1. DNA variation in Ecology and Evolution III- Examples of application of molecular markers

Slide 1

2. Slide 2

Slide 2

Agama atra

Agama atra are rock-dwelling lizards with low dispersal capabilities. Two groups can be distinguished within this species: (1) occupies the dry north-western regions of the Northern Cape Province. They are large-sized animals with a long breeding season. (2) a group that occupies a more southern and eastern distribution in South Africa and is smaller in size with a shorter breeding season.

The aim of this work is to investigate the phylogeographic pattern and infer historical reasons for the present geographic distribution.

39 individuals were samples from 13 populations. The DNA sequence information from two mitochondrial genes were used for the phylogenetic reconstruction.



MATTHEE CONRAD A. and FLEMMING ALEXANDER F. (2002) Population fragmentation in the southern rock agama, Agama atra: more evidence for vicariance in Southern Africa. Molecular Ecology 11, 465:471.

This work was carried out in the Zoology department, University of Stellenbosch.

3. Slide 3

Slide 3

Agama atra results

The phylogenetic reconstruction using different methods (maximum parsimony MP, and maximum likelihood ML ) show congruent results. Three main lineages can be identified, that show geographic distinctiveness; South Central, coinciding with the Great Escarpment; North Central; and Southern Namibia.

These lineages indicate that there is long-term restriction to gene flow between these regions. Beside, the animals with large body size and prolonged breeding season coincide with the North Central clade, whereas the small animals with reduced breeding season are restricted to the South Central clade.

Using the rate of 0.5 -1 % change per million years for 16S rDNA, the clades diverged 2-4 MY ago. The origin of the disjunctive distributions could have been originated from the cycles of dry- wet periods during glacial and interglacial periods respectively, and some of these localities might have served as refugia.

4. Slide 4

Slide 4

Other rock dwelling species, a gecko and a rabbit show a similar pattern of geographic distribution of the maternal lineages. This is indicative of a common causal phenomenon affecting the biota. The appearance of geographical barriers and divergent evolution in these isolated regions is called vicariance.


5. Slide 5

Slide 5

Tilapia hybrids in W Africa

Tilapias are closely related species that underwent a recent evolutionary radiation during the Pleistocene. Many of these species show overlapping geographic distribution and are utilized in aquaculture. Breeding programs in aquaculture usually involve the hybridization between different phenotypes of even species to obtain a certain desired phenotype. Among the most utilized species are Oreochromis aureus and Oreochromis niloticus. The geographic distribution of these species is shown in the map: grey indicates O. niloticus and the dashed line indicates O. aureus. Although these species interbreed well in captivity, no hybrids were detected in the wild. In this work, the authors utilize nuclear and mitochondrial variations along with the morphological identification to test the hypothesis of gene flow between these two species. For this, they utilized the variation at 30 allozyme loci (loci that code for enzymes) and the DNA sequence information for cytochrome b and the mtDNA control region.

Samples were taken in locations in the Nile area and in Western Africa, where O. aureus shares the habitat with O. niloticus.



X. ROGNON and R . GUYOMARD (2003). Large extent of mitochondrial DNA transfer from Oreochromis aureus to O. niloticus in West Africa. Molecular Ecology 12, 435-445.

6. Tilapias hybridization

Slide 6

Tilapia hybrids in W Africa

The slide shows partial results of the paper. The cluster in the 1st figure, on the left, shows the clustering of populations using genetic distance in allozyme polymorphisms between populations. We see that the three O. aureus population (from the Nile River, from the Volta River in Niger and from Senegal cluster together, whereas O. niloticus shows two distinctive clusters: the top one is formed by populations from the Nile area, and the other cluster is formed by western African populations.

The cluster figure on the right shows the clustering of populations using mtDNA polymorphisms. We see that the O. aureus population from Senegal clusters together with O. niloticus from Senegal.

The clustering of all haplotypes shows the situation better: same haplotypes are present in both fish species in Niger and in Senegal.

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Slide 7

Hybridization of tilapias in W Africa

The main results are; shallow divergence between the haplotypes from Senegal, regardless of the species, and a strong divergence between O. niloticus from the Nile and Western Africa. Two possible scenarios can explain this pattern;

ancestral retention

secondary contact followed by introgression

The first case: O. aureus could have originated from O. niloticus in western Africa. This hypothesis agrees with mtDNA data but not with the deeper divergence observed for protein data.

Thus, the most likely explanation is the replacement of mtDNA of one species by the other.

8. Slide 8

Slide 8

Hybridization of tilapias in Southern Africa.

The map is showing the natural distribution of a few tilapia species in Southern Africa. The Nile Tilapia O. niloticus had been introduced in Southern Africa for aquaculture and is documented to have escaped into the natural environment in the Limpopo basin.

An investigation of the hybridization status of O. mossambicus in Southern Africa with mtDNA control region gave surprising and unexpected results.

The network reconstruction using the mtDNA control region of other parapatric species revealed that there is hybridization or introgression with other native species.

The clusters indicate the group of belonging species and the colour represent species. See, for instance, that the yellow genotypes (from O. mossambicus) appear clustering with the native species O. andersonii, O. karongae and the introduced species O. aureus and O. niloticus.

See that it is possible to recognize 3 lineages within O. mossambicus.

Reference: unpublished data of D'Amato et al.

María Eugenia D'Amato, M. Magdalena Esterhuyse, Ben C.W. van der Waal, D. Brink and Filip A.M. Volckaert. Hybridization of the Mozambique tilapia Oreochromis mossambicus in southern Africa evidenced by mitochondrial and microsatellite DNA genotyping.

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Slide 9

Phylogenetics of abalone

Gene families : Slide 17, Class I

The abalones are Gastropoda Molluscs that are distributed worldwide. The phylogenetics of this group has been conflictive and phylogenies with 16S mtDNA and lysine protein and other genes are not completely congruent.

The phylogenetic reconstruction presented here was performed with the DNA sequence information of one highly conserved intron in the haemocyanine gene.

The heomocyanine is equivalent in function to the haemoglobin of mammals. It binds and transports oxygen to the tissue.

Heomocyanines are a family of oxygen-binding proteins that evolved by duplication and fusion (see Lieb and Markl, 2004). Gastropoda show tow isoforms that are immunologically distinct, HtH1 and HtH2. These two forms duplicated approximately 350 Myr ago, before the divergence between limpets and abalones.

In the present work, the phylogeny is intended to be reconstructed from one intronic and flanking the exonic regions of the isoform HtH1. However, to avoid false clustering, the authors have to prove that they sequenced the HtH1 form in all species. For this, they used the ortholog HtH2 of one Haliotis species and another similar isoform in Fisurellidae. The two isoforms show 65% identity within genome.



Bernhard Lieb, Ju¨rgen Markl (2004). Evolution of molluscan hemocyanins as deduced from DNA sequencing. Micron

Klaus Streit, Daniel L. Geiger and Bernhard Lieb (2006). Molecular Phylogeny and the geographic origin of Haliotidae traced by haemocyanin sequences. Journal of Molluscan Studies 72: 105-110.

10. Slide 10

Slide 10

Phylogenetics of abalone

The phylogenetic reconstruction shows that all Haliotis species cluster together, and that the similarity between their HtH1 introns ranges from 80-95% sequence identity, therefore confirming that the sequences utilized in the phylogenetic reconstruction are the paralogs.

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Slide 11

Potential different adaptive values in different populations/ population structures

For population differentiation and Fst statistics See Slide 24, Class II

The threeline grunt Parapristipoma trilineatum is a fish species that lives on the coasts of China and Japan. It is utilized in aquaculture, and the Chinese form was imported to Japan because of its higher growth rate. The higher growth rate indicates different quantitative traits and possibly a different genetic composition. For this, the population genetic structure of this species was investigated from 2 Chinese and 5 Japanese samples using 4 microsatellite loci.

The first table shows that the genetic variability in all population is similar in terms of heterozygosity and number of alleles. The expected heterozygosity (indicated He) do not differ significantly from the Ho (observed heterozygosity).

The second table shows an analysis of heterogeneity of allele frequency distributions among populations (indicated by Fst and the probability that this its value differs significantly from 0). The results show heterogeneity (population structure) between the 2 Chinese populations, and between the Chinese and the Japanese populations. The Japanese populations, however, are not differentiated and can be considered a single stock for management.


Keita Kumagai, Anna A. Barinova, Masamichi Nakajima, and Nobuhiko Taniguchi (2004). Genetic Diversity Between Japanese and Chinese Threeline Grunt (Parapristipoma trilineatum) Examined by Microsatellite DNA Markers. Mar. Biotechnol. 6, 221-228.


12. Slide 12

Slide 12

Population differentiation in the threeline grunt

For microsatellites see Slide 20, Class I.

In this slide we see the pair wise Fst values between all population pairs. These values were utilized as distances to construct a tree (UPGMA) that also reflects the differences between the Chinese populations ( a deep divergence) and the differences between the Japanese and Chinese samples.

Based on these results, recommendations are made to avoid escapees from the fish farms as the introduced fish are distinctively different from the native fish and could disrupt local adaptation.