkani-Covo et al. 2003; Pons and Vogler 2005; Hazkani-Covo and Graur 2007). In honeybee, although the presence of numts has been implicated by previous workers (Pereira and Baker 2004; Kaplan and Linial 2006), no detailed investigation has been performed. The present study is aimed to perform a detailed investigation on genome-wide identification of numts to understand their mode of origin and evolution and to determine their structure and distribution pattern and the underlying mechanisms.
|
Materials and Methods |
|---|
|
TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
|---|
BlastN (Altschul et al. 1990
) search was conducted locally using the complete mtDNA of A. mellifera (accession number L06178; Crozier RH and Crozier YC 1993) as query sequence with honeybee genome sequence (Amel 4.0) (HGSC 2006). Searches were conducted either with or without using the "filter." The use of filter in Blast searches removed many of the authentic numts or parts of numts. To overcome this, Blast was conducted without the use of filter, and the "hits" were manually inspected to identify repeat sequences. This was done by retrieving the hits of both filtered and unfiltered Blast output (multiple alignment) results, formatting them in Excel (using "text to columns" function) and comparing repeat regions by eye. The hits that lacked any flanking mtDNA-like sequences, apart from the repeat sequences, were assumed to be spurious hits and were not considered as numts. The hits with E value below 6 x 10–14 were used for further analyses. This E value was chosen as it was close to that (4.0 x 10–12) for numts identified from African and Africanized bees. These numts were subsequently used for comparative analysis. Partial sequences of many mitochondrial genes of different species and subspecies of honeybee were retrieved from GenBank using keyword searches.
Numts in African honeybees were identified by similar method as above. Genomic trace sequences of A. m. scutellata were obtained from Baylor College of Medicine (http://www.hgsc.bcm.tmc.edu/projects/honeybee/). When trace sequences were used to make Blast database, care was taken to select only hits that contained a mitochondrial sequence as a portion, along with a portion that was not mtDNA-like, within the trace sequence. If the entire trace sequence showed homology to mtDNA, it was not used in further analysis as there was a chance that it could have originated from mitochondrial contaminants. Numts from Africanized honeybees were identified from the genomic sequences of Africanized A. mellifera deposited in GenBank by Tomkins et al. (2002). The trace sequences of A. m. scutellata were first searched with the complete sequence of mtDNA of the honeybee to get a first-hand-information about the possible numts in these sequences. The hits of Amel 4.0 corresponding to the same regions of mtDNA were used as query to perform a second Blast with A. m. scutellata database. The hits of this search were then used as query for reciprocal Blast with Amel 4.0 database to identify the orthologous numts between European and African honeybees. A similar procedure was followed to identify orthologous numts between European and Africanized honeybees. If more than 1 numt of A. m. scutellata showed reciprocal Blast hits with the same Amel 4.0 numt, they were considered as paralogous numts in the African honeybee. However, because the database of A.m. scutellata represented only limited sequences of its genome, multiple numts of Amel 4.0 showing reciprocal Blast hits to the same numt of A.m. scutellata could be forced matches. Those numts were excluded from the paralogy analysis.
The Blast results were obtained in tabular, multiple alignment and also in pairwise hit formats suitable for different types of analysis. All calculations to determine the total number, size, and locations of numts were performed by Microsoft Excel. If 2 or more nuclear sequences showed full-length alignment to exactly the same region of the query mtDNA, they were considered as duplicated numts. In no case, only a portion of the numts was used to check the duplications. If the distances between 2 and more nuclear sequences were "similar" (difference less than 100 bp was allowed) to the distances between the corresponding sequences in mitochondria, then those numts were regarded as fragmentation products of a larger numt. No 2 numts with gap more than the size of complete mtDNA was used. Nonalignment of sequences less than 100 bp was allowed in these comparisons. This is because accumulation of mutations may show lack of homology in small regions (but not the entire gap regions of hundreds of bases) in the intermediate DNA between 2 numts. Numts located in unknown groups were excluded from the gap-comparison analysis. Because these groups have not been assigned to chromosomes, it is not possible to know the gap between a numt of unknown group with numt of known chromosomal location. Also, no comparison was made with numts in neighboring contigs to avoid the risk of mistakes in determining gaps between such numts. Relative orientations of numts were determined by comparing their start and end coordinates. Genetic distances of orthologous numts from honeybee mtDNA (accession number L06178) were calculated using Kimura-2 parameter. Nucleotide diversity and counts of fixed, shared, and exclusive polymorphism were measured by using DNASP 4.0 program (Rozas et al. 2003). Flanking sequences were characterized, for the presence of any conserved motifs, by using DNA Block Aligner (Birney et al. 2004) available at http://www.ebi.ac.uk/Wise2/documentation.html. The nonparametric Spearman test was performed to determine statistical significance of correlations between numt content and chromosome size and also between numt gap and corresponding mitochondrial gap. The chromosome length data published by Beye et al. (2006) were used for comparison with the numt content in each chromosome.
The Official Gene Set Release 1 by BeeBase (http://racerx00.tamu.edu/blast/blast.html) was used for analyzing the position of numts in predicted genes. To determine the positions of numts with respective to the genes of this list, numts identified from the previous version of genome sequences (Amel 2.0) were used. Locations of numts inside nuclear genes were selected only for those genes that were predicted with a probability more than 0.75. The start and end coordinates of predicted nuclear genes were used to determine if any numt was located in the genic or nongenic regions. Similarly, the coordinates of coding regions of the predicted genes were used to determine if a numt was located in the coding regions or in the introns. Numts in coding regions of the gene were verified from expressed sequence tag (EST) evidences. The values of Ka (the number of nonsynonymous substitutions per nonsynonymous site) and Ks (the number of synonymous substitutions per synonymous site) were estimated according to Nei and Gojobori (1986). Pairwise sequence alignment of the predicted gene and the numt was used to identify the codon position for the 1st nucleotide of the inserted numt in that gene. All the synonymous and nonsynonymous sites were determined by using DNASP program.
Phylogenetic analysis was performed by MEGA 3.0 program (Kumar et al. 2004) by Neighbor-Joining method. Bootstrap test of the inferred phylogeny was done with 1,000 replications. Because of length differences of numts, sequences were extended on both sides of the loci to maximize the multiple alignments. The flanking sequences of numts were extracted to see if that was a part of longer numt. This was helpful to identify numt that might contain small insertion/deletions. Those copies were concatenated and then used for the alignment purpose. ClustalW incorporated in MEGA 3.0 was used, with default set of parameters, to perform all multiple alignments.
|
Results |
|---|
|
TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
|---|
Identification of Numts
Nucleotide Blast search with the completely sequenced mtDNA of A. mellifera (accession number L06178), below a cutoff E value of 6 x 10–14, identified a total of 1,380 mitochondrial-like sequences in the genome of honeybee (complete list can be provided upon request). Based on homology, these numts seem to have originated from all over the mitochondrial genome. However, maximum DNA transfer was found to occur from the gene encoding mitochondrial cytochrome c oxidase subunit I (COI), whereas least transfer occurred from ribosomal small RNA gene of mitochondria (data not shown). The transferred numts show extensive length differences ranging from 39 to 863 bp. A total of 226 numts were found located in unknown groups of genome assembly, and the rest (
83%) were found distributed nonuniformly but throughout all the 16 chromosomes (fig. 1). Chromosome 1, the largest chromosome (25.7 Mb) of all in honeybee chromosomes, has the highest amount of numts. The chromosome 16 (6.2 Mb) is about 4 times smaller than chromosome 1 and is considered as the smallest chromosome of honeybee. It contains nearly 3.1 times less numts than chromosome 1. However, the minimal amount of numts is present in chromosome 10. There does not seem to have a statistical significant correlation (Spearman coefficient 0.01602, df = 14) between the numt content in a chromosome and the size of the chromosome (fig. 1). This is in agreement with earlier reports in other species that there is no correlation between genome size and the numt content (Richly and Leister 2004).
|
Blast results in multiple alignment formats revealed, on an average, 9 nuclear copies of varying lengths for each mitochondrial gene fragment. Characterization of the flanking sequences (2 kb in either side) of such paralogous copies did not show any sequence conservation (other than the numts) or presence of any common motifs. This result is consistent with observations in other organisms that there are no hot spots for insertion of a mitochondrial gene in chromosomal DNA (Hazkani-Covo and Graur 2007
).
Together, numts represented about 0.08% of the honeybee nuclear genome. This is much higher than the percentage of numts in other eukaryote genomes including that of human (Bensasson et al. 2001). This may be explained on the basis of exceptionally high rate of recombination in honeybee (Hunt and Page 1995; Beye et al. 2006), which in turn degenerate the transposed DNAs in honeybee genome more rapidly than in other species. This might enable the honeybee genome to harbor these migrated DNAs for a much longer period of time than other organisms, for example, than Drosophila or Anopheles, where the recombination rate is much lower than that of the honeybee.
Origin and Evolution of Numts
The numts showed phylogenetic relationships (fig. 2) that suggested multiple introductions of mitochondrial sequences into the nuclear genome. To obtain the relative age of the ancestor for each group, assuming they have independent origin, the mitochondrial gene sequences of 3 outgroup species Apis dorsata, A. cerana, and A. koschevnikovi were used to determine the extent of nucleotide fixation and sharing with the corresponding numts of A. mellifera. Three groups were randomly selected for this purpose and were subsequently named I, II, and III based on the amount of nucleotide fixation and sharing with the mitochondrial sequences. This was performed for multiple genes of mitochondria and in each case, numts belonging to phylogenetic group I showed an elevated amount of fixed mutations compared with the mtDNA of each outgroup species (table 1 and supplementary fig. 1, Supplementary Material online). The group III numts showed only a minimal amount of fixation of the polymorphisms, whereas group II numts showed an intermediate amount of fixed mutation with mtDNAs. These observations suggest that group I numts might have a most common ancestral DNA that is older than that of group II numts. Similarly, group 2 ancestral DNA is older than that of group III numts but more recent than that of group I numts. These data suggested that numts in honeybee could have derived independently from different mitochondrial origins. Because of differential rate of evolution of mtDNA and nuclear DNA, phylogeny of numts may not correspond to that of the corresponding mtDNAs (supplementary fig. 1, Supplementary Material online). Similar observations have been made in other species as well (Sunnucks and Hales 1996; Bensasson et al. 2001). Apart from fixed and shared mutations with the mtDNAs, numts in honeybee contain numerous recent mutations that are neither shared with mtDNA nor fixed in the nuclear copies. Such mutations in numts contribute to the overall nucleotide diversity of paralogous mitochondrial sequences in the nuclear genome (supplementary fig. 2, Supplementary Material online).
|
|
A comparative analysis was performed between numts of European, African, and Africanized honeybees. Using the honeybee mtDNA (accession number L06178) as query, a total of 15 numts were identified in African honeybee (A.m. scutellata) (supplementary table 1, Supplementary Material online) and 4 from Africanized honeybee DNAs (supplementary table 2, Supplementary Material online) below a threshold E value 4.0 x 10–12. Reciprocal Blast of these numts with the European numts established orthologous relationships only for 9 numts between European and African bees and for 3 between European and Africanized bees (table 2). As expected, in most of the cases, the European numts and European mtDNA were genetically closer than the African numts and European mtDNA (table 2). However, 3 (NADH dehydrogenase [ND2], cytochrome B [cytB], and ATPase 6) out of the 9 African numts and 1 (COII) of the 3 Africanized numts were genetically closer to European mtDNA than the European numts and European mtDNA (table 2). This result suggests that DNA transfer from mitochondria to nuclear genome is probably a continuous process along with the continuing hybridization between European and African honeybees (Schneider et al. 2004
). Moreover, it also supports the dynamic nature of this evolutionary process of gene transfer from organellar genome to nuclear genome in other species (Ricchetti et al. 2004; Timmis et al. 2004). However, due to limited sequence data on African and Africanized bees, no general conclusions can be made in this regard, and further sequence information of African honeybee genome will be useful to test this possibility.
|
Assimilation of mtDNA in Nuclear Genome
Multiple factors including differential codon usage of mtDNA and nuclear DNA (Benne and Sloof 1987
) and differential rate of mutation in nuclear DNA versus mtDNA (Wolfe et al. 1987) may determine the rate of assimilation of numts in the chromosomal DNA (Bensasson et al. 2000, 2001). In honeybee, one of the factors that seem responsible for assimilation of the mitochondrial gene in the chromosome is by fragmentation. At least 260 numts seem to be fragmented products of larger inserts of DNA fragments originated from different parts of the mitochondrial genome (supplementary table 3, Supplementary Material online). This is observed by comparing the physical distances between neighboring numts in chromosomes with those between the homologous sequences in the mtDNA. An example of relative distances and orientations of fragments in mitochondria and in nuclear genome is shown in figure 3. In the majority (78%) of the numts, the gap was larger in the nuclear copies than the distance between their homologous sequences in mtDNA. The 2nd category of numts (n = 223) showed gaps in the nuclear copies that were smaller than the gaps between their mitochondrial homologous sequences. In the 3rd category of numts, however, a total of 260 numts (of size ranging from 46 to 631 bp) were identified that showed similar gaps between the nuclear copies and the homologous sequences in mitochondrial genome. These sequences could be fragmented products of larger numts. The numt gap and corresponding mitochondrial gap showed statistically significant correlation (P < 0.01) assessed by nonparametric Spearman rank-order correlation test (Spearman coefficient 0.2826, df = 158) (fig. 4). Multiple numts (2–5) were found located in cluster that appeared to have undergone fragmentation of larger numts of estimated size ranging from 129 to 7,809 bp (supplementary table 3, Supplementary Material online). In principle, fragmented numts should harbor old mutations as opposed to accumulation of recent mutations in the intact numts. Although it was found true by counting the number of fixed mutations of some of these numts with mtDNA of outgroup honeybee species (data not shown), unavailability of mtDNA sequences corresponding to all the fragmented numts hindered a detailed investigation in this regard. In all fragmentation events, the numts were located in same orientations except just one. The numts located at group 3.6: 45043–44591 and group 6: 51878–51948 were most probably fragmented from an original numt of size 7,809 bp (supplementary table 3, Supplementary Material online), and the fragmented numts were relocated themselves in opposite orientations. The mechanism of fragmentation of numts is not known. Given the fact that there exists no known mechanism for the precise excision of numts, it may be possible that large insertions/deletions may be responsible for such process (Hazkani-Covo and Graur 2007). The complete genome sequences of African honeybee, when that will be available, may be used to compare the fragmented numts to test this hypothesis.
|
|
Duplications of Numts
Of all the scattered numts (n = 785), at least 133 sequences originated from closely spaced regions in the mtDNA but were located away from each other in the nuclear DNA (fig. 3). Also, some scattered numts originated from exactly same locations of mtDNA, and those were considered duplicated numts in the nuclear genome. A total of 68 mtDNA inserts were identified that were duplicated either once or multiple times in the nuclear genome. Of these 68, a total of 58 have been duplicated twice, 7 were duplicated 3 times, 2 were duplicated 4 times, and 1 (tRNA-Glu) was duplicated 7 times (supplementary table 4, Supplementary Material online). Numts of different sizes (42–794 bp) showing homology to mitochondrial ribosomal RNA genes, both small and large subunits, and to those of ND genes (subunits 2, 4, and 5) showed a maximum number of duplications (n = 42 in total) followed by numts of different tRNAs genes (n = 10). The numts that originated from mitochondrial genes coding for COI and COII showed 8 and that from cytB gene showed only 4 duplication events. In some cases, mtDNA fragments of 2 neighboring genes were also found to be duplicated (supplementary table 4, Supplementary Material online). For example, a 126-bp fragment of ATPase 8 and the neighboring tRNA gene (mtDNA positions: 4400–4526) and another 200-bp fragment of ND5 gene and its neighboring tRNA gene (mtDNA positions: 8411–8631) were each duplicated 3 times in the nuclear genome (supplementary table 4, Supplementary Material online). Moreover, there is no reason to assume that numt duplication should be restricted to single genes of mitochondria (Bensasson et al. 2003
; Hazkani-Covo et al. 2003). Duplication of numts was both inter- and intrachromosomal. One-third (n = 26) of the duplication events were restricted to within chromosomes, with distances between them ranging from 61 bp to nearly 480.5 kb (supplementary fig. 3, Supplementary Material online), but others (n = 42) were interchromosomal. In 4 of the intrachromosomally duplicated numts, the distances between the duplicated numt pairs were similar to the length of numts suggesting their canonical mode of duplications. The mitochondrial origins of these numts are 82-bp fragment of tRNA-Tyr gene (66 bp between duplicated numts), 92-bp fragment of tRNA-Asp gene (108 bp between duplicated numts), 184-bp fragment of cytB gene (190 bp between duplicated numts), and 57-bp fragment of the gene coding for rRNA large subunit (61 bp between duplicated numts). Duplicated numts were found inserted either in the same or in the opposite orientations with each other. However, predominantly more duplications (67%) were found in the same orientation than those were in the opposite orientation. In either case, numts that were duplicated just once outnumbered the numts that were duplicated multiple times. However, interestingly, no numt was identified that was duplicated with 1 copy in a gene and another in the nongenic region of the genome. This probably suggests the existence of a differential regulatory mechanism for duplications of numts in genic and nongenic regions in honeybee genome. This is in parallel to observation of differential regulation of insertions of retroelements and inverted repeat transposons in genic and nongenic regions of cereal genomes (Ramakrishna et al. 2002). To understand the relative time of duplications of numts with respect to their differentiation within A. mellifera, orthologous numts with paralogous copies between European and African honeybees were compared (fig. 5). Phylogenetic relationships revealed that the ancestral DNA of ND2 and ATPase 6 numts were inserted into the nuclear genome prior to divergence of these sequences in these subspecies. Then, the sister numt in African subspecies was duplicated. However, in the case of cytB numts, the ancestral DNA was first duplicated in the European honeybee, and one of the duplicated copies was then differentiated in the African subspecies (fig. 5). Thus, it seems that numt duplications occurred at different times with respect to their differentiation within A. mellifera. These results imply that, in honeybee, numt differentiation and duplication are probably not demarked by speciation time frame but may be a continuous process.
|
Insertions/Deletions in Numts
Transfer of mtDNA to the nuclear genome in honeybee, in some cases, is associated with insertion or deletion of small sequences. These included 29 numts in which small sequences have been deleted upon transfer from mitochondria to the nucleus (table 3). The length of the sequences that are deleted varies from 1 to 52 bp, and these sequences are always flanked by 2 identical or highly similar direct repeats. However, only one copy of the repeat is retained in the nuclear copy (fig. 6 and supplementary fig. 4, Supplementary Material online). In some cases, a single-base mutation was observed in one of these motifs at the deletion sites. The motif that contained the extra nucleotide is retained in the numt, and the motif that lacks the specific base is deleted along with the intervening sequences (data not shown). The motifs are mostly AT-rich and of size ranging from 1 to 15 bp (table 3). These direct repeats are hallmarks of sequence variations caused by replication slippage (Noutsos et al. 2005
). Apart from microdeletions in the transferred mtDNA, insertion of small sequences (4–78 bp) was also observed in many (n = 17) numts (table 4). The 2 numts that are separated by the insertion element, however, show homology to 2 overlapping sequences in the mtDNA (fig. 7). Also, upon insertion of the element, the 2 flanking numts often are flipped around the insertion element (supplementary fig. 5, Supplementary Material online). Moreover, unlike the microdeletions in numts, insertions in numts do not always show a common flanking sequence repeats (supplementary fig. 5, Supplementary Material online). However, in general, deletions by replication slippage leave no trace in the resulting sequence, whereas insertion of a small sequence by replication slippage can leave direct repeats at the insertion sites. If this is always the case, then it may be possible that the microdeletion in the numts actually represents insertions in the corresponding mtDNA, and the insertions seen in the numts are in fact the deletions in the corresponding mtDNA. In that case, these specific numts might have transferred to the nucleus before such sequences changes occurred in the honeybee mitochondrial genome. Thus, it seems that replication slippage is a major mechanism by which small sequences are inserted or deleted from mtDNA destined to nucleus.
|
|
|
|
Insertion of Numts in Nuclear Genes
The genomic locations of numts further revealed that at least 50 numts were found located within nuclear genes, those were predicted from the honeybee genome sequence (Official Gene Set Release 1) (table 5). Although some of these host genes had orthologs in D. melanogaster and A. gambiae (supplementary table 5, Supplementary Material online), there was no evidence of presence of any mitochondrial-like sequences in the corresponding orthologous genes in those species (data not shown). Of these 50 numts, 45 were located in the intron regions and only 5 were found in the coding regions of the host genes. Unlike the genes that contain a single numt in the coding sequences, the genes containing intronic numts in many cases were found to harbor more than 1 numt. These were located either in the same or in the different introns within a gene. Also in some cases, the numts were located either in the same or in the different orientation to each other within the gene (table 5). Presence of numts in the introns of nuclear gene may have role in transcriptional changes of these genes due to change of gene length (Chen et al. 2006
). Moreover, numts may also contribute to evolution of these genes based on general hypothesis of intron evolution in eukaryotes (Koonin 2006).
|
Majority (94%) of numts that were located inside predicted nuclear genes showed homology to genes mostly coding for different subunits of cytochrome oxidase or NADH dehydrogenase in the mitochondria (table 5). This suggested that genic numts preferentially originated from protein-coding genes of mitochondria. However, the converse is not true. A numt residing in a nuclear gene is not necessarily derived from a protein-coding gene of mitochondria. This is because 47% of all numts originating from protein-coding genes of mitochondria were also found located in nuclear regions that were nongenic in nature. Similarly, majority of the mitochondrial fragments (88%) that originated from rRNA or tRNA genes were found located in nongenic regions of nuclear genome. Also, in few cases (at least 4), numts were identified that originated from mitochondrial regions containing a portion of protein-coding gene and a neighboring tRNA or rRNA gene (table 5). Combining all genes of mitochondria, 84% of all transposed mtDNA are located in the noncoding regions of nuclear genome.
A total of 5 genes were identified that contained numts in their coding regions. The coding capabilities of these 5 gene sequences are evident from ESTs identified in honeybee (table 5). The numts, that were found located in these coding sequences, were further analyzed for synonymous and nonsynonymous mutations with respect to the mtDNA (supplementary table 6, Supplementary Material online). The polymorphisms in some numts revealed an excess of nonsynonymous over synonymous substitutions as expected for random mutation in the absence of purifying selection. The lack of evidence for purifying selection in these numts indicates that they may be undergoing mutational decay. However, in some genes the synonymous mutations outnumbered the nonsynonymous mutations in the numt locations (supplementary table 6, Supplementary Material online), suggesting that these numts may be under purifying selection. It is unclear why these specific numts be favorably selected as opposed to other numts that have detrimental evolutionary fate. However, it is not quite unexceptional as similar selection is known for retrotransposon inserts in the genomes of other species (Pereira 2004).
|
Discussion |
|---|
|
TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
|---|
The current investigation reports the presence of mtDNA sequences in the nuclear genome (numts) of honeybee. Numts have been studied in other insects such as aphid (Sunnucks and Hales 1996
), grasshopper (Bensasson et al. 2000), and tiger beetles (Pons and Vogler 2005). However, no mitochondrial sequences were detected in the genome of A. gambiae, whereas only a few (6–8) numts were found in the genome of D. melanogaster (Richly and Leister 2004). The sequence identity of numts in the genome depends on the mutation rates of mtDNA and the nuclear DNA of the species. A transposed mitochondrial gene can exist in the nuclear genome for longer evolutionary period (so-called molecular fossils) because of differences in mutation rate of nuclear DNA (Lopez et al. 1994; Arctander 1995; DeWoody et al. 1999; Lu et al. 2002; Olson and Yoder 2002; Woischnik and Moraes 2002). The absolute nucleotide substitution rates in numts are estimated to be 3 x 10–9 to 4 x 10–9 (Li 1997), whereas that of mtDNA is 2 x 10–8 to 2.5 x 10–8 substitutions/site/year (Hasegawa et al. 1985; Brower 1994). The extent of variation of mutation rates of mitochondrial gene and the corresponding numt can affect the existence of the nuclear copy (Blanchard and Lynch 2000; Selosse et al. 2001). This may account for slower or faster rate of assimilation of a mitochondrial gene after migration to the nuclear genome in different species. Mutation rate of nuclear DNA is not significantly different between animals and plants, whereas mutation rate of their mtDNA shows 100-fold difference (Wolfe et al. 1987). Moreover, it is also reported that the mtDNA may be still in the process of transfer to the nucleus in plants, whereas in animals it is in the completion stage (Brennicke et al. 1993). In case of honeybee, it is known that mutation rate of its mtDNA is higher compared with that of Drosophila and Anopheles (Crozier et al. 1989). Moreover, honeybee nuclear genome shows lower rate of evolution compared with these dipteran insects (HGSC 2006). Thus, based on above analogy, it may be possible that rate of nuclear transfer of mtDNA in Anopheles or Drosophila genome is very different than that of honeybee. However, the role of mutation rate of mtDNA on transfer of genes from mitochondria to nucleus is unclear (Yamauchi 2005).
The current study demonstrates predominant transfer of mtDNA to nongenic regions than the genic regions of honeybee genome. It is not clear if this is because of preferential transfer to noncoding regions or simple because of higher chance of having noncoding regions as destinations than coding regions in the genome. In general, 90% of the animal mitochondria genome is composed of genes (Gray 1989, 1992; Boore 1999), and, on the other hand, 95–97% of nuclear genomes are composed of nongenic DNA or so-called junk DNA (Gregory 2001). Thus, the chance of transposition of any fragment of mtDNA to nuclear noncoding region should be much higher than that to the coding nuclear regions, unless there is a preference to transpose to the coding genes. However, it has been shown that there is no correlation between the fraction of noncoding DNA in genome and numt abundance (Richly and Leister 2004). As the current study used the Release 1 gene list, it was not possible to determine the accurate proportion of coding and noncoding regions of honeybee chromosomes. However, as there is no significant correlation of chromosome size and numt content in honeybee, it is unlikely that these sequences are contributing factors for determining the physical length of chromosomes.
Changes in the order of major genes in the honeybee mitochondria compared with that in other insects have been observed (Vlasak et al. 1987; Crozier et al. 1989; Boore 1999). Within A. mellifera, loss of DNA from specific regions of mtDNA such as the COI to COII region has been reported (Crozier et al. 1989). Moreover, in the current study, the COI gene showed the maximum amount of DNA transfer to nucleus. The transfer of a gene from mitochondria to nucleus is believed to develop a dynamic intracellular competition most probably because a smaller mitochondrion can replicate more efficiently than a larger one (Albert et al. 1996). Thus, loss of DNA from the mitochondria, as seen in the honeybee, can influence the rate of gene flow from mitochondria to nucleus. This suggests that the COI region probably migrated to the nucleus faster rate than other regions of the honeybee mtDNA. Mathematical models suggest that apart from the intensity of intracellular competition, rate of numt transfer also depends on the probability of paternal organelle transmission and the effective population size of the species (Yamauchi 2005). Each honeybee colony has one reproductive female, and all the haploid males represent her gametes. Although the effective population size of the honeybee is not known, it has been estimated that the pool of males available to mate with honeybee queens is on the order of 200, and the population is likely to be spatially heterogeneous (Baudry et al. 1998). Moreover, occasional paternal inheritance of mtDNA has also been observed in honeybee (Meusel and Moritz 1993). These factors are expected to affect the overall rate of transfer of genes from mitochondria to nucleus in honeybee.
The discovery of numts in the present study can also be used as a resource to develop numt-based nuclear markers for population studies of honeybee. Numts have been recognized to have potential utility as suitable genetic markers (Bensasson et al. 2001). With the genome sequence of honeybee now in hand, it is possible to use flanking sequences of numts to design site-specific PCR primers that would specifically amplify the required numts. Because of high abundance, numts can be used as genetic markers for high-throughput genotyping/sequencing from different individuals or populations. Comparative analysis of numts in honeybee can be used to understand the evolution of these sequences within and between closely related species and subspecies even when the mtDNA from which they originated became extinct. This is due to the fact that even if the mtDNA lineage to which the most recent ancestor belongs becomes extinct, the nuclear lineage to which the DNA was transferred may still be inherited by present-day individuals (Bensasson et al. 2001). Hence, data from numts may be useful for understanding the ancestral history and speciation the way mtDNA variations are useful (Wakeley and Hey 1997). Moreover, generation of more genomic sequences from African honeybee are helpful in this regard to conduct comparative numt studies between European and African bees and also to study the Africanization of native bees.
|
Supplementary Material |
|---|
|
TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
|---|
Supplementary tables 1–6 and figures 1–5 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
|
Acknowledgements |
|---|
|
TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
|---|
I am thankful to Drs Dan Graur and Greg Hunt for reading the manuscript and for providing useful suggestions. I also greatly appreciate "BeeBase," the official honeybee genome database for the resources.
|
Footnotes |
|---|
William Martin, Associate Editor
|
References |
|---|
|
TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
|---|
Albert B, Godelle B, Atlan A, Paepe RD, Gouyon PH. Dynamics of plant mitochondrial genome: model of a three-level selection process. Genetics (1996) 144:369–382.[Abstract]
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol (1990) 215:403–410.[CrossRef][Web of Science][Medline]
Arctander P. Comparison of a mitochondrial gene and a corresponding nuclear pseudogene. Proc R Soc B (1995) 262:13–29.[Medline]
Arias MC, Sheppard WS. Molecular phylogenetics of honey bee subspecies (Apis mellifera L.) inferred from mitochondrial DNA sequence. Mol Phylogenet Evol (1996) 5:557–566.[CrossRef][Web of Science][Medline]
Arias MC, Sheppard WS. Phylogenetic relationships of honey bees (Hymenoptera:Apinae:Apini) inferred from nuclear and mitochondrial DNA sequence data. Mol Phylogenet Evol (2005) 37:25–35.[CrossRef][Web of Science][Medline]
Baudry E, Solignac M, Garnery L, Gries M, Cornuet J-M, Koeniger N. Relatedness among honeybees (Apis mellifera) of a drone congregation. Proc R Soc Lond B Biol Sci (1998) 265:2009–2014.
Behura SK. Molecular marker systems in insects: current trends and future avenues. Mol Ecol (2006) 15:3087–3113.[CrossRef][Medline]
Benne R, Sloof P. Evolution of the mitochondrial protein synthetic machinery. Biosystems (1987) 21:51–68.[CrossRef][Web of Science][Medline]
Bensasson D, Feldman MW, Petrov DA. Rates of DNA duplication and mitochondrial DNA insertion in the human genome. J Mol Evol (2003) 57:343–354.[CrossRef][Web of Science][Medline]
Bensasson D, Zhang D, Hartl DL, Hewitt GM. Mitochondrial pseudogenes: evolution's misplaced witnesses. Trends Ecol Evol (2001) 16:314–321.[CrossRef][Medline]
Bensasson D, Zhang DX, Hewitt GM. Frequent assimilation of mitochondrial DNA by grasshopper nuclear genomes. Mol Biol Evol (2000) 17:406–415.
Beye M, Gattermeier I, Hasselmann M. Exceptionally high levels of recombination across the honey bee genome. Genome Res (2006) 16:1339–1344. (15 co-authors).
Birney E, Clamp M, Durbin R. Genewise and Genomewise. Genome Res (2004) 14:988–995.
Blanchard JL, Lynch M. Organellar genes: why do they end up in the nucleus? Trends Genet (2000) 16:315–320.[CrossRef][Web of Science][Medline]
Boore JL. Animal mitochondrial genomes. Nucleic Acids Res (1999) 27:1767–1780.
Brennicke A, Grohmann L, Hiesel R, Knoop V, Schuster W. The mitochondrial genome on its way to nucleus: different stage of gene transfer in higher plants. FEBS Lett (1993) 325:140–145.[CrossRef][Web of Science][Medline]
Brower AVZ. Rapid morphological radiation and convergence among races of the butterfly Heliconius erato inferred from patterns of mitochondrial-DNA evolution. Proc Natl Acad Sci USA (1994) 91:6491–6495.
Cameron SA. Multiple origins of advanced eusociality in bees inferred from mitochondrial DNA sequences. Proc Natl Acad Sci USA (1993) 90:8687–8691.
Chen J, Rattner A, Nathans J. Effects of L1 retrotransposon insertion on transcript processing, localization and accumulation: lessons from the retinal degeneration 7 mouse and implications for the genomic ecology of L1 elements. Hum Mol Genet (2006) 15:2146–2156.
Clarke KE, Oldroyd BP, Javier J, Quezada-Euan G, Rinderer TE. Origin of honeybees (Apis mellifera L.) from the Yucatan peninsula inferred from mitochondrial DNA analysis. Mol Ecol (2001) 10:1347–1355.[CrossRef][Medline]
Collet T, Ferreira KM, Arias MC, Soares AE, Del Lama MA. Genetic structure of Africanized honeybee populations (Apis mellifera L.) from Brazil and Uruguay viewed through mitochondrial DNA COI-COII patterns. Heredity (2006) 97:329–335.[CrossRef][Web of Science][Medline]
Crozier RH, Crozier YC. The mitochondrial genome of the honeybee Apis mellifera: complete sequence and genome organization. Genetics (1993) 133:97–117.[Abstract]
Crozier RH, Crozier YC, Mackinlay AG. The CO-I and CO-II region of honeybee mitochondrial DNA: evidence for variation in insect mitochondrial evolutionary rates. Mol Biol Evol (1989) 6:399–411.[Abstract]
DeWoody JA, Chesser RK, Baker RJ. A translocated mitochondrial cytochrome b pseudogene in voles (Rodentia: microtus). J Mol Evol (1999) 48:380–382.[CrossRef][Web of Science][Medline]
Engel MS, Schultz TR. Phylogeny and behavior in honey bees (Hymenoptera:Apidae). Ann Entomol Soc Am (1997) 90:43–53.[Web of Science]
Franck P, Garnery L, Celebrano G, Solignac M, Cornuet JM. Hybrid origins of honeybees from Italy (Apis mellifera ligustica) and Sicily (A. m. sicula). Mol Ecol (2000) 9:907–921.[CrossRef][Medline]
Franck P, Garnery L, Loiseau A, Oldroyd BP, Hepburn HR, Solignac M, Cornuet JM. Genetic diversity of the honeybee in Africa: microsatellite and mitochondrial data. Heredity (2001) 86:420–430.[CrossRef][Web of Science][Medline]
Garnery L, Cornuet JM, Solignac M. Evolutionary history of the honey bee Apis mellifera inferred from mitochondrial DNA analysis. Mol Ecol (1992) 1:145–154.[Medline]
Gray MW. Origin and evolution of mitochondrial DNA. Annu Rev Cell Biol (1989) 5:25–50.[CrossRef][Web of Science][Medline]
Gray MW. The endosymbiont hypothesis revisited. Int Rev Cytol (1992) 141:233–357.[Web of Science][Medline]
Gregory TR. Coincidence, coevolution, or causation? DNA content, cell size and the C-value enigma. Biol Rev (2001) 76:65–101.[Medline]
Hall HG, Muralidharan K. Evidence from mitochondrial DNA that African honey bees spread as continuous maternal lineages. Nature (1989) 339:211–213.[CrossRef][Medline]
Hasegawa M, Kishino H, Yano T. Dating of the human–ape splitting by a molecular clock of mitochondrial DNA. J Mol Evol (1985) 22:160–174.[CrossRef][Web of Science][Medline]
Hazkani-Covo E, Graur D. A comparative analysis of numt evolution in human and chimpanzee. Mol Biol Evol (2007) 24:13–28.
Hazkani-Covo E, Sorek R, Graur D. Evolutionary dynamics of large numts in the human genome: rarity of independent insertions and abundance of post-insertion duplications. J Mol Evol (2003) 56:169–174.[CrossRef][Web of Science][Medline]
[HGSC] Honeybee Genome Sequencing Consortium. Insights into social insects from the genome of the honeybee Apis mellifera. Nature (2006) 443:931–949.[CrossRef][Medline]
Hunt GJ, Page RE Jr. Linkage map of the honey bee, Apis mellifera, based on RAPD markers. Genetics (1995) 139:1371–1382.[Abstract]
Jensen AB, Palmer KA, Boomsma JJ, Pedersen BV. Varying degrees of Apis mellifera ligustica introgression in protected populations of the black honeybee, Apis mellifera mellifera, in northwest Europe. Mol Ecol (2005) 14:93–106.[CrossRef][Medline]
Kaplan N, Linial M. ProtoBee: hierarchical classification and annotation of the honey bee proteome. Genome Res (2006) 16:1431–1438.
Koonin EV. The origin of introns and their role in eukaryogenesis: a compromise solution to the introns-early versus introns-late debate? Biol Direct (2006) doi:10.1186/1745-6150-1-22.
Kumar S, Tamura K, Nei M. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform (2004) 5:150–163.
Li WH. Molecular evolution (1997) Sunderland (MA): Sinauer Associates.
Lopez JV, Yuhki N, Masuda R, Modi W, O'Brien SJO. Numt, a recent transfer and tandem amplification of mitochondrial DNA to the nuclear genome of the domestic cat. J Mol Evol (1994) 39:174–190.[Web of Science][Medline]
Lu XM, Fu YX, Zhang YP. Evolution of mitochondrial cytochrome b pseudogene in genus Nycticelus. Mol Biol Evol (2002) 19:2337–2341.
Meusel MS, Moritz RF. Transfer of paternal mitochondrial DNA during fertilization of honeybee (Apis mellifera L.) eggs. Curr Genet (1993) 24:539–543.[CrossRef][Web of Science][Medline]
Morse RA, Calderone NW. The value of honey bees as pollinators of US crops in 2000 [Internet]. [cited 2007 Aug 1]. Bee Cult (2000) Available from:http://www.masterbeekeeper.org/pdf/pollination.pdf.
Nei M, Gojobori T. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol Biol Evol (1986) 3:418–426.[Abstract]
Noutsos C, Richly E, Leister D. Generation and evolutionary fate of insertions of organelle DNA in the nuclear genomes of flowering plants. Genome Res (2005) 15:616–628.
Olson LE, Yoder AD. Using secondary structure to identify ribosomal numts: cautionary examples from the human genome. Mol Biol Evol (2002) 19:93–100.
Pereira SL, Baker AJ. Low number of mitochondrial pseudogenes in the chicken (Gallus gallus) nuclear genome: implications for molecular inference of population history and phylogenetics. BMC Evol Biol (2004) 4:17.[CrossRef][Medline]
Pereira V. Insertion bias and purifying selection of retrotransposons in the Arabidopsis thaliana genome. Genome Biol (2004) 5:R79.[CrossRef][Medline]
Pinto MA, Rubink WL, Coulson RN, Patton JC, Johnston JS. Temporal pattern of Africanization in a feral honeybee population from Texas inferred from mitochondrial DNA. Evolution Int J Org Evolution (2004) 58:1047–1055.[CrossRef][Web of Science][Medline]
Pinto MA, Rubink WL, Patton JC, Coulson RN, Johnston JS. Africanization in the United States: replacement of feral European honeybees (Apis mellifera L.) by an African hybrid swarm. Genetics (2005) 170:1653–1665.
Pons J, Vogler AP. Complex pattern of coalescence and fast evolution of a mitochondrial rRNA pseudogene in a recent radiation of tiger beetles. Mol Biol Evol (2005) 22:991–1000.
Ramakrishna W, Dubcovsky J, Park YJ, Busso C, Emberton J, SanMiguel P, Bennetzen JL. Different types and rates of genome evolution detected by comparative sequence analysis of orthologous segments from four cereal genomes. Genetics (2002) 162:1389–1400.
Ricchetti M, Tekaia F, Dujon B. Continued colonization of the human genome by mitochondrial DNA. PLoS Biol (2004) 2:E273.[CrossRef][Medline]
Richly E, Leister D. NUMTs in sequenced eukaryotic genomes. Mol Biol Evol (2004) 21:1081–1084.
Rozas J, Sanchez-DelBarrio JC, Messeguer X, Rozas R. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics (2003) 19:2496–2497.
Schneider SS, DeGrandi-Hoffman G, Smith DR. The African honey bee: factors contributing to a successful biological invasion. Annu Rev Entomol (2004) 49:351–376.[CrossRef][Web of Science][Medline]
Selosse M, Albert B, Godelle B. Reducing the genome size of organelles favours gene transfer to the nucleus. Trends Ecol Evol (2001) 16:135–141.[CrossRef][Medline]
Smith DR, Brown WM. Polymorphisms in mitochondrial DNA of European and Africanized honeybees (Apis mellifera). Experientia (1988) 44:257–260.[CrossRef][Web of Science][Medline]
Smith DR, Taylor OR, Brown WM. Neotropical Africanized honey bees have African mitochondrial DNA. Nature (1989) 339:213–215.[CrossRef][Medline]
Sunnucks P, Hales DF. Numerous transposed sequences of mitochondrial cytochrome oxidase I–II in aphids of the genus Sitobion (Hemiptera: aphididae). Mol Biol Evol (1996) 13:510–524.[Abstract]
Thorsness PE, Weber ER. Escape and migration of nucleic acids between chloroplasts, mitochondria and the nucleus. Int Rev Cytol (1996) 165:207–234.[Web of Science][Medline]
Timmis JN, Ayliffe MA, Huang CY, Martin W. Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes. Nat Rev Genet (2004) 5:123–135.[CrossRef][Web of Science][Medline]
Tomkins JP, Luo M, Fang GC, et al, (12 co-authors). New genomic resources for the honey bee (Apis mellifera L.): development of a deep-coverage BAC library and a preliminary STC database. Genet Mol Res (2002) 1:306–316.[Medline]
Vlasak I, Burgschwaiger S, Kreil G. Nucleotide sequence of the large ribosomal RNA of honeybee mitochondria. Nucleic Acids Res (1987) 15:2388.
Wakeley J, Hey J. Estimating ancestral population parameters. Genetics (1997) 145:847–855.[Abstract]
Whitfield CW, Behura SK, Berlocher SH, Clark AG, Johnston JS, Sheppard WS, Smith DR, Suarez AV, Weaver D, Tsutsui ND. Thrice out of Africa: ancient and recent expansions of the honey bee, Apis mellifera. Science (2006) 314:642–645.
Woischnik M, Moraes CT. Pattern of organisation of human mitochondrial pseudogenes in the nuclear genome. Genome Res (2002) 12:885–893.
Wolfe KH, Li WH, Sharp PM. Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast and nuclear DNAs. Proc Natl Acad Sci USA (1987) 84:9054–9058.
Yamauchi A. Rate of gene transfer from mitochondria to nucleus: effects of cytoplasmic inheritance system and intensity of intracellular competition. Genetics (2005) 171:1387–1396.
Zhang DX, Hewitt GM. Nuclear integrations: challenges for mitochondrial DNA markers. Trends Ecol Evol (1996) 11:247–251.[CrossRef]
Zischler H, Geisert H, von Haeseler A, Paabo S. A nuclear ‘fossil’ of the mitochondrial D-loop and the origin of modern humans. Nature (1995) 378:489–492.[CrossRef][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  O. Deusch, G. Landan, M. Roettger, N. Gruenheit, K. V. Kowallik, J. F. Allen, W. Martin, and T. Dagan Genes of Cyanobacterial Origin in Plant Nuclear Genomes Point to a Heterocyst-Forming Plastid Ancestor Mol. Biol. Evol., April 1, 2008; 25(4): 748 - 761. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||