Multiple adaptive losses of alanine-glyoxylate aminotransferase mitochondrial targeting in fruit-eating bats

The enzyme alanine-glyoxylate aminotransferase 1 (AGT) functions to detoxify glyoxylate before it is converted into harmful oxalate. In mammals, mitochondrial targeting of AGT in carnivorous species versus peroxisomal targeting in herbivores is controlled by two signal peptides that correspond to these respective organelles. Differential expression of the mitochondrial targeting sequence (MTS) is considered an adaptation to diet-specific subcellular localization of glyoxylate precursors. Bats are an excellent group in which to study adaptive changes in dietary enzymes; they show unparalleled mammalian dietary diversification as well as independent origins of carnivory, frugivory, and nectarivory. We studied the AGT gene in bats and other mammals with diverse diets and found that the MTS has been lost in unrelated lineages of frugivorous bats. Conversely, species exhibiting piscivory, carnivory, insectivory, and sanguinivory possessed intact MTSs. Detected positive selection in the AGT of ancestral fruit bats further supports adaptations related to evolutionary changes in diet.     

A comparative analysis of the evolutionary relationship between diet and enzyme targeting in bats, marsupials and other mammals

The subcellular distribution of the enzyme alanine:glyoxylate aminotransferase (AGT) in the livers of different mammals appears to be related to their natural diets. Thus, AGT tends to be mitochondrial in carnivores, peroxisomal in herbivores, and both mitochondrial and peroxisomal in omnivores. To what extent this relationship is an incidental consequence of phylogenetic structure or an evolutionarily meaningful adaptive response to changes in dietary selection pressure is unknown. In order to distinguish between these two possibilities, we have determined the subcellular distribution of AGT in the livers of 22 new mammalian species, including members of three orders not studied before. In addition, we have analysed the statistical relationship between AGT distribution and diet in all 77 mammalian species, from 12 different orders, for which the distribution is currently known. Our analysis shows that there is a highly significant correlation between AGT distribution and diet, independent of phylogeny. This finding is compatible with the suggestion that the variable intracellular targeting of AGT is an adaptive response to episodic changes in dietary selection pressure. To our knowledge, this is the first example of such a response being manifested at the molecular and cellular levels across the breadth of Mammalia.     

Adaptive evolution to a high purine and fat diet of carnivorans revealed by gut microbiomes and host genomes

Carnivorous members of the Carnivora reside at the apex of food chains and consume meat‐only diets, rich in purine, fats and protein. Here, we aimed to identify potential adaptive evolutionary signatures compatible with high purine and fat metabolism based on analysis of host genomes and symbiotic gut microbial metagenomes. We found that the gut microbiomes of carnivorous Carnivora (e.g., Felidae, Canidae) clustered in the same clade, and other clades comprised omnivorous and herbivorous Carnivora (e.g., badgers, bears and pandas). The relative proportions of genes encoding enzymes involved in uric acid degradation were higher in the gut microbiomes of meat‐eating carnivorans than plant‐eating species. Adaptive amino acid substitutions in two enzymes, carnitine O‐palmitoyltransferase 1 (CPT1A) and lipase F (LIPF), which play a role in fat digestion, were identified in Felidae‐Candidae species. Carnivorous carnivorans appear to endure diets high in purines and fats via gut microbiomic and genomic adaptations.     

Diet drives convergent evolution of gut microbiomes in bamboo-eating species

Gut microbiota plays a critical role in host physiology and health. The coevolution between the host and its gut microbes facilitates animal adaptation to its specific ecological niche. Multiple factors such as host diet and phylogeny modulate the structure and function of gut microbiota. However, the relative contribution of each factor in shaping the structure of gut microbiota remains unclear. The giant(Ailuropoda melanoleuca) and red(Ailurus styani) pandas belong to different families of order Carnivora. They have evolved as obligate bamboo-feeders and can be used as a model system for studying the gut microbiome convergent evolution. Here, we compare the structure and function of gut microbiota of the two pandas with their carnivorous relatives using 16S rRNA and metagenome sequencing. We found that both panda species share more similarities in their gut microbiota structure with each other than each species shares with its carnivorous relatives. This indicates that the specialized herbivorous diet rather than host phylogeny is the dominant driver of gut microbiome convergence within Arctoidea.Metagenomic analysis revealed that the symbiotic gut microbiota of both pandas possesses a high level of starch and sucrose metabolism and vitamin B12 biosynthesis. These findings suggest a diet-driven convergence of gut microbiomes and provide new insight into host-microbiota coevolution of these endangered species.     

Ecomorphological correlates of craniodental variation in bears and paleobiological implications for extinct taxa: an approach based on geometric morphometrics

Relative warp analyses of landmarks describing cranial and mandibular shape are used for investigating patterns of morphological variation among extant bears (Mammalia, Carnivora, Ursidae) indicative of diet and feeding behavior. These patterns are used for deriving inferences about the autecology of two extinct species previously assumed to have had different dietary preferences, the North American giant, short-faced bear Arctodus simus and the Eurasian cave bear Ursus spelaeus . Results reveal a set of shared craniodental traits among the herbivorous bears, including short and vaulted skulls with well-developed zygomatic arches, lateralized orbits and small canines, concave jaws with a highly positioned condyle, large moment arms for the temporalis and masseter muscles, and long cheek teeth. In contrast, those bears that consume animal resources have long skulls with small zygomatic arches, frontalized orbits and well-developed canines, and long jaws with a deep mandibular symphysis, low muscle leverages, a condyle situated at the level of the tooth row and reduced cheek teeth. The craniodental morphology of omnivorous bears is intermediate between those of faunivores and herbivores. This is also the case of the short-faced bear and the cave bear, which suggests that previous reconstructions of the feeding ecology of these extinct species (highly carnivorous for A. simus and herbivorous for U. spelaeus ) should be revised.     

Primary hyperoxaluria type 1: AGT mistargeting highlights the fundamental differences between the peroxisomal and mitochondrial protein import pathways

Primary hyperoxaluria type 1 (PH1) is an atypical peroxisomal disorder, as befits a deficiency of alanine:glyoxylate aminotransferase (AGT), which is itself an atypical peroxisomal enzyme. PH1 is characterized by excessive synthesis and excretion of the metabolic end-product oxalate and the progressive accumulation of insoluble calcium oxalate in the kidney and urinary tract. Disease in many patients is caused by a unique protein trafficking defect in which AGT is mistargeted from peroxisomes to mitochondria, where it is metabolically ineffectual, despite remaining catalytically active. Although the peroxisomal import of human AGT is dependent upon the PTS1 import receptor PEX5p, its PTS1 is exquisitely specific for mammalian AGT, suggesting the presence of additional peroxisomal targeting information elsewhere in the AGT molecule. This and many other functional peculiarities of AGT are probably a consequence of its rather chequered evolutionary history, during which much of its time has been spent being a mitochondrial, rather than a peroxisomal, enzyme. Analysis of the molecular basis of AGT mistargeting in PH1 has thrown into sharp relief some of the fundamental differences between the requirements of the peroxisomal and mitochondrial protein import pathways, particularly the properties of peroxisomal and mitochondrial matrix targeting sequences and the different conformational limitations placed upon importable cargos.     

Energy digestibility of giant pandas on bamboo‐only and on supplemented diets

Endangered giant pandas (Ailuropoda melanoleuca) are bears (Family Ursidae), within the order Carnivora. They specialize on an herbivorous diet of bamboo yet retain a gastrointestinal tract typical of their carnivorous ancestry. The evolutionary constraints of their digestive tract result in a low extraction efficiency from bamboo (<40% in reported studies). The goal of this study was to determine the energy digestibility of bamboo by giant pandas used in digestibility trials and through subsequent analyses with bomb calorimetry. Seven digestibility trials were conducted (three with bamboo‐only diets and four with supplemental diets). Energy digestibilities ranged from 7.5–38.9% for mixed diets and 9.2–34.0% for bamboo‐only diets. The bamboo‐only trials summarized here represent, to our knowledge, the first empirical data available for energy digestibility on a bamboo diet for giant pandas. Zoo Biol 30:121–133, 2011. © 2010 Wiley‐Liss, Inc.     

Mammalian alanine/glyoxylate aminotransferase 1 is imported into peroxisomes via the PTS1 translocation pathway. Increased degeneracy and context specificity of the mammalian PTS1 motif and implications for the peroxisome-to-mitochondrion mistargeting of AGT in primary hyperoxaluria type 1

Alanine/glyoxylate aminotransferase 1 (AGT) is peroxisomal in most normal humans, but in some patients with the hereditary disease primary hyperoxaluria type 1 (PH1), AGT is mislocalized to the mitochondria. In an attempt to identify the sequences in AGT that mediate its targeting to peroxisomes, and to determine the mechanism by which AGT is mistargeted in PH1, we have studied the intracellular compartmentalization of various normal and mutant AGT polypeptides in normal human fibroblasts and cell lines with selective deficiencies of peroxisomal protein import, using immunofluorescence microscopy after intranuclear microinjection of AGT expression plasmids. The results show that AGT is imported into peroxisomes via the peroxisomal targeting sequence type 1 (PTS1) translocation pathway. Although the COOH-terminal KKL of human AGT was shown to be necessary for its peroxisomal import, this tripeptide was unable to direct the peroxisomal import of the bona fide peroxisomal protein firefly luciferase or the reporter protein bacterial chloramphenicol acetyltransferase. An ill-defined region immediately upstream of the COOH-terminal KKL was also found to be necessary for the peroxisomal import of AGT, but again this region was found to be insufficient to direct the peroxisomal import of chloramphenicol acetyltransferase. Substitution of the COOH-terminal KKL of human AGT by the COOH-terminal tripeptides found in the AGTs of other mammalian species (SQL, NKL), the prototypical PTS1 (SKL), or the glycosomal PTS1 (SSL) also allowed peroxisomal targeting, showing that the allowable PTS1 motif in AGT is considerably more degenerate than, or at least very different from, that acceptable in luciferase. AGT possessing the two amino acid substitutions responsible for its mistargeting in PH1 (i.e., Pro11-- >Leu and Gly170-->Arg) was targeted mainly to the mitochondria. However, AGTs possessing each amino acid substitution on its own were targeted normally to the peroxisomes. This suggests that Gly170-->Arg- mediated increased functional efficiency of the otherwise weak mitochondrial targeting sequence (generated by the Pro11-->Leu polymorphism) is not due to interference with the peroxisomal targeting or import of AGT.     

The effects of diet and digestive cycle on the gastrointestinal tract pH values in the goldfish, Carassius auratus L., Mozambique tilapia, Oreochromis mossambicus (Peters), and channel catfish, Ictalurus punctatus (Rafinesque)

The digestive tract pH values of the teleost fish Oreochromis mossambicus, Carassius auratus and Ictalurus punctatus were determined in fish that were (1) starved, (2) fed herbivorous, omnivorous and carnivorous diets and (3) fed altered pH diets. The digestive tract pH profile was determined throughout an 8 h cycle. The herbivorous or carnivorous nature of the diet did not affect the pH values in any of the species. Significant differences in gut pH values were observed when the fish ingested diets with altered pHs. There was a difference between the starved and baseline fed values in Carassius auratus which was not observed in the other species. The pH values in the digestive tracts of the three species varied during the digestive cycle.     

The diet and growth of 0-group dace, Leuciscus leuciscus (L.), and roach, Rutilus rutilus (L.), in a lowland river

The diets of 0-group dace and roach in the Lower Welsh Dee were studied by stomach contents analysis of seasonal and diel samples. During development, dace preyed on a sequence of food types, initially being mainly carnivorous then becoming nocturnally carnivorous and diurnally omnivorous. Roach showed a similar pattern, although after the first two months of life they were more herbivorous/detritivorous. From the variations in the diets, it is hypothesised that both species are limited by food. This receives circumstantial support from their poor observed growth. The food supply routes by which flow regulation of the River Dee could affect juvenile fish production are noted.     

Noninsect-Based Diet Leads to Structural and Functional Changes of Acidic Chitinase in Carnivora

Acidic chitinase (Chia) digests the chitin of insects in the omnivorous stomach and the chitinase activity in carnivorous Chia is significantly lower than that of the omnivorous enzyme. However, mechanistic and evolutionary insights into the functional changes in Chia remain unclear. Here we show that a noninsect-based diet has caused structural and functional changes in Chia during the course of evolution in Carnivora. By creating mouse-dog chimeric Chia proteins and modifying the amino acid sequences, we revealed that F214L and A216G substitutions led to the dog enzyme activation. In 31 Carnivora, Chia was present as a pseudogene with stop codons in the open reading frame (ORF) region. Importantly, the Chia proteins of skunk, meerkat, mongoose, and hyena, which are insect-eating species, showed high chitinolytic activity. The cat Chia pseudogene product was still inactive even after ORF restoration. However, the enzyme was activated by matching the number and position of Cys residues to an active form and by introducing five meerkat Chia residues. Mutations affecting the Chia conformation and activity after pseudogenization have accumulated in the common ancestor of Felidae due to functional constraints. Evolutionary analysis indicates that Chia genes are under relaxed selective constraint in species with noninsect-based diets except for Canidae. These results suggest that there are two types of inactivating processes in Carnivora and that dietary changes affect the structure and activity of Chia.     

Inhibition of alanine:glyoxylate aminotransferase 1 dimerization is a prerequisite for its peroxisome-to-mitochondrion mistargeting in primary hyperoxaluria type 1

Peroxisome-to-mitochondrion mistargeting of the homodimeric enzyme alanine:glyoxylate aminotransferase 1 (AGT) in the autosomal recessive disease primary hyperoxaluria type 1 (PH1) is associated with the combined presence of a normally occurring Pro(11)Leu polymorphism and a PH1-specific Gly170Arg mutation. The former leads to the formation of a novel NH2-terminal mitochondrial targeting sequence (MTS), which although sufficient to direct the import of in vitro-translated AGT into isolated mitochondria, requires the additional presence of the Gly170Arg mutation to function efficiently in whole cells. The role of this mutation in the mistargeting phenomenon has remained elusive. It does not interfere with the peroxisomal targeting or import of AGT. In the present study, we have investigated the role of the Gly170Arg mutation in AGT mistargeting. In addition, our studies have led us to examine the relationship between the oligomeric status of AGT and the peroxisomal and mitochondrial import processes. The results obtained show that in vitro-translated AGT rapidly forms dimers that do not readily exchange subunits. Although the presence of the Pro(11)Leu or Gly170Arg substitutions alone had no effect on dimerization, their combined presence abolished homodimerization in vitro. However, AGT containing both substitutions was still able to form heterodimers in vitro with either normal AGT or AGT containing either substitution alone. Expression of various combinations of normal and mutant, as well as epitope-tagged and untagged forms of AGT in whole cells showed that normal AGT rapidly dimerizes in the cytosol and is imported into peroxisomes as a dimer. This dimerization prevents mitochondrial import, even when the AGT possesses an MTS generated by the Pro(11)Leu substitution. The additional presence of the Gly170Arg substitution impairs dimerization sufficiently to allow mitochondrial import. Pharmacological inhibition of mitochondrial import allows AGT containing both substitutions to be imported into peroxisomes efficiently, showing that AGT dimerization is not a prerequisite for peroxisomal import.     

An enzyme trafficking defect in two patients with primary hyperoxaluria type 1: peroxisomal alanine/glyoxylate aminotransferase rerouted to mitochondria

Most patients with the autosomal recessive disease primary hyperoxaluria type 1 (PH1) have a complete deficiency of alanine/glyoxylate aminotransferase (AGT) enzyme activity and immunoreactive protein. However a few possess significant residual activity and protein. In normal human liver, AGT is entirely peroxisomal, whereas it is entirely mitochondrial in carnivores, and both peroxisomal and mitochondrial in rodents. Using the techniques of isopycnic sucrose and Percoll density gradient centrifugation and quantitative protein A-gold immunoelectron microscopy, we have found that in two PH1 patients, possessing 9 and 27% residual AGT activity, both the enzyme activity and immunoreactive protein were largely mitochondrial and not peroxisomal. In addition, these individuals were more severely affected than expected from the levels of their residual AGT activity. In these patients, the PH1 appears to be due, at least in part, to a unique trafficking defect, in which peroxisomal AGT is diverted to the mitochondria. To our knowledge, this is the first example of a genetic disease caused by such interorganellar rerouting.     

大熊猫等五种食肉动物血清蛋白和LDH同工酶盘电泳比较

大熊猫(Ailuropoda melanoleuca)是世界珍稀动物之一,近年来,兽类科学工作者对它进行了多方面的研究,也包括生化方面的工作(潘文石等,1982;Sarich,1973),但与其他食肉哺乳动物相比研究较少,而此又为探讨其分类地位所需。本文应用聚丙烯酰胺凝胶盘电泳,对大熊猫、小熊猫(AilurSs fulgens)、黑熊(Selenarctos thibetanus)、家猫及犬等5种食肉动物的血清蛋白和LDH同工酶进行比较分析,目的在于了解这5种动物的血清蛋白图象和LDH同工酶酶谱以及它们之间的谱型、相对活力、迁移率的异同,进而讨论大熊猫的分类地位。     

Why Does the Giant Panda Eat Bamboo? A Comparative Analysis of Appetite-Reward-Related Genes among Mammals

Background

The giant panda has an interesting bamboo diet unlike the other species in the order of Carnivora. The umami taste receptor gene T1R1 has been identified as a pseudogene during its genome sequencing project and confirmed using a different giant panda sample. The estimated mutation time for this gene is about 4.2 Myr. Such mutation coincided with the giant panda''s dietary change and also reinforced its herbivorous life style. However, as this gene is preserved in herbivores such as cow and horse, we need to look for other reasons behind the giant panda''s diet switch.

Methodology/Principal Findings

Since taste is part of the reward properties of food related to its energy and nutrition contents, we did a systematic analysis on those genes involved in the appetite-reward system for the giant panda. We extracted the giant panda sequence information for those genes and compared with the human sequence first and then with seven other species including chimpanzee, mouse, rat, dog, cat, horse, and cow. Orthologs in panda were further analyzed based on the coding region, Kozak consensus sequence, and potential microRNA binding of those genes.

Conclusions/Significance

Our results revealed an interesting dopamine metabolic involvement in the panda''s food choice. This finding suggests a new direction for molecular evolution studies behind the panda''s dietary switch.     

Mandible of the giant panda (Ailuropoda melanoleuca) compared with other Chinese carnivores: functional adaptation

The aim of this study was to understand the mandible of the giant panda in morphometric terms to explore differences between the giant panda and other carnivores distributed in China, in terms of functional adaptation. Twelve mandibular variables were studied using bivariate (allometry) and multivariate (principal components analysis, PCA, and discriminant functional analysis, DFA) tools. When deviations were produced from allometric baselines consisting of all the species studied, the giant panda displayed a much more developed mandibular structure than the bear, leopard, and tiger. This may be related to its specific dietary preference for bamboo, which has very strong fibers. Results also indicate that the mandibular structure among carnivores mainly reflects the differences in their dietary preferences and functional adaptation. Three groups were found referring to dispersal profiles expressed by the first two axes of PCA and DFA: (1) the two panda species – the herbivorous carnivores; (2) the black bear – the omnivorous carnivore; and (3) the tiger and leopard – the hypercarnivores. Nevertheless, a significant separation between the two panda species was also found with the profiles displayed by the first and third axes of DFA. In addition to no close evolutionary relationship and phylogenetic development, a noticeable separation between the two panda species found in DFA analysis may be associated with their variation in consuming different parts of the bamboo plant: the giant panda feeds on stems and the red panda feeds on leaves.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 449–456.     

Marine-freshwater transitions are associated with the evolution of dietary diversification in terapontid grunters (Teleostei: Terapontidae)

The ecological opportunities associated with transitions across the marine-freshwater interface are regarded as an important catalyst of diversification in a range of aquatic taxa. Here, we examined the role of these major habitat transitions and trophic diversification in a radiation of Australasian fishes using a new molecular phylogeny incorporating 37 Terapontidae species. A combined mitochondrial and nuclear gene analysis yielded a well-supported tree with most nodes resolved. Ancestral terapontids appear to have been euryhaline in habitat affiliation, with a single transition to freshwater environments producing all Australasian freshwater species. Mapping of terapontid feeding modes onto the molecular phylogeny-predicted carnivorous dietary habits was displayed by ancestral terapontids, which subsequently diversified into a range of additional carnivorous, omnivorous, herbivorous and detritivorous dietary modes upon transition to freshwater habitats. Comparative analyses suggested that following the freshwater invasion, the single freshwater clade has exhibited an increased rate of diversification at almost three times the background rate evident across the rest of the family. The marine-freshwater transition within Terapontidae appears to have resulted in substantial dietary radiation in freshwater environments, as well as increased lineage diversification rates relative to euryhaline-marine habitats.     

Biomechanical consequences of rapid evolution in the polar bear lineage

The polar bear is the only living ursid with a fully carnivorous diet. Despite a number of well-documented craniodental adaptations for a diet of seal flesh and blubber, molecular and paleontological data indicate that this morphologically distinct species evolved less than a million years ago from the omnivorous brown bear. To better understand the evolution of this dietary specialization, we used phylogenetic tests to estimate the rate of morphological specialization in polar bears. We then used finite element analysis (FEA) to compare the limits of feeding performance in the polar bear skull to that of the phylogenetically and geographically close brown bear. Results indicate that extremely rapid evolution of semi-aquatic adaptations and dietary specialization in the polar bear lineage produced a cranial morphology that is weaker than that of brown bears and less suited to processing tough omnivorous or herbivorous diets. Our results suggest that continuation of current climate trends could affect polar bears by not only eliminating their primary food source, but also through competition with northward advancing, generalized brown populations for resources that they are ill-equipped to utilize.