Phylogeographic patterns of the Moroccan lizard - fingered gecko Saurodactylus brosseti

Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti Daniela Filipa G. M. Rosado MSc in Biodiversity, Genetics and Evolution Faculty of Sciences of University of Porto 2014 Orientador Dr. David James Harris, Researcher, CIBIO Coorientador Dr. Catarina Rato, Researcher, CIBIO Todas as correções determinadas pelo júri, e só essas, foram efetuadas. O Presidente do Júri, Porto, ______/______/_________ FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 2 ACKNOWLEDGMENTS First of all I thank my supervisor James for giving me the opportunity to go on this long journey, for allowing me to go on fieldwork and discover my true passion, for all you did for me, for always being honest even when joking, for making fun of me. In the end, I thank you for making me feel like I am a capable grown-up. You are truly the best “supermarket”. I thank Catarina Rato for all the help through the months, for her patience and kindness and for always smiling. To Pedro, the most special person, I thank you for always being with me, for all the smiles and cuddles, for always believing in the best of me and never let me give up. Ana and Isabel Damas, I am so grateful for your friendship, for your honesty and laughs, for all the stupid things that always make me laugh, for forcing me to get out of the house. If you didn‟t exist, you had to be invented! I thank my friends in Coimbra for all the weekends. Every day spent with you was a lesson, a journey to random places, a tipsy night or an amazing day. Most of all, I thank to André Lages for being the best brother in the whole world, for giving me life lessons, and for listening to all of my meaningless life stories. To everyone in CIBIO, especially the ecology lab, for all the music, jokes and fun. It is the best workplace that anyone could ever dream of. Also, to all the people that cared about me and helped me in the analysis and writing through the last months. To everyone that has ever joined me in Porto night outs and let behind the most remarkable memories. A very special thanks to all the people that was with me on fieldwork, mainly to Henrique, Marina, Amanda, Tavares and Fátima which are the most funny people that I ever worked with; I am proud of being friends with you. PS: Fátima, you are a role model and I wish I will be one third of what you are; Marina, você é massa! For all the support my family gave me throughout all my life; no one have ever put up with so much of my craziness than you: Mom, Dad, Ana, Maria and Inês. FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 3 ABSTRACT Various assessments of phylogeographic patterns have been extensively performed throughout Europe, and general patterns discerned however similar studies remain insufficient for other regions such as North Africa. Morocco has multiple geographical barriers, such as the Moulouya River basin, the Rif Mountains and the Atlas Mountains. The latter divides this country into two bioclimatic regions, which in turn is responsible for the high levels of endemism. It has been reported that genetic subdivision in several species is coincident with the orogeny of the Atlas Mountains around 9 Mya, for example in Natrix maura, Agama impalearis and Myotis nettereri. One very old genus of gecko endemic to Morocco is Saurodactylus, comprising three species: S. mauritanicus (Duméril and Bibron, 1836); S. fasciatus Werner, 1931 and S. brosseti Bons and Pasteur, 1957. A previous study demonstrated high variation within S. brosseti, with 11.4% of genetic diversity for ND4, implying complex phylogeographic patterns. This raises several questions, such as if this might represent a species complex, and how diversity may be divided by the known geological barriers in the region. However, sampling was insufficient to address these issues. In order to assess if phylogenetic and geographical patterns of S. brosseti are related to the orogeny of the Atlas Mountains, additional sampling was needed, and therefore two trips to Morocco were carried out in 2013 and 2014. The field surveys resulted in a short note of new range expansions for some species, and in particular Bufo spinosus, Trapelus boehmei, Tropiocolotes algericus, Acanthodactylus erythrurus, Chalcides polylepis and Scutophis moilensis. Accurate species distribution maps are necessary for any conservation efforts, and this highlights the need for more prospection in the region. For the target species, S. brosseti, two different approaches were combined. For a phylogeographic assessment within the species two mitochondrial and three nuclear genes were sequenced and analyzed. Four main lineages can be differentiated with a level of diversity typically observed between species. The orogeny of the Atlas Mountains occurred at about the same time as these lineages split, and therefore may well have been the barrier that led to the differentiation of these lineages. However, species delimitation approaches were not completely effective at identifying each lineage as a distinct “species”, possibly due to the limited number of specimens included for two of the four lineages. The second approach was to employ a species distribution model to try to identify regions of appropriate habitat were the species may occur. The model identified a large patch of suitable habitat where future sampling effort should be directed. Such studies are crucial for conservation issues; as FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 4 regardless of whether these “lineages” are recognized as species or not, considerable diversity occurs in some small populations to the East of the Atlas that could easily be lost to habitat destruction. The study demonstrates the value of combining fieldwork, molecular analyses and modelling approaches to gain new insights into the evolutionary history of a species. KEYWORDS Phylogeography, Saurodactylus brosseti, Species probability of occurrence, Vicariance, Atlas Mountains FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 5 RESUMO Padrões filogeográficos têm sido extensivamente estudados na Europa. No entanto, estudos semelhantes são limitados noutras regiões, tal como o Norte de África. Marrocos apresenta várias barreiras geográficas, tais como a bacia do Rio Moulouya, as Montanhas Rif e as Montanhas Atlas. Estas últimas dividem o país em duas regiões bioclimáticas, o que por sua vez é responsável pelos altos níveis de endemismo do país. A subdivisão genética de várias espécies, como por exemplo, Natrix maura, Agama impalearis e Myotis nettereri é coincidente com a orogenia do Atlas há cerca de 9 milhões de anos. Saurodactylus é um género de osga muito antigo endémico de Marrocos, incluindo três espécies: S. mauritanicus Duméril and Bibron, 1836; S. fasciatus Werner, 1931 e S. brosseti Bons and Pasteur, 1957. Um estudo realizado demonstrou que S. brosseti apresenta uma grande variação intraespecífica, com 11.4% de diversidade genética para o marcador ND4, o que implica padrões filogeográficos complexos. Este facto suscita várias questões, tais como a existência de um complexo de espécies e de que forma a diversidade observada pode estar dividida pelas barreiras geológicas da região. No entanto, a amostragem mostrou-se insuficiente para responder a estas questões. De modo a compreender a relação dos padrões filogenéticos e geográficos de S. brosseti com a orogénese das Montanhas Atlas, seria necessária amostragem adicional. Como tal, duas viagens de campo a Marrocos, em 2013 e 2014, foram realizadas. As observações feitas resultaram numa anotação científica com a descrição de expansões de distribuição para algumas espécies, em particular Bufo spinosus, Trapelus boehmei, Tropiocolotes algericus, Acanthodactylus erythrurus, Chalcides polylepis e Scutophis moilensis. Para processos de conservação, mapas de distribuição de espécies com maior precisão são necessários realçando a necessidade de uma maior prospeção da região. Duas diferentes abordagens foram combinadas para a espécie em estudo, S. brosseti. Para uma avaliação filogeográfica, dois genes mitocondriais e três nucleares foram sequenciados e analizados, podendo ser distinguidas quatro linhagens com um nível de diversidade tipicamente observado entre espécies. A orogénese do Atlas ocorreu na mesma altura que a separação destas linhagens, indicando a possibilidade de esta ter constituído a barreira que levou à sua diferenciação. Contudo, a abordagem de delimitação de espécies não foi completamente eficaz na identificação de cada linhagem como “espécies” diferentes, possivelmente devido ao número limitado de indivíduos em duas das quatro linhagens. FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 6 A segunda abordagem consistiu na modelação de distribuição de espécies com o objectivo de tentar identificar locais onde o habitat seja adequado à ocorrência de espécies. O modelo identificou um grande fragmento onde amostragem futura deverá ser realizada. Este tipo de estudos é crucial em termos de conservação, independentemente destas linhagens serem reconhecidas como espécies ou não. Pequenas populações a Este do Atlas possuem uma diversidade considerável, podendo estar facilmente sujeitas à destruição de habitat. Esta análise realça o valor de combinar trabalho de campo, análises moleculares e modelação, de forma a obter melhores perspectivas acerca da história evolutiva de uma espécie. PALAVRAS-CHAVE Filogeografia, Saurodactylus brosseti, Probabilidade de ocorrência de espécies, Vicariância, Montanhas Atlas FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 7 TABLE OF CONTENTS Acknowledgments…………………………………………………………………...... 2 Abstract……………………………………………………………………………….. 3 Keywords……………………………………………………………………………… 4 Resumo…………………………………………………………………………………. 5 Palavras-Chave………………………………………………………………………. 6 List of Tables…………………………………………………………………………… 9 List of Figures…………………………………………………………………………. 9 List of Appendix……………………………………………………………………….. 9 List of Abbreviations…………………………………………………………………… 10 General Introduction………………………………………………………………….. 11 Genus Saurodactylus……………………………………………………………... 11 Saurodactylus brosseti…………………………………………………………… 13 Geographic region – Morocco…………………………………………………… 14 Formation of the Atlas Mountains……………………………………………. 16 Phylogeography…………………………………………………………………… 17 Coalescent Theory……………………………………………………………. 18 Phylogenetic data: mitochondrial and nuclear DNA………………………... 18 Phylogenetic Analysis Methods……………………………………………… 20 Biogeography……………………………………………………………………… 21 Geographic distribution……………………………………………………….. 22 Ecological Niche………………………………………………………………. 23 Ecological niche models (ENMs)……………………………………………. 25 Species distribution………………………………………………………….... 25 Models of Species Distribution……………………………………………….. 27 Input Occurrence data…………………………………………………………. 28 Input Environmental Variables……………………………………………….. 29 Accuracy of the output………………………………………………………… 29 Objectives………………………………………………………………………………. 31 Material and methods…………………………………………………………………. 32 Phylogenetic Analysis…………………………………………………………….. 32 Sampling………………………………………………………………………… 32 DNA extraction, amplification and sequencing………………….................. 33 Phylogenetic analysis……………………………………............................... 34 FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 8 Modelling……………………………………………………………………………. 35 Occurrence Data and Environmental Variables……………………………. 35 Species Probability of Occurrence…………………………………………… 36 Manuscripts………………………………………………………………………….... 38 Manuscript I……………………………………………………………………….. 39 Manuscript II……………………………………………………………………….. 50 General Discussion…………………………………………………………………… 64 General References…………………………………………………………………… 67 Appendix….…………………………………………………………………………….. 83 FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 15 semiarid global bioclimates (Bons and Geniez, 1996). In the South of the Atlas the climate is Saharan and an arid bioclimate occurs in almost every region in the east of the Atlas and almost to the shores of the Mediterranean. Here, the summers are characterized for being dry and torrid with cooler winter, with the exception of the oceanic coast where cloudiness is a factor. Precipitation is extremely rare and occurs mostly during the winter; this feature is responsible for the north Sahara climate being included in the Mediterranean region (Bons and Geniez, 1996). Morocco is also known for being quite windy throughout the year, especially during spring. Minimum temperatures at high altitudes can fall below zero degrees in winter, except along coastal strips that have milder conditions. Maximum temperatures in the summer can reach up to 60º C in Saharan regions and 46º C in the remaining territory except in the coast where the climate is more temperate due to the maritime influence. Precipitation generates particular humid biotopes and snowfall has never been recorded in the Saharan regions; in contrast it does snow in the High Atlas, Middle Atlas and highest parts of the Rif Mountains, leaving these areas covered in snow during winter and spring, which are subsequently an important reservoir of water (Bons and Geniez, 1996). The Moulouya, Sebou, Oum-er-Rbia and Tennsitt are the most important rivers originating from these mountains (Schleich et al., 1996). The multitude of bioclimatic regions within Morocco has influenced the dispersion of several Mediterranean species from the North, and some species from the Sahara. This is mainly due to the Atlas Mountains, which also cause the existence of high levels of endemic species of reptiles and amphibians in Morocco (Bons and Geniez, 1996). Indeed, Morocco is included in the Mediterranean Basin, a recognized hotspot of biodiversity (Myers et al., 2000). From the western Mediterranean region, Morocco has one of the richest and most diverse herpetofauna, along with Algeria, with 104 described species of reptiles and amphibians, of which 22 are endemic (Bons and Geniez, 1996). It is well documented how endemism is especially high among amphibians, tortoises and the lizard families Lacertidae and Scincidae (e.g. Cox et al., 2006). In Morocco, endemism is less seen in amphibians, which is not surprising considering the arid and semi-arid habitats predominant in large parts of the region (Cox et al., 2006). The country itself is isolated by several geographical barriers – in the North by the Mediterranean Sea, in the West by the Atlantic Ocean, in the South by the Sahara Desert and in the East by the Moulouya River Valley (Bons and Geniez, 1996). Thus, Morocco presents an ample diversity of geographic barriers and calibration points can be assessed by considering their formation age, in order to try to date phylogeographic breaks. Regarding the Moulouya River Valley, it is sometimes proposed to be a barrier FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 16 preventing dispersal and gene flow of, for example, Testudo graeca (Álvarez et al., 2000), Natrix maura (Barata et al., 2008) and Gerbillus campestris (Nicolas et al., 2014). However, sampling is key – in a study with more extensive sampling of Natrix maura it has been shown that genetic subdivision is not completely coinciding with the Moulouya River Valley (Harris et al., 2003). Evidence of the Atlas Mountain Chain acting as a barrier causing genetic subdivision has been shown in many species, such as Agama impalearis (Brown et al., 2002; Gonçalves et al., 2012), the Acanthodactylus erythrurus group (Fonseca et al., 2009), the freshwater turtle Mauremys leprosa (Fritz et al., 2005 and Fritz et al., 2006), scorpions of the genus Buthus (Habel et al., 2012; Husemann et al., 2012), the scorpion Androctonus mauritanicus (Coelho et al., 2014) and the bat complex Myotis nattereri (Salicini et al., 2013). Formation of the Atlas Mountains The formation of the Atlas Mountains derived from three events – tectonic deformation, extension of the Earth‟s crust and tectonic convergence (Seber and Barazangi, 1996). In the Paleozoic Period, around 300 Mya, occurred the first event where tectonic deformations lead to the formation of the Anti-Atlas; Africa was part of the Gondwana and North America comprising Euroamerica. The Anti-Atlas is a result of the collision between the African and American plates (that formed Pangea), and was initially formed as part of the Alleghanian orogeny (Hatcher, 2008). The second event, an extension of the Earth‟s crust, took place 65 Mya during the Mesozoic period, which separated the African and American continents through rock deposition in the ocean that today forms the High-Atlas. The Middle Atlas is trending northeast-southwest while the High-Atlas trend is west southwest-east northeast corresponding to inverted Mesozoic intracontinental basins; both directions were inherited from initial Triassic rifting (Missenard et al., 2006). Around 35 Mya during the Tertiary period, tectonic convergence of the African and European landmasses occurred in today‟s Strait of Gibraltar area, uplifting the mountain chains that now form the Atlas Mountains. The orogeny of the Atlas is estimated to have occurred around 9 Mya in the late Miocene (Hsu, 1978). Contrarily, there is some evidence based on scattered direct surface suggesting that the uplift of the Middle-Atlas and High-Atlas took place around 7.1 to 5.3 Mya in the post-Miocene (Ayarza et al., 2005). FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 17 Phylogeography Phylogeography is a discipline that relates phylogenies with geographic distributions of species, allowing the assessment of how genes and their history in time affect the current distribution of species in space (Avise, 1998). Early studies using mitochondrial DNA revealed a link between genealogies and geography, which later was also demonstrated for nuclear DNA, becoming the obvious field for microevolution studies (Avise, 2009). The first practical phylogeographic study of a species was conducted using mtDNA in 1979 (Avise et al., 1979), and phylogeography was subsequently recognized as a discipline. The first human study regarding mtDNA variation was conducted a year later (Brown, 1980), and some years after that a phylogeographic study involving several codistributed species was published (Bermingham and Avise, 1986). Some phylogeographic general patterns have been identified and one simple case is species with low dispersal ability. Frequently, populations of these species are highly genealogically different, but with only a few mtDNA haplotypes, while most of the haplotypes are similar and geographically localized (Avise, 2009). Differentiation of these geographically localized haplotypes is usually coincident with historical events preventing dispersal; for example, events such as climatic oscillations in the Quaternary that forced species to refugia, which is known to have led to a lack of gene flow and thus haplotype divergence (e.g. Hewitt, 2004). Even species with high dispersal capabilities can display divergence due to historical events such as the formation of geological barriers (e.g.Jaramillo-Correa et al., 2010; Mirams et al., 2011; Lessios and Robertson, 2013). Dispersal barriers have an impact on the genealogical patterns of lineages or species and split events usually coincide or are more recent than the emergence of the barrier (Avise, 2009). Species with high dispersal ability can also show high genetic differentiation due to philopatry, which is a loyalty behavioral predisposition to particular locations (e.g. Wenink et al., 1996; Karl et al., 2011). On the other hand, not all species present such a defined phylogeographic structure. For instance, recent population expansions are generally defined by a prevalent single haplotype and many other rare haplotypes that are probably derived from a common haplotype enduring separate mutations in time. The widespread haplotype is geographically the source of the expansion (e.g. Watson et al., 1997; de Jong et al., 2011). Should several species have the same ecological or habitat requirements, they tend to show similar genealogical structure. This implies a broader impact of the forces shaping genes and several cases have been documented in Europe (Avise, 2009). With all of this, sometimes history repeats itself and phylogeographic patterns also FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 18 show that. The most common history repetition example is when species are forced to “southern refugia” during glaciations/deglaciations in the Pleistocene, or to multiple refugia (e.g. Hewitt, 2000; Miraldo et al., 2012). Although there are no specific natural rules dictating how genetic lineages are spatially distributed, phylogeography is currently a growing field of study (e.g. Taberlet et al., 1998; Hahn et al., 2014). Coalescent Theory Coalescent allows knowledge of historical population processes and was first described by Kingman (Kigman, 1982), although it has been discovered individually by several authors (reviewed in Rosenberg and Nordborg, 2002; Salemi and Vandamme, 2003). Coalescent is mathematically calculated and traces back the time to ancestry of a genealogy until all lineages coalesce into the most recent common ancestor (MRCA). The absence of selection is typically assumed and the rate at which lineages coalesce depends on the size of the population (Salemi and Vandamme, 2003). Should only genetic drift be a factor, smaller populations will have a faster rate of coalescent lineages; while larger populations will present a slower rate of drift and, consequently, of coalescent lineages. When comparing similarity between more than one population, historical processes can be assessed by the increased or decreased rate of genetic diversity (Salemi and Vandamme, 2003). Selection is, however, always present, and that means genetic diversity is not as random some genotypes have a higher fitness than others (Rosenberg and Nordborg, 2002). When studying a species tree, the branch length represents the time that an ancestral form took to split into its descendants and is measured by the number of mutations that accumulated between that split. The mutations have a rate, which is logically defined by the number of mutations that are expected in each generation (Salemi and Vandamme, 2003). Coalescent methods identify lineages that are evolving independently, testing several alternative hypotheses of divergence by using multilocus data, and can help to explain species diversity (Fujita et al., 2012). Phylogenetic data: mitochondrial and nuclear DNA Linnaeus (1758) started using morphology (the study of the physical aspects an organism) as a way to formulate differences between species which today is still the core of taxonomy. However, modern day micro evolutionary studies are often based on DNA sequence data that allows additional information to be obtained, and is FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 19 particularly useful for assessing the relationships between taxa. Biodiversity as we observe today exists because of variations that have accumulated through diverse mechanisms (mutations, duplications of genes, reorganization of genomes, genetic exchanges (e.g. Faria et al., 2011), allowing organisms to evolve. Species that are more closely related to each other typically have fewer point mutations (or substitutions) between themselves than two species that are not closely related (Salemi and Vandamme, 2003). Since some genes are highly conserved, while others are much more variable, suitable genes can be chosen that are likely to show a suitable level of variation for a variety of different questions (Salemi and Vandamme, 2003). Although there are differences between mitochondrial and nuclear DNA, both provide valuable information and are employed to build phylogenies to understand phylogeography (e.g. Perera and Harris, 2010). Animal mitochondrial DNA (mtDNA) is a circular molecule with typically 37 genes and approximately 17,000 nucleotide base pairs with no introns or large non-coding regions. In higher animals it has a high mutation rate and is usually transmitted maternally. MtDNA is a major tool in phylogeography and it is widely used to study the evolution of species and populations (William et al., 2004). The first utilization of mtDNA variation was conducted with RFLPs (Avise et al., 1979; Brown and Wright, 1979) and later with sequences (Kocher et al., 1989). More recently, it has been concluded by some researchers that studies using mtDNA are not sufficient if more compound questions about the history of populations are to be studied (Godinho et al., 2008). This is because mtDNA provides knowledge of a single locus and it allows researchers to see only a small part of the story; consequently there is an underestimation of diversity and an oversimplification of the evolutionary history (Zhang and Hewitt, 2003). Nuclear DNA has 3 billion base pairs, almost 180,000 more than mtDNA. It also differs in the degree of recombination, ploydy and mutation rate, which is usually slower (William et al., 2004). The rate of substitution in single copy nuclear polymorphic sequences depends on the genic region (Zhang and Hewitt, 2003). However, using nuclear DNA for phylogeography can also bring difficulties related to recombination, heterozygosity, rate variation and also amplification and sequencing (Zhang and Hewitt, 2003). Many studies now include nuclear DNA (Perera and Harris, 2010; Rato et al., 2010), although most available data is still mitochondrial. Nuclear DNA is not going to replace mtDNA in phylogeography, but it will instead help disentangle different aspects of evolutionary history (Zhang and Hewitt, 2003). FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 20 Phylogenetic Analysis Methods Inferring phylogenies is not an easy procedure and several approaches are accessible, although there is no perfect method and getting to the “true” phylogeny is never certain. The most common analysis methods include UPGMA (Sneath and Sokal, 1973), Neighbor-joining (NJ) (Saitou and Nei, 1987), Maximum parsimony (MP) (Fitch, 1971), Maximum likelihood (ML) (Felsenstein, 1981) and Bayesian inference (Huelsenbeck and Ronquist, 2001). The construction of a phylogenetic tree can be separated according to the data they use (character state or distance matrix) or according to the underlying strategy (treeevaluation or clustering) (Salemi and Vandamme, 2003). There are assumptions in each method that need to be carefully taken into consideration, as they may not be valid for the data being used. The most widely used methods are Maximum likelihood and Bayesian inference. Maximum likelihood (ML) uses character state data type and a tree-evaluation method, using a tree topology and a model of evolution to estimate the highest probability. The likelihood is determined by adding all possible nucleotide states in the internal nodes and is optimized with the best combination of evolutionary parameters and branch length possible. This is also done by a given number of trees and the algorithm choses the tree topology that has the maximum likelihood. This process requires time and is computationally challenging. Furthermore, to assess support for actual nodes requires additional analyses, the most common of which is to apply a bootstrap approach, or to use likelihood ratio testes to compare alternative tree topologies. Bayesian methods are in the same group as ML, however they do not search for the best tree. Instead, they try to infer several tree topologies that explain the data, which is called posterior distribution of trees, and has a confidence estimate. The method requires that a prior belief, or prior distribution is given i.e. before the analysis begins, it needs to know what the tree topology is, branch lengths and substitutions model parameters. A technique called Markov chain Monte Carlo (MCMC) is then used to acquire posterior probabilities of the tree. It calculates the likelihood in every “step” and it moves forward and if the likelihood is better than before, until it reaches the maximum value. However, it can also occasionally move backwards, allowing the methodology to overcome local optima. The analysis is typically run multiple times and with a random starting point, again so it does not stop at a local optimum. In the end, the worst trees are discarded as burn-in and posterior probabilities are the posterior values that will be represented in the Bayesian tree (Huelsenbeck and Ronquist, 2001). Bayesian FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 21 inference methods take phylogenetic uncertainty into account, but are still computationally demanding progress. These methods can be performed on separate genes, but the data are usually combined, employing a concatenated approach. However, increase of available molecular data has made it evident that gene trees are not always coincident with species trees (Fujita et al., 2012). This was first thought about when studying the disagreement of gene trees (e.g. Satta et al., 2000) with the realization that is an expected feature, and concatenation approach may lead to an incorrect species tree. One other approach is a coalescent species delimitation approach which allow discordance of the gene tree under genetic drift (Fujita et al., 2012). This approach can also use either ML or Bayesian; each node of the species tree represents a speciation event and each branch is an independent evolutionary lineage (Fujita et al., 2012). Biogeography Biogeography is the science that tries to understand and describe spatial patterns of biodiversity. It studies the past and present distributions of organisms and their associated variation patterns on earth (Brown and Lomolino, 1998). Additionally, it attempts to answer questions such as 1) why does a species or family has such a range; 2) what are the characteristics that allow a species to live in such areas and not others?; 3) what is the role of climate and topography and how do these interact with the species?; 4) how do species change as the environment changes in space?; 5) where was the ancestor of a species found?; and 6) how do historical events shape species distributions? (Brown and Lomolino, 1998). The central question is related to how organisms are distributed and their history on earth. Biogeography is intimately connected to ecology and phylogenetic history (Brown and Lomolino, 1998). It differs from other biological disciplines as it deals with scales of space and time at which experimental manipulations is difficult, serving as a comparative observational science (Brown and Lomolino, 1998). Historical biogeography is important in ecology, and large-scale biogeographical events are also the outcome of ecological processes. Historical biogeography helps to explain largescale patterns of species richness. The ecology of the species is extremely important to better understand patterns of diversity (Wiens and Donoghue, 2004). Also, biogeographical processes determine the composition of the regional species pool (Ricklefs and Schluter, 1993; McPeek and Brown, 2000; Webb et al., 2002), which has a strong effect on the composition of local-scale communities (Morin, 1999). The absence of a certain ecological association from a local community might not be FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 22 described solely by ecology or phylogenetic history of the species, but also by biogeographical patterns of the group or groups comprising the absent ecological association (Ricklefs and Schluter, 1993; Stephens and Wiens, 2004). Geographic distribution The geographical distribution of species is influenced by different factors according to the scale. At a macro-scale, the weather plays a predominant role in influencing geographical distributions of species. Topography, hydrology, geology and weather have an influence at a regional scale; type of soil and hydrology influence at a local scale; and herbivory, micro-disturbances and intraspecific competition have the largest influence at a micro-scale (Brown and Lomolino, 1998). The individual distribution pattern of species depends of the temporal scale of the analysis because the limits of occurrence vary with extinction or recolonization and distribution and abundance expand and contract with environmental variations and anthropogenic activities (Brown and Lomolino, 1998). A fundamental key for biogeography is that each species has a unique geographic range and the ecological processes coupled with historical events provide essential contributions for shaping these ranges. Firstly, how can we measure and define geographic ranges? The easiest and most direct way is with range maps; they are easy to organize and can be later used by other researchers (Brown and Lomolino, 1998). There are three kinds of maps: outline maps, dot maps and contour maps, each reflecting a different aspect. Outline maps depict the supposed limit of the known species distribution as an irregular area (Brown and Lomolino, 1998). It is very prone to inaccuracies, especially if the distribution is not very well understood and if the author has made assumptions based on his/her knowledge. Dot maps are often part of a taxonomic study and plot points on a map correspond to localities where a species has been recorded (Brown and Lomolino, 1998). They can also depict places where verified museum species have been collected and they are, usually, more accurate. However, they can also represent an infinitesimal fraction of the actual distribution, for instance, in the case of sightings of birds, and they do not extrapolate beyond the relatively few sampled locations (Brown and Lomolino, 1998). Contour maps use contour lines to show variation in density, being more informative than the previous types of maps (Brown and Lomolino, 1998). The disadvantage of this method is the lack of available information relating to abundance; despite this, there is a statistical procedure, termed kriging, which interpolates between data points and produces threedimensional landscape depicting variation in abundance within the range (Brown and FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 23 Lomolino, 1998). Databases are also a powerful tool for mapping geographical distributions, although they may contain errors on the spatial location of a species or in species identification (e.g. Lozier et al., 2009). The distribution of species and/or populations is a spatial reflection of their niche. A species will occur when the environmental conditions are most fitting and will be absent in areas where one or more essential resources are missing. Also carrying capacities for a species – maximum population size given the environmental conditions - frequently changes with environmental variation. Ecological Niche There are many definitions of a „niche‟, but all similarly point towards an environment which allows a population or species to survive differing only on the emphasis they put on the key points (Soberón and Nakamura, 2009).The fundamental ecological niche of a species is defined by its biological critical characteristics, including feeding ecology, physiology and reproductive behaviour (Hutchinson, 1957) and describes the abiotic conditions in which it can persist and maintain viable populations (Hutchinson, 1957). However, species are commonly forced to occupy a niche that is a contraction of the fundamental niche, as an effect of pressure from, and interactions with other organisms (Hutchinson, 1957). Also, environmental conditions are not always appropriate for a species in its entire occurrence (Brown and Lomolino, 1998). The fundamental niche is the species‟ niche in the absence of any disturbance, such as interspecific competition, and is determined by the physiological capabilities of the species. The realized niche is where the species is competitively superior as well as more physiologically adapted than competing species (Roughgarden, 1974). Together, organisms occupy a widespread range of environmental conditions but almost every species or lineage inhabit only a limited subset of such conditions, which is determined by intrinsic traits of each organism that are preserved over long evolutionary timescales. Some groups of animals have a wide range across specific regions, for example in the tropics, but fail to colonize other biomes, even if the opportunity to do so has persisted for hundreds of millions of years. Natural populations are exposed to different biotic and abiotic factors including competition, predation, climate and food resources variations (Rundle and Nosil, 2005; Schluter, 2009). This can lead to divergent evolutionary responses and patterns of climatic tolerances (reviewed in Parmesan, 2006), and subsequently evolution of organisms to fulfil new habitats; a process termed niche divergence (e.g. Kozak and Wiens, 2007; Cadena et al., 2012). In contrast, the preservation of ecological similarity FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 24 among populations over time is niche conservatism (Wiens and Graham, 2005; Peterson, 2011), and it can limit adaptation to ecological conditions and promote isolation (e.g. Wiens, 2004; Kozak and Wiens, 2007; Cadena et al., 2012). Although it has been attempted to test conservative evolution before (Bartlein et al., 1989; Ricklefs and Latham, 1992; Peterson and Vargas-Barajas, 1993), ecological niche models have only been tested quantitatively and (Peterson et al. 1999). It was predicted that fundamental niches of species could slowly change under natural selection. Using genetic models it was predicted that niche conservatism rates of adaptation in environments outside the fundamental niche should be frequently slower than extinction process (Houston and McNamara, 1992; Kawecki and Stearns, 1993). The results of a previous study to (Peterson et al., 1999) indicated that ecological niches evolve little at or around the time of a speciation event and that the differences in the ecological niche seem to develop and accumulate later, over the time scale of familial relationships (Peterson, 2011). The types of speciation involved can also be inferred by the conservatism of ecological niches across moderate periods of evolutionary time; for example geographic isolation leads to vicariance. Until recently, evidence for niche conservatism was mixed but a study has shown some structure after setting all evidence in a time-scale (Peterson, 2011). According to this, recent and short-term events, such as distributional shifts at the end of the Pleistocene period, or species invasions present some trend towards conservatism. However, long-term events including differentiation across phylogenies show increasing conservatism breakdown. This means that niche conservatism breaks down over time but at a rate that is still questionable. On timescales in which speciation events are involved, almost all lineages show overall niche conservatism, although no ecological signal associated with speciation is obvious (Peterson, 2011). Some studies have reported niche shifts associated with invasion events in the European plant Centaurea maculosa (Broennimann et al., 2007), the South American fire ant Solenopsis invicta (Fitzpatrick et al., 2007) and the mosquito Aedes albopictus (Medley, 2010), but they may be all a consequence of methodological artefact and not a biological reality (Warren et al., 2008). This difference of opinion also reflects the immature nature of this field and one of the ways forward is a more detailed analysis of niche characteristics and their change through time (Peterson, 2011). Frequent and marked niche change is only seen in phylogenetic history of older groups, and thus estimates of phylogeny give the opportunity to understand the rate of niche change, its speed and how these change events are correlated (Peterson, 2011). During speciation events niche differentiation is expected to be rare, and demonstrations of ecological innovation during this time have to be cautiously studied in order to avoid incorrect conclusion. Moreover, clear FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 31 OBJECTIVES The main aim of this thesis was to determine phylogeographic patterns of variation within S. brosseti. In particular we hoped to determine a) if mtDNA lineages previously identified were also recovered with nuclear markers b) to try to date the origin of these lineages and associate them with known geological events, especially the orogenesis of the Atlas Mountains c) to use species delimitation approaches to assess if S. brosseti may actually correspond to a species complex and d) to extend sampling across the range to assess if additional but previously unsampled lineages might occur. The second aim was to asses which environmental variables might limit the occurrences of S. brosseti in the region, and then use this to predict possible areas where the species might occur but not be recorded. These potential distribution maps should also help to identify possible geographical barriers that shape the distribution of the species. Since these primary aims involved considerable fieldwork, a side aim was to record all the herpetofauna idenfied during the fieldwork, including a DNA barcoding approach when necessary, and to use this to determine possible range extensions to poduce better distribution maps of the herpetofauna of this region. FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 32 MATERIAL AND METHODS In this chapter, a more detailed description of the materials and methods used is offered. Phylogenetic Analysis Sampling Over the years, several fieldtrips have been carried out, where specimens where sampled. Two fieldtrips (May 2013 and May 2014) were conducted and I had the opportunity to be included in both of them, resulting in a sampling of 53 species from 138 localities (see Manuscript II), from which 36 were Saurodactylus brosseti specimens (see Appendix 1 and Fig. 4.). Fig. 4. – Samples used for phylogenetic analyses. The sampling localities included the southernmost known locations in Western Sahara, near Boujdour. Samples were collected by hand and data recorded at the site with GPS and annotations of the basic information about the specimen – DB number (to be included in the reptile database), species, subspecies, date, locality, latitude, longitude, FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 33 altitude, sex, age, snout-vent length (SVL) and, when applicable, pellet, slide, blood, parasites, photo information and observations (e.g. pregnant female). All sample information is available in the reptile database of CIBIO. Additionally, a small piece of each specimen‟s tail tip was collected from live animals and kept in 96% ethanol and photos taken before releasing the animal. Saurodactylus mauritanicus and Saurodactylus fasciatus were used as outgroups, for which samples were already available in the CIBIO database. DNA extraction, amplification and sequencing In the CIBIO laboratory, DNA was extracted from tail tips tissue using a High Salt method (Sambrook et al., 1989). DNA amplification was performed through PCR for two mitochondrial fragments – 12S and ND4 – two nuclear fragments – ACM4 and MC1R – and one intron – BZW1. Mitochondrial genes were chosen so that data produced could be directly compared to an earlier study (Rato and Harris, 2008). ACM4 and MC1R markers were chosen due to its wide use in phylogeographic studies in reptiles (e.g. Rato et al., 2010) and the intron was chosen because they normally present higher mutation rate and consequently higher variation and BZW1 have been used in other members of the Gekkota family (e.g. Fujita et al., 2010). PCR conditions and primers are described on Table 1. Table 1. – Primers names and amplification conditions. Gene 12S ND4 ACM4 BZW1 MC1R Step T (ºC) Time X T (ºC) Time X T (ºC) Time X T (ºC) Time X T (ºC) Time X Initial Denaturation 95º 1' 1 94º 3' 1 94º 5' 1 94º 3' 1 92º 2' 1 Denaturation 95º 48º 72º 15'' 15'' 10'' 35 94º 94º 48º 3' 30'' 30'' 35 94º 94º 55º 5' 30'' 45'' 1 32 94º 94º 62º 3' 30'' 45'' 37 92º 92º 55º 2' 1' 45'' 35 Annealing Extension Final Extension 72º 10' 1 72º 40'' 1 72º 1' 1 72º 1' 72º 1' 1 Primer Forward 12S L ND4 Tg-F Tar1 MC1R F Primer Reverse 12S H LEU Tg-R Tar2 MC1R R Citation Kocher et al. 1989 Arévalo et al. 1994 Gamble et al. 2008 Fujita et al. 2010 Pinho et al. 2010 FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 34 Polymerase Chain Reaction (PCR) amplification was performed with a final 25 µL volume for each sample, according to MyTaq™ protocol (UK) – 15.8 µL of purified water, 5 µL of MyTaq buffer, 1 µL of Forward Primer, 1µL of Reverse Primer, 0.2 µL of Taq, and 2 µL of DNA extraction product. MyTaq™ buffer contains dNTPs, MgCl2, stabilizers and enhancers. PCR were run in a Biometra TProfessional thermal cycler. For each run, two controls were added – a positive control, which is a sample that previously amplified for the gene in question to certify that PCR reaction was effective; and a negative control, which has all reagents except DNA to check for contaminations. To check the success of amplification, 2 µL of PCR product from each sample ran in 2% agarose gel with GelRed Nucleic Acid stain, visualized in an ultraviolet transilluminator. Pictures of the each gel run were taken and saved. Amplification products were sent to Beckman Coulter Genomics (UK) for purification and Sanger sequencing with same primers used in amplification. Phylogenetic analysis Sequences were blasted to the NCBI database on GenBank to confirm the species. Chromatographs were checked and sequences were aligned for posterior phylogenetic analysis using Geneious v 5.6 (Drummond et al., 2012). Alignment was performed using default settings of MAFFT. Nuclear genes were sequenced in both directions to ensure identification of heterozygotes. 12S and ND4 sequences of Saurodactylus brosseti from a previous study (Rato and Harris, 2008) available on GenBank (accession numbers in Appendix 1) were also included in the alignment. jModelTest v2.1.4 (Darriba et al. 2012 – 201) was used to infer which model best fit each data under the Akaike Information Criterion (Cavanaugh 2007 – 205) for separate and concatenated genes in order to decrease the error (Brandley et al. 2005 - 227). Nuclear genes were phased using Seqphase (Flot 2010 - 231) and PHASE (Stephens et al. 2005 – 230) with a threshold of 0.6 and default for all the other parameters. Phylogenetic accuracy can be higher when data sets from different genes are combined into a single phylogenetic analysis (Rokas et al. 2013 - 226). Maximum Likelihood analysis was performed using RAxML v3.0 (Stamatakis, A. 2014 - 229) with 1000 bootstrap replicates for concatenated genes. Bayesian analyses were performed with best fitting models applied to each gene with MrBayes v3.2.2 (Huelsenbeck and Ronquist 2001 – 192) for a concatenated approach (one partition) and *BEAST v1.8.0 (Drummond et al. 2012 – 204) for a coalescent approach (five partitions, all parameters unlinked across partitions, except for 12S and ND4, for which trees were linked into “mitochondrial DNA”). MrBayes analysis began with random starting tree, ran for 10 FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 35 million generations and was sampled every 100 generations. 25.000 (25%) of burn-in trees were discarded and the remaining were used to assess posterior probability values. *BEAST ran three times for 150 million generations with an uncorrelated lognormal relaxed clock with a rate of 0.00701 for 12S (Metallinou et al. 2012 - 225) as calibration point to estimate divergence times. In order to have a species tree, sequences were grouped into four groups, according to the four major clades assessed by MrBayes and RAxML. All runs were combined with LogCombiner v1.8.0 (package of *BEAST) with a burn-in of 10% for each run. Mean genetic distances between the four major clades (given the results from MrBayes and RAxML) were calculated with MEGA6 (Tamura et al. 2013 – 203) for ND4. Consensus trees were visualized in Figtree (v. 1.3.1) and posterior modifications, such as insertion of posterior values and colouring of branches, were performed with Inkscape (v. 0.48). Modelling Occurrence Data and Environmental Variables The input occurrence data for modelling was obtained from geographic coordinates of known occurrence localities (Bons and Geniez, 1996) and the reptile database of CIBIO, in which GPS coordinates were recorded during fieldwork expeditions. In total, 246 points were depicted using ArcMap v 9.3 under the WGS 1984 Datum geographic coordinate system. Twenty current environmental variables were downloaded from WorldClim – Global Climate Data database (Hijmans et al., 2005) (Table 2.). All variables were in 30-arc seconds (approximately 1 Km) resolution tiles: tile 15 and tile 25 in order to comprise all of Morocco and Western Sahara area. Since correlation of environmental variables vary according to its extension, all five variables had to be cut giving Saurodactylus brosseti known distribution range, using ArcMap v9.3. Covariance and correlation matrix were computed using ArcMap with a threshold of 75% - all variables with correlation higher than 0.75 were considered correlated and one of each two correlated variables was withdrawn. Choice of environmental variables was not entirely random, as variables were chosen according to the known limiting factors of the species (Meek, 2008). In the end, five environmental variables were chosen: Annual mean temperature (BIO1), Minimum temperature of coldest month (BIO6), Temperature annual range (BIO7), Precipitation of driest month (BIO14), and Annual precipitation (BIO12). These five variables were also cut according to the distribution of Saurodactylus mauritanicus and Saurodactylus fasciatus. As the distribution of S. brosseti can overlap the distribution of the other two species of the genus, this was FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 36 done to ensure that localities with high probability of occurrence for S. brosseti were not coincident of the localities from either of the other two species. Table 2. – Environmental variables downloaded from WordClim. Code Variable ALT Altitude BIO1 Annual mean temperature BIO2 Mean diurnal range BIO3 Isothermality BIO4 Temperature seasonality BIO5 Maximum temperature of warmest month BIO6 Minimum temperature of coldest month BIO7 Temperature annual range BIO8 Mean temperature of wettest quarter BIO9 Mean temperature of driest quarter BIO10 Mean temperature of warmest quarter BIO11 Mean temperature of coldest quarter BIO12 Annual precipitation BIO13 Precipitation of wettest month BIO14 Precipitation of driest month BIO15 Precipitation seasonality BIO16 Precipitation of wettest quarter BIO17 Precipitation of driest quarter BIO18 Precipitation of warmest quarter BIO19 Precipitation of coldest quarter Species Probability of Occurrence To estimate the presence probability of the three species of Saurodactylus the software Maxent (Phillips and Dudik, 2008) was used. All duplicate presence records from the known occurrence data were removed before the software run to prevent data FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 37 overfitting. All runs were performed with 25 random test percentage, 20 bootstrap replicates and in order to create response curves with jacknife to measure variable importance. The area under the curve (AUC) was used to assess model accuracy. Even though it is not as reliable as it would be expected (Lobo et al. 2007 - 217) this is still the best method. Since Maxent presents its results in a gradient “probability of occurrence” ranging from 0 to 1, a threshold was defined in order to build maps of “suitable or unsuitable” habitat. This was done using the average of the 10 percentile training presence logistic threshold of the 20 replicates for each species. Values above the threshold were considered as “suitable habitat” and values below threshold were considered as “unsuitable habitat”. FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 38 MANUSCRIPTS FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 39 Manuscript I Moroccan herpetofauna: distribution updates including a DNA barcoding approach. Daniela Rosado1,2, David James Harris1, Ana Perera1, Fátima Jorge1,2, Beatriz Tomé1,2, Isabel Damas-Moreira1, Isabel Tavares1,2, Henrique Estrela1,2, Amanda de Sousa1,2, Ana Pereira1,2, Marina Mantovani1,2 and Daniele Salvi1. 1 CIBIO/InBIO Research Centre in Biodiversity and Genetic Resources, InBIO, Universidade do Porto, Campus Agrário de Vairão, Rua Padre Armando Quintas, Nº 7, 4485-661 Vairão, Vila do Conde, Portugal 2 Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, 4099-002 Porto, Portugal Key Words: Reptiles; Amphibians; Morocco; DNA barcoding; Distribution range The Kingdom of Morocco contains some of the richest diversity of herpetofauna in the Maghreb and Western Europe. This fact is clearly related with the considerable topological variation of the region, with the Rif and Atlas Mountains dividing the country into climatically different zones. The first step towards any ecological, conservation or modeling approaches concerning this rich diversity is to develop accurate distribution datasets. Bons & Geniez (1996) presented a detailed assessment of the known diversity at that time. However, various researchers have since then presented data indicating range extension for many species (e.g. Guzman et al., 2007, Harris et al., 2008, Harris et al. 2010, Barnestein et al., 2010; Barata et al., 2011, Beukema et al., 2013, Damas-Moreira et al., 2014), and it is clear that current records are still limited, especially in the Eastern region (Barata et al., 2011, Beukema et al., 2013). At the same time modeling approaches have been employed, which may help to highlight regions in need of further prospection (de Pous et al., 2010). In particular, in an extensive review of the distribution and biogeography of Moroccan amphibians (Beukema et al., 2013), models indicating regions of high probability of occurrence were presented for all species along with greatly improved distribution maps. New records for amphibians can therefore both be compared to these models and can be informative in determining whether distribution maps are stabilizing for such wellstudied groups. Additionally, they can also be used to draw a parallel scenario in groups known to be much harder to locate, such as fossorial species or snakes. FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 40 The accuracy of distribution maps relies on species being correctly identified. This is not always simple – many forms of Moroccan herpetofauna have recently been identified as species complexes (e.g. Rato et al., 2012), and several of these are “cryptic” implying that identification is made using molecular markers. This includes forms of the Podarcis vaucheri complex (Pinho et al., 2007), or lineages within various geckos including Quedenfeldtia (Barata et al., 2012), Ptyodactylus (Perera & Harris, 2010) and Stenodactylus (Metallinou et al., 2012). Other species can be difficult to identify when only juveniles or tadpoles are collected. Additionally many groups such as snakes are often widely sampled as road-killed animals, and in some cases identifying remains to the species level is again difficult. In these cases the use of a DNA “barcoding” approach (Hebert & Gregory, 2005) can be extremely useful. In the present study the authors compile records of two expeditions to Morocco over a combined 5 week period in Spring 2013 and 2014. Sampling covers a wide range of Southern and Eastern Morocco, and was chosen to be complementary to a recent survey of Northern and Central regions (Damas-Moreira et al., 2014). In total 138 localities were sampled and 53 species recorded. GPS coordinates and a detailed list of species per locality are given in Table 1. Photos of most animals are available on request from the authors. Distribution data was compared to published records, and interesting new localities are discussed in the text that follows. In cases where a species diagnosis based on morphological characters could not be made with certainty, a DNA barcoding approach was used. DNA was extracted using standard high-salt methods (Sambrook et al., 1989). PCR was used to amplify a region of the 12S rRNA, using published protocols (Harris et al., 1998). This gene was chosen rather than the classic barcoding COI region since comparative published data for 12S was already available for most of the presumed species. For example 12S rRNA sequences are available for most toad species from Morocco (e.g. Harris & Perera, 2009; de Pous et al., 2013), so this gene could be used to confirm the species diagnosis of tadpoles of this group. All species for which this approach was used are highlighted in Table 1, and are discussed in the text when relevant. New sequences are published in GenBank (accession numbers xxxxx to xxxxx). Overall our findings indicate that even for better-known groups the current distribution maps are imprecise. Several new distribution points and range extensions were found in the Eastern region, where models predicated that more species would be expected (Beukema et al., 2013), or coincided with high probability areas proposed for specific species. This further demonstrates the value of these models, which can be used to guide future prospection in Morocco and other regions. FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 47 Acknowledgments Fieldwork was supported by the Mohamed bin Zayed species conservation fund (grant number 12055077 to DJH), the Percy Sladen Memorial Fund (to DS) and by the British Herpetological Society (to IDM in 2013 and DR in 2014). FJ, DS and AP are supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) under the Programa Operacional Potencial Humano – Quadro de Referência Estratégico Nacional funds from the European Social Fund and Portuguese Ministério da Educação e Ciência (DS: post-doctoral grant SFRH/BPD/66592/2009; AP: contract IF/01257/2012). FJ was funded through a doctoral grant (SFRH/BD/77332/2011) and DJH is supported by FEDER through the compete program, project “Genomics and Evolutionary Biology” co-financed by North Portugal Regional Operational Program (ON.2) under NSRF through the European Regional Development Fund. 110 28.863 -9.755 Tarentola mauritanica, Tropiocolotes algericus 111 28.839 -9.712 Uromastyx nigriventris 112 28.838 -9.721 Uromastyx nigriventris 113 28.809 -9.464 Trapelus boehmei 114 28.688 -9.318 Psammophis schokari, Uromastyx nigriventris 115 28.718 -9.466 Uromastyx nigriventris 116 28.677 -9.472 Uromastyx nigriventris 117 28.719 -10.302 Saurodactylus brosseti, Tarentola mauritanica 118 28.829 -10.412 Tarentola mauritanica 119 28.628 -10.791 Saurodactylus brosseti 120 28.455 -11.038 Psammophis schokari* 121 28.416 -11.398 Saurodactylus brosseti 122 28.221 -11.750 Saurodactylus brosseti 123 28.607 -10.519 Saurodactylus brosseti 124 28.498 -10.478 Ptyodactylus oudrii, Saurodactylus brosseti, Tarentola mauritanica 125 28.499 -10.428 Saurodactylus brosseti 126 27.835 -12.884 Hemorrhois algirus 127 27.820 -11.522 Trapelus boehmei 128 27.004 -13.428 Tarentola chazaliae 129 26.652 -13.652 Tarentola chazaliae 130 26.400 -14.075 Acanthodactylus aureus*, Tarentola chazaliae 131 26.155 -14.418 Acanthodactylus busacki, Saurodactylus brosseti 132 26.075 -14.457 Acanthodactylus busacki, Saurodactylus brosseti, Tropiocolotes algericus 133 26.541 -12.506 Trapelus boehmei 134 26.529 -12.307 Sphenops sphenopsiformis, Tropiocolotes algericus 135 27.051 -11.322 Stenodactylus mauritanicus, Tropiocolotes algericus 136 27.199 -10.694 Acanthodactylus boskianus 137 26.955 -11.664 Tarentola annularis 138 27.067 -13.118 Scutophis moilensis* FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 48 References Barata, M., Perera, A., Harris, D. 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(2010): Genetic variability within the Oudri‟s fan-footed gecko Ptyodactylus oudrii in North Africa assessed using mitochondrial and nuclear DNA sequences. - Molecular Phylogenetics and Evolution; 54 (2): 634–9. Pinho, C., Harris, D. J., & Ferrand, N. (2007): Contrasting patterns of population subdivision and historical demography in three western Mediterranean lizard species inferred from mitochondrial DNA variation. - Molecular Ecology; 16 (6): 1191–205. Rato, C., Carranza, S., & Harris, D. J. (2012): Evolutionary history of the genus Tarentola (Gekkota: Phyllodactylidae) from the Mediterranean Basin, estimated using multilocus sequence data. - BMC Evolutionary Biology; 12 (1): 14. Sambrook J, Fritsch E. F., & Mariatis, T. (1989): Molecular Cloning: a laboratory manual (2nd ed.); Cold Sp FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 50 Manuscript II Atlas Mountains: fuel for speciation? Phylogeographic patterns of Saurodactylus brosseti Daniela Rosado1,2, Catarina Rato1, David James Harris1 1 CIBIO Research Centre in Biodiversity and Genetic Resources, InBIO, Universidade do Porto, Campus Agrário de Vairão, Rua Padre Armando Quintas, Nº 7, 4485-661 Vairão, Vila do Conde, Portugal 2 Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, 4099-002 Porto, Portugal Abstract Phylogeographic assessments in regions such as North Africa are sparse when compared to Europe. However, various geographical barriers of Morocco, especially the Atlas Mountains have been associated with genetic subdivision in several species. Saurodactylus brosseti is a widely distributed species endemic to Morocco and a previous study demonstrated divergence within forms of up to 11.4% for a region of the mitochondrial ND4 gene. This was suggested to be indicative of a possible species complex, although sampling was limited and only mtDNA was employed, which may have unusually high rates of variation in geckos. In order to address these shortcomings, phylogeographic patterns within the species were assessed using two mitochondrial (12S and ND4) and three nuclear markers (ACM4, MC1R and BZW1), and with a greater coverage including apparently isolated southern and eastern populations. Results show four main lineages with high genetic diversity and which split at approximately the same time as the orogenesis of the Atlas Mountains. Species distribution modeling was also employed, identifying a large patch of suitable habitat where future sampling could be directed. The species delimitation approach employed was not completely concordant with the hypothesis that each of the four lineages could correspond to a distinct species. However this may have been due to limited sampling within two lineages. Further assessment, possibly including morphological data, would be valuable prior to any revision of the taxonomy, but the highly distinct lineage in the East clearly is of conservation concern. Keywords: Phylogeography, Saurodactylus brosseti, mitochondrial DNA, nuclear DNA, Species probability of occurrence, Vicariance, Atlas Mountains FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 51 Introduction Assessment of genetic diversity within various species in Europe has led to a strong consensus regarding typical phylogeographic patterns and in particular the role of “southern refugia” during Pleistocene glaciations (Hewitt, 1999). However, similar assessments within North Africa are much scarcer (Barrientos et al., 2014), and have often focused on relationships between North Africa and Europe (reviewed in Husemann et al., 2014). Yet various climatic and geological forces are likely to play a role in structuring diversity in North Africa, and particularly in the Northeastern region that is present day Morocco. For example, how successive expansions and contractions of the Sahara may have affected species is still poorly understood (reviewed in Brito et al., 2013) Further back in time, the genesis of the mountain chains in the region, the Rif and the Atlas Mountains, are expected to play a role as important geological barriers. In particular the orogeny of the Atlas Mountains is estimated to have occurred around 9 million years ago (Mya) in the late Miocene (Hsu, 1978), and separates the region climatically with more Mediterranean conditions to the West and more arid environments to the East. Various studies have indicated a vicariant role for these mountains, causing genetic subdivision in Agama impalearis (Brown et al., 2002; Gonçalves et al., 2012), the Acanthodactylus erythrurus group (Fonseca et al., 2009), the freshwater turtle Mauremys leprosa (Fritz et al., 2005; Fritz et al., 2006), scorpions of the genus Buthus (Habel et al., 2012; Husemann et al., 2012), the scorpion Androctonus mauritanicus (Coelho et al., 2014), and the bat complex Myotis natteri (Salicini et al., 2013). However, although in these cases the Atlas mountains played a role in separating populations, at a smaller scale quite different patterns were observed, with some groups such as scorpions showing many microrefugia (Husemann et al., 2012), and others such as the agamas showing much simpler subdivisions into just two major lineages (Brown et al., 2002). There is therefore a clear need to assess diversity within additional species from the region to try to develop an overall phylogeographic scenario for the region. Saurodactylus is a genus of geckos endemic to the Maghreb region (Bons and Geniez, 1996). It is an extremely old taxon that is estimated to have separated from its sister group almost 100 million years ago (Gamble et al., 2011). It currently comprises three species: Saurodactylus mauritanicus Duméril and Bibron 1836, Saurodactylus fasciatus Werner 1931 and Saurodactylus brosseti Bons and Pasteur 1957. Saurodactylus brosseti is endemic to Morocco, known to occur from Beni Mellal, central Morocco, to the depths of Western Sahara in the south (Figure 1). An assessment of phylogenetic relationships between the three species indicated that each was monophyletic, but that S. brosseti harbored a very high level of mitochondrial diversity, up to 11.4% for the ND4 gene region analyzed (Rato and Harris, 2008). Such a level of diversity is much higher than between many recently described lizard species (e.g. Ahmadzadeh et al., 2013), and was considered to be indicative of a possible species complex. However, sampling in this earlier study was limited, and in particular samples from the known localities of S. brosseti in the very arid regions in Western Sahara and to the East of the Atlas Mountains were not included. Just as molecular data, and particularly mitochondrial DNA sequencing, revolutionized the assessment of shifting distributions with Pleistocene climatic fluctuations, so the current tendency to combine molecular data with ecological niche modelling is transforming our predictions of current and future distributions. With improving data for various climatic variables it is possible to predict species distributions, both under current conditions and for expected future ones. This is particularly valuable in regions like North Africa and for groups such as reptiles were distribution data is still being refined (e.g. Damas-Moreira et al., 2014). Furthermore, this tool has been demonstrated to be effective in the Wall lizards Podarcis, with targeted fieldwork recording specimens in the expected but previously unreported localities (Kaliontzopoulou et al., 2008). FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 52 In this study a phylogeographic assessment of variation within S. brosseti was augmented with an ecological niche modelling approach and a relaxed molecular clock to determine both patterns and timing of subdivisions within the species. This was then used to assess the influence of the different geological events on the evolutionary history of S. brosseti. In particular the aim was to a) determine if the Atlas mountains had caused separation between lineages, b) to assess colonization pathways across the different occupied habitats, c) to determine if the isolated populations were genetically distinct, or were the result of recent fragmentation, possibly associated with climatic changes, and d) to identify other possible regions with high probability of the species occurring. Finally, a species delimitation approach was used to infer if taxonomic changes might be needed, that is if S. brosseti is actually a species complex. Material and methods Phylogenetic Analysis Sampling Two fieldtrips (May 2013 and June 2014) were carried out, from which 36 samples of Saurodactylus brosseti were collected. Samples were collected by hand and data recorded with GPS and annotations of relevant information. In addition, a small piece of the animal tail tip was collected and stored in 96% ethanol and photos were taken before the release of the animal. Available data from previous fieldwork was also used from Saurodactylus brosseti but also S. mauritanicus and S. fasciatus to be used as outgroups. DNA extraction, amplification and sequencing DNA was extracted from tail tips tissue using High Salt method (Sambrook et al., 1989). DNA amplification was performed through PCR for mitochondrial genes (12S and ND4), nuclear genes (ACM4 and MC1R) and one intron (BZW1). PCR conditions and primers were according to the references (Table 1). PCR products were checked with electrophoresis and positive amplification products were sent to Beckman Coulter Genomics (UK) for purification and Sanger sequencing. Table 1 – Primers names and amplification conditions. Gene 12S ND4 ACM4 BZW1 MC1R Step T (ºC) Time X T (ºC) Time X T (ºC) Time X T (ºC) Time X T (ºC) Time X Initial Denaturation 95º 1' 1 94º 3' 1 94º 5' 1 94º 3' 1 92º 2' 1 Denaturation 95º 48º 72º 15'' 15'' 10'' 35 94º 94º 48º 3' 30'' 30'' 35 94º 94º 55º 5' 30'' 45'' 1 32 94º 94º 62º 3' 30'' 45'' 37 92º 92º 55º 2' 1' 45'' 35 Annealing Extension Final Extension 72º 10' 1 72º 40'' 1 72º 1' 1 72º 1' 72º 1' 1 Primer Forward 12S L ND4 Tg-F Tar1 MC1R F Primer Reverse 12S H LEU Tg-R Tar2 MC1R R Citation Kocher et al. 1989 Arévalo et al. 1994 Gamble et al. 2008 Fuijta et al. 2010 Pinho et al. 2010 FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 53 Phylogenetic analysis Confirmation of species was done blasting sequences to the NCBI database on GenBank. Chromatographs were checked and sequences aligned with Geneious v5.6 for posterior phylogenetic analysis. 12S and ND4 sequences of Saurodactylus brosseti from a previously published study (Rato and Harris, 2008) available on GenBank were also included in the alignment. jModelTest v2.1.4 (Darriba et al., 2012 – 201) was used to infer which model best fit each data under the Akaike Information Criterion (Cavanaugh, 2007 – 205) for separate and concatenated genes in order to decrease the error (Brandley et al., 2005 - 227). Nuclear genes were phased using Seqphase (Flot, 2010 - 231) and PHASE (Stephens et al., 2005 – 230) with a threshold of 0.6 and default for all the other parameters. Phylogenetic accuracy can be higher when data sets from different genes are combined into a single phylogenetic analysis (Rokas et al., 2013 - 226). Maximum Likelihood analysis was performed using RAxML v3.0 (Stamatakis, 2014 - 229) with 1000 replicates for concatenated genes. Bayesian analyses were performed with best fitting models applied to each gene with MrBayes v3.2.2 (Huelsenbeck and Ronquist, 2001 – 192) for a concatenated approach (one partition) and *BEAST v1.8.0 (Drummond et al., 2012 – 204) for a coalescent approach (five partitions, all parameters unlinked across partitions, except for 12S and ND4, for which trees were linked into “mitochondrial DNA”). MrBayes analysis began with random starting tree, ran for 10 million generations and was sampled every 100 generations. 25.000 (25%) of burn-in trees were discarded and the remaining were used to assess posterior probability values. *BEAST ran three times for 150 million generations with an uncorrelated lognormal relaxed clock with a rate of 0.00701 for 12S (Metallinou et al., 2012 - 225) as calibration point to estimate divergence times. In order to have a species tree, sequences were grouped into four groups, according to the four major clades assessed by MrBayes and RAxML. All runs were combined with LogCombiner v1.8.0 (package of *BEAST) with a burn-in of 10% for each run. Mean genetic distances between the four major clades (given the results from MrBayes and RAxML) were calculated with MEGA6 (Tamura et al., 2013 – 203) for ND4. Species probability of occurrence Input occurrence data was obtained from geographic coordinates of known occurrence localities (Bons and Geniez, 1996) and CIBIO’s reptile database. In total, 246 points were depicted using ArcMap v9.3 under the WGS 1984 Datum geographic coordinate system. Five environmental variables (Table 2) were chosen according to the species known limiting factors and downloaded from WorldClim – Global Climate Data database (Hijmans et al., 2005 – 147) in 30arc seconds resolution tiles. Since correlation of environmental variables varies according to its extension, all five variables were cut giving Saurodactylus brosseti known distribution range, using ArcMap v9.3. Distribution of S. brosseti can overlap distribution of the other two species of the genus (Bons and Geniez, 1996), and so variables were also cut according to their distribution ranges, ensuring that localities with high probability of occurrence for one species are not coincident of the localities from neither the two other species. To estimate the presence probability of the three species of Saurodactylus the software Maxent (Phillips and Dudik, 2008 – 206) was used. All duplicate presence records from the known occurrence data were removed before the software run to prevent data overfitting. All runs were performed with 25 random test percentage, 20 bootstrap replicates and in order to create response curves with jacknife to measure variable importance. Area under the curve (AUC) was used to assess model accuracy. Even though it is not as reliable as it would be expected (Lobo et al., 2007 - 217) it still the best method. Since Maxent presents its results in a gradient “probability of occurrence” ranging from 0 to 1, a threshold was defined in order to build maps of “suitable or unsuitable” habitat. This FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 54 was done using the average of the 10 percentile training presence logistic threshold of the 20 replicates for each species. Values above the threshold were considered as “suitable habitat” and values below threshold were considered as “unsuitable habitat”. Table 2 – Environmental variables used in the assessment of species distribution probability. Code Variable BIO1 Annual mean temperature BIO6 Minimum temperature of coldest month BIO7 Temperature annual range BIO12 Annual precipitation BIO14 Precipitation of driest month Results From the Bayesian and Maximum Likelihood, four major groups can be distinguished: North, South, Northeast and Anti-Atlas. Northeast and South form a polytomy (Fig. 3), meaning that evolutionary relationships between themselves are not fully resolved. Some of the branches do not have high support. For example, South lineage has little support (posterior value = 0.65) and also in the split into Anti-Atlas and North lineages (posterior value = 0.85). On the other hand, Northeast, Anti-Atlas and North lineages are highly supported (posterior values = 0.99, 1 and 0.98 respectively; bootstrap values = 100 Northeast and 92 for Anti-Atlas lineages). After different groups within the main lineages are considered and spatially plot into the map (Fig. 3, different colors within the main clades), a more or less clear geographical distribution can be seen. South lineage has a geographical distribution partitioned from center of Morocco to the south most geographic known location (color gradient within South lineage); North lineage does not present such perfect division but it is also clear that some geographical structure is present (color gradient within North lineage). Either looking to the phylogeny or to the map into more detail, the region of the Draa Valey between North and South lineages presents lot of variation (Fig. 3); even the Anti-Atlas lineage is covered by that zone. Considering the species tree (Fig. 4), the four lineages are more clearly split, even though most branches do not present high support. Even though, it is evident that divergence occurred at the same time to the North and South of Morocco. Part of the population expanded to the South and has an obvious separation within the lineage that goes further into the south (Fig. 3); the specimens in southernmost locations of Morocco and the ones in Western Sahara are closely related, despite the gap between them. Divergence of the species into its lineages started at roughly the same time, which was in the Miocene around 10 Mya. Atlas Mountains are thought to have started their orogeny around 9 Mya (Hsu 1978, – 22), which then would have had influence on the split of the species. North and Anti-Atlas lineages are estimated to have split around 6 Mya and these two from the Northeast lineage at around 8 Mya. Genetic distances for ND4 show 6.9% of variation between North and Anti-Atlas lineages, 8% of variation between Anti-Atlas and Northeast lineages, and 8.1% divergence between North and Northeast lineages. Higher genetic variation is seen between South and Northeast (11%), North and South (11.3%) and Anti-Atlas and South lineages (11.5%). FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 55 A B C Fig. 1 – Modeling results of species probability of occurrence and habitat suitability for (A) Saurodactylus brosseti, (B) Saurodactylus mauritanicus, and (C) Saurodactylus fasciatus. FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 56 Modeling has predicted new patches of probability of occurrence for Saurodactylus brosseti (Fig. 1). The largest part is in the north of the country, but since it corresponds to the distribution range of Saurodactylus mauritanicus it is not very likely that S. brosseti will occur in that area. In the model prediction for S. mauritanicus there is also areas of suitable habitat for this species in which S. brosseti occur. Other interesting patch of high occurrence probability for S. brosseti is the south most border area of oriental region, which is not of high probability of occurrence for S. mauritanicus, especially by looking at suitable/unsuitable map (Fig. 1). Western Saharan areas present very low probability of occurrence and do not appear as suitable for S. brosseti. Regarding S. fasciatus results, there are no major changes to the actual known distribution range (Fig. 1). AUC showed good fit of the model for the three analyses: 0.955 for S. brosseti, 0.935 for S. mauritanicus and 0.925 for S. fasciatus Fig. 2 – Response curves of Saurodactylus brosseti to the five different environmental variables. The unit used for temperature data are in ºC * 10 and unit used for precipitation data are in mm. FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 63 Tamura K, Stecher G, Peterson D, Filipski A, and Kumar S (2013). MEGA6; Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution, 30, 27252729. FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 64 GENERAL DISCUSSION There are not as many phylogeographic studies of organisms from North Africa compared to Europe, but there are reports of species phylogenies being influenced by geographical barriers, such as the Moulouya River (e.g. Álvarez et al., 2000) or the Atlas Mountains (e.g. Fonseca et al., 2009). Also, climatic fluctuations, especially in the Pleistocene have had an impact in species distribution ranges (Hewitt, 2000). Around 9 Mya the formation of the Atlas Mountains began (Hsu, 1978) and it has had an impact, mainly of a vicariant nature, on several species. In Agama impalearis a simple subdivision into two lineages clearly formed at around 8.5 to 9.4 Mya was reported (Brown et al., 2002). It is suggested that the Atlas Mountains and also the reopening of the Strait of Gibraltar split the Acanthodactylus erythrurs group (Fonseca et al., 2012). Mauremys leprosa (Fritz et al., 2005; Fritz et al., 2006), Myotis natteri (Salicini et al., 2013), Androctonus mauritanicus (Coelho et al., 2014) and various Buthus species (Habel et al., 2012; Husemann et al., 2012) were also biogeographically substructured by the Atlas Mountains, although the latter displays many microrefugia (Husemann et al., 2012). Thus the Atlas Mountains are known to act as both a simple barrier, and a complex region in which multiple endemic forms have evolved, particularly those specialising as high-montane species such as Quedenfeldtia species (Barata et al., 2011) or Atlantolacerta andreanskyi (Barata et al., 2012). The Atlas Mountains have also had an impact and divide the climate of Morocco, which thus presents a much diversified topology, characteristics that together make the kingdom one of the richest in herptofauna diversity. For the reasons presented above, there is a need to assess phylogeographic patterns of the many other species of Morocco. Hewitt extensively studied the effects of the climatic oscillations during the Pleistocene and showed that diversity across many taxa was heavily influenced by those oscillations (Hewitt, 2000). Africa, and specifically the Sahara desert, was not an exception in experiencing climatic variations in the Quaternary Period, suffering from humid and drier periods (Brito et al., 2014); this also had an impact in the flora and fauna of the region. There is an additional need to accurately complete range distribution datasets, mainly in the Eastern region of the country (Beukema et al., 2013). This is the first step towards a complete phylogeographic study and only with a good knowledge of a species distribution range is it possible to truly understand evolutionary history and patterns of diversity within a species. After two fieldtrips to Morocco, a total of 53 different species in 138 localities were sampled. After DNA barcoding techniques were used for species for which identification was not possible in the field, various new range expansions were unveiled FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 65 (Manuscript I). These range extensions, especially for Bufo spinosus, Trapelus boehmei, Tropiocolotes algericus, Acanthodactylus erythrurus, Chalcides polylepis and Scutophis moilensis show that even species that are thought to be well known in terms of their distribution, sometimes are not. Specifically regarding Saurodactylus brosseti, the main focus of this thesis, a previous phylogenetic study, despite its limited sampling, had shown that Saurodactylus brosseti presents high levels of intraspecific variation, up to 11.4% for ND4 (Rato and Harris, 2008). Some recently described species do not present such high variation (e.g. Ahmadzadeh et al., 2013) and so, it represented a possible species complex. The results presented in the previous section again emphasize such diversity, given that results show that S. brosseti presents lot of variation (from 6.9% to 11.5%) between four distinct lineages, which are also geographically structured (Manuscript II). This also demonstrates that by increasing sampling effort it is possible to see more clearly the history of a species. In Rato and Harris (2008), two lineages were shown and that raised questions about the phylogeny of Saurodactylus brosseti; however only after a greater effort to analyse more specimens and with more genes was it possible to understand a new level of structuring in the species. This is very important, meaning that the higher and more accurate the effort is, the better researches can understand biogeographic patterns of species. A plausible explanation for such high differences between lineages is the rise of the Atlas Mountains. Estimated time of divergences showed that the time of the split of the four lineages coincides with the time for the orogeny of the Atlas Mountains. It seems like species expanded North and South at roughly the same time and started to diverge separately. However, the weak support of the tree branches turns makes it difficult to exactly interpret the history of the split. The Northeast lineage could have been an expansion from the South lineages through the east side of the Anti-Atlas or it was originally more related to the North lineage and was after split by the High-Atlas. Furthermore, there is still considerable variation within Northern and Southern lineages (Manuscript II) that is probably due to climatic oscillations during the Pleistocene coupled with the continuity of the Atlas Mountains rising since 9 Mya and onwards. Low support of some branches can be related to high diversity in some lineages, such as the Southern lineage, coupled with low sampling number in other lineages – Northeast and Anti-Atlas. Considering modelling of species distribution, it has been shown to be a useful tool to understand ecological requirements of species and also for directing specific sampling effort. For example, in the Podarcis lizards, modelling was demonstrated to be highly effective, highlighting patches were the species had not been but was later recorded FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 66 (Kaliontzopoulou et al., 2008). In this previous study, the area predicted was small and thus easier to do targeted fieldwork on; the area predicted for Saurodactylus brosseti is much bigger and thus more difficult to prospect extensively. Environmental modelling is a growing and powerful area in which past, present and future historical events can be predicted, making correspondence between environmental conditions and species distribution. Once again, accurate sampling is of great importance in order to get better predictions and model predictions help direct sampling efforts. Hence, the study of niche evolution has gained a major importance in the last few years, focusing on techniques to estimate niche overlap in realized niches between taxa in an explicit spatial context (Broennimann et al., 2012), either based on ordination methods (Hof et al. 2010; Thuiller et al. 2005) or on the output of species distribution models (SDMs) (Guisan and Thuiller, 2005; Warren et al. 2008). More recently, based on the methodology proposed by Evans et al. (2009), SDMs are being combined with calibrated phylogenies in order to study the evolution of climatic niches by reconstructing the ancestral environmental tolerances among lineages (e.g. Ahmadzadeh et al. 2013; Jakob et al. 2010; Smith and Donoghue, 2010). This would also be an interesting future work that could help understand differences in the ecological requirements of the different lineages. Speciation and species delimitation is extremely difficult and it is clear that in order for a lineage or population to be considered a species, integrative frameworks should be used including not only different phylogeny approaches but also morphology (Fujita et al., 2012). Saurodactylus brosseti is a small gecko and morphological assessment is challenging but it could be done in order to support phylogenetic findings. This thesis comprises a phylogeographic analysis from which is clear that the levels of genetic variation within the species is very high. Since only two mitochondrial and three nuclear genes were used and due to small sampling of Northeast and Anti-Atlas lineages, the relations between all four lineages are still not fully understood. A higher number of genes could be sequenced in the future, in order to better estimate the phylogeographic patterns of the species, combined with morphological characters assessment, comprising a full species analysis. 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FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 83 APPENDIX Origin of sample Species Lat Long Accession numbers (12S/ND4/ACM4/BZW1/MC1R) Fieldtrip 2013 Saurodactylus brosseti 30.30 -9.52 Fieldtrip 2013 Saurodactylus brosseti 29.86 -9.50 Fieldtrip 2013 Saurodactylus brosseti 29.86 -9.50 Fieldtrip 2013 Saurodactylus brosseti 29.86 -9.50 Fieldtrip 2013 Saurodactylus brosseti 29.06 -9.93 Fieldtrip 2013 Saurodactylus brosseti 28.63 -10.79 Fieldtrip 2013 Saurodactylus brosseti 28.63 -10.79 Fieldtrip 2013 Saurodactylus brosseti 28.42 -11.40 Fieldtrip 2013 Saurodactylus brosseti 28.22 -11.75 Fieldtrip 2013 Saurodactylus brosseti 28.22 -11.75 Fieldtrip 2013 Saurodactylus brosseti 26.16 -14.42 Fieldtrip 2013 Saurodactylus brosseti 26.07 -14.46 Fieldtrip 2013 Saurodactylus brosseti 28.61 -10.52 Fieldtrip 2013 Saurodactylus brosseti 28.50 -10.48 Fieldtrip 2013 Saurodactylus brosseti 28.50 -10.48 Fieldtrip 2013 Saurodactylus brosseti 28.50 -10.48 Fieldtrip 2013 Saurodactylus brosseti 28.50 -10.43 Fieldtrip 2013 Saurodactylus brosseti 28.50 -10.43 Fieldtrip 2013 Saurodactylus brosseti 28.72 -10.30 Fieldtrip 2013 Saurodactylus brosseti 29.39 -10.17 Fieldtrip 2013 Saurodactylus brosseti 29.65 -9.99 Fieldtrip 2013 Saurodactylus brosseti 30.04 -9.64 FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 84 Fieldtrip 2013 Saurodactylus brosseti 30.04 -9.64 Fieldtrip 2013 Saurodactylus brosseti 30.54 -9.71 Fieldtrip 2013 Saurodactylus brosseti 30.54 -9.71 Fieldtrip 2013 Saurodactylus brosseti 30.54 -9.71 Fieldtrip 2013 Saurodactylus brosseti 31.00 -9.58 Fieldtrip 2013 Saurodactylus brosseti 31.00 -9.58 Fieldtrip 2013 Saurodactylus brosseti 31.12 -9.43 Fieldtrip 2013 Saurodactylus brosseti 31.12 -9.43 Fieldtrip 2013 Saurodactylus brosseti 31.48 -9.76 Fieldtrip 2013 Saurodactylus brosseti 31.48 -9.76 Fieldtrip 2013 Saurodactylus brosseti 31.48 -9.76 Fieldtrip 2013 Saurodactylus brosseti 31.48 -9.76 Fieldtrip 2013 Saurodactylus brosseti 31.48 -9.76 Fieldtrip 2014 Saurodactylus brosseti 31.62 -5.56 Published in Rato and Harris 2008 Saurodactylus brosseti 33.25 -8.50 EU014300/EU014325/-/-/- Published in Rato and Harris 2008 Saurodactylus brosseti 31.62 -8.00 EU014301/EU014326/-/-/- Published in Rato and Harris 2008 Saurodactylus brosseti 31.50 -9.77 EU014311/EU014336/-/-/- Published in Rato and Harris 2008 Saurodactylus brosseti 31.68 -8.85 EU014302/EU014327/-/-/- Published in Rato and Harris 2008 Saurodactylus brosseti 31.68 -8.85 EU014312/EU014337/-/-/- Published in Rato and Harris 2008 Saurodactylus brosseti 31.68 -8.85 EU014304/EU014329/-/-/- Published in Rato and Harris 2008 Saurodactylus brosseti 31.68 -8.85 EU014303/EU014328/-/-/- Published in Rato and Harris 2008 Saurodactylus brosseti 31.07 -8.68 EU014305/EU014330/-/-/- Published in Rato and Harris 2008 Saurodactylus brosseti 30.10 -9.55 EU014306/EU014331/-/-/- Published in Rato and Harris 2008 Saurodactylus brosseti 29.88 -9.60 EU014307/EU014332/-/-/- Published in Rato and Harris 2008 Saurodactylus brosseti 29.88 -9.60 EU014313/EU014338/-/-/- FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 85 Published in Rato and Harris 2008 Saurodactylus brosseti 33.25 -8.50 EU014314/EU014339/-/-/- Published in Rato and Harris 2008 Saurodactylus brosseti 31.89 -6.91 EU014308/EU014333/-/-/- Published in Rato and Harris 2008 Saurodactylus brosseti 31.89 -6.91 EU014309/EU014334/-/-/- Published in Rato and Harris 2008 Saurodactylus brosseti 31.49 -7.98 EU014310/EU014335/-/-/- Reptile DB Saurodactylus brosseti 30.95 -8.25 Reptile DB Saurodactylus brosseti 30.99 -9.04 Reptile DB Saurodactylus brosseti 30.06 -9.09 Reptile DB Saurodactylus brosseti 30.06 -9.09 Reptile DB Saurodactylus brosseti 30.06 -9.09 Reptile DB Saurodactylus brosseti 30.06 -9.09 Reptile DB Saurodactylus brosseti 29.95 -9.01 Reptile DB Saurodactylus brosseti 30.03 -9.05 Reptile DB Saurodactylus brosseti 29.58 -9.40 Reptile DB Saurodactylus brosseti 29.58 -9.40 Reptile DB Saurodactylus brosseti 29.51 -9.06 Reptile DB Saurodactylus brosseti 29.74 -8.96 Reptile DB Saurodactylus brosseti 29.58 -9.40 Reptile DB Saurodactylus brosseti 29.58 -9.40 Reptile DB Saurodactylus brosseti 29.60 -10.03 Reptile DB Saurodactylus brosseti 29.60 -10.03 Reptile DB Saurodactylus brosseti 29.60 -10.03 Reptile DB Saurodactylus brosseti 29.60 -10.03 Reptile DB Saurodactylus brosseti 29.60 -10.03 Reptile DB Saurodactylus brosseti 29.60 -10.03 Reptile DB Saurodactylus brosseti 28.03 -11.36 FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 86 Reptile DB Saurodactylus brosseti 29.48 -10.09 Reptile DB Saurodactylus brosseti 28.54 -10.96 Reptile DB Saurodactylus brosseti 32.53 -7.86 Reptile DB Saurodactylus brosseti 32.53 -7.86 Reptile DB Saurodactylus brosseti 32.53 -7.86 Reptile DB Saurodactylus brosseti 32.53 -7.86 Reptile DB Saurodactylus brosseti 32.53 -7.86 Reptile DB Saurodactylus brosseti 32.66 -7.79 Reptile DB Saurodactylus brosseti 32.66 -7.79 Reptile DB Saurodactylus brosseti 32.66 -7.79 Reptile DB Saurodactylus brosseti 31.62 -5.56 Reptile DB Saurodactylus brosseti 32.14 -6.40 Reptile DB Saurodactylus brosseti 31.75 -8.74 Reptile DB Saurodactylus brosseti 29.09 -9.89 Reptile DB Saurodactylus brosseti 29.09 -9.89 Reptile DB Saurodactylus brosseti 30.42 -9.58 Reptile DB Saurodactylus brosseti 31.68 -8.85 Reptile DB Saurodactylus brosseti 32.13 -6.32 Reptile DB Saurodactylus brosseti 31.10 -8.94 Reptile DB Saurodactylus brosseti 31.10 -8.94 Reptile DB Saurodactylus brosseti 31.89 -7.94 Reptile DB Saurodactylus brosseti 30.81 -7.58 Reptile DB Saurodactylus brosseti 30.81 -7.58 Reptile DB Saurodactylus brosseti 31.33 -9.38 Reptile DB Saurodactylus brosseti 31.28 -9.79 FCUP Phylogeographic patterns of the Moroccan lizard-fingered gecko Saurodactylus brosseti 87 Reptile DB Saurodactylus brosseti 31.26 -9.16 Reptile DB Saurodactylus brosseti 31.26 -9.16 Reptile DB Saurodactylus brosseti 31.51 -8.16 Published in Rato and Harris 2008 Saurodactylus fasciatus 34.77 -5.52 EU014299/EU014343/-/-/- Published in Rato and Harris 2008 Saurodactylus fasciatus 32.60 -7.81 EU014296/EU014340/-/-/- Reptile DB Saurodactylus fasciatus 34.15 -4.83 Reptile DB Saurodactylus mauritanicus 34.90 -3.59 Appendix 1. – Information of the samples used for phylogenetic analyses.