Mitochondrial DNA analysis confirms the existence of two glacial races of rainbow smelt Osmerus mordax and their reproductive isolation in the St Lawrence River estuary (Quebec, Canada)

A phylogeographic analysis of mitochondrial DNA variation was performed in order to test the hypothesis that north-eastern North America has been postglacially recolonized by two races of rainbow smelt Osmerus mordax . This was accomplished by documenting the geographical distribution of two major mtDNA phylogenetic clades among 1290 smelt from 49 lacustrine and anadromous populations covering most of the species' native range. The data set was built by combining previously published results with those generated in this study. The two mtDNA clades showed a geographical dichotomy, independent of life-history types, whereby the more eastern populations were either fixed or largely dominated by one clade and western populations for the other. Such geographical pattern implying a phylogenetic discontinuity provided strong evidence for the persistence of smelt in two distinct glacial refugia as well as their differential postglacial dispersal. The most likely refugium for the so-called Atlantic race was the Atlantic coastal plains, whereas that of the so-called Acadian race was the exundated Grand Banks area. Patterns of postglacial dispersal interpreted from palaeogeographic events suggested that the Atlantic race recolonized northern regions about 5000 years prior the Acadian race. Both races came into contact in the St Lawrence River estuary. While gene flow has been possible, the sympatric occurrence in the estuary of anadromous populations alternatively dominated by one mtDNA clade or the other indicated that reproductive isolation mechanisms between the two races developed within this contact zone. This represents the first evidence of secondary intergradation among distinct races of aquatic organisms in an estuarine environment.     

Postglacial genetic differentiation of reproductive ecotypes of kokanee Oncorhynchus nerka in Okanagan Lake, British Columbia

Okanagan Lake, south-central interior of BC, contains two reproductive ecotypes of kokanee Oncorhynchus nerka ; individuals spawn in tributary streams ('stream-spawners') as well as on shoreline gravel areas ('beach-spawners'). We tested the hypothesis that these sympatric ecotypes comprise a single panmictic population by assaying variation in morphological traits and at allozyme, mitochondrial and minisatellite DNA loci in fish collected from three stream-spawning and two beach-spawning sites. No morphological traits consistently distinguished the reproductive ecotypes with the exception of the number of anal fin rays which was greater in stream-spawning kokanee. Four of 18 allozyme loci screened were polymorphic, but no significant allele frequency differences were detected among populations within ecotypes or between ecotypes. Similarly, allele frequencies at two minisatellite DNA loci were not significantly different among populations or between ecotypes. By contrast, significant differences in the frequencies of mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) haplotypes were detected between stream- and beach-spawners, but not among populations within ecotypes. Further, two RFLPs that distinguished stream- and beach-spawning adults were found in juvenile kokanee sampled from the limnetic zone of Okanagan Lake. The two mtDNA RFLPs and a d-loop sequence variant appear to be unique to Okanagan Lake kokanee because we did not observe these haplotypes in sockeye salmon and kokanee sampled outside of Okanagan Lake. Our data suggest that: (i) there is restricted female-mediated gene flow between stream- and beach-spawning kokanee in Okanagan Lake, (ii) the forms have diverged within the lake basin since the retreat of the Wisconsinian glaciers (< ≊ 11 000 years ago), and (iii) distinct reproductive niches may promote divergence in north temperate freshwater fish faunas.     

Intrinsic reproductive isolation between Trinidadian populations of the guppy, Poecilia reticulata

Although Trinidadian populations of the guppy, Poecilia reticulata, show considerable adaptive genetic differentiation, they have been assumed to show little or no reproductive isolation. We tested this assumption by crossing Caroni (Tacarigua River) and Oropuche (Oropuche R.) drainage populations from Trinidad's Northern Range, and by examining multiple aspects of reproductive compatibility in the F1, F2 and BC1 generations. In open-aquarium experiments, F1 males performed fewer numbers of mating behaviours relative to parental population controls. This is the first documentation of hybrid behavioural sterility within a species, and it suggests that such sterility may feasibly be involved in causing speciation. The crosses also uncovered hybrid breakdown for embryo viability, brood size and sperm counts. In contrast, no reductions in female fertility were detected, indicating that guppies obey Haldane's rule for sterility. Intrinsic isolation currently presents a much stronger obstacle to gene flow than behavioural isolation, and our results indicate that Trinidadian populations constitute a useful model for investigating incipient speciation.     

Relationship between fertilization success and the number of milt donors in rainbow smelt Osmerus mordax (Mitchell): implications for population growth rates

A series of artificial fertilizations using rainbow smelt Osmerus mordax were conducted, each designed to assess the fertilization success of a female's eggs with milt from one to five males. Mean egg fertilization success increased and variance in fertilization success decreased when more males contributed to a constant volume of milt. This may indicate the presence of an Allee effect. The results suggest that potential changes in egg fertilization success due to a decrease in the number of males, in addition to changes in egg production due to a decrease in the number of females, can affect estimates of maximum sustainable harvest rates, and recovery of depleted populations.     

Morphological and genetic differentiation in anadromous smelt Osmerus mordax (Mitchill): disentangling the effects of geography and morphology on gene flow

Morphological analyses were combined with genetic analyses at nine microsatellite loci to examine the determinants of gene flow at 21 spawning locations of rainbow smelt Osmerus mordax along the east coast of Canada. Associations between morphology, geography and gene flow were examined using a computational geometric approach and partial Mantel tests. Significant barriers to gene flow and discontinuities in morphology were observed between Newfoundland and mainland Canada, as well as within Newfoundland samples. On regional scales, contrasting patterns were present with restricted gene flow between Newfoundland populations ( F _ST= c . 0·11) and high gene flow between mainland populations ( F _ST= c . 0·017). Within Newfoundland populations, geographic distance was significantly associated with gene flow ( r = 0·85, P < 0·001) contrasting mainland samples where gene flow was most associated with phenotypic divergence ( r = 0·33, P < 0·001). At large spatial scales, weak ( r = 0·19, P = 0·02) associations between gene flow and geographic distance were observed, and moderate associations were also observed between gene flow and morphology ( r = 0·28, P < 0·001). The presence of significant genetic isolation by distance in Newfoundland samples and the clear discontinuity associated with the Cabot Strait suggest geography may be the primary determinant of gene flow. Interestingly, the association between genetic and morphological divergence within mainland samples and overall, supports the hypothesis that gene flow may be moderated by morphological divergence at larger spatial scales even in high gene flow environments.     

Breeding ecology and evidence of reproductive isolation of a widespread stickleback fish (Gasterosteidae) in Nova Scotia, Canada

A stickleback with brilliant white dorsal breeding colours is widely distributed in north-eastern Nova Scotia, Canada, where it often breeds sympatrically with the threespine stickleback, Gasterosteus aculeatus. Breeding males are highly conspicuous and visible at distances of 20 m or more whereas sympatric G. aculeatus are cryptic and difficult to detect even at 2 m. The white stickleback nests only above the substrate in filamentous algae, where G. aculeatus nests only on the substrate. The white stickleback is smaller in size and more terete than G. aculeatus , but it is morphologically similar in having a complete row of lateral plates and similar lateral plate and gill raker numbers. The white stickleback occurs only in environments where there are filamentous algae (which appears to be an obligatory nesting substrate) and where the water is clear, saline and relatively still. Female choice tests in the laboratory show that the white stickleback is reproductively isolated from G. aculeatus , and field observations on natural spawning support this conclusion. We suggest that the bright breeding colouration may have evolved through sexual selection and/or to advertise unprofitability to predators.     

Post-zygotic reproductive isolation between sympatric taxa in the Chamaecrista desvauxii complex (Leguminosae-Caesalpinioideae)

BACKGROUND AND AIMS: Differences in the mating systems and the mechanisms of reproductive isolation between Chamaecrista desvauxii var. graminea and C. desvauxii var. latistipula were examined in the Chapada Diamantina, Brazil. These taxa occur sympatrically, and their populations demonstrate marked morphological differences. The objective of the present work was to determine if reproductive isolation mechanisms exist between these two populations of C. desvauxii, and to determine the influence of these putative mechanisms on their genetic differentiation. METHODS: Field observations were made of floral biology, phenology and floral visitation, and experiments on intra- and interpopulation pollination and germination rates of the resultant seeds were performed. A genetic examination of the populations was undertaken using four allozyme loci. KEY RESULTS: The varieties examined demonstrated overlapping of flowering periods during the months of June to September. The main pollinator for both varieties was the bee Bombus brevivillus. Both varieties are self-compatible, and a large number of fruits are formed in cross-pollinations with high seed germination rates. Inter-taxa pollinations result in high levels of fruit production, but no seeds are formed. Two of the four loci examined were diagnostic for the varieties, and exclusive high-frequency alleles were encountered at the other loci, leading to a high genetic distance between the two populations (0.495). CONCLUSIONS: Pre-zygotic barriers were not found between the two varieties, and these remain isolated due to post-zygotic events. The two varieties demonstrate marked differences in their morphology, floral biology, phenology and genetic make-up, all of which indicate that they should be treated as two distinct species. A complete revision involving the other varieties of the C. desvauxii complex will be necessary in order to define these two taxa formally.     

Genetic analysis of sympatric char populations in western Alaska: Arctic char (Salvelinus alpinus) and Dolly Varden (Salvelinus malma) are not two sides of the same coin

The North Pacific Ocean has been of great significance to understanding biogeography and speciation in temperate faunas, including for two species of char (Salmonidae: Salvelinus) whose evolutionary relationship has been controversial. We examined the morphology and genetics (microsatellite and mitochondrial DNA) of Arctic char (Salvelinus alpinus) and Dolly Varden char (Salvelinus malma) in lake systems in western Alaska, the eastern and western Arctic, and south of the Alaskan Peninsula. Morphologically, each lake system contained two forms: one (Arctic char) largely confined to lake habitats and characterized by greater numbers of pyloric caeca, gill rakers, and shallower bodies, and another (Dolly Varden) predominated in adjacent stream habitats and was characterized by fewer pyloric caeca, gill rakers, and deeper bodies. MtDNA partial (550 bp) d-loop sequences of both taxa were interspersed with each other within a single 'Bering' clade and demographic inferences suggested historical gene flow from Dolly Varden to Arctic char had occurred. By contrast, the taxa were strongly differentiated in sympatry across nine microsatellite loci in both lakes. Our data show that the two taxa are highly genetically distinct in sympatry, supporting their status as valid biological species, despite occasional hybridization. The interaction between these species highlights the importance of the North Pacific, and Beringia in particular, as an evolutionary wellspring of biodiversity.     

Reproductive isolation among deep-water cichlid fishes of Lake Malawi differing in monochromatic male breeding dress

Male nuptial colour hues are important for the maintenance of reproductive isolation among cichlid fish species, and environmental changes that lead to narrower light spectra can lead to hybridization. However, cichlid species can naturally co-occur in narrow light spectrum habitats, such as turbid shallow lakes and the deep benthic zones of African rift lakes. Closely related species from narrow light spectrum habitats tend to differ little in the palette of male nuptial colours, thus for these taxa differences in colour patterns may be more important than differences in colour hue for species recognition. To investigate this hypothesis we examined morphometric and genetic differentiation among males of four sympatric putative species within the deep-water genus Diplotaxodon. These taxa live in a narrow-light spectrum environment where only blue light is present, and males differ primarily in 'monochromatic' black, white and silver patterning of the body and fins. Significant genetic differentiation was present among taxa in both microsatellite DNA and mitochondrial DNA, including one pair with no significant morphometric differentiation. Thus, these taxa represent reproductively isolated biological species, a result consistent with male nuptial patterning being important for species recognition and assortative mating. As such, we suggest that narrow-light spectra need not always represent barriers to effective visually mediated mate recognition.     

Mechanisms of incomplete prezygotic reproductive isolation in an intertidal snail: testing behavioural models in wild populations

Two morphs (ecotypes) of the marine snail Littorina saxatilis coexist along Galician exposed rocky shores. They hybridize, but gene flow is impeded by a partial prezygotic reproductive barrier, and we have earlier suggested that this is a case of incipient sympatric speciation. To assess the mechanisms of prezygotic reproductive isolation, we estimated deviations from random mating (sexual selection and sexual isolation) of sympatric snails in 13 localities on the shore, and performed mate choice experiments in the laboratory. We also investigated the microdistribution of both morphs over patches of barnacles and blue mussels in the hybridization zone. We used computer simulations to separate the mechanisms contributing to reproductive isolation. On the shores sampled, male–female pairs were strongly assortative both with respect to morphs (mean Yule's V = 0.77) and size (mean Pearson's r = 0.47). In the laboratory, males of both morphs mounted other snails and mated other males and juveniles at random. However, mature females of equal sizes mated assortatively with respect to morph. The two morphs were nonrandomly distributed over barnacle and mussel patches in the hybridization zone. Monte Carlo simulations showed that this microdistribution could explain about half the morph and size relationships in male–female pairs, while a simple rejection mechanism, rejecting the first 1–3 mates if they were of contrasting morphs, accounted for the remaining part of the reproductive isolation, and for parts of the size relationships found between mates. A size discriminant mate choice mechanism may also, to a lesser extent, contribute to the sexual isolation. Sexual selection was observed for female size (larger ones being favoured) and among certain morphs, but distinct biological mechanisms may cause these processes.     

Molecular genetic markers provide no evidence for reproductive isolation among retreat building phenotypes of the net-spinning caddisfly Macrostemum carolina

Larvae of the stream-dwelling, filter-feeding caddisfly Macrostemum carolina construct silken catchnets within protective retreats. In the Savannah River, M. carolina individuals make three different retreats, each with a distinct water entrance hole: (i) at the end of a silken tube; (ii) with a approximately 180 degrees silken backstop; and (iii) flush with the top of the retreat. To resolve whether these different retreats represent alternative behavioural phenotypes within a single panmictic population or fixed phenotypes within three genetically distinct populations or species, we compared the allele frequencies at three polymorphic nuclear loci (allozyme electrophoresis for Gpi, Mpi and Pgm) and the mitochondrial DNA (mtDNA) haplotype frequencies among individuals displaying the three retreat morphs. We also calculated pairwise exact tests of population differentiation using the allozyme and mtDNA allele frequencies. No significant genetic differentiation was detected among caddisflies exhibiting the different retreat morphs. Therefore, these morphs apparently represent a single panmictic population in the Savannah River. Consequently, additional study is required to assess whether this retreat polymorphism is a phenotypically plastic trait under conditional control, or is mediated by alternative alleles at a Mendelian gene or genes (or a combination of the two).     

Sexual conflict does not drive reproductive isolation in experimental populations of Drosophila pseudoobscura

Sexual conflict has been predicted to drive reproductive isolation by generating arbitrary but rapid coevolutionary changes in reproductive traits among allopatric populations. A testable prediction of this proposal is that allopatric populations experiencing different levels of sexual conflict should exhibit different levels of reproductive isolation. We tested this prediction using experimentally evolved populations of the promiscuous Drosophila pseudoobscura. We manipulated sexual conflict by enforcing either monogamy, maintaining natural levels of promiscuity, or elevating promiscuity. Within each treatment, we carried out sympatric and allopatric crosses using replicated populations and examined pre-zygotic (number of mating pairs, mating speed and copulation duration) and post-zygotic (hybrid inviability and sterility) indicators of reproductive isolation. After 50 generations of selection, none of the measures conformed to predictions of sexual conflict driving reproductive isolation. Our results cannot be explained by lack of genetic variation or weak selection and suggest that sexual conflict may not be a widespread engine of speciation.     

Lack of reproductive isolation between populations of the grasshopper Myrmeleotettix maculatus across a steep cline in B-chromosome frequency

The rapid cline in B-chromosome frequency in populations of the grasshopper Myrmeleotettix maculatus in East Anglia, reported by Hewitt & Brown (1970), has not been satisfactorily explained. Test were made of the hypothesis that the cline represented the contact of two groups of populations with differently co-adapted genomes, between which there was reproductive isolation. There was no evidence that females in a cage discriminated between the courtship of males from the same or the opposite side of the cline. Progeny from crosses between populations on opposite sides of the cline showed no reduced survival to hatching, survived to adulthood and had normal meiosis. Thus the evidence available does not support the hypothesis that the cline is the interface of two incompatible genomes. The problems of other models accounting for the cline are discussed.     

Quantification of virus genes provides evidence for seed-bank populations of phycodnaviruses in Lake Ontario,Canada

Using quantitative PCR, the abundances of six phytoplankton viruses DNA polymerase (polB) gene fragments were estimated in water samples collected from Lake Ontario, Canada over 26 months. Four of the polB fragments were most related to marine prasinoviruses, while the other two were most closely related to cultivated chloroviruses. Two Prasinovirus-related genes reached peak abundances of >1000 copies ml⁻¹ and were considered ‘high abundance'', whereas the other two Prasinovirus-related genes peaked at abundances <1000 copies ml⁻¹ and were considered ‘low abundance''. Of the genes related to chloroviruses, one peaked at ca 1600 copies ml⁻¹, whereas the other reached only ca 300 copies ml⁻¹. Despite these differences in peak abundance, the abundances of all genes monitored were lowest during the late fall, winter and early spring; during these months the high abundance genes persisted at 100–1000 copies ml⁻¹ while the low abundance Prasinovirus- and Chlorovirus-related genes persisted at fewer than ca 100 copies ml⁻¹. Clone libraries of psbA genes from Lake Ontario revealed numerous Chlorella-like algae and two prasinophytes demonstrating the presence of candidate hosts for all types of viruses monitored. Our results corroborate recent metagenomic analyses that suggest that aquatic virus communities are composed of only a few abundant populations and many low abundance populations. Thus, we speculate that an ecologically important characteristic of phycodnavirus communities is seed-bank populations with members that can become numerically dominant when their host abundances reach appropriate levels.     

Rapid genetic divergence in postglacial populations of threespine stickleback (Gasterosteus aculeatus): the role of habitat type, drainage and geographical proximity

The goal of this study was to evaluate the role of common ancestry, and of geographical or reproductive isolation, in genetic divergence in populations of threespine sticklebacks (Gasterosteus aculeatus). Using seven DNA microsatellite loci we compared the effects of habitat type, drainage system and geographical proximity on genetic distance among 16 populations situated in an area in Schleswig-Holstein (Germany) that became deglaciated approximately 12 000 years ago. Stickleback population structure correlated only weakly with drainage system, whereas the primary divergence was among habitat types. Phylogenetic analysis revealed that lake (n = 7) and river (n = 5) populations formed two distinct clades (Cavalli-Sforza's and Edwards' chord distance, 82-100% bootstrap support) at approximately equal genetic distances to a third clade, comprising putative estuarine (n = 4) ancestors. Allele frequencies in lake and river populations represented different subsets of the genetically more diverse estuarine populations. In nested amovas approximately twice the genetic variance was distributed among lake vs. river vs. estuarine populations as compared with the combined effects of drainage system and geographical distance. Limited gene flow between habitat types must have been established after postglacial colonization, suggesting ecological hybrid inferiority or behavioural mating barriers between ecotypes. Within estuarine and lake populations, population differentiation followed an isolation-by-distance model. Given the high observed heterozygosities within the 16 study populations (HO = 0.65-0.87), the mean divergence between lake and river population pairs (FST = 0.18 +/- 0.007) would be reached after 300-6000 generations in a stepwise mutation model, depending on the size of N(e). This demonstrates both the utility of hypervariable microsatellites for detecting recent population divergences and the danger of operating at temporal or spatial scales which are beyond their resolution.     

Reproductive isolation and genetic differentiation of ferox trout from sympatric brown trout in Loch Awe and Loch Laggan, Scotland

Molecular marker studies reported here, involving allozymes, mitochondrial DNA and microsatellites, demonstrate that ferox brown trout Salmo trutta in Lochs Awe and Laggan, Scotland, are reproductively isolated and genetically distinct from co-occurring brown trout. Ferox were shown to spawn primarily, and possibly solely, in a single large river in each lake system making them particularly vulnerable to environmental changes. Although a low level of introgression seems to have occurred with sympatric brown trout, possibly as a result of human-induced habitat alterations and stocking, ferox trout in these two lakes meet the requirements for classification as a distinct biological, phylogenetic and morphological species. It is proposed that the scientific name Salmo ferox Jardine, 1835 , as already applied to Lough Melvin (Ireland) ferox, should be extended to Awe and Laggan ferox.     

Morphological differences between,and reproductive isolation of,two populations of the jellyfish Cyanea in Long Island Sound,USA

Two populations of Cyanea, one in the Niantic River estuary and the other in the adjacent Niantic Bay portion of Long Island Sound, were studied for more than a decade. The estuary and the bay are connected by a narrow channel: this and hydrographic features constrain transport between them. Specimens from each site exhibit morphological and seasonal differences comparable to those which distinguish C. capillata from C. lamarckii in European waters. Reproducing River medusae never cooccurred with reproducing Bay medusae. Estuarine jellyfish are sometimes found in the Bay, but reproducing individuals have not been seen there. Reproductively mature Bay jellyfish occasionally appear in the River, but benthic samples show that they do not deposit planulae at this site and thus do not contribute to the next generation of medusae in the River. Such differences suggest an absence of gene flow between these two populations, indicating that the River medusae and Bay medusae might be separate species.     

The population genetics of the apple maggot fly,Rhagoletis pomonella and the snowberry maggot,R. zephyria: implications for models of sympatric speciation

True fruit flies belonging to the Rhagoletis pomonella (Diptera:Tephritidae) sibling species complex possess several attributes consistent with a history of sympatric divergence via host plant shifts. Here, we investigate whether hybridization and genetic introgression is occurring between two members of the group, Rhagoletis pomonella (Walsh), whose primary hosts are domestic apples (Malus pumila) and hawthorns (Crataegus spp., and R. zephyria (Snow) whose host is snowberries (Symphoricarpos spp.). These flies are important because they appear to be at a transition stage between taxa reproductively isolated solely on the basis of host plant-related adaptations and those that have evolved additional non-host dependent sterility and inviability. Observing extensive hybridization and introgression between R. pomonella and R. zephyria would have major repercussions for current models of sympatric speciation.In a survey of allozyme and mitochondrial DNA (mtDNA) variation for 1105 flies collected throughout the northern United States we found two results suggesting that low level hybridization occurs between R. pomonella and R. zephyria. (1) Two flies reared from snowberries and one fly reared from hawthoms had genotypes indicative of them being R. pomonella and R. zephyria, respectively. Rhagoletis pomonella and R. zephyria adults may therefore occasionally frequent each others host plant, providing the opportunity for hybridization. (2) Four flies collected from hawthorns and one from snowberries had genotypes that made them likely to be F1 hybrids.Likelihood analysis revealed the data were also consistent with an hypothesis of shared ancestral alleles (i.e., the pattern of genetic variation could also be explained by R. pomonella and R. zephyria sharing alleles/haplotypes whose origins date to a common ancestor). We estimated that, in the absence of interspecific mating, random assortment of genes within R. pomonella and R. zephyria populations would produce an average of 5.4 flies with genotypes suggesting they were F1 hybrids – a number equivalent to the 5 putative F1 hybrids observed in the study. Our results therefore underscore the difficulty in distinguishing between hypotheses of low level introgression and shared ancestral polymorphism. But even if hybridization is occurring, the data suggest that it is happening at a very low and probably evolutionarily insignificant level (perhaps 0.09% per generation), consistent with sympatric speciation theory. Future tests are discussed that could help resolve the hybridization issue for R. pomonella and R. zephyria.     

Behavioural,ecological and genetic evidence confirm the occurrence of host‐associated differentiation in goldenrod gall‐midges

Host‐associated differentiation (HAD) is considered a step towards ecological speciation and an important mechanism promoting diversification in phytophagous insects. Although the number of documented cases of HAD is increasing, these still represent only a small fraction of species and feeding guilds among phytophagous insects, and most reports are based on a single type of evidence. Here we employ a comprehensive approach to present behavioural, morphological, ecological and genetic evidence for the occurrence of HAD in the gall midge Dasineura folliculi (Diptera: Cecidomyiidae) on two sympatric species of goldenrods (Solidago rugosa and S. gigantea). Controlled experiments revealed assortative mating and strong oviposition fidelity for the natal‐host species. Analysis of mitochondrial DNA showed an amount of genetic divergence between the two host‐associated populations compatible with cryptic species rather than host races. Lower levels of within‐host genetic divergence, gall development and natural‐enemy attack in the S. gigantea population suggest this is the derived host.