Genetics, Vol. 171, 1951-1962, December 2005, Copyright © 2005
doi:10.1534/genetics.105.042770

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Mitochondrial Genome Recombination in the Zone of Contact Between Two Hybridizing Conifers

Chaire de Recherche du Canada en Génomique Forestière et Environnementale and Centre de Recherche en Biologie Forestière, Université Laval, Sainte-Foy, Quebec G1K 7P4, Canada

¹ Corresponding author: Chaire de Recherche du Canada en Génomique Forestière et Environnementale and Centre de Recherche en Biologie Forestière, Pavillon C.-E. Marchand, Université Laval, Sainte-Foy, Quebec G1K 7P4, Canada.
E-mail: bousquet{at}rsvs.ulaval.ca

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
Variation in mitochondrial DNA was surveyed at four gene loci in and around the zone of contact between two naturally hybridizing conifers, black spruce (Picea mariana) and red spruce (P. rubens) in northeastern North America. Most of the mtDNA diversity of these species was found in populations next to or into the zone of contact, where some individuals bore rare mitotypes intermediate between the common mitotypes observed in the allopatric areas of each species. Sequence analysis and tests for mtDNA recombination point to this phenomenon, rather than to recurrent mutation, as the most tenable hypothesis for the origin of these rare mitotypes. From the 10 mitotypes observed, at least 4 would be the product of recombination between 4 of the 5 putative ancestral mitotypes. Tests for cytonuclear disequilibrium and geographical structure of the putative recombinant mitotypes suggest that mtDNA recombination is not frequent and relatively recent on the geological time scale. mtDNA recombination would have been promoted by transient heteroplasmy due to leakage of paternal mtDNA since the Holocene secondary contact between the two species.

Interactions between co-occurring genomes following occasional leakages of paternal mitochondrial DNA (mtDNA) is the most common explanation for the origin of putative recombinant mitotypes (LUNT and HYMAN 1997; LADOUKAKIS and ZOUROS 2001; STÄDLER and DELPH 2002). The production of new mitotypes through recombination of coexisting mtDNA genomes has been observed in animal species in which the leakage of paternal mtDNA is common (BUROKER et al. 1990; LUNT and HYMAN 1997; LADOUKAKIS and ZOUROS 2001; HOARAU et al. 2002) and in artificially induced heteroplasmic plants and fungi (i.e., in somatic hybrids; see XU et al. 1993; LANDGREN and GLIMELIUS 1994; LASER et al. 1997; TOTH et al. 1998 among others). However, despite the increasing number of studies on mitochondrial genome recombination with artificially induced heteroplasmic individuals and somatic hybrids, few examples of the origin of new mitotypes through homologous recombination have been reported in natural populations (but see SAVILLE et al. 1998; STÄDLER and DELPH 2002).

It has been hypothesized that recurrent hybridization events, such as those observed in the zones of contact between closely related species, could provide the opportunity for heteroplasmy and recombination to occur (ROKAS et al. 2003). In hybridizing species, the reproductive barriers are not completely developed, and in some cases, the mechanisms inducing the elimination of male mitochondria could have broken down, leading to the leakage of paternal mitochondria and to transient heteroplasmy (e.g., WAGNER et al. 1991). In plants in general, and in conifers in particular, natural interspecific hybridization is common. Thus, a survey of plant hybrid zones could provide some meaningful examples of mitochondrial DNA recombination occurring in natural populations.

Black spruce (Picea mariana [Mill.] B.S.P.) and red spruce (Picea rubens Sarg.) are two conifer species that interbreed naturally, forming viable hybrids and introgressants in a large region of northeastern North America, notably along the St. Lawrence Valley and in the Maritime provinces of Canada (MANLEY 1972; PERRON and BOUSQUET 1997). These species exhibit a high degree of similarity at the morphological and genetic levels (GORDON 1976; PERRON et al. 1995, 2000; WENG and JACKSON 2000) and presumably represent a progenitor-derivative species pair (PERRON et al. 2000). Thus, these two species should be a suitable hybridizing species pair for detecting mitochondrial genome recombination in natural populations. Black spruce and red spruce do not hybridize with the unique other spruce taxon present in this part of the continent, the sympatric but phylogenetically divergent white spruce (Picea glauca [Moench] Voss) (WRIGHT 1955; GORDON 1976; SIGURGEIRSSON and SZMIDT 1993).

While surveying for mitochondrial diversity within and among natural populations of black spruce across its range, an unexpectedly highly diverse mtDNA multilocus haplotype structure was observed in and around the zone of contact with red spruce in Quebec (JARAMILLO-CORREA et al. 2004). This diversity was much higher than the additive variation detected in zones of suture between genetically different glacial populations. In this study, we have surveyed intensively the mtDNA diversity in part of the zone of contact between black spruce and red spruce. The mitotypes were analyzed at the DNA sequence level to find out if there was any evidence for the involvement of recombination in generating the abundant mtDNA diversity previously observed in this area.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
Population sampling:
Needles from 10 to 50 trees were collected in 41 populations covering a vast zone ranging from the Canadian boreal forest to the mixed-wood montane forest in the eastern United States. The sampled stands included 5 allopatric populations of red spruce and 16 allopatric populations of black spruce for which mtDNA haplotypes and sequences had been previously reported (JARAMILLO-CORREA and BOUSQUET 2003; JARAMILLO-CORREA et al. 2004), and 20 newly sampled populations from the zone of contact between them. These 20 populations differed in the relative abundances of each species (Figure 1; see also Table S1 at http://www.genetics.org/supplemental/). Although difficult to delineate precisely, the zone of contact between black spruce and red spruce could be approximated by the overlap of the natural ranges delineated in BLUM (1990) and VIERECK and JOHNSTON (1990) for red spruce and black spruce, respectively. Additional details such as the location, composition, and the number of trees sampled in each stand are given in Table S1.

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   FIGURE 1.— Minimum-spanning tree showing relationships among the mitotypes found in allopatric populations of black spruce and red spruce and in populations from their zone of contact. Mitotypes are indicated as circles whose sizes are proportional to their relative frequency in the study. Within the circles are the proportions of each mitotype found in each particular zone. The multilocus genotype defining each mitotype is indicated following the nomenclature of JARAMILLO-CORREA et al. (2004). Arrows point to the presumed recombinant mitotypes (see text for details). Dashed lines indicate alternative links creating ambiguities in the topology inferred from a strictly clonal model (represented by the solid lines). Short solid lines indicate the number of presumed mutational steps between the mitotypes observed in our sample.

Numerical analysis:
Given that the mtDNA sequences contained just a few indels and no substitutions, they could be easily aligned visually using BioEdit (HALL 1999). Single-locus sequences were assembled in mitotypes and evolutionary relationships among them were determined with a minimum-spanning tree generated with the program TCS (CLEMENT et al. 2000). The presence of loops and alternative links within a minimum-spanning tree can be produced by recombination and/or recurrent mutation (TEMPLETON and SING 1993). To determine whether the uncertainties observed in the spanning tree could be accounted for by recombination, we examined our data in three different ways. First, we applied the four-gametic criterion defined in HUDSON and KAPLAN (1985), implying that recombination within a segment flanked by two polymorphic loci can be inferred when all four possible combinations of alleles are found. For every pair of polymorphic sites, we looked for the presence of all possible combinations of "alleles" (i.e., AA, AB, BA, and BB) in individuals bearing different mitotypes within the same population. Second, we calculated the number of mitotypes expected under a model of clonality (no recombination) and under a model of random assortment (free recombination) following the methods of MAYNARD SMITH and SMITH (1998). This method estimates the probability of obtaining the observed number of haplotypes (mitotypes) in the data set under a given mutation model. It is based on the fact that if there is no recombination and no recurrent mutation, no homoplasies would be detected in the most parsimonious tree derived from the data set (MAYNARD SMITH and SMITH 1998). We initially determined the minimal number of mutational changes necessary to generate all the polymorphisms observed in all mitotypes. We then estimated the number of effective sites by using white spruce sequences (P. glauca [Moench] Voss) as outgroup. For the clonal model, we started from the putative ancestral mitotype and assembled new mitotypes by simulating the mutational changes above at sites chosen randomly with replacement. For the free recombination model, we randomly assembled mitotypes by choosing with replacement among the variable positions detected in the sequence alignments. The number of homoplasies observed in the most parsimonious tree derived from the original data set was finally compared to the mean number of homoplasies observed when using the simulated data sets. The distribution of data under both null hypotheses was determined by 1000 Monte Carlo iterations. The third method used was the phylogenetic test of linkage disequilibrium described by BURT et al. (1996). This approach aimed to quantify the levels of phylogenetic homoplasy relative to the expected rates under a model of complete linkage and under a model of free recombination between markers. In this test, the length of the phylogenetic tree generated from the original data set was compared to the length of the trees expected under each model.

Given that most of the mitotypes forming the loops and alternative links in the minimum-spanning tree were mostly located in populations in or near the zone of contact between black spruce and red spruce (see RESULTS), we decided to test whether there was a significant association between those putative recombinant mitotypes and hybrid or introgressant nuclear genotypes in this particular region. To do so, we estimated the normalized cytonuclear disequilibrium value, D* (ASMUSSEN and BASTEN 1994, 1996), using the computer program kindly provided by C. J. Basten (North Carolina State University). Diagnostic randomly amplified polymorphic DNA (RAPD) and expressed sequence tag polymorphism (ESTP) markers between black spruce and red spruce (PERRON et al. 1995, 2000; PERRY and BOUSQUET 1998) were used to determine the nuclear background of all sampled individuals. Trees were classified as black spruce, red spruce, or hybrids or introgressants on the basis of the multilocus genotypes derived from these markers (see PERRON et al. 1995, 2000 for more details). Given that both black spruce and red spruce are anemophilous species characterized by outcrossing mating systems, each with moderate-to-low levels of population structure (e.g., RAJORA et al. 2000; PERRY and BOUSQUET 2001), we pooled and considered all the trees sampled in and around the zone of contact as a single population. To simplify calculations of cytonuclear disequilibrium, a two-locus linkage test was conducted by considering the three above-mentioned classes of nuclear multilocus genotypes and two classes of mitotypes: putative ancestral and putative recombinant mitotypes (see RESULTS for more details).

To test whether the geographic distribution of each mitotype was clumped or followed a random pattern in the populations in and around the zone of contact, we scored the number of trees bearing each particular mitotype in each population and then estimated the variance-to-mean ratio (ID) and the Green's index (GI), following the methods of LUDWIG and REYNOLDS (1988) and KREBS (1999). These indices were used because some of the mitotypes observed were too rare to perform a formal spatial autocorrelation analysis for each of them (see RESULTS). These tests are based on the assumption that if a particular mitotype is randomly dispersed among populations, as could be determined by a Poisson distribution (LUDWIG and REYNOLDS 1988), one would expect that the mean number of individuals bearing this particular mitotype in each population would equal the variance among populations (thus ID = 1 and GI = 0; KREBS 1999).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
Mitochondrial DNA diversity and tests of recombination:
All four mtDNA loci were polymorphic and displayed a total of 10 different indels. These indels varied between 5 and 20 bp and were distributed among the four mitochondrial regions surveyed as shown in Table 1. The remaining positions of these genes were monomorphic. No substitutions were detected among the 120 individuals sequenced for the four mtDNA loci investigated. The combination of the polymorphisms detected revealed the same 10 mitotypes previously reported in black spruce (JARAMILLO-CORREA et al. 2004). Sequencing of individuals bearing the same mitotype in different populations did not reveal any homoplasy of DNA fragment length. Four of the observed polymorphisms were exclusive to two mitotypes: one insertion at the locus nad1 intron b/c and two insertions at the locus nad5 intron 1 were exclusive to mitotype VIII, and a second repeat of a GC-rich motif at the locus nad7 intron 1 was exclusive to mitotype X (Table 1).

The homoplasy test (MAYNARD SMITH and SMITH 1998) revealed that only 6 mitotypes were expected under a strict clonal model in which mutation was the sole source of variation. No more than 2 of these 6 mitotypes (a mean value of 0.91 calculated from 1000 Monte Carlo iterations) could be produced by recurrent mutation events (Table 2). On the other hand, a maximum of 54 mitotypes (mean value of 48.2) would be expected under a free recombination model, but only 7 of them would be the product of recurrent mutations. When we repeated the calculations considering that the loci nad1 intron b/c and nad5 intron 1 were always linked, as indicated by our data (Table 1), a maximum of 20 mitotypes (mean value of 16.4) would be expected (Table 2). About 6 of these mitotypes would be the product of recurrent mutations (Table 2).

Distribution of mitotype diversity and association with the zone of contact:
Among the 10 mitotypes observed in this study, only 3 (I, IV, and VIII) were observed in allopatric populations of black spruce located far away from the zone of contact with red spruce (Figure 2; Table 3; Table S1 at http://www.genetics.org/supplemental/). Only one mitotype (mitotype IV) was predominant in allopatric populations of red spruce, while the remaining 7 mitotypes (II, III, V, VI, VII, IX, and X) were rare and exclusively observed in populations in and around the zone of contact between the two species (Figure 2; Table 3; Table S1). It must be noted that 2 of the most abundant mitotypes, I and IV, were also predominant in this region. The high and singular mitotype diversity observed in this zone suggests that the putative recombination phenomenon should be restricted to populations in and around the zone of contact between black spruce and red spruce.

View larger version (60K): [in this window] [in a new window] [Download PPT slide]   FIGURE 2.— Distribution of mitotypes in allopatric populations of black spruce and red spruce and in populations from their zone of contact. Population numbers correspond to those in Table S1 at http://www.genetics.org/supplemental/. The shaded region represents the approximate zone of contact (following BLUM 1990 and VIERECK and JOHNSTON 1990).

To infer recombinant and nonrecombinant types, we considered the arrangement of the mitotypes on the minimum-spanning tree (Figure 1) and their relative abundance (Figure 2; Table 3; Table S1 at http://www.genetics.org/supplemental/; and relative mitotype abundances on Figure 1) and, following the assumptions of CRANDALL and TEMPLETON (1993), we determined two possible scenarios that were used to calculate the cytonuclear disequilibrium and the dispersion indices. In the scenario 1, we assumed that the ancestral mitotypes were the three more widespread forms (I, IV, and VIII) and two of the mitotypes intermediate between them, III and VII (Figure 1). Mitotype III was marginally more abundant than other intermediate mitotypes in the zone of contact, and it was also found at low frequency in some allopatric populations of red spruce and black spruce (Figures 1 and 2; Table 3), supporting the hypothesis that it is ancestral. Mitotype VII was not widely distributed in the zone of contact and was absent from most of the allopatric populations of both species (Figure 2; Table 3). However, it was intermediate between two terminal mitotypes, I and VIII, in the minimum-spanning tree (Figure 1), supporting the hypothesis that it is ancestral. It is also noteworthy that one of the two shortest paths linking the most widespread forms, I, IV, and VIII, involves mitotypes III and VII, further suggesting that they are ancestral (Figure 1). The remaining four mitotypes, II, V, VI, and IX, which were less abundant in and around the zone of contact than mitotypes III and VII (Figure 2; Table 3), would have been derived more recently and were assumed to be recombinant types. In scenario 2, we assumed that only the three most widespread mitotypes (I, IV, and VIII) were ancestral, while the remaining ones (II, III, V, VI, VII and IX) were all recombinant. In both scenarios, mitotype X was assumed to be the result of a recent mutation event and a direct derivative of mitotype IV. This mitotype was observed in a single individual and was characterized by a second repeat of a GC motif at the locus nad7 intron 1. Consequently, it was not considered in the upcoming analyses. It must be noted that all these assumptions are not intended to be absolute; they are meant only to test some hypotheses that could lead to further studies.

The cytonuclear disequilibrium test performed following the scenario 1 (mitotypes I, III, IV, VII, and VII were the ancestral types, while mitotypes II, V, VI, and IX were the recombinant types) revealed that, in the zone of contact, red spruce trees carried the putative ancestral mitotypes (mostly mitotypes III and IV) more frequently than expected, and the putative recombinant mitotypes less frequently than expected (Table 4). There were no particular trends for black spruce or for the hybrid and introgressant trees under this scenario (Table 4). For scenario 2 (mitotypes I, IV, and VIII were the ancestral types and mitotypes II, III, V, VI, VII, and IX were the recombinant types), the results of the cytonuclear disequilibrium did not reveal any particular association between the nuclear and mitochondrial backgrounds in the trees in and around the zone of contact (Table 4).

View larger version (52K): [in this window] [in a new window] [Download PPT slide]   FIGURE 3.— Distribution of the putative recombinant mitotypes in populations in and around the zone of contact between black spruce and red spruce (represented by shading). Solid dots denote the presence of a given mitotype in a given population and solid crosses denote its absence. Two indices of dispersion (ID and GI; LUDWIG and REYNOLDS 1988; KREBS 1999) are given at the top right corner of each mitotype map (see text for details). Significant departures from random dispersion: *P < 0.05; **P < 0.01; ***P < 0.001.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

The former two tests have previously been shown to be powerful for detecting recombination under conditions of low sequence divergence (MAYNARD SMITH 1999), such as observed for plant mitochondrial exons and introns (PALMER and HERBORN 1988; LAROCHE et al. 1997). However, the possible presence of mutational hot spots within the regions analyzed could increase recurrent mutations and produce new but undetected haplotypes (MARSHALL et al. 2001). Such a phenomenon would lead to an underestimation of the number of expected mitotypes. Sequence comparisons among related species have shown that polymorphisms in organellar genomes are sometimes located in mutational hot spots, such as for the chloroplast genome in grasses (Poaceae; MORTON and CLEGG 1993) and in species from the genus Clusia (HALLE et al. 2004). When comparing extensively the sequences of three of the four mtDNA regions surveyed in this study (the SSU rRNA V1 region, nad1 intron b/c, and nad5 intron 1) among different species of the genus Picea and with other conifers, the indel sites defining mitotypes in black spruce and red spruce were monomorphic in the other spruces and conifers surveyed (JARAMILLO-CORREA et al. 2003). Alternatively, the sites detected as polymorphic in different conifers for the loci nad1 intron b/c, nad5 intron 1, and nad7 intron 1 (i.e., SENJO et al. 1999; MITTON et al. 2000; SPERISEN et al. 2001; RICHARDSON et al. 2002; BURBAN and PETIT 2003; GODBOUT et al. 2005) were monomorphic in black spruce and red spruce (data not shown). Such comparisons imply that the indels observed in black spruce and red spruce are not located in mutational hot spots or hypervariable sites. The low mutation rates estimated for exons and introns of various mitochondrial genes in angiosperms (LAROCHE et al. 1997) also suggest a low occurrence of mutational hot spots in transcribed regions of the plant mitochondrial genome. Altogether, this multiple evidence suggests that recombination instead of recurrent mutation should be the driving force responsible for the high mitotype diversity observed in the zone of contact between black spruce and red spruce.

Putative causes for mtDNA recombination:
Previous studies suggested three main phenomena to explain the origin of putative recombinant mitotypes: intragenomic recombination (i.e., recombination between genomes within an organelle), the presence of sublimons, and recombination among different genomes in heteroplasmic cells (e.g., SAVILLE et al. 1998; ANDERSON et al. 2001; STÄDLER and DELPH 2002; WOLFE and RANDLE 2004). Intragenomic recombination from interactions between repeated elements has been widely reported in the plant mitochondrial genome of different species (PALMER and HERBON 1988; ALBERT et al. 1998). However, with such intragenomic recombination, one would expect the excision or duplication of regions >5000 bp long (ALBERT et al. 1998). In our data set, there were no signs of duplication or deletion of fragments of >20 bp long. Thus, even if the regions surveyed were implicated in intragenic recombination events, these phenomena alone cannot explain the recurrent occurrence of the small indels underlying the different mitotypes observed (see Table 1). In addition, such a phenomenon would not be expected to occur exclusively in the zone of contact between black spruce and red spruce, but also in other natural populations of both black spruce and red spruce.

Another process that could lead to the mitochondrial variation observed in our data set is the occurrence of subgenomic molecules at a very low stoichiometry known as "sublimons" (SMALL et al. 1987). Such sublimons might allow for the long-term coexistence of two or more copies of different genome regions within a particular cell lineage (ARRIETA-MONTIEL et al. 2001). If such sublimons are selected for in the zone of contact between black spruce and red spruce, they could increase their frequency and eventually replace the predominant mitotype without recombination. However, even if these sublimons represent the origin of the rarer mitotypes observed in this zone of contact, the question of how these sublimons had originated remains. Given the indel composition of the rarer mitotypes (see Table 1), and thus of the presumed sublimons, recombination in heteroplasmic lineages arises as the most plausible explanation.

Previous investigations of mtDNA diversity in plants and fungi have suggested recombination mediated by heteroplasmy as the most plausible explanation to account for the origin of putative recombinant mitotypes (SAVILLE et al. 1998; ANDERSON et al. 2001; STÄDLER and DELPH 2002). In plants, several studies have shown that the interaction between different coexisting mitochondrial genomes can produce new mitotypes without involving recurrent mutation. In somatic hybrids, where the mitochondria of two different taxa co-occur in the same cytoplasm, the interactions and exchanges between the coexisting genomes have resulted in new mitotypes (i.e., XU et al. 1993; LANDGREN and GLIMELIUS 1994). Similar results have been obtained with intergeneric crosses and with gynodioecious plants where heteroplasmy is common (i.e., LASER et al. 1997; MCCAULEY et al. 2005).

In hybrid zones, heteroplasmy could be promoted by the lack of incomplete reproductive isolation (ROKAS et al. 2003). The geographic distribution of mitotypes in this study suggests that some mitochondrial forms could be the product of recombination mediated by heteroplasmy in the zone of contact between black spruce and red spruce. The rare mitotypes, most of them intermediate forms between mitotypes I and IV (Table 1; Figure 1), were observed exclusively in and around the zone of contact between these two species, coinciding with a suture zone between mitotypes I and IV (Figure 1; JARAMILLO-CORREA et al. 2004). In allopatric areas occupied exclusively by black spruce, two other suture zones between the geographically structured mitotypes I, IV, and VIII were previously detected (JARAMILLO-CORREA et al. 2004). However, none of the rare mitotypes was observed in these suture zones or in any other allopatric populations of black spruce or red spruce located far away from the zone of contact between the two species (Figure 1; Table 3; Table S1 at http://www.genetics.org/supplemental/; see also JARAMILLO-CORREA et al. 2004 for central and western Canada). These distribution patterns suggest that introgressive hybridization between black spruce and red spruce is a major contributing factor to the presence of recombinant mitotypes in and around their zone of contact.

In plants, heteroplasmy seems to be a rare phenomenon, which is usually associated with paternal leakage in natural populations (FAURON et al. 1995; COYER et al. 2004; MCCAULEY et al. 2005). In conifers, some exceptional cases of paternal leakage have been detected in controlled crosses between Pinus banksiana and P. contorta (WAGNER et al. 1991) and in embryos from Pseudotsuga menziesii (OWENS and MORRIS 1991; MOGENSEN 1996). In our study, we did not detect any double-banded mtDNA phenotype indicative of heteroplasmy or biparental inheritance among the 834 adult trees analyzed. Previous studies on cytoplasmic DNA recombination (e.g., SAVILLE et al. 1998; MARSHALL et al. 2001; STÄDLER and DELPH 2002) have also invoked heteroplasmy as the driving force for cpDNA or mtDNA recombination, but they have also failed to detect heteroplasmic individuals in natural populations. These studies and our data indicate that heteroplasmy is a rare phenomenon, which would be very difficult to detect in vivo. The lack of apparent heteroplasmy in the adult trees sampled in this study could be due to the fact that heteroplasmic cells are selected out during the early stages of the plant (MOGENSEN 1996), potentially leading to homoplasmic adult individuals. Further studies on hybrid embryos and seedlings could lead to the detection of heteroplasmic individuals.

The role of hybridization:
Considering the geographic distribution of mitotypes in this study (Figure 2; Table 3; Table S1 at http://www.genetics.org/supplemental/), and the possibility that recombination is a rare and relatively recent phenomenon, two predictions were worth testing. First, if mtDNA recombination is indeed rare and is prompted by heteroplasmy due to infrequent paternal leakage during hybridization, as discussed above, we would not expect a close association between hybrid or introgressant nuclear genotypes and mtDNA recombinant types in and around the zone of contact between black spruce and red spruce. Second, we would expect a clumped spatial structure for the mtDNA recombinant types. In other words, while the geographic distribution of mitotypes suggests that mtDNA recombination is restricted to populations in and around the zone of contact between the two species (see Figure 1), a lack of association between recombinant mitotypes and hybrid or introgressant trees and a clumped distribution of recombinant types within this zone of contact would indicate how rare and recent the recombination phenomenon is.

The cytonuclear disequilibrium test showed no evidence that the hybrid or introgressant trees bore the putative recombinant mitotypes more often than expected (Table 4), suggesting that mtDNA recombination is not a frequent phenomenon. The fact that, under scenario 1, most red spruce trees did not harbor putative recombinant mitotypes (see Table 4) could further suggest that, once produced, trees bearing recombinant mitotypes are mostly pollinated by black spruce. This hypothesis needs still to be tested in the field, although evidence has been shown previously that introgressive hybridization might be asymmetrical with prevalent gene flow originating from black spruce (PERRON and BOUSQUET 1997). It is likely that the power of the cytonuclear disequilibrium test might have been diminished by pooling hybrids and introgressants with different genotypes (F₁ hybrids, F₂ hybrids, backcrosses, etc.) during the analysis. Such a procedure could "dilute" the association, if any, between nuclear hybrid genotypes and recombinant mitotypes. However, this hypothesis is likely only if mtDNA recombination mediated by heteroplasmy is a phenomenon that occurs only in F₁ hybrids. It has been previously shown that only a small proportion of the introgressants and hybrids in mixed populations from the zone of contact between black spruce and red spruce represent putative F₁ hybrids (PERRON and BOUSQUET 1997). If these F₁ hybrids undergo more or less extensive backcrossing, the cytonuclear disequilibrium between recombinant mitotypes and F₁ hybrid genotypes would decay rapidly in a matter of a few generations. However, even when restricting the cytonuclear disequilibrium test to putative F₁ hybrids, no disequilibrium was observed between hybrid nuclear genotypes and recombinant mitotypes under both tested scenarios (data not shown), thus reinforcing the idea that mtDNA recombination is not a frequent phenomenon.

The estimated dispersion indices showed that individuals bearing mitotypes II, V, VI, VII, and IX were not randomly distributed and were significantly clustered in some populations (Figure 3), while individuals bearing the most widespread and presumably ancestral mitotypes (I, III, IV, and VIII) were randomly distributed (data not shown). This spatial structure, together with the lack of cytonuclear disequilibrium and the low frequency of the putative recombinant mitotypes in populations in and around the zone of contact between the two species, suggests that mtDNA recombination is a restricted and localized phenomenon on the geographical scale and a rare phenomenon on the geological time scale. It has probably occurred only a limited number of times since the secondary contact between black spruce and red spruce during the Holocene.

The results of this study support the hypothesis of homologous recombination of the mitochondrial genome promoted by heteroplasmy and occasional leakage of paternal mtDNA in the zone of contact between these two naturally hybridizing species. Further tests with controlled crosses or somatic hybrids could help determine the exact role of hybridization between black spruce and red spruce in generating mtDNA recombination. Our data could also imply that recombination in the plant mitochondrial genome could be more common than previously suspected, as discussed for the plastid genome by WOLFE and RANDLE (2004). In addition to the present evidence from the zone of contact between black spruce and red spruce, recombination has been previously inferred in the mitochondrial genome of the gynodioecious plant S. acaulis (STÄDLER and DELPH 2002) and it has also been suspected in a population from the zone of contact between Larix gmelinii and L. sibirica in Russia (SEMERIKOV and LASCOUX 2003). As for cpDNA, apparent recombinant chlorotypes have been observed in Microseris (VIJVERBERG and BACHMANN 1999) and in Pinus contorta (MARSHALL et al. 2001). Thus, clonality is not always the rule and cytoplasmic recombination should be considered when conducting phylogenetic and phylogeographic inferences, especially when discordant topologies arise from the use of different genes or genomes (POSADA and CRANDALL 2002; WOLFE and RANDLE 2004).

	ACKNOWLEDGEMENTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
We are especially grateful to M. Perron (from Ministère des Ressources Naturelles et de la Faune de Québec) for his help in collecting some of the samples and for providing some of the original RAPD and ESTP data from populations in the zone of contact between black spruce and red spruce. We also thank I. Giguère, S. Plante, and S. Senneville for laboratory and field assistance, I. Gamache for her help on spatial analysis, and C. Basten for providing his disequilibrium program. We have appreciated greatly the thoughtful and constructive comments from R. J. Petit, J. Beaulieu, D. Khasa, L. Bernier, and two anonymous reviewers on earlier drafts of the manuscript. This research was supported by grants from Fonds Québécois de la Recherche sur la Nature et les Technologies and the National Sciences and Engineering Research Council of Canada to J.B. and an international fellowship from the Québec Ministry of Education to J.P.J.-C.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 

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