Answers.Body: <p>The exact answer to your question is bacteria do not have a mitochondrion, therefore <em>cox1</em> is not a universal gene combining both Archaebacteria and bacteria. In contrast 16S is universal for these two kingdoms AND it is also single copy. Moreover, bacteria have diverse tropisms beyond the Krebs or Calvin cycles. Extensive studies have been performed on 16S and it provides an excellent taxonomic target in bacteria.</p>
  <p>18S is a problematic target in eukaryotes because it can paralogous and that is not an ideal target in taxonomic studies, but has nicely conserved primer site: thus single copy genes are required/preferred. In addition, 18S is prone to indels (insertions/deletions) and these are difficult to model, align and understand the underlying molecular mechanism or selection advantage.</p>
  <p>Every animal on earth has a mitochondrion and therefore <em>cox1</em>. Mitochondrion genes are guaranteed to be single copy in higher eukaryotes and <em>cox1</em> has been extensively sequenced throughout metazoans. Thereby barcoding is appropriate because there's an exact reference to identify a given species is usually available. It has been extremely successful in insects which are the most biodiverse and species rich taxonomic Class on earth: there are more insects than all other higher eukaryotic species combined.</p>
  <hr />
  <p>In answer to the comments ...</p>
  <ul>
  <li><p>Whether mitochondrion were originally bacteria is a very difficult question and remains a theory (compartmentalisation theory - very old). I have not seen any rigorous phylogenetic support for this.</p>
  </li>
  <li><p><em>cox1</em> (COI) or <em>cox2</em> (COI) is not universal to bacteria, e.g. <a href="https://www.sciencedirect.com/science/article/pii/S0005272820301547" rel="nofollow noreferrer">here</a>. Its primary use is for aerobic organisms (oxygen consuming). Thus <em>cox1</em> has a strong Krebs cycle association but there are a multitude of anaerobic pathways.</p>
  </li>
  </ul>
  <p>Anaerobic bacteria don't necessarily need <em>cox1</em>/<em>cox2</em> and there are a lot of obligate anaerobic bacteria in addition to non-obligatory anaerobic bacteria (which can do both).</p>
  <p>Marine metazoan are obligate aerobic organisms.</p>
  <ul>
  <li><p>There is no reason to suppose bacteria  are strictly  single copy <em>cox1</em>. mtDNA is tiny and hence copy number heavily constrained, bacterial genomes by contrast are much larger and could easily accommodate duplication events.</p>
  </li>
  <li><p>There isn't much work on bacterial <em>cox1</em>, it has been pursued to attempt to augment antibiotic therapy. <a href="https://www.sciencedirect.com/science/article/pii/S0005272820301547" rel="nofollow noreferrer">Degli Esposti (2020)</a> is the closest I know to a comprehensive discussion on the bacterial <em>cox</em> genes.</p>
  </li>
  </ul>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2023-02-22T23:00:52.753Z
Answers.Text: Thanks for your answer. A few questions:
  • I know prokaryotes don't have mitochondria, but that shouldn't make a difference since mitochondria are descendants of bacteria, right? As far as I understand, both eukaryotes, bacteria and archaea have the COI gene.
  • What do you mean by "diverse tropisms beyond the Krebs or Calvin cycles" and how does it matter?
  • I had not considered paralogy, but now that you bring it up I understand that it must be problematic to have different variants of the same marker gene in the same individual. But do paralogues of COI really exist in bacteria/archaea?
Answers.UserDisplayName: Joel
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2023-02-22T23:07:26.950Z
Answers.Text: I'm primarily interested in marine metazoan diversity, so COI is what I'm being told to use. But I just fancied what a lovely story it would be if those universal COI primers could be designed to work on prokaryotes as well, and then it got me trying to find out why not. If you know any paper that discusses this, I'd be happy for it!
Answers.UserDisplayName: Joel
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2023-02-23T00:40:23.690Z
Answers.Text: @Joel addressed in comments.
Answers.UserDisplayName: M__
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2023-02-23T14:27:29.020Z
Answers.Text: Thank you @M__. You bring back parts of my biology education that I'd forgotten, and now it all makes sense. If I'll do prokaryotic metabarcoding, I can now rest assured that 16S is the correct choice. But I still hope for someone to find a universal marker gene that does work for eukaryotes and prokaryotes alike :)
Answers.UserDisplayName: Joel
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2023-02-22T15:00:32.380Z
Answers.LastActivityDate: 2023-02-23T09:32:46.120Z
Answers.LastEditDate: 2023-02-23T09:32:46.120Z
Answers.UserDisplayName: M__
Body: <p>I'm a beginner in bioinformatics working with environmental metabarcoding, and while reading up on different marker genes, I understand that COI is preferably used for animals thanks to its higher variability over 18S (although it depends on which taxonomic resolution fits best and which reference data is available). What I don't understand though, and can't find an answer to, is why COI metabarcoding isn't used for bacteria and archaea as well. Why is 16S preferred, although bacteria and archaea clearly have the COI gene? Is it not variable enough? Or are the primer binding sites too variable? Something else?</p>
  
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2023-02-22T14:49:33.460Z
Comments.Text: Hi @Joel welcome to the site. This is a reasonable question and I'm happy to answer it, however could you state the taxonomic groups in your study?
Comments.UserDisplayName: M__
Tags: <phylogenetics><phylogeny><barcode>
Title: Why is COI metabarcoding not used for prokaryotes?
ThreadText: # Why is COI metabarcoding not used for prokaryotes?
  
  (Asked by: username_0 on 2023-02-22)
  
  I'm a beginner in bioinformatics working with environmental metabarcoding, and while reading up on different marker genes, I understand that COI is preferably used for animals thanks to its higher variability over 18S (although it depends on which taxonomic resolution fits best and which reference data is available). What I don't understand though, and can't find an answer to, is why COI metabarcoding isn't used for bacteria and archaea as well. Why is 16S preferred, although bacteria and archaea clearly have the COI gene? Is it not variable enough? Or are the primer binding sites too variable? Something else?
  
  **Tags:** phylogenetics, phylogeny, barcode
  
  > **Comments:**
  > **username_1**: Hi @username_0 welcome to the site. This is a reasonable question and I'm happy to answer it, however could you state the taxonomic groups in your study?  
  
  ---
  
  ## 1 answers
  
  ### Answer by username_1 on 2023-02-22. Score: 3 | Accepted answer
  
  The exact answer to your question is bacteria do not have a mitochondrion, therefore *cox1* is not a universal gene combining both Archaebacteria and bacteria. In contrast 16S is universal for these two kingdoms AND it is also single copy. Moreover, bacteria have diverse tropisms beyond the Krebs or Calvin cycles. Extensive studies have been performed on 16S and it provides an excellent taxonomic target in bacteria.
  
  18S is a problematic target in eukaryotes because it can paralogous and that is not an ideal target in taxonomic studies, but has nicely conserved primer site: thus single copy genes are required/preferred. In addition, 18S is prone to indels (insertions/deletions) and these are difficult to model, align and understand the underlying molecular mechanism or selection advantage.
  
  Every animal on earth has a mitochondrion and therefore *cox1*. Mitochondrion genes are guaranteed to be single copy in higher eukaryotes and *cox1* has been extensively sequenced throughout metazoans. Thereby barcoding is appropriate because there's an exact reference to identify a given species is usually available. It has been extremely successful in insects which are the most biodiverse and species rich taxonomic Class on earth: there are more insects than all other higher eukaryotic species combined.
  
  ---
  
  In answer to the comments ...
  
  * Whether mitochondrion were originally bacteria is a very difficult question and remains a theory (compartmentalisation theory - very old). I have not seen any rigorous phylogenetic support for this.
  * *cox1* (COI) or *cox2* (COI) is not universal to bacteria, e.g. [here](https://www.sciencedirect.com/science/article/pii/S0005272820301547). Its primary use is for aerobic organisms (oxygen consuming). Thus *cox1* has a strong Krebs cycle association but there are a multitude of anaerobic pathways.
  
  Anaerobic bacteria don't necessarily need *cox1*/*cox2* and there are a lot of obligate anaerobic bacteria in addition to non-obligatory anaerobic bacteria (which can do both).
  
  Marine metazoan are obligate aerobic organisms.
  
  * There is no reason to suppose bacteria are strictly single copy *cox1*. mtDNA is tiny and hence copy number heavily constrained, bacterial genomes by contrast are much larger and could easily accommodate duplication events.
  * There isn't much work on bacterial *cox1*, it has been pursued to attempt to augment antibiotic therapy. [Degli Esposti (2020)](https://www.sciencedirect.com/science/article/pii/S0005272820301547) is the closest I know to a comprehensive discussion on the bacterial *cox* genes.
  
  > **Comments:**
  > **username_0**: Thanks for your answer. A few questions: • I know prokaryotes don't have mitochondria, but that shouldn't make a difference since mitochondria are descendants of bacteria, right? As far as I understand, both eukaryotes, bacteria and archaea have the COI gene. • What do you mean by "diverse tropisms beyond the Krebs or Calvin cycles" and how does it matter? • I had not considered paralogy, but now that you bring it up I understand that it must be problematic to have different variants of the same marker gene in the same individual. But do paralogues of COI really exist in bacteria/archaea?  
  > **username_0**: I'm primarily interested in marine metazoan diversity, so COI is what I'm being told to use. But I just fancied what a lovely story it would be if those universal COI primers could be designed to work on prokaryotes as well, and then it got me trying to find out why not. If you know any paper that discusses this, I'd be happy for it!  
  > **username_1**: @username_0 addressed in comments.  
  > **username_0**: Thank you @username_1. You bring back parts of my biology education that I'd forgotten, and now it all makes sense. If I'll do prokaryotic metabarcoding, I can now rest assured that 16S is the correct choice. But I still hope for someone to find a universal marker gene that does work for eukaryotes and prokaryotes alike :)  
  
  ---
  
  