We examined the genetic diversity on a microgeographic scale of Rhinichthys atratulus (Eastern Blacknose Dace) in Allyn Brook, a small tributary in the upper Coginchaug River drainage in Connecticut. By looking at gene flow on a microgeographic scale among populations that had no physical barriers to migration, we tested the null hypothesis that the populations should be homogeneous. We resolved seven polymorphic microsatellite loci and one mitochondrial gene, nd2, in three adjacent populations (<0.5 km) in Allyn Brook and compared these populations to the two closest populations (>5 km) in the Coginchaug River. A dam from the 1920’s in lower Allyn Brook has isolated Allyn-Brook populations from Coginchaug-River populations. Allyn Brook was selected because there are only three riffle habitats in the brook and, therefore, there can be no immigration from upstream populations. Each population has private ( i.e., unique) alleles and haplotypes, and there are significant genetic differences between all sites. The Allyn Brook populations are almost as different from one another as they are from the distant populations in the Coginchaug River from which they have been isolated for more than 80 years. These results point to in situ evolution and little migration or gene flow among populations on a microgeographic scale. This raises interesting questions for conservation of genetic diversity of stream fishes.
One of the prominent features of the Anthropocene has been the disruption of continuous habitat for the world’s biota. Understanding how fragmented landscape features affect the maintenance and production of genetic diversity across space and time is critical for conservation biology Anderson, Epperson, Fortin, Holderegger, James, Rosenberg, Scribner and Spear [
Population genetic structure and migration behavior of riverine fishes have also been studied for ecosystems in which no obvious physical barriers were present. Genetic variation among Poecilia reticulata (Guppy) in Trinidad correlated positively with geographical distance; populations separated by waterfalls were more genetically divergent than those that were not [
In our studies of the population structure and ecology of the small riffle-dwelling fish, Rhinichthys atratulus (Eastern Blacknose Dace), we have found large degrees of eco-genetic variation among populations in rivers in Connecticut and in the eastern United States [
The present study examines genetic differences among and between populations of R. atratulus on a microgeographic scale (
because there are no obvious physical barriers to gene flow and movement of fishes among the habitats should prevent the establishment of novel genetic diversity. This is critical because we must be able to distinguish the effects of a naturally patchy environment on a local level from anthropogenic fragmentation of ecosystems.
In this paper we: 1) compare the genetic diversity among R. atratulus from adjacent habitats that are isolated in a single small stream (see below); 2) we estimate the migration and mutation rate for each of the populations; 3) we estimate whether the number of individuals in the populations are stable; and 4) we compare the populations from the study sites (
Allyn Brook, a tributary of the Coginchaug River in central Connecticut, USA (
Physical and chemical characterizations of the three habitats are given in Appendix 1. Conductivity, pH, dissolved oxygen, and water temperature were measured using a Yellow Springs Instrument (YSI) probe Model 556 MPS. At three points along each riffle canopy cover was estimated using a GRS Densitometer. Stream flow velocity was estimated with a Price-Gurley meter at 60% of the depth. Flow and depth were recorded approximately every meter along the riffle. The riffle slope was measured using a tripod and surveyors rod with a vertical tolerance of ±5 cm. There were no significant differences among the three sites in physical or chemical characteristics.
Rhinichthys atratulus (
Individuals were collected in summer 2016 and 2017, with the number of individuals collected per site as follows: AB = 34; AB1 = 14; and AB2 = 27 (Appendix 1). Samples were collected via electrofishing. Upper caudal fins clips were
taken and stored in 95% ethanol for subsequent DNA extraction and analysis. Permissions for collecting and handling individuals for scientific study were governed by: CT Scientific Collection Permits SC - 13023 and SC - 17031; IACUC 2015 - 1212 - Chernoff - A, IACUC 2017 - 1212 - Chernoff - A.
DNA was extracted from fin clips using the DNeasy blood and tissue kit (QIAGEN Sciences, MD, USA) using the protocol from [
The primers and PCR protocol for amplification of 14 microsatellite loci from non-coding regions are from Kraczkowski and Chernoff [
DnaSP v5 10.01 was used to calculate nucleotide and haplotype diversity as well as neutrality tests Tajima’s D, Fu and Li’s D and Fu’s F [
H = ( N / N − 1 ) ( 1 − ∑ x i 2 ) (1)
where x i 2 is the frequency of each haplotype within a sample and N is the sample size. Nucleotide diversity (π) is the average number of nucleotide differences per nucleotide site between two DNA sequences in all possible pairs in the sample population [
π = ∑ x i x j τ i j (2)
where xi and xj are the respective frequencies in the ith and jth sequences, and τij is the number of nucleotide differences per nucleotide site in the ith and jth sequences and summed over all sequences in the sample.
Arlequin 3.5 (with input files generated in DnaSP) was used to generate a haplotype network and to test null hypotheses of genetic homogeneity with analyses of molecular variance (AMOVAs).
The programs PGDSpider [
We used STRUCTUREv2.3.4 to generate a-posteriori Bayesian-likelihood classifications for each individual based upon the genetic signatures of all seven microsatellite loci [
The program Migrate v3.2.1 was used to estimate: the number of migrants per generation [
Eleven haplotypes were identified from the 72 individuals that were sequenced for the nd2 gene from the three Allyn Brook populations. Haplotype 1, hypothesized as the ancestral haplotype of the populations that recolonized the Connecticut River Basin [
Statistics describing haplotype and nucleotide diversity were similar among all sites (Appendix 2). Tests of neutrality (Tajima’s D, Fu’s F, and Fu and Li’s D) were not significant (P > 0.05, Appendix 2), indicating that there have not been recent bottlenecks, population expansions or non-random eco-evolutionary forces [
The parsimony network (
Frequency in Population | |||
---|---|---|---|
Haplotype ID Number | AB | AB1 | AB2 |
1 | 0.0312 | 0.0714 | 0.154 |
9 | 0.156 | 0.143 | 0.192 |
10 | 0.281 | 0.357 | 0.154 |
14 | 0.0625 | 0 | 0.0769 |
37 | 0.0625 | 0 | 0 |
44 | 0.344 | 0.357 | 0.308 |
47 | 0.0312 | 0 | 0 |
48 | 0.0312 | 0 | 0 |
49 | 0 | 0.0714 | 0.0385 |
50 | 0 | 0 | 0.0385 |
51 | 0 | 0 | 0.0385 |
# Haplotypes | 8 | 5 | 8 |
# Private Haplotypes | 3 | 0 | 2 |
# Alleles for 7 Loci | 59 | 43 | 50 |
Mean Alleles ± S.D. | 8.4 ± 6.4 | 6.1± 4.2 | 7.1 ± 5.1 |
# Private Alleles | 10 | 3 | 6 |
Sample Size | 34 | 14a | 27a |
aN = 13 and 26 for two of seven microsatellite loci, respectively.
AB | AB1 | AB2 | |
---|---|---|---|
Rhca15b | 0.286 | 0.286 | 0.286 |
Rhca16 | 0.500 | 1.000 | 0.750 |
Rhca20 | 0.467 | 0.400 | 0.333 |
Bd165 | 0.667 | 0.600 | 0.500 |
Ca3 | 0.288 | 0.382 | 0.342 |
Ca12 | 0.500 | 0.500 | 0.417 |
Bd174 | 0.500 | 0.385 | 0.482 |
N | 34 | 14a | 27a |
Mean | 0.458 | 0.508 | 0.444 |
s.d. | 0.134 | 0.239 | 0.156 |
aN = 13 and 26 for two of seven microsatellite loci, respectively.
CT, unpubl. data). When the haplotype network (
Seventy-five individuals were sequenced for seven polymorphic microsatellite loci. The frequencies of all alleles for each locus and site are given in Appendix 3. With the exception of Rhca15b and Rhca16, the allelic diversity was large both within and among sites (
There were seven significant deviations from Hardy-Weinberg (H-W) equilibrium (Appendix 4). Three significant deviations from H-W are due to heterozygote deficits. Three inbreeding coefficients for the four deficits range from 0.13 to 0.80. As the rate of inbreeding increases, the value of the coefficient approaches 1.0 [
Garza-Williamson statistics estimate whether populations have reduced recently in size or have gone through bottlenecks (Garza and Williamson 2001). For seven or more loci, values less than 0.68 indicate that the population has gone through a recent reduction in size [
Global AMOVA’s were calculated among Allyn Brook sites as well as between the two Coginchaug River sites and Allyn Brook sites (
The maximum likelihood classification model for Allyn Brook populations with the best fit was for two genetic groups (k = 2;
Microsatellites | nd2 | |||||
---|---|---|---|---|---|---|
Comparisons | Sum of squares | Variance components | Percent Variation | Sum of squares | Variance components | Percent Variation |
AB v AB1 | 17.578 | 0.2211 | 9.45281*** | 0.491 | 0.00769 | 0.63979 |
AB v AB2 | 31.797 | 0.2645 | 11.490*** | 2.21 | 0.03828 | 3.1326*** |
AB1 v AB2 | 6.339 | 0.0857 | 4.37*** | 1.035 | 0.02541 | 2.1352* |
All AB sites | 40.544 | 0.0215 | 9.6136*** | 2.668 | 0.0279 | 2.307*** |
CR sites vs. AB sites | 174.392 | 0.3654 | 14.684*** | 21.846 | 0.1443 | 13.72*** |
*P < 0.01, ** P < 0.001, *** P < 0.0001.
The following are the percentage of individuals with a > 50% probability of belonging to the one genetic group (shown as red in
We calculated the classification probabilities by adding microsatellite data from populations in the nearby Coginchaug River (CR and CRA), the two closest sampled populations to Allyn Brook populations; the Coginchaug and the Allyn Brook populations have been separated from each other for at least 80 years because of the dam (
The number of migrants per generation was estimated from both the nd2 and microsatellite data (
The population with the lowest θ, or estimated population-scaled mutation rate, for microsatellites was AB1 (θ = 0.091) and for nd2 was AB2 (θ = 0.0036). Using AB1 as an example, this means that we expect there will be 9.1 mutations per 100 individuals in the next generation over all sampled loci. AB and AB2 had the highest θ’s for microsatellites (θ = 0.098); AB1 was highest for nd2 (θ = 0.0.0042). The θ’s for microsatellites were an order of magnitude larger than those for nd2, indicating more rapid evolution of the former.
The values of M are relative within data type (
We examined the structure among populations of R. atratulus, in order to assess gene flow on a microgeographic scale. Importantly due to a dam placed near the mouth of Allyn Brook in the 1920’s [
Microsatellites | nd2 | |||
---|---|---|---|---|
Migrants per Generation | M | Migrants per Generation | M | |
AB2ÞAB | 3.54 | 36.02 | 1.078 | 300.4 |
AB1ÞAB | 2.958 | 30.098 | 3.82 | 58.6 |
ABÞAB2 | 6.573 | 67.339 | 10.667 | 343.3 |
ABÞAB1 | 1.949 | 21.345 | 2.081 | 909.6 |
AB2ÞAB1 | 8.533 | 93.456 | 2.867 | 262.6 |
AB1ÞAB2 | 1.467 | 15.023 | 1.101 | 913.1 |
Maximum Likelihood | −1213.46 | −5799.16 |
populations in the main river for more than 80 years. Furthermore, there are no physical barriers to movement among the riffles that we sampled and the three riffles are the only remaining habitat for our study species.
We discovered a number of private alleles and haplotypes suggesting that migration and gene flow among populations was insufficient to prevent in situ evolution and the establishment of novel genes in the populations. We expected the middle site, AB1, to display the greatest effects of migration and geneflow. In fact, AB1 has no private haplotypes, which could have been due to small sample size. But AB1 had private alleles though the fewest (
Maximum-likelihood analyses of migration provide evidence for limited gene flow through the exchange of less than 11 individuals per generation among all sites, with slightly fewer individuals on average migrating upstream than downstream (
The results of AMOVAs and Bayesian a posteriori classifications of Allyn Brook individuals show that all three populations differed significantly in their genetic signatures consistent with low levels of estimated migration and gene flow among populations (
There is only weak evidence to suggest a relationship between geographic distance and genetic differences. At the largest geographic scale, the genetic distances between Allyn Brook populations and Coginchaug River populations would be consistent with an isolation by distance model. Within Allyn Brook, there are only three populations and mathematically no relationship could be calculated. However, the transition between the two microsatellite genetic signatures identified in the Bayesian classification analysis (
We present six hypotheses for why gene flow is low even on such a small geographic scale: 1) The fish are not moving between populations; 2) fish are moving but not mating and, therefore, not exchanging genetic material; 3) migrating fish are preyed upon in the long sandy channels between riffle habitats; 4) fish migrate but upon some density threshold of encounters with neighboring dace return to the original site (F. Cohan, pers. comm.); 5) only individuals that possess common haplotypes or microsatellite alleles migrate, or individuals with private haplotypes or alleles have minimal rates of migration, such that we cannot detect migration with genetic data; or 6) all fish are migrating, but private haplotypes and alleles are selected against in the new habitat. We dismiss hypothesis 6 as unlikely because statistical tests failed to identify non-random processes (Appendix 2) and the microsatellite loci are for the most part in Hardy-Weinberg equilibrium (Appendix 4). The four significant heterozygote deficits were likely due to inbreeding (inbreeding coefficients ranged from 0.13 to 0.80). Because the riffle habitats of the Allyn Brook sites are virtually identical in size, substrate, chemistry, forest overhang and flow, we dismiss hypothesis 6—there should not be significant differences in any selection regimes among riffle sites. At this time there is no evidence to support or refute hypotheses 2, 4 and 5.
There is some support in the literature for hypothesis 1. The restricted movement paradigm states that resident stream fishes tend to be relatively sedentary [
Lonzarich, Lonzarich and Warren Jr [
It is also possible that R. atratulus are preyed upon in the runs between riffle habitats (hypothesis 3). In observations of the fishes in Allyn Brook, there were high numbers of large (>18 cm) Semotilus corporalis (Fallfish), known as a predator of R. atratulus [
Larson, Hoffman et al. 2002 inferred in a study over several that R. obtusus (Western Blacknose Dace; the sister group of R. atratulus) (Kraczkowski and Chernoff 2014) had established a robust population in a neighboring creek in Arkansas. They labelled the suspected emigres as “dispersers”. Clearly, R. atratulus is capable of migrating, even long distances, for example, the post-glacial colonization of the Connecticut River basin from south of New York [
The genetic differentiation of populations on a microgeographic scale has important implications for conservation of biodiversity. The Evolutionary Significant Unit (ESU), first proposed by Ryder (1986) has been expanded upon importantly by Moritz [
At a microgeographic scale, there does not seem to be a point where populations of Rhinichthys atratus are genetically homogeneous. This is demonstrated for both microsatellite and haplotype data. There were large numbers of private alleles and haplotypes within each of the sampled populations. The high numbers of private haplotypes and alleles indicate low rates of migration and gene flow and high in situ evolution within populations. Maximum likelihood estimations of the population genetic models developed by Beerli [
We are grateful to Joel LaBella, Laurie Kenney, Valerie Marinelli, Virginia Harris, Diane Meredith, Blanche Meslin and Suzanne Bussolari for all the help with field gear, arranging and accounting for supplies, shipping samples, etc., that we so depend upon. We are grateful to Fred Cohan for his insightful comments on a draft of the manuscript. SL, LB, SK, DM, and NN received funding from undergraduate internship programs of the College of the Environment and the College of Integrated Sciences. BC received from Schumann Funds, Biology Department and College of the Environment, and project grants from Academic Affairs, Wesleyan University to undertake this study. All of the procedures involving the collection and handling of fishes were humane and ethical, and were approved by state and animal care and use committees: CT Scientific Collection Permits SC-13023 and SC-17031; IACUC 2015-1212-Chernoff-A, IACUC 2017-1212- Chernoff-A.
The authors declare no conflicts of interest regarding the publication of this paper.
Loomis, S.J., Anatone, K., Bither, L., Kang, S.J., Neri, N., Machado, D., Kraczkowski, M.L. and Chernoff, B. (2020) Microgeographic Variation and Inter-Riffle Migration of Rhinichthys atratulus (Pisces: Cyprinidae) in a Small Connecticut Stream, United States. Open Journal of Ecology, 10, 460-481. https://doi.org/10.4236/oje.2020.107030
Appendix 1. Collection information from Allyn Brook localities. Negative values indicate western longitudes. The conductance of the water is given in millisiemens/cm (mS). Water chemistry and flow were measured over four days in August 2018.
Appendix 2. Diversity statistics and tests of neutrality and non-random eco-evolutionary processes.
Appendix 3. Allele frequencies for the seven polymorphic microsatellite loci from three Allyn Brook localities. Diploid sample sizes (2N) follow: AB = 68; AB1 = 28; AB2 = 54.
Appendix 4. Observed and expected heterozygosity and tests of Hardy-Weinberg equilibrium at each site for seven polymorphic microsatellite loci.
*P < 0.01, ** P < 0.001, *** P < 0.0001.