Received: 16 February 2019 Revised: 7 May 2019 Accepted: 21 May 2019 DOI: 10.1111/gbb.12582 ORIGINAL ARTICLE Evolution of ASPM coding variation in apes and associations with brain structure in chimpanzees Sheel V. Singh1 | Nicky Staes1,2,3 | Elaine E. Guevara1 | Steven J. Schapiro4 | John J. Ely5 | William D. Hopkins4 | Chet C. Sherwood1 | Brenda J. Bradley1 1Center for the Advanced Study of Human Paleobiology, Department of Anthropology, Abstract The George Washington University, Studying genetic mechanisms underlying primate brain morphology can provide Washington, District of Columbia insight into the evolution of human brain structure and cognition. In humans, loss-of- 2Behavioural Ecology & Ecophysiology Group, Department of Biology, University of Antwerp, function mutations in the gene coding for ASPM (Abnormal Spindle Microtubule Antwerp, Belgium Assembly) have been associated with primary microcephaly, which is defined by a 3Centre for Research and Conservation, Royal Zoological Society of Antwerp, Antwerp, significantly reduced brain volume, intellectual disability and delayed development. Belgium However, less is known about the effects of common ASPM variation in humans and 4 Michael E. Keeling Center for Comparative other primates. In this study, we characterized the degree of coding variation at Medicine and Research, University of Texas MD Anderson Cancer Center, Bastrop, Texas ASPM in a large sample of chimpanzees (N = 241), and examined potential associa- 5MAEBIOS, Alamogordo, New Mexico tions between genotype and various measures of brain morphology. We identified and genotyped five non-synonymous polymorphisms in exons 3 (V588G), Correspondence Sheel Singh, Center for the Advanced Study of 18 (Q2772K, K2796E, C2811Y) and 27 (I3427V). Using T1-weighted magnetic reso- Human Paleobiology, The George Washington nance imaging of brains, we measured total brain volume, cerebral gray and white University, 800 22 St. NW, Suite 6000, Washington, DC 20052. matter volume, cerebral ventricular volume, and cortical surface area in the same Email: [email protected] chimpanzees. We found a potential association between ASPM V588G genotype and Funding information cerebral ventricular volume but not with the other measures. Additionally, we found George Washington University Luther Rice that chimpanzee, bonobo, and human lineages each independently show a signature Foundation; Office of Multidisciplinary Activities, Grant/Award Number: 1542848; of accelerated ASPM protein evolution. Overall, our results suggest the potential NSF INSPIRE, Grant/Award Number: SMA- effects of ASPM variation on cerebral cortical development, and emphasize the need 1542848; National Chimpanzee Brain Resource, Grant/Award Number: NIH for further functional studies. These results are the first evidence suggesting ASPM NS092988; James S. McDonnell Foundation, variation might play a role in shaping natural variation in brain structure in nonhuman Grant/Award Number: 220020293 primates. KEYWORDS apes, brain size, genetics, neurobiology, ventricular volume 1 | INTRODUCTION proximate mechanisms underlying human brain expansion, numerous studies have investigated evolutionary changes of specific genes The expansion of the cerebral cortex in relation to body size is a underlying the size and structure of the human cerebral cortex.7-11 remarkable feature that distinguishes humans from other primates.1,2 Of particular interest are genes that are associated with primary Studying genetic mechanisms underlying variation in primate brain microcephaly (Microcephaly Primary Hereditary or MCPH), a congeni- morphology can provide insight into the evolution of human brain tal disorder that involves a reduction in brain size that is more than size, neural reorganization and cognition.3-6 To elucidate the two standard deviations below the mean.12 In humans, MCPH is © 2019 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society Genes, Brain and Behavior. 2019;1–9. wileyonlinelibrary.com/journal/gbb 1 2 SINGH ET AL. strongly correlated with mild-to-moderate intellectual disability, but ASPM may be unique to the hominoid lineage and is suggested to no further neurological deficits or malformations are present.13-15 have been an important mechanism in the evolutionary history of ASPM (Abnormal Spindle Microtubule Assembly) is a protein- human brain size. coding gene located on chromosome 1q31.3 in humans that is associ- ASPM's evolution in modern humans seems to have been driven ated with MCPH5, one of the most frequently occurring forms of by positive selection, causing recently emerged ASPM haplotypes to MCPH.16,17 Yet, the extent to which ASPM affects natural variation in increase in frequency.29,30,33 It is possible that human haplogroup var- brain morphology remains contentious. Magnetic Resonance Imaging iants arose in Eurasia and are still in the process of dispersion. (MRI) data have shown that loss of function non-synonymous genetic Although ASPM shows strong signatures of adaptive evolution, studies mutations and single nucleotide variants (SNVs) in the protein-coding examining ASPM variation have yielded mixed and controversial region of ASPM are responsible for reduced brain volumes in results in humans, as significant effects are largely associated with humans18,19; however, other studies have also shown the oppo- microcephaly and not common DNA variants in the general site.15,20,21 Individuals with ASPM mutations have shown impairment population.18,21,23,34 of various cognitive and behavioral abilities,14,18,22 though the direct Despite the major interest in ASPM for its potential role in the impact of such deficits has ranged from mild to severe. In addition, evolution of the human brain, little research has been conducted adaptive evolution of ASPM has been associated with variation in IQ regarding within-species inter-individual genetic variation in other pri- in certain human studies.23 mates. Therefore, the aims of this study were to identify coding varia- Studies that measure neuroanatomical abnormalities associated tion in ASPM in chimpanzees (Pan troglodytes), one of the closest with ASPM loss-of-function mutations find that the degree of reduc- living relatives of humans, and to examine the potential association of tion differs among various brain regions. For example, while volume such variation with measure of brain structure. Additionally, we and surface area of many cortical regions are impacted in microceph- sought to reexamine signatures of positive selection in ASPM among aly, hippocampal volume and surrounding medial temporal lobe struc- hominoids based on newly available data that increase depth of cover- tures remain relatively preserved.18 The differential impact of ASPM age and now includes bonobos. on brain volumetric measures may be due to the gene's role in the regulation of neuronal migration. In fetuses, ASPM is preferentially 2 | MATERIAL AND METHODS expressed during cerebral cortical neurogenesis.14,24 In mice, expres- sion of the ASPM homolog is reduced postnatally, suggesting that the 2.1 | Subjects gene is expressed in neural progenitor cells which solely produce neu- rons, rather than proliferative pools that generate both neurons and This study used DNA samples for a total of 241 adult chimpanzees glia.14,25 In addition, studies in mice show that ASPM is also important (Pan troglodytes verus). All subjects were members of the colonies of for maintaining a cleavage plane orientation, which promotes symmet- apes residing at the Yerkes National Primate Research Center ric proliferative divisions of apical progenitors in the brain.26,27 This (YNPRC) (N = 50 females and 44 males, ages ranging 6-53 years old) role suggests that downregulation of the gene during neurogenesis and the Michael E. Keeling Center for Comparative Medicine and promotes asymmetric divisions, therefore limiting the expansion of Research of The University of Texas MD Anderson Cancer Center the neocortex in particular.26 These findings support the role of ASPM (KCCMR) (N = 84 females and 63 males, ages ranging from 8-51 years in embryonic cortical neural progenitor proliferation and suggest that old). American Psychological Association and National Institute of ASPM protein changes can contribute to variation in the development Health guidelines for the ethical use of chimpanzees in research were of the cerebral cortical morphology of the brain. adhered to during all aspects of this research. Evolutionary changes in the coding sequence of ASPM have also been shown to be associated with brain size evolution in nonhuman 2.2 | DNA extraction and genotyping primates and other mammals.28-31 For example, acceleration of non- synonymous substitutions in ASPM is significantly related to The reference genome Pan_tro_3.0 for chimpanzees is largely based on encephalization quotient (EQ) in cetaceans, suggesting that the gene asingleP. t. verus male, but includes additional lower-coverage genomic may have contributed to relative brain size expansion.31 Odontocete sequence data from seven other individuals.35,36 We used these com- lineages with increased EQ show significant evidence of positive bined data for eight individuals and five previously deposited sequences selection; however, absolute brain and body mass do not relate to from Genbank37 (Accession numbers: NM_001008994 XM_514079, ASPM selection rates.31 In addition, ASPM variation is related to neo- AY497016 AY365047, XM_016948364, XR_001715725, XM_ natal brain size in non-primate