1. Diversification - non-vertebrates
Contributed papers - Oklahoma B
Good afternoon everybody. Welcome to this session on non-invertebrate diversification. My name is Vincent Smith and I’ll be your chair for this afternoon. I think we have a very interesting line-up of 6 talks, which will bring us through to 5pm. Just before we start a quick reminder to the speakers. I will alert you when three minutes are remaining by indicating like this (indicate), and then again if necessary at 1 minute by standing up.
3.30 PM PM
Chair - Vince Smith

2. A hitchhikers guide to the Galápagos
Co-phylogeography of Galápagos mockingbirds and their parasites
Its now 3.30 so we should get started with our first speaker which is myself in a talk co-authored by Jan Stefka, and our talk title is “A hitchhikers guide to the Galapagos: the cophylogeny of Galapagos mockingbirds and their ectoparasites”. Jan has been doing a Marie Curie Postdoctoral fellowship with me for the past 2 years and I should point out that most of the work I’m presenting today is his.
Jan Stefka
& Vince Smith

3. The Galapágos archipelago
BALTRA
South Seymour
BARTOLOMÉ
Bartholomew
ESPAÑOLA
Hood
FERNANDINA
Narborough
FLOREANA
Charles
GENOVESA
Tower
ISABELA
Albemarle
MARCHENA
Bindloe
NORTH SEYMOUR
PINZÓN
Duncan
PINTA
Abingdon
RÁBIDA
Jervis
SAN CRISTÓBAL
Chatham
SANTA CRUZ
Indefatigable
SANTA FÉ
Barrington
SANTIAGO
San Salvador
c. 1-2 MYA
7.000 YA
Further SE
up to 80 MYA?
The Galapagos Islands at some level are probably familiar to everyone in this room and barley need an introduction. They are an archipelago of volcanic islands distributed around the equator in the Pacific Ocean, approximately 500 nautical miles off the west of continental Ecuador. The group consists of 15 main islands, 3 smaller islands, and 107 rocks and islets, which are spread out over a distance of 220 km (137 mi).
The archipelago is located on the Nazca Plate, which is moving east/southeast, diving under the South American Plate. It is also atop the Galapagos hotspot that is creating new volcanic islands. Consequently the islands are geologically young, the youngest being Isabela and Fernandina in the NW which are still being formed. The oldest island is thought to have formed between 5 million and 10 million years ago.
c. 2-3 MYA
5-9 MYA
100
(km) 60
(m)

4. The progression rule
Patterns of colonization & diversification linked to geological history
Youngest
Islands
Oldest
Shallowest
node coalescences
Deepest
Consequently the patterns of colonization & diversification of the endemic flora and fauna on these islands is closely linked to their geological history, with the youngest islands in the SE, and therefore the flora and fauna of these islands typically hosting taxa that have only recently coalesced. Where as islands in the NE are much older and play host to taxa showing more basal node coalescences.
100
(km) 60
(m)

5. Galapágos endemics ISABELA 100 (km) 60 (m) FERNANDINA
BALTRA
South Seymour
BARTOLOMÉ
Bartholomew
ESPAÑOLA
Hood
FERNANDINA
Narborough
FLOREANA
Charles
GENOVESA
Tower
ISABELA
Albemarle
MARCHENA
Bindloe
NORTH SEYMOUR
PINZÓN
Duncan
PINTA
Abingdon
RÁBIDA
Jervis
SAN CRISTÓBAL
Chatham
SANTA CRUZ
Indefatigable
SANTA FÉ
Barrington
SANTIAGO
San Salvador
This phenomenon, known as the progression rule has been studies in a range of organisms have colonized the Galapagos Islands that have radiated throughout the archipelago to form unique assemblages. Most of the terrestrial Galapagos fauna have diversified in parallel to the geological formation of the islands, particularly for taxa with very low vagility and on large islands with diverse habitats. Ecology and habitat specialization appear to be critical in speciation both within and between islands. Some of the best studies examples of this include the giant Galapagos tortoises, Galapagos terrestrial snails and Darwin’s finches.
100
(km) 60
(m)

6. Galapágos mockingbirds
One group that until recently was surprisingly poorly studied is the four species of mockingbirds that inhabit the Galapagos. These are endemic to the Gal疳agos Islands and are descended from the Ecuadorian Long-tailed Mockingbird. Arguably these birds had a greater influence than any other animal on Darwin’s theory of evolution because they were the first species that Darwin noticed distinct differences among when he looked from island to island. Interestingly enough they are one of the few groups that Darwin bothered to label properly by island, and this image here shows some of the specimens from the NHM collections that he collected, with Darwin’s original labels attached.
Mimus spp.

7. Host systematics traditional taxonomy mtDNA phylogeny S.East West
Middle +
North
West
mtDNA (Arbogast et al, 2006), colonization MYA
mtDNA phylogeny
Host systematics
traditional taxonomy
Host mockingbirds
Mimus sp.
M. parvulus
M. melanotis
San Cristobal
Morphologically there are four nominal species of Galapagos mockingbirds, each with distinct geographic distributions. The Hood mockingbird (Mimus macdonaldi) inhabits Española, the San Cristóbal mockingbird (M. melanotis) is found on the island of the same name and the Floreana mockingbird M. trifasciatus is present on two islets adjacent to Floreana. The rest of the archipelago is populated by the Galápagos mockingbird (M. parvulus).
A recent study by Arbogast et al. showed that the mitochondrial data only partially fit the traditional taxonomy of Galápagos mockingbirds. Despite belonging to three different nominal species, birds from the eastern islands of the archipelago (Española, San Cristóbal, and Genovesa) possess similar mtDNA haplotypes, while populations from Isabela in the west of the archipelago, are genetically more divergent from other M. parvulus populations than previously expected.
M. trifasciatus
Floreana
M. macdonaldi
Hood

8. Galapágos mockingbirds
I first became involved in a study of these birds when I was asked to identify some samples of ectoparasites collected from these birds. Paquita Hoeck at the Zoological Museum, University of Zurich, Switzerland as part of a project with Lukas Keller were collecting blood samples and ectoparasites from mockingbirds from 14 if the islands between 2004 and 2008.
Mist nets & potter traps
Wing vein puncture
Ectoparasites collected by dust ruffling
14 Islands

9. Mockingbird ectoparasites
Amblyceran louse
Myrsidea nesomimi
11 islands
Ischnoceran louse
Brueelia galapagensis
6 (smaller) islands
Myrsidea
The samples showed that three species of ectoparasite were present across most of the mockingbird population. Two of these were parasitic lice bellowing to the two major suborders of feather lice. The one at the top here is the amblyceran louse Myrsidea nesomimi which was collected form birds on 11 islands. The second louse Brueelia galapagensis, which belongs to a different suborder could only be found on birds on 6 islands. We also collected large numbers of Analygid mites – at least 4 different species, although only one species was widespread and present on 11 of the samples islands.
Brueelia
Analgid mite
Analges sp.
11 islands
Analges

10. Questions What are the evolutionary histories of these lineages
Are the host & ectoparasite evolutionary histories congruent?
Where there is discordance, can we explain it
Biogeography
To what extent do these diversifications match the successional origins of the islands (progression rule)
Analges
Brueelia
Myrsidea
Mockingbirds
Given that these are obligate permanent ectorparasites of the mockingbirds, whose life cycle and distribution means they are closely tied to their hosts, we then set out to recover the revolutionary history of these lineages; find out whether these evolutionary histories are congruent; in cases where they are not, seek to explain these discrepancies; and finally give the close association of these lineage, to what extent does their diversification match the successional origin of the islands?

11. Data
Homologous 1050 bp fragment COI sequenced in the mockingbirds and all 3 parasite taxa + outgroups
Complementary nuclear EF1α sequenced in Brueelia & Analges (not informative in Myrsidea)
400 Mimus individuals covering the 11 sampled islands genotyped using microsatellites
Analyses
NJ, ML and BI phylogenetic analyses
Haplotype network built using TCS
Mockingbird microsats analysed via Bayesian clustering algorithm in Structure
*BEAST to compare mutation rates, infer a multi-species tree from gene trees, & estimate dates of speciation
GeoPhylo and Google Earth to visualize genetree congruence
We did this by compiling 3 different data sets: we sequenced a homologous 1050 bp fragment of mitochondrial COI for the mockingbirds and all 3 parasite taxa + outgroups. Generated complementary nuclear EF1α sequences in Brueelia & Analges (this nuclear gene was not informative for the Myrsidea); and we genotyped 400 mockingbird individuals using microsatellites.
We then used a standard range of phylogenetic analyses on the COI and EF1-a data; built haplotype networks using TCS; the Mockingbird microsats were analysed using the Bayesian clustering algorithm in Structure; *BEAST was used to compare mutation rates, infer a multi-species tree from gene trees, of the different lineages & estimate dates speciation. Finally we used a program called GeoPhylo to converts the phylogenies of each lineage and the geographic coordinates of the collection points into a KML file that can be viewed in Google Earth. This was done to help visualize congruence between our individual genetrees.
What I’m going to do now is walk you through some of the key points for each of these lineages. These slides show a Maximum likelihood phylogeny, excluding outgroups; a maps of the islands with a colour scheme this is consistent across all figures and the haplotype networks.

12. Mockingbirds COI ML tree 107 sequences (25 haplotypes)
Island populations often single haplotypes (Floreana)
Largely congruent with traditional taxonomy
Basal split separates SE pop. from rest
No regular migration between islands (Structure analysis)
Incongruence with ectos must have another explanation (eg ancestral polymorphism)
So first off the mockingbirds. Of the 107 individuals sequenced we found 25 haplotypes. Each Island populations was usually a single haplotypes, and in the case of the endangered Floreana mockingbird population this has a single haplotype despite being on 2 islets, although several haplotypes are shared between two or three islands in the central and North-West part of the archipelago.
These data are largely congruent with traditional taxonomy (exceptions are not reflected in the microsat data) and the basal split separates SE pop. from the rest. As with previous studies this fits well with the progression rule with Islands or groups of islands form well supported monophyletic clades. This suggests there is no regular migration between islands. Given the obligate permanent nature of the ectoparasites on these mockingbirds, this suggests that any incongruence with obligate permanent ectoparasites must have another explanation (for example ancestral polymorphism rather than the ectoparasites switching between hosts). SE

13. Feather mites (Analges)SE
COI
Next up the feature mites. From the 86 Analges sequences we identified 71 haplotypes. Genetically this makes the mites the most diverse mockingbird ectoparasite, both in terms of number of haplotypes and their nucleotide diversity. The pattern of haplotype distribution in Analges shows an extreme level of diversification, with each island (with the exception of GbE) comprising an exclusive set of haplotypes. Analges also shows extremely high levels of allelic variability in EF1α sequences. The distribution of genetic variation is geography dependent, showing clear structure between the South-East and North- West, and topologically this broadly maps to the host phylogeny. Analges also showed the fastest mutation rate of all the taxa sampled at approximately nine time the rate of Mimus.
86 sequences (71 haplotypes), most diverse ecto.
Haplotypes exclusive to each island (*GbE)
Broadly maps to host phylogeny, SE root
Fastest mutation rate (9x Mimus)

14. Amblyceran lice (Myrsidea)
COI
98 sequences (37 haplotypes)
Broadly maps to host phylogeny, basal SE clade
Island groups monophyletic & well supported
But less pop. structure than mites
Several haplotypes shared across islands
Champion & Santa Fe relationship (recent migration perhaps by unknown louse vector)
Mutation rate approx 2x Mimus
The Myrisdea feature lice show less population structure than in Analges. 37 haplotypes were identified from 98 sequences, but again this broadly maps to the host phylogeny, and broadly fits the progression rule with haplotyes from the SE islands locted at the root of the tree. Generally the Island groups are monophyletic and well supported, but the reduced population structure means that Several haplotypes shared across islands. Interestingly one haplotype was shared between Champion (a small islet off Floreana) and Santa Fe, suggesting a recent migration event, perhaps by unknown louse vector).
The mutation rate in Myrisdea is approx twice that of Mimus.

15. Ischnoceran lice (Brueelia)SE NW
45 sequences (8 haplotypes)
Very low levels of genetic diversity
Island populations comprise 1-3 haplotypes
Some genetic isolation between islands
Dispersal via hitchhiking on hippoboscids?
B. galapagensis ‘contaminant’ on Small Ground Finch
Inter-island migration of Small Ground Finch with hippoboscids carrying lice?
Geospiza fuliginosa
Genetically Brueelia is the most conserved taxon with only a few SNP mutations spread across the whole 1050bp COI fragment. So much so that we did not attempt to build a COI phylogeny. Just 8 haplotypes could be identified from the sequences.
Each island population is made up of one to three haplotypes. Nevertheless, several Brueelia populations contain exclusive haplotypes . This was also recovered from the EF1-a data, which confirms that the deepest genetic separation in Brueelia lies between the SE islands and the rest of the archipelago.
A possible explanation for this reduced genetic structure compared to the other ectoparasites comes in the form of a particular behaviour exhibited by these lice. Brueelia are commonly found in a phoretic association with hippoboscid flies. These flies are commonly associated with birds and are very common on mockingbirds in the Galapagos. Its possible that this species of Brueelia is hitchhiking between the Galapagos Islands attached to the abdomens of hippoboscids. However, the only way that flies can travel between the islands is if they are attached to birds and we know this can’t be travelling on mockingbirds. Interestingly this species of Brueelia has been occasionally found on Small Ground Finches and we know that these birds do exhibit some migration between the islands. Therefore its possible that inter-island migration of Small Ground Finch with hippoboscids carrying lice, that subsequently get back on to mockingbirds, may explain why the levels of genetic structure in Brueelia are so low.

16. Cophylogeny Wide confidence intervals on node ages
*BEAST (node age & multi-species tree)
Single calibration - Espanola (mean 2.9 Mya, SD 0.9)
Wide confidence intervals on node ages
Indicative of the sequence of speciation
SE split 1.53 Mya on multi-species tree
Multi-species tree agrees with traditional Mockingbird taxonomy & geological history
Incongruence best seen in Google Earth visualization
Given the lack of genetic structure in Brueelia we excluded it from any cophylogeetic analysis, and focused on the Mimus, Analges and Myrsidea genetrees, for which sampling was available on all 11 islands.
To reconcile the evolutionary history of the parasites and their hosts, a putative 杜ulti-species� tree was reconstructed in *BEAST. This allows simultaneous estimation of phylogeny and node age. Typically reconciling host and parasite phylogenies would be done using a somewhat different approach. For example using software like TreeMap, TreeFitter or ParaFit. The problem is that these methods are based on reconciling single host and parasite trees. There is also a more practical problem some of this software is so old that it simply won't run on recent operating systems.
Needless to say, this single calibration led to wide confidence intervals on node ages, which cannot be taken as exact ages. Nevertheless, they are indicative of the sequence of speciation. This analysis put the split between the SE and NW radiation 1.53Mya. The multi-species tree agrees with traditional Mockingbird taxonomy, recognising all 4 mockingbird species and is also compatible with the geological history of the islands. Posterior probabilities of all nodes are relatively high, with values for all but two above 0.8.
Despite several incongruences between individual tree genealogies observed for particular taxa, the independent phylogenetic signals become evident when sequences are jointly analysed. This is best seen when individual genealogies are contrasted with the resulting species tree in Figure 5c, or using a Google Earth visualisation.

17. Geophylogeny GeoPhylo ML gene trees Lat. long. data KML file
Google Earth
Unfortunately there is not an easy way for me to show you this visualisation without running Google Earth, but anyone familiar with Google Earth will be familiar with the general principles. This produces a 3 dimensional visualisation that you can zoom and rotate to explore the individual genealogies of each lineage, and the multispecies tree.
To create this we used the online GeoPhylo tool that converts ML phylogenies of Mimus, Analges and Myrsidea, and a multispecies tree into KML files that can be opened and explored in Google Earth.
Google Earth

18. Summary
Evolutionary histories of Mimus & 2 ectoparasites (Analges & Myrsidea) broadly congruent
These diversifications can be explained by the successional origins of the islands (progression rule) and co-diversification of ectoparasite lineages
Low genetic variability & lack of co-phylogeographic congruence in one ectoparasite lineage (Brueelia)
May be explained by life history traits of Brueelia linked to phoretic dispersal
To sum up, the evolutionary histories of Mimus & 2 ectoparasites (Analges & Myrsidea) broadly congruent. These diversifications can be explained by the successional origins of the islands (progression rule) and codiversifiation of ectoparasite lineages. Low genetic variability & lack of co-phylogeographic congruence in one ectoparasite lineage (Brueelia). This may be explained by life history traits of Brueelia linked to phoretic dispersal (hitch-hiking) on hippoboscid flies.
Very shortly you should be able to read more about this work in a paper that Jan and I have written that has been accepted for publication by BMC Evolutionary Biology.
Read more shortly at:
Stefka et al A hitchhikers guide to the Galapagos: co-phylogeography of Galapagos mockingbirds and their parasites. BMC Evolutionary Biology (accepted pending revision)

19. Acknowledgements
Paquita Hoeck and Lukas Keller (Zoological Museum, University of Zurich, Switzerland) who provided host and parasite samples and some microsat. data.
European Union FP7 Marie Curie Fellowship program.
...and Douglas Adams
Finally a few quick acknowledgements. I want to thank Paquita Hoeck and Lukas Keller based at the Zoological Museum in the University of Zurich. Paquita and Lukas provided the host and parasite samples and some of the microsat. data.
I also want to thank the European Union framework 7 Marie Curie Fellowship program which funded this work.
And finally and Douglas Adams who provided the inspiration for our title. Thank you very much.