Research Ideas and Outcomes :
Research Idea
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Corresponding author: Katherine H McLean (katherine.mclean@auckland.ac.nz)
Academic editor: Editorial Secretary
Received: 14 Jun 2023 | Accepted: 30 Apr 2024 | Published: 13 May 2024
© 2024 Katherine McLean
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
McLean K (2024) Raising the Neanderthal (molecules) from the dead: a proposal for in vivo resurrection of Neanderthal haemoglobin for the investigation of biochemical adaptations for cold tolerance. Research Ideas and Outcomes 10: e107983. https://doi.org/10.3897/rio.10.e107983
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Since the first discoveries of Neanderthal fossils, their derived characteristics, such as increased robusticity, have engaged researchers. Adaptation to cold environments has been hypothesised to explain such traits and this hypothesis has driven the majority of discourse. This proposal seeks to examine this hypothesis and locate evidence of Neanderthals being physiologically adapted to cold at the biomolecular level. Haemoglobin is a biomolecule that has been previously demonstrated to adapt to cold in some species, driven by the inhibition of the protein’s function by low temperatures. Neanderthal haemoglobin is extinct; however, using pre-sequenced genomic data, I propose to resurrect Neanderthal haemoglobin so I can examine the consequences of lowered temperature on its function. This project could potentially detect signs of cold adaptation in the Neanderthal globin genes and provide empirical evidence for the cold adaptation hypothesis.
Homo neanderthalensis, haemoglobin, molecular resurrection, adaptation, evolution
Neanderthals (Homo neanderthalensis) have long been hypothesised to have possessed thermoregulatory adaptations to their Ice Age environments due to traits such as skeletal robusticity and expanded ribcages (
I propose shifting the focus to molecular genetics. I theorise that, if Neanderthals were biologically adapted to the cold, they will possess inherited molecular-level adaptations. To locate potential molecular adaptations in Neanderthals and diagnose their evolutionary purpose, I plan to utilise a method that has not yet been exploited in palaeoanthropology: the functional analysis of inferred ancestral gene products. The resurrection and functional analysis of extinct genes can help link molecular adaptation events to historical environmental changes (
The Neanderthal is thus an ideal subject.
Haemoglobin is a strong candidate molecule for this study as it is highly adaptable to environmental alteration and functionally dependent on temperature (
My research into Neanderthal haemoglobin cold adaptation would first involve identifying the globin genes using contemporary human genomes as a comparison. Observed differences may be indicative of evolutionary processes. After noting where the Neanderthal sequences differ from those of Homo sapiens, I would ascertain if nucleotide changes were likely to have been driven by positive selection. The globin genes for both species would be subcloned into expression vectors and the products expressed in cell culture. The now tangible Neanderthal haemoglobin could then be functionally tested under various environmental conditions.
If my hypothesis is correct, then I might expect to see amino acid alterations in the Neanderthal globin genes that lower the effect of temperature on haemoglobin-oxygen affinity and facilitate the release of oxygen at cold temperatures. Functional testing of the expressed molecule at various temperatures may differentiate increased oxygen affinity specifically for thermoregulation from increased oxygenation for alternate purposes. Ultimately, I aim to generate novel evidence of Neanderthal thermoregulatory mechanisms for cold adaptation by uniting palaeoanthropology and molecular resurrection.
The proposed focus is situated within the broader context of hominin adaptability. It is therefore necessary to discuss the nature of molecular adaptation in genus Homo and order Primates, as this will be intimately related to how haemoglobin may adapt and express in Homo neanderthalensis. To proceed, one must develop detailed understandings of three core concepts: the selective pressures experienced by Neanderthals, the known effects of such pressures on the haemoglobin protein, and the limitations and abilities of genome sequencing and gene expression technologies.
Neanderthals shared swathes of Eurasia with Homo sapiens until approximately 40,000 years ago (
Neanderthals likely utilised behavioural adaptations to their new Arctic environment (
In contemporary humans, biological thermoregulatory adaptations are not restricted to morphology and physiology. Inherited molecular-level adaptations, such as those that help Inuit cope with low-carbohydrate diets (
Not all research aligns with the cold-adapted hypothesis (
Furthermore, the argument put forward by
One candidate I have identified as a plausible site for genetic evidence of cold adaptation in Neanderthals is the haemoglobin molecule. This is due to haemoglobin being:
The human globin genes are organised into two clusters located on separate chromosomes, the alpha-globin cluster and the beta-globin cluster (
Haemoglobin’s ability to offload oxygen to respiring cells is inhibited at lower temperatures (
Haemoglobin is a highly adaptable molecule. As the protein responsible for oxygen transport in almost all vertebrates (
Haemoglobin Adaptation in Order Primates. Haemoglobin adaptation has been observed in both humans and non-human primates. Over 60 human haemoglobin variants have amino acid replacements that increase oxygen affinity or blood concentration (
Most of these human haemoglobin adaptions are single-base point mutations, which can occur in short evolutionary timeframes (
Haemoglobin adaptation has also been described in a non-human primate. Researchers identified mutations in the haemoglobin sequences of Macaca fascicularis (
Haemoglobin Adaptation to Cold. Cold-adapted haemoglobin has been identified in various taxa, from fish to mammals. The haemoglobins of several Arctic mammals, including polar bears and reindeer, have been found to possess lowered enthalpy values, decreasing the effect of temperature change on haem-oxygen affinity (
In an alternative approach to the low-temperature oxygenation problem, polar fishes developed haemoglobins with increased oxygen-binding properties (
Whilst cold-adapted haemoglobin has not yet been identified in humans, signals of strong natural selection for other cold-relevant alleles, specifically those that limit hepatic fatty acid oxidation, have been found in Inuit populations (
The same evolutionary processes have influenced development throughout time and environmental change consistently leads to evolutionary innovation (
Ancient DNA (aDNA) refers to any DNA isolated from palaeo- or archaeological specimens (
The next step in palaeogenetics was to identify regions of potential evolutionary importance and analyse their functional consequences (
Research is now producing results that would have once been considered science fiction. Extinct gene products have been expressed from a range of species (see, for example,
A study by
Simply identifying differences between extinct genes and living analogues does not inform whether selection caused these differences. Discerning whether ancient differences were caused by positive directional selection is integral to linking potential adaptations to causative environmental changes. Neutral evolutionary theory must be excluded to claim that an observed mutation is adaptive (
Detecting positive selection can be achieved by searching for signatures left behind by selective sweeps (
Neanderthal adaptation, haemoglobin genomics, and molecular resurrection are disparate subjects. However, when brought together, a gap in the literature appears. While the expression of extinct genes and gene products has become increasingly common in the biological sciences, palaeoanthropologists have remained focused on reconstructing anatomy and energetics. Researchers have not yet resurrected extinct hominin gene products for any purpose, as the relevant technologies have only been recently developed—let alone for testing hypotheses about ancient evolutionary events. I plan to unite these subjects to test my hypotheses that: 1) Neanderthals were indeed a cold-adapted species; 2) this cold adaptation included inherited genetic traits; and 3) haemoglobin is one of the Neanderthal genetic products affected by this adaptation.
My proposed methods consist of seven steps:
I will obtain annotated copies of human globin gene sequences via a genomic database, most likely from the databases run by the United States National Center for Biotechnology Information (NCBI). I will obtain an annotated copy of the Neanderthal genome via the Neandertal Genome Project Browser, run by the Department of Evolutionary Genetics at the Max Planck Institute for Evolutionary Anthropology. The Neanderthal data will be a compilation of four primary specimens along with various fragments (
Due to the secondary nature of my data, I will not need to conduct extraction, amplification, or sequencing myself and, thus, will not need to apply for approval from my institution's relevant ethics committees. I will, however, need to verify that all human genetic information I use was previously obtained with appropriate ethical approval from equivalent institutions.
As I am working within an Aotearoa New Zealand context, my research must be performed in accordance with the 1940 Treaty of Waitangi (
I will next move to identify the Neanderthal alpha- and beta-globin gene sequences. These will consist of a series of loci that need to be individually located. Due to the strong similarities between human globin sequences and those of other great apes, along with the equivalence between Neanderthal and Homo sapiens chromosomes (
I will align the alpha- and beta-globin sequences for Neanderthals and modern humans and produce consensus sequences that represent the most frequent bases at each locus—eliminating sub-species-level variation. Finalised consensus sequences will be compared and I will note the position of any mutations. Since template damage can affect aDNA sequencing (
I will utilise statistical tests to ascertain whether observed nucleotide differences were likely driven by positive directional selection. Neutral evolutionary theory will function as my null hypothesis. To search for the signatures left behind by selective sweeps, I will utilise a binomial test to compute summary statistics on the likelihood of my observations occurring in the absence of selection. A variety of other approaches to identifying LD and positive selection may also be beneficial, including a Bayesian framework (
I will conduct empirical structure-function analyses to investigate whether observed changes alter protein behaviour in a manner consistent with selection. I will obtain amino-acid sequences for the globin genes of other mammalian species via a cross-organism database, align the sequences, and note shared, deleted, or substituted amino acids. This will establish whether Neanderthal alterations are common across species and identify conserved regions. Nucleotide alterations at points that fundamentally alter a protein’s physiochemical properties rarely occur by chance and, if they do, are promptly eliminated from populations if they provide no fitness benefit (
I will use the normal human adult haemoglobin-expression plasmid pHE2 to express modern human haemoglobin (
I will measure the oxygen binding equilibria for the expressed proteins while exposing them to stepwise increases in temperature at various pH levels. Analysis of measured cooperative binding will utilise the Hill–Langmuir equation, an essential tool for determining the degree of cooperativity (
Finally, molecular models will be constructed to locate potential structural causes of observed functional differences.
This identification of structural bases for observed functional differences is predicated upon finding differences between the Neanderthal and human haemoglobins. Regardless, this project would still be the first expression of an extinct hominin gene product. Even if I fail to identify thermoregulatory adaptations in Neanderthal haemoglobin, this work would be a stepping-stone for further research into palaeoanthropological gene resurrection.
As this proposal was created while the author was a post-graduate student, this work would not have been possible without the academic support of the Biological Anthropology Department at the University of Auckland | Waipapa Taumata Rau, specifically Prof. Judith Littleton, Dr Nicholas Malone, and Dr Heather Battles.