Thesis
Reference
Insights on the History of Seasonally Dry Tropical Forests in South America: Inferences from the Genetic Structure of the trees Astronium
urundeuva (Anacardiaceae) and Geoffroea spinosa (Fabaceae)
CAETANO WYLER, Sofia Alexandra
Abstract
Les Forêts Tropicales Décidues ou Semi-décidues (FTDS) constituent, en Amérique du Sud, un important ecosystème. La distribution des sept nuclei isolés autour du Bassin Amazonien a été suggérée comme étant le résultat de la fragmentation récente d'une formation plus continue, qui a atteint son extension maximale pendant les périodes glaciaires du Pléistocène. Ce travail vise en particulier à tester ce scénario, ainsi que l'hypothèse alternative de dispersion à longue distance. Dans ce contexte, la répartition de la diversité génétique est décrite pour deux arbres, Astronium urundeuva (Allemão) Engler (Anacardiaceae; Syn: Myracrodruon urundeuva F.F. and M.F. Allemão) et Geoffroea spinosa Jacq. (Fabaceae). Les deux espèces sont spécifiques des FTDS, et ont été choisies parce qu'elles présentent, entre autres caractéristiques, quelques traits contrastés très importants:
A. urundeuva est dioïque, distribuée de façon continue dans l'Est de l'Amérique du Sud, et ses graines se dispersent par le vent; G. spinosa, au contraire, est une espèce hermaphrodite qui est distribuée de façon [...]
CAETANO WYLER, Sofia Alexandra. Insights on the History of Seasonally Dry Tropical Forests in South America: Inferences from the Genetic Structure of the trees
Astronium urundeuva (Anacardiaceae) and Geoffroea spinosa (Fabaceae). Thèse de doctorat : Univ. Genève, 2008, no. Sc. 3946
DOI : 10.13097/archive-ouverte/unige:101781 URN : urn:nbn:ch:unige-1017814
Available at:
http://archive-ouverte.unige.ch/unige:101781
Disclaimer: layout of this document may differ from the published version.
1 / 1
BIBLIOTHEQUE SCIENCES II
Division Biologle 30, quai Ernest-Ansermet
1211 GENEVE4
Insights on the history of Seasonally Dry Tropical Forests in South America:
Inferences from the genetic structure of the trees
Astronf um urundeuva (Anacardiaceae) and
Geoffroea spinosa (Fabaceae)
presentee a la Faculte des Sciences de l'Universite de Geneve pour obtenir le grade de Docteur es sciences, mention biologie
par
Soffa Alexandra RAMOS CAETANO de
tisbonne (P17)
These N° 3 946
Geneve 2008
Insights on the history of Seasonally Dry Tropical Forests in South America:
Inferences from the genetic structure of the trees
Astronium urundeuva (Anacardiaceae) and
Geoffroea spinosa (Fabaceae)
THESE
presentee a la Faculte des Sciences de l'Universite de Geneve pour obtenir le grade de Docteur es sciences, mention biologie
par
Sofia Alexandra RAMOS CAETANO de
Lisbonne (PT)
These N° 3 946 Geneve
2008
(Departement de botanique et de· biologie ve_getale), Messieurs L. EXCOFFIER, docteur (University of Bern - Zoological Institute - Computational and Molecular Population Genetics Lab - Bern, Switzerland). et T. PENNINGTO�, docteur (Royal Botanic Garden Edinburgh - Tropical Diversity Section - Edinburgh. United Kingdom), autorise !'impression de la presente these, sans exprimer d'opinion sur les propositions qui y sont enoncees.
Geneve. le 4 fevrier 2008
These - 3946 -
TABLE OF CONTENTS
ACKNOWLEDGEMENTS SUMMARY
RESUME
CHAPTER I -INTRODUCTION
1.1 General introduction 1.2 Objectives
1. 3 Organisation of the manuscript 1 .4 State of the art
CHAPTER 11 -MOLECULAR MARKERS
2.1 Introduction
2.2 Chloroplast Spacers 2.3 Microsatellite Markers
3
5
7 9 11 12 14 15 21 23 24 25 2.3.1 Primer Note in MolEcol Notes for Astronium urundeuva 29 2.3.2 Primer Note in MolEcol Notes for Geoffroea spinosa 32
CHAPTER 111-ASTRONIUM URUNDEUVA 37
3.1 Presentation of Astronium urundeuva 39
3.2 Differentiation among three closely related Astronium species 43
3.2.1 Research article in press in Candollea 45
3.3 Genetic structure within and between populations of Astronium urundeuva 68
3.2.2 Research article submitted to MolEcol 69
CHAPTER IV - GEOFFROEA SPINOSA 99
4.1 Presentation of Geoffroea spinosa 101
4.2 Genetic structure within and between populations 104
4.2.1 Material and Methods 105
4.2.2 Results 107
4.2.3 Discussion 114
4. 3 Colonisation of the Galapagos Islands 118
4.3.1 Research article to be submitted to PNAS 121
CHAPTER V - PHYLOGEOGRAPHY 145
5.1 Introduction 147
5.2 Comparative. Phylogeography of Astronium urundeuva and Geoffroea spinosa 149 5.2.1 Chapter in press within the Book Seasonally Dry Tropical Forests 151
CHAPTER VI -GENERAL DISCUSSION, CONCLUSIONS AND PERSPECTIVES 187
6.1 General discussion 189
6.2 Conclusions 193
6.3 Perspectives 195
BIBLIOGRAPHIC REFERENCES 199
APPENDIX 219
1 - Book Chapter published in 2006 with preliminary results 221
2 - Supplementary data for Candollea 241
3 - Supplementary data for Molecular Ecology 245
4 -Supplementary data for G. spinosa Microsatellites 251
5 -The Approximate Bayesian Computation 257
6 - Photos 263
ACKNOWLEDGEMENTS
La realisation de cette these n 'aurait pas ete possible sans l 'aide et les encouragements de nombreuses personnes,
a
qui je tiens temoigner ma profonde gratitude. Leur soutien a pris des formes tres diverses, que ce soit au niveau technique, scientifique ou humain, et j'espere n'oublier personne dans les prochaines lignes.Tout d'abord, je tiens
a
remercier Yamama Naciri de m'avoir accueillie dans son labo et d'avoir dirige cette these avec,a
la fois de la competence, de la patience et surtout de la tenacite. La qualite de ce travail doit beaucoupa
son encadrement dynamique et motivant,a
son haut savoirfaire et
a
sa rigueur scientifique. Je la remercie egalement pour l'amitie qu'elle m'a temoignee au cours de ces annees, en partageant avec moi de nombreuses experiences qui seront pour toujours enregistrees dans mon creur.Je remercie le Professeur Rodolphe Spichiger, d'avoir accepte la direction de cette these, et d'avoir apporte ses connaissances floristiques et systematiques sans lesquels ce travail aura it ete amoindri.
Je le remercie egalement d'avoir corrige les differentes versions de ce manuscrit et de m'avoir accorde son soutien et sa confiance.
Taus mes remerciements
a
Laurent Excoffier et Estella Poloni d'avoir fait partie du comite de these.Leurs commentaires au cours des deux reunions et des rencontres hors des CJB ont permis d'ameliorer significativement cette recherche. Un merci particulier
a
Laurent Excoffier qui a aimablement accepte d'etre membre de mon jury de these et d'apporter ainsia
la discussion ses competences statistiques en genetique des populations. Je le remercie egalement de m'avoir accorde l'acces gratuit au cluster CMPGa
Berne. Merci IIJe remercie Toby Pennington pour son soutien au cours de cette etude, ainsi que ses patientes relectures et corrections des differents articles qui font partie de ce manuscrit. Je le remercie egalement de faire partie du jury de these et d'apporter ainsi ses connaissances floristiques dans la discussion de ces resultats. Thank you II
Je tiens aussi
a
remercier Darien Prado, qui a fait preuve d'une grande disponibilite pour m'aidera
mener
a
bien ce travail, par les lectures et relectures minutieuses des differents articles et de!'ensemble de la these. Je le remercie aussi pour son enthousiasme, sa gentillesse et son amitie.
Muchas Gracias 11
Sur le terrain, ii y a vraiment beaucoup de gens qui ont apporte leur soutien et je tiens
a
lesremercier tous. Argentine: Luis Oakley et Darien Prado pour l'echantillonnage et pour le superbe voyage; Bolivie: Luis Oakley ainsi que Stephan Beck pour l'echantillonnage; Bresil: Ary Oliveira-Filho pour l'obtention du permis de recolte et d'exportation et !'organisation sur place de la mission;
Rubens dos Santos pour sa bonne compagnie pendant les longs kilometres de route et ses performances sur le terrain; Dulcineia de Carvalho de m'avoir accueillie dans son labo, mais aussi
chez elle pendant mon sejour; les equipes des labos de Floristique et de Biologie Moleculaire avec qui j'ai partage des moments vraiment tres sympas; Warley dos Santos pour l'echantillonnage dans le Mato Grosso; Galapagos : Galapagos Diving pour le transport entre les iles, meme si les conditions ont ete parfois difficiles; Edie Rosero pour l'echantillonnage a Floreana et Famille Cruz pour l'accueil; le Parque Nacional du Galapagos et la Fondation Charles Darwin; Paraguay: Fatima Mereles pour !'organisation sur place de la mission; Monica Soloaga et sa maman pour l'amitie et l'accueil en debut de sejour; Karen Elizeche pour l'echantillonnage et sa maman pour l'accueil donne en fin de sejour; Patricia Silveira pour son travail de diplome; Perou: Aniceto Daza pour l' echantillonnage.
Je tiens ensuite a remercier tous les membres du labo aux CJB, soit d'avoir participe directement a l'obtention des donnees, Regine Niba, Lara Turin, Luisa Schneider, Oliver Perron, soit d'avoir ete de bons conseils et pour les nombreuses discussions, Mathieu Perret, Jean-Franc;:ois Manen, Fadil Adjiva.
Je remercie pareillement Nicolas Wyler et Mathias Currat d'avoir partage leurs respectives connaissances informatiques, Nicolas dans les SIG et Mathias dans la methode ABC. Cette these a ete beaucoup amelioree avec ces deux outils, et mon acces aces mondes paralleles n'aurait pas pu se faire sans eux.
Tous mes remerciements vont aussi a mes collegues des CJB. L'ambiance qui regne dans l'institut constitue un environnement de travail fort agreable et je vous remercie vivement pour tous les moments partages. Je remercie particulierement le present directeur, Dr. Pierre-Andre Loizeau, de m'avoir accueillie dans son institut et de m'avoir accorde les moyens de terminer cette these dans les meilleurs delais.
Ce travail a ete finance par les Fonds National Suisse de la Recherche Scientifique (3100A0/100806- 1 & 2) et les CJB. D'autres institutions ont egalement participe avec des bourses pour les frais de voyage: Societe Academique de Geneve, Societe de Physique et d'Histoire Naturelle and Societe Suisse de Zoologie.
Merci enfin a mes amis et a ma famille qui m'ont soutenu tout au long de ce chemin, en ayant supporte (des fois avec beaucoup de difficulte) ce long sejour si loin. La distance n'a pas toujours ete facile a gerer, mais je vous remercie d'avoir ete la. Obrigada !! Meme si la these parait parfois une tongue course en solitaire, contre vents et marees, cette traversee ne serait pas possible sans Nico. Merci.
A vous tous, un grand MERCI ! ! A big THANK YOU, to you all I!
A todos, muchas GRACIAS I!
A todos, um muito OBRIGADA 11
SUMMARY
Seasonally Dry Tropical Forests (SDTF) are one of the important ecosystems in South America, whose distribution around the Amazon Basin over seven isolated nuclei has been hypothesized to have been attained by recent fragmentation of a more continuous formation that reached a maximum extension during dry Pleistocene glacial periods. In the present study, this particular scenario is tested against the alternative hypothesis of isolation through long-distance dispersal. In this context, the patterns of genetic diversity of two unrelated trees, Astronium urundeuva (Allemao) Engler (Anacardiaceae; Syn:
Myracrodruon urundeuva F.F. and M.F. Allemao) and, Geoffroea spinosa Jacq. (Fabaceae), are described. Both species are specific to the SDTF, and they were chosen because they present, among other requirements, some contrasting important features: A. urundeuva is a dioecious species, relatively continuously distributed in eastern South America, with seeds being wind-dispersed; G. spinosa is a hermaphrodite species that is distributed over five disjunct areas, with seeds dispersed by small mammals or by water courses. The genetic patterns of the two species were assessed by sequencing chloroplast intergenic spacers and by microsatellite genotyping, as these two markers resolve complementary levels of differentiation.
Because tree species belonging to the genus Astronium can sometimes be difficult to identify on the sole basis of the morphological traits of the leaves, a preliminary study using a Bayesian assignment procedure was developed in order to assess the degree of differentiation of A. urundeuva with its two sister species, A. balansae Engl. and A.
fraxinifolium Schott. The three species were unambiguously identified at the molecular level, and the analyses within A. urundeuva could be safely undertaken, since possible bias issued from a mixed sampling could be ruled out. The patterns observed for A. urundeuva suggested former vicariance of an ancestral population, followed by progressive differentiation of the two main lineages due to isolation at the range extremes (Chaco and Caatinga). It is further suggested that, more recently, a rapid range expansion occurred, allowing the two lineages to meet at the limits of the Caatinga nucleus (secondary contact).
The data ob�ained for G. spinosa seemed to correspond to a two-level structure that probably reflects different times of differentiation. The Pacific nucleus corresponded
to a much more ancient vicariance, whilst the differentiation among the Chacoan group and the Caatinga seems rather recent. Whether these groups represent a real regional level of differentiation was, therefore, questioned. The particular unresolved status of G.
sp;nosa in the Galapagos Islands was further investigated through an Approximate Bayesian Computation (ABC) approach by testing two different colonisation scenarios, human introduction versus ·natural dispersal. Results clearly show that statistically human introduction is the most sustained scenario.
Although independent in their evolution, A. urundeuva and G. spinosa share some pieces of history that help clarifying the past distribution patterns of the SDTF in South America. The main purpose of the present study was attained, at least to a certain extent, and for eastern South America, the vicariance scenario is strongly supported. The present data favours the recent existence of a continuous SDTF formation that stretched from the Brazilian Caatinga to Chaco region, and that got subsequently fragmented as the climate became more humid.
RESUME
Les Forets Tropicales Decidues ou Semi-decidues (FTDS) constituent, en Amerique du Sud, un important ecosysteme. La distribution des sept nuclei isoles autour du Bassin Amazonien a ete suggere comme etant le resultat de la fragmentation recente d'une formation plus continue, qui a atteint son extension maximale pendant les periodes glaciaires du Pleistocene. Ce travail vise en particulier
a
tester ce scenario, ainsi que l'hypothese alternative de dispersiona
longue distance. Dans ce contexte, la repartition de la diversite genetique est decrite pour deux arbres, Astron;um urundeuva (Allemao) Engler (Anacardiaceae; Syn: Myracrodruon urundeuva F.F. and M.F. Allemao) et Geoffroea spinosa Jacq. (Fabaceae). Les deux especes sont specifiques des FTDS, et ont ete choisies parce qu'elles presentent, entre autres caracteristiques, quelques traits contrastes tres importants: A. urundeuva est dioi"que, distribuee de fac;:on continue dans l'est de l' Amerique du Sud, et ses graines se dispersent par le vent; G. spinosa, au contraire, est une espece hermaphrodite qui est distribuee de fac;:on discontinue sur cinq regions, et ses graines sont dispersees par des petits mammiferes ou par les cours d'eau. Les patterns genetiques des deux especes ont ete etablis au moyen du sequenc;:age de fragments intergeniques du chloroplaste et par le genotypage de microsatellites, puisque ces deux types de marqueurs resolvent des niveaux complementaires de differenciation.Les arbres du genre Astronium sont parfois difficiles
a
identifier sur la seule base des traits morphologiques de leurs feuilles. De ce fait, une etude preliminaire a ete effectuee, en appliquant une methode Bayesienne, de fac;:ona
estimer le degre de differentiation entre A. urundeuva et deux especes voisines, A. balansae Engl. et A. frax;nifoUum Schott qui sont trouvees en sympatrie. Les trois especes ont ete identifiees sans aucune ambigui"te, et les analyses au sein d' A. urundeuva ont pu etre entreprises sans risque, car le possible biais dOa
un echantillonnage melange a ete ecarte. Les donnees observees pour A.urundeuva suggerent une fragmentation ancienne d'une population ancestrale, suivie de la differentiation progressive des deux lignages due
a
un isolement aux deux extremes de la distribution de l'espece (Chaco et Caatinga). ll est egalement suggere que plus recemment, ces deux lignages se sont repandus tres rapidement, permettant un contact secondaire au niveau de la limite sud du nucleus Caatinga.Les donnees obtenues pour G. spinosa correspondent
a
une structurea
deux niveaux qui reflete probablement des periodes isolees de differenciation. Le nucleus Pacifiquecorrespond a une fragmentation beaucoup plus ancienne, tandis que la differenciation entre le groupe du Chaco et celui de la Caatinga semble plus recente. Le fait que ces deux groupes puissent ne pas representer un veritable niveau de differentiation a meme ete questionne. Par ailleurs, comme le statut de G. spinosa aux Galapagos est encore inconnu (espece introduite ou native), une methode Bayesienne (Approximate Bayesian Computation; ABC) a ete utilisee pour tester deux scenarios differents de colonisation, introduction humaine versus dispersion naturelle. Les resultats montrent clairement que l'introduction humaine est plus soutenue statistiquement.
Quoique independantes dans leur evolution, A. urundeuva et G. spinosa partagent quelques fragments d'histoire, dont la comprehe.nsion est importante pour clarifier l'histoire de la distribution des FTDS en Amerique du Sud. Le but principal de ce travail a ete atteint, en tout cas jusqu'a un certain point. Pour l'est du continent, le scenario de vicariance est vivement soutenu. Les donnees presentees ici favorisent done l'existence recente d'une formation FTDS plus continue qui s'est etendue de la Caatinga dans le nord
est du Bresil jusqu'a la region du Chaco, et qui s'est ensuite fragmentee au moment ou le climat est devenu plus humide.
CHAPTER I
INTRODUCTION
1 . 1 GENERAL INTRODUCTION 1211 GENEVE 4
In the last decades, biological diversity has received increasing attention from biologists, ecologists, conservationists and politicians interested in understanding the threats it is currently facing, and in delineating the necessary approaches to preserve it. This diversity is not, however, arbitrarily distributed over the planet's surface, being instead organized into different biomes that interact in more or Less complex relationships (Olson et al. , 2001 ). Therefore, assessing the way biodiversity is distributed necessarily requires to understand the origin, history and present-day maintenance of the different biomes, and ultimately of the individual species they are composed of (Pennington et al. , 2004a). In this perspective, characterizing the distribution of genetic diversity, and understanding the present and ancient ecological and evolutionary processes through which it arises, is one of the most promising research axes. This is, clearly, the scope of population genetics and phylogeogeography that aim at explaining the mechanisms controlling the geographic distribution of the genetic diversity (Avise et al. , 1 987).
In this study, the theoretical principles of both disciplines are combined in order to trace back the Long-term history of a particular ecosystem in South America, the Seasonally Dry Tropical Forests (SDTF). In this context, the patterns of genetic diversity within two unrelated trees, Astronium urundeuva (Allemao) Engler (Anacardiaceae; Syn:
Myracrodruon urundeuva F.F. and M.F. Allemao) and Geoffroea spinosa Jacq. (Fabaceae), are described. In a general way, trees have already been pointed out as particularly suited to investigate the factors that shape the evolution of ecosystems, due to their intrinsic attributes (Petit and Hampe, 2006): great Longevity; high levels of genetic diversity within populations but Little differentiation among populations; Large population sizes; Low speciation and mutation rates. For the present study, the two tree species have been selected among four other candidate species, because they satisfied some fundamental requirements: specificity to the SDTF, wide distribution throughout the SDTF, good taxonomic definition, relatively easy recognition in the field (in the absence of flowers and fruits, although some prudence for A. urundeuva is still kept), relative abundance and improbable dispersion by humans.
1 . 2 OBJECTIVES
The main purpose of the present study is to test whether the current areas of SDTF are refugial fragments of a previously larger and more continuous SDTF (Prado and Gibbs, 1 993), which may have been spread across large areas of the Amazon basin during the late Pleistocene (Pennington et al., 2000). The alternative hypothesis is that these wide distributions of SDTF species would have been attained by long-disiance dispersal (Gentry, 1 982; Naciri et al., 2006). In the last years, several authors have attempted to test these hypotheses, most of them by assessing the floristic links among species assemblages of SDTF areas (Pennington et al., 2000; Linares-Palomino et al., 2003; Bridgewater et a[; , 2004; Pennington et al., 2004b; Spichiger et al., 2004; Oliveira-Filho et al., 2006; Queiroz, 2006). Herein, the hypothesis of a previously more widespread SDTF formation will be tested by exploring the chloroplast and nuclear genetic structures within the two selected SDTF specific tree species across their range. In this perspective, the following goals have been defined:
1 . Describe the distribution of the genetic variability and structure within each species. Two hierarchical levels will be tested: the area level (putative refugia) and the population within area level, but other configurations could also be tested, depending on the results. Comparable amounts of genetic differentiation between and within areas are expected in the case of a recent fragmentation of the SDTF into distinct areas. Otherwise, a much higher differentiation between areas should be observed under the long-distance dispersal hypothesis, due to founding events. Because of the inherent features of each genetic marker, the genetic structure is expected to be more pronounced with the chloroplast spacers.
2. Compare the phylogeographic patterns of both species. The way chloroplast haplotypes are spatially distributed has been shown to be strongly influenced by the demographic regime of colonisation (Ibrahim et al. , 1 996; Le Corre et al., 1 997). In a
"diffusive model", populations spread as an advancing front that leads to large areas in which the same alleles are predominant. At the other extreme, isolated events of long
distance dispersal, following a leptokurtic progression, are expected to dramatically increase the patchiness observed. Moreover, when diffusion and long distance dispersal are associated (stratified dispersal), more complex spatial patterns are observed, often with an apparent mixing between a clinal trend and a patchy structure.
3. Reconstruct the potential routes of colonisation. The colonisation routes can be followed, particularly in plants, by examining the patterns of genetic variability distribution, when considering neutral markers (Comes and Kadereit, 1998). Because the levels of diversity progressively diminish with distance to the source population, along with a geographical axis (Wijsman and Cavalli-Sforza, 1984), identifying the direction of the dispersal should still be possible. Moreover, and although caution should be taken when interpreting gene genealogies, the way the differentiated haplotypes are organized in space is also very suggestive when retracing colonisation routes.
4. Identify the centre of genetic variability for each species. A common correlation is usually made between the centre of genetic diversity for a species (that is the region displaying the highest genetic variability), the centre of origination of the species itself, and the centre of dispersal (Epperson, 2003). This generalisation has been proved to be false in some cases (e.g., centres of diversity that correspond to admixture areas and not to the origin of populations; Comps et al., 2001; Walter and Epperson, 2001 ), and higher levels of genetic diversity in well defined geographical areas may simply reflect more stable population demography and larger population sizes (Comes and Kadereit, 1998). Identifying each species' geographical origin is not within the scope of this study. However, and considering that each species possesses individual evolutionary histories, identifying a common centre of genetic diversity, and possibly a common centre of dispersal, would greatly contribute to understand the processes behind the SDTF distribution patterns.
The approach to be used here, focusing upon the distribution of the genetic variability of several tree-species to assess more general historical patterns for a given ecosystem, has been widely used across Europe and North America (e.g, Huntley et al., 1989; Taberlet et al. , 1998; Lessa et al., 2003). Whilst the studies conducted in the northern continents were largely centred on recolonisation of empty territories after the last glacial maximum (LGM), the colonisation history in South America involves several replacement events among the different ecosystems. Therefore, more complicated and imprecise patterns are expected for the SDTF, reflecting the combination between the two colonisation scenarios, recent fragmentation versus long-distance dispersal, in the context of vegetation shifts.
Keeping these particularities in mind, the following general questions will be addressed:
},>, What can be inferred from the observed patterns of the two species on the SDTF history? Do the two species show the same history? To what extent do the observed patterns allow wider inferences on the ecosystem history?
},>, Why do the SDTF have a patchy distribution around the Amazon Basin? Is there an evidence of a former larger and more continuous formation, or have the disjunct populations resulted from rare and isolated events of long-distance dispersal?
},>, When did the scattered pattern originate? If the current distributions have been attained by vicariance rather than dispersal, did this fragmentation occur after the Quaternary ice ages, or is it more ancient?
).- Where was the widespread SDTF located? If expansion of the SDTF existed, did it extend into the Amazon Basin or was the continuous formation restricted to eastern South America?
� How does the differential gene flow, through pollen and seeds dispersal, interfere in the observed structure? Is there differential structuring using nuclear or chloroplast markers?
},>, What is the extant connection between the SDTF nuclei? Does the current genetic structure within and between areas evidence isolation of the different areas?
1 . 3 ORGANISATION OF THE MANUSCRIPT
To achieve the above objectives, different but complementary approaches have been adopted, which are organised in two ways: population genetics within each species, and comparitive phylogeography. This manuscript is, therefore, organized in the following way:
within this first introductory chapter a bibliographic revision gathering relevant theoretical aspects about the central question of the SDTF in South America is presented (1.4).
Chapter II describes the advantages of using a combination of two types of molecular tools, namely chloroplast intergenic spacers (2.2) and microsatellite markers (2.3) that have been published as primer notes in Molecular Ecology Notes for both species (Caetano et al., 2005; Naciri-Graven et al. , 2005).
In the following two chapters, I was interested in discussing separately the genetic patterns obtained within Astronium urundeuva (Chapter Ill) and Geoffroea spinosa
(Chapter IV). For Astronium urundeuva, the floristic and vegetative features are first described, followed by the presentation of its geographical range and description of the sampling strategy (3.1 ). Because differentiating A. urundeuva from two closely related species, A. fraxinifolium and A. balansae, can sometimes be difficult, a novel Bayesian approach was applied that is the subject of a paper in press in Candollea (3.2). Finally, the genetic variability within A. urundeuva across its whole range (3.3) is described and discussed in the light of the SDTF history, in shape of a paper submitted to Molecular Ecology.
Floristic and vegetative characteristics of Geoffroea spinosa, along with a description of its distribution range and the sampling performed are presented in section 4.1. The way the genetic diversity and structure obtained with microsatellites is geographically distributed is then discussed (4.2). Finally, the status of the species in the Galapagos Islands is considered (4.3), in shape of a paper draft that will soon be submitted to the Proceedings of the National Academy of Sciences. Here, both scenarios of colonisation, namely over
water dispersal versus human introduction, are confronted through simulations applying the Approximate Bayesian Computation method.
Chapter V compares the phylogeographic patterns of the two species. These results are presently in press within an edited volume composed of chapters prepared by the participants of the meeting "Symposium on Latin American Seasonally Dry Tropical Forests" that took place at Stanford University in December 2006.
Within Chapter VI, a synthesis of the results and the different discussions is performed, in order to link the different chapters and supply general conclusions. Furthermore, some perspectives are also presented and discussed.
1 .4 STATE OF THE ART
South America has high physical, climatological and biological diversity. It includes environments ranging from dry desert to humid rainforest that are organised in very complex patterns. Consequently, the vegetation distribution presents a major patchiness, and there are currently many reasons to explain its particular highly complex history. Part of the difficulty in interpreting the present patterns derives from the overlapping, in a time perspective, of the most relevant events that structured vegetation (Figure 1.1;
Burnham and Graham, 1999). These authors have outlined four major events to explain the
mosaic of habitats observed:
i) The geographic isolation of South America that persisted as an island from early Jurassic until the Late Pliocene (3-2 million years ago; MYA).
ii) The uplift of the Andes, which extended from 1 5 MY A to the present, with important consequences in drainage systems. Interestingly, young rising events have been suggested for the central and northern Andean plateaus, with more than half of the modern elevation having occurred between 1 0 and 2 MYA (Gregory-Wodzicki, 2000).
iii) The emergence of the Panamanian land bridge between North and South America is also very recent, dating from the late Pliocene, about 3 to 3 . 5 MYA.
iv) The climatic fluctuations during the Quaternary epoch, during which the ice sheets and the permafrost remained restricted to high altitudes and never reached the lowlands. Conditions during the last glacial maximum (LGM) have been shown to be both drier (van der Hammen and Absy, 1 994) and cooler (Colinvaux et al., 1 996).
The particular effect of the latter event on the distribution of the several South American biomes has received increasing attention in the last decade. Whereas in Europe and North America some general recolonisation patterns could be established (Taberlet et al., 1 998;
Swenson and Howard, 2005), the situation is by far more poorly understood in South America (Coltrinari, 1 993; Servant et al. , 1 993). For instance, current disagreement still exists about which climatic factor, drought or cooling, most influenced the biomes distribution (Pennington et al. , 2000). It is, nevertheless, generally accepted that these climatic changes have driven substitutions between ecosystems, instead of recolonisation of empty territories in comparison with what has been described in the northern continents.
In this perspective, and in order to explain the huge species diversity in the Amazon Basin, the "Refuge Theory" has been established. According to this theory, the observed diversity would have resulted from consecutive speciation events, favoured by successive fragmentations and subsequent isolation of the Amazonian rainforests in pocket areas (Haffer, 1 982; Prance, 1 982a; Connor, 1 986). This proposition depends upon the validation of a replacement model, according to which rainforest species distributions would have been temporarily fragmented within some sort of drier vegetation matrix. Until recently, a form of grass-rich savanna has been assumed to be the substituting vegetation
(Bush, 1 994; Colinvaux et al., 1 996; Hoorn, 1 997), but the lack of grass pollen in the few pollen cores from the Amazon Basin (Haberle and Maslin, 1 999) have led authors to directly reject the whole Refuge Theory (Connor, 1 986; Colinvaux et al., 1 996; Colinvaux, 1997;
Hoorn, 1 997). More recently, the debate went, however, further and the replacement theory has gained some new support (Haberle and Maslin, 1 999; Cowling et al., 2001 ; Ledru et al., 2001 ), with the introduction of an ecological niche modelling approach, based on the occurrence patterns of 1 7 species of birds and woody plants (Bonaccorso et al., 2006). The authors were, indeed, able to confirm the fragmentation of Amazon species' ranges during the Pleistocene, but could not clearly resolve the trends of the savanna species. A second form of dry-adapted vegetation, the SDTF, has been proposed as replacement vegetation (Pennington et al. , 2000). This suggestion was based upon several key studies: first the detailed description of the patterns of distribution for over 80 SDTF taxa (Prado and Gibbs, 1 993), second the recognition of the SDTF as a real phytogeographic unit (Prado, 2000), and third pollen cores that were claimed as SDTF species (Pennington et al., 2000).
ERA PERIOD EPOCH EVENTS
Holocene
1 Lat Clitate
Quaternary 1 0'000 YA · Present
1 .8 MYA · Present Pleistocene ch
+ges
1 .8 M · 10'000 YA bridge
.., Pliocene J•
•
u � 5.8 · 1 .8 MYA
-�
c. Miocene urft
� 23.8 · 5.8 MYA
::E Tertiary Oligocene
65 · 1 .8 MYA 33.7 · 23.8 MYA
Eocene
54.8 - 33.7 MYA
Pleocene Isolation
65 - 54.8 MYA
Maastrichtian
70.6 - 65 MYA
Campanian
83.5 - 70.6 MYA
Santonian
u � 85.8 - 83.5 MYA
::E Coniacian
89.3 - 85.8 MYA
Cretaceous Turonian
144 - 65 MYA 93.5 - 89.3 MYA
Cenomanian '
99.6 - 93.5 MYA
Albian
1 12.0 - 99.6 MYA
Figure 1 . 1 Time scale for the mid-Cretaceous period to present showing the major events responsible for the structuring of the neotropical vegetation patterns (adapted from Burnham and Graham, 1 999).
Seasonally Dry Tropical Forests occur where the rainfall is less than 1600 mm/yr, with a period of at least 5-6 months receiving less than 100 mm (Gentry, 1995). SDTF are tree
dominated ecosystems (Murphy and Lugo, 1986), characterised by the almost complete absence of grasses in the ground layer, and the presence of deciduous or semi-deciduous vegetation during the dry season (Mooney et al., 1995). Flowering and fruiting is seasonal, occurring synchronously for many species between the dry and wet seasons. Conspicuous flowers and wind-dispersed fruits and seeds are very frequent (Pennington et al. , 2006b).
Moreover, SDTF occur on fertile soils with moderate to high pH and nutrient levels, and low levels of aluminium. Because such soils are very suitable for agriculture, dry forest destruction has been observed in many areas (Miles et al., 2006). Moreover, and according to these authors, a variety of factors, mostly resulting from human activity, represent major threats to practically all tropical dry forests.
Four major SDTF areas can be found in South America: north-eastern Brazil (the Caatinga nucleus), two areas in the periphery of the Chaco domain defined by Prado and Gibbs (1993) as the Misiones and the Piedmont nuclei, and the Caribbean coasts of the Colombia and Venezuela. Other smaller and more isolated SDTF areas occur in dry valleys of the Andes and northern Bolivia, Peru, southern Ecuador and Colombia (Figure 1.2; Pennington et al., 2000). Isolated smaller remnants occurring as enclaves surrounded by a different vegetation have also been reported: within the Cerrado (a savanna biome that covers the central Brazilian plateau; Furley and Ratter, 1988), and within the Chaco (woodlands and xeromorphic forests that occur on the less well-drained soils of Paraguay, Argentina and Bolivia; Spichiger et al., 1995).
On the basis of the close floristic links between distant SDTF areas in South America, Prado and Gibbs (1993) hypothesized the "Pleistocenic Arc". This theory proposes that present
day disjunct distribution patterns of the SDTF represent "vestiges of a once extensive and largely contiguous seasonal woodland formation, which may have reached its maximum extension during a dry-cool climatic period ea. 18,000-12,000 BP, coinciding with the contraction of the humid forest" (Prado and Gibbs, 1993). According to these authors, the more continuous expanse of SDTF stretched from the Brazilian Caatinga to the SDTF in the peripheral areas of the Chaco domain, and possibly also reached the dry inter-Andean valleys of Bolivia, Peru and Ecuador. More recently, Pennington et al. (2000) went further to suggest that SDTF species may have also penetrated into the Amazon basin during glacial periods. These authors found support to their proposal from the pollen core from
northwest Amazon, which was claimed to be equally consistent with SDTF (against a rainforest biome), and argued that distinguishing these two ecosystems is not straightforward. This is owed to the fact that most pollen types cannot be identified to the species level, and because most families are found simultaneously in both SDTF and rainforests.
•soTF
•Chaco Cerrado
' •
. .
"'°---==4l--'-=:l---CiQOl<JlomUe,:
Figure 1 .2 Schematic distributions of the SDTF, the Cerrado and the Chaco in South America. For the SDTF the different nuclei are numbered: 1 . Caatinga; 2. Misiones; 3. Chiquitano; 4. Piedmont;
5. Bolivian, Peruvian and Ecuadorean inter-Andean valleys; 6. Pacific coast of Peru and Ecuador and the Galapagos Islands; 7. Caribbean coast of Colombia and Venezuela and the Dutch Antilles Islands (adapted from Pennington et al. , 2000).
The hypothesis concerning the expansion of the SDTF during the driest-coolest phases of the Pleistocene epoch and subsequent fragmentation into the current pocket areas has been increasingly debated, as testified by the production of two edited volumes within the three last years (Pennington et al. , 2006a; Dirzo et al. , in press). Alternatively, other authors have invoked that the currently scattered distribution of the SDTF species in South America could be the result of rare, isolated events of long-distance dispersal (Gentry, 1 982; Naciri et al., 2006 - Appendix 1 ). In the next chapters (Ill to VI), the former scenarios will be discussed in light of the genetic data obtained for both A. urundeuva and G.
spinosa. The observed patterns will be put in parallel, in order to infer the most parsimonious colonisation history for the SDTF in South America.
CHAPTER II
MOLECULAR MARKERS
2. 1 INTRODUCTION
In the past two decades, the understanding of plant biogeography has been substantially improved by the introduction of various molecular marker systems. Different types of markers have been developed to evaluate polymorphism, but increased attention has been given to the DNA-based techniques in the detection of variation within plants (Nybom, 2004). DNA markers have acted as multifaceted fundamental tools in the recent advances observed in various fields, among which are population genetics, phyloge<!>graphy, phylogeny, taxonomy, ecology, conservation, evolution, physiology, embryology, genetic engineering and plant breeding. The discovery of PCR (polymerase chain reaction; Mullis and Faloona, 1 987) was a landmark in this effort and proved to be a fundamental step in the whole process. Ideal DNA markers should satisfy several essential conditions: be polymorphic; show codominant inheritance that allows the determination of the homozygous and heterozygous states in diploid systems; present a selectively neutral behaviour in such a way that environmental conditions or management practices exercise no pressures; be available with an easy and fast assay; be highly reproducible; and allow an easy exchange and comparison of data among different model organisms. It seems obvious that it is practically impossible to fulfil all the above criteria, and, therefore, the selection of the suitable marker system relies on a compromise that depends on the type of study to be undertaken (Weising et al., 1 995).
The biggest dilemma that plant population biologists interested in phylogeography are confronted with is probably the detection of phylogenetically informative intraspecific variation. Uniparentally inherited markers, such as chloroplast DNA, have been traditionally used in phylogeographic and evolutionary analyses (e.g. , McCauley, 1 995;
Petit, 1 997; Dutech et al., 2000; Cavers et al., 2003), but more recently, however, neutral nuclear loci have been proposed as good information source for phylogeographic processes (Luikart et al. , 2003). Microsatellites are of particular promise, providing new opportunities to test the effect of the colonisation process on patterns of genetic structure (e.g. , Heuertz et al. , 2004; Song et al. , 2006). In fact, the potential of phylogeography can only be fully appreciatted if the patterns described are based on the combination of different molecular markers that resolve complementary levels of information (Schaal et al. , 1 998). Accordingly, and for the purposes of the present study, two different molecular tools have been selected, chloroplast intergenic spacers and nuclear microsatellites.
2.2 CHLOROPLAST SPACERS
The use of the chloroplast genome for population genetic and phylogeographic studies have been motivated by the same attractive characteristics that have made mitochondrial DNA such a widely used marker in animal studies (Avise and Lansman, 1 983; Ennos et al. ,
1 999) . The haploid chloroplast DNA is characterized by a reduced effective population size when compared to the nuclear DNA, an assumed lack of recombination and a generally uniparental inheritance. In fact, the mode of plastid transmission seems not that simple, and the strict inheritance of the chloroplast is being more and more questioned (Lee et al. , 1 988; Johnson and Palmer, 1 989; Reboud and Zeyl, 1 994). However, and despite the few exceptions according to which the chloroplast is paternally (Masoud et al. , 1 990;
Testolin and Cipriani, 1 997) or even bi parentally inherited (Hansen et al. , 2007), maternal inheritance remains the most common mode of plastid transmission among angiosperms (Harris, 1 991 ). On the other hand, comparative studies of chloroplast DNA also suggest that rearrangements can be mediated by intra or intermolecular recombination (Ogihara et al. ,
1 988). Short inverted repeats found i n the chloroplast have been proved to function as hotspots of intermolecular recombination (Kawata et al. , 1 997), and other authors have found recombination signatures using chloroplast microsatellites (Marshall et al. , 2001 ) .
Herein, a strict maternal inheritance and n o recombination will be assumed for the two tree species, because no means to test the chloroplast inheritance were available"
Analysis of genetic variation using chloroplast markers allows the elucidation of historical factors influencing genetic variation (Schaal et al., 1 998), and because of the restricted nature of gene flow through seed dispersal, it is more likely to detect the effects of population history in present-day genetic patterns with chloroplast DNA than with nuclear markers (Ennos et al. , 1 999) . The contemporary gene flow does, of course, have an influence in the distribution of genetic variability, but the effects of historical and recent factors can often be easily differentiated (Templeton et al. , 1 995; Schaal et al. , 1 998).
Although widely used, chloroplast markers present some limitations at the intraspecific level due to the often low rate of evolution and the scarcity of informative characters (Ennos et al. , 1 999). This problem has been partially solved with the use of intergenic spacers and introns of chloroplast DNA (Taberlet et al. , 1 991 ; Demesure et al. , 1 995;
Dumolin-Lapegue et al. , 1 997b; Hamilton, 1 999a) in studies focusing on the relationships among and within plant populations (Mousadik and Petit, 1 996; Dumolin-Lapegue et al. ,
1 997a; Caron et al., 2000; Cavers et al., 2003). The common accumulation of insertions and deletions (indels) in these parts of the genome appears to evolve more rapidly than the changes in gene sequence, and proved to supply important reliable information at the intraspecific level (Gielly and Taberlet, 1 994; Provan et al., 2001 ; Hamilton et al., 2003;
lngvarsson et al., 2003; Kress and Erickson, 2007).
Although several research efforts have been conducted to find noncoding regions that reliably amplify within a broad group of angiosperm species (Grivet et al., 2001 ; Duminil et al., 2002), discovering informative variation is still a meticulous process involving the investigation of a great amount of regions. A straightforward approach is the use of chloroplast DNA-based datasets, but the probability of identifying variation at a given locus through DNA sequencing is still low, due to the small portions of DNA targeted by such technique (Schaal et al., 1 998). It is, nevertheless, undeniable that the direct sequencing of cpDNA loci allows an improved access to the different types of mutations, allowing a clear evaluation of their respective contribution to the evolution of a given species. At the beginning of this study, a preliminary survey of several chloroplast spacers · was undertaken, where universal primers available in the literature were tested not only for amplification consistency among the two key species (in order to have comparable data), but also for the amount of informative characters through sequencing. Satisfying results were obtained with three loci: trnH-psbA, trnS-trnG (Hamilton, 1 999a) and trnL-trnF (Taberlet et al., 1 991 ). These will be used in different combinations in the following chapters.
2.3 MICROSATELLITE MARKERS
In the past few years, a wide variety of techniques have been developed, among which the highly variable nuclear microsatellite markers, which proved to complement to a great extent o.ur understanding of plant evolution (e.g. , Latouche-Halle et al., 2004; Walter and Epperson, 2005; Truong et al., 2006; Craft et al., 2007). Microsatellites are short tandem repeats of one to six nucleotides that are frequent and widely distributed through the nuclear genome (Jame and Lagoda, 1 996). A survey of the presence, abundance and ubiquity of microsatellites in higher plants revealed that, within 34 species, di and trinucleotide repeats were uncovered at a frequency of one every 50 kb, with AT repeats
being by far the most frequently observed class of di nucleotide microsatellites (Morgante and Olivieri, 1 993).
Because microsatellites are codominant, it is possible to distinguish the copies inherited from both parents and, hence, to define homozygous and heterozygous individuals. This feature is particularly important when standard population genetics formulae for estimating among- and within population diversity are to be applied (Nybom, 2004). In a general way, microsatellites are more frequent in non-coding regions (but see Ranum and Day, 2002), and, therefore, are often assumed to be neutral markers. However, this assumption is not always valid (Nielsen et al., 2006), as microsatellites can sometimes suffer from hitchhiking effects, due to association with functional genes or coding regions.
Whenever loci are under stabilizing selection, estimates of differentiation among populations will be deflated, whilst an inflation of the genetic differentiation measures is, on the other hand, expected if loci are submitted to differential (spatial) selection (Beaumont and Balding, 2004; Beaumont, 2005).
Slippage and proofreading errors during DNA replication are usually the origin of microsatellite mutations, which primarily change the number of repeats and consequently the length of the sequence (Eisen, 1 999). Very high mutation rates are often associated with these markers, which vary greatly among species, ranging from 5x1 0·5 in Drosophila (Schlotterer et al. , 1 998) to 10·3 in human (Brinkmann et al., 1 998). In plants, mutation rates have been estimated from inbred lines (maize, wheat, chickpea) and have been reported to vary from 7. 7x10·4 to 1 0·2 (Udupa and Baum, 2001 ; Thuillet et al., 2002;
Vigouroux et al. , 2002). Moreover and even within species, mutation rates vary widely among loci ( di Rienzo et al., 1 998), depending on the microsatellite length (Primmer et al. , 1 996) and motif (Schlotterer and Tautz, 1 992). In population genetics, mutation rate is a critical parameter because it allows access to the history of a population o� of a portion of the genome, through variability at microsatellite loci. However, little is known about the variance of this parameter within tree species. The different mutation models used for microsatellites are also of great importance for analysing microsatellite evolution and for the inferences using real datasets: i) Infinite Allele Model (1AM; Kimura and Crow, 1 964), where any mutation creates a new allele; ii) Stepwise Mutation Model (SMM; Kimura and Ohta, 1978), where mutation occurs by loss or gain of a single unit of the repeat motif; iii) Two Phase Model (TPM; di Rienzo et al. , 1 994), where a proportion of the mutations introduce the gain or the loss of more than one repeat, in addition to more frequent single-step mutations; and iv) K-Allele Model (KAM; Crow and Kimura, 1 970), where an
exact number of k possible allelic states can be found.
The major drawback of these markers has been attributed to time and money involved in the finding of species-specific oligonucleotides in the flanking regions of the microsatellites. Valuable markers require, therefore, the identification of a fragment with multiple versions (alleles) adjacent to more stable stretches, where specific primers are designed. If mutations arise in the primer region, amplification fails to occur, resulting in genotyping errors (null alleles; Callen et al., 1993) that may substantially bias the subsequent analyses. This may, indeed, represent a potential difficulty when using primer sequences in other congeneric species, but it depends primarily on the evolutionary history of the species in question. In this perspective, the usefulness of these markers for the identification of close related species has already been reported (Duminil et al. , 2006).
However, caution should still be taken in such studies, because microsatellites can be substantially different in length and/or structure among different taxa, the so-called
"ascertainment effect" (van Treuren, 1998). Moreover, even within the same species, no guarantees exist that microsatellite markers will efficiently amplify everywhere, especially if widespread samples with ancient evolutionary histories are considered. Given the wide distribution range of the samples used in this study, particular attention will be given to the effec:,t of such null alleles on the observed structure. Another quite common problem reported with microsatellites is allelic dropout (Falush et al. , 2007), which generally occurs because detection of the different alleles is size-based toward the shortest alleles that are preferentially amplified during the PCR process. Finally, a problem also frequently associated with microsatell ites is homoplasy (Selkoe and Toonen, 2006), which corresponds to the confusion between identity by state and identity by descent. In fact, due to the high mutation rates often associated to these markers, alleles from different origins can evolve in a convergent way, resulting in the same allele being genotyped in divergent populations, and ultimately in a "false" decrease of the genetic structure.
Although homoplasy has been shown to represent only significant problem in very few population genetics studies (Estoup et al. , 2002), special attention will be given to this artefact here. In fact, the effects of homoplasy can be relatively significant in the case where the different SDTF regions correspond to ancient vicariance events, as alleles would have had sufficient time to evolve in a convergent way.
At the beginning of the present study, no microsatellites primers had been published for any of the key species before the beginning of this study, but, under the supervision of Y.
Naciri, several loci had already been identified for both Astronium urundeuva and
