Molecular identification of critically endangered European eels (Anguilla anguilla) in US retail outlets.

The European eel (Anguilla anguilla) has declined by over 90% since the early 1980s and has been listed as critically endangered. Yet, despite strict export bans from the European Union, the European eel is still sold illegally in many countries. Efforts to monitor the trade of European eels have been primarily concentrated in Asian markets where concerningly high rates of European eel have been reported. Comparably fewer studies have assessed the identities of eel samples from the United States (US), despite the obvious implications for eel conservation. To address this knowledge gap, we purchased 137 eel products (134 freshwater eels and three saltwater eels) from grocers, sushi bars, and restaurants in nine states across the US from 2019 to 2021. Seven samples (5.2%) labeled as freshwater eels (or "unagi") were identified as European eels using a combination of mitochondrial (cytochrome b) and nuclear (18S rRNA) restriction digestion assays, a fast and inexpensive molecular tool for seafood identification that can identify hybrids between European eels (A. anguilla) and American eels (A. rostrata). No hybrids between European and American eels were found and all seven samples identified with restriction digestion as European eels were confirmed by sequencing of cytochrome b and 18S rRNA. Frequency of European eels in US markets did not significantly correlate with state or retail type. Although illegal eel exports are likely reaching US consumers, the frequency of European eel samples in this study of the US market is much lower than found in other non-European countries.

Multiple and Extra-Pair Mating in a Pair-Living Hermaphrodite, the Intertidal Limpet Siphonaria gigas.

Pair-living is a common social system found across animal taxa, and the relationship between pair-living and reproduction varies greatly among species. Siphonaria gigas, hermaphroditic pulmonate gastropods, often live in pairs in the rocky intertidal zone of the tropical Eastern Pacific. Combining genetic parentage analysis using four polymorphic microsatellite loci with behavioral observations from a 10-week field study, we provide the first description of the mating system of a Siphonaria species incorporating genetic data. S. gigas mated both within-pair and extra-pair and three out of four paired S. gigas individuals produced egg masses with extra-pair paternity. Multiple paternity was detected, but at a relatively low frequency (19% of egg masses) compared to other marine gastropods. Behavioral data indicate one potential advantage of pair-living: paired S. gigas produced almost twice as many egg masses as their solitary counterparts over four reproductive cycles. These observations, together with constraints on the movement of S. gigas, suggest that pairing may ensure mate access and increase reproductive success.

Apareamiento múltiple y extrapareja en un hermafrodita que vive en pareja, la lapa intermareal Siphonaria gigas (Multiple and extra-pair mating in a pair-living hermaphrodite, the intertidal limpet Siphonaria gigas). Vivir en pareja es un sistema social común que se encuentra en los taxones de animales, y la relación entre la vida en pareja y la reproducción varía mucho entre las especies. Siphonaria gigas, gasterópodos pulmonados y hermafroditas, a menudo viven en parejas en la zona rocosa intermareal del Pacífico oriental tropical. Combinando el análisis de parentesco genético utilizando cuatro loci de microsatélites polimórficos con observaciones de comportamiento de un estudio de campo de 10 semanas, proporcionamos la primera descripción del sistema de apareamiento de una especie Siphonaria que incorpora datos genéticos. S. gigas se apareó tanto dentro como fuera de la pareja, y tres de cada cuatro individuos de S. gigas emparejados produjeron masas de huevo con paternidad extrapareja. Se detectó paternidad múltiple, pero a una frecuencia relativamente baja (19% de las masas de huevo) en comparación con otros gasterópodos marinos. Los datos de comportamiento indican una ventaja potencial de la vida en pareja: S. gigas emparejado produjo casi el doble de masas de huevo que sus contrapartes solitarias durante cuatro ciclos reproductivos. Estas observaciones, junto con el movimiento restringido de S. gigas, sugieren que el emparejamiento puede garantizar el acceso de pareja y aumentar el éxito reproductivo. Translated to Spanish by YE Jimenez (yordano_jimenez@brown.edu).

Geographic origin and timing of colonization of the Pacific Coast of North America by the rocky shore gastropod Littorina sitkana.

The demographic history of a species can have a lasting impact on its contemporary population genetic structure. Northeastern Pacific (NEP) populations of the rocky shore gastropod Littorina sitkana have very little mitochondrial DNA (mtDNA) sequence diversity and show no significant population structure despite lacking dispersive planktonic larvae. A contrasting pattern of high mtDNA diversity in the northwestern Pacific (NWP) suggests that L. sitkana may have recently colonized the NEP from the NWP via stepping-stone colonization through the Aleutian-Commander Archipelago (ACA) following the end of the last glacial 20,000 years ago. Here, we use multi-locus sequence data to test that hypothesis using a combination of descriptive statistics and population divergence modeling aimed at resolving the timing and the geographic origin of NEP populations. Our results show that NEP populations share a common ancestor with a population of L. sitkana on the Kamchatka Peninsula ∼46,900 years ago and that NEP populations diverged from each other ∼21,400 years ago. A more recent population divergence between Kamchatka and NEP populations, than between Kamchatka and other populations in the NWP, suggests that the ACA was the most probable dispersal route. Taking into account the confidence intervals for the estimates, we conservatively estimate that L. sitkana arrived in the NEP between 107,400 and 4,100 years ago, a range of dates that is compatible with post-glacial colonization of the NEP. Unlike other congeners that are relatively abundant in the Pleistocene fossil record of the NEP, only one report of L. sitkana exists from the NEP fossil record. Although broadly consistent with the molecular data, the biogeographic significance of these fossils is difficult to evaluate, as the shells cannot be distinguished from the closely-related congener L. subrotundata.

Discovery and optimization of novel benzothiophene-3-carboxamides as highly potent inhibitors of Aurora kinases A and B.

Aurora kinases as regulators of cell division have become promising therapeutic targets recently. Here we report novel, low molecular weight benzothiophene-3-carboxamide derivatives designed and optimized for inhibiting Aurora kinases. The most effective compound 36 inhibits Aurora kinases in vitro in the nanomolar range and diminishes HCT 116 cell viability blocking cytokinesis and inducing apoptosis. According to western blot analysis, the lead molecule inhibits Aurora kinases equipotently to VX-680 (Tozasertib) and similarly synergizes with other targeted drugs.

Novel compounds with potent CDK9 inhibitory activity for the treatment of myeloma.

Cyclin-dependent kinases (CDKs) and Polo-like kinases (PLKs) play key role in the regulation of the cell cycle. The aim of our study was originally the further development of our recently discovered polo-like kinase 1 (PLK1) inhibitors. A series of new 2,4-disubstituted pyrimidine derivatives were synthesized around the original hit, but their PLK1 inhibitory activity was very poor. However the novel compounds showed nanomolar CDK9 inhibitory activity and very good antiproliferative effect on multiple myeloma cell lines (RPMI-8226).

Formation of the Isthmus of Panama.

The formation of the Isthmus of Panama stands as one of the greatest natural events of the Cenozoic, driving profound biotic transformations on land and in the oceans. Some recent studies suggest that the Isthmus formed many millions of years earlier than the widely recognized age of approximately 3 million years ago (Ma), a result that if true would revolutionize our understanding of environmental, ecological, and evolutionary change across the Americas. To bring clarity to the question of when the Isthmus of Panama formed, we provide an exhaustive review and reanalysis of geological, paleontological, and molecular records. These independent lines of evidence converge upon a cohesive narrative of gradually emerging land and constricting seaways, with formation of the Isthmus of Panama sensu stricto around 2.8 Ma. The evidence used to support an older isthmus is inconclusive, and we caution against the uncritical acceptance of an isthmus before the Pliocene.

Out-of-the tropics or trans-tropical dispersal? The origins of the disjunct distribution of the gooseneck barnacle Pollicipes elegans.

BACKGROUND: Studying species with disjunct distributions allows biogeographers to evaluate factors controlling species ranges, limits on gene flow, and allopatric speciation. Here, we use phylogeographic and population genetic studies of the barnacle Pollicipes elegans to discriminate between two primary hypotheses about the origin of disjunct distributions of extra-tropical populations: trans-tropical stepping-stone colonization versus an out-of-the tropics origin. RESULTS: Nucleotide diversity peaked in the centre of the species' range in samples from El Salvador and was lower in samples from higher latitudes at Mexico and Peru. Haplotypes from El Salvador samples also had a deeper coalescent, or an older time to a most recent common ancestor. A deep phylogeographical break exists between Mexico and all samples taken to the south (El Salvador and Peru). Isolation-with-migration analyses showed no significant gene flow between any of the three regions indicating that the difference in genetic differentiation among all three regions is explained primarily by differences in population separation times. Approximate Bayesian Computation model testing found strong support for an out-of-the tropics origin of extra-tropical populations in P. elegans. CONCLUSIONS: We found little evidence consistent with a stepping-stone history of trans-tropical colonization, but instead found strong evidence for a tropical origin model for the largely disjunct distribution of P. elegans. Sea surface temperature and habitat suitability are likely mechanisms driving decline of populations in tropical regions, causing the disjunct distribution.

Seafood substitutions obscure patterns of mercury contamination in Patagonian toothfish (Dissostichus eleginoides) or "Chilean sea bass".

Seafood mislabeling distorts the true abundance of fish in the sea, defrauds consumers, and can also cause unwanted exposure to harmful pollutants. By combining genetic data with analyses of total mercury content, we have investigated how species substitutions and fishery-stock substitutions obscure mercury contamination in Patagonian toothfish (Dissostichus eleginoides), also known as "Chilean sea bass". Patagonian toothfish show wide variation in mercury concentrations such that consumers may be exposed to either acceptable or unacceptable levels of mercury depending on the geographic origins of the fish and the allowable limits of different countries. Most notably, stocks of Patagonian toothfish in Chile accumulate significantly more mercury than stocks closer to the South Pole, including the South Georgia/Shag Rocks stock, a fishery certified by the Marine Stewardship Council (MSC) as sustainably fished. Consistent with the documented geography of mercury contamination, our analysis showed that, on average, retail fish labeled as MSC-certified Patagonian toothfish had only half the mercury of uncertified fish. However, consideration of genetic data that were informative about seafood substitutions revealed a complex pattern of contamination hidden from consumers: species substitutions artificially inflated the expected difference in mercury levels between MSC-certified and uncertified fish whereas fishery stock substitutions artificially reduced the expected difference in mercury content between MSC-certified and uncertified fish that were actually D. eleginoides. Among MSC-certified fish that were actually D. eleginoides, several with exogenous mtDNA haplotypes (i.e., not known from the certified fishery) had mercury concentrations on par with uncertified fish from Chile. Overall, our analysis of mercury was consistent with inferences from the genetic data about the geographic origins of the fish, demonstrated the potential negative impact of seafood mislabeling on unwanted mercury exposure for consumers, and showed that fishery-stock substitutions may expose consumers to significantly greater mercury concentrations in retail-acquired fish than species substitutions.

Selection and demographic history shape the molecular evolution of the gamete compatibility protein bindin in Pisaster sea stars.

Reproductive compatibility proteins have been shown to evolve rapidly under positive selection leading to reproductive isolation, despite the potential homogenizing effects of gene flow. This process has been implicated in both primary divergence among conspecific populations and reinforcement during secondary contact; however, these two selective regimes can be difficult to discriminate from each other. Here, we describe the gene that encodes the gamete compatibility protein bindin for three sea star species in the genus Pisaster. First, we compare the full-length bindin-coding sequence among all three species and analyze the evolutionary relationships between the repetitive domains of the variable second bindin exon. The comparison suggests that concerted evolution of repetitive domains has an effect on bindin divergence among species and bindin variation within species. Second, we characterize population variation in the second bindin exon of two species: We show that positive selection acts on bindin variation in Pisaster ochraceus but not in Pisaster brevispinus, which is consistent with higher polyspermy risk in P. ochraceus. Third, we show that there is no significant genetic differentiation among populations and no apparent effect of sympatry with congeners that would suggest selection based on reinforcement. Fourth, we combine bindin and cytochrome c oxidase 1 data in isolation-with-migration models to estimate gene flow parameter values and explore the historical demographic context of our positive selection results. Our findings suggest that positive selection on bindin divergence among P. ochraceus alleles can be accounted for in part by relatively recent northward population expansions that may be coupled with the potential homogenizing effects of concerted evolution.

Density drives polyandry and relatedness influences paternal success in the Pacific gooseneck barnacle, Pollicipes elegans.

BACKGROUND: Polyandry is a common mating strategy in animals, increasing female fitness through direct (material) and indirect (genetic) benefits. Most theories about the benefits of polyandry come from studies of terrestrial animals, which have relatively complex mating systems and behaviors; less is known about the potential benefits of polyandry in sessile marine animals, for which potential mates may be scarce and females have less control over pre-copulatory mate choice. Here, we used microsatellite markers to examine multiple paternity in natural aggregations of the Pacific gooseneck barnacle Pollicipes elegans, testing the effect of density on paternity and mate relatedness on male reproductive success. RESULTS: We found that multiple paternity was very common (79% of broods), with up to five fathers contributing to a brood, though power was relatively low to detect more than four fathers. Density had a significant and positive linear effect on the number of fathers siring a brood, though this relationship leveled off at high numbers of fathers, which may reflect a lack of power and/or an upper limit to polyandry in this species. Significant skew in male reproductive contribution in multiply-sired broods was observed and we found a positive and significant relationship between the proportion of offspring sired and the genetic similarity between mates, suggesting that genetic compatibility may influence reproductive success in this species. CONCLUSIONS: To our knowledge, this is the first study to show high levels of multiple paternity in a barnacle, and overall, patterns of paternity in P. elegans appear to be driven primarily by mate availability. Evidence of paternity bias for males with higher relatedness suggests some form of post-copulatory sexual selection is taking place, but more work is needed to determine whether it operates during or post-fertilization. Overall, our results suggest that while polyandry in P. elegans is driven by mate availability, it may also provide a mechanism for females to ensure fertilization by compatible gametes and increase reproductive success in this sessile species.

Characterization of microsatellite loci and repeat density in the gooseneck barnacle, Pollicipes elegans, using next generation sequencing.

Pollicipes elegans is a commercially important and biogeographically significant rocky-shore gooseneck barnacle found along the eastern Pacific coasts of Peru, El Salvador, and Mexico. Little is known about its reproductive biology, and no genetic resources exist despite its growing importance as a fisheries species in the region. Next generation sequencing methods can provide rapid and cost-effective development of molecular markers such as microsatellites, which can be applied to studies of paternity, parentage, and population structure in this understudied species. Here, we used Roche 454 pyrosequencing to develop microsatellite markers in P. elegans and made genomic comparisons of repeat density and repeat class frequency with other arthropods and more distantly related taxa. We identified 13 809 repeats of 1-6 bp, or a density of 9744 bp of repeat per megabase queried, which was intermediate in the range of taxonomic groups compared. Comparison of repeat class frequency distributions revealed that P. elegans was most similar to Drosophila melanogaster rather than the more closely related crustacean Daphnia pulex. We successfully isolated 15 polymorphic markers with an average of 9.4 alleles per locus and average observed and expected heterozygosities of 0.501 and 0.597, respectively. Four loci were found to be out of Hardy-Weinberg equilibrium, likely due to the presence of null alleles. A preliminary population genetic analysis revealed low but significant differentiation between a Peruvian (n = 47) and Mexican (n = 48) population (F(ST) = 0.039) and markedly reduced genetic diversity in Peru. These markers should facilitate future studies of paternity, parentage, and population structure in this species.

The complex analytical landscape of gene flow inference.

Gene flow estimation is essential for characterizing local adaptation, speciation potential and connectivity among threatened populations. New model-based population genetic methods can resolve complex demographic histories, but many studies in fields such as landscape genetics continue to rely on simple rules of thumb focused on gene flow to explain patterns of spatial differentiation. Here, we show how methods that use gene genealogies can reveal cryptic demographic histories and provide better estimates of gene flow with other parameters that contribute to genetic variation across landscapes and seascapes. We advocate for the expanded use and development of methods that consider spatial differentiation as the product of multiple forces interacting over time, and caution against a routine reliance on post-hoc gene flow interpretations.

Demographic processes underlying subtle patterns of population structure in the scalloped hammerhead shark, Sphyrna lewini.

Genetic diversity (θ), effective population size (N(e)), and contemporary levels of gene flow are important parameters to estimate for species of conservation concern, such as the globally endangered scalloped hammerhead shark, Sphyrna lewini. Therefore, we have reconstructed the demographic history of S. lewini across its Eastern Pacific (EP) range by applying classical and coalescent population genetic methods to a combination of 15 microsatellite loci and mtDNA control region sequences. In addition to significant population genetic structure and isolation-by-distance among seven coastal sites between central Mexico and Ecuador, the analyses revealed that all populations have experienced a bottleneck and that all current values of θ are at least an order of magnitude smaller than ancestral θ, indicating large decreases in N(e) (θ = 4N(e)µ), where µ is the mutation rate. Application of the isolation-with-migration (IM) model showed modest but significant genetic connectivity between most sampled sites (point estimates of Nm = 0.1-16.7), with divergence times (t) among all populations significantly greater than zero. Using a conservative (i.e., slow) fossil-based taxon-specific phylogenetic calibration for mtDNA mutation rates, posterior probability distributions (PPDs) for the onset of the decline in N(e) predate modern fishing in this region. The cause of decline over the last several thousand years is unknown but is highly atypical as a post-glacial demographic history. Regardless of the cause, our data and analyses suggest that S. lewini was far more abundant throughout the EP in the past than at present.

Divergence genetics analysis reveals historical population genetic processes leading to contrasting phylogeographic patterns in co-distributed species.

Coalescent samplers are computational time machines for inferring the historical demographic genetic processes that have given rise to observable patterns of spatial genetic variation among contemporary populations. We have used traditional characterizations of population structure and coalescent-based inferences about demographic processes to reconstruct the population histories of two co-distributed marine species, the frilled dog whelk, Nucella lamellosa, and the bat star, Patiria miniata. Analyses of population structure were consistent with previous work in both species except that additional samples of N. lamellosa showed a larger regional genetic break on Vancouver Island (VI) rather than between the southern Alexander Archipelago as in P. miniata. Our understanding of the causes, rather than just the patterns, of spatial genetic variation was dramatically improved by coalescent analyses that emphasized variation in population divergence times. Overall, gene flow was greater in bat stars (planktonic development) than snails (benthic development) but spatially homogeneous within species. In both species, these large phylogeographic breaks corresponded to relatively ancient divergence times between populations rather than regionally restricted gene flow. Although only N. lamellosa shows a large break on VI, population separation times on VI are congruent between species, suggesting a similar response to late Pleistocene ice sheet expansion. The absence of a phylogeographic break in P. miniata on VI can be attributed to greater gene flow and larger effective population size in this species. Such insights put the relative significance of gene flow into a more comprehensive historical biogeographic context and have important implications for conservation and landscape genetic studies that emphasize the role of contemporary gene flow and connectivity in shaping patterns of population differentiation.

Small-molecule inhibitors of NADPH oxidase 4.

NOX enzymes are the major contributors in many oxidative damage related diseases. Unfortunately, at present no specific NOX inhibitor is available. Here, we describe the discovery and development of novel NOX4 inhibitors. Compound libraries were tested in a cell-based assay as a primary screen, monitoring H2O2 production. Twenty-four compounds inhibited Nox4 activity with low-micromolar IC(50) values of which three were selected for further drug development.

The 'Expansion-Contraction' model of Pleistocene biogeography: rocky shores suffer a sea change?

Approximately 20,000 years ago the last glacial maximum (LGM) radically altered the distributions of many Northern Hemisphere terrestrial organisms. Fewer studies describing the biogeographic responses of marine species to the LGM have been conducted, but existing genetic data from coastal marine species indicate that fewer taxa show clear signatures of post-LGM recolonization. We have assembled a mitochondrial DNA (mtDNA) data set for 14 co-distributed northeastern Pacific rocky-shore species from four phyla by combining new sequences from ten species with previously published sequences from eight species. Nuclear sequences from four species were retrieved from GenBank, plus we gathered new elongation factor 1-alpha sequences from the barnacle Balanus glandula. Results from demographic analyses of mtDNA for five (36%) species (Evasterias troschelii, Pisaster ochraceus, Littorina sitkana, L. scutulata, Xiphister mucosus) were consistent with large population expansions occurring near the LGM, a pattern expected if these species recently recolonized the region. However, seven (50%) species (Mytilus trossulus, M. californianus, B. glandula, S. cariosus, Patiria miniata, Katharina tunicata, X. atropurpureus) exhibited histories consistent with long-term stability in effective population size, a pattern indicative of regional persistence during the LGM. Two species of Nucella with significant mtDNA genetic structure showed spatially variable demographic histories. Multilocus analyses for five species were largely consistent with mtDNA: the majority of multilocus interpopulation divergence times significantly exceeded the LGM. Our results indicate that the LGM did not extirpate the majority of species in the northeastern Pacific; instead, regional persistence during the LGM appears a common biogeographic history for rocky-shore organisms in this region.

It's about time: divergence, demography, and the evolution of developmental modes in marine invertebrates.

Differences in larval developmental mode are predicted to affect ecological and evolutionary processes ranging from gene flow and population bottlenecks to rates of population recovery from anthropogenic disturbance and capacity for local adaptation. The most powerful tests of these predictions use comparisons among species to ask how phylogeographic patterns are correlated with the evolution and loss of prolonged planktonic larval development. An important and largely untested assumption of these studies is that interspecific differences in population genetic structure are mainly caused by differences in dispersal and gene flow (rather than by differences in divergence times among populations or changes in effective population sizes), and that species with similar patterns of spatial genetic variation have similar underlying temporal demographic histories. Teasing apart these temporal and spatial patterns is important for understanding the causes and consequences of evolutionary changes in larval developmental mode. New analytical methods that use the coalescent history of allelic diversity can reveal these temporal patterns, test the strength of traditional population-genetic explanations for variation in spatial structure based on differences in dispersal, and identify strongly supported alternative explanations for spatial structure based on demographic history rather than on gene flow alone. We briefly review some of these recent analytical developments, and show their potential for refining ideas about the correspondence between the evolution of larval developmental mode, population demographic history, and spatial genetic variation.