Temporal and spatial patterns of spawning site use by two cavity-spawning darters in a Wisconsin stream

Synopsis There was partial spatial and complete temporal overlap of spawning by Etheostoma flabellare and E. nigrum in Marsh Creek, Wisconsin. E. flabellare tended to spawn at shallower depths than E. nigrum. Both species spawned over a greater range of depths in an area with few spawning sites (cavities under rocks or logs) than in an area where spawning sites were abundant. In the laboratory E. flabellare were able to prevent E. nigrum from occupying cavities they would have otherwise occupied. E. nigrum appear to be competitively inferior to E. flabellare in terms of spawning-site acquisition and retention, but possess several attributes that may allow them to persist in the presence of the latter.     

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.     

Seasonal colour and antipredator behaviour in Etheostoma (Percidae)

This study examined how colour varies across season and sex in the fantail darter Etheostoma flabellare and the banded darter Etheostoma zonale. Etheostoma flabellare has male‐only parental care and exhibited slight sexual dimorphism in overall colour, with no discernible effect of season on colour; whereas E. zonale does not have parental care and exhibited substantial sexual dimorphism in colour, but only in the breeding season. Additionally, antipredator behaviour of E. zonale was compared between males that were fully coloured during the breeding season and males that were partially coloured at that time, but the effects of colour and season were not consistent across males.     

Critical current speeds and microhabitats of the benthic fishes Percina roanoka and Etheostoma flabellare

Synopsis Critical current speed (maximum current speed at which benthic stream fishes are able to hold station without active swimming) was determined in a laboratory flow chamber for Percina roanoka and Etheostoma flabellare. Mean critical current speeds differed significantly among adult P. roanoka, adult E. flabellare, and juvenile E. flabellare, and were consistent with microhabitat differences among these darters in natural environments. Behavior of E. flabellare enhanced its station-holding ability, but tests with preserved individuals suggested that morphological differences explained more of the difference in critical current speeds of the darters. On a smooth surface where grasping of the substrate was not possible, use of pectoral fins did not significantly enhance the ability of darters to maintain position in flowing water.     

Feeding ecology of three predacious aquatic insects and two fish in a riffle of the Speed River,Ontario

The food habits of two fish (Etheostoma flabellare and Rhinichthys atratulus) and three predacious aquatic insects (Nigronia serricornis, Paragnetina media and Agnetina capitata) were examined for one year in a riffle of the Speed River, Ontario. Adult E. flabellare fed primarily on net-spinning caddisflies whereas juvenile E. flabellare relied heavily on chironomids. Rhinichthys atratulus had a more varied diet composed of chironomids, Antocha larvae (Tipulidae) and net-spinning caddisflies. Two of the aquatic insect predators consumed large numbers of net-spinning caddisflies; however, P. media contained large percentages of Hydropsyche and Cheumatopsyche whereas N. serricornis fed primarily on non-hydropsychid net-spinners. The diet of A. capitata was composed largely of chironomids and mayflies. Schoener's diet overlap index () suggested that large E. flabellare and P. media had similar diets; however, E. flabellare ingested much larger prey than P. media. Adult E. flabellare contained a significantly greater proportion of larger prey items in comparison with benthic samples whereas similar comparisons with other predators showed no selection for larger prey. These data demonstrate that E. flabellare was size selective given the range of prey sizes available whereas the other predators were not. Also, there was little dietary overlap among the fish and invertebrate predators suggesting a partitioning of resources.     

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.     

Limiting similarity and the intensity of competitive effects on the mottled sculpin,Cottus bairdi,in experimental stream communities

The identification of potential competitors has been driven by the concept of limiting similarity. Lacking are explicit tests of interaction strength among morphologically similar and dissimilar species. I used the mottled sculpin, Cottus bairdi, as a focal species in an artificial stream experiment designed to compare the effect of intraspecific competition to interspecific cometition from two very different species: a congener, the Kanawha sculpin (C. carolinae ssp.), and an unrelated species, the fantail darter (Etheostoma flabellare). The differences in morphology between these two species generate specific predictions under limiting similarity regarding the likelihood of competition and its relative strength: the congener should be a more important potential competitor. Increased fish density had a strong effect on the multivariate response of survival and growth, and on the relative condition of C. bairdi, indicating competition. The effect of additional C. bairdi or Kanawha sculpins were roughly equal, but the effect of E. flabellare was significantly greater. The most important potential impact on C. bairdi came from interspecific competition by a species that is smaller and very different in morphology, contrary to predictions based on limiting similarity.     

Comparative genetic isolation patterns for multiple headwater fishes in three geographic regions

Headwater‐resident fishes may be prone to a high rate of isolation and a pronounced hierarchical genetic structure due to a combination of niche preference and dendritic effects of river networks. Genetic isolation patterns were compared using microsatellites in six headwater fishes, Fundulus olivaceus, Semotilus atromaculatus, Erimyzon claviformis, Etheostoma artesiae, Etheostoma whipplei and Etheostoma parvipinne, in three geographic regions that included drainages of small, medium and large sizes in the southern United States. All species showed hierarchical nesting of genetic populations and there were clear and mostly consistent differences between species and regions that were identified through summary statistics derived from two independent analyses. For species comparisons, a high isolation grouping (increased number of isolated genetic clusters or sites within regions) and a low‐isolation grouping (decreased number of clusters or sites) were identified. Species group placement was related to niche breadth along the river continuum and presumed abundance and variability of preferred microhabitats, with increased headwater specialization among species being associated with placement in the high‐isolation grouping. There was a weakly significant positive effect of drainage size on the number of isolated clusters or sites across all species. Regional patterns were shared in two species, with the region containing the smallest drainages having lower rates of isolation in both datasets. This study shows the effects of regional and species characteristics on genetic isolation for headwater species, which are especially prone to isolation due to spatial, dendritic effects of river networks.     

Riverscape genetics of the orangethroat darter complex

Freshwater darters belonging to the orangethroat darter species complex, or Ceasia, are widely distributed in the Central and Southern United States, with ranges that span both glaciated and unglaciated regions. Up to 15 species have been recognized in the complex, with one, Etheostoma spectabile, having a widespread northern distribution and another, Etheostoma pulchellum, having a sizeable southern distribution. The other species in the complex have much more restricted distributions in unglaciated regions of the Central Highlands. We sampled 384 darters from 52 sites covering much of the range of Ceasia and evaluated patterns of genetic diversity, genetic structure, and pre- and post-glacial patterns of range contraction and expansion. We anticipated finding much stronger signals of genetic differentiation and diversification in unglaciated regions, given the higher species diversity and levels of endemism reported there. Surprisingly, microsatellite genotyping revealed two well-differentiated genetic clusters of E. spectabile in samples from glaciated regions, one confined to the Illinois River basin and another found in the Wabash drainage and Great Lakes tributaries. This suggests that there was expansion from two isolated glacial refugia, with little subsequent post-glacial gene flow. Fish collected from throughout the unglaciated region were less genetically differentiated. Fish assigned to Etheostoma burri and Etheostoma uniporum based on collection sites and morphological characters were not genetically differentiated from E. spectabile samples from the region. Hybridization and introgression occurring in the Central Highlands may confound genetic delineation of species in this region of high endemism and diversity.     

Temperature selection and critical thermal maxima of the fantail darter,Etheostoma flabellare,and johnny darter,E. nigrum,related to habitat and season

Synopsis Riffle dwelling fantail darters (Etheostoma flabellare) selected lower temperatures in winter (19.3°C) compared to pool dwelling johnny darters (E. nigrum; 22.0°C. A similar trend was evident in summer tests (fantail darters, 20.3°C; johnny darters, 22.9°C). Summer tested animals selected higher temperatures than winter tested animals maintained at the same acclimation temperature and photoperiod. When tested together in the same gradient, both species appeared not to thermoregulate, but tended to avoid each other. Critical thermal maxima (CTMax) did not differ between seasons for either species (fantail darters, 31.1°C winter, 31.3°C summer; johnny darters, 30.9°C winter, 30.5°C summer). Differences in the thermal responses of these darters correlated with differences in their respective habitats.     

Convergence, divergence, and the effect of congeners on body size ratios in stream fishes

Abstract Using collections from the years 1892–1999, I determined maximum standard length within each of 1030 populations of riffle-inhabiting darters ( Etheostoma spp.) representing five species from 788 sites in Illinois. Each site contained one to four riffle-inhabiting species of Etheostoma . Based on maximum-sized individuals in each collection, I calculated a ratio of standard lengths for all sympatric species pairs. Null models were developed using random pairings of body size measurements from sites with only one species of riffle Etheostoma to test whether body size ratios of sympatric species pairs are larger, indicating divergence, or smaller, indicating convergence, than ratios generated from allopatric populations. Results suggest that two of nine species pairs tend to converge in body size when sympatric. This suggests that convergence, in some cases, may facilitate the persistence of similar species in diverse communities. When variation in sympatric congener number is accounted for, the E. caeruleum : E. spectabile interaction results in significantly increasing size ratios as congener number increases. Etheostoma caeruleum and E. spectabile are the most ecologically similar and evolutionarily closely related species in the dataset. The divergence within this pair, associated with sympatric congener number, suggests that the degree of divergent character displacement may be indirectly enhanced by community structure.     

Distribution,community structure and production of fishes in the upper Speed River,Ontario: a preimpoundment study

Synopsis The upper Speed River, on which a dam was under construction, was surveyed through fourteen qualitative and six quantitative collections to determine the distribution of fishes in the river above Guelph, Ontario and in a tributary, Luteral Creek. At the quantitative localities the removal method was used to estimate density and standing crop of all species. At three of these localities age and growth of the dominant species were determined and total ecological production calculated. Two approaches to the estimation of production for the 0–1 age class were compared and found to result in differences of 33–39% in the estimates of total production, thus illustrating the extent to which methodology may affect such estimates The dominant species in the river were, Semotilus atromaculatus, Notropis cornutus, Pimephales notatus, Rhinichthys atratulus, Hypentelium nigricans, Catostomus commersoni, Ambloplites rupestris, Micropterus dolomieui and Etheostoma flabellare. Density ranged from 11126 to 74765 individuals per hectare and standing crop from 32.4 to 190.0 kg ha⁻¹. Production values at the three localities were 15, 19 and 54 kg ha⁻¹ yr⁻¹ , and are low compared with estimates for other fluvial systems. Comparison with a 1951 survey of the Speed River revealed considerable changes in the composition and distribution of fish species. The ranges of several cold-water species had contracted towards the headwaters, whereas several warm-water species had extended their ranges up the river     

A holistic approach to taxonomic evaluation of two closely related endangered freshwater mussel species, the oyster mussel Epioblasma capsaeformis and tan riffleshell Epioblasma florentina walkeri (Bivalvia: Unionidae)

Species in the genus Epioblasma have specialized life historyrequirements and represent the most endangered genus of freshwatermussels (Unionidae) in the world. A genetic characterizationof extant populations of the oyster mussel E. capsaeformis andtan riffleshell E. florentina walkeri sensu late was conductedto assess taxonomic validity and to resolve conservation issuesfor recovery planning. These mussel species exhibit pronouncedphenotypic variation, but were difficult to characterize phylogeneticallyusing DNA sequences. Monophyletic lineages, congruent with phenotypicvariation among species, were obtained only after extensiveanalysis of combined mitochondrial (1396 bp of 16S, cytochrome-b,and ND1) and nuclear (515 bp of ITS-1) DNA sequences. Incontrast, analysis of variation at 10 hypervariable DNA microsatelliteloci showed moderately to highly diverged populations basedon F_ST and R_ST values, which ranged from 0.12 to 0.39 and 0.15to 0.71, respectively. Quantitative variation between specieswas observed in fish-host specificity, with transformation successof glochidia of E. capsaeformis significantly greater (P<0.05)on greenside darter Etheostoma blennioides, and that of E. f.walkeri significantly greater (P<0.05) on fantail darterEtheostoma flabellare. Lengths of glochidia differed significantly(P<0.001) among species and populations, with mean sizesranging from 241 to 272 µm. The texture and colourof the mantle-pad of E. capsaeformis sensu stricto is smoothand bluish-white, whereas that of E. f. walkeri is pustuledand brown, with tan mottling. Based on extensive molecular,morphological and life history data, the population of E. capsaeformisfrom the Duck River, Tennessee, USA is proposed as a separatespecies, and the population of E. f. walkeri from Indian Creek,upper Clinch River, Virginia, USA is proposed as a distinctsubspecies. (Received 23 February 2005; accepted 16 January 2006)     

Evolutionarily independent genes and genomes and insights on genetic structure, evolution and conservation of the collis group of darters

Comparing variation across evolutionarily independent characters, notably nuclear and mitochondrial genes, yields a more robust estimate of diversification than is generally recovered from individual characters. Patterns of variation across multiple molecular markers from the mitochondrial ( 16SrRNA , cytochrome b ) and nuclear ( ldhA6 and aldB ) genomes were examined from six populations of Etheostoma collis and two populations of Etheostoma saludae , species aligned in the collis groups. Phylogenetic analyses revealed that sequence variation among individuals from the Roanoke, Tar and Neuse Rivers and the Catawba and Pee Dee Rivers, respectively, form highly supported, deeply divergent clades. Relationships of alleles sampled from Saluda River E. saludae and Cape Fear River E. collis to these lineages are unresolved, but all groups are reciprocally monophyletic for both nuclear and mtDNA loci. Phylogenetic analyses suggest that historical factors have had a strong influence on the distribution of genetic variation among populations. Genetic variation within the collis group is consistent with all previously proposed taxonomic hypotheses for the collis group, providing no taxonomic insights. From a conservation standpoint, each population of the collis group is an ESU, thereby warranting a drainage-specific management strategy.     

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.     

Ecological and evolutionary consequences of early ontogenies of darters (Etheostomatini)

Synopsis The ecological classification of fishes into reproductive guilds is based on the premises that (1) reproductive styles and early ontogeny are closely related, and (2) both are correlated with the ecology of a species. A comparison of early ontogenies of logperch (Percina caprodes), rainbow darter (Etheostoma caeruleum), and fantail darter (E. flabellare) confirmed these premises, and provided possible explanations for diversity within the Etheostomatini. Young logperch have limited vitelline circulation, hatch while still poorly developed, and therefore must drift from oxygen rich lotic habitats to lentic habitats where small planktonic prey are available. Young rainbow and fantail darters have extensive vitelline plexuses, are well developed at transition to first feeding, and begin feeding on aquatic insects. Thus there is no necessity for a drift interval. As a result, the latter species are adapted for stream life. Interspecific differences in reproductive styles and early ontogenies may have contributed to speciation of darters by allowing partitioning of breeding sites and food resources for young. In addition, reduced drift dispersal and small stream habitation may have indirectly contributed to speciation by reducing genetic exchange among populations.     

Modelling patterns of coexistence of three congeneric headwater fishes

1. Mechanisms driving patterns of occurrence and co-occurrence among North American freshwater fishes are poorly understood. In particular, the influence of biotic interactions on coexistence among stream reaches and their effects on regional species distribution patterns is not well understood for congeneric headwater fishes.
2. Occupancy models provide a useful framework for examining patterns of co-occurrence while also accounting for imperfect detection. Occupancy models may be extended to test for evidence that a dominant species influences the occurrence of a subordinate species and thus evaluate support for the hypothesis that species interactions drive patterns of coexistence.
3. We examined patterns of occurrence and co-occurrence at the stream-reach scale among three species of darters (Percidae: Etheostomatinae) that occupy headwater streams within a Gulf Coastal Plain drainage in the south-eastern U.S.A. We assessed species occurrences at 97 sites in first- to third-order streams on one occasion each and used data from four sub-reaches sampled with equal effort at each site to estimate species-specific detection probabilities. Following sampling, a suite of habitat variables was collected at three equidistant points along each of the three transects established within a sub-reach. Coarse (stream-segment, catchment, network) scale variables were also incorporated using geospatial data. Single-species and two-species occupancy models were used to examine patterns of occupancy and coexistence.
4. The occupancy of each species was influenced by distinct habitat variables. Goldstripe darters (Etheostoma parvipinne) were constrained by a stream size gradient, groundwater input appeared to influence the occurrence of Yazoo darters (Etheostoma raneyi), and local habitat heterogeneity (e.g. variation in depth and current velocity) appeared to influence the occupancy of redspot darters (Etheostoma artesiae).
5. We found no evidence that the presence of one species influenced the occurrence of another within a stream-reach based on two-species occupancy models. Rather, species co-occurrences were best explained as independent occurrences within a stream-reach according to species-specific habitat associations.
6. Occupancy modelling may provide a suitable framework for evaluating the influence of biotic interactions among congeneric stream fishes along species-specific habitat gradients at the stream reach scale. Our study offers insight into how habitat variation can influence coexistence of potential competitors across a large river system.

     

Investigation of plasmid-mediated resistance in E. coli isolated from healthy and diarrheic sheep and goats

Escherichia coli is zoonotic bacteria and the emergence of antimicrobial-resistant strains becomes a critical issue in both human and animal health globally. This study was therefore aimed to investigate the plasmid-mediated resistance in E. coli strains isolated from healthy and diarrheic sheep and goats. A total of 234 fecal samples were obtained from 157 sheep (99 healthy and 58 diarrheic) and 77 goats (32 healthy and 45 diarrheic) for the isolation and identification of E. coli. Plasmid DNA was extracted using the alkaline lysis method. Phenotypic antibiotic susceptibility profiles were determined against the three classes of antimicrobials, which resistance is mediated by plasmids (Cephalosporins, Fluoroquinolone, and Aminoglycosides) using the disc-diffusion method. The frequency of plasmid-mediated resistance genes was investigated by PCR. A total of 159 E. coli strains harbored plasmids. The isolates antibiogram showed different patterns of resistance in both healthy and diarrheic animals. A total of (82; 51.5%) E. coli strains were multidrug-resistant. rmtB gene was detected in all Aminoglycoside-resistant E. coli, and the ESBL-producing E. coli possessed different CTX-M genes. Similarly, fluoroquinolone-resistant E. coli possessed different qnr genes. On the analysis of the gyrB gene sequence of fluoroquinolone-resistant E. coli, multiple point mutations were revealed. In conclusion, a high prevalence of E. coli with high resistance patterns to antimicrobials was revealed in the current study, in addition to a wide distribution of their resistance determinants. These findings highlight the importance of sheep and goats as reservoirs for the dissemination of MDR E. coli and resistance gene horizontal transfer.     

Biogeographic origin and radiation of Cuban Eleutherodactylus frogs of the auriculatus species group,inferred from mitochondrial and nuclear gene sequences

We studied phylogenetic relationships of the Eleutherodactylus auriculatus species group to infer colonization and diversification patterns in this endemic radiation of terrestrial frogs of the genus Eleutherodactylus in the largest of the Greater Antilles, Cuba. An initial screening of genetic diversity based on partial sequences of the 16S rRNA gene in almost 100 individuals of all species of the group and covering the complete known geographic range of their occurrence found most species endemic to small ranges in the eastern Cuban mountains while a single species was widespread over most of Cuba. Our molecular phylogeny, based on 3731 bp of four mitochondrial and one nuclear gene, suggests that most cladogenetic events within the group occurred among clades restricted to the eastern mountains, which acted as refugia and facilitated the diversification in this group. Our results reveal two separate colonization events of Central and Western Cuba and allow inferring the timing of the subsequent diversification events that occurred between 11 and 2 Mya. Because populations previously assigned to E. auriculatus represent four genetically strongly divergent lineages that also differ in their advertisement calls, we propose that E. auriculatus as currently recognized comprises four species. The difficulties in assigning the name auriculatus to any of these four species, and the fact that E. principalis is nested within one of them, stress the need for a thorough taxonomic revision of this group.