Multiple bursts of pancreatic ribonuclease gene duplication in insect-eating bats Huihui Xu a , Yang Liu a , Fanxing Meng a , Beibei He b , Naijian Han c , Gang Li c , Stephen J. Rossiter d, ⁎, Shuyi Zhang a, ⁎ a Institute of Molecular Ecology and Evolution, Institutes for Advanced Interdisciplinary Research, East China Normal University, Shanghai 200062, China b School of Life Sciences, East China Normal University, Shanghai 200062, China c Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China d School of Biological and Chemical Sciences, Queen Mary, University of London, London E1 4NS, UK abstract article info Article history: Accepted 1 April 2013 Available online 1 May 2013 Keywords: RNASE1 Chiroptera Gene duplication Positive selection Pancreatic ribonuclease gene (RNASE1) was previously shown to have undergone duplication and adaptive evolution related to digestive efficiency in several mammalian groups that have evolved foregut fermentation, including ruminants and some primates. RNASE1 gene duplications thought to be linked to diet have also been recorded in some carnivores. Of all mammals, bats have evolved the most diverse dietary specializations, mainly including frugivory and insectivory. Here we cloned, sequenced and analyzed RNASE1 gene sequences from a range of bat species to determine whether their dietary adaptation is mirrored by molecular adapta- tion. We found that seven insect-eating members of the families Vespertilionidae and Molossidae possessed two or more duplicates, and we also detected three pseudogenes. Reconstructed RNASE1 gene trees based on both Bayesian and maximum likelihood methods supported independent duplication events in these two families. Selection tests revealed that RNASE1 gene duplicates have undergone episodes of positive selec- tion indicative of functional modification, and lineage-specific tests revealed strong adaptive evolution in the Tadarida β clade. However, unlike the RNASE1 duplicates that function in digestion in some mammals, the bat RNASE1 sequences were found to be characterized by relatively high isoelectric points, a feature previously suggested to promote defense against viruses via the breakdown of double-stranded RNA. Taken together, our findings point to an adaptive diversification of RNASE1 in these two bat families, although we find no clear evidence that this was driven by diet. Future experimental assays are needed to resolve the func- tions of these enzymes in bats. © 2013 Published by Elsevier B.V. 1. Introduction Vertebrate pancreatic ribonuclease (RNase1) is a secretory en- zyme that belongs to the ribonuclease A superfamily (Beintema and Kleineidam, 1998). Its biological function in vertebrates is to degrade pathogenic RNA and thus protect organisms as part of the immune system (Sorrentino, 2010; Sorrentino and Libonati, 1994). Previous studies have uncovered high expression levels of RNase in the pan- creas of ruminants and Old World colobine monkeys, both of which have evolved foregut fermentation (Barnard, 1969; Beintema, 1990; Beintema et al., 1973). Accordingly it has been proposed that RNase in these taxa might serve an enzymatic function to digest symbiotic bacteria in their foreguts so allowing bacterial derived nitrogen to be utilized efficiently (Barnard, 1969; Beintema, 1990). Subsequent work has revealed that colobine monkeys possess two or more RNASE1 genes and that the gene duplicate products are functionally suited to a low pH (Zhang, 2006; Zhang et al., 2002). Spe- cifically, amino acid replacements in pancreatic RNASE1 have resulted in a greater overall negative charge, and the consequent reduction in isoelectric point (pI) is thought to be an adaptation to the acidified environment of the foregut (Zhang, 2006). The origin of digestive RNASE1 genes in Asian and African colobines appears to be indepen- dent, providing compelling evidence that natural selection has shaped RNase1 evolution for its ability to break down the RNA of cellulolytic bacteria (Yu et al., 2010; Zhang, 2006). Similar RNASE1 duplications have been reported in ruminants (Breukelman et al., 1998, 2001; Kleineidam et al., 1999), as well as other herbivorous species such as the guinea pig (Van den Berg et al., 1977). Yet RNASE1 gene duplica- tions are not restricted to herbivores; multiple copies have also been documented in some carnivores, which have also been attributed to dietary efficiency (Yu and Zhang, 2006). Gene 526 (2013) 112–117 Abbreviations: RNASE1, pancreatic ribonuclease gene; RNase1, pancreatic ribo- nuclease; RNase, ribonuclease; AGT, alanine-glyoxylate aminotransferase 1; GLUT4, glucose transporter 4. ⁎ Corresponding authors. E-mail addresses: s.j.rossiter@qmul.ac.uk (S.J. Rossiter), syzhang@bio.ecnu.edu.cn (S. Zhang). 0378-1119/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.gene.2013.04.035 Contents lists available at SciVerse ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene