Phylogeny and temporal diversification of darters (Percidae: Etheostomatinae)

Discussions aimed at resolution of the Tree of Life are most often focused on the interrelationships of major organismal lineages. In this study, we focus on the resolution of some of the most apical branches in the Tree of Life through exploration of the phylogenetic relationships of darters, a species-rich clade of North American freshwater fishes. With a near-complete taxon sampling of close to 250 species, we aim to investigate strategies for efficient multilocus data sampling and the estimation of divergence times using relaxed-clock methods when a clade lacks a fossil record. Our phylogenetic data set comprises a single mitochondrial DNA (mtDNA) gene and two nuclear genes sampled from 245 of the 248 darter species. This dense sampling allows us to determine if a modest amount of nuclear DNA sequence data can resolve relationships among closely related animal species. Darters lack a fossil record to provide age calibration priors in relaxed-clock analyses. Therefore, we use a near-complete species-sampled phylogeny of the perciform clade Centrarchidae, which has a rich fossil record, to assess two distinct strategies of external calibration in relaxed-clock divergence time estimates of darters: using ages inferred from the fossil record and molecular evolutionary rate estimates. Comparison of Bayesian phylogenies inferred from mtDNA and nuclear genes reveals that heterospecific mtDNA is present in approximately 12.5% of all darter species. We identify three patterns of mtDNA introgression in darters: proximal mtDNA transfer, which involves the transfer of mtDNA among extant and sympatric darter species, indeterminate introgression, which involves the transfer of mtDNA from a lineage that cannot be confidently identified because the introgressed haplotypes are not clearly referable to mtDNA haplotypes in any recognized species, and deep introgression, which is characterized by species diversification within a recipient clade subsequent to the transfer of heterospecific mtDNA. The results of our analyses indicate that DNA sequences sampled from single-copy nuclear genes can provide appreciable phylogenetic resolution for closely related animal species. A well-resolved near-complete species-sampled phylogeny of darters was estimated with Bayesian methods using a concatenated mtDNA and nuclear gene data set with all identified heterospecific mtDNA haplotypes treated as missing data. The relaxed-clock analyses resulted in very similar posterior age estimates across the three sampled genes and methods of calibration and therefore offer a viable strategy for estimating divergence times for clades that lack a fossil record. In addition, an informative rank-free clade-based classification of darters that preserves the rich history of nomenclature in the group and provides formal taxonomic communication of darter clades was constructed using the mtDNA and nuclear gene phylogeny. On the whole, the appeal of mtDNA for phylogeny inference among closely related animal species is diminished by the observations of extensive mtDNA introgression and by finding appreciable phylogenetic signal in a modest sampling of nuclear genes in our phylogenetic analyses of darters.     

The evolution of male nuptial colour in a sexually dimorphic group of fishes (Percidae: Etheostomatinae)

To test hypotheses explaining variation in elaborate male colouration across closely related species groups, ancestral‐state reconstructions and tests of phylogenetic signal and correlated evolution were used to examine the evolution of male body and fin colouration in a group of sexually dichromatic stream fishes known as darters (Percidae: Etheostomatinae). The presence or absence of red–orange and blue–green male colour traits were scored across six body regions in 99 darter species using a recently estimated amplified fragment length polymorphism (AFLP) phylogeny for comparative analyses. Ancestral‐state reconstructions infer the most recent common ancestor of darters to lack red–orange colour and possess blue–green colour on different body regions, suggesting variation between species is due to independent gains of red–orange and losses of blue–green. Colour traits exhibit substantial phylogenetic signal and are highly correlated across body regions. Comparative analyses were repeated using an alternative phylogenetic hypothesis based on one mitochondrial and two nuclear genes, yielding similar results to analyses based on the AFLP phylogeny. Red–orange colouration in darters appears to be derived; whereas, blue–green appears to be ancestral, which suggests that different selection mechanisms may be acting on these two colour classes in darters.     

AFLPs support deep relationships among darters (Percidae: Etheostomatinae) consistent with morphological hypotheses

Recent attention has focused on the efficacy of amplified fragment length polymorphisms (AFLPs) for resolving deep evolutionary relationships. Here we show that AFLPs provide resolution of deep relationships within the family Percidae that are more consistent with previous morphological hypotheses than are relationships proposed by previous molecular analyses. Despite in silico predictions, we were able to resolve relatively ancient divergences, estimated at >25 MA. We show that the most distantly related species share the fewest fragments, but suggest that large data sets and extensive taxon sampling are sufficient to overcome this obstacle of the AFLP technique for deep divergences. We compare genetic distances estimated from mitochondrial DNA with those from AFLPs and contrast traditional PAUP^* Nei–Li AFLP genetic distances with a recently proposed method utilizing the Dice equation with constraining nucleotides.     

Temporal patterns of diversification and microendemism in Eastern Highland endemic barcheek darters (Percidae: Etheostomatinae)

Eastern North America is the location of the world's most species-rich temperate freshwater fish fauna. Hypotheses regarding the geographic and temporal scale of teleost diversification in this region have not been broadly investigated using absolute divergence time estimates among the constituent lineages. This study used time-calibrated molecular phylogenies estimated from mitochondrial and nuclear genes to investigate the temporal and geographic signatures of diversification within barcheek darters, a clade of allopatrically distributed species endemic to the Eastern Highlands. Results from divergence time estimates using an uncorrelated lognormal model suggest that the barcheek darters are an ancient group with a crown node estimate of 16.3 mya, 95% highest posterior density (HPD): [12.4, 20.5], and the clade is characterized by substantial intraspecific divergence times within several species. In particular, the Caney Fork endemic Etheostoma basilare comprises five strongly supported and deeply divergent clades with a most recent common ancestor estimated at 8.0 mya, 95% HPD: [5.6, 10.7]. These results are concordant with the hypothesis that geologically stable areas of eastern North America have facilitated both the generation and preservation of lineages across a substantial breadth of evolutionary time, and that allopatric speciation in darters has occurred at much smaller spatial scales than previously realized.     

The influence of life‐history strategy on genetic differentiation and lineage divergence in darters (Percidae: Etheostomatinae)

Recent studies determined that darters with specialized breeding strategies can exhibit deep lineage divergence over fine geographic scales without apparent physical barriers to gene flow. However, the extent to which intrinsic characteristics interact with extrinsic factors to influence population divergence and lineage diversification in darters is not well understood. This study employed comparative phylogeographic and population genetic methods to investigate the influence of life history on gene flow, dispersal ability, and lineage divergence in two sympatric sister darters with differing breeding strategies. Our results revealed highly disparate phylogeographic histories, patterns of genetic structure, and dispersal abilities between the two species suggesting that life history may contribute to lineage diversification in darters, especially by limiting dispersal among large river courses. Both species also showed striking differences in demographic history, indicating that extrinsic factors differentially affected each species during the Pleistocene. Collectively, our results indicate that intrinsic and extrinsic factors have influenced levels of gene flow among populations within both species examined. However, we suggest that life‐history strategy may play a more important role in lineage diversification in darters than previously appreciated, a finding that has potentially important implications for understanding diversification of the rich North American freshwater fish fauna.     

Evolution of the premaxillary fraenum and substratum in snubnose darters and allies (Percidae: Etheostoma)

Darters (Percidae: Etheostomatinae), a species‐rich group of North American freshwater fishes, vary in the presence of a premaxillary fraenum, a strip of skin that connects the premaxillary bones to the snout, and it is hypothesized that this trait is a trophic adaptation to particular substrata. Ancestral state reconstructions and analyses of phylogenetic associations between presence of the premaxillary fraenum and preferred stream substratum were conducted in a clade of closely related darters (snubnose darters and allies) that vary in morphology and habitat preferences. The most recent common ancestor of this clade was inferred to possess a fraenum and to inhabit rocky substrata, consistent with previous hypotheses, but a significant correlation between fraenum presence and substratum type across the phylogeny was not found.     

Rapid allopatric speciation in logperch darters (Percidae: Percina)

Theory predicts that clades diversifying via sympatric speciation will exhibit high diversification rates. However, the expected rate of diversification in clades characterized by allopatric speciation is less clear. Previous studies have documented significantly higher speciation rates in freshwater fish clades diversifying via sympatric versus allopatric modes, leading to suggestions that the geographic pattern of speciation can be inferred solely from knowledge of the diversification rate. We tested this prediction using an example from darters, a clade of approximately 200 species of freshwater fishes endemic to eastern North America. A resolved phylogeny was generated using mitochondrial DNA gene sequences for logperches, a monophyletic group of darters composed of 10 recognized species. Divergence times among logperch species were estimated using a fossil calibrated molecular clock in centrarchid fishes, and diversification rates in logperches were estimated using several methods. Speciation events in logperches are recent, extending from 4.20 +/- 1.06 million years ago (mya) to 0.42 +/- 0.22 mya, with most speciation events occurring in the Pleistocene. Diversification rates are high in logperches, at some nodes exceeding rates reported for well-studied adaptive radiations such as Hawaiian silverswords. The geographic pattern of speciation in logperches was investigated by examining the relationship between degree of sympatry and the absolute age of the contrast, with the result that diversification in logperches appears allopatric. The very high diversification rate observed in the logperch phylogeny is more similar to freshwater fish clades thought to represent examples of sympatric speciation than to clades representing allopatric speciation. These results demonstrate that the geographic mode of speciation for a clade cannot be inferred from the diversification rate. The empirical observation of high diversification rates in logperches demonstrates that allopatric speciation can occur rapidly.     

The effect of phylogeny on interspecific body shape variation in darters (Pisces: Percidae)

We conducted a geometric morphometric analysis of interspecific body shape variation among representatives of 31 species of darters (Pisces: Percidae) to determine whether there is evidence of a phylogenetic effect in body shape variation. Cartesian transformation grids representing relative shape differences of individual species and subspecies revealed qualitative similarities within most traditionally recognized taxonomic groups (genera and subgenera). Canonical variates analysis and a UPGMA cluster analysis were conducted to explore further the relationships among body shapes of species; both analyses revealed patterns of variation consistent with the interpretation that shape is associated with taxonomic affinities. Normalized Mantel statistics revealed a significant positive association between body shape differences and phylogenetic interrelatedness for each of four recent phylogenetic hypotheses, providing evidence of a phylogenetic effect. This result is somewhat surprising, however, given the largely incompatible nature of these four phylogenies. We provide evidence that this result may be due to (1) the inclusion of multiple sets of closely related species to represent the traditionally recognized genera and subgenera within each phylogeny and/or (2) the inclusion of several species with relatively divergent shapes and their particular positions within the phylogenies relative to one another or to the other species of darters.     

Female-specific color is a signal of quality in the striped plateau lizard (Sceloporus virgatus)

Recent theoretical and empirical studies confirm that male matechoice and/or female–female mate competition can be expressedin the absence of sex-role reversal. Such reproductive patternsmay select for the evolution of female traits that indicatefemale phenotypic or genotypic quality among non–role-reversedspecies. Although attention to the evolution and function offemale ornaments is increasing, additional focus is needed onfemale-specific ornaments (those not expressed in conspecificmales) and on nonavian systems in order to gain a broad understandingof how selection acts directly on ornamentation of female animals.In the striped plateau lizard, Sceloporus virgatus, only femalesdevelop orange throat patches during the reproductive season.The color peaks in expression near the time of ovulation andappears to stimulate male courtship. Here, I examine whetherthis female-specific ornament can be used by males to reliablyassess female phenotypic quality. Using multivariate regressionanalyses, I show that the area of the orange color patch predictsbody condition and mite load, the chroma (i.e., saturation)of the color patch predicts body size, and both patch area andchroma reliably predict average egg mass. Thus, female reproductivecolor may function as a condition-dependent signal, indicatingphenotypic quality to potential mates.     

Spawning behavior of snubnose darters (Percidae) in natural and laboratory environments

Eight species of snubnose darters (genus Etheostoma) were observed and filmed to compare the spawning behavior in the field and in aquaria. These darters exhibited substrate searching, male lateral quivers, and head jabbing behavior in both the field and aquaria. In streams, snubnose darters attached eggs singly to cobble and larger rock substrates, while in aquaria several species were found to attach eggs to fine aquarium gravel as well as cobble and larger rocks. In general, these results support previous aquarium studies of snubnose darters. Information on spawning behavior in snubnose darters as well as darters of the closely related subgenus Etheostoma is synthesized in order to address the phylogenetic affinities of some species in this group of fish.     

Phylogenetic Correlation Between Male Nuptial Color and Behavioral Responses to Color Across a Diverse and Colorful Genus of Freshwater Fish (Etheostoma spp., Teleostei: Percidae)

Sexual selection theory predicts that preferences in both sexes select for the elaboration of male nuptial coloration, with empirical evidence supporting these predictions. Empirical studies are often limited in their taxonomic inclusiveness, however, and typically do not examine how male and female preferences contribute to macroevolutionary patterns of male color variation across multiple lineages in a clade. This study examined color preferences in a group of dichromatic freshwater fishes known as darters (genus Etheostoma) that vary in the presence of male coloration. The strengths of attraction to black, blue, gray, and red models were tested in females of 18 species, in addition to males of five species. We found a positive association between the presence of red or orange on the body and the amount of time associating with red models, suggesting color variation is at least in part due to variation in female preferences between species. Males also spent more time associating with colors that most closely resembled conspecifics, suggesting that preferential responses to color in males also can contribute to the diversity of nuptial coloration in darters.     

Effect of spatial pattern and microhabitat on pit construction and relocation in Myrmeleon hyalinus (Neuroptera: Myrmeleontidae) larvae

Abstract.  1. In two sets of enclosure experiments, we studied the spatial pattern, relocation rates, pit construction rates, and microhabitat preference of Myrmeleon hyalinus larvae.
2. We showed that M. hyalinus larvae actively prefer shady sites and often relocate to shady areas when exposed to the sun. This behaviour may constitute a life-saving strategy in desert environments.
3. The initial spatial pattern in a cluster of antlion larvae did not affect the final pattern, relocation rate, or the pathway moved while relocating. We interpret this finding to mean that the spatial pattern of M. hyalinus larvae is mainly influenced by factors operating at large time-scales, such as exploitation competition and physical-microclimatological constraints, rather than those operating at shorter time-scales, such as interference competition.
4. In contrast, the likelihood of pit construction was positively correlated with nearest neighbour distance, possibly as a result of interference competition.
5. Pit construction rates were constant throughout the experiment, while relocation rates decreased with time.
6. Contrary to previous studies, we found no correlation between body mass and spatial position inside the cluster, suggesting that these differences are the products of slower processes (e.g. exploitation and body condition).     

Living on the bottom: Kinematics of benthic station‐holding in darter fishes (Percidae: Etheostomatinae)

Darters represent a substantial radiation of freshwater fishes that live in close association with the substrate in North American streams and rivers. A key feature of any darter species is therefore its ability to stay in place or to “hold station” in flowing water. Here, we quantify the station‐holding performance of two morphologically divergent darter species, the fantail darter Etheostoma flabellare and the Missouri saddled darter Etheostoma tetrazonum. We also characterize the primary kinematic responses of the two species when holding station in flow speeds ranging from 4 to 56 cm s^?1 in a flow tank on either plexiglas or small rock substrate. We then present a series of hypotheses about the potential hydrodynamic and functional consequences of the observed postural changes and the links among morphology, posture, and station‐holding performance. On both substrates, E. tetrazonum was able to hold station at higher flow speeds than E. flabellare. On rocks, E. tetrazonum slipped at an average speed of 55.7 cm s^?1 whereas E. flabellare slipped at 40.2 cm s^?1. On plexiglas, E. tetrazonum slipped at an average speed of 24.7 cm s^?1 whereas E. flabellare slipped at 23.1 cm s^?1. We measured body and fin positions of the two species from individual frames of high‐speed video while holding station on rocks and plexiglas. We found that on both substrates, the two species generally exhibited similar kinematic responses to increasing flow: the head was lowered and angled downward, the back became more arched, and the median and caudal fin rays contracted as water flow speed increased. The ventral halves of the pectoral fins were also expanded and the dorsal halves contracted. These changes in posture and fin position likely increase negative lift forces thereby increasing substrate contact forces and reducing the probability of downstream slip. J. Morphol., 2010. © 2009 Wiley‐Liss, Inc.     

The accumulation of reproductive barriers during speciation: postmating barriers in two behaviorally isolated species of darters (Percidae: Etheostoma)

Identifying the manner in which reproductive barriers accumulate during lineage divergence is central to establishing general principles of species formation. One outstanding question is which isolating mechanisms form the first complete barrier to gene flow in a given lineage or under a particular set of conditions. To identify these initial reproductive barriers requires examining lineages in very early stages of divergence, before multiple reproductive barriers have evolved to completion. We quantified the strength of three postmating barriers in a pair of darter species and compared these estimates to each other and to the strength of behavioral isolation (BI) reported in a previous study. Results reveal no evidence of gametic incompatibility but intermediate levels of conspecific sperm precedence and hybrid inviability. As BI is nearly complete, our analysis comparing the strength of multiple reproductive barriers implicates the evolution of mate choice as central to both the origin and maintenance of these species. Further examination of ecological isolation and hybrid sterility is necessary to determine the role of these barriers in darter speciation.