Phylogeography and population structure of the European tree frog (Hyla arborea) for supporting effective species
conservation
THESIS
Submitted in partial fulfilment of the requirements for the degree
DOCTOR OF PHILOSOPHY (PhD)
awarded by the University of Veterinary Medicine Hannover
by
Astrid Krug Bruchsal, Germany
Hannover 2012
PD Dr. Heike Hadrys, Dr. Stefan Könemann (until 09.03.2011) Prof. Dr. Miguel Vences
1st Evaluation: Prof. Dr. Heike Pröhl
University of Veterinary Medicine Hannover Institute of Zoology
PD Dr. Heike Hadrys
University of Veterinary Medicine Hannover Division of Ecology and Evolution
Prof. Dr. Miguel Vences
Technical University of Braunschweig Division of Evolutionary Biology Zoological Institute
2nd Evaluation: Dr. Robert Jehle University of Salford
School of Environment & Life Sciences Ecosystems and Environment Research Centre
Date of oral exam: 8th of November 2012
Astrid Krug was sponsored by the Scholarship Programme of the German Federal Environmental Foundation (DBU) # 20007/899.
Research funds were provided by the German Federal Environmental Foundation (DBU), Heidehof-Stiftung # 57129.01.2/3.10, and “Hans-Schiemenz-Fonds“ - Deutsche Gesellschaft für Herpetologie und Terrarienkunde (DGHT).
Table of Contents
Summary..………1
Zusammenfassung………3
1 General introduction………...………...…5
1.1 Global amphibian decline………6
1.2 Conservation genetics………..6
1.3 The European tree frog………7
1.3.1 Characteristics……….7
1.3.2 Distribution……….9
1.3.3 Conservation status and major threats………...10
1.3.4 Conservation genetics in the European tree frog………...11
1.4 Aims of the study………...12
1.4.1 Phylogeography in Germany and adjacent areas………...12
1.4.2 Management Units in Lower Saxony and adjacent areas………..12
2 Defining units for conservation management for the European tree frog (Hyla arborea) in Lower Saxony and adjacent areas………14
2.1 Abstract………..15
2.2 Introduction………16
2.3 Materials and methods………...17
2.3.1 Sample collection and preparation……….17
2.3.2 Statistical analysis………..20
2.3.2.1 Historic structure: Analysis of mtDNA………..20
2.3.2.2 Recent structure: Analysis of microsatellites……….21
2.3.2.3 Biogeographic zones………..22
2.4 Results………23
2.4.1 Mitochondrial sequence analysis………...23
2.4.2 Microsatellite analysis………24
2.4.3 Biogeographic zones………..29
2.5 Discussion………..30
2.5.1 Genetic structure and conservation units………...31
2.5.2 Genetic diversity………33
2.5.3 Future goals………33
2.5.4 Conclusion and implications for conservation management……….34
2.6 Acknowledgement……….34
3 Phylogeographic structure of the European tree frog (Hyla arborea) in its German distribution area………36
3.1 Abstract………..37
3.2 Introduction………38
3.3 Material and methods……….39
3.3.1 Sample collection and preparation……….39
3.3.2 Statistical analysis………..41
3.3.2.1 Analysis of mtDNA in Germany………41
3.3.2.2 Analysis of microsatellites in Germany……….41
3.3.2.3 Analysis of mtDNA in the European context……….42
3.4 Results………43
3.4.1 Analysis of mtDNA in Germany………43
3.4.2 Analysis of microsatellites in Germany……….46
3.4.3 Analysis of mtDNA in the European context……….49
3.5 Discussion………..51
3.5.1 Distinct genetic lineages in the European tree frog? ……….52
3.5.2 Phylogrographic structures of the tree frog in Germany………52
3.5.3 Genetic diversity………53
3.5.4 Conclusion……….54
3.6 Acknowledgement……….54
4 General discussion……….………...56
4.1 Future goals………58
5 References……….60
6 Appendix………...70
Affidavit………87
7 Acknowledgment………..88
List of Abbreviations
°C degree Celsius
µl microliter
µM micromolar
bp base pairs
cyt b cytochrome b
DNA deoxyribonucleic acid
dNTP’s deoxynucleotide triphosphates ESU evolutionary significant unit
IUCN International Union for Conservation of Nature
km kilometre
min minute
mM millimolar
mtDNA mitochondrial DNA
MU management unit
ng nanogram
nuDNA nuclear DNA
P probability
PCR polymerase chain reaction
s second
SD standard deviation Taq Thermus aquaticus
U enzyme unit
List of Figures and Tables
Figure 1.1: Calling tree frog male
Figure 1.2: Distribution map of the European tree frog (Hyla arborea)
Figure 2.1: Current distribution of the European tree frog in Lower Saxony and adjacent areas
Figure 2.2: Haplotype network cyt b Lower Saxony
Figure 2.3: Distribution of cyt b haplotypes in Lower Saxony Figure 2.4: Isolation by distance plots
Figure 2.5: LnPD and delta K
Figure 2.6: STRUCTURE bar plot for K = 7
Figure 2.7: GENELAND map of estimated cluster membership for K = 7 Figure 2.8: Most important barriers to gene flow
Figure 3.1: Haplotype network of cytochrome b Germany Figure 3.2: Haplotype distribution and physical map of Germany Figure 3.3: LnPD and delta K
Figure 3.4: Distribution of distinct genetic clusters K = 4 Figure 3.5: Haplotype network of cytochrome b Europe Figure 3.6: Distribution of cyt b haplotypes in Europe
Table 2.1: Overview of sample sites Lower Saxony Table 2.2: Pairwise Dest values and pairwise FST values Table 3.1: Overview of sample sites Germany
Astrid Krug
Phylogeography and population structure of the European tree frog (Hyla arborea) for supporting effective species conservation
Many amphibian species around the world are threatened by consequences of habitat degradation and fragmentation. The European tree frog (Hyla arborea) has suffered from dramatic population declines in the last decades and has therefore been categorised as threatened in many Red Data lists. Conservation measures are conducted at many places. To support such measures I conducted molecular studies on two geographic levels to reveal phylogeographic structures and genetic diversity, which are important for effective species conservation management.
In Lower Saxony in Germany the current distribution of the tree frog is very patchy with some main occurrences in the lowlands. In order to define management units I sampled 237 individuals at 14 sites (~ 3 - 250 km apart from each other) across the tree frog
distribution area in Lower Saxony and adjacent areas. All samples were genotyped with eight microsatellite loci and twelve sites were sequenced for an mtDNA cytochrome b fragment.
While all but one of the microsatellite pairwise Dest and FST values showed significant genetic differentiation (Dest: 0 - 0.46, FST: 0 - 0.18), Bayesian analyses suggested seven distinct genetic clusters. The cytochrome b haplotype distribution highlights the former connection of the currently fragmented populations along the river Elbe.
However, to reveal genetic structuring at higher geographic levels, as could have been generated e.g. by different postglacial colonisation routes, I conducted the second study with a sampling network of 31 sites across the tree frogs’ distribution area in Germany. 372
individuals were again analysed by mtDNA cytochrome b sequences and eight microsatellite loci. Sequence divergence between sample sites was low, varying between 0 and 0.4 % (overall 0.2 %), and no distinct genetic lineages were found. Nonetheless, a clear North-South partitioning was revealed by both molecular markers with the Central German Uplands as likely barrier. Furthermore, the influence of the major rivers such as Elbe, Rhine, and Danube on the phylogeographic structure was revealed.
In general the genetic diversity was relatively high in both studies. Therefore, each of the sampled tree frog occurrences should have the potential to maintain or recover to a stable population size when applying appropriate local conservation measures. For new resettlement
projects, the identified genetic structures should be considered when choosing source populations. Where possible, reconnection of originally linked occurrences that are now separated in different conservation units due to habitat fragmentation and genetic drift should be facilitated.
Astrid Krug
Phylogeographie und Populationsstruktur des Europäischen Laubfroschs (Hyla arborea) zur Unterstützung eines effektiven Artenschutzes
Weltweit sind viele Amphibienarten, hauptsächlich durch die Folgen von Habitat-
Degradierung und Fragmentierung, gefährdet. Der Europäische Laubfrosch (Hyla arborea) hat in den letzten Jahrzehnten immense Bestandsrückgänge erfahren und wurde daher in vielen Roten Listen als gefährdet eingestuft. Naturschutzmaßnahmen werden bereits vielerorts durchgeführt. Um solche Maßnahmen zu unterstützen, habe ich molekulargenetische Studien auf zwei geografischen Ebenen durchgeführt zur Aufdeckung phylogeographischer Strukturen sowie der genetischen Diversität, welche für ein effektives Artenschutzmanagement wichtig sind.
In Niedersachsen ist die aktuelle Verbreitung des Laubfroschs sehr verinselt mit einigen Schwerpunktvorkommen im Tiefland. Um Management Units zu beschreiben, habe ich 237 Individuen an 14 Probestellen (~ 3 - 250 km voneinander entfernt) im
niedersächsischen sowie angrenzenden Verbreitungsgebiet gesammelt. Alle Proben wurden mit acht Mikrosatelliten-Loci genotypisiert und zwölf Probestellen wurden für ein mtDNA Cytochrome b Fragment sequenziert.
Während bis auf einen alle paarweisen Mikrosatelliten Dest und FST Werte signifikant unterschiedlich waren (Dest: 0 - 0.46, FST: 0 - 0.18), wiesen die Bayesischen Analysen sieben unterschiedliche genetischen Cluster auf. Die Verbreitung der Cytochrom b Haplotypen hebt die ehemalige Verbindung, zurzeit fragmentierter Populationen, entlang der Elbe hervor.
Um übergeordnete genetische Strukturen aufzudecken, wie sie durch unterschiedliche nacheiszeitliche Besiedlungslinien entstanden sein können, habe ich die zweite Studie mit einem Netzwerk von 31 Probeorten im deutschen Laubfrosch Verbreitungsgebiet
durchgeführt. 372 Individuen wurden wiederum mithilfe von mtDNA Cytochrome b Sequenzen und acht Mikrosatelliten-Loci analysiert. Sequenz Divergenzen zwischen Probeorten waren gering und variierten zwischen 0 und 0,4 % (gesamt: 0,2 %). Es wurden keine unterschiedlichen genetischen Linien gefunden.
Dennoch konnte eine klare Nord-Süd Unterteilung mit den deutschen Mittelgebirgen als wahrscheinliche Barriere in beiden molekularen Markern aufgezeigt werden. Des
Weiteren wurde der Einfluss von großen Flüssen wie z.B. Elbe, Rhein und Donau auf die phylogeographische Struktur deutlich.
Im Allgemeinen war die genetische Diversität in beiden Studien relativ hoch. Daher sollte jedes der beprobten Laubfroschvorkommen das Potential besitzen, eine stabile Populationsgröße wieder zu erlangen bzw. sie zu erhalten, wenn geeignete
Naturschutzmaßnahmen vor Ort durchgeführt werden. Im Fall von neuen
Wiederansiedlungsprojekten sollten bei der Wahl der Spenderpopulation die gefundenen genetischen Strukturen berücksichtigt werden. Wo es möglich ist, sollte eine
Wiedervernetzung von ursprünglich in Verbindung gewesener Vorkommen, welche heute durch Habitatfragmentierung und genetische Drift in unterschiedliche Conservation Units eingeteilt wurden, durchgeführt werden.
1
General introduction
1.1 Global amphibian decline
In the recent years, increasing attention has been paid to amphibian research and conservation.
The reason for this is that there has been a dramatic worldwide decline in amphibians observed in the last decades. In the 2008 IUCN Red List, nearly one-third (32.4 %) of the 6,260 assessed amphibian species were classified as globally threatened or extinct and there is strong evidence that the pace of extinctions is increasing. The data also demonstrate that amphibians are far more threatened than either birds (12 %) or mammals (23 %). (Stuart et al., 2004; IUCN, 2012)
Amphibians are often considered as bioindicators for the condition of the biosphere.
Their highly permeable skin used for respiration and hydration and their specialized ecology which makes them dependent on both aquatic and terrestrial habitats cause them to be particularly susceptible to environmental perturbations.
Habitat loss, fragmentation and degradation are the most significant threats to amphibians (e.g. Beebee and Griffiths, 2005; Temple and Cox, 2009). Numerous
consequences of the intensification of agriculture, draining of wetlands, river regulation, and impassable barriers such as urban areas and roads are involved in this decrease. (e.g.
Hitchings and Beebee, 1997; Ray et al., 2002; Wood et al., 2003). Because most amphibian species have low dispersal abilities they are particularly affected by the severe effects of habitat fragmentation (see review Cushman, 2006). Pollution, invasive species and the rapid dispersion of diseases such as the chytrid fungus (Batrachochytrium dendrobatidis) and the Ranavirus (FV3) have also accelerated the decline of amphibians (Berger et al., 1998; Daszak et al., 1999; Fisher et al., 2009). This alarming situation induced many studies to investigate the different factors and their correlations to find effective solutions to prevent or reverse amphibian declines (Alford and Richards, 1999; Blaustein and Kiesecker, 2002; Beebee and Griffiths, 2005; Blaustein et al., 2012).
1.2 Conservation genetics
Molecular genetic methods have become a tool of increasing importance for species
conservation (e.g. Frankham, 2005; Schwartz et al., 2007). Highly diverse molecular markers allow assessing the conservation status and the extinction risk of populations by measuring
different parameters such as genetic diversity, connectivity, inbreeding, and the effective population size. These methods enable researchers to assess which populations urgently need supportive measures and which populations are or are not the best for effective reintroduction and restocking measures. For example inadvertently using an already inbred population for reintroductions can lead to reduced fitness in the new colonies, as happened in South Australian koalas (Seymour et al., 2001). Additionally, crossinghighly genetically
differentiated populations can lead to a hybrid breakdown and thus reduced fitness (e.g. Huff et al., 2011). For this purpose populations can be assigned to Management Units (MUs), identified as sets of populations with distinct allele frequencies (Moritz, 1994a; Moritz, 1994b) to which conservation efforts should be directed.
Phylogeographic analyses, mostly based on sequencing of mitochondrial DNA (cyt b, d-loop, COI), can reveal distinct genetic lineages, evolved by different glacial refugia or postglacial colonisation routs. For example two distinct Rana temporaria lineages were described in Eastern and Western parts of Europe forming a contact zone in northern Germany. Furthermore, they found evidence for an Irish glacial refugium (Palo et al., 2004;
Schmeller et al., 2008; Teacher et al., 2009). The boundaries of such contact zones and genetic lineages are important to know for conservation management to decide the right strategies and not to accidentally mix distinct clades. Another study clarified the status of Pelophylax lessonae in Southern England (Snell et al., 2005). Originally the species has been considered as an introduction into Britain, with Italy as most likely source. However, the genetic analyses supported its native status in Britain with a possible colonisation route via Poland and Hungary. This has prompted a programme for re-establishing the clade in England.
1.3 The European tree frog 1.3.1 Characteristics
The family of the tree frogs (Hylidae) is highly diverse, currently containing 40 genera and 901 species (Frost, 2011). In Europe six species of the genus Hyla occur: Hyla arborea (Linnaeus, 1758; European tree frog), Hyla intermedia (Boulenger, 1882; Italian tree frog), Hyla meridionalis (Boettger, 1874; Mediterranean tree frog), Hyla molleri (Bedriaga, 1890;
Figure 1.1: Calling tree frog male (Foto: Michael Werner)
Iberian tree frog), Hyla orientalis (Bedriaga, 1890; Shelkovnikov's tree frog), and Hyla sarda (De Betta, 1853; Tyrrhenian tree frog ). In Germany only the European tree frog, also known as the Common tree frog, can be found.
The European tree frog is one of the smallest European anurans (Figure 1.1). The body length of adult individuals ranges from 27 mm to 50 mm (Tester, 1990; Friedl and Klump, 1997). Their dorsal skin is smooth and bright green. Depending on temperature,
“mood”, and substrate, the coloration varies from yellowish to green, grey, or dark brown. The ventral side and the inner surface of the limbs are whitish to light grey
with granular skin. On both sides a dark lateral stripe goes from the nostrils over the
tympanum to the inguinal region, where it forms the inguinal loop. Characteristic for the tree frogs are their finger- and toe tips expanded into microscopic structured discs, which enable them to climb smooth plants. Males can be detected by the yellowish to brownish subgular vocal sac, while females have a white and smooth throat.
The breeding season starts between late March and early May and ends between early June and mid-July (Schneider, 1966; Schneider, 1971; Tester, 1990; Grosse, 1994). The breeding ponds are characterized by rich submerged vegetation, shallow areas and exposure to the sun (Grosse and Nöllert, 1993). While males spend several nights at the breeding site, females typically stay for only one night. Friedl and Klump (2005) observed that the duration of male chorus attendance reflects male quality. Females deposit several clumps with a total clutch size of 150 to 450 eggs (Clausnitzer and Clausnitzer, 1984). In Eastern Europe clutch sizes up to 1000 eggs per female were observed (Bannikov et al., 1985). The majority of the tadpoles in Central Europe complete metamorphosis between June and August.
After the mating season the tree frogs migrate to their summer habitat, usually within the radius of 500 m of the breeding site. Single individuals, especially juveniles migrate greater distances up to 3400 m (Fog, 1993). They can be found in trees, bushes, perennial plants or riparian vegetation. Important are sunny places with a moist microclimate and a
complex vegetation structure (Stumpel, 1993). In the autumn the frogs migrate to their winter habitat. Deciduous and mixed forests with dense layers or piles of leaves and brushwood, copses, crevices and caves offer frost free places for hibernation (Nöllert and Nöllert, 1992;
Geiger, 1998; Grosse, 2009).
The lifespan of European tree frogs can reach in the wild 4 - 6 years (Stumpel and Hanekamp, 1986; Tester, 1990; Friedl and Klump, 1997). In captivity ages up to 22 years have been reported (Bannikov et al., 1985). Year-to-year survival rates were found to range between 20 and 44 % and in cold winters even lower (Tester, 1990; Friedl and Klump, 1997).
Tester (1990) determined a population turn over rate of only three years.
1.3.2 Distribution
Hyla arborea is widely distributed across the European continent (Figure 1.2). It occurs from North West Iberia and France eastwards to Western Russia and the Caucasian region, and southwards to the Balkans and Turkey. Except for southern and eastern Denmark and extreme southern parts of Sweden it is absent from Scandinavia (Kaya et al., 2009). It is a lowland species that has been recorded at a maximum altitude of 1,000 m a.s.l. in the Carpathian Mountains (Zavadil, 1993).
Figure 1.2: Distribution map of the European tree frog (Hyla arborea), modified from IUCN (2009).
1.3.3 Conservation status and major threats
The European tree frog is listed on Appendix II of the Berne Convention and on Annex IV of the EU Natural Habitats Directive. Therefore, it is subject to a strict protection system.
Although the species is listed in the IUCN Red List in the Least Concern category, the overall population shows a decreasing trend (Kaya et al., 2009). While the species is common in suitable habitats in parts of its range, it has been reported to be fragmented and in significant decline over large parts of its Western European distribution (e.g. Fog, 1995; Baker, 1997;
Gasc et al., 1997). The species is protected by national legislation in many countries. In the German Red Lists it is categorised in five states as Vulnerable, in six states as Endangered, in three states as Critically Endangered, and in one state (Berlin) as Extinct (Bast et al., 1992;
Podloucky and Fischer, 1994; Bitz and Simon, 1996; Jedicke, 1996; Laufer, 1999; Rau et al., 1999; Schlüpmann and Geiger, 1999; Nöllert et al., 2001; Beutler and Rudolph, 2003; Klinge,
2003; Brandt and Feuerriegel, 2004; Meyer and Buschendorf, 2004; Schneeweiß et al., 2004;
Kühnel et al., 2005; Flottmann et al., 2008)
The European tree frog is affected by habitat fragmentation and habitat degradation (Grosse, 1994; Tester and Flory, 1995; Pellet et al., 2004a; Pellet et al., 2004b). The loss of calling and breeding sites and the introduction of fish (Filoda, 1981; Clausnitzer, 1983;
Bronmark and Edenhamn, 1994) are the main reasons for population decline.
1.3.4 Conservation genetics in the European tree frog
Some conservation genetics studies on the European tree frog have already been conducted.
One of the first studies used allozymes to investigate the status tree frogs in Sweden (Edenhamn et al., 2000). Low genetic variation was found in comparison with continental populations. The development of species specific microsatellite-primers by Arens (2000) and Berset-Brändli (2008) prompted more studies that assessed the genetic status of tree frog populations in Denmark, the Netherlands, and Switzerland (Andersen et al., 2004; Arens et al., 2006; Angelone and Holderegger, 2009; Dubey et al., 2009; Angelone et al., 2011). Most of these populations suffered from habitat loss and habitat fragmentation which was apparent in the genetic data. The lowest genetic diversity was found in the Danish populations on Lolland and was associated with an increased larval mortality (Andersen et al., 2004).
However, in Switzerland the genetic analyses provided compelling evidence for the success of conservation and connectivity measures in the Reuss valley, leading to an enhanced tree frog migration among breeding sites within distances up to 4 km (Angelone and Holderegger, 2009). In a study in 2005 I started to shed light on the genetic situation of tree frogs in
Germany. Bayesian analyses indicated that the tree frog occurrences near Hannover were fragmented into four genetically distinct clusters. However, the genetic variation was relatively high compared to the values in the adjacent countries. Moreover, within the Hannover region, I identified a potential source population for an introduced and previously unknown population in southwest Hannover (Krug and Pröhl, submitted).
A phylogenetic study of the circum-Mediterranean Hyla species was carried out by Stöck et al. (2008). Their data suggest the Balkan region as a possible Pleistocene refugium with the subsequent colonisation of Middle and Western Europe by a single genetic lineage.
1.4 Aims of the study
The alarming situation in the European tree frog has evoked a great number of conservation measures. My aim is to support such measures with genetic analyses on different geographical levels by providing information for effective and successful management. My thesis consists of two geographic levels of analyses and interpretation which I will describe briefly.
1.4.1 Phylogeography in Germany and adjacent areas
For several European amphibian species different postglacial colonisation routes have been described as forming contact zones in the German region amongst others (e.g. Weitere et al., 2004; Schmeller et al., 2008). The borders of such lineages are important for making correct decisions and to develop effective strategies in conservation management of threatened species and therefore need to be delineated for the European tree frog. Stöck et al. (2008) made a first attempt with a European wide analysis. Since their purpose was a wide-ranging phylogenetic study only few sample sites from Germany were included (four individuals from three sites in Germany). Important genetic structures could have been overlooked. Therefore, my goal is – with a more extensive sampling network – to analyse the phylogeographic structure, i.e. the existence of distinct genetic lineages of tree frogs in Germany. The results will be discussed in the context of already published mitochondrial data of other European sample sites.
1.4.2 Management Units in Lower Saxony and adjacent areas
In Lower Saxony the current distribution of the European tree frog is very patchy with some larger occurrences in the lowlands. Severe declines have been observed mainly in the second part of the last century (Manzke and Podloucky, 1995). In some places measures for
conservation management have already been implemented and initial success has been observed (e.g. Clausnitzer, 2004 (Celle); Buschmann et al., 2006 (Steinhuder Meer); LaReG, 2007 (Braunschweig); Richter and Mügge, 2012 (Diepholz)). Units for conservation
management need to be delineated for the European tree frog in order to support these conservation activities.
The second aim of this study is to perform a large scale conservation genetic survey of the European tree frog across its distribution in Lower Saxony and adjacent areas and to obtain insight into contemporary as well as historical processes. My special interest is to assess genetic diversity and to define units for conservation management.
2
Defining units for conservation management for the
European tree frog (Hyla arborea) in Lower Saxony
and adjacent areas
2.1 Abstract
The European tree frog Hyla arborea has suffered from dramatic population declines in the last decades and has therefore been categorised as threatened in many Red Data lists. In Lower Saxony in Germany the current distribution of the tree frog is very patchy with some main occurrences in the lowlands. For supporting effective conservation measures this study aims to assess genetic diversity and to define units for conservation management.
Across the tree frog distribution area in Lower Saxony and adjacent areas 237 individuals were sampled at 14 sites (~ 3 - 250 km apart from each other). All samples were genotyped with eight microsatellite loci and twelve sites were sequenced for an mtDNA cytochrome b fragment.
While all but one of the microsatellite pairwise Dest and FST values showed significant genetic differentiation (Dest: 0 - 0.46, FST: 0 - 0.18), Bayesian analyses indicated common structures forming seven distinct genetic clusters. The cytochrome b haplotype distribution highlights the former connection of the currently fragmented populations along the river Elbe.
Since genetic diversity was relatively high, each of the sampled tree frog occurrences should have the potential to recover to a stable population size when applying appropriate local conservation measures. For new resettlement projects identified genetic structures should be considered for the choice of source populations. Where possible, it would be preferable to reconnect originally linked occurrences that are now separated in different conservation unites due to habitat fragmentation and genetic drift.
2.2 Introduction
Amphibian populations around the world are seriously affected by severe declines in the last decades. In Europe habitat loss, fragmentation and degradation are the most significant threats to amphibians (Temple and Cox, 2009). For most of these species intensive conservation efforts are needed to prevent them from further decline and to regain a favourable
conservation status.
Stabilization of weakened populations can be achieved e.g. by improving the habitat, constructing new breeding ponds and connecting populations to stable networks (e.g. Hyla arborea: Tester and Flory, 2004; Bombina bombina: Brockmüller and Drews, 2009; Triturus cristatus and Pelobates fuscus: Rannap et al., 2009). However, in cases where extreme fragmentation isolates populations, reconnection is difficult. Highly isolated populations already suffering from inbreeding and low genetic diversity can be strengthened by
introducing translocated individuals from other populations with the aim of increasing fitness and genetic diversity (e.g. Vipera berus: Madsen et al., 1999). This is a difficult task to undertake because mixing different gene pools can in the best case result in a higher fitness of the offspring (hybrid vigor), but it could also result in outbreeding-effects with fitness
depression (hybrid breakdown) in subsequent generations which may drive the population into further decline. For example the introduction of individuals into a small inbred
population of Florida panthers (Puma concolor coryi) led to a higher survival rate of hybrid offspring and helped to recover the population (Pimm et al., 2006). On the other hand, mixed- source reintroductions of slimy sculpins (Cottus cognatus) have led to outbreeding depression in second-generation descendents. In this case, source populations were genetically
differentiated by an FST of 0.32 (Huff et al., 2011).
Therefore, to minimize negative effects when translocations are necessary or mixed- source introductions are planned, it is essential to reveal genetic structures for effective
species conservation management. Potential hidden barriers and units for conservation need to be delineated.
Units for conservation management have been defined by using genetic analysis for several endangered species such as koalas (Lee et al., 2010), harbour porpoises (Wiemann et al., 2010) or Larch Mountain salamander (Wagner et al., 2005). These studies showed that in all these species limited migration due to natural and anthropogenic barriers formed genetic
distinct units. For purposes of conservation management the studies recommended all handling each unit individually.
The European tree frog is a species that showed long-term decline in much of its Western European distribution, mainly caused by habitat loss, fragmentation and degradation.
In Lower Saxony in Germany the current distribution of the tree frog is very patchy with some main occurrences in the lowlands (Figure 1). Severe declines have been observed mainly in the second part of the last century (Manzke and Podloucky, 1995). At some places measures for conservation management are already implemented and first successes have become apparent (e.g. Clausnitzer, 2004 (Celle); Buschmann et al., 2006 (Steinhuder Meer);
LaReG, 2007 (Braunschweig); Richter and Mügge, 2012 (Diepholz)).
For supporting such conservation activities, units for conservation management need to be delineated for the European tree frog. Most genetic analyses of the European tree frog have been conducted on a very local level and measured the genetic structure and diversity in more or less fragmented metapopulation systems (e.g. Edenhamn et al., 2000; Andersen et al., 2004; Arens et al., 2006; Angelone and Holderegger, 2009; Dubey et al., 2009). The aim of my study was to perform a large scale conservation genetic survey of the European tree frog across its distribution in Lower Saxony and adjacent areas. To allow insight on contemporary as well as historical processes I used eight microsatellite loci and mtDNA cytochrome b sequences. My specific aim was to assess genetic diversity and to define units for conservation management.
2.3 Materials and methods
2.3.1 Sample collection and preparation
Fourteen sites were sampled across the tree frog distribution in Lower Saxony and adjacent distributions in North Rhine Westphalia and Saxony Anhalt. I chose one sample site in each main occurrence of the tree frog in this region (Figure 2.1). In the occurrence near Hannover however, I sampled four sites: two in the west of Hannover (KZ, KO) and two in the east of Hannover (KH, BH) for a comparison with small scaled spatial distances. In total 237 individuals were sampled with 5 - 22 individuals per sample site (see Table 2.1). Genetic material was collected by tips of tadpole tails and by buccal swabs of adult frogs. The adults
were collected from the choruses during the breeding season in spring 2005 and 2008.
Tadpoles were sampled in summer 2007. DNA from the tail clips was fixed in 99 % ethanol and extracted using a proteinase K digestion followed by a Phenol-Chlorophorm protocol (Sambrook et al., 1989) and stored at -20 °C. DNA was extracted from the buccal swabs with an Invisorb Spin Swab Kit (Invitek) following the manufacturer’s protocol and stored at -20
°C.
Figure 2.1:Current distribution of the European tree frog in Lower Saxony and adjacent areas on the basis of TK25-quadrants (grey squares) (1994-2010 in Lower Saxony (NLWKN, 2011), 1993-2006 in North Rhine Westphalia (LANUV and NRW, 2011) and 1990-2000 in Saxony Anhalt (Meyer et al., 2004)). Dashed lines denote state borders, dots denote sample sites.
Table 2.1: Overview of sample sitesa : Samples from adult frogs,t : samples from tadpoles, Ho: observed heterozygosity, He: expected heterozygosity, SD: standard deviation, FIS: inbreeding coefficient with bold values for significant difference after 1000 permutations, R: mean allelic richness over all loci, h: haplotype diversity, π: nucleotide diversity, N: number of sampled individuals, (♀): number of females included when adult frogs were sampled, NA: mean number of alleles over all loci IDSample site mean Ho ± SDmean He ± SDFISNARhπ [%] N (♀) QUQuakenbrück a 0.813 ± 0.2420.754 ± 0.137-0.0894.130.000.005 (0) WKWesterkappeln a 0.703 ± 0.2110.598 ± 0.157-0.1913.630.540.068 (0) EKEspelkamp a 0.800 ± 0.1570.764 ± 0.085-0.0505.505.360.000.0012 (0) KZKananohe Zentrum a 0.709 ± 0.1950.679 ± 0.129-0.0455.134.680.000.0020 (1) KOKananohe Ost a 0.739 ± 0.2200.692 ± 0.085-0.0724.504.46- - 11 (1) KHKolshorn a 0.778 ± 0.1390.720 ± 0.089-0.0836.505.420.530.1320 (0) BHBeinhorn a 0.731 ± 0.1360.701 ± 0.089-0.0445.634.91- - 20 (0) BABassumt 0.788 ± 0.1030.759 ± 0.090-0.0385.755.210.410.0520 RURuschwedel a 0.765 ± 0.1120.740 ± 0.070-0.0355.504.900.680.1418 (4) WGWolfsburg-Gifhorn a 0.810 ± 0.1570.800 ± 0.074-0.0137.506.520.610.0820 (1) STStrothe a/t 0.762 ± 0.1610.728 ± 0.130-0.0486.635.640.190.0221 ANAmt Neuhaus a 0.744 ± 0.1460.747 ± 0.0840.0046.505.490.660.1522 (0) SWSalzwedel t 0.631 ± 0.2280.705 ± 0.1750.1086.135.160.570.1120 PWPevestorfer Wiesen a 0.819 ± 0.1250.766 ± 0.084-0.0706.505.570.420.0520 (0)
For microsatellite analyses 5 - 22 individuals from each sample site were used. A total of eight polymorphic microsatellite loci (WHA1-9, WHA1-20, WHA1-25, WHA1-60, WHA1- 67, WHA1-103, WHA1-104, and WHA1-140) previously isolated by Arens et al. (2000) were amplified following the authors’ protocol, except for the annealing temperature for WHA1- 20, which was changed to 64.6 °C. PCR products were genotyped using the capillary
sequencer MegaBace 1000 (Amersham Bioscience). Allele scoring was performed using the software Genetic Profiler v. 2.2.
Fragments of 901 bp of cytochrome b (cyt b) of 5 - 20 individuals from each sample site, except KZ and BH in the Hannover population, were amplified via PCR using the primers MVZ 15-L (5′- GAACTAATGGCCCACACWWTACGNAA -3′) and Cytb AR-H (TAWAAGGGTCTT CTACTGGTTG) from Moritz (1992) and Goebel (1999). The PCR reaction (25 µl) consisted of 20 - 100 ng DNA, 1 µl of each primer (10 µM), 0.8 µl dNTP’s (10 mM 5PRIME), 2.5 µl 10x advanced Buffer (5PRIME), 1.25 U Taq DNA Polymerase (5PRIME), and 17.45 µl H2O. PCR conditions were as follows: an initial denaturation at 94 °C for 3:00 min; 35 cycles at 94 °C for 45 s, annealing temperature of 50 °C for 45 s, extension at 65 °C for 1:00 min. The PCR products were sent to the Macrogen Company (Seoul, South Korea) for purification and sequencing with an ABI3730XL genetic analyzer (Applied Biosystems).
2.3.2 Statistical analysis
2.3.2.1 Historic structure: Analysis of mtDNA
Both directions of the cytochrome b sequences were assembled using the computer software SeqMan™ II (DNASTAR, Inc., Konstanz, Germany). Multiple sequence alignments were performed in MEGA 4 (Tamura et al., 2007) using the Muscle algorithm (Edgar, 2004) and all variable sites were confirmed by visual inspection of the chromatograms. The same program was used to calculate p-distances between sample sites (Tamura et al., 2004).
Haplotype diversity (h) and nucleotide diversity (π) (Nei, 1987) were determined with ARLEQUIN v. 3.11 (Excoffier et al., 2005). A haplotype network of the cyt b data set was constructed via the statistical parsimony analysis of the program TCS 1.21 (Clement et al., 2000) using the default settings.
2.3.2.2 Recent structure: Analysis of Microsatellites
Microsatellite-data were checked for null alleles, stuttering and allelic dropout using MICRO- CHECKER (Van Oosterhout et al., 2004). The program FSTAT v. 2.9.3 (Goudet, 1995) was used to test for genotypic disequilibrium of all pairs of loci in each sample and to calculate average allelic richness per population, which measures the number of alleles per locus corrected for different sample sizes. For the calculation of average allelic richness sample sites with less than ten individuals (QU and WK) were disregarded.
For each sample site and locus the observed and expected heterozygosity (Nei, 1987), and deviation from Hardy-Weinberg equilibrium (HWE) (Guo and Thompson, 1992) were determined with ARLEQUIN v. 3.11 (Excoffier et al., 2005). GENEPOP v. 4.1 (Rousset, 2008) was used to test for a global deviation from HWE in each sample site. I calculated the
inbreeding coefficient FIS per sample site (Weir and Cockerham, 1984) using GENETIX v. 4.05 (Belkhir et al., 1996-2004) and tested the significance with a permutations test (1,000
permutations).
Genetic differentiation between the sample sites was calculated as pairwise FST values (Weir and Cockerham, 1984) in ARLEQUIN (Excoffier et al., 2005). However, since FST depends on marker variability and has been shown to be an imprecise estimate for genetic differentiation when applying microsatellites (Hedrick, 2005; Jost, 2008), I additionally calculated pairwise Dest (Jost, 2008), a substitute measure of genetic differentiation, using the R package DEMEtics (Gerlach et al., 2010). Significance was calculated by 10,000
bootstraps.
In order to test for isolation by distance I conducted a mantel test for correlation between pairwise genetic distances (FST and Dest) and pairwise geographic distances, implemented in IBDWS 3.23 (Jensen et al., 2005). As proposed by Rousset (1997) for populations in two-dimensional habitats, geographical distance was log-transformed and genetic distance was expressed as FST /(1 − FST) respectively Dest /(1 − Dest). Significance for r ≤ 0 was assessed via 10,000 bootstraps. Sample site QU was omitted from these analyses because of the small sample size (N = 5). The linear geographic distances among sample sites were calculated in ArcView GIS 3.3 using the Distance Matrix extension (Jenness, 2005).
Two Bayesian clustering models were conducted to infer genetic clusters. First I used STRUCTURE (Pritchard et al., 2000). The aim of this method is to define clusters of individuals
on the basis of their genotypes at multiple loci using a Bayesian procedure. It attempts to find population clusters by reducing linkage disequilibrium and deviations from the Hardy-
Weinberg equilibrium within inferred clusters. The user specifies a priori the number of population clusters (K) and estimates the log likelihood Pr(X|K) for this model. For finding the most likely number of genetic clusters the log likelihood Pr(X|K) is always calculated for a series of K values.
All STRUCTURE runs used 50,000 iterations after a burn-in period of 50,000. Because of the patchy distribution of the tree frog occurrences in this region and the large distances between most sample sites, I used the assumption of the no-admixture model and independent allele frequencies. This prior means that allele frequencies are expected in different clusters to be reasonably different from each other. Twenty runs were performed for each K. The range of possible Ks tested was from 1 to 14, according to the number of sampled breeding sites. I calculated the average log likelihood Pr(X|K) (given by the estimated Ln Prob of data = Ln P(D) in the software result output) for each K across all runs. Since it is not always
straightforward to detect the true number of K, I included the ΔK statistics proposed by Evanno (2005), using Structure Harvester v.0.6.8 (Earl and vonHoldt, 2012).
Secondly, I applied GENELAND version 3.2.2 (Guillot et al., 2005a; Guillot et al., 2005b). This software uses again a Bayesian method to detect population structure but considers spatial information of the individuals. The number of genetic clusters (K) was determined by independently running the MCMC ten times, allowing K to vary from 1 to 14 to verify the consistency of the inferred K. The number of MCMC iterations was set to
100,000 per run with a thinning of 100. The uncertainty of spatial coordinates was set to 0 km and the uncorrelated frequency model was used without the assumption of null alleles.
2.3.2.3 Biogeographic zones
To identify biogeographical boundaries or zones where genetic differences between pairs of populations were largest I used the Monmonier’s algorithm as implemented in Barrier 2.2 (Manni et al., 2004). I computed the first three barriers based on cytochrome b data (p- distances) and on microsatellites (FST values). For the microsatellite data I tested the robustness of the barriers by 100 bootstrapped FST-matrices calculated via the R-package
Hierfstat (Goudet, 2005). I then considered only barriers with more than 50% support. To assess significances in this study I applied sequential Bonferroni corrections (Rice, 1989) to all multiple comparisons.
2.4 Results
2.4.1 Mitochondrial sequence analysis
I revealed 11 haplotypes of the cytochrome b fragment which differed by ten variable sites and nine parsimony informative sites (Figure 2.2; Appendix 1 and 2). Most haplotypes were unique to one sample site except haplotype Hy-1, Hy-2, and Hy-5. Hy-1 (blue) and Hy-5 (red) showed a broad distribution almost over the complete sampling area. Haplotype Hy-2 (green) was restricted to five sample sites in the north east (Figure 2.3).
P-distances were low, varying between 0 and 0.4 % (Appendix 3). The highest estimates of mtDNA diversity were found in RU (h = 0.68, π = 0.14 %) and AN (h = 0.66, π = 0.15 %). The lowest values were found in QU, EK, and KZ (all: h = 0, π = 0 %).
Figure 2.2: Haplotype network of 11 distinct haplotypes of cyt b of Hyla arborea (901 bp) in Lower Saxony and adjacent areas. Each haplotype is represented by one circle and colour. The size of the circles corresponds to the haplotype frequency. Lines between haplotypes denote mutational steps between sequences.
Figure 2.3:Distribution of cyt b haplotypes in the sample area of Lower Saxony and adjacent areas. Each haplotype is represented by one colour corresponding to the colours in the haplotype network Figure 2.2.
2.4.2 Microsatellite analysis
The eight microsatellite markers examined were polymorphic with seven to sixteen alleles per locus. The analysis using Micro-Checker uncovered signs of null alleles for the locus WHA1- 67 in the sample site KO and for the locus WHA1-140 in the sample site SW. As null alleles for the two loci were found at a single sample site only, I did not adjust for null alleles.
Furthermore this analysis revealed no evidence for large allele dropout or scoring errors due to stuttering.
Deviation from Hardy-Weinberg-Equilibrium was found for WHA1-60 with a significant heterozygosity excess in the sample sites KH, BH and AN. For WHA1-104 a deficiency was found in KH. The global test for HWE over all loci in each population resulted
in no significant deviation. Significant values for the inbreeding coefficient FIS were obtained for the sample sites SW (FIS = 0.108), WK (FIS = -0.191), PW (FIS = -0.070) and KH (FIS
= -0.083). No Linkage (genetic) disequilibrium was found between any pair of loci.
Since Berset-Brändli et al. (2007) found the locus WHA1-6 to be sex linked with a suppressed recombination in males, I tested its influence on the outcome of all analyses.
Excluding the sex-linked locus increased the FIS values slightly. Only sample site SW showed now significant signs for inbreeding (FIS: 0.130). Expected heterozygosity values however did not change remarkably after excluding WHA1-60 (mean He locus WHA1-60 included: 0.73;
excluded: 0.72). An influence of WHA1-60 on the results of all other analyses was not evident. Therefore, I decided to keep this locus in the analyses.
Genetic differentiation calculated as pairwise Dest- and pairwise FST values were significant in all cases except between the two sample sites in the West of Hannover KZ and KO (Table 2.2). In general Dest values were higher than FST values.
The Mantel test for Isolation by distance showed a significant but low correlation between genetic and geographic distances (Figure 2.4; Dest: r = 0.40, P = 0.0007; FST: r = 0.40, P = 0.0003). Indicating, that genetic differentiation is partially explained by geographic distance among sites.
Table 2.2: Pairwise Dest values (below matrix) and pairwise FST values (upper matrix) between sample sites; bold = significant difference after sequential Bonferroni correction. QUWKEKKZKOKHBHBARUWGSTANSWPW QU00.1140.0530.1210.1250.1170.1100.0460.1120.0540.0870.0910.0910.067 WK0.19800.1080.0870.1210.1270.1630.1040.1800.1130.0890.1340.1540.153 EK0.1360.28500.0510.0430.0750.0850.0650.0850.0680.0930.0710.1000.074 KZ0.3510.2230.1470-0.0010.0870.1050.0750.1250.0880.0880.0970.0960.096 KO0.3870.3020.145-0.00400.0850.1070.0730.1180.0880.0870.0910.0910.096 KH0.4490.3480.2550.2740.27000.0360.0600.0630.0720.0580.0940.1080.086 BH0.4190.4260.2750.3130.3250.09600.0810.0920.0800.0750.1170.1180.092 BA0.2190.2800.2570.2480.2520.2090.26600.0680.0370.0710.0790.0780.070 RU0.4260.4640.2920.3560.3610.1940.2770.22900.0600.0670.0730.0990.068 WG0.1880.3460.3070.3310.3340.2700.2840.1500.22600.0500.0730.0820.063 ST0.3270.2550.3460.2870.3010.1960.2450.2560.1900.18300.0620.1020.068 AN0.2960.3720.2570.3030.3120.3120.3710.2820.2500.2930.23300.1070.084 SW0.2960.3970.3350.2590.2480.3760.3860.2660.3230.2870.3600.38700.079 PW0.2730.4130.2990.2570.2920.3200.3100.2760.2580.2590.2330.3050.2670
Figure 2.4:Isolation by distance plots. (a) Dest /(1 - Dest) versus log geographic distance and (b) FST /(1 - FST) versus Log geographic distance. Lines are the RMA (reduced major axis) regression.
For the STRUCTURE analysis both approaches to determine the correct number of genetic clusters, the ΔK statistics by Evanno (2005) and the average log likelihood- values Pr(X|K), peaked clearly at K = 7 (Figure 2.5; Figure 2.6). One main cluster in the West of Hannover consisted of the sample sites WK, EK, KZ, KO (red). The sample sites KH and BH in the East of Hannover formed an extra cluster (blue) separated from the sites in the West. Sample site WG was admixed with large parts of the cluster build by BA (yellow) and RU and ST (green).
The sample sites in the East – SW, PW and AN were separated in three further clusters (orange, grey and light-blue) whereas the latter formed one cluster together with QU in the West of Lower Saxony.
Figure 2.5:Mean values of estimated Ln probability of data (LnPD) for each K (a) and delta K (b)
Figure 2.6: STRUCTURE bar plot for K = 7; QU, WK, EK etc. = sample sites, separated by fine black lines. Each individual is represented by a single vertical line broken into K-coloured segments, with lengths proportional to each of the K-inferred clusters.
In consistency with the STRUCTURE result, in the GENELAND analysis the highest average log posterior probability was found for seven genetic clusters. Cluster 1: WK, EK, KZ, KO;
cluster 2: KH, BH; cluster 3: QU, BA, WG; cluster 4: RU, ST; cluster 5: SW; cluster 6: AN;
and cluster 7: PW. This is the same clustering as found by the Structure analysis except for the assignment of sample site QU. (Figure 2.7)
Figure 2.7: Map of estimated cluster membership for K = 7 by GENELAND analysis. Each cluster is shaded in a single colour.
2.4.3 Biogeographic zones
The most significant genetic discontinuities among sampled locations were estimated on the basis of microsatellite FST-values and cyt b -p-distances. Obtained barriers are marked with lines in figure 2.8. Two barriers were supported by both markers (microsatellites and cyt b).
One barrier separated KZ from KH (KO and BH were not considered in sequence analysis), and a second barrier was found between WK and EK.
Figure 2.8: Most important barriers to gene flow from the BARRIER analysis (Manni et al., 2004). Red Lines indicate most significant barriers to gene flow (> 50 % bootstrap support) estimated on microsatellite FST-values.
Significance of barriers are given as percent bootstrap. Barriers estimated on the basis of cyt b p-distances are marked with grey lines ranked I-III, in order of decreasing magnitude. In green the Delaunay triangulation, in blue the Voroni tessellation between sample sites, used to calculate borders to neighbouring sample sites.
2.5 Discussion
I analysed the broad scaled genetic structure and variation of the Lower Saxonian tree frog occurrences to aid conservation management implementation. Cyt b sequences showed low variation but distinct geographic-genetic pattern was revealed. Using microsatellite analysis I found seven distinct genetic clusters. Genetic diversity was high in most sample sites.
For supporting measures of effective conservation management, the identification of conservation units or management units (MUs) is critical (Palsbøll et al., 2007).
“Management Units are defined as demographically independent breeding units and are identified as populations having distinctive allele frequencies” (Moritz, 1994b). Their recognition is fundamental to proper short-term management (Moritz, 1994a). The genetic clusters revealed by STRUCTURE and GENELAND can more or less been used to delineate such
management units as conducted e.g. for Koalas in the area of Sidney (Lee et al., 2010).
Palsbøll et al. (2007) propose that “MU status should only be assigned when the observed estimate of genetic divergence is significantly greater than a predefined threshold value.”
Unfortunately, corresponding studies are missing especially for amphibians defining such a specific threshold value. The only study that defined MUs in an anuran species on a
comparable geographic scale was conducted by Dolgener et al. (2012) for yellow bellied toads (Bombina bombina) by applying microsatellites and d-loop sequences. Based on cyt b sequences and RAPD loci Wagner et al. (2005) defined two distinct conservation units in Larch Mountain salamanders in the North and South of the Columbia River. However, since he found distinct monophyletic groups in cyt b the revealed units should be considered on the level of ESU’s (evolutionary significant units) rather than as management units.
2.5.1 Genetic structure and conservation units
Microsatellite pairwise Dest and FST values showed all significant genetic differentiation except the two closest sites in the West of Hannover. However, mtDNA and Bayesian
analyses of the microsatellites still display distinct relationships between currently fragmented occurrences.
In the Northeast of Lower Saxony, the distribution of the cyt b haplotype Hy-2 (green in Figure 2.3) highlights the former connection of the populations along the river Elbe. In both Bayesian analyses of the microsatellites the localities RU and ST form a common cluster. In earlier distribution maps, a sparse but nearly continuous distribution up into the 1980s was apparent in this area (Appendix 5). However, today both populations are separated by a large gap in the distribution forming demographically independent breeding units and should be considered as separate management units.
Interestingly AN, PW, and SW which, in current and former distributions constitute a relatively well connected area, present distinct genetic clusters in the Bayesian analyses.
Furthermore, a major barrier to gene flow was revealed around SW and PW. The significant FIS values found for SW indicate that the separation of this site may have resulted from genetic drift caused by inbreeding. For now AN, PW, and SW should be regarded as separate management units. Nonetheless, efforts should be orientated to a reconnection of this area.
In contrast, the Western occurrences show a relatively strong geographic structure.
Because of the common cluster of KZ, KO, EK, and WK in the Bayesian analyses of microsatellites one could assume that there was migration among these populations at least until the recent past. This would imply that these occurrences could be considered as one management unit. However, on the basis of the completely different haplotypes in EK and KZ and the large geographic distance between each other for now both occurrences should be handled as different management units. A male biased dispersal could be a possible
explanation for the genetic pattern. However, there are no specific studies on the phenomenon of different dispersal abilities in European tree frogs. A molecular analysis of further sample sites of the tree frog occurrence near EK would be helpful to clarify the relationships between both sites.
Additionally, WK should be regarded as an extra management unit considering the observed barriers. The low sample size of five individuals in QU does not allow it to be assigned as a distinct conservation unit in this locality. A more intense sampling in this area is required.
A clear assignment to a distinct management unit is warranted for KH and BH in the East of Hannover. Microsatellite and sequence data show a clear separation of the occurrences in the West of Hannover, underlined by strong barriers. Interestingly the genetic divergence of the sample sites KZ and KO in the West of Hannover and the sample sites KH and BH in the East of Hannover into two different clusters is similar to the divergence among other occurrences in lower Saxony separated by much larger geographic distance. This observation raises the question of how fast and by which means such genetic differentiation could be generated?
One possibility is that recently constructed motorways in combination with genetic drift due to population bottlenecks contribute to population differentiations which are apparent not only in microsatellite allele frequencies but also in mitochondrial haplotype frequencies. One central question here is whether these relatively young barriers (motorways expanded in the 1960ies, and dense urban areas) are the only reasons leading to this
differentiation or whether more ancient and natural circumstances, as can be hypothesised by the mtDNA data, also played a role? Maybe the assumption that these populations were formerly linked (Manzke and Podloucky, 1995) need to be reassessed.
Rather inconclusive is the assignment of WG. In the STRUCTURE analysis this site appears to be an admixture of the adjacent clusters. Very interesting is the similarity between BA and WG apparent in genetic distances and in Bayesian analyses – although they are 140 km apart from each other. A possible explanation is their location in the area of influence of the “Aller Urstromtal” (ice-marginal valley). It is possible that these populations have been connected in the past by migration along this “valley”, which is still evident by the high genetic similarity. However, an anthropogenic influence by translocation of individuals can not be excluded. I would assign BA to its own management unit because of the apparent current demographic independence of WG.
2.5.2 Genetic diversity
Values of expected heterozygosity (He) as a measure for genetic diversity of the sampled populations was found to vary between 0.60 and 0.80 (mean: 0.73). This is comparable but mostly higher as values found in studies on the European tree frog in neighbouring countries.
Here, fragmented as well as more continuous distributions have been investigated. Mean He values are found between 0.39 - 0.59 in the Netherlands (Arens et al., 2006) and 0.54 - 0.68 in Switzerland (Angelone and Holderegger, 2009; Dubey et al., 2009). The lowest values were found in Denmark on the island of Lolland (0.35 - 0.50). This population had already suffered reduced fitness, indicated by increased larval mortality (Andersen et al., 2004). Such low values have not been reached by any of my sampled populations.
2.5.3 Future goals
Actually it is not known whether certain genetic distances are originally high or low, whether they are driven by natural environmental effects or by anthropogenic effects such as
motorway-barriers, or are e.g. the effect of unknown translocations. The question remains how fast in the case of European tree frogs the revealed genetic differentiations arises, especially, if one considers the populations in the West and East of Hannover which are relatively close to each other. Other sites in Lower Saxony are geographically distant but show similar genetic differentiation. This raises the next question: whether the genetic
differentiation at this level has an impact on the ecological differentiation in the tree frog.
Breeding and fitness tests between populations that have genetically diverged to different degrees would be of interest in this respect.
2.5.4 Conclusion and implications for conservation management
The values for heterozygosity in the surveyed areas are relatively high, and therefore I would not expect fitness-depression to occur in most H. arborea populations in Lower Saxony.
Measures for population recovery should be in the first instance constructing 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)). Even small and fragmentary relict populations can recover to strong populations of high constancy (Schwartze, 2002).
However, if translocations of individuals are necessary e.g. to recover very small and inbred populations or for reintroduction measures, the revealed genetic structures of the Bayesian analyses by GENELAND and STRUCTURE and the identified barriers should be considered, especially as long as it is not known which degree of genetic differentiation could already be enough to cause effects of outbreeding depression. This is an urgent aspect to investigate in future studies. Nonetheless, in the long run a reconnection of originally linked occurrences, which at present are separated into different MUs in consequence of habitat fragmentation and genetic drift, should be achieved.
2.6 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, Landkreis Diepholz, Landkreis Gifhorn, Landkreis Lüneburg, Landkreis Osnabrück, Landkreis Stade, Landkreis Uelzen, Region Hannover, Stadt Wolfsburg, Sachsen Anhalt. I am especially grateful to Annika Ruprecht,
Christina Akman, Frank Weihmann, Günter Krug, Heike Pröhl, Irena Czycholl, Ivonne Meuche, Jana Kirchhoff, Kim Jochum, Matei Balborea, Michael Weinert, and Wiebke Feindt for help during field work. Finally I thank our technician Sabine Sippel for her assistance in the molecular lab.
3
Phylogeographic structure of the European tree frog
(Hyla arborea) in its German distribution area
3.1 Abstract
Knowledge about the existence of different genetic lineages within endangered species is important for conservation management. To assess the phylogeographic structure of the European tree frog across its distribution area in Germany, 372 individuals were sampled at 31 sites and sequence analyses of a mitochondrial gene fragment (cytochrome b) and analyses of eight microsatellite loci were carried out. Sequence divergence between sample sites was low varying between 0 and 0.4 % (overall: 0.2 %) and no distinct genetic lineages were found. Nonetheless, a clear North-South partitioning could be revealed by both molecular markers with the Central German Uplands as probable barrier. Furthermore, the influence of the major rivers such as Elbe, Rhine, and Danube on the phylogeographic structure could be revealed. Concerning future conservation measures, the identified genetic structures should be considered, especially for the choice of individuals if resettlements should be necessary.
