DNA fingerprints from hypervariable mitochondrial genotypes

Conventional surveys of restriction-fragment polymorphisms in mitochondrial DNA of menhaden fish (Brevoortia tyrannus/patronus complex) and chuckwalla lizards (Sauromalus obesus) revealed exceptionally high levels of genetic variation, attributable to differences in mtDNA size as well as in restriction sites. The observed probabilities that any two randomly drawn individuals differed detectably in mtDNA genotype were 0.998 and 0.983 in the two species, respectively. Thus, the variable gel profiles provided unique mtDNA "fingerprints" for most conspecific animals assayed. mtDNA fingerprints differ from nuclear DNA fingerprints in several empirical respects and should find special application in the genetic assessment of maternity.      

Morphological and genetic investigations of two western Gulf of Mexico menhadens (Brevoortia spp.)

Gulf menhaden Brevoortia patronus , the main target of a commercial fishing industry in the Gulf of Mexico, and finescale menhaden Brevoortia gunteri , a non-targeted congeneric, have historically been difficult to differentiate. In this study, meristic counts resulted in two of three characteristics that clearly distinguished species, and genetic differences included 1) near fixation for different alleles between species at two of five loci and 2) significantly large divergence ( F _ST) values between species at four loci. Implications for the correct identification of these species and their putative hybrids are discussed.     

Food resource partitioning and trophic morphology of Brevoortia gunteri and B. patronus

The menhadens Brevoortia gunteri and B. patronus are sympatric and morphologically similar. The two species are planktophagous and exhibited significant ( P < 0·0001) food resource partitioning, with phytoplankton predominating in B. patronus stomachs and zooplankton in B. gunteri. The branchial apparatus of both species is of the typical alosine form. Brevoortia patronus has significantly more branchiospinules per mm (=19·47) and significantly longer gill rakers (=13·35 mm) than B. gunteri (=14·11, =12·01 mm respectively). B. patronus is characterized by a gill raker system forming a fine-meshed filter capable of retaining smaller food items. The results support the hypothesis that food resource partitioning is related to different morphological features of the branchial apparatus.     

Molecular evidence of hybridization in Florida's sheoak (Casuarina spp.) invasion

The presence of hybrids in plant invasions can indicate a potential for rapid adaptation and an added level of complexity in management of the invasion. Three Casuarina tree species, Casuarina glauca , Casuarina cunninghamiana and Casuarina equisetifolia , native to Australia, are naturalized in Florida, USA. Many Florida Casuarina trees are considered unidentifiable, presumably due to interspecific hybridization. We collected tissue from over 500 trees from Australia and Florida and genotyped these using amplified fragment length polymorphisms. Our goal was to determine the exact identity of the Florida species, including any putative hybrid combinations. In Australia, we found high assignment values to the three parental species, and no evidence of hybridization. In Florida, we found many trees with strong assignment to any one of the three species, as well as 49 trees with assignment values intermediate to C. glauca and C. equisetifolia , suggesting hybridization between these species. One population of 10 trees had assignment values intermediate to C. cunninghamiana and C. glauca , suggesting additional hybridization. For 69 of these putative hybrid and parental types, we sequenced a low-copy intron of nuclear G3pdh , and these sequences indicated that some Florida trees contain heterozygotic combinations of C. glauca and C. equisetifolia haplotypes. The presence of novel hybrids in the Florida invasion may enhance evolution of invasive traits in these species. Novel Casuarina hybrids in Florida have no coevolutionary history with any insects or diseases, which may be problematic for biological control efforts.     

The occurrence of mountain hare mitochondrial DNA in wild brown hares

If interspecific hybrids are fertile and backcross to either parental species, transmission of mitochondrial DNA over the species barrier can occur. To investigate if such transmission has occurred between the brown hare Lepus europeus Pall and the mountain hare L. timidus L. in Scandinavia, an analysis of genetic variation in mitochondrial DNA from 36 hares, collected from 15 localities, was performed. Sequence divergence of mtDNA between species was estimated at 8 ± 1% (SD). Intraspecific mtDNA sequence divergence varied between 0.09 and 0.38% in brown hares and 0.10 and 1.44% in mountain hares. In six out of 18 brown hares examined, two different haplotypes of mountain hare origin were detected, demonstrating a transmission of mtDNA haplotypes from mountain hares to brown hares. The results indicate that interspecific hybridization between the two species occurs in wild populations.     

Extensive hybridization in hawksbill turtles (Eretmochelys imbricata) nesting in Brazil revealed by mtDNA analyses

Bahia state hosts over 90% of hawksbill (Eretmochelys imbricata) nests registered in the main nesting sites monitored by Projeto Tamar-IBAMA in Brazil. The genetic diversity of this hawksbill population (n=119) was assayed through the analyses of 752 bp of the mitochondrial DNA control region in nesting females. Seven distinct haplotypes, defined by 125 polymorphic sites, were found. Most of the individuals (n=67) display four typical hawksbill haplotypes, 50 individuals display two haplotypes characteristic of the loggerhead turtle (Caretta caretta) and two individuals had a haplotype affiliated with the olive ridley (Lepidochelys olivacea). These results demonstrate hybridization between the hawksbills and two species that nest along the Bahia coast. Of special interest is the high occurrence of loggerhead × hawksbill hybrids (42%), which display loggerhead mtDNA haplotypes but are characterized morphologically as hawksbills. The true hawksbill haplotypes present only three variable sites and low genetic diversity values (h=0.358±0.069; π=0.0005±0.0001). The occurrence of several nesting individuals with identical mtDNA from another species may also suggest a long history of introgression between species producing likely F2 or further generation hybrids. Marine turtle hybrids have been previously reported, but the high frequency observed in Bahia is unprecedented. Such introgression may influence evolutionary pathways for all three species, or may introduce novel morphotypes that develop apart from the parental species. The presence of a unique hybrid swarm has profound conservation implications and will significantly influence the development and implementation of appropriate management strategies for these species.     

Calling behavior and directional hybridization between two toads (Bufo microscaphus x B. woodhousii) in Arizona

Male calling effort and mitochondrial DNA (mtDNA) variation were examined in a breeding chorus of toads from a hybrid zone between Bufo microscaphus and B. woodhousii in central Arizona. The chorus comprised 50 B. microscaphus and 17 hybrids, identified on the basis of morphology and advertisement calls; no pure B. woodhousii were observed. Males produced advertisement calls throughout the early evening, even when relatively large numbers of males (>50) were present at the chorus; active searching and satellite tactics were not observed. Calling efforts (call duration x call rate) of hybrids (23.9%, n = 8) and B. microscaphus (24.9%, n = 19) were similar and comparable to call efforts of B. woodhousii (21.9%, n = 10) from a different site. Moreover, repeatabilities of calling effort were significant (r = 0.45) for hybrid males, but not for B. microscaphus and B. woodhousii. Thus, calling behavior of hybrid males was neither significantly reduced nor more variable than that of their parental species. The distribution of mtDNA haplotypes revealed directional introgression is occurring between male B. microscaphus and female B. woodhousii: All 17 hybrids possessed B. woodhousii mtDNA. The proximate mechanism driving hybridization appears to involve common male (B. microscaphus) and rare female (B. woodhousii) matings as B. woodhousii expands its range.     

Gene regulatory divergence among species estimated by altered developmental patterns in interspecific hybrids

Disturbances in the schedules of gene expression in developing interspecific fish hybrids have been used to draw inferences about the extent of gene regulatory divergence between species and about the degree to which this gene regulatory divergence is correlated with structural gene divergence, as estimated by genetic distance. Sperm from each of 10 different species representing six genera within the family Centrarchidae was used to fertilize eggs of the Florida largemouth bass (Micropterus salmoides floridanus). The genetic distances (D; Nei 1978) between the parental species used to form the hybrids ranged from 0.133 to 0.974. The developmental success and temporal patterns of gene expression of each of the hybrids were compared with those of the Florida largemouth bass. As the genetic distance between the paternal species and the Florida largemouth bass increased, there was a general decline in developmental success in the hybrid embryos as demonstrated by the observed reductions in the percentage of hatching and by progressively earlier and more extensive morphological abnormalities. Concomitantly, progressively more marked alterations in developmental schedules of expression of 15 enzyme loci occurred in the hybrids as the genetic distance between parental species increased. However, observed deviations from this trend for a few species may represent an uncoupling of the rates and modes of evolution of structural genes from those for genes regulating developmental processes.      

Mitochondrial DNA variation in the hybridizing fire-bellied toads, Bombina bombina and B. variegata

Using five restriction enzymes, geographical variation of mitochondrial DNA (mtDNA) in Bombina bombina and B. variegata was studied in samples from 20 locations. Each restriction enzyme produced a species-specific fragment pattern. B. bombina haplotypes A and B were closely related to each other. In contrast, haplotypes A and B of B. variegata formed two distinct lineages. A very distinctive haplotype (C) was found in the Carpathian Mountains, whereas two other haplotypes, D and E (differing by a single AvaI site), were present in western Europe and the Balkans, respectively. Populations polymorphic for haplotypes D and E occurred in the central Balkans where the haplotypes could replace each other clinally. mtDNA sequence divergence between B. bombina and B. variegata was estimated as 6.0-8.1% and 4.7-5.2% between type C and types D/E of B. variegata. The latter divergence is contrary to allozyme and morphological data that place the western and Carpathian B. v. variegata together (Nei's D = 0.07) and separate them from the Balkan subspecies B. v. scabra (Nei's D = 0.18). Broad interspecific correlation among morphology, allozymes and mtDNA types in European fire-bellied toads argues that, despite continuous hybridization (interrupted perhaps during Pleistocene glacial maxima), little or no mtDNA introgression between the species has occurred outside the narrow hybrid zones that separate these parapatric species.     

REPRODUCTIVE ISOLATION AND INTROGRESSION BETWEEN NOTROPIS CORNUTUS AND NOTROPIS CHRYSOCEPHALUS (FAMILY CYPRINIDAE): COMPARISON OF MORPHOLOGY,ALLOZYMES, AND MITOCHONDRIAL DNA

Hybrid zones in fluvial fishes may be heterogeneous from drainage to drainage. The comparison of data from morphology, allozymes, and mitochondrial DNA (mtDNA) indicates variability in the causes and degree of restriction of gene flow between Notropis cornutus and Notropis chrysocephalus. Allozyme marker loci show frequency-dependent introgression; i.e., the rarer species, whichever it is at a particular locality, tends to exhibit a higher proportion of introgressed alleles. Unlike allozymes, introgression of mtDNA haplotypes varies geographically. In westward-flowing Michigan drainages, N. cornutus mtDNA haplotypes are more common in F₁ hybrids and backcrosses, independent of parental frequencies. In eastward-flowing Michigan drainages, N. chrysocephalus mtDNA is more common in F₁ hybrids and backcrosses; this pattern may be due to local ecological effects or frequency-dependent introgression. Morphological data alone are not sufficient to distinguish all classes of hybrids. The lack of concordance of morphological, allozymic, and mtDNA introgression patterns implies operation of one or two factors: 1) geographically variable patterns of selection against different hybrid and backcross combinations or 2) genetic differences between Michigan populations inhabiting eastward- and westward-flowing drainage systems accumulated during historical isolation.     

Chloroplast and mitochondrial molecular tests identify European×Japanese larch hybrids

Hybrids between European and Japanese larches combine the properties of both parental species (drought resistance, canker resistance, stem straightness) and exhibit a fast growth rate. They are produced in seed orchards, generally by natural pollination. Seeds are collected and used for afforestation as interspecific hybrids. However, there are no convenient tests to assess the interspecific hybrid proportion. In the present study, we developed diagnostic molecular markers suitable for the individual identification of hybrids, whatever their developmental stage. Our strategy involved testing a combination of maternally inherited markers from the mitochondrial genome (mtDNA) and paternally inherited markers from the chloroplast genome (cpDNA). Hybrids were then identified by the presence of a mitochondrial sequence inherited from one parental species and a chloroplast sequence inherited from the other parental species. To achieve this aim, markers discriminating both parental species were first sought. Amplifications of mitochondrial and chloroplast sequences were performed using specific PCR primers. After testing 33 primer pairs in combination with nine restriction enzymes, we detected one mitochondrial marker, f13 which was amplified in Japanese larch and absent in European larch, and one chloroplast marker, ll-TaqI which showed different restriction patterns depending on the species. A restriction fragment of 601 bp was obtained in Japanese larch while two fragments of 120 bp and 481 bp were observed in European larch. These patterns were found in all 197 individuals tested from the two pure species. These markers were then used for the evaluation of the hybrid proportion in a seed lot produced from seed orchards; this was assessed as between 43% and 53% depending on the parental species. The male and female parental species could be determined for each progeny.Communicated by D.B. Neale     

Complete mitochondrial DNA sequence analysis of Bison bison and bison–cattle hybrids: Function and phylogeny

Complete mitochondrial DNA (mtDNA) genomes from 43 bison and bison-cattle hybrids were sequenced and compared with other bovids. Selected animals reflect the historical range and current taxonomic structure of bison. This study identified regions of potential nuclear–mitochondrial incompatibilities in hybrids, provided a complete mtDNA phylogenetic tree for this species, and uncovered evidence of bison population substructure. Seventeen bison haplotypes defined by 66 polymorphic sites were discovered, whereas 728 fixed differences and 86 non-synonymous mutations were identified between bison and bison–cattle hybrid sequences. The potential roles of the mtDNA genome in the function of hybrid animals and bison taxonomy are discussed.     

PATERNAL LEAKAGE OF MITOCHONDRIAL DNA IN A FUCUS (PHAEOPHYCEAE) HYBRID ZONE1

Eukaryotic mitochondria are mostly uniparentally (maternally) inherited, although mtDNA heteroplasmy has been reported in all major lineages. Heteroplasmy, the presence of more than one mitochondrial genome in an individual, can arise from recombination, point mutations, or by occasional transmission of the paternal mtDNA (=paternal leakage). Here, we report the first evidence of mtDNA paternal leakage in brown algae. In Denmark, where Fucus serratus L. and Fucus evanescens C. Agardh have hybridized for years, we found eight introgressed individuals that possessed the very distinct haplotypes of each parental species. The finding of heteroplasmy in individuals resulting from several generations of backcrosses suggests that paternal leakage occurred in earlier generations and has persisted through several meiotic bottlenecks.     

Genetic Evidence for Natural Hybridization between Species of Dioecious Ficus on Island Populations1

Natural hybrids between Ficus septica and two closely related dioecious species, F. fistulosa and F. hispida, were confirmed using amplified fragment length polymorphisms (AFLP) and chloroplast DNA markers. Ficus species have a highly species‐specific pollination mutualism with agaonid wasps. Therefore, the identification of cases in which breakdown in this sophisticated system occurs and the circumstances under which it happens is of interest. Various studies have confirmed that Ficus species are able to hybridize and that pollinator‐specificity breakdown can occur under certain conditions. This study is the first example in which hybrid identity and the presence of hybrids in the natural distribution of parental species for Ficus have been confirmed with molecular markers. Hybrid individuals were identified on three island locations in the Sunda Strait region of Indonesia. These findings support Janzen's (1979) hypothesis that breakdown in pollinator specificity is more likely to occur on islands. We hypothesized that hybrid events could occur when the population size of pollinator wasps was small or had been small in one of the parental species. Later generation hybrids were identified, indicating that backcrossing and introgression did occur to some extent and that therefore, hybrids could be fertile. The small number of hybrids found indicated that there was little effect of hybridization on parental species integrity over the study area. Although hybrid individuals were not common, their presence at multiple sites indicated that the hybridization events reported here were not isolated incidences. Chloroplast DNA haplotypes of hybrids were not derived solely from one species, suggesting that the seed donor was not of the same parental species in all hybridization events.     

Genetic characterization of hybridization and introgression between anadromous rainbow trout (Oncorhynchus mykiss irideus) and coastal cutthroat trout (O. clarki clarki)

Interspecific hybridization represents a dynamic evolutionary phenomenon and major conservation problem in salmonid fishes. In this study we used amplified fragment length polymorphisms (AFLP) and mitochondrial DNA (mtDNA) markers to describe the extent and characterize the pattern of hybridization and introgression between coastal rainbow trout (Oncorhynchus mykiss irideus) and coastal cutthroat trout (O. clarki clarki). Hybrid individuals were initially identified using principle coordinate analysis of 133 polymorphic AFLP markers. Subsequent analysis using 23 diagnostic AFLP markers revealed the presence of F1, rainbow trout backcross, cutthroat trout backcross and later-generation hybrids. mtDNA analysis demonstrated equal numbers of F1 hybrids with rainbow and cutthroat trout mtDNA indicating reciprocal mating of the parental types. In contrast, rainbow and cutthroat trout backcross hybrids always exhibited the mtDNA from the recurrent parent, indicating a male hybrid mating with a pure female. This study illustrates the usefulness of the AFLP technique for generating large numbers of species diagnostic markers. The pattern of hybridization raises many questions concerning the existence and action of reproductive isolating mechanisms between these two species. Our findings are consistent with the hypothesis that introgression between anadromous populations of coastal rainbow and coastal cutthroat trout is limited by an environment-dependent reduction in hybrid fitness.     

Analysis of mitochondrial DNA from somatic hybrid cell lines of Saccharum officinarum (sugarcane) and Pennisetum americanum (pearl millet)

Mitochondrial DNA (mtDNA) from cell suspension cultures of two intergeneric somatic hybrids of Pennisetum americanum (pearl millet) + Saccharum officinarum (sugarcane) was examined by restriction endonuclease digestion and hybridization with sorghum mtDNA cosmids. The mtDNA of one somatic hybrid was indistinguishable from that of pearl millet, while the second exhibited a combination of parental mtDNAs, suggesting mitochondrial fusion. Several novel, possibly recombinant, mtDNA restriction fragments were detected in this hybrid, which may have resulted from intergenmic recombination.Florida Agriculture Experiment Station Journal Series No: 8090.     

The expression of human glycerol-3-phosphate dehydrogenase in human/rodent somatic-cell hybrids

Our previous studies using rodent/human somatic-cell hybrids suggested that the expression of human mitochondrial glycerol-3-phosphate dehydrogenase (GPDM) is dependent on the presence of human mitochondria. This has now been tested directly by analysis of GPDM activity in a series of nine hybrid-cell lines, four segregating human chromosomes and five losing rodent chromosomes (reverse segregants). The chromosome composition of the hybrids was deduced from analysis of biochemical markers and examination of G- and G11-banded metaphase spreads and the mitochondrial content was determined by Southern blot analysis, using cloned mouse and human mtDNA sequences as probes. We found that the mtDNA species present in these hybrids correlated exactly with the pattern of chromosome segregation such that the conventional hybrids contained rodent mtDNA and the reverse segregants human mtDNA. However, the pattern of GPDM expression was not directly correlated with the species of chromosomes or mitochondria present: all the hybrids showed strong rodem GPDM activity and two from each class of hybrid also showed human GPDM activity but the other hybrids were negative for human GPDM. We conclude that rodent GPDM readily integrates into human mitochondria, that the expression of rodent GPDM is not dependent on the presence of rodent mitochondria, and that GPDM is not coded by mtDNA. Human GPDM either is not capable of being inserted into the rodent mitochondrial membrane or is regulated in some way in the hybrid cells by an unidentified rodent factor.     

Mitochondrial genomes in intraspecies mammalian cell hybrids display codominant or dominant/recessive behavior

A unique type of nonstochastic mitochondrial DNA (mtDNA) segregation was found in mammalian cells. In human cell hybrids isolated from the fusion of HeLa cells with 23, GM639, A549, or 293 cells, HeLa mtDNA was always lost from the hybrids, whereas both parental mtDNAs were maintained in hybrids of HeLa X 143BTK-. Similar phenomena were observed in mouse cell hybrids isolated by the fusion of cells with different mtDNA types. Types 1, 2, and 3, can be distinguished from each other by restriction fragment-length polymorphisms. The mouse cell hybrids between cells with type 1 and type 2 mtDNA always lost type 2 mtDNA, whereas the hybrids between cells with type 2 and type 3 mtDNA retained both types stably. These observations suggest that either a codominant or a dominant/recessive relationship may be present in intraspecies mitochondrial genomes of human and mouse cells. When the mitochondrial genomes in cell hybrids are codominant, stochastic segregation occurs while nonstochastic segregation occurs when they are in the dominant/recessive relationship. These concepts may help elucidate organelle heredity in animals.     

Interspecific hybridization in Daphnia: distinction and origin of hybrid matrilines

Three coexisting Daphnia species belonging to the D. longispina group (D. galeata, D. hyalina, and D. cucullata) form species-hybrid complexes by producing interspecific hybrids in several lakes in Germany and The Netherlands. To evaluate the genetic consequences of interspecific hybridization, I studied the patterns of mitochondrial DNA (mtDNA) sequence variation. The directionality of interspecific hybridization and divergence of hybrids from parental species was tested, using the DNA sequences of a segment of mtDNA. Via the polymerase chain reaction, it was possible to investigate single animals and even single resting eggs. A species-specific marker was established, using restriction patterns of amplified cytochrome b segments. mtDNA genotypes of hybrids revealed unidirectional mitochondrial gene flow for two hybrids, which were investigated by using multiple clones. No evidence for introgression of mtDNA was found. On the basis of a phylogenetic analysis, the species exhibit considerable distinctness, whereas differences between clones within species and between hybrids and maternal species tend to be very low. These results indicate a recent origin of hybrids and suggest that the radiation of the D. longispina group occurred > 5 Mya.      

A high frequency of intergenomic mitochondrial recombination and an overall biased segregation of B. campestris or recombined B. campestris mitochondria were found in somatic hybrids made within Brassicaceae

Mitochondrial segregation and rearrangements were studied in regenerated somatic hybrids from seven different species combinations produced using reproducible and uniform methods. The interspecific hybridizations were made between closely or more distantly related species within the Brassicaceae and were exemplified by three intrageneric, two intergeneric and two intertribal species combinations. The intrageneric combinations were represented by Brassica campestris (+) B. oleracea, B. napus (+) B. nigra and B. napus (+) B. juncea (tournefortii) hybrids, the intergeneric combinations by B. napus (+) Raphanus sativus and B. napus (+) Eruca sativa hybrids, and the intertribal combinations by B. napus (+) Thlaspi perfoliatum and B. napus (+) Arabidopsis thaliana hybrids. In each species combination, one of the two mitochondrial genotypes was B. campestris since the B. napus cultivar used in the fusions contained this cytoplasm. Mitochondrial DNA (mtDNA) analyses were performed using DNA hybridization with nine different mitochondrial genes as probes. Among the various species combinations, 43–95% of the hybrids demonstrated mtDNA rearrangements. All examined B. campestris mtDNA regions could undergo intergenomic recombination since hybrid-specific fragments were found for all of the mtDNA probes analysed. Furthermore, hybrids with identical hybrid-specific fragments were found for all probes except cox II and rrn18/rrn5, supporting the suggestion that intergenomic recombination can involve specific sequences. A strong bias of hybrids having new atp A-or atp9-associated fragments observed in the intra- and intergeneric combinations could imply that these regions contain sequences that have a high reiteration number, which gives them a higher probability of recombining. A biased segregation of B. campestris-or B. campestris-like mitochondria was found in all combinations. A different degree of phylogenetic relatedness between the fusion partners did not have a significant influence on mitochondrial segregation in the hybrids in this study.