Muscle cellularity, growth performance and growth-related gene expression of juvenile climbing perch Anabas testudineus in response to different eggs incubation temperature.

Although indigenous climbing perch (Anabas testudineusis) is a highly valuable species, slow growth pattern during the culture period impeding its commercial success in aquaculture. In many fish species, it has been demonstrated that incubation temperature of eggs influenced the muscle development and growth rates, which persisted throughout the subsequent larval and juvenile phases. Therefore, this study aimed to investigate whether different incubation temperature of eggs prior to hatching can stimulate the muscle development, growth, and growth-related gene expression of the slow-growing indigenous species of climbing perch. The fertilized eggs of A. testudineus from an artificial breeding program were incubated under control temperature of 24 °C (IT24), 26 °C (IT26), 28 °C (IT28), and 30 °C (IT30) in 10L glass aquaria with four replicated units for each temperature treatment. After hatching, the larvae from each incubated temperature were separately reared at ambient temperature for 10 days in aquarium, 20 days in hapas, and the next 42 days in cages, totaling 72 days post-hatching (dph). The hatching rates were found significantly (P < 0.05) higher in IT28 compared to the other incubation temperature treatments. After 72 dph, the growth performances (%length gained, %weight gained and SGR) were found to be significantly highest (P < 0.05) in the IT28, followed by the treatments IT30, IT26, and IT24, respectively. Survival rate (73 ± 1.257%) was also found to be highest in the same treatment. The rate of new muscle fiber formation was identified to be significantly highest (P < 0.05) in IT28 followed by the IT26, IT30 and IT24, respectively. The relative mRNA expression level of GHRH, IGF1, IGF2 and PRL was also significantly highest in the IT28 (P < 0.05) compared to other treatments. Results from the present study clearly suggested that 28 °C is the optimum eggs incubation temperature of the native strain of A. testudineus for its highest growth performances in captive breeding condition.

Differences in morphological growth trajectories between greenside darter Etheostoma blennioides populations from two distinct drainages.

The greenside darter Etheostoma blennioides is a small-bodied benthic stream fish that occurs in multiple drainages of the eastern USA. Previous studies have revealed some morphological differentiations among greensides from isolated drainage systems but growth relationships among functional morphological characteristics have not been examined within and between populations. Specifically, we tested for differences in functional morphology and allometric distinctions in morphological growth trajectories between greenside populations from two drainages, the Osage River and White River. Morphological differences between individuals of the two drainages included more dorso-ventrally compressed bodies, longer snouts and larger jaw features in White River darters. Furthermore, body depth, snout length and jaw width grow at significantly different trajectories in individuals of the two drainages. Individuals of the White River drainage (generally a higher gradient stream system) become comparatively more streamlined and develop proportionately larger feeding related traits during growth. These results suggest that individuals of both drainages share a similar morphology during early life but deviate during continued development and this may be partially due to environmentally induced plastic response. This study represents a novel approach for comparing morphological development among fish populations and presents environmental factors (e.g., stream gradient and elevation) that potentially influence functional characteristics.

Fish size effect on sagittal otolith outer shape variability in round goby Neogobius melanostomus (Pallas 1814).

Round goby Neogobius melanostomus (Pallas 1814) has become a significant component in the diet of piscivorous fish from the Pomeranian Bay (Bornholm Basin, Baltic Sea). Proper identification of fish species in the diet of predators is significant in biological studies of fish and other aquatic animal species, and, with regard to N. melanostomus, it is important to the knowledge of trophic web structures in areas this species has invaded. A total of 142 individuals of N. melanostomus, measuring 16-174 mm standard length, were examined. Seventy-two fishes were caught during monitoring surveys in fishing grounds, whereas 70 were found in the stomachs of European perch Perca fluviatilis, pike-perch Sander lucioperca and Baltic cod Gadus morhua. The objective of the present study was to analyse the sagittal otoliths to identify variations in outer shape with increases in fish length; expand and correct descriptions of the sagitta, lapillus and asteriscus otoliths; and evaluate the relationships among otolith dimensions and fish standard length. The otoliths were described morphologically. The analysis of the outer shape of sagittal otoliths using Fourier analysis and multivariate statistics exhibited great phenotypic variability that was associated with fish length, including within pairs in individuals and/or among individuals in length classes. In addition, the asterisci and lapilli of N. melanostomus from selected specimens, which were described for the first time with regard to fish length, were found to be less variable compared to sagittal otoliths. This study presents the first analysis of intrapopulation phenotypic plasticity of N. melanostomus sagittal otolith morphology as it is linked to fish size.

Photoreceptors and eyes of pikeperch Sander lucioperca, pike Esox lucius, perch Perca fluviatilis and roach Rutilus rutilus from a clear and a brown lake.

The photoreceptors and eyes of four fish species commonly cohabiting Fennoscandian lakes with different light transmission properties were compared: pikeperch Sander lucioperca, pike Esox lucius, perch Perca fluviatilis and roach Rutilus rutilus. Each species was represented by individuals from a clear (greenish) and a humic (dark brown) lake in southern Finland: Lake Vesijärvi (LV; peak transmission around 570 nm) and Lake Tuusulanjärvi (LT; peak transmission around 630 nm). In the autumn, all species had almost purely A2-based visual pigments. Rod absorption spectra peaked at c.526 nm (S. lucioperca), c. 533 nm (E. lucius) and c. 540 nm (P. fluviatilis and R. rutilus), with no differences between the lakes. Esox lucius rods had remarkably long outer segments, 1.5-2.8-fold longer than those of the other species. All species possessed middle-wavelength-sensitive (MWS) and long-wavelength-sensitive (LWS) cone pigments in single, twin or double cones. Rutilus rutilus also had two types of short-wavelength sensitive (SWS) cones: UV-sensitive [SWS1] and blue-sensitive (SWS2) cones, although in the samples from LT no UV cones were found. No other within-species differences in photoreceptor cell complements, absorption spectra or morphologies were found between the lakes. However, E. lucius eyes had a significantly lower focal ratio in LT compared with LV, enhancing sensitivity at the expense of acuity in the dark-brown lake. Comparing species, S. lucioperca was estimated to have the highest visual sensitivity, at least two times higher than similar-sized E. lucius, thanks to the large relative size of the eye (pupil) and the presence of a reflecting tapetum behind the retina. High absolute sensitivity will give a competitive edge also in terms of short reaction times and long visual range.

Agonistic character displacement of genetically based male colour patterns across darters.

Agonistic character displacement (ACD) occurs when selection to avoid maladaptive interspecific aggression leads to the evolution of agonistic signals and/or associated behavioural biases in sympatry. Here, we test for a pattern consistent with ACD in male colour pattern in darters (Percidae: Etheostoma). Male colour pattern has been shown to function in male-male competition rather than female mating preferences in several darter species. Additionally, males bias their aggression towards conspecific over heterospecific males in sympatry but not in allopatry, consistent with divergent ACD in male behavioural biases. We use a common garden approach to show that differences in male colour pattern among four closely related darter species are genetically based. Additionally, we demonstrate that some aspects of male colour pattern exhibit enhanced differences in sympatric compared to allopatric populations of two darter species, consistent with ACD. However, other male colour traits are more similar between species in sympatry compared with allopatry, indicating that not all signal components are under strong divergent selection in sympatry. This study provides evidence that interspecific male-male aggressive interactions alone can promote elaborate male signal evolution both between and within species. We discuss the implications this has for male-driven ACD and cascade ACD.

Spawning-stock characteristics and migration of a lake-bound population of the endangered Macquarie perch Macquaria australasica.

The intrapopulation variability in the size and age structure of the spawning stock and migration of the threatened Macquarie perch Macquaria australasica in Lake Dartmouth was investigated between 2008 and 2016. Sampling centred on the core reproductive period (October-December) when mature fish migrate from the lake into riverine habitat to spawn. Spawning fish were predominantly large, spanning a broad age structure, with a high proportion of fish (25%) aged 15-30 years. The overall median size of spawning fish did not change for males or females during the 9 year study period. The size of the smaller mature male fish did change in some years suggesting a small proportion of male M. australasica matured at age 1+ and 2+. Acoustic telemetry employed over 3 years showed that M. australasica were most likely to be in the spawning reach from October to mid-December, migrated to this reach annually and moved large distances throughout the lake all year, with no evidence for any spatial structuring. Mature fish sometimes occupied the spawning reach for several months after the core reproductive period, which increased their vulnerability to recreational fishing. Males tagged in the lake were seldom recorded in the spawning reach, presumably because a high proportion had not yet entered the spawning stock despite their size suggesting maturity. Maintaining a broad age and size-structure of the spawning stock of long-lived iteroparous fish species is crucial for recruitment stability and population persistence. Overexploitation of the spawning stock has probably contributed to previous population declines in the lake as well as the collapse of other M. australasica populations in south-eastern Australia.

Feeding range of age 1+ year Eurasian perch Perca fluviatilis in the Baltic Sea.

Using the widespread Eurasian perch Perca fluviatilis as a model organism, feeding ranges were investigated using stable-isotope ratios (δ15 N and δ13 C) and body condition. Differences were found between closely located sampling sites in a littoral area without obvious migration barriers, indicating that individual fish had small feeding ranges. Body condition differences between sampled stations were consistent over 4 years. Such sedentary behaviour is important to consider in, e.g. fisheries management and environmental monitoring, as local catch regulations may be meaningful or geographic stability in sampling locations may reduce noise in data.

Chemicals released by predation increase the growth rate of yellow perch, Perca flavescens.

Water-soluble factors associated with walleye Sander vitreus predation on either yellow perch Perca flavescens or fathead minnows Pimephales promelas markedly increased the growth rate of P. flavescens. The findings suggest that P. flavescens possess an inducible growth-promoting mechanism regulated by water-born chemicals. It may be possible to increase the growth rate of farm-raised P. flavescens by manipulating this system.

Three new species of Plagioporus Stafford, 1904 from darters (Perciformes: Percidae), with a redescription of Plagioporus boleosomi (Pearse, 1924) Peters, 1957.

A form of Plagioporus Stafford, 1904 is described from the intestine of three North American species of darters (Perciformes: Percidae) from River West Twin, Wisconsin, USA, that we consider to be conspecific with Plagioporus boleosomi (Pearse, 1924) Peters, 1957 based on similarities in the sucker ratio, extent of the forebody, shape and position of the testes, vitellarium distribution and terminal genitalia. Three new species of Plagioporus are described from the intestine of darters as follows: Plagioporus fonti n. sp. from Percina nigrofasciata Agassiz in Florida, USA, Plagioporus limus n. sp. from Etheostoma squamosum Distler in Arkansas, USA and Plagioporus aliffi n. sp. from Etheostoma blennioides newmanni Miller in Arkansas, USA. Morphologically Plagioporus fonti n. sp., Plagioporus limus n. sp. and Plagioporus aliffi n. sp. are most similar to one another and to P. boleosomi, Plagioporus lepomis Dobrovolny, 1939 and 'P. etheostomae', a nomen nudum for a species described from Etheostoma blennioides Rafinesque in Kentucky, USA, all of which are collectively distinguished from congeners in having a combination of confluent vitellarium in the post-testicular space and absence of vitelline follicles with their entire length distributed in the forebody. Plagioporus fonti n. sp., P. limus n. sp. and P. aliffi n. sp. are respectively distinguished from one another and their closest congeners in having the anterior extent of the vitellarium in the anterior half of forebody to slightly anterior to the ventral sucker as opposed to one approximately at the level of the posterior margin of the ventral sucker, possession of an excretory vesicle reaching the anterior testis as opposed to one only reaching the posterior testis and having a longer than wide oral sucker and a wider than long ventral sucker. A Bayesian inference (BI) analysis of partial 28S rDNA sequences was conducted using the three new species and 24 sequences of opecoelids retrieved from GenBank, including ten species of Plagioporus. Plagioporus aliffi n. sp., Plagioporus fonti n. sp. and P. boleosomi comprised a moderately supported sister group to a clade containing all species of Plagioporus except Plagioporus limus n. sp. and Plagioporus shawi (Mcintosh, 1939) Margolis, 1970. Plagioporus limus and in turn P. shawi were resolved as sister to all other congeners with high and moderate support, respectively.

The accumulation of reproductive isolation in early stages of divergence supports a role for sexual selection.

Models of speciation by sexual selection propose that male-female coevolution leads to the rapid evolution of behavioural reproductive isolation. Here, we compare the strength of behavioural isolation to ecological isolation, gametic incompatibility and hybrid inviability in a group of dichromatic stream fishes. In addition, we examine whether any of these individual barriers, or a combined measure of total isolation, is predicted by body shape differences, male colour differences, environmental differences or genetic distance. Behavioural isolation reaches the highest values of any barrier and is significantly greater than ecological isolation. No individual reproductive barrier is associated with any of the predictor variables. However, marginally significant relationships between male colour and body shape differences with ecological and behavioural isolation are discussed. Differences in male colour and body shape predict total reproductive isolation between species; hierarchical partitioning of these two variables' effects suggests a stronger role for male colour differences. Together, these results suggest an important role for divergent sexual selection in darter speciation but raise new questions about the mechanisms of sexual selection at play and the role of male nuptial ornaments.

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.

Warming alters the body shape of European perch Perca fluviatilis.

The consequences of elevated temperature on body shape were investigated by comparing European perch Perca fluviatilis from the Forsmark area of the Baltic Sea to P. fluviatilis from a nearby Biotest enclosure. The Biotest is a man-made enclosure within the Baltic Sea that has received warm water from a nuclear power plant since 1980, resulting in temperatures that are elevated 5-10 °C relative to the surrounding Baltic Sea. Sampled fish ranged from young-of-the-year to 14 years. Geometric morphometrics and multivariate statistical analysis revealed significant morphological differences between individuals of P. fluviatilis from these two habitats. Most importantly, relative shape changed with size, with small individuals of P. fluviatilis from Biotest being characterized by a deeper body shape and a larger caudal peduncle than the smaller Baltic individuals. In large specimens, smaller differences were found with Biotest individuals being more slender than Baltic individuals. These results show that, in order to have a full understanding of the biological effects of elevated temperatures, studies that cover the entire size range of organisms will be important. Apart from the direct influence of temperature on growth rate and body shape, other ecological factors affected by temperature are discussed as possible contributors to the observed differences between the two populations.

A population genetic window into the past and future of the walleye Sander vitreus: relation to historic walleye and the extinct "blue pike" S. v. "glaucus".

BACKGROUND: Conserving genetic diversity and local adaptations are management priorities for wild populations of exploited species, which increasingly are subject to climate change, habitat loss, and pollution. These constitute growing concerns for the walleye Sander vitreus, an ecologically and economically valuable North American temperate fish with large Laurentian Great Lakes' fisheries. This study compares genetic diversity and divergence patterns across its widespread native range using mitochondrial (mt) DNA control region sequences and nine nuclear DNA microsatellite (µsat) loci, examining historic and contemporary influences. We analyze the genetic and morphological characters of a putative endemic variant- "blue pike" S. v. "glaucus" -described from Lakes Erie and Ontario, which became extinct. Walleye with turquoise-colored mucus also are evaluated, since some have questioned whether these are related to the "blue pike". RESULTS: Walleye populations are distinguished by considerable genetic divergence (mean FST mtDNA = 0.32 ± 0.01, µsat = 0.13 ± 0.00) and substantial diversity across their range (mean heterozygosity mtDNA = 0.53 ± 0.02, µsat = 0.68 ± 0.03). Southern populations markedly differ, possessing unique haplotypes and alleles, especially the Ohio/New River population that houses the oldest haplotype and has the most pronounced divergence. Northern formerly glaciated populations have greatest diversity in Lake Erie (mean heterozygosity mtDNA = 0.79 ± 0.00, µsat = 0.72 ± 0.01). Genetic diversity was much less in the historic Lake Erie samples from 1923-1949 (mean heterozygosity mtDNA = 0.05 ± 0.01, µsat = 0.47 ± 0.06) than today. The historic "blue pike" had no unique haplotypes/alleles and there is no evidence that it comprised a separate taxon from walleye. Turquoise mucus walleye also show no genetic differentiation from other sympatric walleye and no correspondence to the "blue pike". CONCLUSIONS: Contemporary walleye populations possess high levels of genetic diversity and divergence, despite habitat degradation and exploitation. Genetic and previously published tagging data indicate that natal homing and spawning site philopatry led to population structure. Population patterns were shaped by climate change and drainage connections, with northern ones tracing to post-glacial recolonization. Southerly populations possess unique alleles and may provide an important genetic reservoir. Allelic frequencies of Lake Erie walleye from ~70-90 years ago significantly differed from those today, suggesting population recovery after extensive habitat loss, pollution, and exploitation. The historic "blue pike" is indistinguishable from walleye, indicating that taxonomic designation is not warranted.

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.

Phylogenetic and coalescent strategies of species delimitation in snubnose darters (Percidae: Etheostoma).

The rapid accumulation of multilocus data sets has led to dramatic advances in methodologies for estimating evolutionary relationships among closely related species, but relatively less advancement has been made in methods for discriminating between competing species delimitation hypotheses. Multilocus data sets provide an advantage in testing species delimitation scenarios because they offer a direct test of species monophyly and aid in the biological interpretation of such phenomena as allele-sharing and deep coalescent events. Most species tree estimation methods that are designed to analyze multilocus data sets require the a priori assignment of individuals to species categories and therefore do not provide a strategy to directly test competing species delimitation scenarios. An approach was recently proposed that utilizes a coalescent-based species tree estimation method to inform species delimitation decisions by comparing likelihood scores that measure the fit of gene trees within a given species tree. We use a multilocus nuclear and mitochondrial DNA sequence data set to both reexamine a recently proposed species delimitation scenario in the Etheostoma simoterum species complex and test the utility of species tree estimation methods in testing species delimitation hypotheses. Descriptions of species in the E. simoterum species complex of snubnose darters, a group of six teleost freshwater fish species, are based largely on male nuptial coloration. Most of the putative species are nonmonophyletic at every examined locus. Using a novel combination of Bayesian-estimated gene tree topologies, Bayesian phylogenetic species tree inferences, coalescent simulations, and examination of phenotypic variation, we assess the occurrence of shared alleles among species, and we propose that results from our analyses support a three-species rather than a six-species delimitation scenario in the E. simoterum complex. We found that comparing likelihood scores from the species tree estimation approach used across many potential delimitation scenarios resulted in a systematic bias toward over-splitting in the E. simoterum complex and failed to support a species delimitation scenario that was consistent with geography, phenotype, or any previous species delimitation hypothesis. Despite common expectations, we demonstrate that application of molecular approaches to species delimitation can result in the recognition of fewer, instead of a larger number of species. In addition, our analyses highlight the importance of phenotypic character information in providing an independent assessment of alternative species delimitation hypotheses in the E. simoterum species complex.

Indirect trophic interactions with an invasive species affect phenotypic divergence in a top consumer.

While phenotypic responses to direct species interactions are well studied, we know little about the consequences of indirect interactions for phenotypic divergence. In this study we used lakes with and without the zebra mussel to investigate effects of indirect trophic interactions on phenotypic divergence between littoral and pelagic perch. We found a greater phenotypic divergence between littoral and pelagic individuals in lakes with zebra mussels and propose a mussel-mediated increase in pelagic and benthic resource availability as a major factor underlying this divergence. Lakes with zebra mussels contained higher densities of large plankton taxa and large invertebrates. We suggest that this augmented resource availability improved perch foraging opportunities in both the littoral and pelagic zones. Perch in both habitats could hence express a more specialized foraging morphology, leading to an increased divergence of perch forms in lakes with zebra mussels. As perch do not prey on mussels directly, we conclude that the increased divergence results from indirect interactions with the mussels. Our results hence suggest that species at lower food web levels can indirectly affect phenotypic divergence in species at the top of the food chain.

Genetic and morphometric differences demonstrate fine-scale population substructure of the yellow perch Perca flavescens: need for redefined management units.

Whole-body morphometrics and 15 nuclear DNA microsatellite loci were analysed for 158 Perca flavescens collected during the spawning season from four spawning locations in central Lake Erie, two along the northern shore and two along the southern shore, to evaluate fine-scale variation (spanning 17-94 km). Results showed significant morphological and genetic differences among P. flavescens from the four locations. The magnitudes of differences were unrelated to geographic distance, demonstrating spatially heterogeneous levels of genetic divergence. These results linked morphometric and genetic variation, showing a discontinuity of scale between currently defined management units and population structure of P. flavescens in Lake Erie, and support that P. flavescens might exist as one or more metapopulations. Findings demonstrate the value of using complementary techniques for evaluating population structure.

Nannoperca pygmaea, a new species of pygmy perch (Teleostei: Percichthyidae) from Western Australia.

A new species of pygmy perch (Percichthyidae) from south-western Australia is described on the basis of 15 specimens collected from the Hay River system. Nannoperca pygmaea sp. nov. differs from the sympatric congener N. vittata (Castelnau) by the absence of dark pigment on the ventral surface anterior to the anus, the possession of thin latero-ventral stripes, generally fewer dorsal rays and fewer anal rays, hind margin of scales on caudal peduncle without distinct pigment, and a more pronounced spot (ocellus) that is surrounded by a halo at the termination of the caudal peduncle. The new species is distinguished from congeners Nannoperca australis Günther, N. oxleyana Whitley and N. variegata Kuiter and Allen in possessing an exposed and serrated preorbital bone and jaws that may just reach to below the anterior margin of the eye, versus a smooth and hidden preorbital and the jaws reaching to at least below the pupil; and from the remaining congener, N. obscura (Klunzinger) in possessing a distinct haloed ocellus at base of caudal fin versus an indistinct barring, as well as a dark spot behind operculum, and the lack of dusky scale margins. It differs from the other sympatric pygmy perch found in the region, N. balstoni Regan, by the presence of an exposed rear edge of the preorbital (vs. hidden under skin), fewer transverse scale rows (13 vs. 15-16), small mouth (rarely reaching eye vs. reaching well beyond eye), ctenoid (vs. cycloid) body scales, generally fewer pectoral rays and smaller maximum size. Allozyme analyses unequivocally demonstrate that sympatric populations of N. pygmaea sp. nov. and N. vittata belong in different genetic lineages, display no genetic intermediates, and are diagnosable by fixed allozyme differences at 15 different loci. Due to its extremely restricted range, where it is known from only 0.06 km2, N. pygmaea sp. nov. requires urgent legislative protection.

[Intraspecies variation of reproductive strategies in river perch (Perca fluviatilis)].

The variation of reproductive strategies in different populations of river perch and intrapopulation groups differing in the rate of growth have been analyzed. Using a bioenergetic approach, the origin and differences between these groups have been studied. It was shown that bioenergetic mechanisms of regulation of the sizes and weight of the eggs lead to formation of the strategy of forced reproduction in the slow-growing group of perch, unlike the reproductive strategy of the fast-growing group of perch characterized by a later reaching of sexual maturity at a greater relative weight of gonads and spawning of larger eggs, which provides for a longer lifespan of larvae. The great number of age groups in the spawning part of this group compensates for a decrease in reproduction in years with unfavorable conditions.

Incipient speciation in allopatric Etheostoma rupestre (Percidae: Etheostomatinae) lineages, with the description of three new subspecies.

In recent years, new species descriptions for the North American darters have proliferated. Most species concepts accepted by contemporary ichthyologists require that a valid species be both monophyletic and diagnoseable, yet many lineages exhibit modal or range differences in morphological characteristics without individuals being diagnosable. Such scenarios present difficulties with regards to proper taxonomic recognition of divergent lineages and often prohibit appropriate conservation action. Following the example of recent authors, we provide meristic, geometric morphometric, and pigmentation data to support the recognition of three subspecies of Etheostoma rupestre, a species endemic to the Mobile Basin. These morphological data cohere with previous genetic work for E. rupestre. The nominate subspecies Etheostoma rupetsre rupestre (Tsais Rock Darter) is endemic to the Tombigbee River and Black Warrior River watersheds in Alabama and Mississippi and is characterized by having lower numbers of lateral blotches, lower range and mean of lateral line scales, lower modal number of scales above the lateral line, and lower degrees of nape squamation than other subspecies. Etheostoma rupestre piersoni (Shamrock Darter), ssp. nov., is endemic to the Cahaba and Alabama River Watersheds in Alabama and is characterized by intermediate counts of lateral blotches and higher scale counts and nape squamation than E. r. rupestre. Etheostoma rupestre uphapeense (Jade Darter), ssp. nov., is restricted to several small, disjunct populations in the Coosa and Tallapoosa watersheds in Alabama, Georgia, and Tennessee. Etheostoma r. uphapeense is characterized by having a higher mean number of lateral blotches than both other subspecies and higher scale counts than E. r. rupestre. While E. r. rupestre and E. r. piersoni are widespread and abundant within their respective ranges, E. r. uphapeense has a disjunct range and is often uncommon where it occurs. Etheostoma r. uphapeense should be monitored where it occurs to discern population trends.