PERMANENT GENETIC RESOURCES: Microsatellite DNA primers for the candy darter, Etheostoma osburni and variegate darter, Etheostoma variatum, and cross-species amplification in other darters (Percidae)

In order to investigate a potential hybrid zone between the candy darter, Etheostoma osburni, and variegate darter, Etheostoma variatum, and examine population variation within E. osburni, a suite of primers for 15 polymorphic microsatellite loci were developed. The average number of alleles per locus was 5.5 in E. osburni and 7.6 in E. variatum, and the average observed heterozygosities were 62.5% and 71.4%, respectively. There were no deviations from Hardy-Weinberg equilibrium and no observed linkage disequilibrium after Bonferroni correction. The utility of these primers was also tested in 11 species of darters representing all four genera of darters. Success of cross-species amplification was largely consistent with phylogenetic relationships of darters.     

Molecular phylogeny of the snubnose darters, subgenus Ulocentra (genus Etheostoma, family Percidae)

Snubnose darters comprise one of the largest subgenera of the percid genus Etheostoma. Many species are described based on differences in male breeding coloration. Few morphological synapomorphies have been proposed for the subgenus and their relatives, making it difficult to delineate monophyletic clades. The phylogenetic relationships of the 20 snubnose darter species of the subgenus Ulocentra and 11 members of its proposed sister subgenus Etheostoma were investigated with partial mitochondrial DNA sequences including 1033 bp encompassing the entire mitochondrial control region, the tRNA-Phe gene, and part of the 12S rRNA gene. Two hypotheses on the relationship and monophyly of the two subgenera were evaluated. Both maximum-parsimony and neighbor-joining analyses supported monophyly of the subgenus Ulocentra and resolved some species-level relationships. The banded darter, E. zonale, and its sister taxon, E. lynceum, were not closely related to the snubnose darters and appear to be diverged from the other members of the subgenus Etheostoma, fitting their former distinction as the recognized subgenus Nanostoma. The sister group to Ulocentra appears to be a restricted species assemblage within the subgenus Etheostoma containing E. blennioides, E. rupestre, E. blennius, and the E. thalassinum species group. The placement of the harlequin darter, E. histrio, is problematic, and it may represent a basal member of Ulocentra or of the restricted subgenus Etheostoma. Despite recent estimates of divergence times between nominal Ulocentra taxa, each species exhibits its own unique set of mtDNA haplotypes, providing no direct evidence for current genetic exchange between species. The nominal taxa of snubnose darters thus appear to be evolving independently from each other and therefore constitute valid species under the Phylogenetic Species Concept.     

Phylogeography of the greenside darter complex, Etheostoma blennioides (Teleostomi: Percidae): a wide-ranging polytypic taxon

The greenside darter, Etheostoma blennioides (Teleostomi: Percidae), is a wide-ranging polytypic taxon that occurs throughout eastern North America. A previous morphological study recognized four subspecies (blennioides, newmanii, gutselli, and pholidotum), several morphological races, and three zones of morphological intergradation. We generated complete cytochrome b (1140bp) sequence data for 51 individuals from across the range of the greenside darter inclusive of all of the currently recognized taxa to assess genetic variation and taxonomic boundaries. Both maximum parsimony and mixed model Bayesian analyses resulted in two strongly supported deeply divergent clades including (1) a Tennessee River drainage clade, and (2) an Ohio River and Great Lakes basins, Interior Highlands, and Atlantic slope clade. Etheostoma blennius, a closely related congener, nested within the Tennessee River clade of E. blennioides, rendering the complex paraphyletic. Test of alternative topologies failed to support the current taxonomic designations. The inclusion of nuclear sequence data from intron 1 of the S7 ribosomal protein (523bp) from a subset of the populations was included to independently test whether the currently recognized taxa conform to distinct evolutionary lineages and also to clarify potential issues associated with ancestral hybridization. Although the nuclear data was less variable than the mitochondrial data, the monophyly of several of the subspecies could not be rejected.     

Phylogenetic relationships among Boleosoma darter species (Percidae: Etheostoma)

Darters represent a species rich group of North American freshwater fishes studied in the context of their diverse morphology, behavior, and geographic distribution. We report the first molecular phylogenetic analyses of the Boleosoma darter clade that includes complete species sampling. We estimated the relationship among the species of Boleosoma using DNA sequence data from a mitochondrial (cytochrome b) and a nuclear gene (S7 ribosomal protein intron 1). Our analyses discovered that the two Boleosoma species with large geographic distributions (E. nigrum and E. olmstedi) do not form reciprocally monophyletic groups in either gene trees. Etheostoma susanae and E. perlongum were phylogenetically nested in E. nigrum and E. olmstedi, respectively. While analysis of the nuclear gene resulted in a phylogeny where E. longimanum and E. podostemone were sister species, the mitochondrial gene tree did not support this relationship. Etheostoma vitreum was phylogenetically nested within Boleosoma in the mitochondrial DNA and nuclear gene trees. Our analyses suggest that current concepts of species diversity underestimate phylogenetic diversity in Boleosoma and that Boleosoma species likely provide another example of the growing number of discovered instances of mitochondrial genome transfer between darter species.     

Characterization of a contemporaneous hybrid zone between two darter species (Etheostoma bison and E. caeruleum) in the Buffalo River System

Hybrid zones have long intrigued evolutionary biologists and provide a natural laboratory to explore the evolution of reproductive isolation (speciation). Molecular characterization of hybrid zone dynamics can provide insight into the strength of reproductive isolation as well as the underlying evolutionary processes shaping gene flow. Approximately one-third of darter species naturally hybridize making this species-rich North American freshwater teleost fish clade an ideal system to investigate the extent and direction of hybridization. The objective of this study was to use diagnostic microsatellite markers to calculate genetic hybrid index scores of two syntopic, but distantly related darter species, Etheostoma bison and Etheostoma caeruleum. A combination of hybrid index scores, assignment tests, and mitochondrial haplotype profiles uncovered mixed ancestry in approximately 6 % of sampled adult individuals, supporting contemporaneous hybridization that was previously undocumented in E. bison. Moreover, hybrids were not limited to the F₁ generation, but encompassed the entire suite of hybrid categories (F₁, F₂ and backcross hybrids). The low number of hybrids assigned to each hybrid category represents a bimodal hybrid zone, suggesting reproductive isolation is strong (but incomplete) and also advocates for the ability of hybrids to produce second-generation hybrids and backcross into both parental species, mediating introgression across species boundaries. To this end, cytonuclear profiles of the sampled parental species and hybrids were consistent with bidirectional gene flow, although there was an overall trend of asymmetric hybridization between E. caeruleum females and E. bison males. The spatiotemporal variation in hybridization rates and resulting cytonuclear patterns expanded on in this study provide a comparative genetic framework on which future studies can begin to elucidate the underlying processes that not only generate a mosaic hybrid zone, but maintain the distinctness of species in the face of gene flow.     

Landscape genetic patterns of the rainbow darter Etheostoma caeruleum: a catchment analysis of mitochondrial DNA sequences and nuclear microsatellites

Catchment population structure and divergence patterns of the rainbow darter Etheostoma caeruleum (Percidae: Teleostei), an eastern North American benthic fish, are tested using a landscape genetics approach. Allelic variation at eight nuclear DNA microsatellite loci and two mitochondrial DNA regions [cytochrome (cyt) b gene and control region; 2056 aligned base pairs (bp)] is analysed from 89 individuals and six sites in the Lake Erie catchment (Blanchard, Chagrin, Cuyahoga and Grand Rivers) v. the Ohio River catchment (Big Darby Creek and Little Miami River). Genetic and geographic patterning is assessed using phylogenetic trees, pair‐wise F_ST analogues, AMOVA partitioning, Mantel regression, Bayesian assignment, 3D factorial correspondence and barrier analyses. Results identify 34 cyt b haplotypes, 22 control region haplotypes and 137 microsatellite alleles whose distributions demonstrate marked genetic divergence between populations from the Lake Erie and Ohio River catchments. Etheostoma caeruleum populations in the Lake Erie and Ohio River catchments diverged c. 1·6 mya during the Pleistocene glaciations. Greater genetic separations characterize the Ohio River populations, reflecting their older habitat age and less recent connectivity. Divergence levels within the Lake Erie catchment denote more recent post‐glacial origins. Notably, the western Lake Erie Blanchard River population markedly differs from the three central basin tributary samples, which are each genetically distinguishable using microsatellites. Overall relationships among the Lake Erie sites refute a genetic isolation by geographic distance hypothesis. Etheostoma caeruleum populations thus exchange few genes and have low migration among tributaries and catchments.     

Incomplete behavioural isolation and asymmetric female preference in darter sister species (Percidae: Etheostoma)

In this study, female preferences and behavioural isolation were estimated in a pair of allopatric sister species, Etheostoma duryi and Etheostoma flavum. Dichotomous mate preference trials were conducted to determine whether females prefer to associate with conspecific over heterospecific males and free‐spawning assays were conducted to determine whether those preferences translated into behavioural isolation. Dichotomous mate choice trials revealed asymmetric female preference, as female E. flavum preferred conspecific males, whereas female E. duryi showed no preference. Free‐spawning assays indicated that behavioural isolation remains incomplete between E. duryi and E. flavum (I_B = 0·19). In addition to female mating preferences, male behaviour also appeared to influence mating outcomes as male E. flavum consistently courted conspecific females more often in free‐spawning assays whereas male E. duryi did not. The data therefore suggest that despite marked divergence in male nuptial colour, divergence in female preferences between these species may not be sufficient to maintain species boundaries upon secondary contact. These results contrast with similar work in a sympatric darter species pair and may be explained by considering the contributions of reinforcement and differences in colour pattern as well as colour value.     

Isolation and identification of eight microsatellite loci in the Cherokee darter (Etheostoma scotti) and their variability in other members of the genera Etheostoma, Ammocrypta, and Percina

The Cherokee darter Etheostoma scotti is a federally threatened fish endemic to the Etowah River system of northwest Georgia. In order to analyse the population structure and genetic diversity of this fish, eight tetranucleotide microsatellite genetic markers were developed. The marker set was applied to 13 additional darter species to test cross-species amplification and polymorphism. Successful amplification was obtained for all eight loci in each of the 13 other species of darters, with between seven and eight polymorphic loci per species.     

Twenty-three microsatellite DNA loci for population genetic studies and parentage assignment in orangethroat darter, Etheostoma spectabile

The genus Etheostoma is a species-rich and ecologically important group of fishes in North America. The orangethroat darter (Etheostoma spectabile) is widely distributed and abundant in headwater streams throughout the central Midwest, and is an excellent model for ecological and mating system studies. We developed 23 novel, polymorphic, and independent microsatellite loci for E. spectabile. We found from two to 14 alleles per locus, and observed heterozygosities ranged from 0.39 to 1.0. These markers, in combination with others isolated from Etheostoma taxa, will be useful for ecological and evolutionary studies in the genus.     

Range expansion by invasion: genetic characterization of invasion of the greenside darter (Etheostoma blennioides) at the northern edge of its distribution

Species introductions in freshwater ecosystems are often complex processes, yet an understanding of the nature of the introduction can inform management and conservation actions. The greenside darter (Etheostoma blennioides), until recently a species of special concern, expanded its Canadian range and is now common and widespread in the Grand River watershed (GRW). This is despite there being no evidence of greenside darter in the GRW prior to 1990. The goal of this study was to genetically characterize the GRW greenside darter introduction. Greenside darter were sampled in the GRW, the three known native watersheds in Canada, and one site from Ohio. We measured genetic diversity and population structure, and tested for population bottlenecks using eight microsatellite loci. Genotype assignment was used to identify possible introduction sources. Populations in the GRW showed similar genetic diversity to native watershed populations with no evidence for recent or historical population bottlenecks. Genotype assignment showed that one of the Canadian watersheds and the Ohio site were not potential sources of the GRW greenside darter, whereas the Thames River watershed was the most likely source. Substantial population genetic structure exists among the sample sites in the GRW. Clearly, the current widespread and abundant distribution of the greenside darter in the GRW is not the result of recent expansion of an existing native population, but rather multiple introductions into at least three sites in the GRW, followed by rapid population growth. Although the GRW E. blennioides is introduced, it harbours considerable genetic diversity and represents an important northern range extension for this species.     

Characterization of eight polymorphic microsatellite DNA markers for the greenside darter,Etheostoma blennioides (Percidae)

The greenside darter, Etheostoma blennioides is a small benthic fish found in fast‐flowing streams in eastern North America. In Canada, this species is native to three, and introduced into one, Great Lakes tributaries in southwestern Ontario. It is currently listed as a species of Special Concern. To characterize population genetic structure and diversity in the Canadian populations of greenside darter, eight polymorphic microsatellite markers were developed for the species. The polymerase chain reaction primers were tested between 32 and 60 individuals from the Sydenham River and yielded a high number of alleles (four to 42 per locus), and observed heterozygosities ranging from 0.14 to 0.82.     

Alarm pheromone recognition in a Missouri darter assemblage

The a priori null hypotheses that responses to donor, orangethroat darter Etheostoma spectabile , johnny darter Etheostoma nigrum , logperch Percina caprodes , slenderhead darter Percina phoxocephala and blackside darter Percina maculata , extracts would be absent in the three test species, orangethroat darter, johnny darter and logperch, was rejected. This was because orangethroat and johnny darter responded with decreased activity to skin extracts derived from conspecifics and from other members of the assemblage. In contrast, logperch were notable by their lack of a detectable response to any species. Similarities and differences in habitat utilization are seen as a possible basis for these responses.     

Systematics and population genetics of the coldwater (Etheostoma ditrema) and watercress (Etheostoma nuchale) darters,with comments on the Gulf darter (Etheostoma swaini) (Percidae: subgenus Oligocephalus)

Current taxonomy of the Etheostoma asprigene species group recognizes four species, two of which (Etheostoma ditrema Ramsey and Suttkus and Etheostoma nuchale Howell and Caldwell) are restricted to isolated springs and spring-fed stream systems above the Fall Line of the Mobile Basin of Alabama, Georgia, and Tennessee. Previous studies of morphological and biochemical variation between disjunct populations of E. ditrema support the presence of multiple independent lineages. Unfortunately, the lack of phylogenetic methodology has made it impossible to distinguish historically differentiated non-sister taxa from currently intergrading sister lineages.The current study examines members of the E. asprigene species group, particularly E. ditrema and E. nuchale, from throughout their respective ranges for products of 44 presumptive gene loci. Highly significant levels of genetic subdivision (F_IT) and genetic differentiation (F_ST) are observed both within and between species of this group, supporting the presence of previously unrecognized diversity within this clade. Phylogenetic evaluation of polyallelic loci supports the specific recognition of E. nuchale and three taxa currently under the name E. ditrema: nominal E. ditrema (upper Coosa River system), E. sp. cf. E. ditrema (Coldwater Spring), and E. sp. cf. E. ditrema (central Coosa River system) based on either fixed allelic products or significant allele frequency differences. The Gulf darter, Etheostoma swaini, is also identified as a composite of at least three diagnosable evolutionary species, two of which (Black Warrior River system above and Cahaba River system below the Fall Line) are more closely related to E. nuchale and species of the E. ditrema complex than to remaining E. swaini. It is recommended that future conservation efforts with these highly geographically restricted and imperiled species incorporate these patterns of genetic subdivision into management and recovery plans.     

Early development of johnny darter,Etheostoma nigrum,and fantail darter,E. flabellare,with a discussion of its ecological and evolutionary aspects

Synopsis Fantail darters,Etheostoma flabellare, have larger eggs with greater yolk volume than johnny darters,E. nigrum, and have a longer embryo period. This longer embryo period is associated with accelerated finfold differentiation, producing a larger, better formed individual at the onset of exogenous feeding. Developmental differences among these two species and the rainbow darter,Etheostoma caeruleum, primarily in timing of ontogenetic events, are described in detail. These differences in timing are considered largely early developmental phenomena as they affect embryos, larvae, and aievins but apparently not juveniles or adults. Demographic aspects of early life history were assessed using data from studies of Ontario streams. Differences in early survival among species appear to play an important role in recruitment of mature females, but cannot be attributed to parental care or egg size differences.     

Microsatellite DNA markers for the rainbow darter,Etheostoma caeruleum (Percidae), and their potential utility for other darter species

The rainbow darter Etheostoma caeruleum is a small fish in the perch family (Percidae) that is adapted to fast‐flowing streams in eastern North America. It is relatively sensitive to habitat degradation and is widely used as a sentinel of stream condition. To provide a complementary tool for assessing the integrity of stream ecosystems, 16 highly polymorphic tetranucleotide microsatellite markers were identified for these darters. Between four and 16 loci were found to be useful in five congeneric species. These markers will be useful for characterizing population genetic structure and diversity of rainbow darters and related fishes.     

Nest defense and aggressive interactions between a small benthic fish (the johnny darter Etheostoma nigrum) and crayfish

Synopsis Aggression by nest-guarding male johnny darters, Etheostoma nigrum, against intruding crayfish was investigated in laboratoy experiments and field observations. In the laboratory, darter success in chasing crayfish, Orconectes rusticus, from the nest site was inversely related to crayfish size. Small crayfish (less than 15 mm carapace length) were routinely evicted from the nest area by nips directed at the posterior end of the abdomen. Although such aggressive behavior was less successful against larger crayfish, even the largest crayfish tested (carapace length 30–32 mm) were chased from the nest area in 33% of the trials. Those large crayfish that entered nests often remained despite repeated attacks by the male johnny darter and egg predation was observed. In a small Ohio stream, openings to johnny darter nests were generally between 7 and 13 mm. Thus crayfish with a carapace height greater than 13 mm (corresponding to a carapace length greater than 29 mm) would not be able to enter johnny darter nests. In field observations, male Johnny darters successfully defended nests against another crayfish species (Orconectes sanborni, carapace length 12–29 mm). Together, aggressive behavior and small size of nest entrances allow the johnny darter to successfully reproduce in areas with abundant crayfish. The Unit is sponsored jointly by the United States Fish and Wildlife Service, The Ohio Department of NaturalResources, and The Ohio State University.     

Early development of the rainbow darter,Etheostoma caeruleum,according to the theory of saltatory ontogeny

Synopsis The early development of rainbow darter, Etheostoma caeruleum, was examined from an ecological perspective. Steps and thresholds of ontogeny to completion of body squammation are defined, and related to environmental factors. Rainbow darter eggs are about 2 mm diameter, considerably larger than those of related logperch (Percina caprodes). The embryonic vitelline respiratory plexus is much more extensive. The pelagic interval characteristic of logperch and ancestral percids is eliminated and onset of exogenous feeding is delayed. The larger larvae of the rainbow darter can begin feeding directly on aquatic insects, and complete their life cycle in streams. Therefore, shifts in the timing of important thresholds (e.g. exogenous feeding) are ecologically important. Furthermore, early maturation and/or delayed bone and scale formation may be responsible for reductions in the lateralis system and scalation in this and other darter species.     

Reproductive isolation between two darter species is enhanced and asymmetric in sympatry

Robust reproductive isolation was found between the rainbow darter Etheostoma caeruleum and the orangethroat darter Etheostoma spectabile, as more offspring were produced when conspecific males and females were crossed as compared with heterospecific crosses. Furthermore, fewer eggs resulted from heterospecific crosses involving sympatric E. spectabile females than those using allopatric E. spectabile females, while a similar pattern was not observed in heterospecific crosses using E. caeruleum females. These results suggest that reinforcement, i.e. selection for pre‐zygotic reproductive barriers driven by reduced hybrid fitness, may have contributed to the evolution and maintenance of reproductive barriers between these potentially hybridizing species in sympatry.     

Reinforcement in the banded darter Etheostoma zonale: The effect of sex and sympatry on preferences

Reinforcement occurs when selection against hybrid offspring strengthens behavioral isolation between parental species and may be an important factor in speciation. Theoretical models and experimental evidence indicate that both female and male preferences can be strengthened upon secondary contact via reinforcement. However, the question remains whether this process is more likely to affect the preferences of one sex or the other. Males of polygynous species are often predicted to exhibit weaker preferences than females, potentially limiting the ability for reinforcement to shape male preferences. Yet, in darters (Percidae: Etheostoma), male preference for conspecific mates appears to arise before female preferences during the early stages of allopatric speciation, and research suggests that male, but not female, preferences become reinforced upon secondary contact. In the current study, we aimed to determine whether the geographically widespread darter species Etheostoma zonale exhibits a signature of reinforcement, by comparing the strength of preference for conspecific mates between populations that are sympatric and allopatric with respect to a close congener, E. barrenense. We examined the strength of preference for conspecifics for males and females separately to determine whether the preferences of one or both sexes have been strengthened by reinforcement. Our results show that both sexes of E. zonale from sympatric populations exhibit stronger conspecific preferences than E. zonale from allopatric populations, but that female preferences appear to be more strongly reinforced than male preferences. Results therefore suggest that reinforcement of female preferences may promote behavioral isolation upon secondary contact, even in a genus that is characterized by pervasive male mate choice.     

Microsatellite DNA primers for the endangered vermilion darter,Etheostoma chermocki,and cross‐species amplification in other darters (Percidae: Etheostoma)

The endangered vermilion darter (Etheostoma chermocki) is endemic to the Black Warrior River system in the Mobile Basin in Alabama. Restoration and conservation of this species require an understanding of its population genetic structure, which can be characterized using microsatellite DNA. Nine microsatellite loci were developed; eight loci were polymorphic. Although observed heterozygosity was lower than expected heterozygosity in most polymorphic loci, only one locus showed significant deviation from Hardy–Weinberg equilibrium. These nine markers were tested in an additional 24 species of Etheostoma and appear to have sufficient allelic variation to be useful in studies of population genetic structure.