Transcript Leucetta chagosensis

Phylogeography of Leucetta chagosensis
(Porifera, Calcarea)
Christoph Flucke, Jens Kurz, Rasmus Liedigk,
Zdenka Valenzova
1.Introduction
Leucetta chagosensis is the most dominant sponge in specialized habitats like
overhangs and shaded areas. There is a restricted capability of sponge larvae
to disperse.
Therefore it occurs a distribution through founder effects and vicariance. It is
important to understand the distribution of L. chagosensis in the Pacific Ocean,
to fit new samples into an existing phylogeny (Wörheide 2002) and compare
geographical distribution.
Aims: 1) sequence crucial parts of genes in available samples
2) build a phylogenetic tree
3) try to explain the phylogeographic changes in the genom
2.Materials and Methods
DNA extraction, PCR and sequencing reaction:
The ITS 1, 5.8S, ITS 2 regions (Fig.1) from rDNA sequences from 26 specimens
of Leucetta chagosensis from 13 localities (Fig.2) were investigated. The
extraction was performed with the Qiagen DNeasy Tissue extraction kit
according to the manufacturer instructions. ITS regions 1 and 2 and the 5.8S
region were amplified (~900 bp) with oligonucleotide primers RA2 (forward)
connecting to the 3‘ terminus of the 18S subunit and ITS2.2 (reverse)
connecting to the 5‘ terminus of the 28S rRNA subunit (Wörheide, 1998).
Biotaq-Polymerase was used to perform PCR-amplifications in a total volume of
25 µl. PCR-products were purified using a Silica-based method (modified from
Boyle, JS and Lew, 1995). After Cycle Sequencing Reaktion templates were
sequenced in capillary sequencer.
Phylogenetic analysis:
Sequences were assembled using the program CodonCodeAligner. Multiple alignments
were created in the SeaView alignment editor. BLAST searches were run to confirm that
all sequences are from Leucetta chagosensis. To find the best model of DNA substitution
the program Modeltest was used. The parameters of this model were used for a
Maximum Likelihood analysis in RAxML (Bootstrap analysis 500 replicates) and a
Bayesian analysis in MrBayes.
Fig.1: ITS region
Fig.2: Distribution of the four major clades
in the Pacific Ocean.
3. Results
bp
By the chosen analysis
methods a PCR signal
5000
could be established at
3000
12 of 25 examined
1500
samples. Figure 3 show
1000
exemplary the detected
signals for five examined
samples. All detected
Fig. 3: show the detected PCR signals
signals lye at the 800 for the examined samples 1, 2,
3, 9 and 10
1000bp band height of
the used 100bp marker.
These are signs for a fully isolated ITS region sequencelength, wich included
the 18S rRNA gene, ITS1 region, 5.8S rRNA gene, ITS2 region and the 28S
rRNA gene.
After purification and sequence process, 6 of 12 detected PCR signals could be used for a
secondary phylogenetic contemplation. A sequence comparison showed that the six used
samples were a Pericharax and five Leucetta chagosensis sequences. For a reconstruction
of the phylogenetic connections, like figure 4 and 5, 18 Leucetta chagosensis sequences
and one Leucetta micrographis sequence as a defined outgroup, were used from the NCBI
nucleotide - online database. All listed sequences are a result of a nucleotide comparison
from the five detected Leucetta chagosensis sequences by this Site. Figure 4 and 3 show
the phylogenetic trees from the RAxML and MrBayes program where the isolated ITS
sequences (Contig numbers) are compared with the NCBI sequences (AF numbers).
4.Discussion
Leucetta micrographis and Pericharax sp. were confirmed to be outgroups as
predicted.
According to both phylograms, there seem to be four major clades:
1.Indonesia (Sulawesi, Bali, Sangalaki)
2.Vanuatu a) Vanuatu, SGBR, Kakaban, Samoa
b) Vanuatu, Samoa (MrBayes)
3.NGBR (N/C/SGBR, Taiwan, Guam)
4.SGBR (C/SGBR)
The borders between these clades are not strict, especially not for the SGBR
group, which is found also in other clades. The reliance of the data, which
states the Kakaban sample belongs to the Vanuatu clade is very poor.
According to MrBayes the Fiji taxon is an outgroup and the Vanuatu clade is
divided into a Vanuatu and a Vanuatu/Samoa clade. The model of RAxML is
therefore preferred, because it makes geographically more sense. The theory
of four major Leucetta clades in the pacific ocean (Wörheide et al, 2002) is
well supported.
Fig.4: RAxML Phylogram
Fig.5: MrBayes Phylogram
References
Wörheide et al. 2002. Phylogeography of western Pacific Leucetta
‚chagosensis‘ from ribosomal DNA sequences: implications for population
history and conservation of the GBR (Australia). Molecular Ecology 11: 17531768.
Wörheide et al. 2008. Deep genetic divergences among Indo-Pacific populations of
Leucetta chagosensis: founder effects, vicariance or both? BMC Evolutionary Biology
8: 24.
Wörheide et al. 2004. Intragenomic variation of the rDNA ITS: implications for
phylogenetic studies. Molecular Phylogenetics and Evolution 33: 816-830.
Wörheide et al. 2005. Biodiversity, molecular ecology and phylogeography of marine
sponges: patterns, implications and outlooks. Integr. Comp. Biol. 45: 377-385.