The genome of Anoplarchus purpurescens (Stichaeidae) reflects its carnivorous diet.

Digestion is driven by digestive enzymes and digestive enzyme gene copy number can provide insights on the genomic underpinnings of dietary specialization. The "Adaptive Modulation Hypothesis" (AMH) proposes that digestive enzyme activity, which increases with increased gene copy number, should correlate with substrate quantity in the diet. To test the AMH and reveal some of the genetics of herbivory vs carnivory, we sequenced, assembled, and annotated the genome of Anoplarchus purpurescens, a carnivorous prickleback fish in the family Stichaeidae, and compared the gene copy number for key digestive enzymes to that of Cebidichthys violaceus, a herbivorous fish from the same family. A highly contiguous genome assembly of high quality (N50 = 10.6 Mb) was produced for A. purpurescens, using combined long-read and short-read technology, with an estimated 33,842 protein-coding genes. The digestive enzymes that we examined include pancreatic α-amylase, carboxyl ester lipase, alanyl aminopeptidase, trypsin, and chymotrypsin. Anoplarchus purpurescens had fewer copies of pancreatic α-amylase (carbohydrate digestion) than C. violaceus (1 vs. 3 copies). Moreover, A. purpurescens had one fewer copy of carboxyl ester lipase (plant lipid digestion) than C. violaceus (4 vs. 5). We observed an expansion in copy number for several protein digestion genes in A. purpurescens compared to C. violaceus, including trypsin (5 vs. 3) and total aminopeptidases (6 vs. 5). Collectively, these genomic differences coincide with measured digestive enzyme activities (phenotypes) in the two species and they support the AMH. Moreover, this genomic resource is now available to better understand fish biology and dietary specialization.

When digestive physiology doesn't match "diet": Lumpenus sagitta (Stichaeidae) is an "omnivore" with a carnivorous gut.

What an animal ingests and what it digests can be different. Thus, we examined the nutritional physiology of Lumpenus sagitta, a member of the family Stichaeidae, to better understand whether it could digest algal components like its better studied algivorous relatives. Although L. sagitta ingests considerable algal content, we found little evidence of algal digestion. This fish species has a short gut that doesn't show positive allometry with body size, low amylolytic activity that actually decreases as the fish grow, no ontogenetic changes in digestive enzyme gene expression, elevated N-acetyl-glucosaminidase activity (indicative of chitin breakdown), and an enteric microbial community that is consistent with carnivory and differs from members of its family that consume and digest algae. Hence, we are left concluding that L. sagitta is not capable of digesting the algae it consumes, and instead, are likely targeting epibionts on the algae itself, and other invertebrates consumed with the algae. Our study expands the coverage of dietary and digestive information for the family Stichaeidae, which is becoming a model for fish digestive physiology and genomics, and shows the power of moving beyond gut content analyses to better understand what an animal can actually digest and use metabolically.

Genomic and biochemical evidence of dietary adaptation in a marine herbivorous fish.

Adopting a new diet is a significant evolutionary change, and can profoundly affect an animal's physiology, biochemistry, ecology and genome. To study this evolutionary transition, we investigated the physiology and genomics of digestion of a derived herbivorous fish, Cebidichthys violaceus. We sequenced and assembled its genome (N50 = 6.7 Mb) and digestive transcriptome, and revealed the molecular changes related to digestive enzymes (carbohydrases, proteases and lipases), finding abundant evidence of molecular adaptation. Specifically, two gene families experienced expansion in copy number and adaptive amino acid substitutions: amylase and carboxyl ester lipase (cel), which are involved in the digestion of carbohydrates and lipids, respectively. Both show elevated levels of gene expression and increased enzyme activity. Because carbohydrates are abundant in the prickleback's diet and lipids are rare, these findings suggest that such dietary specialization involves both exploiting abundant resources and scavenging rare ones, especially essential nutrients, like essential fatty acids.

Comparative Transcriptomics Reveals Patterns of Adaptive Evolution Associated with Depth and Age Within Marine Rockfishes (Sebastes).

The genetic underpinnings that contribute to ecological adaptation and speciation are not completely understood, especially within marine ecosystems. These evolutionary processes can be elucidated by studying adaptive radiations, because they provide replicates of divergence within a given environment or time-frame. Marine rockfishes (genus Sebastes) are an adaptive radiation and unique model system for studying adaptive evolution in the marine realm. We investigated molecular evolution associated with ecological (depth) and life history (lifespan) divergence in 2 closely related clades of Sebastes. Brain transcriptomes were sequenced via RNA-Seq from 3 species within the subgenus Pteropodus and a pair of related congeners from the subgenus Sebastosomus in order to identify patterns of adaptive evolution. De novo assemblies from these transcriptomes were used to identify 3867 orthologous clusters, and genes subject to positive selection were identified based on all 5 species, depth, and lifespan. Within all our analyses, we identified hemoglobin subunit α to be under strong positive selection and is associated with the depth of occurrence. In our lifespan analysis we identified immune function genes under positive selection in association with maximum lifespan. This study provides insight on the molecular evolution of rockfishes and these candidate genes may provide a better understanding of how these subgenera radiated within the Northeast Pacific.

Ambient CO2, fish behaviour and altered GABAergic neurotransmission: exploring the mechanism of CO2-altered behaviour by taking a hypercapnia dweller down to low CO2 levels.

Recent studies suggest that projected rises of aquatic CO2 levels cause acid-base regulatory responses in fishes that lead to altered GABAergic neurotransmission and disrupted behaviour, threatening fitness and population survival. It is thought that changes in Cl(-) and HCO3 (-) gradients across neural membranes interfere with the function of GABA-gated anion channels (GABAA receptors). So far, such alterations have been revealed experimentally by exposing species living in low-CO2 environments, like many oceanic habitats, to high levels of CO2 (hypercapnia). To examine the generality of this phenomenon, we set out to study the opposite situation, hypothesizing that fishes living in typically hypercapnic environments also display behavioural alterations if exposed to low CO2 levels. This would indicate that ion regulation in the fish brain is fine-tuned to the prevailing CO2 conditions. We quantified pH regulatory variables and behavioural responses of Pangasianodon hypophthalmus, a fish native to the hypercapnic Mekong River, acclimated to high-CO2 (3.1 kPa) or low-CO2 (0.04 kPa) water. We found that brain and blood pH was actively regulated and that the low-CO2 fish displayed significantly higher activity levels, which were reduced after treatment with gabazine, a GABAA receptor blocker. This indicates an involvement of the GABAA receptor and altered Cl(-) and HCO3 (-) ion gradients. Indeed, Goldman calculations suggest that low levels of environmental CO2 may cause significant changes in neural ion gradients in P. hypophthalmus. Taken together, the results suggest that brain ion regulation in fishes is fine-tuned to the prevailing ambient CO2 conditions and is prone to disruption if these conditions change.

Gonadal transcriptomics elucidate patterns of adaptive evolution within marine rockfishes (Sebastes).

BACKGROUND: The genetic mechanisms of speciation and adaptation in the marine environment are not well understood. The rockfish genus Sebastes provides a unique model system for studying adaptive evolution because of the extensive diversity found within this group, which includes morphology, ecology, and a broad range of life spans. Examples of adaptive radiations within marine ecosystems are considered an anomaly due to the absence of geographical barriers and the presence of gene flow. Using marine rockfishes, we identified signatures of natural selection from transcriptomes developed from gonadal tissue of two rockfish species (Sebastes goodei and S. saxicola). We predicted orthologous transcript pairs, and estimated their distributions of nonsynonymous (Ka) and synonymous (Ks) substitution rates. RESULTS: We identified 144 genes out of 1079 orthologous pairs under positive selection, of which 11 are functionally annotated to reproduction based on gene ontologies (GOs). One orthologous pair of the zona pellucida gene family, which is known for its role in the selection of sperm by oocytes, out of ten was identified to be evolving under positive selection. In addition to our results in the protein coding-regions of transcripts, we found substitution rates in 3' and 5' UTRs to be significantly lower than Ks substitution rates implying negative selection in these regions. CONCLUSIONS: We were able to identify a series of candidate genes that are useful for the assessment of the critical genes that diverged and are responsible for the radiation within this genus. Genes associated with longevity hold potential for understanding the molecular mechanisms that have contributed to the radiation within this genus.

Why Do Some Lineages Radiate While Others Do Not? Perspectives for Future Research on Adaptive Radiations.

Understanding the processes that drive phenotypic diversification and underpin speciation is key to elucidating how biodiversity has evolved. Although these processes have been studied across a wide array of clades, adaptive radiations (ARs), which are systems with multiple closely related species and broad phenotypic diversity, have been particularly fruitful for teasing apart the factors that drive and constrain diversification. As such, ARs have become popular candidate study systems for determining the extent to which ecological features, including aspects of organisms and the environment, and inter- and intraspecific interactions, led to evolutionary diversification. Despite substantial past empirical and theoretical work, understanding mechanistically how ARs evolve remains a major challenge. Here, we highlight a number of understudied components of the environment and of lineages themselves, which may help further our understanding of speciation and AR. We also outline some substantial remaining challenges to achieving a detailed understanding of adaptation, speciation, and the role of ecology in these processes. These major challenges include identifying factors that have a causative impact in promoting or constraining ARs, gaining a more holistic understanding of features of organisms and their environment that interact resulting in adaptation and speciation, and understanding whether the role of these organismal and environmental features varies throughout the radiation process. We conclude by providing perspectives on how future investigations into the AR process can overcome these challenges, allowing us to glean mechanistic insights into adaptation and speciation.

Minimal overall divergence of the gut microbiome in an adaptive radiation of Cyprinodon pupfishes despite potential adaptive enrichment for scale-eating.

Adaptive radiations offer an excellent opportunity to understand the eco-evolutionary dynamics of gut microbiota and host niche specialization. In a laboratory common garden, we compared the gut microbiota of two novel derived trophic specialist pupfishes, a scale-eater and a molluscivore, to closely related and distant outgroup generalist populations, spanning both rapid trophic evolution within 10 kya and stable generalist diets persisting over 11 Mya. We predicted an adaptive and highly divergent microbiome composition in the trophic specialists reflecting their rapid rates of craniofacial and behavioral diversification. We sequenced 16S rRNA amplicons of gut microbiomes from lab-reared adult pupfishes raised under identical conditions and fed the same high protein diet. In contrast to our predictions, gut microbiota largely reflected phylogenetic distance among species, rather than generalist or specialist life history, in support of phylosymbiosis. However, we did find significant enrichment of Burkholderiaceae bacteria in replicated lab-reared scale-eater populations. These bacteria sometimes digest collagen, the major component of fish scales, supporting an adaptive shift. We also found some enrichment of Rhodobacteraceae and Planctomycetia in lab-reared molluscivore populations, but these bacteria target cellulose. Overall phylogenetic conservation of microbiome composition contrasts with predictions of adaptive radiation theory and observations of rapid diversification in all other trophic traits in these hosts, including craniofacial morphology, foraging behavior, aggression, and gene expression, suggesting that the functional role of these minor shifts in microbiota will be important for understanding the role of the microbiome in trophic diversification.

Comparative transcriptomics reveal tissue level specialization towards diet in prickleback fishes.

Beyond a few obvious examples (e.g., gut length, amylase activity), digestive and metabolic specializations towards diet remain elusive in fishes. Thus, we compared gut length, δ13C and δ15N signatures of the liver, and expressed genes in the intestine and liver of wild-caught individuals of four closely-related, sympatric prickleback species (family Stichaeidae) with different diets: Xiphister mucosus (herbivore), its sister taxon X. atropurpureus (omnivore), Phytichthys chirus (omnivore) and the carnivorous Anoplarchus purpurescens. We also measured the same parameters after feeding them carnivore or omnivore diets in the laboratory for 4 weeks. Growth and isotopic signatures showed assimilation of the laboratory diets, and gut length was significantly longer in X. mucosus in comparison to the other fishes, whether in the wild, or in the lab consuming the different diets. Dozens of genes relating to digestion and metabolism were observed to be under selection in the various species, but P. chirus stood out with some genes in the liver showing strong positive selection, and these genes correlating with differing isotopic incorporation of the laboratory carnivore diet in this species. Although the intestine showed variation in the expression of hundreds of genes in response to the laboratory diets, the liver exhibited species-specific gene expression patterns that changed very little (generally <40 genes changing expression, with P. chirus providing an exception). Overall, our results suggest that the intestine is plastic in function, but the liver may be where specialization manifests since this tissue shows species-specific gene expression patterns that match with natural diet.

Withering syndrome induced gene expression changes and a de-novo transcriptome for the Pinto abalone, Haliotis kamtschatkana.

In the abalone and Candidatus Xenohaliotis californiensis (Ca. Xc) system, the Ca. Xc bacterium infects abalone digestive tissues and leads to extreme starvation and a characteristic "withering" of the gastropod foot. First identified in black abalone in California after an El Niño event, withering syndrome (WS) has caused large declines in wild black and captive white abalone on the northeastern Pacific coast, but disease resistance levels are species-, and possibly population-specific. This study compared gene expression patterns in the digestive gland of Ca. Xc-exposed and unexposed (control) Pinto abalone (Haliotis kamtschatkana), a particularly susceptible species. Lab-induced Ca. Xc infections were followed over 7 months and RNAseq was used to identify differential gene expression. Exposed Pinto abalone showed distinct changes in expression of 68 genes at 3 and 7 months post-infection relative to those in control animals. Upregulation of an orexin-like receptor (which is involved in feeding signaling) and a zinc peptidase-like region (many amino peptidases are zinc peptidases) in animals infected for 7 months indicates that animals with Ca. Xc infection may be starving and upregulating processes associated with feeding and digestion. Other groups of differentially expressed genes (DEGs) were upregulated or downregulated across control and exposed individuals over the 7-month experiment, including DEG groups that likely correspond to early disease state and to general stress response of being held in captivity. No patterns emerged in genes known to be involved in molluscan immune response, despite this being an expectation during a 7-month infection; digestion-related genes and unannotated DEGs were identified as targets for future research on potential immune response to WS in abalone.

Characterization, Selection, and Trans-Species Polymorphism in the MHC Class II of Heermann's Gull (Charadriiformes).

The major histocompatibility complex (MHC) enables vertebrates to cope with pathogens and maintain healthy populations, thus making it a unique set of loci for addressing ecology and evolutionary biology questions. The aim of our study was to examine the variability of Heermann's Gull MHC class II (MHCIIB) and compare these loci with other Charadriiformes. Fifty-nine MHCIIB haplotypes were recovered from sixty-eight Heermann's Gulls by cloning, of them, twelve were identified as putative true alleles, forty-five as unique alleles, and two as pseudogenes. Intra and interspecific relationships indicated at least two loci in Heermann's Gull MHCIIB and trans-species polymorphism among Charadriiformes (coinciding with the documented evidence of two ancient avian MHCIIB lineages, except in the Charadriidae family). Additionally, sites under diversifying selection revealed a better match with peptide-binding sites inferred in birds than those described in humans. Despite the negative anthropogenic activity reported on Isla Rasa, Heermann's Gull showed MHCIIB variability consistent with population expansion, possibly due to a sudden growth following conservation efforts. Duplication must play an essential role in shaping Charadriiformes MHCIIB variability, buffering selective pressures through balancing selection. These findings suggest that MHC copy number and protected islands can contribute to seabird conservation.

Elevated Gene Copy Number Does Not Always Explain Elevated Amylase Activities in Fishes.

Amylase activity variation in the guts of several model organisms appears to be explained by amylase gene copy number variation. We tested the hypothesis that amylase gene copy number is always elevated in animals with high amylolytic activity. We therefore sequenced the amylase genes and examined amylase gene copy number in prickleback fishes (family Stichaeidae) with different diets including two species of convergently evolved herbivores with the elevated amylase activity phenotype. We found elevated amylase gene copy number (six haploid copies) with sequence variation among copies in one herbivore (Cebidichthys violaceus) and modest gene copy number (two to three haploid copies) with little sequence variation in the remaining taxa, which included herbivores, omnivores, and a carnivore. Few functional differences in amylase biochemistry were observed, and previous investigations showed similar digestibility among the convergently evolved herbivores with differing amylase genetics. Hence, the phenotype of elevated amylase activity can be achieved by different mechanisms (i.e., elevated expression of fewer genes, increased gene copy number, or expression of more efficient amylase proteins) with similar results. Phylogenetic and comparative genomic analyses of available fish amylase genes show mostly lineage-specific duplication events leading to gene copy number variation, although a whole-genome duplication event or chromosomal translocation may have produced multiple amylase copies in the Ostariophysi, again showing multiple routes to the same result.

A transcriptomic scan for positively selected genes in two closely related marine fishes: Sebastes caurinus and S. rastrelliger.

Comparative genomic analyses can provide valuable insight into functional evolutionary divergence among closely related species. Here we employ a comparative evolutionary analysis of expressed sequence tags (ESTs) from two closely related species of marine fishes (genus Sebastes--rockfish). Sebastes is a highly diverse group of marine fishes that inhabit a wide array of marine habitats and the study of this group can provide insights into speciation in the marine environment. ESTs were developed for S. caurinus (23,668 from brain, kidney, and spleen tissues) and S. rastrelliger (11,207 from brain and pituitary tissues). Following assembly we were able to identify, with high confidence, 257 orthologous sequence pairs between the two species through a reciprocal best hit blast search. An analysis of functional divergence between orthologs revealed that 19.46% had Ka/Ks values greater than 0.5 and 8.17% had Ka/Ks values greater than one, identifying a large pool of candidate genes to further study adaptive divergence in the group. Genes with elevated Ka/Ks values belonged to the following functional categories: immune function, metabolism, longevity, and reproductive behavior, indicating that adaptive divergence in these functional groups may be important in the diversification of this group of fishes. This study provides the ground work to better understand the molecular evolution of genes involved in a radiation of marine fishes.

An integrated approach to fast and informative morphological vouchering of nematodes for applications in molecular barcoding.

Molecular surveys of meiofaunal diversity face some interesting methodological challenges when it comes to interstitial nematodes from soils and sediments. Morphology-based surveys are greatly limited in processing speed, while barcoding approaches for nematodes are hampered by difficulties of matching sequence data with traditional taxonomy. Intermediate technology is needed to bridge the gap between both approaches. An example of such technology is video capture and editing microscopy, which consists of the recording of taxonomically informative multifocal series of microscopy images as digital video clips. The integration of multifocal imaging with sequence analysis of the D2D3 region of large subunit (LSU) rDNA is illustrated here in the context of a combined morphological and barcode sequencing survey of marine nematodes from Baja California and California. The resulting video clips and sequence data are made available online in the database NemATOL (http://nematol.unh.edu/). Analyses of 37 barcoded nematodes suggest that these represent at least 32 species, none of which matches available D2D3 sequences in public databases. The recorded multifocal vouchers allowed us to identify most specimens to genus, and will be used to match specimens with subsequent species identifications and descriptions of preserved specimens. Like molecular barcodes, multifocal voucher archives are part of a wider effort at structuring and changing the process of biodiversity discovery. We argue that data-rich surveys and phylogenetic tools for analysis of barcode sequences are an essential component of the exploration of phyla with a high fraction of undiscovered species. Our methods are also directly applicable to other meiofauna such as for example gastrotrichs and tardigrades.