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Hill, GE, Havird JC, Sloan DB, Burton RS, Greening C, Dowling DK.  2019.  Assessing the fitness consequences of mitonuclear interactions in natural populations. Biological Reviews. 94:1089-1104.   10.1111/brv.12493   AbstractWebsite

Metazoans exist only with a continuous and rich supply of chemical energy from oxidative phosphorylation in mitochondria. The oxidative phosphorylation machinery that mediates energy conservation is encoded by both mitochondrial and nuclear genes, and hence the products of these two genomes must interact closely to achieve coordinated function of core respiratory processes. It follows that selection for efficient respiration will lead to selection for compatible combinations of mitochondrial and nuclear genotypes, and this should facilitate coadaptation between mitochondrial and nuclear genomes (mitonuclear coadaptation). Herein, we outline the modes by which mitochondrial and nuclear genomes may coevolve within natural populations, and we discuss the implications of mitonuclear coadaptation for diverse fields of study in the biological sciences. We identify five themes in the study of mitonuclear interactions that provide a roadmap for both ecological and biomedical studies seeking to measure the contribution of intergenomic coadaptation to the evolution of natural populations. We also explore the wider implications of the fitness consequences of mitonuclear interactions, focusing on central debates within the fields of ecology and biomedicine.

Harada, AE, Burton RS.  2019.  Ecologically relevant temperature ramping rates enhance the protective heat shock response in an intertidal ectotherm. Physiological and Biochemical Zoology. 92:152-162.   10.1086/702339   AbstractWebsite

Thermal stress experiments are essential for understanding organisms' thermal limits and the physiological processes that contribute to establishing those limits. Experiments typically employ either an abrupt transfer to near-lethal temperatures or a gradually increasing thermal exposure. In the current study, we used three populations of the intertidal copepod Tigriopus californicus that are known to differ in upper thermal tolerance to investigate the effects of gradual versus abrupt thermal exposures on survivorship, developmental time, and heat shock protein gene expression. The developmental rate of nauplii was unaffected following the gradual exposure, whereas developmental time slowed by similar to 2 d (similar to 20%) following an abrupt exposure. The gradual exposure also improved survivorship in comparison to the abrupt exposure. Furthermore, the heat shock protein genes hsp70 and hspb1 showed greater upregulation during the gradual thermal exposure compared to the abrupt exposure. Though the differences in response to each thermal regime varied in magnitude among the different populations, the types of responses were very similar (i.e., following the gradual exposure survivorship increased, developmental time showed no effect, and heat shock protein gene upregulation during the exposure increased). Therefore, the enhanced protective effect of the heat shock response during gradual exposures appears to be conserved within the species despite population-level differences in thermal tolerance. Thus, an ecologically relevant thermal exposure likely enables improved cellular protective mechanisms by allowing for an effective and timely heat shock response, which plays a role in mitigating the effects of thermal stress and thereby enhances tolerance to elevated temperatures.

Lima, TG, Burton RS, Willett CS.  2019.  Genomic scans reveal multiple mito-nuclear incompatibilities in population crosses of the copepod Tigriopus californicus. Evolution. 73:609-620.   10.1111/evo.13690   AbstractWebsite

The evolution of intrinsic postzygotic isolation can be explained by the accumulation of Dobzhansky-Muller incompatibilities (DMI). Asymmetries in the levels of hybrid inviability and hybrid sterility are commonly observed between reciprocal crosses, a pattern that can result from the involvement of uniparentally inherited factors. The mitochondrial genome is one such factor that appears to participate in DMI in some crosses but the frequency of its involvement versus biparentally inherited factors is unclear. Here we assess the relative importance of incompatibilities between nuclear factors (nuclear-nuclear) versus those between mitochondrial and nuclear factors (mito-nuclear) in a species that lacks sex chromosomes. We used a Pool-seq approach to survey three crosses among genetically divergent populations of the copepod, Tigriopus californicus, for regions of the genome that are affected by hybrid inviability. Results from reciprocal crosses suggest that mito-nuclear incompatibilities are more common than nuclear-nuclear incompatibilities overall. These results suggest that in the presence of very high levels of nucleotide divergence between mtDNA haplotypes, mito-nuclear incompatibilities can be important for the evolution of intrinsic postzygotic isolation. This is particularly interesting considering this species lacks sex chromosomes, which have been shown to harbor a particularly high number of nuclear-nuclear DMI in several other species.

Harada, AE, Healy TM, Burton RS.  2019.  Variation in thermal tolerance and its relationship to mitochondrial function across populations of Tigriopus californicus. Frontiers in Physiology. 10   10.3389/fphys.2019.00213   AbstractWebsite

Variation in thermal tolerance plays a key role in determining the biogeographic distribution of organisms. Consequently, identifying the mechanistic basis for thermal tolerance is necessary for understanding not only current species range limits but also the capacity for range limits to shift in response to climate change. Although variation in mitochondrial function likely contributes to variation in thermal tolerance, the extent to which mitochondrial function underlies local thermal adaptation is not fully understood. In the current study, we examine variation in thermal tolerance and mitochondrial function among three populations of the intertidal copepod Tigriopus californicus found across a latitudinal thermal gradient along the coast of California, USA. We tested (1) acute thermal tolerance using survivorship and knockdown assays, (2) chronic thermal tolerance using survivorship of nauplii and developmental rate, and (3) mitochondrial performance at a range of temperatures using ATP synthesis fueled by complexes I, II, and I&II, as well as respiration of permeabilized fibers. We find evidence for latitudinal thermal adaptation: the southernmost San Diego population outperforms the northernmost Santa Cruz in measures of survivorship, knockdown temperature, and ATP synthesis rates during acute thermal exposures. However, under a chronic thermal regime, survivorship and developmental rate are more similar in the southernmost and northernmost population than in the mid-range population (Abalone Cove). Though this pattern is unexpected, it aligns well with population-specific rates of ATP synthesis at these chronic temperatures. Combined with the tight correlation of ATP synthesis decline and knockdown temperature, these data suggest a role for mitochondria in setting thermal range limits and indicate that divergence in mitochondrial function is likely a component of adaptation across latitudinal thermal gradients.

Duke, EM, Harada AE, Burton RS.  2018.  Large interannual variation in spawning in San Diego marine protected areas captured by molecular identification of fish eggs. Marine Ecology Progress Series. 604:199-210.   10.3354/meps12738   AbstractWebsite

Long-term monitoring of marine ecosystems is critical to assessing how global processes such as natural environmental variation and climate change affect marine populations. Ichthyoplankton surveys provide one approach to such monitoring. We conducted weekly fish egg collections off the Scripps Institution of Oceanography (SIO) Pier (La Jolla, CA, USA) for 3 yr (2014 to 2017) and added a second sampling site near the La Jolla kelp forest for 1 yr (2017). Fish eggs were identified using DNA barcoding and data were compared to previous work from SIO Pier surveys from 2012 to 2014. We documented large interannual variability in fish egg abundance associated with climatic fluctuations, including an El Nino event captured during our sampling years. Overall egg abundance was reduced by >50% during periods of anomalously warm water in 2014 to 2016. Fish egg abundance rebounded in 2017 and was accompanied by a phenological shift of peak spawning activity. We found that interannual fish egg abundance may be linked with upwelling regimes and winter temperatures. Across the period of joint sampling, we found no distinct differences in community composition between the SIO Pier (soft bottom) and kelp forest habitat we sampled (2 km distant). Long-term monitoring of fish spawning can contribute to our understanding of how natural environmental variation, such as El Nino events, affects fish reproductive activity. This understanding may extend to trends in marine resource availability associated with climate and aid in evaluating the efficacy of existing management efforts.

Tsuboko-Ishii, S, Burton RS.  2018.  individual culturing of tigriopus copepods and quantitative analysis of their mate-guarding behavior. Jove-Journal of Visualized Experiments.   10.3791/58378   AbstractWebsite

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a potential mate to form a pair. While this phenomenon has attracted interest of researchers, methods for its analysis have not been well described. Here we describe procedures for: 1) individual culturing and staging of Tigriopus juveniles and adults, and 2) video-based analysis of their mate-guarding behavior. The culturing method enables experimental control of paring experience of animals as well as the ability to track their development before behavioral tests. The analysis method allows quantitative evaluation of several aspects of the mate-guarding behavior, including capturing attempts by males and swimming trajectory of mate-guarding pairs. Although these methods were originally established for ethological studies on Tigriopus, with proper modifications they can also be applied to studies of other zooplankton in different research fields, such as physiology, toxicology, and ecological genetics.

Tangwancharoen, S, Moy GW, Burton RS.  2018.  Multiple modes of adaptation: Regulatory and structural evolution in a small heat shock protein gene. Molecular Biology and Evolution. 35:2110-2119.   10.1093/molbev/msy138   AbstractWebsite

Thermal tolerance is a key determinant of species distribution. Despite much study, the genetic basis of adaptive evolution of thermal tolerance, including the relative contributions of transcriptional regulation versus protein evolution, remains unclear. Populations of the intertidal copepod Tigriopus californicus are adapted to local thermal regimes across their broad geographic range. Upon thermal stress, adults from a heat tolerant southern population, San Diego (SD), upregulate several heat shock proteins (HSPs) to higher levels than those from a less tolerant northern population, Santa Cruz (SC). Suppression of a specific HSP, HSPB1, significantly reduces T. californicus survival following acute heat stress. Sequencing of HSPB1 revealed population specific nucleotide substitutions in both promoter and coding regions of the gene. HSPB1 promoters from heat tolerant populations contain two canonical heat shock elements (HSEs), the binding sites for heat shock transcription factor (HSF), whereas less tolerant populations have mutations in these conserved motifs. Allele specific expression of HSPB1 in F1 hybrids between tolerant and less tolerant populations showed significantly biased expression favoring alleles from tolerant populations and supporting the adaptive divergence in these cis-regulatory variants. The functional impact of population-specific nonsynonymous substitutions in HSPB1 coding sequences was tested by assessing the thermal stabilization properties of SD versus SC HSPB1 protein variants. Recombinant HSPB1 from the southern SD population showed greater capacity for protecting protein structure under elevated temperature. Our results indicate that both regulatory and protein coding sequence evolution within a single gene appear to contribute to thermal tolerance phenotypes and local adaptation among conspecific populations.

Barreto, FS, Watson ET, Lima TG, Willett CS, Edmands S, Li WZ, Burton RS.  2018.  Genomic signatures of mitonuclear coevolution across populations of Tigriopus californicus. Nature Ecology & Evolution. 2:1250-1257.   10.1038/s41559-018-0588-1   AbstractWebsite

The copepod Tigriopus californicus shows extensive population divergence and is becoming a model for understanding allopatric differentiation and the early stages of speciation. Here, we report a high-quality reference genome for one population (similar to 190 megabases across 12 scaffolds, and similar to 15,500 protein-coding genes). Comparison with other arthropods reveals 2,526 genes presumed to be specific to T. californicus, with an apparent proliferation of genes involved in ion transport and receptor activity. Beyond the reference population, we report re-sequenced genomes of seven additional populations, spanning the continuum of reproductive isolation. Populations show extreme mitochondrial DNA divergence, with higher levels of amino acid differentiation than observed in other taxa. Across the nuclear genome, we find elevated protein evolutionary rates and positive selection in genes predicted to interact with mitochondrial DNA and the proteins and RNA it encodes in multiple pathways. Together, these results support the hypothesis that rapid mitochondrial evolution drives compensatory nuclear evolution within isolated populations, thereby providing a potentially important mechanism for causing intrinsic reproductive isolation.

Ahern, ALM, Gómez-Gutiérrez J, Aburto-Oropeza O, Saldierna-Martínez RJ, Johnson AF, Harada AE, Sánchez-Uvera AR, Erisman B, Castro Arvizú DI, Burton RS.  2018.  DNA sequencing of fish eggs and larvae reveals high species diversity and seasonal changes in spawning activity in the southeastern Gulf of California. Marine Ecology Progress Series. 592:159-179. AbstractWebsite

ABSTRACT: Ichthyoplankton studies can provide valuable information on the species richness and spawning activity of fishes, complementing estimations done using trawls and diver surveys. Zooplankton samples were collected weekly between January and December 2014 in Cabo Pulmo National Park, Gulf of California, Mexico (n = 48). Ichthyoplankton is difficult to identify morphologically; therefore the DNA barcoding method was employed to identify 4388 specimens, resulting in 157 operational taxonomic units (OTUs) corresponding to species. Scarus sp., Halichoeres dispilus, Xyrichtys mundiceps, Euthynnus lineatus, Ammodytoides gilli, Synodus lacertinus, Etrumeus acuminatus, Chanos chanos, Haemulon flaviguttatum and Vinciguerria lucetia were the most abundant and frequent species recorded. Noteworthy species identified include rare mesopelagic species such as the giant oarfish Regalecus glesne and highly migratory and commercially important species such as black skipjack Euthynnus lineatus and yellowfin tuna Thunnus albacares. Spawning activities showed distinct seasonal patterns, with the highest abundance of ichthyoplankton recorded during spring, highest species richness during summer (90 OTUs) and lowest species richness during winter (28 OTUs). A total of 7 OTUs were recorded throughout the year (4.5%), 10 OTUs during 3 seasons (6.5%), 36 OTUs in 2 seasons (23%) and 104 OTUs were recorded in 1 season (66%). The study found eggs and/or larvae of 47 species that were not previously reported in Cabo Pulmo National Park. The results will allow resource managers to compare shifting populations and spawning patterns of species that may be affected by both conservation efforts and broader oceanographic changes associated with climate change.

Ostersetzer-Biran, O, Lane N, Pomiankowski A, Burton R, Arnqvist G, Filipovska A, Huchon D, Mishmar D.  2017.  The first mitochondrial genomics and evolution SMBE-satellite meeting: A new scientific symbiosis. Genome Biology and Evolution. 9:3054-3058.   10.1093/gbe/evx227   AbstractWebsite

The central role of the mitochondrion for cellular and organismal metabolism is well known, yet its functional role in evolution has rarely been featured in leading international conferences. Moreover, the contribution of mitochondrial genetics to complex disease phenotypes is particularly important, and although major advances have been made in the field of genomics, mitochondrial genomic data have in many cases been overlooked. Accumulating data and new knowledge support a major contribution of this maternally inherited genome, and its interactions with the nucleus, to both major evolutionary processes and diverse disease phenotypes. These advances encouraged us to assemble the first Mitochondrial Genomics and Evolution (MGE) meeting-an SMBE satellite and Israeli Science foundation international conference (Israel, September 2017). Here, we report the content and outcome of the MGE meeting (; last accessed November 5, 2017).

Tsuboko-Ishii, S, Burton RS.  2017.  Sex-specific rejection in mate-guarding pair formation in the intertidal copepod, Tigriopus californicus. Plos One. 12   10.1371/journal.pone.0183758   AbstractWebsite

Securing a potential mate is one of the most important processes in sexual reproduction of animals. Intertidal copepods of the genus Tigriopus show mate-guarding behavior where a male captures a female and continues to clasp her for up to two weeks prior to copulation. Although these copepods form a mate-guarding pair between a male and a female with high accuracy, interactions between the sexes in pair formation have not been well described and the mechanism allowing successful male-female pair formation is not yet understood. In this study, we performed experiments with Tigriopus californicus to analyze the behavior of both a capturer (male) and a captured individual (female or male) in formation of a guarding pair. While capturer males were attracted by both females and males, capture of virgin males was terminated in a significantly shorter time than that of virgin females. However, when presented freshly killed females or males, regardless of the sex of the body, capturer males continued to clasp the body for a comparable time as in an attempt on a living female. Our results suggest that a sex-specific rejection signal actively sent by captured males prevents male-male pair formation. Experiments also suggest that mated females reject an attempt of pair formation. To our knowledge, this is the first study to suggest involvement of active rejection by a captured individual in facilitation of reproductively successful malefemale guarding pair formation in the genus Tigriopus.

Pereira, RJ, Sasaki MC, Burton RS.  2017.  Adaptation to a latitudinal thermal gradient within a widespread copepod species: the contributions of genetic divergence and phenotypic plasticity. Proceedings of the Royal Society B-Biological Sciences. 284   10.1098/rspb.2017.0236   AbstractWebsite

Understanding how populations adapt to heterogeneous thermal regimes is essential for comprehending how latitudinal gradients in species diversification are formed, and how taxa will respond to ongoing climate change. Adaptation can occur by innate genetic factors, by phenotypic plasticity, or by a combination of both mechanisms. Yet, the relative contribution of such mechanisms to large-scale latitudinal gradients of thermal tolerance across conspecific populations remains unclear. We examine thermal performance in 11 populations of the intertidal copepod Tigriopus californicus, ranging from Baja California Sur (Mexico) to British Columbia (Canada). Common garden experiments show that survivorship to acute heat-stress differs between populations (by up to 3.8 degrees C in LD50 values), reflecting a strong genetic thermal adaptation. Using a split-brood experiment with two rearing temperatures, we also show that developmental phenotypic plasticity is beneficial to thermal tolerance (by up to 1.3 degrees C), and that this effect differs across populations. Although genetic divergence in heat tolerance strongly correlates with latitude and temperature, differences in the plastic response do not. In the context of climate warming, our results confirm the general prediction that low-latitude populations are most susceptible to local extinction because genetic adaptation has placed physiological limits closer to current environmental maxima, but our results also contradict the prediction that phenotypic plasticity is constrained at lower latitudes.

Pierce, NT, Navarro MO, Gaasterland T, Burton RS.  2017.  Effect of low pH and low oxygen conditions on developmental gene expression and hatching of Doryteuthis opalescens embryos. Integrative and Comparative Biology. 57:E132-E132. AbstractWebsite
Gleason, LU, Burton RS.  2016.  Regional patterns of thermal stress and constitutive gene expression in the marine snail Chlorostoma funebralis in northern and southern California. Marine Ecology Progress Series. 556:143-159.   10.3354/meps11850   AbstractWebsite

Southern California (USA) populations of the intertidal snail Chlorostoma funebralis occupy warmer climates than northern California populations, and southern populations are more thermally tolerant and have unique transcriptomic responses to heat stress compared to northern populations. To investigate how climate affects body temperature patterns for C. funebralis, iButton temperature loggers encased in empty C. funebralis shells (robosnails) were deployed at 3 northern and 3 southern California sites for 1.5 mo in the late summer and early fall of 2014, typically when maximum annual temperatures are reached. Measurements revealed that southern, thermally tolerant populations experienced higher average daily maximum and absolute maximum temperatures than northern, less tolerant populations, and that robosnails in southern, but not northern, California exceeded temperatures that cause 100% mortality. Similarly, the probability of a site reaching 27 degrees C, the temperature that induces the heat shock response in C. funebralis, was 3 times higher at the southern compared to the northern sites. To determine whether these exposures to stressful temperatures are related to gene expression differences, we then tested for a correlation between the probability of reaching 27 degrees C and the constitutive (non-induced) expression of genes previously implicated as pre-adapted in southern California populations. We identified 222 genes (including 14 involved in ubiquitin protein degradation, a response to heat stress) with a significant correlation. The results demonstrate how combining in situ temperature and transcriptome data can increase our understanding of thermal adaptation and better inform predictions regarding the impact of future climate change.

Gleason, LU, Burton RS.  2016.  Genomic evidence for ecological divergence against a background of population homogeneity in the marine snail Chlorostoma funebralis. Molecular Ecology. 25:3557-3573.   10.1111/mec.13703   AbstractWebsite

The balance between natural selection, gene flow and genetic drift is difficult to resolve in marine invertebrates with extensive dispersal and fluctuating population sizes. The intertidal snail Chlorostoma funebralis has planktonic larvae and previous work using mtDNA polymorphism reported no genetic population structure. Nevertheless, recent studies have documented differences in thermal tolerance and transcriptomic responses to heat stress between northern and southern California, USA, populations. To gain insight into the dynamics influencing adaptive divergence, we used double-digest restriction site-associated DNA (ddRAD) sequencing to identify 1861 genomewide, quality-filtered single-nucleotide polymorphism (SNP) loci for C.funebralis collected from three northern and three southern California sites (15 individuals per population). Considering all SNPs, there was no evidence for genetic differentiation among populations or regions (average F-ST=0.0042). However, outlier tests revealed 34 loci putatively under divergent selection between northern and southern populations, and structure and SNP tree analyses based on these outliers show clear genetic differentiation between geographic regions. Three of these outliers are known or hypothesized to be involved in stress granule formation, a response to environmental stress such as heat. Combined with previous work that found thermally tolerant southern populations show high baseline expression of stress response genes, these results further suggest that thermal stress is a strong selective pressure across C.funebralis populations. Overall, this study increases our understanding of the factors constraining local adaptation in marine organisms, while suggesting that ecologically driven, strong differentiation can occur at relevant loci in a species with planktonic larvae.

Stewart, JD, Beale CS, Fernando D, Sianipar AB, Burton RS, Semmens BX, Aburto-Oropeza O.  2016.  Spatial ecology and conservation of Manta birostris in the Indo-Pacific. Biological Conservation. 200:178-183.   10.1016/j.biocon.2016.05.016   AbstractWebsite

Information on the movements and population connectivity of the oceanic manta ray (Manta birostris) is scarce. The species has been anecdotally classified as a highly migratory species based on the pelagic habitats it often occupies, and migratory behavior exhibited by similar species. As a result, in the absence of ecological data, population declines in oceanic manta have been addressed primarily with international-scale management and conservation efforts. Using a combination of satellite telemetry, stable isotope and genetic analyses we demonstrate that, contrary to previous assumptions, the species appears to exhibit restricted movements and fine scale population structure. M. birostris tagged at four sites in the Indo-Pacific exhibited no long-range migratory movements and had non-overlapping geographic ranges. Using genetic and isotopic analysis, we demonstrate that the observed movements and population structure persist on multi-year and generational time scales. These data provide the first insights into the long-term movements and population structure of oceanic manta rays, and suggest that bottom-up, local or regional approaches to managing oceanic mantas could prove more effective than existing, international-scale management strategies. This case study highlights the importance of matching the scales at which management and relevant ecological processes occur to facilitate the effective conservation of threatened species. (C) 2016 Elsevier Ltd. All rights reserved.

Brandão, MC, Freire AS, Burton RS.  2016.  Estimating diversity of crabs (Decapoda: Brachyura) in a no-take marine protected area of the SW Atlantic coast through DNA barcoding of larvae. Systematics and Biodiversity. 14:288-302.: Taylor & Francis   10.1080/14772000.2016.1140245   Abstract

DNA barcoding was used to identify crab larvae from the Marine Biological Reserve of Arvoredo, encompassing a coastal archipelago off the SW Atlantic coast (27°S, 48°W). Partial mitochondrial COI or 16S rRNA gene sequences were obtained for 488 larvae, leading to the identification of 20 species. The COI sequences generated 13 barcode index numbers (BINs) within Barcode of Life Data Systems (BOLD), among which 11 were concordant with single species. DNA from ∼ 6% of the larvae did not amplify using the primers tested; based on external morphological characteristics, these larvae represented four possible additional operational taxonomic units (OTUs) at the family level. Intraspecific variation for the COI and 16S rRNA genes was found to be < 2.6% and < 2.1% respectively (Kimura 2-parameter distance), whereas interspecific divergence ranged from 7.9% to 21.5% and 6.4% to 14.5%, respectively. These results imply that both genes are suitable for use in species identification of brachyuran crabs of this area. Molecular identification of this group successfully enabled the diagnosis of larvae of closely related species, including congeners in Mithrax, Achelous and Callinectes. In addition, eight out of 20 species recognized represent new records for the reserve suggesting that the brachyuran fauna in the area has been underestimated based on traditional biodiversity measures. The availability of primers suited to the targeted species, and the development of a taxonomically comprehensive DNA barcoding database are the major recommendations to improve the accuracy and feasibility of using DNA barcoding for species identification of SW Atlantic brachyuran crabs.

Pereira, RJ, Barreto FS, Pierce NT, Carneiro M, Burton RS.  2016.  Transcriptome-wide patterns of divergence during allopatric evolution. Molecular Ecology. 25:1478-1493.   10.1111/mec.13579   AbstractWebsite

Recent studies have revealed repeated patterns of genomic divergence associated with species formation. Such patterns suggest that natural selection tends to target a set of available genes, but is also indicative that closely related taxa share evolutionary constraints that limit genetic variability. Studying patterns of genomic divergence among populations within the same species may shed light on the underlying evolutionary processes. Here, we examine transcriptome-wide divergence and polymorphism in the marine copepod Tigriopus californicus, a species where allopatric evolution has led to replicate sets of populations with varying degrees of divergence and hybrid incompatibility. Our analyses suggest that relatively small effective population sizes have resulted in an exponential decline of shared polymorphisms during population divergence and also facilitated the fixation of slightly deleterious mutations within allopatric populations. Five interpopulation comparisons at three different stages of divergence show that nonsynonymous mutations tend to accumulate in a specific set of proteins. These include proteins with central roles in cellular metabolism, such as those encoded in mtDNA, but also include an additional set of proteins that repeatedly show signatures of positive selection during allopatric divergence. Although our results are consistent with a contribution of nonadaptive processes, such as genetic drift and gene expression levels, generating repeatable patterns of genomic divergence in closely related taxa, they also indicate that adaptive evolution targeting a specific set of genes contributes to this pattern. Our results yield insights into the predictability of evolution at the gene level. See also the Perspective by Renaut and Dion-Cote

Bonin, CA, Goebel ME, O'Corry-Crowe GM, Burton RS.  2016.  Impacts of ecology and behavior on Antarctic fur seal remating and relatedness. Journal of Experimental Marine Biology and Ecology. 476:72-77.   10.1016/j.jembe.2015.12.008   AbstractWebsite

Antarctic fur seals (Arctocephalus gazella) are polygynous and both sexes are typically faithful to a breeding site. These characteristics could promote remating among individuals over time, leading to increased relatedness levels and negatively affecting genetic diversity. To examine this issue, the reproductive output of 55 females was monitored annually for 12 years and their pups were sampled (n = 280) and genotyped using 17 microsatellite markers. A full likelihood pedigree inference method was used to confirm maternities inferred in the field and estimate the number of full sibling pups born across years. Relatedness coefficients were estimated for pairs of individuals in the pedigree and compared to simulated values for each relationship category. There were nine cases where a female mated with the same male twice and one case where a female mated with the same male three times over the study period. The observed relatedness coefficients estimated among the sampled pups matched the simulated distribution for half-siblings. In addition, no first order relatives were found among the fur seal mothers studied, nor did observed relatedness coefficient distributions differ significantly from simulated values. Together, these results suggest a low remating rate and a negligible effect of remating on pair-wise relatedness. Territorial male replacement over time as well as female small-scale movements, driven by suitable pupping habitat, likely contribute to the low remating frequency observed in Antarctic fur seals. (C) 2015 Elsevier B.V. All rights reserved.

Harada, AE, Lindgren EA, Hermsmeier MC, Rogowski PA, Terrill E, Burton RS.  2015.  Monitoring spawning activity in a Southern California marine protected area using molecular identification of fish eggs. Plos One. 10   10.1371/journal.pone.0134647   AbstractWebsite

In order to protect the diverse ecosystems of coastal California, a series of marine protected areas (MPAs) have been established. The ability of these MPAs to preserve and potentially enhance marine resources can only be assessed if these habitats are monitored through time. This study establishes a baseline for monitoring the spawning activity of fish in the MPAs adjacent to Scripps Institution of Oceanography (La Jolla, CA, USA) by sampling fish eggs from the plankton. Using vertical plankton net tows, 266 collections were made from the Scripps Pier between 23 August 2012 and 28 August 2014; a total of 21,269 eggs were obtained. Eggs were identified using DNA barcoding: the COI or 16S rRNA gene was amplified from individual eggs and sequenced. All eggs that were successfully sequenced could be identified from a database of molecular barcodes of California fish species, resulting in species-level identification of 13,249 eggs. Additionally, a surface transport model of coastal circulation driven by current maps from high frequency radar was used to construct probability maps that estimate spawning locations that gave rise to the collected eggs. These maps indicated that currents usually come from the north but water parcels tend to be retained within the MPA; eggs sampled at the Scripps Pier have a high probability of having been spawned within the MPA. The surface transport model also suggests that although larvae have a high probability of being retained within the MPA, there is also significant spill-over into nearby areas outside the MPA. This study provides an important baseline for addressing the extent to which spawning patterns of coastal California species may be affected by future changes in the ocean environment.

Barreto, FS, Schoville SD, Burton RS.  2015.  Reverse genetics in the tide pool: knock-down of target gene expression via RNA interference in the copepod Tigriopus californicus. Molecular Ecology Resources. 15:868-879.   10.1111/1755-0998.12359   AbstractWebsite

Reverse genetic tools are essential for characterizing phenotypes of novel genes and testing functional hypotheses generated from next-generation sequencing studies. RNA interference (RNAi) has been a widely used technique for describing or quantifying physiological, developmental or behavioural roles of target genes by suppressing their expression. The marine intertidal copepod Tigriopus californicus has become an emerging model for evolutionary and physiological studies, but this species is not amenable to most genetic manipulation approaches. As crustaceans are susceptible to RNAi-mediated gene knock-down, we developed a simple method for delivery of gene-specific double-stranded RNA that results in significant suppression of target gene transcription levels. The protocol was examined on five genes of interest, and for each, at least 50% knock-down in expression was achieved. While knock-down levels did not reach 100% in any trial, a well-controlled experiment with one heat-shock gene showed unambiguously that such partial gene suppression may cause dramatic changes in phenotype. Copepods with suppressed expression of heat-shock protein beta 1 (hspb1) exhibited dramatically decreased tolerance to high temperatures, validating the importance of this gene during thermal stress, as proposed by a previous study. The application of this RNAi protocol in T.californicus will be invaluable for examining the role of genes putatively involved in reproductive isolation, mitochondrial function and local adaptation.

Barreto, FS, Pereira RJ, Burton RS.  2015.  Hybrid dysfunction and physiological compensation in gene expression. Molecular Biology and Evolution. 32:613-622.   10.1093/molbev/msu321   AbstractWebsite

The formation of new species is often a consequence of genetic incompatibilities accumulated between populations during allopatric divergence. When divergent taxa interbreed, these incompatibilities impact physiology and have a direct cost resulting in reduced hybrid fitness. Recent surveys of gene regulation in interspecific hybrids have revealed anomalous expression across large proportions of the genome, with 30-70% of all genes exhibiting transgressive expression (i.e., higher or lower levels compared with both parental taxa), and these were mostly in the direction of downregulation. However, as most of these studies have focused on pairs of species exhibiting high degrees of reproductive isolation, the association between regulatory disruption and reduced hybrid fitness prior to species formation remains unclear. Within the copepod species Tigriopus californicus, interpopulation hybrids at F-2 or later generations show reduced fitness associated with mitochondrial dysfunction. Here we show that in contrast to studies of interspecific hybrids, only 1.2% of the transcriptome is transgressively expressed in F3+ interpopulation hybrids of T. californicus, and nearly 80% of these genes are overexpressed rather than underexpressed; remarkably, none of these genes are among those showing divergent expression between parentals, nor is magnitude of transgressive gene expression in hybrids dependent on levels of protein sequence divergence. Moreover, many genes with transgressive expression are components of functional pathways impacted by mitonuclear incompatibilities in hybrid T. californicus (e.g., oxidative phosphorylation and antioxidant response). Our results suggest that hybrid breakdown at early stages of speciation may result from initial incompatibilities amplified by the cost of compensatory physiological responses.

Gleason, LU, Burton RS.  2015.  RNA-seq reveals regional differences in transcriptome response to heat stress in the marine snail Chlorostoma funebralis. Molecular Ecology. 24:610-627.   10.1111/mec.13047   AbstractWebsite

To investigate the role of gene expression in adaptation of marine ectotherms to different temperatures, we examined the transcriptome-wide thermal stress response in geographically separated populations of the intertidal snail Chlorostoma funebralis. Snails from two southern (heat tolerant) and two northern (heat sensitive) populations were acclimated to a common thermal environment, exposed to an environmentally relevant thermal stress and analysed using RNA-seq. Pooling across all populations revealed 306 genes with differential expression between control and heat-stressed samples, including 163 significantly upregulated and 143 significantly downregulated genes. When considered separately, regional differences in response were widely apparent. Heat shock proteins (Hsps) were upregulated in both regions, but the magnitude of response was significantly greater in northern populations for most Hsp70s, while the southern populations showed greater upregulation for approximately half of the Hsp40s. Of 177 stress-responsive genes in northern populations, 55 responded to heat stress only in northern populations. Several molecular chaperones and antioxidant genes that were not differentially expressed in southern populations showed higher expression under control conditions compared with northern populations. This suggests that evolution of elevated expression of these genes under benign conditions preadapts the southern populations to frequent heat stress and contributes to their higher thermal tolerance. These results indicate that evolution has resulted in different transcriptome responses across populations, including upregulation of genes in response to stress and preadaptation of genes in anticipation of stress (based on evolutionary history of frequent heat exposure). The relative importance of the two mechanisms differs among gene families and among populations.

Tangwancharoen, S, Burton RS.  2014.  Early life stages are not always the most sensitive: heat stress responses in the copepod Tigriopus californicus. Marine Ecology Progress Series. 517:75-83.   10.3354/meps11013   AbstractWebsite

Because of their complex life histories, different life stages of many marine invertebrates may be exposed to varying environmental challenges. Ultimately, the life stage that is least tolerant of its environment will determine the species' abundance and distribution. The intertidal copepod Tigriopus californicus lives in high intertidal pools along the Pacific coast of North America. Unlike most other invertebrates, the different life stages of T. californicus all share the same tidepool habitat. To determine physiological tolerances of various life history stages of this species, we examined responses to acute heat stress in nauplii, copepodids, and adults from 6 populations along a latitudinal gradient. Results show that early developmental stages (nauplii and cope podids) are generally more tolerant than adults. Our results contrast with the widely accepted generalization that larval forms are more sensitive to physical stressors than adults. As previously observed in adults, nauplii and copepodids from southern populations survive higher temperatures than those from northern populations. Acute heat stress was found to delay development but did not affect adult size. We hypothesize that variation in the thermal tolerance of early life stages among intertidal species reflects ecological differences in larval habitats: where larvae remain in the intertidal zone and experience the same high temperatures as adults, selection will favor high larval thermal tolerance, while in species with planktonic larvae, the buffered temperature regime of the water column might relax such selection, and thermal tolerance will be highest in the more exposed intertidal adults.

Bonin, CA, Goebel ME, Hoffman JI, Burton RS.  2014.  High male reproductive success in a low-density Antarctic fur seal (Arctocephalus gazella) breeding colony. Behavioral Ecology and Sociobiology. 68:597-604.   10.1007/s00265-013-1674-7   AbstractWebsite

Understanding how population density influences mating systems may lead to important insights into the plasticity of breeding behavior, but few natural systems allow for such studies. Antarctic fur seals (Arctocephalus gazella) provide an interesting model system because they breed in colonies of varying densities. Previous studies have largely focused on a high-density site at Bird Island, South Georgia. Here, 13 highly polymorphic microsatellite loci were used to conduct a genetic analysis of a low-density breeding colony of this species at Livingston Island, approximately 1,600 km south of South Georgia. The majority of adults seen ashore (n = 54) were sampled together with every pup born (n = 97) over four consecutive years. Paternities were confidently assigned for 34 out of the 97 pups. Two out of 23 sampled males accounted for the paternity of 28 % of all pups sampled during the study and 82 % of the pups with an assigned father. Moreover, a full likelihood pedigree inference method assigned a further eight paternities to a single unsampled male seal that is inferred to have held a territory during the season before the study began. The most successful males in our study easily surpassed the previous record for the total number of pups sired per male seal for the species. Furthermore, we identified two triads of full siblings implying that their parents remated in three consecutive years. These findings suggest that territorial male fur seals may achieve greater success in monopolizing access to breeding females when population density is relatively low.