Genetic parentage in the indigo bunting: a study using DNA fingerprinting

Summary Parentage of nestlings in a North Carolina population of indigo buntings (Passerina cyanea) was analyzed using DNA fingerprinting. Three minisatellite DNA probes (wild type M13, Jeffreys' 33.15 and 33.6) were used to analyze nuclear DNA isolated from muscle biopsies of 63 nestlings, their parents, and other local adults. Each probe detected approximately 15 scorable fragments per individual, with 18%–39% overlap between probes. The proportion of bands shared (using all fragments over all three probes) between presumably unrelated adults averaged 0.23. Of the 63 offspring analyzed, 35 had at least one fragment not present in either putative parent. The distribution of offspring with novel fragments was distinctly bimodal. The lower mode (offspring with 0, 1, or 2 novel fragments, N=41) fit a Poisson distribution, a pattern expected if mutation (estimated rate per fragment= 0.01) were the source of the novel fragments. The remaining 22 offspring had more novel fragments than could be explained by mutation alone (minimum of four independent fragments across all three probes, =8.2). A low band-sharing proportion with the resident male ( =0.24) and high band-sharing with the resident female ( =0.60) implicated extra-pair fertilizations as the source of all 22. Thus in this sample, 35% of all nestlings came from extra-pair fertilizations and none from intra-specific brood parasitism. Of 25 broods sampled, 12 (48%) had at least one excluded offspring. In 3 broods all of the offspring excluded the resident male. Band-sharing proportions between excluded nestlings within a brood could not distinguish between single and multiple extra-pair paternity. Although young males tended to be excluded less often than older males, wing length and weight were not associated with the frequency of exclusion. Weight and wing length of females also were not associated with involvement in EPCs. Six of the 22 excluded offspring (in 3 broods) shared a large proportion of bands and had fewer than four novel fragments when compared to the fingerprints of a neighboring territorial male, implicating those males as actual fathers. The parentage of the remaining 16 offspring (in 9 broods) could not be clearly assigned because (1) one or more neighbors were not sampled or (2) difficulties in scoring across gels prevented confident alignment of fingerprint bands, and insufficient DNA was obtained from muscle samples to allow reanalysis of potential actual fathers on the same gel as excluded nestlings.     

Intraspecific brood parasitism in the moorhen: parentage and parasite-host relationships determined by DNA fingerprinting

Parasitic female moorhens (Gallinula chloropus) lay from one to six eggs in the nests of conspecific neighbours. DNA fingerprinting was used to show that parasitic eggs could be correctly identified when they appeared in addition to or outside the host’s laying sequence. Moorhen hosts accept all parasitic eggs laid after the 2nd day of their laying period. To understand why moorhen hosts tolerate parasitic eggs, we tested two hypotheses. (1) The quasi-parasitism hypothesis: females lay their eggs in the evening when the host males are normally in attendance at the nest, so host males may allow parasitic females to lay in their nests in exchange for fertilizing their eggs. However, DNA fingerprinting showed that all the parasitic eggs were sired by the parasites’ mates. Parasitic moorhens frequently continue laying a clutch in their own nest, without a break in the laying sequence after a parasitic laying bout. The eggs laid by brood parasites in their own nests were also sired by their own mates. Therefore this hypothesis was rejected. (2) The kin selection hypothesis: if one or both members of the host pair are close relatives of the parasite, the costs of rearing parasitic chicks will be to some degree offset by inclusive fitness benefits. We examined the genetic relationships between parasites and their hosts using DNA fingerprinting and genealogical data. Natal philopatry by both sexes was relatively common in this population, and the probability that a neighbour of either sex was a first-order relative (parent-offspring) was calculated as 0.18. Although first-order relatives were not preferentially chosen as hosts over individuals that were not first-order relatives, even through random host selection there is almost a one-in-five chance that brood parasites in this population are closely related to their hosts. This may facilitate host tolerance of parasitic eggs. Other hypotheses are also discussed. Received: 3 February 1995/Accepted after revision: 27 August 1995     

Evolutionary relationships of deep-sea mussels inferred by mitochondrial DNA sequences

In order to elucidate the evolutionary process of deep-sea Bathymodiolus mussels, we investigated the phylogenetic relationships of 16 species worldwide by analyzing nucleotide sequences of the mitochondrial COI and ND4 genes. Deep-sea mussels were clustered into three groups by basal trichotomous divergence. The first was composed of four species found in Japanese waters and one species from the Gulf of Mexico, which contain methanotrophic endosymbiotic bacteria. The second included nine species distributed in the West and East Pacific, Indian, and Atlantic Oceans. Members of the second group were trichotomously divided into the Indo-West Pacific, Atlantic, and East Pacific subclusters. The Indo-West Pacific subcluster was composed of three very closely related species with mutual genetic distances at the intraspecific level (av. 0.019 in COI and 0.009 in ND4 relative to av. 0.156 in COI and 0.265 in ND4 among Bathymodiolus species other than Cluster A species), suggesting some gene flow among these species. The third consisted of two West Pacific species. Species in the second and third groups contain mainly thioautotrophic endosymbionts, including some species harboring both methanotrophs and thioautotrophs.     

Extreme mitochondrial DNA divergence within populations of the deep-sea gastropod Frigidoalvania brychia

The deep sea supports a diverse and highly endemic invertebrate fauna, the origin of which remains obscure. Little is known about geographic variation in deep-sea organisms or the evolutionary processes that promote population-level differentiation and eventual speciation. Sequence variation at the 16 S rDNA locus was examined in formalin-preserved specimens of the common upper bathyal rissoid Frigidoalvania brychia (Verrill, 1884) to examine its population genetic structure. The specimens came from trawl samples taken over 30 years ago at depths of 457-1,102 m at stations in the Northwest Atlantic south of Woods Hole, Massachusetts, USA. Near the upper boundary of its bathymetric range (500 m), extremely divergent haplotypes comprising three phylogenetically distinct clades (average uncorrected sequence divergence among clades ~23%, ~3% within clades) were found at stations separated by a maximum distance of ~80 km, suggesting the presence of high levels of intraspecific divergence or the possibility of morphologically cryptic species. Only one of these clades was found at two stations in the mid- to lower part of F. brychia's depth distribution (800-1,100 m), suggesting lower clade diversity with increasing depth, although among-sample divergence, with a single exception, was minimal. One station was genetically divergent from all others sampled, containing a unique suite of haplotypes including two found only at this site. Steep vertical selective gradients, major oceanographic changes during the late Cenozoic, and habitat fragmentation by submarine canyons might have contributed to an upper bathyal region that is highly conducive to evolutionary change.     

Cetacean DNA fingerprinting using the oligonucleotide (GGAT)4 as a probe

Since the discovery in 1985 of hypervariable minisatellite regions in human DNA, DNA fingerprinting has been widely used in animals and plants. Although many different probes have been developed recently, most investigators still use 33.15 and 33.6 human polycore minisatellites for hybridization studies of cetaceans. This animal order shows considerably less variability than humans, and thus some limitations have to be taken into account when using these probes. The present study demonstrates that the short repetitive tandem oligonucleotide (GGAT)₄ hybridizes with cetacean (Globicephala macrorhynchus andGrampus griseus) DNA, yielding individual-specific patterns of bands or DNA fingerprints. Freedom of design and shorter hybridization times of oligonucleotides allow an increase in the number of usable probes used, helping to overcome some of the problems found when only human polycore minisatellites are used.     

Mitochondrial DNA analysis of the genetic relationships among populations of scad mackerel (Decapterus macarellus, D. macrosoma, and D. russelli) in South-East Asia

The genetic relationships among South-East Asian populations of the scad mackerels Decapterus macarellus, D. macrosoma and D. russelli (Pisces: Carangidae) were investigated. In 1995 and 1996, 216 fish were sampled in seven localities spanning the seas of Indonesia and were examined for restriction-site polymorphisms using ten restriction enzymes for the mitochondrial (mt) DNA control region, amplified by the polymerase chain-reaction. The inferred phylogeny of haplotypes led to the recognition of three distinct mitochondrial lineages or phylads consistent with the distinctions of current taxonomy. All 15 mtDNA haplotypes found in D. macarellus and all 9 haplotypes found in D. macrosoma were arranged as star-like clusters, suggesting recent evolutionary history. In contrast, the phylad formed by 6 haplotypes in D. russelli from the Sulawesi Sea exhibited diffuse topology, suggesting that ancestral lineages of this species have been retained to the present. Average nucleotide-divergence estimates between haplotypes of different phylads were between 0.042 and 0.135, suggesting ancient separation, in consistency with published allozyme data. High levels of haplotype diversity, but no geographical heterogeneity, was detected within D. macarellus from the Molucca Sea and the Banda Sea. Populations of D. macrosoma exhibited both significant differences between adjacent regions (Sunda Strait and Java Sea), and broadscale genetic homogeneity from the South China Sea to the Sulawesi Sea via the Java Sea and Makassar Strait. The geographic isolation of the D. macrosoma population sampled in the Sunda Strait suggests that this region constitute a sharp transition zone between the Indian Ocean and the Sunda Shelf. Near-monomorphism of haplotypes and low nucleotide diversity (d _X) were observed in the samples of D. macrosoma from the continental shelf (haplotype-diversity estimates, h, = 0.00 to 0.25 ± 0.08 and d _X = 0.000 to 0.002). This was in contrast to the comparatively high haplotype and nucleotide diversities observed in other pelagic fish species including D. macarellus (h = 0.82 ± 0.05, d _X = 0.012 to 0.015) and D. russelli (h = 0.63 ± 0.12, d _X = 0.016), and in the oceanic D. macrosoma population sampled in the Sunda Strait (h = 0.67 ± 0.31, d _X = 0.005). We hypothesise that this may be the consequence of recent and perhaps repeated bottleneck events that have affected the D. macrosoma population sampled on the continental shelf. Received: 29 September 1997 / Accepted: 3 September 1999     

Phylogenetic relationships of coral-associated gobies (Teleostei,Gobiidae) from the Red Sea based on mitochondrial DNA data

Bryaninops, Gobiodon, Paragobiodon and Pleurosicya are the most abundant genera of coral-associated gobies. These genera are adapted to live among coral, while other small reef gobies (e.g., the genus Eviota) show no obligate association with this living substrate. Thirteen coral-associated species and two Eviota species were sampled from different regions of the Red Sea, along with four populations/species of Gobiodon from the Indian and western Pacific Oceans. A molecular phylogenetic analysis was performed using partial sequences of 12S rRNA, 16S rRNA and cytochrome b mitochondrial genes, 1,199 base pairs in total. Several clades were consistently resolved in neighbor joining-, maximum parsimony-, maximum likelihood and Bayesian analyses. While each of the four genera Gobiodon, Paragobiodon, Bryaninops and Pleurosicya proved to be monophyletic, their relative position in the phylogeny did not support an emergence of coral-associated gobiids as a monophyletic assemblage. Instead, two separate monophyletic sub-groups were discovered, the first comprising Gobiodon and Paragobiodon, and the second Bryaninops and Pleurosicya. Our molecular phylogenetic examinations also revealed one unassigned species of Gobiodon from the Maldives as a distinct species and confirmed three putative and yet unassigned species from the Red Sea. Moreover, the uniformly black colored species of Gobiodon are not monophyletic but have evolved independently within two distinct species groups. Genetic distances were large in particular within Pleurosicya and Eviota. Estimated divergence times suggest that coral-associated gobies have diversified in parallel to their preferred host corals. In particular, divergence times of Gobiodon species closely match those estimated for their typical host coral genus Acropora.     

Alternative reproductive tactics in male common shrews: relationships between mate-searching behaviour,sperm production,and reproductive success as revealed by DNA fingerprinting

The common shrew (Sorex araneus) is a solitary small mammal with a promiscuous mating system. Previous studies of this species suggest that females typically mate multiply, and that males may adopt alternative mate-searching tactics. We studied two generations of common shrews in a population near Oxford, England. Males were found to adopt two different mate-searching tactics. Those classed as type A occupied relatively small exclusive ranges during March, and made repeated long-distance movements to visit female ranges around the time of first oestrus in April. Males classed as type B established large overlapping ranges in areas of relatively high female density during March, and maintained these ranges throughout April. Type B males were larger than type A males at an early stage of sexual maturation, but there was no difference in the adult body size of the two types of male. Type A males had significantly higher epididymal sperm counts than type B males. Paternity analyses of litters born during the first year of the study reveal that the mean number of offspring fathered by type B males was greater than the mean number fathered by type A males. It is concluded that different mate-searching tactics may be conditional upon the timing of sexual maturation. Differences in sperm production are discussed in relation to sperm competition theory.     

Mitochondrial DNA polymorphisms reveal additional genetic heterogeneity within the Leptasterias hexactis (Echinodermata: Asteroidea) species complex

Sea stars of the Leptasterias hexactis complex are important members of the rocky intertidal community of the temperate and subarctic North American Pacific coasts. Recent attempts to determine the genetic structure and taxonomic relationships of these sea stars have not been completely successful, due to the combination of extreme morphological variability and relative scarcity of taxonomically-informative genetic (allozyme) polymorphisms. The present study used mitochondrial DNA restriction-site polymorphisms for PCR (polymerase chain-reaction)-amplified products to provide additional information about this complex. Five restriction-site haplotypes were identified in southern Alaska and in the Puget Sound region that were allozymically distinct. Because the allozymic differences (Nei's unbiased genetic differences >0.08) are maintained in sympatry, the haplotypes behave evolutionarily as conventional species. Although most haplotypes show deficiencies of heterozygotes for polymorphic allozyme loci, there is no evidence for the existence of cryptic species within any of the haplotypes. Rather than consisting of a few polytypic and cosmopolitan species, the L. hexactis complex is apparently composed of numerous species with more restricted ranges. Determining the exact ranges and taxonomic relationships of the various species will require additional study.     

Levels of polyandry and intracolonial genetic relationships in Apis florea

DNA was extracted from worker and drone pupae of each of five colonies of the dwarf honey bee Apis florea. Polymerase chain reactions (PCR) were conducted on DNA extracts using five sets of primers known to amplify microsatellite loci in A. mellifera. Based on microsatellite allele distributions, queens of the five colonies mated with at least 5–14 drones. This is up to 3 times previous maximum estimates obtained from sperm counts. The discrepancy between sperm count and microsatellite estimates of the number of matings in A. florea suggests that despite direct injection of semen into the spermatheacal duct, either A. florea drones inject only a small proportion of their semen, or queens are able to rapidly expel excess semen after mating. A model of sexual selection (first proposed by Koeniger and Koeniger) is discussed in which males attempt to gain reproductive dominance by increasing ejaculate volume and direct injection of spermatozoa into the spermatheca, while queens attempt to maintain polyandry by retaining only a small fraction of each male's ejaculate. It is shown, at least in this limited sample, that the effective number of matings is lower in A. florea than in A. mellifera.     

Analysis of the genetic structure of European eel (Anguilla anguilla) using microsatellite DNA and mtDNA markers

The spawning population of European eel (Anguilla anguilla L.) has been considered panmictic on the basis of genetic markers and morphometric studies. This hypothesis was tested by screening glass eel from five locations (Ireland, Italy, Morocco, Sweden and U.K.), belonging to two cohorts at the cytochrome b (cyt b) locus (392 bp) of the mitochondrion and at five nuclear microsatellite loci. Seventeen cyt b haplotypes were detected, of which ten were singletons; the most common haplotype occurred in 47% of all fish. Haplotype number increased significantly with latitude. Phylogeographical structure based on the cytoplasmic marker was weak (F_ST=0.014) and non-significant. Close similarity was revealed between British and Irish glass eel populations, and weak differentiation among the British/Irish, Atlantic Moroccan, Italian and Swedish Baltic populations, respectively. No hierarchical genetic structure was obvious. Levels of genetic variation detected with five microsatellites were much higher levels than found with allozymes in previous studies (mean number of alleles per locus=11.1; mean expected heterozygosity=0.68). Overall among-population microsatellite variance was low but significant (F_ST=0.004), and caused by the linked microsatellite loci Aan03 and Aan04. The Hardy-Weinberg-Castle equilibrium and the absence of gametic disequilibria at these loci in the Moroccan population might point to its genetic isolation, although the impact of just two out of five loci is puzzling. Given the weak differentiation typical for marine species and the limitations of our data, the results should be interpreted with caution. However, combined with recent evidence from a related study, the paradigm that the European eel constitutes a panmictic population becomes difficult to maintain.     

Close genetic similarity of Atlantic and Pacific skipjack tuna (Katsuwonus pelamis) demonstrated with restriction endonuclease analysis of mitochondrial DNA

Restriction endonuclease analysis of mitochondrial DNA indicated a surprisingly high degree of genetic similarity between skipjack tuna (Katsuwonus pelamis) from the Atlantic and Pacific Oceans. The present results (1983) support the findings of previous morphological and electrophoretic studies. Evidently, since the uplift of the Panamá land bridge about 3.1 million years ago, there has been continued genetic contact between Atlantic and Pacific skipjack tuna, presumably via the Southern Ocean.     

Phylogenetic relationships among species of Calyptogena (Bivalvia: Vesicomyidae) collected around Japan revealed by nucleotide sequences of mitochondrial genes

Phylogenetic relationships among the seven species of deep-sea giant clams Calyptogena (Bivalvia: Vesicomyidae) collected around Japan were examined using parts of nucleotide sequences of mitochondrial genes for cytochrome oxidase I (COI) and cytochrome oxidase III (COIII) and the encoded amino acid sequences. The seven species were C. soyoae (Sagami Bay), C. fausta (Suruga Bay), C. kaikoi (Nankai Trough), C. nautilei (Nankai Trough), C. phaseoliformis (Japan Trench), C. solidissima (Minami-Ensei Knoll, Okinawa Trough) and Calyptogena sp. (Iheya Ridge, Okinawa Trough). A clear phylogenetic split was observed between one group of three species (C. kaikoi, C. phaseoliformis and C. fausta) and the remaining species. This clustering corresponds to the two previously described subgenera within the genus Calyptogena (Calyptogean and Ectenagena) with the exception of the placement of C. nautilei, which had been placed in the subgenus Ectenagena. Genetic distances between two haplotypes of C. soyoae were 0.043 for the COI region and 0.055 for the COIII region, and three amino acid substitutions were detected with the COIII region. Calyptogena sp. from the Iheya Ridge could be distinguished from one of the two haplotypes (type A) of C. soyoae by only a single nucleotide substitution, a result that suggests that Calyptogena sp. of the Iheya Ridge diverged from C. soyoae after the two haplotypes had diverged, and it is now isolated from C. soyoae in Sagami Bay.     

Lack of population subdivision among the minke whales (Balaenoptera acutorostrata) from Icelandic and Norwegian waters based on mitochondrial DNA sequences

The minke whale (Balaenoptera acutorostrata) is subject to commercial whaling, but stock identification and assessment are still uncertain. Mitochondrial DNA (mtDNA) sequences were determined to examine the population structure of minke whales from the central and northeastern parts of the North Atlantic, as well as the Antarctic regions IV and V. The analyses include 345 nucleotide positions of the control region of 110 individuals, and 250 nucleotide positions of the NADH dehydrogenase subunit 2 gene for a representative selection of North Atlantic minke whales. Maximum parsimony analyses and sequence divergence calculations did not reveal any genetic differentiation between individuals from the central and northeastern parts of the North Atlantic. These results do not support the International Whaling Commission's separation of minke whales in this area into different management units, and they are in conflict with previously reported results from allozyme analyses. Comparison of minke whale control region sequences showed that the sequence diversity of North Atlantic minke whales is substantially lower (0.0065) than that of Antarctic minke whales (0.0166), and clearly demonstrated that individuals from these two areas represent genetically distinct populations.     

Investigating stock structure and trophic relationships among island-associated dolphins in the oceanic waters of the North Atlantic using fatty acid and stable isotope analyses

Short-beaked common dolphins (Delphinus delphis) and Atlantic spotted dolphins (Stenella frontalis) are the two most abundant cetacean species in the oceanic waters of Madeira and the Azores. They are of similar size, occur in similar habitats and are regularly observed in mixed-species groups to forage together. Genetic analyses suggested that, within each species, dolphins ranging around both archipelagos belong to the same panmictic population. We tested the hypotheses that (1) within each species, individuals from the two archipelagos belong to a single ecological stock; (2) between species, common and spotted dolphins have distinct trophic niches; using fatty acid (FA) and stable isotope (SI) analyses. Fatty acids and stable isotopes were analysed from 86 blubber and 150 skin samples of free-ranging dolphins, respectively. Sex-related differences were not significant, except for common dolphin FA profiles. In S. frontalis, FA and SI differences between archipelagos suggested that individuals belonged to different ecological stocks, despite the existence of gene flow between the two archipelagos. In D. delphis, differences were more pronounced, but it was not possible to distinguish between stock structure and a seasonal effect, due to differential sampling periods in the Azores and Madeira. Inter-specific comparisons were restricted to the Azores where all samples were collected during summer. Differences in FA proportions, noticeably for FA of dietary origin, as well as in nitrogen SI profiles, confirmed that both species feed on distinct resources. This study emphasizes the need for an integrated approach including both genetic and biochemical analyses for stock assessment, especially in wide-ranging marine top predators.     

Comparison of allozyme and mitochondrial DNA spatial differentiation in the limpet Patella vulgata

Allozyme and restriction enzyme analysis of mtDNA was used to study variation in samples from British populations of the marine limpet Patella vulgata in two regions. South Wales and Northeast England. Allozyme analysis revealed significant differences in allele frequencies among samples. However F _ST (population differentiation) values were no higher between than within regions, indicating that genetic heterogeneity was localised and not related to geographic separation. For mtDNA, samples from South Wales exhibited higher haplotype diversity values than samples from Northeast England. In addition there were substantial differences in the haplotype distribution between regions. The value of , the haploid analogoue of F _ST , was low within regions (=0.09) but high between regions (=0.44). The estimated difference in migration rate for allozymes and mtDNA exceed the neutral expectation, unless it is assumed that there are influential differences in the magnitude of female and male gametic dispersal.     

A molecular genetic analysis of social structure, dispersal, and interpack relationships of the African wild dog (Lycaon pictus )

The African wild dog is a highly social, pack-living predator of the African woodland and savannah. The archetypal wild dog pack consists of a single dominant breeding pair, their offspring, and non-breeding adults who are either offspring or siblings of one of the breeding pair. Non-breeding adults cooperate in hunting, provisioning and the protection of young. From these observations follows the prediction that the genetic structure of wild dogs packs should resemble that of a multigenerational family, with all same-sexed adults and offspring within a pack related as sibs or half-sibs. Additionally, a higher kinship between females from neighboring packs should be evident if females tend to have small dispersal distances relative to males. We test these predictions through analysis of mitochondrial DNA control region sequences and 14 microsatellite loci in nine wild dog packs from Kruger National Park, Republic of South Africa. We show that as predicted, African wild dog packs generally consist of an unrelated alpha male and female, subdominant close relatives, and offspring of the breeding pair. Sub-dominant wild dogs occasionally reproduce but their offspring rarely survive to 1 year of age. Relatedness influences the timing and location of dispersal events as dispersal events frequently coincide with a change in pack dominance hierarchy and dispersers often move to areas with a high proportion of close relatives. Received: 22 February 1996 / Accepted after revision: 16 November 1996