To investigate the evolution of HMGCS2, we used a previously published whole genome alignment to inspect the gene sequence and the surrounding locus across 70 placental mammals (Sharma and Hiller, 2017). Surprisingly, we discovered that three independent lineages (cetaceans, pteropodids and the African savanna elephant) exhibit large deletions that remove HMGCS2 exons or gene-inactivating mutations that shift the HMGCS2 reading frame and destroy conserved splice site dinucleotides (Figure 1B). All three lineages have a deletion of exon one that encodes the mitochondrial targeting domain; such a deletion causes HMG-CoA synthase-2 deficiency in human individuals (Pitt et al., 2015). Other mutations affect exons encoding key residues required for HMG-CoA synthase catalytic activity and leave little of the coding sequence intact. Together with the deletion of the promoter region in pteropodids, the elephant and the sperm whale (Figure 1—figure supplement 1), this shows that three mammalian lineages lost the enzyme that is required for ketogenesis.

In cetaceans and pteropodids, the remnants of the once-functional HMGCS2 gene are located in a conserved genomic context with REG4 upstream and PHGDH downstream. In elephant, the three remaining HMGCS2 exons also occur in the same genomic locus adjacent to the conserved PHGDH gene, but inversions that already happened in the ancestor of elephants and the closely related manatees rearranged the locus upstream of HMGCS2 (Figure 1—figure supplement 2). These rearrangements were succeeded by a large deletion in the elephant lineage that removed the first five HMGCS2 exons together with the REG4 gene.

To rule out that the gene-inactivating mutations are sequencing or genome assembly errors, we validated all smaller mutations and exon deletions with unassembled sequencing reads from the SRA and TRACE archives using blastn. All 22 mutations in cetaceans were confirmed by at least 30 reads, with no support for the non-gene-inactivating allele (Figure 1B, Supplementary file 1). This includes the deletion of exon one that exhibits shared breakpoints in the toothed and baleen whale lineages (Figure 1—figure supplement 3), which strongly suggests that this deletion and thus HMGCS2 loss already occurred before the split of the main cetacean lineages (Figure 1C). This is further supported by the 2 bp frameshifting insertion in exon two that is shared between killer whale and minke whale, and was later deleted in dolphin and sperm whale.

In pteropodid bats, the ~4.5 kb deletion that removed coding exon two is validated by sequencing reads and is shared between both flying foxes (Figure 1B), suggesting that HMGCS2 was already lost in their common ancestor. Using the HMGCS2 sequence of the David’s myotis bat, we detected no evidence for the presence of the deleted HMGCS2 exons in unassembled sequencing reads of both flying fox species, while we readily found all exons of the HMGCS1 paralog, showing that the search is sufficiently sensitive. In the Egyptian fruit bat, HMGCS2 is entirely removed by a large deletion between the REG4 and PHGDH genes, which we validated with an independent PacBio assembly (Figure 1—figure supplement 4). Consistent with ongoing gene erosion, the 1 bp deletion in exon three is heterozygous in the black flying fox (Figure 1B).

To rule out that the partial gene deletion in the African savanna elephant is an assembly error, we used the manatee HMGCS2 sequence. Sensitive blastn searches found no significant hits for the deleted HMGCS2 exons or the deleted neighboring REG4 gene in the unassembled sequencing reads of two different savanna elephant individuals (Cortez et al., 2014). In contrast, the three remaining HMGCS2 exons as well as all exons of the paralogous HMGCS1 could be recovered. We also investigated related elephant species, making use of recently published sequence data from the African forest elephant and the Asian elephant (Palkopoulou et al., 2018; Reddy et al., 2015). Further, we queried sequence data from two American mastodons, extinct Elephantimorpha that split from elephants 28–24 Mya (Rohland et al., 2007). As for the savanna elephant, the three remaining HMGCS2 exons and entire HMGCS1 gene were found in all three species, while the deleted HMGCS2 exons and the REG4 gene were not found (Figure 1B). Parsimony suggests that the deletion, which removed large parts of HMGCS2, occurred prior to the divergence of mastodons and the elephant species.

We further found that the remaining HMGCS2 sequence evolves under relaxed selection in cetaceans, pteropodids and Elephantimorpha (p<3e-3, Supplementary file 2). Together with the conserved genomic context, the lack of any evidence of a remaining functional HMGCS2 in unassembled reads and the validated gene-inactivating mutations, we conclude that the main ketogenesis enzyme is lost in three independent mammalian lineages. Finally, we considered the possibility that HMGCS1, the cytosolic HMG-CoA synthase, may compensate for HMGCS2 loss, which would require HMGCS1 to be localized in the mitochondria, where ketogenesis happens in other species. We found that the HMGCS1 protein of cetaceans, pteropodids and elephant does not possess a mitochondrial targeting domain. Furthermore, an analysis of available liver RNA-seq data from the minke whale and Egyptian fruit bat provides no indication of alternative or novel exons in HMGCS1 that could encode such a targeting signal. Thus, HMGCS1 does not seem to be capable of compensating for the loss of HMGCS2, suggesting that ketogenesis is lost in cetaceans, pteropodids and Elephantimorpha.

Next, we investigated whether the loss of HMGCS2 is associated with the loss of other enzymes in the ketogenesis pathway (Figure 1A). ACAT1 and HMGCL do not exhibit inactivating mutations in cetaceans, pteropodids and the elephant, likely because the respective enzymes are not only required for the production of ketone bodies but are also involved in leucine and isoleucine metabolism. In contrast to these two pleiotropic genes, BDH1 is only involved in converting acetoacetate into the ketone body d-β-hydroxybutyrate (Figure 1A). We found that BDH1 exhibits several inactivating mutations and evolved under relaxed selection in cetaceans and pteropodids (Figure 1—figure supplement 5, Supplementary file 2). Overall, this suggests that the loss of HMGCS2 is only associated with the loss of non-pleiotropic genes in the ketogenesis pathway.

The 59 other mammals, for which the genome assembly fully covered the HMGCS2 locus (Figure 1B), do not exhibit inactivating mutations in this gene. Consistent with the presence of a functional gene, we further estimated an average non-synonymous/synonymous (dN/dS) ratio of 0.16, which indicates that HMGCS2 evolves under strong purifying selection in other mammals.

The observation that HMGCS2 is well-conserved in the majority of mammals is consistent with ketogenesis being an important metabolic process. However, the recurrent loss of HMGCS2 raises the question of which energy source is used by the brain during fasting. Consistent with the loss of ketogenesis in cetaceans, bottlenose dolphins do not produce ketone bodies after fasting for 3 days but are nevertheless able to maintain high blood glucose levels over this entire period (Ridgway, 2013). It was suggested that dolphins maintain high glucose levels by synthesis of glucose from non-carbohydrates (gluconeogenesis), in particular from glucogenic amino acids that are abundant in their diet (Ridgway, 2013). This suggests that ketogenesis became dispensable in dolphins and that HMGCS2 was lost as a consequence of relaxed or no selection to maintain this gene. Similarly, the loss of ketogenesis in pteropodid fruit bats may be a consequence of the relatively constant availability of fruit year-round, which provides large quantities of glucose. This is in agreement with molecular dating, which estimates that the loss of HMGCS2 happened rather late in the lineage leading to the fruit bat clade and may even have occurred independently after the split of the frugivorous flying foxes and the Egyptian fruit bat (Figure 2A). Consistent with lack of ketone bodies as alternative fuel, Egyptian fruit bats that were fasted for more than 24 hours in captivity frequently died (van der Westhuyzen, 1978). Thus, like HMG-CoA synthase-2 deficient human individuals, these bats are sensitive to starvation. Hence, while ketogenesis may have been lost under ancestral conditions of constantly available, glucose-rich food, the loss of HMGCS2 may now represent a disadvantage, which will be of interest to ongoing conservation efforts for ecologically and economically important species in the pteropodid family. In contrast to cetaceans and fruit bats, little is known about how elephants respond to fasting; however, the following observation is consistent with the loss of ketogenesis. During musth, when elephant males experience longer periods of fasting and can lose 10% of their body weight, their blood becomes slightly more alkaline (Rasmussen and Perrin, 1999). This is contrary to an increased blood acidity that would be expected from an increasing production of acidic ketone bodies.

Figure 2 Download asset Open asset HMGCS2 loss and brain size evolution. (A) In pteropodids, molecular dating estimates that the loss of HMGCS2 happened 29–18 Mya and thus may overlap the split of the flying foxes and the Egyptian fruit bat. It is not possible to resolve whether gene loss happened before or after the split as HMGCS2 is completely deleted in the Egyptian fruit bat. While horseshoe bats and other insectivorous bat lineages have brains not larger than expected for their body size (encephalization quotient (EQ) <1), brain size has increased in the lineage leading to the fruit bats that have EQ values > 1 (Stephan et al., 1981). Thus, brain size expansion presumably predates the loss of ketogenesis. (B) HMGCS2 was already lost in the cetacean ancestor before the split of toothed and baleen whales ~ 36 Mya, as inferred from shared inactivating mutations in exons 1, 2 and 8. Molecular dating further estimates that the loss of this gene happened early on the cetacean branch 50–47 Mya. The cetacean ancestor had a brain slightly larger than expected for its body size with an EQ of 1.4. While EQ values increased and decreased in several cetacean lineages, brain size has greatly expanded in dolphins, reaching an EQ of 3.7 (Montgomery et al., 2013). Thus, brain size expansion in dolphins occurred after the loss of ketogenesis. (C) Early proboscids such as Moeritherium, an extinct lineage that split from other proboscids ~ 43 Mya, had brains about 20% of the size expected for a mammal of the same body size, and thus an EQ of 0.2 (Shoshani et al., 2006). Exact EQ values of Palaeomastodons are not known; however, fossils have a small braincase, which indicates a low EQ (Sanders et al., 2010; Benoit, 2015). In contrast, mastodons that diverged from elephants ~ 27 Mya had brains about twice as large as expected from their body size (EQ 2.2), similar to extant elephants (Shoshani et al., 2006). This suggests that brain size expansion happened in a period between 37 and 27 Mya. Molecular dating indicates that HMGCS2 loss happened between 45 and 42 Mya, suggesting that the loss of ketogenesis precedes brain size expansion in the elephant lineage. Divergence times of extinct proboscid lineages were taken from (Shoshani and Tassy, 2013) and (Rohland et al., 2007). Supporting Information. https://doi.org/10.7554/eLife.38906.009

Given the importance of ketogenesis to provide energy to the brain during starvation, it is noteworthy that species in all three HMGCS2-loss lineages generally have large relative brain sizes (Stephan et al., 1981; Boddy et al., 2012). For example, the encephalization quotient (EQ), measuring the ratio between the observed brain size and the size expected for a mammal of the same body weight, is 3.7 for the bottlenose dolphin (Montgomery et al., 2013). Compared to human, dolphins and elephants are also among the few mammals that have a higher degree of neocortex folding, a measure that positively correlates with neuron number (Manger et al., 2012; Lewitus et al., 2014). Furthermore, while powered flight imposes a constraint on body and brain size in bats, pteropodid fruit bats exhibit a well-developed visual brain system and have brains nearly twice as large as that of insectivorous vesper bats of equal body weight (Stephan et al., 1981). Species in all three lineages also exhibit cognitive behaviors that are regarded as a sign of intelligence, exemplified by vocal learning and, in dolphins and elephants, by complex social structures, tool use and self-recognition (Krützen et al., 2005; Foerder et al., 2011; Poole et al., 2005; Prat et al., 2015; Plotnik et al., 2006). Thus, the loss of HMGCS2 in independent large-brained species suggests that ketone bodies are not strictly required to fuel large mammalian brains during fasting.

Finally, the timing of HMGCS2 loss has implications for understanding the general preconditions for brain size expansion during the evolution of mammals. While the loss of HMGCS2 in pteropodids likely happened after brain size expansion in this lineage (Figure 2A), shared inactivating mutations show that HMGCS2 was already inactivated in the cetacean ancestor, and thus prior to a period of brain size expansion that resulted in the large brains of dolphins (Boddy et al., 2012; Montgomery et al., 2013) (Figure 2B). For the elephant lineage, we used molecular dating to estimate that HMGCS2 was lost around 45–42 Mya (Supplementary file 3). Thus, like in toothed whales, the loss of this gene likely occurred prior to the period that led to large relative brain sizes in modern elephants (Shoshani et al., 2006) (Figure 2C). Consequently, while ketogenesis was likely a crucial factor for brain size increase in humans (Cunnane and Crawford, 2003; Wang et al., 2014), the loss of ketogenesis has not prohibited drastic evolutionary brain size expansion in two other mammalian lineages.

In conclusion, we have identified three independent losses of HMGCS2 in placental mammals. While this may contribute to starvation sensitivity in fruit bats, cetaceans and elephants can withstand periods of fasting. Hence, alternative strategies to fuel large brains during fasting have evolved at least twice, revealing flexibility in the energy metabolism of mammals. Finally, the timing of HMGCS2 loss indicates that ketogenesis is not a universal precondition for the evolution of large mammalian brains. More generally, our results further highlight the potential of comparative gene analyses (Emerling and Springer, 2014; Meredith et al., 2009; Castro et al., 2014; Albalat and Cañestro, 2016; Lopes-Marques et al., 2017; Hecker et al., 2017; Gaudry et al., 2017; Sharma et al., 2018a; Sharma et al., 2018b; Meyer et al., 2018; Emerling et al., 2018) to reveal novel insights into the evolution of metabolic, physiological or morphological phenotypes.