loading-tree.qmd

# Loading a Phylogenetic Tree {#sec-itol-load}

## Introduction

As noted in @sec-introduction, you will generally encounter phylogenetic trees as a plain text file in one of a handful of common file formats, such as:

- [`newick`](https://en.wikipedia.org/wiki/Newick_format), file ending `.new`, `.nwk`, `.newick`
- [`NEXUS`](https://en.wikipedia.org/wiki/Nexus_file), file ending `.nex`, `.nexus`
- [`phylip`](https://en.wikipedia.org/wiki/PHYLIP), file ending `.phy`, `.phylip`

Whichever software tool you use to read and visualise your tree, you will need to _load_ your data into that tool. In `iToL` you need to upload the data to the server. In `figtree` or `dendroscope` you would open the file in the application. Using `ggtree` in `R`, you would write code to open the file and run commands to visualise it.

::: { .callout-note }
In this workshop we will be using the online `iToL` service to visualise and interpret trees. This does not require you to install any software on your machine. The `iToL` service is available at the link below.

- [**`iToL`**](https://itol.embl.de/)
:::

![`iToL` landing page at [https://itol.embl.de](https://itol.embl.de).](assets/images/itol-landing.png){#fig-itol-landing width=80%}

## Load your tree data

Your tree data is in a file called `tree_newick.nwk`. This tree file describes a phylogeny with 13 samples, and it should be downloaded to your computer from the link below.

::: { .callout-note }
If you single-click on the link below, the tree file will open in your browser.

If you right-click on the link, you will see a _context menu_ with the option `Save link as…` (or similar, depending on your operating system/browser). This will allow you to save the file to your computer.
:::

- [**`tree_newick.nwk`**](https://raw.githubusercontent.com/sipbs-compbiol/BM211-Workshop-5/main/assets/trees/tree_newick.nwk)

::: { .callout-important title="Task" }
Download the tree file `tree_newick.nwk` to your computer.
:::

::: { .callout-tip collapse="true" }
## I need a hint!

Right-click on the link above and use `Save link as…` to save the file on your computer.
:::

::: { .callout-important title="Task" }
Upload the tree file `tree_newick.nwk` to `iToL`.
:::

::: { .callout-tip collapse="true" }
## I need a hint!

- Click on the `Upload` button (@fig-itol-upload-button) to see the tree file upload page
- Follow the instructions on the page

![`iTol` tree file upload button.](assets/images/itol-upload-button.png){#fig-itol-upload-button width=-80%}
:::

::: { .callout-warning collapse="true" }
## Help! I'm stuck!

- Click on the `Upload` button (@fig-itol-upload-button) to see the tree file upload page
- Enter a name for the tree in the `Tree name:` field.
- Click on the `Browse` button, and select your downloaded `tree_newick.nwk` file. The name of the file will appear next to the button.
- Click on the `Upload` button

![`iToL` tree file upload page.](assets/images/itol-tree-upload.png){#fig-itol-tree-upload width=80%}
:::

::: { .callout-caution collapse="true" }
## Check your result

After uploading the tree, `iTol` should present the default tree view, as in (@fig-itol-tree-newick-01).

![The default `iToL` view of the uploaded `tree_newick.nwk` tree.](assets/images/itol-tree-newick-01.png){#fig-itol-tree-newick-01 width=80%}
:::

## Understanding your tree

Considering the `newick` file contents you saw in @sec-introduction (and shown below), you might expect that the tree shows relationships between species represented by the letters `A`-`M` in that file:

```text
(((((((A:4,B:4):6,C:5):8,D:6):3,E:21):10,((F:4,G:12):14,H:8):13):13,((I:5,J:2):30,(K:11,L:11):2):17):4,M:56);
```

The tree `iTol` presents at first (@fig-tree-newick-01) shows these relationships. 

![Rectangular phylogram representation of the `tree_newick.nwk` file.](assets/images/tree-newick-01.png){#fig-tree-newick-01 width=80%}

- the tree is a set of _bifurcating branches_ that spread out from the most recent common ancestor (MRCA) and represent an **estimated** evolutionary history
- this is a _rooted tree_, so the most recent common ancestor (MRCA) is considered to be represented at the very far left of the tree, at its _root_
  - **in this tree, _ancestors_ are on the left, and _descendants_ are on the right**
- the horizontal dimension in this plot shows the amount of genetic change since the MRCA - less change towards the left-hand side, more towards the right
  - **this tree is a _phylogram_, which means that the lengths of the branches are meaningful and represent the amount of genetic change**

::: { .callout-note }
Branch lengths are usually drawn in units of _substitutions per site_ - the estimated total number of nucleotide substitutions, divided by (_normalised to_) the length of the sequence.

You may sometimes see alternative units being used, such as estimated time, or the percentage of sites that have changed.
:::

::: { .callout-important collapse="true" }
## Question 01

In @fig-tree-newick-01 which species shows the most genetic change since the MRCA?

- `A`
- `E`
- `G`
- `K`
:::

### Trees and time

The general relationship between the phylogram you have made and time is shown in @fig-tree-newick-02.

![`iToL` phylogram annotated with _root_, _leaves_, _bifurcations_, and the relationship between ancestors and descendents.](assets/images/tree-newick-02.png){#fig-tree-newick-02 width=80%}

As the tree represents genetic change, and the rate of genetic change may not be constant in all sequences or organisms, we can't immediately interpret branch lengths in terms of _elapsed time_. But, when the sequences/organisms used to build the tree are known to exist _now_, we can say that the leaves of the tree approximately represent the current date.

::: { .callout-note }
There are phylogenetic techniques that allow us to convert genetic distances into approximate times, but they are beyond the scope of this workshop.
:::

- **currently existing species (`A`-`M`) - the input for building the tree - are shown at the _leaves_** (or _leaf nodes_) of the tree, at the ends of the _branches_
- the branching events (_bifurcations_) show how evolutionary lineages split, as the amount of genetic change since the MRCA increases from left to right; they represent events in evolution where a population divides into two subgroups the go on to have distinct genetic histories
  - **in a biological context, this process is often referred to as _speciation_**
  - **the order of bifurcation since the MRCA can be interpreted as the order in which speciation events occurred**
- the amount of genetic change between branching events is represented by the length of the branch that separates them; the scale bar allows you to interpret the branch length quantitatively

::: { .callout-important collapse="true" }
## Question 02

@fig-tree-newick-01 implies that which speciation event was the earliest in time?

- the speciation separating `A` and `B`
- the speciation separating `B` and `C`
- the speciation separating `B` and `D`
- the speciation separating `C` and `E`
:::

### Ancestry and history

Phylogenetic trees represent patterns of shared ancestry and history between lineages. For instance, in @fig-tree-newick-03 common and unique ancestors of `A`, `B` and `C` are indicated, as are common and unique histories of `I` and `J`.

![`iToL` phylogram annotated with unique and shared ancestries and histories. Ancestors are single points on a branch (represented here as boxes), whereas histories are the entire set of ancestors represented by a branch.](assets/images/tree-newick-03.png){#fig-tree-newick-03 width=80%}

::: { .callout-important collapse="true" }
## Question 03

In @fig-tree-newick-01 which of the following accumulated the greatest amount of genetic change?

- the unique history of `E`
- the shared history of `I` and `J`
- the shared history of `I`, `J`, `K`, and `L`
- the unique history of `D`
:::

### Clades

A _clade_ is a grouping on the tree that includes a common ancestor and all of its descendants. We call a group with these properties _monophyletic_ (i.e. it comprises a single phylum). @fig-tree-newick-04 shows examples of clades in the `iToL` tree you generated. 

![`iToL` phylogram annotated with _clades_, groupings on the tree that include a common ancestor and all its descendants. Clades can be _nested_ inside each other as a hierarchy. Clade4 (`A`, `B`, `C`) is completely contained within Clade3 (`A`, `B`, `C`, `D`, `E`).](assets/images/tree-newick-04.png){#fig-tree-newick-04 width=80%}

::: { .callout-important collapse="true" }
## Question 04

In @fig-tree-newick-01 which of the following groups of leaf nodes form a _clade_?

1. (`J`, `K`, `L`)
2. (`F`, `G`, `H`)
3. (`D`, `E`)
4. (`K`, `L`)
:::

## Summary

::: { .callout-note title="Well Done!"}
After successfully working through this section you should be able to:

- upload a phylogenetic tree into `iToL`
- explain the meaning of branching events and branch lengths in a phylogenetic tree, and interpret the speciation events in a tree
- explain how a phylogenetic tree represents history and ancestry
- explain the concept of a _clade_ in phylogenetics, and identify one on a phylogenetic tree
:::

::: { .callout-important }
**Please answer the questions below in the formative quiz on MyPlace**

- [MyPlace formative quiz]({{< var myplace.quiz1 >}})
:::