Effects of temperature acclimation on the upper thermal tolerance of two Arctic fishes.

The thermally dynamic nearshore Beaufort Sea, Alaska, is experiencing climate change-driven temperature increases. Measuring thermal tolerance of broad whitefish (Coregonus nasus) and saffron cod (Eleginus gracilis), both important species in the Arctic ecosystem, will enhance understanding of species-specific thermal tolerances. The objectives of this study were to determine the extent that acclimating broad whitefish and saffron cod to 5°C and 15°C changed their critical thermal maximum (CTmax) and HSP70 protein and mRNA expression in brain, muscle and liver tissues. After acclimation to 5°C and 15°C, the species were exposed to a thermal ramping rate of 3.4°C · h-1 before quantifying the CTmax and HSP70 protein and transcript concentrations. Broad whitefish and saffron cod acclimated to 15°C had a significantly higher mean CTmax (27.3°C and 25.9°C, respectively) than 5°C-acclimated fish (23.7°C and 23.2°C, respectively), which is consistent with trends in CTmax between higher and lower acclimation temperatures. There were species-specific differences in thermal tolerance with 15°C-acclimated broad whitefish having higher CTmax and HSP70 protein concentrations in liver and muscle tissues than saffron cod at both acclimation temperatures. Tissue-specific differences were quantified, with brain and muscle tissues having the highest and lowest HSP70 protein concentrations, respectively, for both species and acclimation temperatures. The differences in broad whitefish CTmax between the two acclimation temperatures could be explained with brain and liver tissues from 15°C acclimation having higher HSP70a-201 and HSP70b-201 transcript concentrations than control fish that remained in lab-acclimation conditions of 8°C. The shift in CTmax and HSP70 protein and paralogous transcripts demonstrate the physiological plasticity that both species possess in responding to two different acclimation temperatures. This response is imperative to understand as aquatic temperatures continue to elevate.

Comparison of Two Total Mercury Screening and Assessment Methods in Fishes: Biopsy Punch and Dried Muscle Samples.

Rapid and effective quantification of total mercury concentrations ([THg]) in fish muscle is an important part of ongoing monitoring to provide reliable and near real-time public health guidance. Methods for quantifying THg in fish muscle frequently require the use of large sample mass and numerous preparation steps. Wet (aka fresh weight) biopsy punch samples of fish muscle have been used to quantify THg directly, without drying and homogenization. Both methods have advantages and disadvantages. We compare the use of fresh weight biopsy punches for quantifying THg to using larger, dried homogenized samples. The [THg] determination for the two sampling methods was EPA method 7473. Three separate biopsy punch samples and a large muscle sample were taken from each fish and analyzed on a Direct Mercury Analyzer. There were no statistical differences between mean log transformed wet weight [THg] from biopsy punches and homogenized muscle across all samples or within individual species. Similarly, across the range of [THg] (7.5-612.7 ng/g ww), linear regression of [THg] from biopsy punch and homogenized muscle samples was not different from a 1:1 linear relationship. Linear regression statistics of [THg] with fish fork length produced similar results for both biopsy punch and homogenized muscle samples. However, the coefficient of variation among biopsy punch replicates for individual fish was frequently above the acceptable threshold of 15%. We recommend biopsy punches be used as an effective tool for broad-scale rapid monitoring of fish resources for Hg, while homogenized muscle samples be used for fine-scale ecological and health questions.

Ecotoxicology of mercury concentrations in arctic lamprey (Lethenteron camtschaticum).

Arctic lamprey (Lethenteron camtschaticum) is an important dietary resource for rural and indigenous communities in parts of Alaska, with some commercial use. As with many fish species harvested for human consumption, there are concerns regarding mercury concentrations ([Hg]) in Arctic lamprey that may impact human health. To date, information regarding the life cycle and diet of Arctic lamprey is scarce, with no published studies examining [Hg] in Arctic lamprey tissues. Our goals were to investigate the feeding ecology of Arctic lamprey from the Bering Sea, determine how diet and potential dietary shifts might influence [Hg] in muscle, and determine if current [Hg] may pose a human health risk. The mean total [Hg] in Arctic lamprey muscle (n = 98) was 19 ng/g wet-weight. Log transformed total [Hg] were not associated with any measured biological variables including length, mass, δ13C values, or δ15N values. A stable isotope mixing model estimated that capelin (Mallotus villosus) accounted for 40.0 ± 4.0% of the Arctic lamprey diet, while Pacific sand lance (Ammodytes hexapterus) and Pacific herring (Clupea pallasii) accounted for 37.8 ± 3.1% and 22.2 ± 3.5% respectively. Finally, diet percentage compositions shifted based on size class (i.e., medium versus large). These results indicated that feeding location, bioaccumulation, and biomagnification are not important drivers of [Hg] in Arctic lamprey and current [Hg] do not pose a human health risk. Taken together, this research further expands our knowledge of Arctic lamprey trophic ecology in the eastern Bering Sea.

Freshwater and anadromous fishing in Ice Age Beringia.

While freshwater and anadromous fish have been critical economic resources for late prehistoric and modern Native Americans, the origin and development of fishing is not well understood. We document the earliest known human use of freshwater and anadromous fish in North America by 13,000 and 11,800 years ago, respectively, from primary anthropogenic contexts in central Alaska (eastern Beringia). Fish use appears conditioned by broad climatic factors, as all occurrences but one are within the Younger Dryas chronozone. Earlier Bølling-Allerød and later early Holocene components, while exhibiting similar organic preservation, did not yield evidence of fishing, suggesting that this was a response to changing environmental factors, perhaps reductions in higher ranked resources such as large terrestrial mammals. Late Pleistocene and recent Indigenous peoples harvested similar fish taxa in the region (salmon, burbot, whitefish, and pike). We characterize late Pleistocene fishing in interior Beringia as an important element of broad-spectrum foraging rather than the intensive communal fishing and storage common among recent peoples.

Genomics reveal the origins and current structure of a genetically depauperate freshwater species in its introduced Alaskan range.

Invasive species are a major threat to global biodiversity, yet also represent large-scale unplanned ecological and evolutionary experiments to address fundamental questions in nature. Here we analyzed both native and invasive populations of predatory northern pike (Esox lucius) to characterize landscape genetic variation, determine the most likely origins of introduced populations, and investigate a presumably postglacial population from Southeast Alaska of unclear provenance. Using a set of 4329 SNPs from 351 individual Alaskan northern pike representing the most widespread geographic sampling to date, our results confirm low levels of genetic diversity in native populations (average 𝝅 of 3.18 × 10-4) and even less in invasive populations (average 𝝅 of 2.68 × 10-4) consistent with bottleneck effects. Our analyses indicate that invasive northern pike likely came from multiple introductions from different native Alaskan populations and subsequently dispersed from original introduction sites. At the broadest scale, invasive populations appear to have been founded from two distinct regions of Alaska, indicative of two independent introduction events. Genetic admixture resulting from introductions from multiple source populations may have mitigated the negative effects associated with genetic bottlenecks in this species with naturally low levels of genetic diversity. Genomic signatures strongly suggest an excess of rare, population-specific alleles, pointing to a small number of founding individuals in both native and introduced populations consistent with a species' life history of limited dispersal and gene flow. Lastly, the results strongly suggest that a small isolated population of pike, located in Southeast Alaska, is native in origin rather than stemming from a contemporary introduction event. Although theory predicts that lack of genetic variation may limit colonization success of novel environments, we detected no evidence that a lack of standing variation limited the success of this genetically depauperate apex predator.

Vulnerability of Pacific salmon to invasion of northern pike (Esox lucius) in Southcentral Alaska.

The relentless role of invasive species in the extinction of native biota requires predictions of ecosystem vulnerability to inform proactive management strategies. The worldwide invasion and range expansion of predatory northern pike (Esox lucius) has been linked to the decline of native fishes and tools are needed to predict the vulnerability of habitats to invasion over broad geographic scales. To address this need, we coupled an intrinsic potential habitat modelling approach with a Bayesian network to evaluate the vulnerability of five culturally and economically vital species of Pacific salmon (Oncorhynchus spp.) to invasion by northern pike. This study was conducted along 22,875 stream km in the Southcentral region of Alaska, USA. Pink salmon (O. gorbuscha) were the most vulnerable species, with 15.2% (2,458 km) of their calculated extent identified as "highly" vulnerable, followed closely by chum salmon (O. keta, 14.8%; 2,557 km) and coho salmon (O. kisutch, 14.7%; 2,536 km). Moreover, all five Pacific salmon species were highly vulnerable in 1,001 stream km of shared habitat. This simple to implement, adaptable, and cost-effective framework will allow prioritizing habitats for early detection and monitoring of invading northern pike.

The rise and fall of the ancient northern pike master sex-determining gene.

The understanding of the evolution of variable sex determination mechanisms across taxa requires comparative studies among closely related species. Following the fate of a known master sex-determining gene, we traced the evolution of sex determination in an entire teleost order (Esociformes). We discovered that the northern pike (Esox lucius) master sex-determining gene originated from a 65 to 90 million-year-old gene duplication event and that it remained sex linked on undifferentiated sex chromosomes for at least 56 million years in multiple species. We identified several independent species- or population-specific sex determination transitions, including a recent loss of a Y chromosome. These findings highlight the diversity of evolutionary fates of master sex-determining genes and the importance of population demographic history in sex determination studies. We hypothesize that occasional sex reversals and genetic bottlenecks provide a non-adaptive explanation for sex determination transitions.

Addressing incomplete lineage sorting and paralogy in the inference of uncertain salmonid phylogenetic relationships.

Recent and continued progress in the scale and sophistication of phylogenetic research has yielded substantial advances in knowledge of the tree of life; however, segments of that tree remain unresolved and continue to produce contradicting or unstable results. These poorly resolved relationships may be the product of methodological shortcomings or of an evolutionary history that did not generate the signal traits needed for its eventual reconstruction. Relationships within the euteleost fish family Salmonidae have proven challenging to resolve in molecular phylogenetics studies in part due to ancestral autopolyploidy contributing to conflicting gene trees. We examine a sequence capture dataset from salmonids and use alternative strategies to accommodate the effects of gene tree conflict based on aspects of salmonid genome history and the multispecies coalescent. We investigate in detail three uncertain relationships: (1) subfamily branching, (2) monophyly of Coregonus and (3) placement of Parahucho. Coregoninae and Thymallinae are resolved as sister taxa, although conflicting topologies are found across analytical strategies. We find inconsistent and generally low support for the monophyly of Coregonus, including in results of analyses with the most extensive dataset and complex model. The most consistent placement of Parahucho is as sister lineage of Salmo.

Ecological drivers of mercury concentrations in fish species in subsistence harvests from Kotzebue Sound, Alaska.

The State of Alaska assesses human exposure to mercury (Hg) via fish consumption producing consumption guidelines for fish tailored for children and women of childbearing age. Under these guidelines, unrestricted consumption is suggested for many fish species, while limited consumption is recommended for others. Subsequent questions have arisen regarding ecological drivers influencing [Hg] in fishes consumed by Alaskans. This community-assisted public health study evaluates [Hg] in fishes from Kotzebue Sound to examine factors that may drive observed [Hg]. We examined eight species of subsistence harvested fish (least cisco, chum salmon, Pacific herring, humpback whitefish, sheefish, starry flounder, Pacific tomcod, and fourhorn sculpin) from Kotzebue Sound. We report total Hg concentrations ([THg]) and monomethyl Hg+ concentrations ([MeHg+]) in the context of various factors (such as species, fork length, carbon and nitrogen stable isotope (δ15N or δ13C)) values that may influence [Hg] and [MeHg+]. Across all 297 fish, [THg] ranged from 3.4 - 235.2 ng/g ww. [THg] was positively correlated with fork length in six of eight fish species, as well as with trophic level (indicated by δ15N values) in five species. [MeHg+] was positively correlated with fork length in four species, and with δ15N values over all specimens examined, and specifically for three individual species. In six of the seven species analyzed, %MeHg was >80% of [THg]. This value decreased with fork length in three species, with no relationship for δ15N values in any species. Among top ranked models based on Akaike Information Criterion correction (AICc), fork length was more frequently included as an explanatory factor for [Hg] than δ15N or δ13C values. The food web magnification factor for [THg] was 11.3, and 12.6 for [MeHg+]. Biomagnification is likely driving [THg] and [MeHg+] over the entire food web, while within species, bioaccumulation is likely a stronger driver of [THg] and [MeHg+] than feeding ecology or trophic position. The [THg] for all species fell within the established unrestricted consumption guideline of 200 ng/g weight wet as established by the State of Alaska's fish consumption guidelines for Hg.

Mercury concentrations in marine species from the Aleutian Islands: Spatial and biological determinants.

Several species found in the Bering Sea show significant spatial variation in total mercury concentrations ([THg]) longitudinally along the Aleutian Island chain. We assessed [THg] in other members of the Bering Sea food web to better understand the factors shaping regional differences. [THg] and stable carbon and nitrogen isotope ratios (δ15N and δ13C values) were measured in muscle tissue from 1052 fishes and cephalopods from parts of the Bering Sea and North Pacific Ocean adjacent to the Aleutian Islands. The spatial distribution of the samples enabled regional comparisons for 8 species of fish and one species of cephalopod. Four species showed higher mean length-standardized [THg] in the western Aleutian Islands management area. [THg] in yellow Irish lord were very different relative to those observed in other species and when included in multi-species analyses drove the overall regional trends in mean [THg]. Multi-species analyses excluding measurements for yellow Irish lord showed mean length-standardized [THg] was greater in the western Aleutian Islands than in the central Aleutian Islands management area. Linear regression of [THg] and δ15N values showed a significant and positive relationship across all species, varying between regions and across species. Isotopic space of all species was significantly different between the western Aleutian Islands and central Aleutian Islands, driven largely by δ13C values. Stable isotope values observed follow the same regional trend of lower trophic taxa reported in the literature, with significantly lower δ13C values in the western Aleutian Islands. We conclude that there are regional differences in carbon and nitrogen stable isotope ecology, as well as species-specific feeding ecology that influence [THg] dynamics in part of the marine food web along the Aleutian Island chain. These regional differences are likely contributors to the observed regional variations of [THg] in some high-level predators found in these regions.

Genetic variation and population structure among larval Lethenteron spp. within the Yukon River drainage, Alaska.

The absence of information on genetic variation and population structure of lampreys Lethenteron spp. in the eastern part of their distribution limits our understanding of the migration ecology and spatial population genetic structure of the species. We examined genetic variation within and among three aggregations of Lethenteron spp. larvae in the Yukon River drainage, Alaska, using microsatellite genotypes. A total of 120 larval lampreys were genotyped at eight microsatellite loci. Global FST was 0.053 (95% CI 0.021-0.086), while pairwise FST values ranged from 0.048-0.057. Model-based Bayesian clustering analyses with sample locality priors (LOCPRIOR) identified three distinct, but admixed, genetic clusters that corresponded with the three aggregations. Estimates of contemporary gene flow indicate substantial reciprocal migration among sites consistent with no or low-fidelity natal homing. These results are largely in agreement with previous reports of historic and contemporary gene flow among Lethenteron spp. in other parts of their geographic distribution.

Early-branching euteleost relationships: areas of congruence between concatenation and coalescent model inferences.

Phylogenetic inference based on evidence from DNA sequences has led to significant strides in the development of a stable and robustly supported framework for the vertebrate tree of life. To date, the bulk of those advances have relied on sequence data from a small number of genome regions that have proven unable to produce satisfactory answers to consistently recalcitrant phylogenetic questions. Here, we re-examine phylogenetic relationships among early-branching euteleostean fish lineages classically grouped in the Protacanthopterygii using DNA sequence data surrounding ultraconserved elements. We report and examine a dataset of thirty-four OTUs with 17,957 aligned characters from fifty-three nuclear loci. Phylogenetic analysis is conducted in concatenated, joint gene trees and species tree estimation and summary coalescent frameworks. All analytical frameworks yield supporting evidence for existing hypotheses of relationship for the placement of Lepidogalaxias salamandroides, monophyly of the Stomiatii and the presence of an esociform + salmonid clade. Lepidogalaxias salamandroides and the Esociformes + Salmoniformes are successive sister lineages to all other euteleosts in the majority of analyses. The concatenated and joint gene trees and species tree analysis types produce high support values for this arrangement. However, inter-relationships of Argentiniformes, Stomiatii and Neoteleostei remain uncertain as they varied by analysis type while receiving strong and contradictory indices of support. Topological differences between analysis types are also apparent within the otomorph and the percomorph taxa in the data set. Our results identify concordant areas with strong support for relationships within and between early-branching euteleost lineages but they also reveal limitations in the ability of larger datasets to conclusively resolve other aspects of that phylogeny.

Littorally adaptive? Testing the link between habitat, morphology, and reproduction in the intertidal sculpin subfamily Oligocottinae (Pisces: Cottoidea).

While intertidal habitats are often productive, species-rich environments, they are also harsh and highly dynamic. Organisms that live in these habitats must possess morphological and physiological adaptations that enable them to do so. Intertidal fishes are generally small, often lack scales, and the diverse families represented in intertidal habitats often show convergence into a few general body shapes. However, few studies have quantified the relationship between phenotypes and intertidal living. Likewise, the diversity of reproductive traits and parental care in intertidal fishes has yet to be compared quantitatively with habitat. We examine the relationship of these characters in the sculpin subfamily Oligocottinae using a phylogenetic hypothesis, geometric morphometrics, and phylogenetic comparative methods to provide the first formal test of associations between fish phenotypes and reproductive characters with intertidal habitats. We show that the ability to live in intertidal habitats, particularly in tide pools, is likely a primitive state for Oligocottinae, with a single species that has secondarily come to occupy only subtidal habitats. Contrary to previous hypotheses, maximum size and presence of scales do not show a statistically significant correlation with depth. However, the maximum size for all species is generally small (250 mm or less) and all show a reduction in scales, as would be expected for an intertidal group. Also contrary to previous hypotheses, we show that copulation and associated characters are the ancestral condition in Oligocottinae, with copulation most likely being lost in a single lineage within the genus Artedius. Lastly, we show that body shape appears to be constrained among species with broader depth ranges, but lineages that occupy only a narrow range of intertidal habitats display novel body shapes, and this may be associated with habitat partitioning, particularly as it relates to the degree of wave exposure.

Trophic ecology of introduced populations of Alaska blackfish (Dallia pectoralis) in the Cook Inlet Basin, Alaska.

Introduced non-native fishes have the potential to substantially alter aquatic ecology in the introduced range through competition and predation. The Alaska blackfish (Dallia pectoralis) is a freshwater fish endemic to Chukotka and Alaska north of the Alaska Range (Beringia); the species was introduced outside of its native range to the Cook Inlet Basin of Alaska in the 1950s, where it has since become widespread. Here we characterize the diet of Alaska blackfish at three Cook Inlet Basin sites, including a lake, a stream, and a wetland. We analyze stomach plus esophageal contents to assess potential impacts on native species via competition or predation. Alaska blackfish in the Cook Inlet Basin consume a wide range of prey, with major prey consisting of epiphytic/benthic dipteran larvae, gastropods, and ostracods. Diets of the introduced populations of Alaska blackfish are similar in composition to those of native juvenile salmonids and stickleback. Thus, Alaska blackfish may affect native fish populations via competition. Fish ranked third in prey importance for both lake and stream blackfish diets but were of minor importance for wetland blackfish.

Beringian sub-refugia revealed in blackfish (Dallia): implications for understanding the effects of Pleistocene glaciations on Beringian taxa and other Arctic aquatic fauna.

BACKGROUND: Pleistocene climatic instability had profound and diverse effects on the distribution and abundance of Arctic organisms revealed by variation in phylogeographic patterns documented in extant Arctic populations. To better understand the effects of geography and paleoclimate on Beringian freshwater fishes, we examined genetic variability in the genus Dallia (blackfish: Esociformes: Esocidae). The genus Dallia groups between one and three nominal species of small, cold- and hypoxia-tolerant freshwater fishes restricted entirely in distribution to Beringia from the Yukon River basin near Fairbanks, Alaska westward including the Kuskokwim River basin and low-lying areas of Western Alaska to the Amguema River on the north side of the Chukotka Peninsula and Mechigmen Bay on the south side of the Chukotka Peninsula. The genus has a non-continuous distribution divided by the Bering Strait and the Brooks Range. We examined the distribution of genetic variation across this range to determine the number and location of potential sub-refugia within the greater Beringian refugium as well as the roles of the Bering land bridge, Brooks Range, and large rivers within Beringia in shaping the current distribution of populations of Dallia. Our analyses were based on DNA sequence data from two nuclear gene introns (S7 and RAG1) and two mitochondrial genome fragments from nineteen sampling locations. These data were examined under genetic clustering and coalescent frameworks to identify sub-refugia within the greater Beringia refugium and to infer the demographic history of different populations of Dallia. RESULTS: We identified up to five distinct genetic clusters of Dallia. Four of these genetic clusters are present in Alaska: (1) Arctic Coastal Plain genetic cluster found north of the Brooks Range, (2) interior Alaska genetic cluster placed in upstream locations in the Kuskokwim and Yukon river basins, (3) a genetic cluster found on the Seward Peninsula, and (4) a coastal Alaska genetic cluster encompassing downstream Kuskokwim River and Yukon River basin sample locations and samples from Southwest Alaska not in either of these drainages. The Chukotka samples are assigned to their own genetic cluster (5) similar to the coastal Alaska genetic cluster. The clustering and ordination analyses implemented in Discriminant Analysis of Principal Components (DAPC) and STRUCTURE showed mostly concordant groupings and a high degree of differentiation among groups. The groups of sampling locations identified as genetic clusters correspond to geographic areas divided by likely biogeographic barriers including the Brooks Range and the Bering Strait. Estimates of sequence diversity (θ) are highest in the Yukon River and Kuskokwim River drainages near the Bering Sea. We also infer asymmetric migration rates between genetic clusters. The isolation of Dallia on the Arctic Coastal Plain of Alaska is associated with very low estimated migration rates between the coastal Alaska genetic cluster and the Arctic Coastal Plain genetic cluster. CONCLUSIONS: Our results support a scenario with multiple aquatic sub-refugia in Beringia during the Pleistocene and the preservation of that structure in extant populations of Dallia. An inferred historical presence of Dallia across the Bering land bridge explains the similarities in the genetic composition of Dallia in West Beringia and western coastal Alaska. In contrast, historic and contemporary isolation across the Brooks Range shaped the distinctiveness of present day Arctic Coastal Plain Dallia. Overall this study uncovered a high degree of genetic structuring among populations of Dallia supporting the idea of multiple Beringian sub-refugia during the Pleistocene and which appears to be maintained to the present due to the strictly freshwater nature and low dispersal ability of this genus.

Two New Species of Marine Saprotrophic Sphaeroformids in the Mesomycetozoea Isolated From the Sub-Arctic Bering Sea.

The genus Sphaeroforma previously encompassed organisms isolated exclusively from animal symbionts in marine systems. The first saprotrophic sphaeroformids (Mesomycetozoea) isolated from non-animal hosts are described here. Sphaeroforma sirkka and S. napiecek are also the first species in the genus possessing endogenous DNA-containing motile propagules and central vacuoles, traits that have previously guided morphological differentiation of sphaeroformids from the genus Creolimax. Phylogenetic analysis of DNA sequences from the 18S rRNA and the ITS1-5.8S--ITS2 loci firmly place S. sirkka and S. napiecek within Sphaeroforma, extending the number of known species to six within this genus. The discovery of these species increases the geographical range, cellular variation and life history complexity of the sphaeroformids.

Mercury and methylmercury distribution in tissues of sculpins from the Bering Sea.

Fish skeletal muscle is often used to monitor mercury concentrations and is used by regulatory agencies to develop fish consumption advisories. However, the distribution of mercury species (MeHg+ and THg) in muscle tissue and other organs is not well understood in a number of fish species. Here we evaluate the spatial distribution of THg and MeHg+ in skeletal muscle and internal organs (heart, liver, and kidney) of 19 sculpin representing three species: Myoxocephalus scorpius (shorthorn sculpin n = 13), Myoxocephalus jaok (plain sculpin, n = 4), and Megalocottus platycephalus (belligerent sculpin, n = 2). Four subsamples of muscle were taken along the lateral aspect of each fish, from muscle A (cranial) to muscle D (caudal). Using Games-Howell post hoc procedure to compare mean concentrations of all tissues, muscle samples were significantly different from internal organs, although there was no difference between muscle-sampling locations. THg concentrations (ww) were higher in muscle (muscle A through D mean ± SD, 0.30 ± 0.19 mg/kg) than that in heart (0.06 ± 0.05 mg/kg), kidney (0.08 ± 0.06 mg/kg), and liver (0.09 ± 0.08 mg/kg). Percent MeHg+ decreased with age in both skeletal muscle and organs (p < 0.05). In contrast to some previous reports for other fish species, this study found significantly higher THg concentrations in muscle than in the liver. This study highlights the importance of using muscle samples when evaluating potential Hg exposure in risk assessments for piscivorous wildlife and human populations, and assumptions related to organ mercury concentrations should be examined with care.

Mitochondrial genomic investigation of flatfish monophyly.

We present the first study to use whole mitochondrial genome sequences to examine phylogenetic affinities of the flatfishes (Pleuronectiformes). Flatfishes have attracted attention in evolutionary biology since the early history of the field because understanding the evolutionary history and patterns of diversification of the group will shed light on the evolution of novel body plans. Because recent molecular studies based primarily on DNA sequences from nuclear loci have yielded conflicting results, it is important to examine phylogenetic signal in different genomes and genome regions. We aligned and analyzed mitochondrial genome sequences from thirty-nine pleuronectiforms including nine that are newly reported here, and sixty-six non-pleuronectiforms (twenty additional clade L taxa [Carangimorpha or Carangimorpharia] and forty-six secondary outgroup taxa). The analyses yield strong support for clade L and weak support for the monophyly of Pleuronectiformes. The suborder Pleuronectoidei receives moderate support, and as with other molecular studies the putatively basal lineage of Pleuronectiformes, the Psettodoidei is frequently not most closely related to other pleuronectiforms. Within the Pleuronectoidei, the basal lineages in the group are poorly resolved, however several flatfish subclades receive consistent support. The affinities of Lepidoblepharon and Citharoides among pleuronectoids are particularly uncertain with these data.

Molecular data do not provide unambiguous support for the monophyly of flatfishes (Pleuronectiformes): a reply to Betancur-R and Ortí.

Betancur-R and Ortí (2014) offer a criticism of our recent examination of the monophyly of extant flatfishes (Pleuronectiformes; Campbell et al., 2013). We welcome this opportunity to examine and respond to the main issues presented in Betancur-R and Ortí (2014). Briefly, this debate centers on the question of whether or not analyses of the available evidence support a stable and confident conclusion regarding a sister group relationship between the two recognized pleuronectiform suborders: Psettodoidei (four species) and Pleuronectoidei (>700 species). In Campbell et al. (2013), we reported results based on sequences from six nuclear genes compatible with monophyly of Pleuronectoidei but not with that of Pleuronectiformes. In our analyses, the most closely related percomorph family to the Pleuronectoidei was resolved to be the Centropomidae. In Campbell et al. (2013), we also provided a critical review of the morphological evidence in favor flatfish monophyly showing that this evidence requires a careful re-examination where it concerns psettodoids. Here we present our perspective on the issues raised in Betancur-R and Ortí (2014).

Phylogeny of the Elopomorpha (Teleostei): evidence from six nuclear and mitochondrial markers.

The Elopomorpha (eels and relatives) is a morphologically diverse group of predominantly marine teleost fishes comprising about 1000 species placed in 25 families. It is one of the three major living teleost lineages along with the Osteoglossomorpha and Clupeocephala. Among a few morphological synapomorphies that have been offered as evidence for the monophyly of the Elopomorpha, the remarkable leptocephalus larvae stand out. Several studies aiming at reconstructing the elopomorph phylogeny using morphological or molecular characters led to inconsistent results. In this study, we have tested previous hypotheses regarding inter- and intra-relationships of the Elopomorpha using a multi-locus dataset composed of three nuclear and three mitochondrial genes. Our analyses were based on likelihood phylogenetic reconstruction methods using different character-weighting data matrices and gene partition schemes to assess reliability of our findings. Our results confirm the respective monophyly of the Elopomorpha, Osteoglossomorpha and Clupeocephala. The majority of our analyses identify the Elopomorpha as the sister group of a clade containing the rest of the Teleostei. Within the Elopomorpha, the Elopiformes is the sister group of the remaining taxa. The Albuliformes sensuForey et al. (1996) and the Notacanthiformes are never sister-taxa in our phylogenetic trees, in contradiction with the recent mitogenomic hypothesis and current classification. Our results place the Notacanthiformes as the sister group of the Anguilliformes, including Saccopharyngiformes. Among anguilliforms, the families Congridae and Muraenesocidae are not monophyletic. The recently discovered anguilliform "living fossil" family Protanguillidae is not the sister group of the remaining Anguilliformes, instead, the sister group of the Synaphobranchidae. Based on the results presented here, we propose a revised classification for the Elopomorpha, comprised of four orders, including a resurrected Notacanthiformes but surrendering the Saccopharyngiformes. Within Anguilliformes, we recognized four monophyletic suborders named Protanguilloidei, Muraenoidei, Anguilloidei, and Congroidei.