Acetylesterase and cinnamoyl ester hydrolase activities were demonstrated in culture supernatant of the anaerobic ruminal fungus Neocallimastix patriciarum. A cDNA expression library from N. patriciarum was screened for esterases using p-naphthyl acetate and a model cinnamoyl ester compound. cDNA clones representing four different esterase genes (bnaA-D) were isolated. None of the enzymes had cinnamoyl ester hydrolase activity, but two of the enzymes (BnaA and BnaC) had acetylxylan esterase activity. bnaA, bnaB and bnaC encode proteins with several distinct domains. Carboxy-terminal repeats in BnaA and BnaC are homologous to protein-docking domains in other enzymes from Neocallimastix species and another anaerobic fungus, a Piromyces sp. The catalytic domains of BnaB and BnaC are members of a recently described family of Ser/His active site hydrolases [Upton, C. & Buckley, J. T. (1995). Trends Biochem Sci 20, 178-1791. BnaB exhibits 40% amino acid identity to a domain of unknown function in the CelE cellulase from Clostridium thermocellum and BnaC exhibits 52% amino acid identity to a domain of unknown function in the XynB xylanase from Ruminococcus flavefaciens. BnaA, whilst exhibiting less than 10 O/ O overall amino acid identity to BnaB or BnaC, or to any other known protein, appears to be a member of the same family of hydrolases, having the three universally conserved amino acid sequence motifs. Several other previously described esterases are also shown to be members of this family, including a rhamnogalacturonan acetylesterase from Aspergillus aculeatus. However, none of the other previously described enzymes with acetylxylan esterase activity are members of this family of hydrolases.

36The rumen is the hallmark organ of ruminants and hosts a diverse ecosystem of 37 microorganisms that facilitates efficient digestion of plant fibers. We used 897 38 transcriptomes from three Cetartiodactyla lineages: ruminants, camels and cetaceans, 39 as well as data from ruminant comparative genomics and functional assays to explore 40 the genetic basis of rumen origin and evolution. Comparative analyses reveal that the 41 rumen and the first-chamber stomachs of camels and cetaceans shared a common 42 tissue origin from the esophagus. The rumen recruited genes from other tissues/organs 43 and up-regulated many esophagus genes to aquire functional innovations involving 44 epithelium absorption, improvement of the ketone body metabolism and regulation of 45 microbial community. These innovations involve such genetic changes as 46 ruminant-specific conserved elements, newly evolved genes and positively selected 47 genes. Our in vitro experiements validate the functions of one enhancer, one 48 positively selected gene and two newly evolved antibacterial genes. Our study 49 provides novel insights into the origin and evolution of a complex organ. 50 3Evolutionary biology has a long history of trying to understand how complex organs 51 evolve 1 . The origin of some notable organs has been central to animal evolution, e.g. 52 the eyes of animals 2,3 , electric organs of fishes 4 , mammalian placenta 5,6 and ruminant 53 headgear 7 . Another remarkable organ innovation found in mammals are the 54 multi-chambered stomachs found in the Cetartiodactyla lineages, including Tylopoda 55 (e.g. camels), Tayassuidae (e.g. peccaries), Hippopotamidae (e.g. hippos), Cetacea 56 (e.g. whales) and Ruminantia (Fig. 1). Among these, ruminants have the most complex 57 digestive system in herbivores, allowing efficient uptake of nutrients from plant 58 material by providing a microbial fermentation ecosystem in the highly specialized 59 rumen 8 . Camels (Tylopoda) have three-chambered stomachs and are also sometimes 60 called "pseudo-ruminants" due to their similar ruminating behavior and microbial 61 fermentation taking place in their first-chamber (FC) stomach 9 . The whales (Cetacea) 62 form the sister group of the Ruminantia 10 , however the FC of their four-chambered 63 stomach is mainly used as a temporary storage chamber for ingested food and for 64 mechanical grinding of food items 11 . With the rumen, ruminants obtained a unique 65 evolutionary advantage through superior utilization of short chain fatty acids (SCFAs) 66 from microbial fermentation, which significantly promoted the expansion and 67 diversification of ruminant taxa 12 . The evolutionary innovation of the rumen is 68 therefore interesting not only in its functional complexity and uniqueness, but also 69 because it has greatly benefited humans by providing high-quality nutrition in the shape 70 of highly productive ruminant livestock species 13,14 . 71The anatomical predecessor from which the rumen evolved has been proposed to 72 4 be the esophagus 15 , yet the...