Animal phylogeny

The animal kingdom comprises 34 large groups, or phyla

- species within a phylum share a basic body plan, or set of characters that allows them to be grouped

Relationships between phyla are difficult to determine, and remain controversial after 150 years of work

- lack of homologous characters between phyla

- simultaneous radiation of most groups during Cambrian

hard to tell which groups are sister taxa, or who came first

“Traditional” Animal Phylogeny

- acoelomates, pseudocoelomates branch off first- 2 deep divisions: protostomes + deuterostomes

i.e.,wrong

Who is related to whom?Mollusc Annelid Arthropod

share obvious segmentation

common ancestor, segmented?

Who is related to whom?Mollusc Annelid Arthropod

share trochophore larval stage

common ancestor,with trochophore?

Molecular Phylogeny re-wrote the book

Molluscs Annelids Arthropods Nematodes

ancestor withtrochophorelarva

ancestorwith acuticle

Lophotrochozoa Ecdysozoa

…Protostomes were divided into 2 major sub-groups

Based on analysis ofDNA, geneorder…

Animal phylogeny: re-written by genetics

Initial molecular phylogenetic studies compared genes that were highly conserved across phyla

Compare a gene that is so important, it does not change much even over long periods of time

- that way, creatures as different as a person and a sponge still have sequences that are similar enough to compare

Small subunit ribosomal RNA gene (18S rRNA) is strongly conserved by selection; permits comparisons across very distantly related organisms

Lophophorate Placement: 18S rRNA data

Halanych et al. 1995

Comparison of the highly-conserved 18S rRNA gene sequence showed that all 3 lophophorates group with protostomes, not with the deuterostomes as previously thought

Proposed clade within protostomes: the Lophotrochozoa

- lophophorates, molluscs + annelids; excludes arthropods

- no synapomorphy; either have a lophophore, or a trochophore larva

Long-Branch Attraction ProblemSome taxa have fast-evolving DNA

Often drop out at base of tree, clustered with:

(a) primitive animals

(b) other fast-evolvers, whom they may not be related to

Early molecular studies found that nematodes dropped out near the cnidarians, suggesting they were basal bilaterians

- this supported morphological analysis that said nematodes lacked a true coelom, so were basal to (more primitive than)

other Bilaterians

Long-Branch Attraction ProblemSome taxa have fast-evolving DNA

Often drop out at base of tree, clustered with:

(a) primitive animals

(b) other fast-evolvers, whom they may not be related to

This is an artifact (a false result) of how computer programs analyze DNA sequences, called long-branch attraction

- sequences that are fast-evolving give very long branches on trees, which tend to “attract” other long branches

sequences that are very different (fast mutating) get lumped together with other fast-evolving sequences

Long-Branch Attraction Problem

Turned out that most nematodes have very fast-mutating DNA, so give long branches and tend to fall out near the base of any tree

A slow-evolving nematode DNA sequence grouped with the arthropods, contradicting the older hypothesis that “pseudo- coelomates” were basal (= primitive) to other bilaterians

Ecdysozoa: clade of molting protostomes

Later analyses used 18S sequences to group molting pseudocoelomates such as nematodes & priapulids with the arthropods

- clade was named Ecdysozoa, to reflect the synapomorphy of molting a cuticle to grow

All subsequent analyses have supported this split within the protostomes

Traditional vs. Molecular Trees

Molecular studies found a hidden deep division in protostomes that conventional morphology had never suggested

Also moved lophophorates out of Deuterostomia

Morphology Molecular

Next-generation sequencingRecent animal phylogenies have relied on recent advances in sequencing technology and computational analysis

1) reverse-transcribe all mRNA in a tissue sample into DNA

- this gives you a cDNA library of all the protein-coding genes that were being expressed in that tissue, which is called the transcriptome, or an EST library

2) use 454 pyrosequencing (“next-generation sequencing”) to get partial sequences for thousands of genes

- you get many short sequences (~300 bp) which are hopefully overlapping, allowing computer to align them and infer the whole gene sequence from the pieces

- 400 million nucleotides can be sequenced in 10 hr

Phylogenomicstop represents the complete gene sequence, inferred fromthe aligned, overlapping pieces from individual runs

each sequence is one piece of this particular gene, assembled by the computer based on the parts where they overlap (i.e., where the sequences are identical)

PhylogenomicsRecent animal phylogenies have relied on recent advances in sequencing technology and computational analysis

3) computer algorithms are then used to piece together each gene sequence

4) the corresponding amino acid sequences are then used to compute the phylogenetic tree

PhylogenomicsProblems with next-generation sequencing approaches to phylogenomics:

- any given gene sequence may be incomplete- some genes may not be expressed in a given tissue,

so they will not get sequenced- cost limits how many runs you can do, hence how many sequences you can obtain for a given species

lots of missing data in the final dataset: the sequence ofany particular gene may be available for only some of the species you are trying to put into a phylogeny

PhylogenomicsProblems with next-generation sequencing approaches to phylogenomics:

- contamination from symbionts or food

- determining whether copies of a gene from different taxa are orthologs, meaning copies of the same gene and not

a related but different gene

- some genes exits as families of similar genes, related by decent from one ancestral gene

Phylogenomicsmade-up example: say animals have Actin 1, Actin 2, and Actin 3 genes, descended from one ancestral Actin gene

ancestralActin gene

actin actin actin 1 2 3

gene duplication:3 versions of actinwere present in ancestral mollusc, with similar but different amino acids

(present in 1st bilaterian)

GAFLSM.. GAFGSW.. TALLMM..

GAFLSM..

ancestralamino acids

red letters = amino acids that changed by mutation since the 3 versions appeared in the ancestral mollusc

Phylogenomicsmade-up example: say there’s Actin 1, Actin 2, and Actin 3 genes in animals, descended from an ancestral Actin gene

chitonsquidclamsnail

GAFLSM.. MAFGSW.. TALLMM..

GAFLSM.. MAFGSW.. TALLMM..

AAFPMM.. GWFGSP.. KRLLMY..

AAFPMM.. GAFLSP.. KRLLMQ..

3 “Actin” genes inancestral mollusc

actin actin actin 1 2 3GAFLSM.. GAFGSW.. TALLMM..

diverge over timein each lineage

related lineages should have more similar amino acids for each gene ortholog (copy of same original version)

clam

snail

squid

chiton

PhylogenomicsDetermining whether copies of a gene are orthologs

- what you DON’T want: to align the copies of Actin 1 from squid, clam and chiton with Actin 2 from chiton !!

GAFLSM.. MAFGSW.. TALLMM..

GAFLSM.. MAFGSW.. TALLMM..

AAFPMM.. GWFGSP.. KRLLMY..

AAFPMM.. GAFLSP.. KRLLMQ..

actin actin actin 1 2 3

this is a paralog of actin 1 gene: a divergent copy that exists alongside actin 1 in the genome

chitonsquidclamsnail

Phylogenomics

GAFLSM..

GAFLSM..

AAFPMM..

GAFLSP..

“actin 1” (or so you think..)

reallyactin 2!

chitonsquidclamsnail chiton

squid

snail

clam

at these 3 positions, “snail” now looksmore related to chiton + squid becauseit has the same amino acids as they do!

false information

wrong phylogeny

Determining whether copies of a gene are orthologs

- what you DON’T want: to align the copies of Actin 1 from squid, clam and chiton with Actin 2 from chiton !!

Hejnol et al. 2009

Most recent animal phylogenyused 1,487 genes

> 270,000 amino acid positions were analyzed

complete data set could not even be fully analyzed due to requirement for huge amount of computer time

- reduced 844-gene dataset used to test stability of proposed relationships

Most recent animal phylogenyused 1,487 genes

- each gene only had to be present in 18 of the

94 included species (lots of missing data)

- phoronid: only 2 genes used in analysis!

indicates that ctenophores are most basal animal – even more distant than sponges !?!

Do you buy it?

Why or why not?

Are you surprised the position of phoronids is still unknown, if only 2 genes were used to place Phoronis in this tree?

The rules of what makes a grouping “significant” (meaning we can really trust it is correct) state that you need bootstrap support of over 70%

These are the numbers in black to the left of a node = 100%

Of the major clades that group many phyla together (shaded boxes, or names on the left of the tree), in which can we be really confident?

*

Based on the rules of what makes a grouping count as “significant” (meaning we can really trust it), only the following clades are secure:

- Bilateria

- Protostomia

- Deuterostomia

None of the other named clades of phyla I have been using were “significantly” supported

Does it surprise you that after 100 years of effort, in the age of whole-genome sequencing, we still do not really know what phyla are sister to what other phyla??

What could be done to try to better resolve the relationships among phyla? What kinds of things can you think of that would help to improve this tree either in terms of data or methods?

Taxonomic rank “phylum” is used to lump animals into groups based on body plan (arrangement of morphological traits)

Most phyla are pretty different from each other; which phyla are sister groups remains controversial

Body plan is the product of development, when genetic information is converted into tissues/organs, relative position, numbers, shape...

- why are there only a handful of different body plans?

“Why should there be so much variety and so little real novelty?”- Darwin, 1872

Body Plan evolution

Body Plan evolution

Holland (1998) proposed 6 major developmental transitions during the evolution of the Animal Kingdom

- meaning, times when big changes in developmental control mechanisms resulted in major changes to body plan

- changes in development = differences in control genes (Hox) and genes they boss around (whose expression they control)

1 multicellularity

2 symmetry

3 bilateral, 3 germ layers

4 axis flip

5 6

Transition 1 - origin of multicellularity

- cell layers, cell adhesion, spatially controlled differentiation

- genes encoding cadherins, collagen, lectin proteins- transcription factors controlling cell differentiation

- in fact, sponge homeobox genes correspond to genes in higher animals controling cell differentiation, not spatial organization

Transition 2 - origin of symmetry + tissues

- tissues (2 or more cell types working together) - nerves (genes for ion channels, neurotransmitters, etc) - spatial information: body axis formation

Body Plan evolution

1 multicellularity

2 symmetry

3 bilateral, 3 germ layers

4 axis flip

5 6

Transition 3 - origin of bilateral symmetry + 3 germ layers

- nerve chord (running down belly, or down back)

- complete digestive system

- 3 germ layers (ectoderm, endoderm, mesoderm)

- Bilateral symmetry, meaning several body axes

anterior-posterior (head tail)

dorsal-ventral (back front)

left-right

proximal-distal (near the center out toward tips)

Body Plan evolution

Body Plan evolution

Duplication of ancestral gene cluster Hox genes (ectoderm) and ParaHox genes (endoderm), needed for 3-layer embryo with a complete gut

This gene duplication may have spurred Cambrian Explosion, when all modern phyla appear at about same time in fossils

Hox genes originally patterned ectoderm of triploblast phyla

ParaHox gene cluster: Gsx --- Xlox --- Cdx

Gsx affects anterior end of gut, near mouth

Xlox patterns middle of complete gut (pancreas)

Cdx patterns the end of gut, near anus

endodermpatterning

a) duplication of ancestral Hox-like gene into a cluster of related genes (happened before step #2, cnidarians)

b) duplication of ancestral cluster of Hox-like genes (now 2 sets)

c) divergence of double cluster into 2 different clusters: 1) ParaHox genes, for patterning endoderm

2) Hox genes, controlling ectoderm

ParaHox

Hox

Gsx Xlox Cdx

Happened at step #3, before most animal phyla diverged

(head) (tail)

(genes diverge over time)

Transition 4 - dorsal-ventral axis inverted in protostomes

- front-to-back axis flipped in ancestor of protostomes such that they have nerve chord on ventral side, not dorsal

worm you

gut gut

nervechord

nerve chord (backbone)dorsal

ventral

Body Plan evolution

Body Plan evolution

Transition 5 - origin of the vertebrates

- migrating neural crest cells, key to development of our complex central nervous systems

- new cell types (i.e., osteoblasts that build bones)

- tetraploidy (= 4 copies) of Hox cluster: double-duplication

Transition 6 - after hagfish, rest of vertebrates got:

- wandering mesoderm cells

- 2 pairs of appendages (tetrapods)

- cranial arches become jaws

Body Plan evolution

Transitions 5 & 6 involved major gene duplication events

- whole groups of genes were duplicated via tetraploidy of the genome (four copies of everything!)

- some genes copies were lost after duplication

- other copies, especially of developmental genes, were kept

gene duplication produces new master control genes that can take on new roles, producing changes in body plans

step #5: double-duplication of Hox cluster in vertebrate ancestor

(fly)

mouse

ParaHox

Hox

Gsx Xlox Cdx

1st duplication

2nd duplication