Formatotrophs

Last updated

Formatotrophs are organisms that can assimilate formate or formic acid to use as a carbon source or for reducing power. [1] Some authors classify formatotrophs as one of the five trophic groups of methanogens, which also include hydrogenotrophs, acetotrophs, methylotrophs, and alcoholotrophs. [2] Formatotrophs have garnered attention for applications in biotechnology as part of a "formate bioeconomy" in which synthesized formate could be used as a nutrient for microoganisms. [3] [4] Formate can be electrochemically synthesized from CO2 and renewable energy, and formatotrophs may be genetically modified to enhance production of biochemical products to be used as biofuels. [5] Technical limitations in culturing formatotrophs have limited the discovery of natural formatotrophs and impeded research on their formate-metabolizing enzymes, which are of interest for applications in carbon sequestration and astrobiology.

Contents

Etymology

Formatotrophs gain their name from Latin formica, meaning "ant" [6] (formic acid having been named for its presence as a chemical defense in ants) and from Greek trophikos, meaning "pertaining to nourishment or food." [7]

Natural formatotrophs and their ecological role

Formatotrophs perform key metabolic processes through syntrophic relationships. In these relationships, formate is harvested for energy or carbon metabolism in diverse environments. These reactions are of particular importance in biogeochemical process related to carbon cycling and transfer of reducing agents such as hydrogen, acting as a keystone with abiotic formate. [8] Some methanogenic organisms convert formate into hydrogen and bicarbonate, providing hydrogen for other methanogens. Formate can be assimilated by formatotrophs in syntrophic associations with methanogens present during oxidation of formate; otherwise, formate oxidation would not be energetically sufficient to support growth and is thermodynamically disfavored (△G = +1.3 kJ /mol). So at least one methanogenic partner microorganism must be present to remove hydrogen. Some microorganisms, such as Desulfurococcus amylolyticus , are able to convert formate into carbon dioxide, acetate, citrate, and ethanol. [9]

Formate oxidation equation

Examples of natural formatotrophs

Confocal laser scanning microscopy images showing different microbial morphotypes in chimney samples from the Lost City hydrothermal field Confocal laser scanning microscopy images showing different microbial morphotyes in chimney samples.webp
Confocal laser scanning microscopy images showing different microbial morphotypes in chimney samples from the Lost City hydrothermal field

Recent metagenomic studies indicate widespread presence of potential formatotrophs in the Lost City hydrothermal field , an area of alkaline hydrothermal chimneys in the Atlantic Ocean, where serpentinization reactions of rock matter form calcium carbonate structures, hydrogen, methane, formate and other components. The exteriors of the chimneys are usually coated in biofilm. [14] [15] Harsh environmental conditions limit the development of microorganisms because chemical reactions keep concentrations of dissolved inorganic carbon low, indicating that carbon dioxide is not the primary carbon source. [16] Thus, initial studies hypothesized that formate was the main carbon source due the high concentrations of formate (36 to 158 μM) found in the field. [17] The metabolism of microbial communities in the hydrothermal field are largely unknown due to difficulties with laboratory isolation and culture. Metagenomic and genomic evidence supports the assimilation of formate in the Lost City chimneys as the main carbon source. [18] Metagenome assembled genomes (MAGs) determined that the most abundant genome was in the Methanosarcinales, which did not present metabolic pathways related with formate metabolism, and Chloroflexota (formerly Chloroflexi) MAGs were five times less abundant. [19]

The biofilm formed over the chimneys in the Lost City provides a glimpse of one possible carbon cycle that may have been in operation in the early days of life on earth, in an ecosystem based on geochemical reactions. [20] Similarly, studies of carbon assimilation strategies in ultrabasic groundwater explored the chemosynthesis microbial reactions in wells drilled into the ultramafic Coast Range Ophiolite Microbial Observatory (CROMO) and found that the microbial communities present in those aquifers use the products of serpentinization, including formate and methane, as carbon sources.

C. necator is one of the most well-studied aerobic formatotrophs. It can use carbon dioxide, formate, and hydrogen as carbon and energy sources and has a denitrification process. It is a model microorganism studied for production of polyhydroxyalkanoate, a compound of interest in bioplastic engineering. It has been gaining particular attention to be used as a chassis for metabolic engineering for the synthesis of alcohols and other bio-based compounds. A significant limitation for further engineering with this strain is the limited cell density that can be achieved in chemically defined media. [21]

Formate assimilation metabolic pathways

Natural metabolic pathways for formate assimilation include the reductive pentose phosphate pathway, serine pathway, reductive acetyl-CoA pathway in acetogens, reductive acetyl-CoA pathway in methanogens, and glycine pathway. The reductive pentose phosphate pathway uses 11 formate molecules to produce 1 acetyl-CoA, whereas the reductive acetyl-CoA pathway uses only 4. [22]

Table 1: Summary of metabolic pathways for formate assimilation[ citation needed ]
PathwayAmount of formate required to synthesize acetyl-CoA
Reductive pentose phosphate pathway11 formate molecules (4 for NADPH regeneration and 7 for ATP production)
Serine pathway7 formate molecules (1 assimilated, 3 to provide NADPH, and 3 for ATP generation)
Reductive acetyl-CoA pathway in acetogens and methanogens4 formate molecules (1 assimilated, 3 to provide NADPH)

Formatotrophs for carbon sequestration

The low ionization potential of formate makes it a good electron donor to provide reducing power to microorganisms. To sequester carbon, the production of formate by electrosynthesis — an abiotic process — could be integrated with a biotic process that uses it as a carbon source. Formatotrophic microorganism could feasibly be used to produce valuable chemicals. [11] Few formatotrophs have been studied, and thus most research into fermentation of formate is focused on the development of synthetic pathways or matching enzymes from different microorganism to create totally new pathways [23] and on the improvement of enzymes by directed evolution techniques. There are many pathways that could potentially assimilate formate for the production of biofuels, other biosynthetic products or single-cell protein, whether by using existing formate-fixing reactions or by designing novel enzymes. [24]

The US Department of Energy, US National Renewable Energy Laboratory and US Advanced Research Projects Agency–Energy have set up funding opportunities to improve formate assimilation with C. necator. [25]

Microorganism growing in serpentinization systems are of interest to understand carbon cycling between abiotic and biotic systems. These studies have further applications in astrobiology and studies of evolution and the emergence of life. [26]

Related Research Articles

<span class="mw-page-title-main">Metabolism</span> Set of chemical reactions in organisms

Metabolism is the set of life-sustaining chemical reactions in organisms. The three main functions of metabolism are: the conversion of the energy in food to energy available to run cellular processes; the conversion of food to building blocks of proteins, lipids, nucleic acids, and some carbohydrates; and the elimination of metabolic wastes. These enzyme-catalyzed reactions allow organisms to grow and reproduce, maintain their structures, and respond to their environments. The word metabolism can also refer to the sum of all chemical reactions that occur in living organisms, including digestion and the transportation of substances into and between different cells, in which case the above described set of reactions within the cells is called intermediary metabolism.

Primary nutritional groups are groups of organisms, divided in relation to the nutrition mode according to the sources of energy and carbon, needed for living, growth and reproduction. The sources of energy can be light or chemical compounds; the sources of carbon can be of organic or inorganic origin.

Methanogens are microorganisms that produce methane as a metabolic byproduct in hypoxic conditions. They belong to the domain Archaea and are members of the phylum Euryarchaeota. Methanogens are common in wetlands, where they are responsible for marsh gas, and can occur in the digestive tracts of animals including ruminants and humans, where they are responsible for the methane content of belching and flatulence. In marine sediments, the biological production of methane, termed methanogenesis, is generally confined to where sulfates are depleted below the top layers. Methanogens play an indispensable role in anaerobic wastewater treatments. Other methanogens are extremophiles, found in environments such as hot springs and submarine hydrothermal vents as well as in the "solid" rock of Earth's crust, kilometers below the surface in the deep biosphere.

<span class="mw-page-title-main">Biological carbon fixation</span> Series of interconnected biochemical reactions

Biological carbon fixation, or сarbon assimilation, is the process by which living organisms convert inorganic carbon to organic compounds. These organic compounds are then used to store energy and as structures for other biomolecules. Carbon is primarily fixed through photosynthesis, but some organisms use chemosynthesis in the absence of sunlight. Chemosynthesis is carbon fixation driven by chemical energy rather than from sunlight. 

<span class="mw-page-title-main">Sulfate-reducing microorganism</span> Microorganisms that "breathe" sulfates

Sulfate-reducing microorganisms (SRM) or sulfate-reducing prokaryotes (SRP) are a group composed of sulfate-reducing bacteria (SRB) and sulfate-reducing archaea (SRA), both of which can perform anaerobic respiration utilizing sulfate (SO2−
4) as terminal electron acceptor, reducing it to hydrogen sulfide (H2S). Therefore, these sulfidogenic microorganisms "breathe" sulfate rather than molecular oxygen (O2), which is the terminal electron acceptor reduced to water (H2O) in aerobic respiration.

Methylotrophs are a diverse group of microorganisms that can use reduced one-carbon compounds, such as methanol or methane, as the carbon source for their growth; and multi-carbon compounds that contain no carbon-carbon bonds, such as dimethyl ether and dimethylamine. This group of microorganisms also includes those capable of assimilating reduced one-carbon compounds by way of carbon dioxide using the ribulose bisphosphate pathway. These organisms should not be confused with methanogens which on the contrary produce methane as a by-product from various one-carbon compounds such as carbon dioxide. Some methylotrophs can degrade the greenhouse gas methane, and in this case they are called methanotrophs. The abundance, purity, and low price of methanol compared to commonly used sugars make methylotrophs competent organisms for production of amino acids, vitamins, recombinant proteins, single-cell proteins, co-enzymes and cytochromes.

An acetogen is a microorganism that generates acetate (CH3COO−) as an end product of anaerobic respiration or fermentation. However, this term is usually employed in a narrower sense only to those bacteria and archaea that perform anaerobic respiration and carbon fixation simultaneously through the reductive acetyl coenzyme A (acetyl-CoA) pathway (also known as the Wood-Ljungdahl pathway). These genuine acetogens are also known as "homoacetogens" and they can produce acetyl-CoA (and from that, in most cases, acetate as the end product) from two molecules of carbon dioxide (CO2) and four molecules of molecular hydrogen (H2). This process is known as acetogenesis, and is different from acetate fermentation, although both occur in the absence of molecular oxygen (O2) and produce acetate. Although previously thought that only bacteria are acetogens, some archaea can be considered to be acetogens.

<span class="mw-page-title-main">Mixed acid fermentation</span> Biochemical conversion of six-carbon sugars into acids in bacteria

In biochemistry, mixed acid fermentation is the metabolic process by which a six-carbon sugar is converted into a complex and variable mixture of acids. It is an anaerobic (non-oxygen-requiring) fermentation reaction that is common in bacteria. It is characteristic for members of the Enterobacteriaceae, a large family of Gram-negative bacteria that includes E. coli.

Microbial metabolism is the means by which a microbe obtains the energy and nutrients it needs to live and reproduce. Microbes use many different types of metabolic strategies and species can often be differentiated from each other based on metabolic characteristics. The specific metabolic properties of a microbe are the major factors in determining that microbe's ecological niche, and often allow for that microbe to be useful in industrial processes or responsible for biogeochemical cycles.

In biology, syntrophy, syntrophism, or cross-feeding is the cooperative interaction between at least two microbial species to degrade a single substrate. This type of biological interaction typically involves the transfer of one or more metabolic intermediates between two or more metabolically diverse microbial species living in close proximity to each other. Thus, syntrophy can be considered an obligatory interdependency and a mutualistic metabolism between different microbial species, wherein the growth of one partner depends on the nutrients, growth factors, or substrates provided by the other(s).

Hydrogen-oxidizing bacteria are a group of facultative autotrophs that can use hydrogen as an electron donor. They can be divided into aerobes and anaerobes. The former use hydrogen as an electron donor and oxygen as an acceptor while the latter use sulphate or nitrogen dioxide as electron acceptors. Species of both types have been isolated from a variety of environments, including fresh waters, sediments, soils, activated sludge, hot springs, hydrothermal vents and percolating water.

<span class="mw-page-title-main">Wood–Ljungdahl pathway</span> A set of biochemical reactions used by some bacteria

The Wood–Ljungdahl pathway is a set of biochemical reactions used by some bacteria. It is also known as the reductive acetyl-coenzyme A (acetyl-CoA) pathway. This pathway enables these organisms to use hydrogen as an electron donor, and carbon dioxide as an electron acceptor and as a building block for biosynthesis.

<i>Methanobacterium</i> Genus of archaea

Methanobacterium is a genus of the Methanobacteria class in the Archaea kingdom, which produce methane as a metabolic byproduct. Despite the name, this genus belongs not to the bacterial domain but the archaeal domain. Methanobacterium are nonmotile and live without oxygen, which is toxic to them, and they only inhabit anoxic environments.

Methanobrevibacter smithii is the predominant archaeon in the microbiota of the human gut. M. smithii has a coccobacillus shape. It plays an important role in the efficient digestion of polysaccharides by consuming the end products of bacterial fermentation. Methanobrevibacter smithii is a single-celled microorganism from the Archaea domain. M. smithii is a methanogen, and a hydrogenotroph that recycles the hydrogen by combining it with carbon dioxide to methane. The removal of hydrogen by M. smithii is thought to allow an increase in the extraction of energy from nutrients by shifting bacterial fermentation to more oxidized end products.

Sulfur is metabolized by all organisms, from bacteria and archaea to plants and animals. Sulfur can have an oxidation state from -2 to +6 and is reduced or oxidized by a diverse range of organisms. The element is present in proteins, sulfate esters of polysaccharides, steroids, phenols, and sulfur-containing coenzymes.

Sulfurimonas is a bacterial genus within the class of Campylobacterota, known for reducing nitrate, oxidizing both sulfur and hydrogen, and containing Group IV hydrogenases. This genus consists of four species: Sulfurimonas autorophica, Sulfurimonas denitrificans, Sulfurimonas gotlandica, and Sulfurimonas paralvinellae. The genus' name is derived from "sulfur" in Latin and "monas" from Greek, together meaning a “sulfur-oxidizing rod”. The size of the bacteria varies between about 1.5-2.5 μm in length and 0.5-1.0 μm in width. Members of the genus Sulfurimonas are found in a variety of different environments which include deep sea-vents, marine sediments, and terrestrial habitats. Their ability to survive in extreme conditions is attributed to multiple copies of one enzyme. Phylogenetic analysis suggests that members of the genus Sulfurimonas have limited dispersal ability and its speciation was affected by geographical isolation rather than hydrothermal composition. Deep ocean currents affect the dispersal of Sulfurimonas spp., influencing its speciation. As shown in the MLSA report of deep-sea hydrothermal vents Campylobacterota, Sulfurimonas has a higher dispersal capability compared with deep sea hydrothermal vent thermophiles, indicating allopatric speciation.

<i>Methanococcus maripaludis</i> Species of archaeon

Methanococcus maripaludis is a species of methanogenic archaea found in marine environments, predominantly salt marshes. M. maripaludis is a non-pathogenic, gram-negative, weakly motile, non-spore-forming, and strictly anaerobic mesophile. It is classified as a chemolithoautotroph. This archaeon has a pleomorphic coccoid-rod shape of 1.2 by 1.6 μm, in average size, and has many unique metabolic processes that aid in survival. M. maripaludis also has a sequenced genome consisting of around 1.7 Mbp with over 1,700 identified protein-coding genes. In ideal conditions, M. maripaludis grows quickly and can double every two hours.

Syntrophococcus sucromutans is a Gram-negative strictly anaerobic chemoorganotrophic Bacillota. These bacteria can be found forming small chains in the habitat where it was first isolated, the rumen of cows. It is the type strain of genus Syntrophococcus and it has an uncommon one-carbon metabolic pathway, forming acetate from formate as a product of sugar oxidation.

Methanocaldococcussp. FS406-22 is an archaea in the genus Methanocaldococcus. It is an anaerobic, piezophilic, diazotrophic, hyperthermophilic marine archaeon. This strain is notable for fixing nitrogen at the highest known temperature of nitrogen fixers recorded to date. The 16S rRNA gene of Methanocaldococcus sp. FS406-22, is almost 100% similar to that of Methanocaldococcus jannaschii, a non-nitrogen fixer.

<span class="mw-page-title-main">Hydrothermal vent microbial communities</span> Undersea unicellular organisms

The hydrothermal vent microbial community includes all unicellular organisms that live and reproduce in a chemically distinct area around hydrothermal vents. These include organisms in the microbial mat, free floating cells, or bacteria in an endosymbiotic relationship with animals. Chemolithoautotrophic bacteria derive nutrients and energy from the geological activity at Hydrothermal vents to fix carbon into organic forms. Viruses are also a part of the hydrothermal vent microbial community and their influence on the microbial ecology in these ecosystems is a burgeoning field of research.

References

  1. ↑ Fabarius JT, Wegat V, Roth A, Sieber V (April 2021). "Synthetic Methylotrophy in Yeasts: Towards a Circular Bioeconomy". Trends in Biotechnology. 39 (4): 348–358. doi: 10.1016/j.tibtech.2020.08.008 . PMID   33008643. S2CID   222143629.
  2. ↑ Barbera AC, Vymazal J, Maucieri C (2019). "Greenhouse gases formation and emission.". Encyclopedia of Ecology. Vol. 2 (Second ed.). pp. 329–333. doi:10.1016/B978-0-12-409548-9.10895-4. ISBN   9780444641304. S2CID   133696333.
  3. ↑ Bar-Even A (22 March 2016). "Synthetic metabolism and the formate bio-economy concept:addressing humanity's grand challenges". Imperial College London. Retrieved 2021-11-18.
  4. ↑ Kensy F (21 June 2021), Formate Bioeconomy – A new way for sustainable biomanufacturing? , retrieved 2021-11-18
  5. ↑ Al-Tamreh, Shaima A.; Ibrahim, Mohamed H.; El-Naas, Muftah H.; Vaes, Jan; Pant, Deepak; Benamor, Abdelbaki; Amhamed, Abdulkarem (September 2021). "Electroreduction of Carbon Dioxide into Formate: A Comprehensive Review". ChemElectroChem. 8 (17): 3207–3220. doi:10.1002/celc.202100438. ISSN   2196-0216.
  6. ↑ "Formica". Online Etymology Dictionary. Retrieved 2021-12-22.
  7. ↑ "Trophus". Online Etymology Dictionary. Retrieved 2021-12-22.
  8. ↑ Morris BE, Henneberger R, Huber H, Moissl-Eichinger C (May 2013). "Microbial syntrophy: interaction for the common good". FEMS Microbiology Reviews. 37 (3): 384–406. doi: 10.1111/1574-6976.12019 . PMID   23480449.
  9. 1 2 Ergal I, Reischl B, Hasibar B, Manoharan L, Zipperle A, Bochmann G, et al. (March 2020). "Formate Utilization by the Crenarchaeon Desulfurococcus amylolyticus". Microorganisms. 8 (3): 454. doi: 10.3390/microorganisms8030454 . PMC   7143981 . PMID   32210133.
  10. ↑ Carere CR, Hards K, Wigley K, Carman L, Houghton KM, Cook GM, Stott MB (2021). "Growth on Formic Acid Is Dependent on Intracellular pH Homeostasis for the Thermoacidophilic Methanotroph Methylacidiphilum sp. RTK17.1". Frontiers in Microbiology. 12: 651744. doi: 10.3389/fmicb.2021.651744 . PMC   8024496 . PMID   33841379.
  11. 1 2 Yishai O, Lindner SN, Gonzalez de la Cruz J, Tenenboim H, Bar-Even A (December 2016). "The formate bio-economy". Current Opinion in Chemical Biology. Energy Mechanistic Biology. 35: 1–9. doi:10.1016/j.cbpa.2016.07.005. PMID   27459678.
  12. ↑ Bae SS, Kim TW, Lee HS, Kwon KK, Kim YJ, Kim MS, et al. (January 2012). "H2 production from CO, formate or starch using the hyperthermophilic archaeon, Thermococcus onnurineus". Biotechnology Letters. 34 (1): 75–79. doi:10.1007/s10529-011-0732-3. PMID   21898132. S2CID   13057806.
  13. ↑ Crowther GJ, Kosály G, Lidstrom ME (July 2008). "Formate as the main branch point for methylotrophic metabolism in Methylobacterium extorquens AM1". Journal of Bacteriology. 190 (14): 5057–5062. doi:10.1128/JB.00228-08. PMC   2447001 . PMID   18502865.
  14. 1 2 Lecoeuvre A, Ménez B, Cannat M, Chavagnac V, Gérard E (March 2021). "Microbial ecology of the newly discovered serpentinite-hosted Old City hydrothermal field (southwest Indian ridge)". The ISME Journal. 15 (3): 818–832. Bibcode:2021ISMEJ..15..818L. doi:10.1038/s41396-020-00816-7. PMC   8027613 . PMID   33139872.
  15. ↑ Lang SQ, Brazelton WJ (February 2020). "Habitability of the marine serpentinite subsurface: a case study of the Lost City hydrothermal field". Philosophical Transactions. Series A, Mathematical, Physical, and Engineering Sciences. 378 (2165): 20180429. Bibcode:2020RSPTA.37880429L. doi:10.1098/rsta.2018.0429. PMC   7015304 . PMID   31902336.
  16. ↑ Lang SQ, Früh-Green GL, Bernasconi SM, Brazelton WJ, Schrenk MO, McGonigle JM (January 2018). "Deeply-sourced formate fuels sulfate reducers but not methanogens at Lost City hydrothermal field". Scientific Reports. 8 (1): 755. Bibcode:2018NatSR...8..755L. doi:10.1038/s41598-017-19002-5. PMC   5768773 . PMID   29335466.
  17. ↑ McGonigle JM, Lang SQ, Brazelton WJ (April 2020). "Genomic Evidence for Formate Metabolism by Chloroflexi as the Key to Unlocking Deep Carbon in Lost City Microbial Ecosystems". Applied and Environmental Microbiology. 86 (8): e02583–19. Bibcode:2020ApEnM..86E2583M. doi:10.1128/AEM.02583-19. PMC   7117926 . PMID   32033949.
  18. ↑ McGonigle JM, Lang SQ, Brazelton WJ (April 2020). "Genomic Evidence for Formate Metabolism by Chloroflexi as the Key to Unlocking Deep Carbon in Lost City Microbial Ecosystems". Applied and Environmental Microbiology. 86 (8). Bibcode:2020ApEnM..86E2583M. doi:10.1128/AEM.02583-19. PMC   7117926 . PMID   32033949.
  19. ↑ Singleton, Caitlin M.; Petriglieri, Francesca; Kristensen, Jannie M.; Kirkegaard, Rasmus H.; Michaelsen, Thomas Y.; Andersen, Martin H.; Kondrotaite, Zivile; Karst, Søren M.; Dueholm, Morten S.; Nielsen, Per H.; Albertsen, Mads (2021-03-31). "Connecting structure to function with the recovery of over 1000 high-quality metagenome-assembled genomes from activated sludge using long-read sequencing". Nature Communications. 12 (1): 2009. doi:10.1038/s41467-021-22203-2. ISSN   2041-1723. PMC   8012365 . PMID   33790294.
  20. ↑ "NOAA Ocean Explorer". oceanexplorer.noaa.gov. Retrieved 2021-11-18.
  21. ↑ Polès-Lahille, Aurore; Paillet, Margaux; Da Silva, Aurélie; Kadi, Nora; Basque, Eric; Thuet, Flavien; Balbuena, David; Ribault, Sébastien (2013-12-04). "How to assess chemically defined media and feeds from 9 suppliers on CHO cells producing mAb". BMC Proceedings. 7 (6): P100. doi: 10.1186/1753-6561-7-S6-P100 . ISSN   1753-6561.
  22. ↑ "5.12G: The Acetyl-CoA Pathway". Biology LibreTexts. 2017-05-09. Retrieved 2024-05-18.
  23. ↑ Mao W, Yuan Q, Qi H, Wang Z, Ma H, Chen T (August 2020). "Recent progress in metabolic engineering of microbial formate assimilation". Applied Microbiology and Biotechnology. 104 (16): 6905–6917. doi:10.1007/s00253-020-10725-6. PMID   32566995. S2CID   219958811.
  24. ↑ Bar-Even A (July 2016). "Formate Assimilation: The Metabolic Architecture of Natural and Synthetic Pathways". Biochemistry. 55 (28): 3851–3863. doi:10.1021/acs.biochem.6b00495. PMID   27348189.
  25. ↑ Johnson CW (2021-03-15). "BETO 2021 Peer Review - Improving Formate Upgrading by Cupriavidus Necator 2.3.2.111". OSTI   1772964.{{cite journal}}: Cite journal requires |journal= (help)
  26. ↑ Fani, Renato (September 2012). "The Origin and Evolution of Metabolic Pathways: Why and How did Primordial Cells Construct Metabolic Routes?". Evolution: Education and Outreach. 5 (3): 367–381. doi:10.1007/s12052-012-0439-5. ISSN   1936-6434.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.