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

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Elevated Gene Copy Number Does Not Always Explain Elevated Amylase Activities in Fishes 000 Elevated Gene Copy Number Does Not Always Explain Elevated Amylase Activities in Fishes † Donovan P. German1,*, Keywords: digestive enzyme, evolution, diet, specialization, † Dolly M. Foti1, genetics. Joseph Heras1 Hooree Amerkhanian1 Brent L. Lockwood2 1Department of Ecology and Evolutionary Biology, University of California, Irvine, California 92697; 2Department of Introduction Biology, University of Vermont, Burlington, Vermont 05405 Digestive enzymes provide the chemical mechanism by which Accepted 4/18/2016; Electronically Published 5/24/2016 animals digest different components of their food, and, thus, the activity levels of digestive enzymes are commonly used as Online enhancements: supplemental tables and figures. ametricofananimal’s digestive ability (Krogdahl et al. 2005; Karasov and Douglas 2013). Among the suite of digestive en- zymes synthesized by vertebrates, the carbohydrase a-amylase frequently shows correlations between dietary substrate con- ABSTRACT centration and activity level, even when analyzed in a phylo- Amylase activity variation in the guts of several model organisms genetic context in fishes (Horn et al. 2006; German et al. 2010), appears to be explained by amylase gene copy number variation. birds (Kohl et al. 2011), primates (Perry et al. 2007), and canids We tested the hypothesis that amylase gene copy number is al- (Axelsson et al. 2013). a-amylase (a(1 → 4)-D-glucan gluca- ways elevated in animals with high amylolytic activity. We there- nohydrolase, EC 3.2.1.1) catalyzes the endohydrolysis of a(1 → 4)- fore sequenced the amylase genes and examined amylase gene glucosidic linkages in starch and similar molecules, producing copy number in prickleback fishes (family Stichaeidae) with dif- oligo- and disaccharides, which are subsequently hydrolyzed to ferent diets including two species of convergently evolved her- monomeric glucose by intestinal disaccharidases (Ferey-Roux bivores with the elevated amylase activity phenotype. We found et al. 1998; Krogdahl et al. 2005). In fishes, herbivores and om- elevated amylase gene copy number (six haploid copies) with se- nivores have the highest activity levels of amylase in their diges- quence variation among copies in one herbivore (Cebidichthys tive tracts, and carnivores have the lowest, showing the impor- violaceus) and modest gene copy number (two to three haploid tance of soluble polysaccharides in the nutrition of herbivores copies) with little sequence variation in the remaining taxa, which and omnivores (Zemke-White and Clements 1999; Horn et al. included herbivores, omnivores, and a carnivore. Few functional 2006; German et al. 2004, 2010; also see fig. 1). Moreover, her- differences in amylase biochemistry were observed, and previous bivorous and omnivorous prickleback fishes (family Stichaeidae) investigations showed similar digestibility among the conver- have been shown to have elevated amylase gene expression and gently evolved herbivores with differing amylase genetics. Hence, enzyme activities even when fed diets practically devoid of starch, the phenotype of elevated amylase activity can be achieved by thus suggesting that amylase genes are expressed constitutively in different mechanisms (i.e., elevated expression of fewer genes, in- some species (German et al. 2004; Kim et al. 2014). creased gene copy number, or expression of more efficient amy- Elevated amylase activity may in fact represent a digestive lase proteins) with similar results. Phylogenetic and comparative specialization in herbivores and omnivores, allowing for effi- genomic analyses of available fish amylase genes show mostly cient starch digestion (German et al. 2004, 2010, 2015; Skea lineage-specific duplication events leading to gene copy number et al. 2005, 2007; Karasov and Douglas 2013), yet we do not variation, although a whole-genome duplication event or chro- know the molecular underpinnings of these abilities. Elevated mosomal translocation may have produced multiple amylase cop- gene copy number appears to explain increased amylase activ- ies in the Ostariophysi, again showing multiple routes to the same ities in Drosophila (Shibata and Yamazaki 1995; Inomata et al. result. 1997; Inomata and Yamazaki 2000), salivary amylase in humans (Perry et al. 2007), and pancreatic amylase in mice (Sugino 2007) and dogs (Axelsson et al. 2013). But do these limited examples (an *Corresponding author; e-mail: [email protected]. insect and three mammals) represent the only mechanism by † These authors contributed equally to this work. which elevated amylase activities are achieved in animals and in fi Physiological and Biochemical Zoology 89(4):000–000. 2016. q 2016 by The shes in particular? With the examples above in mind, we for- University of Chicago. All rights reserved. 1522-2152/2016/8904-5160$15.00. mulated three questions regarding amylase genetics and evolu- DOI: 10.1086/687288 tion and how these relate to digestion in fishes: (1) Are differences This content downloaded from 128.200.169.189 on May 24, 2016 12:09:36 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c). 000 D.P.German,D.M.Foti,J.Heras,H.Amerkhanian,andB.L.Lockwood Figure 1. Phylogenetic relationships of the polyphyletic family Stichaeidae based on 2,100 bp of cytb,16s,andtomo4c4 genes (Kim et al. 2014). Bayesian posterior probabilities are indicated on nodes. Species used in this study are shown in bold and indicated with two asterisks. H p herbivory, O p omnivory, C p carnivory. Evolution of herbivory (dashed lines) and omnivory (dotted lines) is shown. Numbers in parentheses show number of taxa evaluated at that branch. Boxes highlight alleged families or subfamilies within the polyphyletic family Stichaeidae, with Cebidichthyidae (top), Xiphisterinae (middle), and Alectriinae (bottom) all highlighted (Chereshnev et al. 2013; Kim et al. 2014). Xiphisterinae is recognized as Xiphisteridae by Chereshnev et al. (2013). Pancreatic amylase activity values are mean 5 SEM. Amylase ’ p ! activities were compared among taxa with ANOVA, followed by Tukey s HSD (ANOVA, F4, 32 23.50, P 0.001). Values not sharing a superscript letter are significantly different. A color version of this figure is available online. in amylase activities among taxa always underpinned by differ- et al. 2004; German et al. 2004, 2014, 2015), whole-animal ences in amylase gene copy number? (2) What are the functional (Horn et al. 1986), and ecological (Horn 1983; Horn et al. 1983) consequences of increases in amylase gene copy number leading levels. Hence, with dietary diversity, ontogenetic dietary shifts, to increased expression of amylase genes and thus elevated am- convergent evolution of herbivory, and sister taxa with differ- ylase activity? (3) Across fishes, have changes in amylase gene ent diets, the pricklebacks are a highly appropriate study system copy number happened via lineage-specific duplication events, for understanding dietary specialization and the mechanisms as appears to be common in other vertebrates (Perry et al. 2007; underlying pancreatic amylase activity variation in particular. Axelsson et al. 2013), or by another mechanism, such as whole- We focused on members of two parts of the stichaeid phy- genome duplication (WGD; Ohno 1970; Christoffels et al. 2004; logeny: the Cebidichthyidae (Chereshnev et al. 2013; Kim et al. Kasahara 2007; Glasauer and Neuhauss 2014) or chromosomal 2014), which features the evolution of herbivory in Cebidi- translocation (Fraser et al. 2005)? chthys violaceus, and the Xiphisterinae (Chereshnev et al. 2013; Answering the above questions is crucial in the process of un- Kim et al. 2014), which features omnivory in two species (Phy- derstanding whether this widespread phenomenon—a match tichthys chirus and Xiphister atropurpureus), and the evolu- between amylase activity and diet—is truly adaptive and always tion of herbivory in Xiphister mucosus. For comparison, we arises by the same mechanism (i.e., changes in gene copy num- included two carnivorous stichaeids, Dictyosoma burgeri and ber). We therefore examined amylase genetics and potential Anoplarchus purpurescens, as carnivory is the basal dietary functional consequences in prickleback fishes with different condition of the family (fig. 1). Our study had five parts. First, diets and evolutionary histories (fig. 1). Prickleback fishes have we sequenced the amylase genes and examined sequence di- been studied in the context of niche specialization spanning versity of these genes in six stichaeid species (fig. 1). Second, we several layers of biological organization, including the isoto- measured gene copy number in five of the species and expres- pic (Saba 2004), molecular (Kim et al. 2014), biochemical (Chan sion of amylase genes in the two herbivores, C. violaceus and This content downloaded from 128.200.169.189 on May 24, 2016 12:09:36 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c). Gene Copy Number and Amylase Activities 000 X. mucosus. Because expression is impacted by regulatory ele- man et al. (2015). Because gut fullness can affect digestive en- ments,ofteninthe50 flanking region of a gene (Ma et al. 2004; zyme expression (Karasov and Douglas 2013), we confirmed that Wiebe et al. 2007), we sequenced the 50 flanking regions (in- each individual had gut content masses on par with those ob- cluding their putative promoters) of C. violaceus and X. mu- served for these species in previous investigations (German cosus
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