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.

Reptilian digestive efficiency: Past, present, and future.

Digestion and assimilation of nutrients and energy is central to survival. At its most basic level, investigations of digestion in animals must examine digestive efficiency, or how much of a given meal (i.e., energy) or a specific nutrient an organism can acquire from its food. There are many studies examining this in reptiles, but there is large variation in methodology, and thus, in the conclusions drawn from the gathered data. The majority rely on ratio-based analyses that can jeopardize the reliability of their findings. Therefore, we reviewed the literature to identify common themes in the digestive efficiency data on reptiles. Due to the sheer number of available studies, we largely focused on lizards, but included data on all reptilian groups. As an example of what the current data can reveal, we performed a meta-analysis of digestive efficiency in lizards as a function of temperature using regression analyses. We detected a weak positive trend of soluble carbohydrate digestibility as a function of temperature, but no similar trend in broad-scale digestive efficiency, and propose that these patterns be reevaluated with non-ratio data. We conclude with calls to end conducting analyses on ratios and instead employ covariate methods, for more studies of reptilian digestive efficiency and related processes using consistent methodology, more representation of each population (e.g., many studies focus on males only), and more detailed studies examining the effects of temperature on digestion (since the current data are inconclusive).

Elevated aerial temperature modulates digestive enzyme activities in Mytilus californianus.

The marine intertidal mussel Mytilus californianus aggregates to form beds along the Pacific shores of North America. As a sessile organism it must cope with fluctuations in temperature during low-tide aerial exposure, which elevates maintenance costs and negatively affects its overall energy budget. The function of its digestive gland is to release enzymes that break apart ingested polymers for subsequent nutrient absorption. The effects of elevated aerial warming acclimation on the functioning of digestive gland enzymes are not well studied. In this study we asked whether digestive gland carbohydases and proteases could be overstimulated in warm condition to possibly mitigate the costs related to the heat-shock response. We compared mussels acclimated to a + 9 °C heat-shock during daily low-tide aerial exposure to mussels acclimated to isothermal tidal conditions in a simulated intertidal system. The results showed fairly consistent activities of cellulase, trypsin, and amino-peptidase across tidal variation and between thermal treatments; however, amylase activity was lower in warmed versus cool mussels across low and high-tide. We also observed the expression of heat-shock genes in gill tissue during warm tidal conditions, suggestive that moderate temperatures during aerial exposure can induce a stress response.

Reference genome of the Monkeyface Prickleback, Cebidichthys violaceus.

Pricklebacks (Family Stichaeidae) are generally cold-temperate fishes most commonly found in the north Pacific. As part of the California Conservation Genomics Project (CCGP), we sequenced the genome of the Monkeyface Prickleback, Cebidichthys violaceus, to establish a genomic model for understanding phylogeographic patterns of marine organisms in California. These patterns, in turn, may inform the design of marine protected areas using dispersal models based on forthcoming population genomic data. The genome of C. violaceus is typical of many marine fishes at less than 1 Gb (genome size = 575.6 Mb), and our assembly is near-chromosome level (contig N50 = 1 Mb, scaffold N50 = 16.4 Mb, BUSCO completeness = 93.2%). Within the context of the CCGP, the genome will be used as a reference for future whole genome resequencing projects, enhancing our knowledge of the population structure of the species and more generally, the efficacy of marine protected areas as a primary conservation tool across California's marine ecosystems.

Proximate Drivers of Population-Level Lizard Gut Microbial Diversity: Impacts of Diet, Insularity, and Local Environment.

Diet has been suggested to be an important driver of variation in microbiota composition in mammals. However, whether this is a more general phenomenon and how fast changes in gut microbiota occur with changes in diet remains poorly understood. Forty-nine years ago, ten lizards of the species Podarcis siculus were taken from the island of Pod Kopiste and introduced onto the island of Pod Mrcaru (Croatia). The introduced population underwent a significant dietary shift, and their descendants became omnivorous (consuming up to 80% plant material during summer). Variation in their gut microbiota has never been investigated. To elucidate the possible impact on the gut microbiota of this rapid change in diet, we compared the microbiota (V4 region of the 16S rRNA gene) of P. siculus from Pod Mrcaru, Pod Kopiste, and the mainland. In addition, we explored other drivers of variation in gut microbiota including insularity, the population of origin, and the year of sampling. Alpha-diversity analyses showed that the microbial diversity of omnivorous lizards was higher than the microbial diversity of insectivorous lizards. Moreover, omnivorous individuals harbored significantly more Methanobrevibacter. The gut microbial diversity of insectivorous lizards was nonetheless more heterogeneous. Insectivorous lizards on the mainland had different gut microbial communities than their counterparts on the island of Pod Kopiste. Bacillus and Desulfovibrio were more abundant in the gut microbiota from insular lizards compared to mainland lizards. Finally, we showed that the population of origin was also an important driver of the composition of the gut microbiota. The dietary shift that occurred in the introduced population of P. siculus has had a detectable impact on the gut microbiota, but other factors such as insularity and the population of origin also contributed to differences in the gut microbial composition of these lizards, illustrating the multifactorial nature of the drivers of variation in gut microbiota. Overall, our data show that changes in gut microbiota may take place on ecological timescales. Yet, diet is only one of many factors driving variation in gut microbiota across populations.

Sustained changes in digestive physiology and microbiome across sequential generations of zebrafish fed different diets.

Alterations to ratios of protein and fiber in an organism's diet have been shown to structurally and functionally alter its individual digestive physiology. However, it is unclear how these dietary changes may affect phenotypic changes across generations. We utilized feeding trials, morphological analyses, enzyme activities, and 16S rRNA sequencing of the gut microbiome of zebrafish (Danio rerio) to determine how variations to fiber and protein concentrations, kept consistent across sequential generations, affect phenotypic changes. Our results show that Parental (P) and first generation (F1) fish did not differ from each other in terms of their intestine length, intestine mass, enzyme activity levels, and microbial community composition for any of the three experimental diets (high-protein/low-fiber, moderate-protein/fiber, and low-protein/high-fiber). However, each of the three experimental diets for the P and F1 fish, as well as the ancestral diet fish, did have distinct microbial community structure from one another. This indicates that there is a strong dietary effect on digestive physiology and gut microbial community and that these effects are consistent when the diet is kept homogenous across generations.

Abalone under moderate heat stress have elevated metabolic rates and changes to digestive enzyme activities.

Abalone around the world are subject to increasing frequency of marine heatwaves, yet we have a limited understanding of how acute high temperature events impact the physiology of these commercially and ecologically important species. This study examines the impact of a 5 °C temperature increase over ambient conditions for six weeks on the metabolic rates, digestive enzyme activities in the digestive gland, and digestive efficiency of Red Abalone (Haliotis rufescens) and Paua (H. iris) on their natural diets. We test the hypothesis that abalone digestive function can keep pace with this increased metabolic demand in two separate experiments, one for each species. H. iris had higher food intake in the heat treatment. Both species had higher metabolic rates in the heat treatment with Q10 = 1.73 and Q10 = 2.46 for H. rufescens and H. iris, respectively. Apparent organic matter digestibility, protein digestibility, and carbohydrate digestibility did not differ between the heat treatment and the ambient (control) treatment in either experiment. H. rufescens exhibited higher maltase, alanine-aminopeptidase, and leucine-aminopeptidase activities in the heat treatment. Amylase, ß-glucosidase, trypsin, and alkaline phosphatase activities in the digestive gland tissue did not differ between temperature treatments. H. iris exhibited lower amylase and ß-glucosidase activities in the heat treatment, while maltase, trypsin, leucine-aminopeptidase, and alkaline phosphatase activities did not differ between treatments. We conclude that over six weeks of moderate heat stress both abalone species were able to maintain digestive function, but achieved this maintenance in species-specific ways.

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.

Shark spiral intestines may operate as Tesla valves.

Looking to nature for inspiration has led to many diverse technological advances. The spiral valve intestine of sharks has provided the opportunity to observe the efficiency of different valve systems. It is supposed that the spiral intestine present in sharks, skates and rays slows the transit rate of digesta through the gut and provides increased surface area for the absorption of nutrients. In this investigation, we use a novel technique-creating three-dimensional reconstructions from CT scans of spiral intestines-to describe the morphology of the spiral intestine of at least one species from 22 different shark families. We discuss the morphological data in an evolutionary, dietary and functional context. The evolutionary analyses suggest that the columnar morphology is the ancestral form of the spiral intestine. Dietary analyses reveal no correlation between diet type and spiral intestine morphology. Flow rate was slowed significantly more when the two funnel-shaped spiral intestines were subjected to flow in the posterior to anterior direction, indicating their success at producing unidirectional flow, similar to a Tesla valve. These data are available to generate additional three-dimensional morphometrics, create computational models of the intestine, as well as to further explore the function of the gastrointestinal tract of sharks in structural and physiological contexts.

Gut microbiomes of sympatric Amazonian wood-eating catfishes (Loricariidae) reflect host identity and little role in wood digestion.

Neotropical wood-eating catfishes (family Loricariidae) can occur in diverse assemblages with multiple genera and species feeding on the same woody detritus. As such, they present an intriguing system in which to examine the influence of host species identity on the vertebrate gut microbiome as well as to determine the potential role of gut bacteria in wood digestion. We characterized the gut microbiome of two co-occurring catfish genera and four species: Panaqolus albomaculatus, Panaqolus gnomus, Panaqolus nocturnus, and Panaque bathyphilus, as well as that of submerged wood on which they feed. The gut bacterial community did not significantly vary across three gut regions (proximal, mid, distal) for any catfish species, although interspecific variation in the gut microbiome was significant, with magnitude of interspecific difference generally reflecting host phylogenetic proximity. Further, the gut microbiome of each species was significantly different to that present on the submerged wood. Inferring the genomic potential of the gut microbiome revealed that the majority of wood digesting pathways were at best equivalent to and more often depleted or nonexistent within the catfish gut compared to the submerged wood, suggesting a minimal role for the gut microbiome in wood digestion. Rather, these fishes are more likely reliant on fiber degradation performed by microbes in the environment, with their gut microbiome determined more by host identity and phylogenetic history.

Rapid Dietary Shift in Podarcis siculus Resulted in Localized Changes in Gut Function.

AbstractNatural dietary shifts offer the opportunity to address the nutritional physiological characters required to thrive on a particular diet. Here, we studied the nutritional physiology of Podarcis siculus, with populations on Pod Mrcaru, Croatia, that have become omnivorous and morphologically distinct (including the development of valves in the hindgut) from their insectivorous source population on Pod Kopiste. We compared gut structure and function between the two island populations of this lizard species and contrasted them with an insectivorous mainland out-group population in Zagreb. On the basis of the adaptive modulation hypothesis, we predicted changes in gut size and structure, digestive enzyme activities, microbial fermentation products (short-chain fatty acids [SCFAs]), and plant material digestibility concomitant with this dietary change. The Pod Mrcaru population had heavier guts than the mainland population, but there were no other differences in gut structure. Most of the enzymatic differences we detected were between the island populations and the out-group population. The Pod Mrcaru lizards had higher amylase and trehalase activities in their hindguts compared with the Pod Kopiste population, and the Pod Kopiste lizards had greater SCFA concentrations in their hindguts than the omnivorous Pod Mrcaru population. Interestingly, the differences between the Pod Mrcaru and Pod Kopiste populations are primarily localized to the hindgut and are likely influenced by microbial communities and a higher food intake by the Pod Mrcaru lizards. Although subtle, the changes in hindgut digestive physiology impact the digestibility of plant material in adult lizards-Pod Mrcaru lizards had higher digestibility of herbivorous and omnivorous diets fed over several weeks in the laboratory than did their source population.

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.

Seagrass digestion by a notorious 'carnivore'.

What an animal consumes and what an animal digests and assimilates for energetic demands are not always synonymous. Sharks, uniformly accepted as carnivores, have guts that are presumed to be well suited for a high-protein diet. However, the bonnethead shark (Sphyrna tiburo), which is abundant in critical seagrass habitats, has been previously shown to consume copious amounts of seagrass (up to 62.1% of gut content mass), although it is unknown if they can digest and assimilate seagrass nutrients. To determine if bonnetheads digest seagrass nutrients, captive sharks were fed a 13C-labelled seagrass diet. Digestibility analyses, digestive enzyme assays and stable isotope analyses were used to determine the bonnethead shark's capacity for digesting and assimilating seagrass material. Compound-specific stable isotope analysis showed that sharks assimilated seagrass carbon (13.6 ± 6.77‰ δ13C mean ± s.d. for all sharks and all amino acid types analysed) with 50 ± 2% digestibility of seagrass organic matter. Additionally, cellulose-component-degrading enzyme activities were detected in shark hindguts. We show that a coastal shark is digesting seagrass with at least moderate efficiency, which has ecological implications due to the stabilizing role of omnivory and nutrient transport within fragile seagrass ecosystems.

The beach-spawning California grunion Leuresthes tenuis eats and digests conspecific eggs.

Apparent egg cannibalism was investigated in the beach-spawning California grunion Leuresthes tenuis. Three hypotheses were tested to determine whether L. tenuis regularly consumes and efficiently digests conspecific eggs. First, examination of the gut contents of adults collected at four spawning sites over two seasons showed that the intestines of most fish from all the sites (57-87%, n ≥ 30, each site) contained L. tenuis eggs. The two other hypotheses focused on digestion of the eggs. First, the force required to crush cannibalized eggs was significantly less than that for uncannibalized eggs (fertilized or unfertilized), indicating that ingestion weakens the egg chorions. Second, conspecific eggs fed to fish held in the laboratory visibly degraded as they passed through the gut. The eggs lost c. half of their protein content and about two-thirds of their lipid content as they passed from proximal to distal regions of the gut, indicating that digestion occurred. Digestive enzyme activities of the gut further confirmed that L. tenuis can break down the contents of ingested eggs. Trypsin activity decreased and aminopeptidase activity increased posteriorly along the gut, whereas amylase and lipase activities exhibited less clear patterns by gut region. As far as is known, this study is the first to show that L. tenuis is an egg cannibal.

The effects of protein and fiber content on gut structure and function in zebrafish (Danio rerio).

Chemical reactor theory (CRT) suggests that the digestive tract functions as a chemical reactor for processing food. Presumably, gut structure and function should match diet to ensure adequate nutrient and energy uptake to maintain performance. Within CRT, dietary biochemical composition is the most important factor affecting gut structure and function in vertebrates. We fed Danio rerio (zebrafish) diets ranging from high- to moderate- to low-quality (i.e., ranging from high-protein, low-fiber to low-protein, high-fiber), and observed how gut length and surface area, as well as the activity levels of digestive enzymes (amylase, maltase, trypsin, aminopeptidase, and lipase) shifted in response to these dietary changes. Fish on the low-quality diet had the longest guts with the largest intestinal epithelial surface area and enterocyte cellular volumes. Fish on the moderate-quality diet had intermediate values of most of these parameters, and fish on the high-quality diet, the lowest. These data largely support CRT. Digestive enzyme activity levels were generally elevated in fish fed the moderate- and low-quality diets, but were highest in the fish fed the moderate-quality diet, suggesting that a diet with protein levels closest to that of the natural diet of D. rerio (they are omnivorous in nature) may elicit the best gut performance. However, fish fed the carnivore diet reached the largest terminal body size. Our results support CRT in terms of gut structure; however, our enzyme results do not necessarily agree with CRT and largely depend on which enzyme is discussed. In particular, the evidence for lipase activities being elevated in the fish fed the low-protein, high-fiber diet perhaps reflects a lipid-scavenging mechanism in fish consuming high-fiber foods rather than CRT.

Modulation of digestive physiology and biochemistry in Mytilus californianus in response to feeding level acclimation and microhabitat.

The intertidal mussel Mytilus californianus is a critical foundation species that is exposed to fluctuations in the environment along tidal- and wave-exposure gradients. We investigated feeding and digestion in mussels under laboratory conditions and across environmental gradients in the field. We assessed whether mussels adopt a rate-maximization (higher ingestion and lower assimilation) or a yield-maximization acquisition (lower ingestion and higher assimilation) strategy under laboratory conditions by measuring feeding physiology and digestive enzyme activities. We used digestive enzyme activity to define resource acquisition strategies in laboratory studies, then measured digestive enzyme activities in three microhabitats at the extreme ends of the tidal- and wave-exposure gradients within a stretch of shore (<20 m) projected sea-ward. Our laboratory results indicated that mussels benefit from a high assimilation efficiency when food concentration is low and have a low assimilation efficiency when food concentration is high. Additionally, enzyme activities of carbohydrases amylase, laminarinase and cellulase were elevated when food concentration was high. The protease trypsin, however, did not increase with increasing food concentration. In field conditions, low-shore mussels surprisingly did not have high enzyme activities. Rather, high-shore mussels exhibited higher cellulase activities than low-shore mussels. Similarly, trypsin activity in the high-shore-wave-sheltered microhabitat was higher than that in high-shore-wave-exposed. As expected, mussels experienced increasing thermal stress as a function of reduced submergence from low to high shore and shelter from wave-splash. Our findings suggest that mussels compensate for limited feeding opportunities and thermal stress by modulating digestive enzyme activities.

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.