Restriction site heteroplasmy in the mitochondrial DNA of Brycon opalinus ( Cuvier , 1819 ) ( Characiformes , Characidae , Bryconiae )

Homoplasmy is a feature usually found in the mtDNA of higher animal taxa. On the other hand, the presence of two classes of mtDNA in the same cell or organism is rare and may appear in length or site variation. Data from mtDNA RFLP analysis of Brycon opalinus populations (Cuvier, 1819; Characiformes, Characidae, Bryconinae) revealed site heteroplasmy from endonuclease NheI digestion. Southern blotting hybridization was used to survey a total of 257 specimens with 24 restriction enzymes. Three different restriction fragment patterns of mtDNA were obtained from NheI digestion. Two individuals from hatchery broodstock were found to have two of them. NheI digests of heteroplasmic individuals yielded two fragments of approximately 1180 and 1260 bp. Despite the low frequency of this type of heteroplasmy in the whole B. opalinus population, the presence of site heteroplasmy in this species supports the evidence of this phenomenon in lower vertebrate groups. Correspondence


Introduction
Mitochondrial DNA (mtDNA) has been a useful tool for population and evolution studies (1).Although attention has been focused on inter-individual variation, the nature and extent of intra-individual variation, or heteroplasmy of the mtDNA, cannot be ignored (2).
Homoplasmy is a feature usually found in the mtDNA of higher animal taxa (3).mtDNA polymorphism used in phylogeography and phylogeny analysis of related species is based on losses and gains of restric-tion sites by means of base substitution or insertion or deletion of a few base pairs (4).However, this polymorphism can also occur in the same organism due to mtDNA sequence differences.This phenomenon, known as heteroplasmy, consists of length or site variation, i.e., the presence of different mtDNA lengths or different nucleotide orders, respectively, in the same cell.
While site heteroplasmy is rare (5), length heteroplasmy is frequent in natural populations.Heteroplasmy seems to be related to length variants (6) and is commonly found in natural populations of fish, such as, shortnose sturgeon (7), sturgeon (8), percid fish species (9), brook stickleback (10), Atlantic cod (11), Walleye (12), and striped bass (13).Length heteroplasmy may arise from mechanisms of replication slippage, duplication or deletion and is frequently found in tandem repeated structures in the D-loop region (14).
Brycon is a genus comprising more than 60 neotropical species (15) distributed in Central and South America.We have previously analyzed mtDNA variability in natural and captive populations of Brycon opalinus (16).Data from RFLP analysis of B. opalinus populations demonstrated an unusual pattern of site heteroplasmy in samples from captive broodstock and from two rivers.Thus, in the present study we describe and report the frequency of mtDNA site heteroplasmy in populations of B. opalinus.Total DNA was extracted (17) from each individual and was screened by 24 restriction enzymes.The DNA fragments produced by the endonucleases were immobilized by Southern transfer (18) and the mtDNA frag-ments were detected by hybridization with [P 32 ]-labeled homologous probes produced in our laboratory from B. opalinus mtDNA.

Results and Discussion
The presence of two mtDNAs of different lengths is common and may be the result of polymerase slippage in the repeat units during mtDNA replication.Intra-individual mtDNA differences at a single base are usually thought to be found in human diseases associated with mtDNA mutations (19).
Petri et al. (20) showed an extensive sequence heteroplasmy in the mtDNA control region from a European bat species whose mtDNA sequence variation within an individual was as high as that detected in the entire population.Intra-individual sequence variation has been observed in normal humans (21,22) and in other mammals (23).It seems that site heteroplasmy may be a principle, rather than an exception (24).
Few cases of restriction site heteroplasmy have been observed in fish.Bentzen et al. (25), using restriction enzyme analysis, assessed mtDNA variation in samples of American shad (Alosa sapidissima) captured in 14 rivers in Canada.The survey of the shad populations showed site heteroplasmy in ten individuals from different rivers, which indicated that this form of heteroplasmy might have originated more than once.Gold and Richardson (26) also produced evidence of site heteroplasmy in the marine species Sciaenops ocellatus.In the cited study one of 750 individuals showed site heteroplasmy involving the NcoI and XbaI restriction enzymes.Brzuzan and Ciesielski (27), working on formalin-preserved individual of the species Coregonus albula from an extinct population, found nucleotide substitutions and site heteroplasmy compared to existing populations.
RFLP analysis of B. opalinus showed that six of 24 restriction enzymes were informative (ApaI, AvaII, EcoRI, HincII, HpaI and NheI) (16).The restriction enzyme NheI digestion revealed three different fragment patterns, NheI-A, NheI-B, and NheI-C, as a result of restriction site losses and gains.Pattern A originated pattern B by losing F 4 and B originated C by losing F 5 .Thus, pattern C was possibly produced from pattern A by losing F 4 and F 5 (Figure 1).
Two of 80 samples of B. opalinus broodstock from the hydroelectric power plant hatchery showed a different pattern besides NheI-A, -B and -C.The two samples mentioned above showed both the NheI-A of 1180 bp and NheI-B of 1260 bp in the same individual (Figure 2).This new pattern was initially misinterpreted as a consequence of incomplete digestion by the NheI enzyme since there is no evolutionary explanation for the appearance of this fragment pattern and the sum of the bands exceeds the total mtDNA length of approximately 16,300 (± 500) bp in B. opalinus species.In order to determine if the appearance of fragments NheI-A and NheI-B in the same individual was a result of incomplete digestion, the samples were digested five times with larger quantities of the NheI restriction enzyme and the incubation time was prolonged.Even under these experimental conditions the resulting fragments continued to be the same, with the intensity of both bands remaining unchanged.In the case of incomplete digestion, a difference in intensity between the two fragments would be expected.
The site heteroplasmy observed in individuals of B. opalinus might have originated from a germ line cell through an independent mutational event followed by an incomplete segregation of the two fragments produced by the NheI endonuclease.Another possibility is the contribution of paternal mtDNA, as observed in mussels (28) and mammals (29).This, however, may not be the case since the paternal mtDNA contribution is rarely fixed in the mtDNA present in an organism (30).According to Birky et al. (31), heteroplasmy is commonly found in germ line cells, and arises in somatic cells when more than one type of mtDNA molecule remains after embryonic development.The site heteroplasmy observed in the B. opalinus fish species, although restricted to a captive population, is further evidence of the occurrence of this phenomenon in lower vertebrate group.

A
total of 257 specimens were sampled between 1997 and 1998 in the Paraíba do Sul basin located between the north of São Paulo State and the south of Rio de Janeiro State in southeastern Brazil.Wild populations were sampled from seven tributary rivers in the Paraibuna do Sul basin.A sampling of 80 individuals was taken from the broodstock of the Paraibuna Hydroelectric Power Company (CESP) Hatchery in the State of São Paulo.

Figure 1 .
Figure 1.Three mutational patterns revealed by the endonuclease NheI showing restriction site gains or losses.The pattern A generates B by loosing F 4 restriction site and B generates C by loosing F 5 restriction site.

Figure 2 .
Figure 2. Restriction site heteroplasmy in Brycon opalinus mtDNA.Restriction fragment patterns obtained by Southern blotting from NheI digestion.Lane 1 is the λ HindIII DNA marker.Lanes 2, 3 and 4 are the different fragment pattern of NheI digestion.Individuals in lanes 5 and 6 are heteroplasmic for NheI showing two classes (2 and 3) in the same individual.