The great difference in body size between the largest and smallest carnivores, with more than 130,000 times in body mass and 50 times in length, is significantly shocking in mammals. Reconstructions incorporating fossil data supported small body size (<5kg) for the ancestors of the species in Caniformia [24]. Though a few studies have been conducted to reveal molecular mechanisms for body size variation within species (such as the domestic dog), the genetic mechanisms underlying the huge differences in body size among the living carnivores are not well understood. In the present study, we scanned the whole genomes of 20 carnivores covering contrasting body size and identified that 337 genes were significantly related with both head body length and body mass. Selective pressure analyses revealed different evolutionary patterns in the extremely large and small species. Furthermore, fixed amino acid changes have also been identified in extremely small-body-sized carnivores and suggest the potential functions of these loci to restrict body size growth. All these results provide a genetic basis for studying the body size evolution in carnivores.
Obesity-related genes contributing to increasing body size of carnivores
In carnivores, some species of Pinnipedia and Fissipedia have evolved relatively large body size, containing some extremely large species such as walrus, northern sea lion, weddell seal and polar bear, even weighed exceed 350kg. Due to the semi-aquatic life habits, species in Pinnipedia had evolved a large body size to increase body surface area to reduce the rapid heat loss in water [25]. Similarly, the polar bear is the largest extant bear to adapt to the cold Arctic regions, which are averagely weighed 372 kg [26]. Interestingly, polar bears and seals have relatively thick subcutaneous fat, which accounted for more than 30% of their body weight and were much higher than that of other wild carnivores [27-29]. It has been suggested that pinnipeds and polar bears are covered with a thick layer of fat to protect them from cold environment. In general, obesity refers to certain degree of overweight and a thick fat layer, which is a state caused by excessive fat accumulation [30], and percent body fat ≥ 25% for men and ≥ 30% for women is an indication of obesity [31].
Fourteen positively correlated BSAGs were identified in our study and variations of these genes were reported to cause obesity (BRAP, CHCHD5, CPT1C, GPR1, LDLR, MAP2K5, PLEKHS1, SLC30A8, ST3GAL2, STX16, ZFHX3, ZGRF1, ZNF395 and ZPLD1). For instance, SNPs in BRAP (BRCA1 associated protein) were shown to associate with obesity and other metabolic abnormalities [32]. And STX16 (syntaxin 16) encoded a protein that is a member of the syntaxin or t-SNARE family and deletion in this gene may cause obesity and macrosomia in human [33]. Our result revealed that MAP2K5 (mitogen-activated protein kinase kinase 5) was enriched in the GO cluster of “positive regulation of growth (GO:0045927)” and genetic variations in this gene were reported to cause childhood obesity [34]. Furthermore, the evolutionary rates of 14 obesity-related BASGs in large carnivores were higher than that of small ones. Specially, three obesity-related BSAGs (i.e. ST3GAL2, ZGRF1 and ZPLD1) were determined rapid evolution in extremely large carnivores. For instance, the evolutionary rate of ZPLD1 (zona pellucida like domain containing 1) gene is 0.68632 in the extremely large carnivores, 5.5 times of that identified in the control group. Deletions in ZPLD1 has been proved to contribute to genetic susceptibility of common childhood obesity [35]. And it was reported that ST3GAL2-null mice have an increase of 50% in fat mass and 9% in lean body mass [36]. For ZGRF1 (Zinc finger GRF-type containing 1), this gene encodes a protein that contains GRF zinc finger (zf-GRF) and transmembrane domains, and a recent genome-wide and exome chip association study revealed its association with adiposity [37]. Thus, the 14 obesity-related BSAGs identified in this study may contribute to increased body size and accumulated body fat for the large carnivores.
Molecular evidence for the Peto’s Paradox in carnivores?
Animal gigantism is a recurring phenomenon that is seemingly influenced by available resources and natural selection [19]. Being larger brings an array of advantages for an organism, however, there also exists associated biological tradeoffs, including the increased risk of developing cancer due to with more cells [38]. Surprisingly, empirical cancer rates did not vary with body size, large and long-lived animals have a lower risk of suffering cancer than smaller, shorter-lived animals, and this phenomenon was called the Peto’s Paradox [20, 22, 39]. Recent years, the Peto’s Paradox has been studied in many large mammals, such as the bowhead whale and humpback whale [40, 41]. Their genomes provided the following two major evidences related to cancer suppression: 1) multiple duplications of tumor suppressor genes; 2) positive selection in genes related to cancer and aging. It was suggested that large species might have evolved multiple mechanisms to suppress cancer.
Carnivores are relatively long-lived mammals, and generally speaking, species in Pinnipedia have a longer lifespan than species in Fissipedia. Some extremely large species in carnivores, such as walrus and polar bears were reported to live 40 or more years in the wild [42]. Within particular taxa in carnivores, body size and lifespan also seem to be positively correlated [21]. For instance, the sea otter (about 27.4kg) may live as long as 27 years in the wild, as compared with the ferret (about 975.6g) which has lifespan much shorter as 11.1 years [21]. The relatively large polar bears (maximum lifespan: 43.8 years) and brown bears (maximum lifespan: 40 years) also live longer than the smaller giant pandas (maximum lifespan: 36.8 years). However, the molecular mechanism for maintaining the longevity of the large carnivores is not very clear, and there have been very few relevant studies on cancer in carnivores so far.
In our study, it was interestingly identified a total of 100 BSAGs in carnivores which were related to cancer control process, including tumor suppressor, DNA repair, as well as immunity. In addition, we also identified 15 cancer-related REGs in extremely large carnivores. Among them, 21 BSAGs were determined as tumor suppressor genes in previous studies. For example, the APC (APC regulator of WNT signaling pathway) gene encoded a multidomain protein that played a crucial role in tumor suppression by antagonizing the WNT signaling pathway. Variants in APC would induce various kinds of cancer such as colorectal cancer, pancreatic cancer and so on [43, 44]. The DSC3 (Desmocollin 3) gene is required for cell adhesion and desmosome formation, and it has been proved that this gene had a tumor suppressive activity through inhibition of AKT pathway in colorectal cancer [45]. ZFHX3 (zinc finger homeobox 3) was essential to regulate myogenic and neuronal differentiation, and it was reported to function as a tumor suppressor in several cancers [46]. The evolutionary rates of these three genes are significantly positively correlated with both body size parameters, suggesting the higher evolutionary rate in large carnivores than small ones. Meanwhile, two tumor suppressor genes, ADAM11 and TEP1, exhibited rapid evolution in the extremely large carnivores. The evolutionary rate of ADAM11 is 0.48527 in the extremely large carnivores, 15.4 times of that identified in the background group. And this gene has been identified as tumor suppressor gene in human breast cancer [47]. The TEP1 (Telomerase associated protein 1) product was a component of the ribonucleoprotein complex responsible for telomerase activity. And alterations in TEP1 has been confirmed to cause several types of human tumors including brain, breast, prostate and lung cancer [48]. These two genes all had relatively higher evolutionary rates in extremely large carnivores and suggested enhanced ability for suppressing cancer.
Additionally, 16 BSAGs were found to be related with “DNA repair” and it was well known that deficits in DNA repair capacity might lead to genetic instability and carcinogenesis [49]. Recent study revealed that loss of a single allele of the CLSPN (claspin) gene in mice was sufficient to drive earlier tumorigenesis [50]. And HELQ (Helicase, POLQ like) played a critical role for replication-coupled DNA repair, germ cell maintenance and tumor suppression in mammals [51]. Importantly, 18 immunity related genes were identified in BSAGs of carnivores, among which three genes exhibited elevated evolutionary rates (MAGT1, RFXANK and SKAP2). For instance, ITK (IL2 inducible T cell kinase) functions downstream of the T-cell receptor and was important for T-cell activation, development, differentiation, and production of many pro-inflammatory cytokines [52]. Moreover, ITK has been found to be prevalent in all main types of human neoplasia [53, 54]. And the evolutionary rate of MAGT1 (magnesium transporter 1) was 0.33127 in extremely large carnivores, 6.1 times of that in background group. Loss of MAGT1 would disrupt T cell signaling and lead to a novel human primary immunodeficiency [55], and furthermore, overexpression of MAGT1 was associated with development and metastasis of colorectal cancer [56]. Here, we obtained 100 cancer-related genes that were significantly associated with body size evolution in carnivores including 15 cancer-related REGs that identified in extremely large group, which might protect them from cancer invasion, special for large and long-lived species. These results might provide novel molecular evidence for the Peto’s Paradox with regard to carnivores.
Fixed amino acid changes in extremely small bod-sized carnivores contributing to growth restriction
There are some extremely small species in carnivores, such as meerkat and ferret with body mass less than 1 kg and body length less than 50 cm. The smaller size brings multiple superiority to species. For instance, smaller carnivores could take great advantage of food resources that are not available to some large animals to ensure survival when the environment changes dramatically and have more free energy and time to engage in activities that increase mating and reproductive success [15-17].
Compared with other carnivores in our dataset, six fixed amino acid changes from six genes were identified in extremely small-body-sized group: CDC7 (S513L), ENG (V367I), LIG4 (S784P), MMP2 (S579T), TSPAN8(S178T), and POLE (E701D). These six genes have been shown to be related with the phenotype of reduced body size. CDC7 (cell division cycle 7) was very conserved through mammalian evolution but a fixed amino acid change (S513L) was identified in extremely small carnivores. CDC7 played essential roles in initiation of mitotic DNA replication, and previous study showed that CDC7-/- or low expression of CDC7 protein would lead to reduced body size with decreased cell proliferation in mice [57]. Importantly, the fixed changes (S513L) were located in the protein kinase domain that functioned as an on/off switch for many cellular processes including metabolism, cell cycle progression, transcription [58]. Another fixed change (V367I) was examined in the Zona_pellucida domain of ENG (endoglin) that was reported Eng -/- mice three times smaller than wild type mice at E10.5 of the embryonic period [59]. The unique amino acid mutation (S784P) was determined in the key domain (DNA ligase IV domain) of the LIG4 (DNA ligase 4) gene that was reported mutations in humans or mice would cause growth failure and microcephaly, and this might be the result of activation of the DNA damage response, leading to a large amount of apoptosis during development [60]. Fixed amino acid mutation was separately found in the MMP2 (S579T) and POLE (E701D) gene. Previous studies have shown MMP2 (matrix metallopeptidase 2) knockout in mice and mutations of POLE (DNA polymerase epsilon, catalytic subunit) in human could cause short stature [61, 62]. Finally, we found a fixed difference at site 178 of the Tetraspannin domain in TPSAN8 between extremely small carnivores and others, this gene was reported that genetic ablation of in mice caused a reduction (-15.6%) in the body weight of male fed a normal chow diet [63]. Furthermore, the changes of S513L in CDC7 and S784P in LIG4 exhibit transitions of polarity might cause radical changes in the three-dimensional structure and function of proteins [64]. These six unique changes examined in the extremely small carnivores might have restricted body size growth. Of course, function experiments are need to further test if these changes cause growth retardation.