1. 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

2. “Traditional” Animal Phylogeny - acoelomates, pseudocoelomates branch off first - 2 deep divisions: protostomes + deuterostomes i.e., wrong

3. Who is related to whom? MolluscAnnelidArthropod share obvious segmentation common ancestor, segmented?

4. Who is related to whom? MolluscAnnelidArthropod share trochophore larval stage common ancestor, with trochophore?

5. Molecular Phylogeny re-wrote the book Molluscs Annelids Arthropods Nematodes ancestor with trochophore larva ancestor with a cuticle Lophotrochozoa Ecdysozoa …Protostomes were divided into 2 major sub-groups Based on analysis of DNA, gene order…

6. 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

7. 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

8. Long-Branch Attraction Problem Some 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

9. Long-Branch Attraction Problem Some 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

10. 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

11. 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

12. 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

13. Next-generation sequencing Recent 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

14. Phylogenomics top represents the complete gene sequence, inferred from the 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)

15. Phylogenomics Recent 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

16. Phylogenomics Problems 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 of any particular gene may be available for only some of the species you are trying to put into a phylogeny

17. Phylogenomics Problems 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

18. Phylogenomics made-up example: say animals have Actin 1, Actin 2, and Actin 3 genes, descended from one ancestral Actin gene ancestral Actin gene actin actin actin 1 2 3 gene duplication: 3 versions of actin were present in ancestral mollusc, with similar but different amino acids (present in 1 st bilaterian) GAFLSM.. GAFGSW.. TALLMM.. GAFLSM.. ancestral amino acids red letters = amino acids that changed by mutation since the 3 versions appeared in the ancestral mollusc

19. Phylogenomics made-up example: say there’s Actin 1, Actin 2, and Actin 3 genes in animals, descended from an ancestral Actin gene chiton squid clam snail GAFLSM.. MAFGSW.. TALLMM.. AAFPMM.. GWFGSP.. KRLLMY.. AAFPMM.. GAFLSP.. KRLLMQ.. 3 “Actin” genes in ancestral mollusc actin actin actin 1 2 3 GAFLSM.. GAFGSW.. TALLMM.. diverge over time in each lineage related lineages should have more similar amino acids for each gene ortholog (copy of same original version) clam snail squid chiton

20. Phylogenomics 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 !! 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 chiton squid clam snail

21. Phylogenomics GAFLSM.. AAFPMM.. GAFLSP.. “actin 1” (or so you think..) really actin 2! chiton squid clam snail chiton squid snail clam at these 3 positions, “snail” now looks more related to chiton + squid because it 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 !!

22. Hejnol et al. 2009 Most recent animal phylogeny used 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

23. Most recent animal phylogeny used 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!

24. 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?

25. 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? *

26. 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

27. 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?

28. 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

29. 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)

30. 1 multicellularity 2 symmetry 3 bilateral, 3 germ layers 4 axis flip 5 6

31. 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

32. 1 multicellularity 2 symmetry 3 bilateral, 3 germ layers 4 axis flip 5 6

33. 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

34. 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 endoderm patterning

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

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

37. 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)

38. 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 nerve chord nerve chord (backbone) dorsal ventral Body Plan evolution

39. 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

40. 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

41. step #5: double-duplication of Hox cluster in vertebrate ancestor (fly) mouse ParaHox Hox Gsx Xlox Cdx 1 st duplication 2 nd duplication