3 Phylogeographic structure of the European tree frog (Hyla arborea) in its
3.3 Material and methods
3.5.4 Conclusion
No different genetic lineages were revealed for the European tree frog. Thus there is no risk of unintentionally mixing lineages that have been separated for at least several 100,000 years when conducting responsible-minded translocations for species conservation. Nonetheless, the detected genetic structures and main migration barriers should be considered in future conservation measures. This is important because differentiation caused by substantially shorter time spans can lead to a reduced fitness in the offspring. (e.g. Holleley et al., 2011).
As mentioned in my previous study (chapter 2), as long as the degree of genetic differentiation which could cause outbreeding depression is unknown, translocations of individuals should not be conducted without thoroughly assessing the potential consequences for the receiver population. In general heterozygosity values were high in the sampled
populations and indicate no alarming situation concerning reduced fitness as a potential consequence of lost genetic diversity.
3.6 Acknowledgement
This research was supported by grants from the German Federal Environmental Foundation (DBU), Heidehof-Stiftung, and “Hans-Schiemenz-Fonds“ - Deutsche Gesellschaft für Herpetologie und Terrarienkunde (DGHT). I thank the following Nature conservation authorities for permission for tree frog collection: the biosphere reserve Niedersächsische Elbtalaue, Kreis Minden Lübbecke, Kreis Steinfurt, Kreis Soest, Landkreis Diepholz, Landkreis Gifhorn, Landkreis Lüneburg, Landkreis Osnabrück, Landkreis Stade, Landkreis
Uelzen, Region Hannover, Stadt Gera, Stadt Wolfsburg, Oberbayern, Niederbayern, Haßberge, Baden-Württemberg, Brandenburg, Rheinland-Pfalz, Sachsen, Sachsen Anhalt, Schleswig-Holstein. I am especially grateful to Annika Ruprecht, Axel Kwet, Christina Akman, Frank Weihmann, Günter Krug, Hans-Dieter Bast, Heike Pröhl, Herbert Schnabel, Hubert Laufer, Irena Czycholl, Ivonne Meuche, Jana Kirchhoff, Johannes Penner, Kim Jochum, Kristine Heißler, Matei Balborea, Michael Weinert, Oscar Brusa, Oscar Klose, Thomas Schoger-Ohnweiler, Wiebke Feindt, and Wolf-Rüdiger Große for help during field work or sending samples. Finally I thank our technician Sabine Sippel for her assistance in the molecular lab and Sönke von den Berg for technical support.
4
General discussion
The aim of this doctoral thesis was to investigate phylogeographic structures of an endangered amphibian species, the European tree frog (Hyla arborea), on different geographic scales. I will summarise the main outcomes of my analyses and discuss their importance for species conservation management.
On the large scale geographic level encompassing sample sites across the German distribution area my aim was to assess the potential for the presence of distinct genetic lineages thought to have evolved due to postglacial recolonisation of the continent from multiple refugia. Distinct postglacial genetic clades or lineages were previously detected for other amphibian species such as Rana arvalis (Babik et al., 2004), Rana temporaria (Palo et al., 2004), and
Salamandra salamandra (Steinfartz et al., 2000). However, different genetic lineages could not be revealed for the European tree frog in my studies.
In contrast to the previous studies, differentiation at the mitochondrial gene Cyt b was low.
The main haplotype was the same that was found to occur between Western France and Albania (Stöck et al., 2008; Stöck et al., 2011) supporting the hypothesis of Stöck et al.
(2008), who suggest that a single mtDNA lineage spread from a potential Pleistocene refugium in the Balkan region.
Nonetheless, some phylogeographic structure in the German distribution could be detected by cyt b and microsatellite data. One haplotype and its descendants were found only in Northern part of Germany and adjacent areas in the Netherlands (Stöck et al., 2011) with the Central German Uplands as a distribution border. Additionally, the Bayesian
microsatellite analysis supports the separation of Southern sample sites to those in the North.
Furthermore, the influence of major rivers such as Rhine, Main, Elbe, and Danube and mountain ranges in the Southwest was evident.
Since no different genetic lineages were revealed for the European tree frog there is no risk of unintentionally mixing lineages that have been separated for at least several 100,000 years. However, for species conservation measures such as translocation of individuals from larger to smaller populations, the detected genetic structures and main migration barriers should be considered. This is important because differentiation caused by substantially shorter time spans can lead to a reduced fitness in the offspring. (e.g. Holleley et al., 2011).
Therefore, I conducted the study on a medium scale level on the distribution of the tree frog in Lower Saxony and adjacent areas. My aim was to describe management units (MUs) and genetic diversity for supporting effective conservation management for the tree frog in Lower Saxony.
Cyt b sequences showed low differentiation but a distinct geographic-genetic pattern was revealed. Using microsatellite analysis I found seven distinct genetic clusters. As a consequence of the patchy distribution of the tree frog in this area most sample sites were assigned to individual management units. Also for the occurrences in the Eastern part of the sampling area along the river Elbe where the former and present-day distribution has been more continuous, I found a current separation in distinct MUs. Although I recommend treating each genetic cluster as one or more management units, in the long run the originally linked occurrences which are presently separated into different MUs as a consequence of habitat fragmentation and genetic drift, should be reconnected.
In general heterozygosity values as a measure for genetic diversity were high, not only in Lower Saxony, but also in most German sample sites. This suggests that reduced fitness as a potential consequence of lost genetic diversity is not a current problem in my study area.
Therefore, inital measures for population recovery should be to construct networks of breeding sites. There are several reports that the European tree frog responds well to new suitable water bodies or their restoration and often colonises them the following breeding season (e.g. Hansen, 2004 (in DK); Zumbach, 2004 (in CH)) .
However, if translocations of individuals are necessary e.g. to recover very small and inbred populations or for reintroduction measures, the revealed genetic structures and the identified barriers should be considered, especially as long as it is not known what degree of genetic differentiation can cause effects of outbreeding depression.
4.1 Future goals
For future studies I emphasize the urgent need of breeding and fitness tests between
populations that have genetically diverged to different degrees. Because, as is discussed for both studies, it is currently not known what degree of genetic differentiation could be enough to cause effects of outbreeding depression.
The inference of management units was conducted in this study in an area where the distribution of the European tree frog was very patchy. Consequentially almost each sample site was assigned to a separate management unit. For comparison it would be interesting to reveal the structure of the management units in an area with more continuous distribution of the European tree frog such as in Mecklenburg-Western Pomerania or in parts of Bavaria.
In this study cyt b was not very differentiated and the main haplotype is widely distributed across Europe. Most other haplotypes are unique for one sample site and are typically only one mutation step apart from the main haplotype. Historic relationships of populations could be assessed for few sites only. The suitability of microsatellites is limited for the purpose of historic relationships since artefacts like homoplasy could lead to incorrect similarities. Therefore, for future studies in the European tree frog the analysis of the
hypervariable mtDNA d-loop would probably give better resolution for the historic structure and relationships aspects, at least for the delineation of management units.
Although the cyt b differentiation was low, I was able to reveal distinct
phylogeographic structures with importance for conservation measures. Similar studies including other amphibian species are important since this taxon is highly endangered.
Breeding experiments could shed light on the association between the degree genetic
differentiation and in- or outbreeding depression. Especially, more studies are necessary that reveal that relationship between barriers to gene flow, degree of habitat fragmentation, genetic divergence and fitness consequences and long-term survivorship.
5
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