MASARYK UNIVERSITY FACULTY OF SCIENCE DEPARTMENT OF BOTANY AND ZOOLOGY
Phylogeny of squirrels (Sciurus, Rodentia) based on supertrees reconstruction
Bachelor thesis
Patrícia Pečnerová
Supervisor: Mgr. NATÁLIA MARTÍNKOVÁ, Ph.D. BRNO 2011 BIBLIOGRAFICKÝ ZÁZNAM
Autor: Patrícia Pečnerová Prírodovedecká fakulta, Masarykova univerzita Ústav botaniky a zoológie
Názov práce: Fylogenéza veveríc (Sciurus, Rodentia) pomocou rekonštrukcie superstromov
Študijný program: Biológia
Študijný odbor: Systematická biológia a ekológia
Vedúci práce: Mgr. Natália Martínková, Ph.D.
Rok obhajoby: 2011
Kľúčové slová: Sciurini, stromové veverice, fylogenéza, superstromy BIBLIOGRAPHIC ENTRY
Author: Patrícia Pečnerová Faculty of Science, Masaryk university Department of Botany and Zoology
Title of thesis: Phylogeny of squirrels (Sciurus, Rodentia) based on supertrees reconstruction
Degree programme: Biology
Field of study: Systematic biology and ecology
Supervisor: Mgr. Natália Martínková, Ph.D.
Year of defence: 2011
Keywords: Sciurini, tree squirrels, phylogeny, supertrees ACKNOWLEDGEMENT
I would like to express my thanks to Dr. Natália Martínková, who supervised my thesis, provided me with advise, help and support, dedicated a lot of time to me and has taught me essentials of scientific work. PREHLÁSENIE
Prehlasujem, že som túto bakalársku prácu napísala samostatne a výhradne s použitím citovaných prameňov. Súhlasím s uložením tejto bakalárskej práce v knižnici Ústavu botaniky a zoológie Prírodovedeckej fakulty MU v Brne, prípadne v inej knižnici MU, s jej verejným požičiavaním a využitím pre vedecké, vzdelávacie alebo iné verejne prospešné účely, a to za predpokladu, že prevzaté informácie budú riadne citované a nebudú využívané komerčne.
Brno 5. mája 2011 ...... Patrícia Pečnerová ABSTRACT
Since molecular data are used to infer phylogeny, many taxonomic ambiguities have been discussed in the group of tree squirrels of the tribe Sciurini. In this study, I worked with extensive published molecular material to establish phylogenetic relationships and discover where the tribe originated. I used mitochondrial and nuclear DNA sequences and through Bayesian analysis and six different methods of supertree reconstruction, I determined the phylogeny of Sciurini squirrels. A total of 17 included species represented all currently recognized genera and wide geographic distribution of the group. Results of this work supported findings of recent molecular studies. Instead of five currently recognized genera, only two of them were supported and the genus Sciurus was paraphyletic. Species of Microsciurus, Rheithrosciurus and Syntheosciurus occured in lineages with Sciurus species. Originally assigned subgeneric level of these taxa would be more appropriate. Furthermore, supertrees revealed routes of dispersal of the tribe, as species were clustered according to their geographic distribution. Early divergence of palearctic species indicated that the genus Sciurus evolved in Eurasia, crossed land bridge in Beringia and dispersed across North America, entering South America after formation of the Isthmus of Panama. In South America, Microsciurus and Syntheosciurus diversified, while ancestors of Rheithrosciurus colonized Borneo from Eurasia. Relatively recent colonization of the tropics by Sciurini squirrels demonstrates that hypotheses associated with tropical origin as an explanation for latitudinal gradient in species richness were not relevant. Higher diversification rate in the tropics was probably a more accurate explanation. ABSTRAKT
Odkedy sa molekulárne dáta využívajú na stanovenie fylogenézy, vrámci skupiny stromových veveríc tribu Sciurini sa pojednáva o mnohých taxonomických nejasnostiach. Pracovala som s rozsiahlym molekulárnym materiálom, aby som stanovila fylogenetické vzťahy a odhalila, kde skupina vznikla. Použila som mitochondriálne a jadrové DNA sekvencie, verejne dostupné, a pomocou Bayesiánskej analýzy a šiestich rôznych metód rekonštrukcie superstromov som určila fylogenézu veveríc tribu Sciurini. Zahrnutých bolo 17 druhov, ktoré reprezentovali všetky súčasne rozoznávané rody a rozsiahle geografické rozšírenie skupiny. Výsledky tejto práce podporili objavy súčasných molekulárnych štúdií. Namiesto piatich aktuálne rozoznávaných rodov boli v našej štúdii potvrdené iba dva a rod Sciurus bol parafyletický. Druhy rodov Microsciurus, Rheithrosciurus a Syntheosciurus sa objavili v líniach spolu s druhmi rodu Sciurus, pôvodne pridelená úroveň podrodov by bola pre tieto taxóny vhodnejšia. Navyše, superstromy odhalili smery, ktorými sa tribus rozširoval, pretože druhy boli zoskupené podľa geografického rozšírenia. Ranné odštiepenie palearktických druhov naznačilo, že rod Sciurus sa vyvinul v Eurázii, prekročil pevninský most v Beringii a rozšíril sa cez Severnú Ameriku, vstúpiac do Južnej Ameriky po vytvorení Panamskej šije. V Južnej Amerike sa oddelili rody Microsciurus a Syntheosciurus, zatiaľ čo predkovia rodu Rheithrosciurus kolonizovali Borneo z Eurázie. Pomerne nedávna kolonizácia trópov vevericami tribu Sciurini dokazuje, že neboli platné hypotézy spojené s tropickým pôvodom ako vysvetlením gradientu v druhovej bohatosti podľa nadmorskej výšky. Vyššie diverzifikačné tempo v trópoch bolo, pravdepodobne, vhodnejšie vysvetlenie. TABLE OF CONTENTS
1. Introduction...... 9 1.1. Phylogenetics...... 9 1.2. Ecology and zoogeography of tree squirrels...... 9 1.3. Taxonomic background...... 11 1.4. Evolutionary history...... 12 1.5. Latitudinal gradient in species richness...... 13 1.5.1. Geometrical constraints...... 13 1.5.2. Origin-related interpretations...... 14 1.5.3. Effects of evolutionary rates...... 15 1.6. Aims...... 19 2. Materials and methods...... 20 3. Results...... 23 4. Discussion...... 25 4.1. Phylogenetic reconstruction...... 25 4.2. Explanation of recent zoogeography...... 26 4.3. Tropical species richness...... 26 4.4. Conclusions and implications...... 27 References...... 28 Appendix...... 36 9 1. INTRODUCTION
1.1. Phylogenetics
According to one of the basic principles of biological evolution, all living organisms have evolved from a common ancestor (DARWIN 2006). Lineages of descendants of this single ancestor are depicted in a tree diagram creating basic phylogenetic units, phylogenetic trees (GAUCHER et al. 2010). Tree-like forms with genealogical content have origins in work of Charles Darwin (MOOERS & HEARD 1997, BAUM 2008). HUSON & BRYANT (2005) characterize phylogenetic tree as "a leaflabeled tree that represents the evolutionary history of a set of taxa, possibly with branch lengths, either unrooted or rooted". Root is the point of the oldest common ancestor. With respect to existence or non-existence of a common ancestor, the tree can be rooted or not (GAUCHER et al. 2010). Branches and nodes are of major importance in a phylogenetic tree. Nodes correspond to diversifying events and branches connect them with possibility to indicate the evolutionary time (WHELAN et al. 2001). Longer branches indicate more time of speciation and separation of taxa if mutation rate is similar along branches. The position of a node with respect to root reflects when in evolutionary history the taxon separated. The taxa closer to the root are in a basal position and are more similar to the common ancestor. Meanwhile, the most recently diverged taxa overcame more speciation events and diversified. Terminal nodes (leaves or tips) are specifically identified in a labeled tree (MOOERS & HEARD 1997). Clade, or a lineage, is a monophyletic group in the tree, a group including the ancestor and all its descendants (BAUM 2008). Taxa in this group have common evolutionary history as they all diverged from a single point, the specific node of the tree.
1.2. Ecology and zoogeography of tree squirrels
Tribe Sciurini represents a group of tree squirrels found in deciduous, coniferous and tropical forests that are generally diurnal (NOWAK 1999). Sciurini squirrels, in comparison with other squirrels of the family Sciuridae, are of average size, except the small genera of Microsciurus Allen, 1985 and Syntheosciurus Bangs, 1902 and the large Rheithrosciurus Gray, 1867. The geographic range of 37 species of this tribe comprises Eurasia, North and South America. Geographic distribution and classification are further described in Tab 1. 10 Tab 1. Geographic distribution and classification of the tribe Sciurini according to the classification of WILSON & REEDER (2005) and the geographic distribution defined by the IUCN (IUCN v2010.4 2010). Species marked with "*" sign were included in this study. Genus Subgenus Species Ecozone Geographic Range Microsciurus M. alfari * Neotropic Central, South America M. flaviventer * Neotropic South America M. mimulus Neotropic Central, South America M. santanderensis Neotropic Central, South America Rheithrosciurus R. macrotis * Indomalaya Borneo Island Sciurus Sciurus S. alleni Nearctic/Neotropic Mexico S. arizonensis Nearctic Mexico, United States S. aureogaster Nearctic/Neotropic Mexico S. carolinensis * Nearctic Canada, United States S. colliaei Nearctic/Neotropic Mexico S. deppei Neotropic Mexico, Central America S. lis * Palearctic Japan S. nayaritensis Nearctic/Neotropic Mexico, United States S. niger * Nearctic Canada, United States S. oculatus Nearcitc/Neotropic Mexico S. variegatoides * Neotropic Mexico, Central America S. vulgaris * Palearctic Eurasia S. yucatanensis Neotropic Mexico, Central America Otosciurus S. aberti * Nearctic Mexico, United States Guerlinguetus S. aestuans * Neotropic South America S. gilvigularis Neotropic South America S. granatensis * Neotropic Central, South America S. ignitus * Neotropic South America S. pucheranii Neotropic South America S. richmondi Neotropic South America S. sanborni Neotropic South America S. stramineus * Neotropic South America Tenes S. anomalus * Palearctic Asia Hadrosciurus S. flammifer Neotropic South America S. pyrrhinus Neotropic South America Hesperosciurus S. griseus * Nearctic Mexico, United States Urosciurus S. igniventris Neotropic South America S. spadiceus Neotropic South America Syntheosciurus Sy. brochus * Neotropic Central America Tamiasciurus T. douglasii Nearctic Canada, United States T. hudsonicus * Nearctic Canada, United States T. maernsi Nearctic/Neotropic Mexico 11 1.3. Taxonomic background
MOORE (1959) distinguished two separate tribes based primary on cranial characters: Sciurini and Tamiasciurini. This classification was modified by BLACK (1963) who used fossil record to reconstruct the phylogenetic relationships. BLACK (1963) included Tamiasciurus Trouessart, 1880 in the tribe Sciurini. This position was later confirmed by inmunological studies (HIGHT et al. 1974, ELLIS & MAXSON 1980) and protein electrophoresis (HAFNER et al. 1994). MOORE (1959) also assigned generic status to Microsciurus and Syntheosciurus, both previously treated as subgenera. These works are basal for current taxonomy. Despite important findings brought by molecular studies, taxonomic structure of the group used nowadays is in many ways still similar to the one suggested by MOORE (1959) and modified by BLACK (1963). Only the most recent publications (MERCER & ROTH 2003, HERRON et al. 2004, STEPPAN et al. 2004, ROTH & MERCER 2008, OSHIDA et al. 2009) appeal for a taxonomic revision. According to current systematics, the tribe Sciurini consists of five genera: Microsciurus, Rheithrosciurus, Sciurus Linnaeaus, 1758, Syntheosciurus and Tamiasciurus (WILSON & REEDER 2005). However, in research of MERCER & ROTH (2003), HERRON et al. (2004) and STEPPAN et al. (2004) Microsciurus appears nested in the Sciurus clade, demonstrating that Microsciurus should belong within a paraphyletic Sciurus. VILLALOBOS & CERVANTES-REZA (2007) identify the monotypic genus Syntheosciurus as sister to Sciurus variegatoides, what indicates another impeachement of the monophyletic status of the genus Sciurus. Tamiasciurus consists of three species: T. douglasii, T. hudsonicus and T. maernsi (WILSON & REEDER 2005). This genus extends through North American continent to Alaska in the north (ARBOGAST et al. 2001). It was regarded as tribe Tamiasciurini (MOORE 1959) but recent research (BLACK 1963, THORINGTON et al. 1998, MERCER & ROTH 2003) suggested its close relationship with Sciurus. Species of the genus Microsciurus can be found in Central and South America. There are currently four recognized species: M. alfari, M. flaviventer, M. mimulus and M. santanderensis (WILSON & REEDER 2005). MOORE (1959) created subtribe Microsciurina with Microsciurus as a genus. According to protein and molecular data, previous classification as subgenus of the genus Sciurus might be more accurate (HAFNER et al. 1994, MERCER & ROTH 2003, HERRON et al. 2004, STEPPAN et al. 2004). 12 Syntheosciurus is a monotypic genus with its only species Sy. brochus located in Costa Rica and Panama (WILSON & REEDER 2005). Information about this genus is lacking, particularly data on its phylogenetic relationships. From the taxonomic point of view, Syntheosciurus contains two subspecies despite some work treating S. poaensis with species rank (WILSON & REEDER 2005). Rheithrosciurus macrotis is the single species of another monotypic genus Rheithrosciurus. This squirrel of above average size is endemic to Borneo (WILSON & REEDER 2005). MOORE (1959) included Rheithrosciurus in the tribe Sciurini and this position seams to be confirmed by molecular data (MERCER & ROTH 2003). The genus Sciurus is the largest genus of the tribe with its 28 species and broad distribution in Eurasia, North and South America. However, the allocation is characterized by remarkable differences in species diversity in distant parts of the area. While three palearctic species cover area expanding from the western parts of Europe to Russia, Mongolia, northeast China and Hokkaido of Japan in the east, remaining 25 species are located in the Americas. The vast majority of the New World species can be found in Central and northern part of South America (WILSON & REEDER 2005). OSHIDA & MASUDA (2000) based their study on mitochondrial cytochrome b gene sequences and found out that Sciurus species form two clusters with large genetic distances, the Old World Sciurus and the New World Sciurus. Nevertheless, only two species from Eurasia and four species from Americas were examined. To test the monophyly of the Old World Sciurus, samples of another palearctic species had to be analyzed (Sciurus anomalus). OSHIDA et al. (2009) studied all three palearctic species, two nearctic and two neotropic species. Their work did not confirm monophyly of the Old world cluster. S. anomalus was included in the New World cluster but the genetic distances between S. anomalus and both clusters were similar. Consequently, three branches can be recognized: S. anomalus; S. lis + S. vulgaris; the New World species (OSHIDA et al. 2009).
1.4. Evolutionary history
Evolutionary history of the tribe Sciurini initiated about 13 million years ago (Mya) according to molecular-clock tree, using changes in the third nucleotide codon position of the IRBP gene (MERCER & ROTH 2003). MERCER & ROTH (2003) and STEPPAN et al. (2004) determined the genus Tamiasciurus as basal taxon of this group. Tamiasciurus species are distributed in North America what signifies that diversification and primal extension of the 13 tribe was in North America. However, OSHIDA et al. (2009) suggested that the genus Sciurus diverged in Eurasia and later crossed Beringia to colonize Americas. From these findings we can assume that there were several migrations through Beringia in the early history of this group. The genus Rheithrosciurus deserves additional consideration. Its unique distribution on Borneo still lacks explanation. Most recently, American origin was suggested by molecular analysis with R. macrotis positioned among New World species of Sciurus (MERCER & ROTH 2003).
1.5. Latitudinal gradient in species richness
HILLEBRAND (2004) demonstrated that the increase of biodiversity with decreasing latitude is a general pattern related to different groups of organisms and habitats, with the same trends on Northern and Southern Hemisphere. He related the pattern to factors such as body mass, trophic level, geographic area and, due to global generalism, probably temperature as well. Throughout the history, hypotheses and research on distribution of species richness have accumulated. This broad range of possible causes includes climate change (DYNESIUS & JANSSON 2000), area size (ROSENZWEIG 1995, CHOWN & GASTON 2000), temperature (ROHDE 1992, ALLEN et al. 2006), evolutionary time (FISCHER 1960, STEPHENS & WIENS 2003), tropical origin and niche conservatism (WIENS & DONOGHUE 2004, WIENS et al. 2006) and others. There is a lack of consensus in the hypotheses categorization. Several criteria have been used to simplify the model classification (ROHDE 1992; WILLIG et al. 2003, MITTELBACH et al. 2007). Here, I divided latitudinal gradient explanations in three categories: geometrical constraints, origin-related interpretations and effects of evolutionary rates.
1.5.1. Geometrical constraints
The application of geometric model is a special approach, the mid-domain effect (COLWELL & LEES 2000). This pattern deals with range size and geographic boundaries and assumes the most significant species richness in the middle. COLWELL & LEES (2000) characterize the mid-domain effect as "the increasing overlap of species ranges towards the centre of a shared geographic domain due to geometric boundary constraints in relation to the 14 distribution of species range sizes and midpoints". This topic was previously studied (COLWELL & HURTT 1994, WILLIG & LYONS 1998) with several models included. WILLIG & LYONS (1998) used a stochastic model based on the binomial distribution, proposed as an adequate response to species richness diversity. They compared predicted and observed species distribution and implemented various latitudinal constraints. Analyses yielded statistically significant results for both studied groups, bats and marsupials.
1.5.2. Origin-related interpretations
From the biological point of view, hypotheses concerning species distribution are oriented on the origin of the group with time, area or ecologic conditions as the main effecting factors: dispersal restricted by physiological limits (JANZEN 1967), time-for-speciation effect (STEPHENS & WIENS 2003), tropical conservatism hypothesis (WIENS & DONOGHUE 2004, WIENS et al. 2006). JANZEN (1967) demonstrated that organisms used to homogeneous environmental and climatic conditions are less prone to dispersal and their ability to colonize new regions is reduced. More stable conditions in tropics can represent a barrier for overcoming the more intensive seasonal climate changes in temperate zones. KLEIDON & MOONEY (2000) studied influence of climatic factors on growth rates and global biodiversity. They created a simulation model and found a narrow correlation between climatic restrictions and species richness allocation. With increase of stress (accumulation of climatic restrictions, decrease of number of "good growing days") decreases diversity and as the stress is least significant in tropics, the highest levels of biodiversity are restricted to tropical regions (KLEIDON & MOONEY 2000). Associations between irregular distribution of global biodiversity and time are extensive (PIANKA 1966, FARRELL et al. 1992, STEPHENS & WIENS 2003, WIENS & DONOGHUE 2004, FINE & REE 2006). FISCHER (1960) concluded his broad-scale review with an idea that latitudinal diversity gradient is caused by more time for diversification and evolutionary processes in tropical parts of the world as these regions have been more stable throughout the evolutionary history (e.g. in comparison with temperate regions affected by glaciations). This result now represents the main idea of the hypothesis of the time-for- speciation effect (STEPHENS & WIENS 2003). Work of WIENS & DONOGHUE (2004) used physiological barriers of dispersal 15 (JANZEN 1967, KLEIDON & MOONEY 2000) and time-for-speciation effect (STEPHENS & WIENS 2003) as a part of their tropical conservatism hypothesis. The other part is phylogenetic niche conservatism, previously developed in several studies (FARRELL et al. 1992, PETERSON et al. 1999, WEBB et al. 2002). Niche conservatism reflects the tendency of organisms to be bounded to their ancestral habitat. The ability of some taxa to expand and enter new habitats can be determined by the ancestral niche in the case of niche conservatism (WIENS & DONOGHUE 2004). This hypothesis predicts that organisms prefer habitats to which they are preadapted. The tropical conservatism hypothesis implies all models to explain the latitudinal gradient in species richness. Moreover, WIENS & DONOGHUE (2004) pointed out that in the recent past, tropics occupied more extensive areas, and, as a consequence, many recent species have originated in the tropics. There are only two objections contradicting the original hypothesis (WIENS & DONOGHUE 2004): restrictions in dispersal are caused by temperate seasonality (not by cold temperatures in higher latitudes) and WIENS et al. (2006) were unable to provide a significant test of the role of regression of tropics in diversity gradient. FINE & REE (2006) studied "time-integrated species-area effect" which is closely related to the tropical conservatism hypothesis. According to their work, only integrated action of time and area has a remarkable influence on species richness.
1.5.3. Effects of evolutionary rates
Except for the above-mentioned explanations of the latitudinal species diversity gradient, there are hypotheses concerning differences in evolutionary rates among distant parts of the world. These hypotheses deal either with latitudinal variations in speciation rates or latitudinal variations in extinction rates. Work of MITTELBACH et al. (2007) represents a structured summary of models dealing with latitudinal gradient and contains an extensive part about evolutionary rate models. In this work, I focus on several causes of variations in evolutionary rates: geographic area, climatic change, genetic drift, mutation rate (changing with temperature, especially in ectotherms), biotic interactions. Geographic area Geographic range size is often studied as a factor stimulating latitudinal diversity gradient (TERBORGH 1973, ROSENZWEIG 1995, CHOWN & GASTON 2000, JABLONSKI & 16 ROY 2003). These studies share basic principles regarding geographic range and species richness: geographic range size is positively correlated with speciation rates and negatively correlated with extinction rates. But there is no correlation between species range size and size of ecoclimatic zone according to CHOWN & GASTON (2000). The reason is that the area model presumes larger range sizes for species in the tropics in comparison with range sizes of species from higher latitudes. However, this model takes into consideration equal numbers of species and the species biodiversity is remarkably higher in the tropics so this assumption cannot be tested (CHOWN & GASTON 2000). Higher speciation rates in tropical regions are caused by the possibility to be limited by some kind of barrier, which increases in correlation with range size. In other words, larger areas are more prone to allopatric speciation. The opposite case is the relationship with the extinction rate. Species with more dispersed geographic range are not exposed to catastrophies which could cause extinction as much as species with smaller ranges. The greater the geographic range size, the more global and destructive catastrophe would be needed to make the species go extinct. Important factor is that species with larger ranges should be more resistant to disturbances because within their range, more variable conditions occur. As CHOWN & GASTON (2000) explained, "species at low latitudes might have large ranges for geographic reasons, whereas those at high latitudes might have large ranges for climatic reasons". This opinion of CHOWN & GASTON (2000) further explains why the tropics can be considered as "museums" of diversity. On the other hand, there are still discussions about tropics as "museum" or "cradle" of biodiversity STEBBINS (1974, in CHOWN & GASTON 2000). To give an example representing the "cradle" opinion, JABLONSKI (1993) found a significantly higher number of ordinal originations in tropical post-Palaeozoic marine benthic invertebrates. Contrary to the above-mentioned studies, JABLONSKI & ROY (2003) found that factors modifying geographic ranges attenuate speciation rates too. GAVRILETS et al. (2000) claim that increased range size (and consequently population size) induce higher population density and dispersal ability, this means reduce speciation.
Climatic change This model is based on regular deviations in Earth's orbit, the Milankovitch oscillations (JANSSON & DYNESIUS 2002). These oscillations appear on a time scale of 10 to 100 thousand years and are caused by interactions with planets of the Solar System (JANSSON & 17 DYNESIUS 2002). Variations of Earth's axis have implications for ecological conditions and distribution of species on Earth. DYNESIUS & JANSSON (2000) identified the changes of geographic ranges caused by Milankovitch oscillations as "orbitally forced species range dynamics" (ORD), in subsequent work (JANSSON & DYNESIUS 2002) renamed to orbitally forced range dynamics. Differences in ORD among regions induce variations of species diversity. Larger ORD leads to generalism, lower levels of specialization, increased dispersal ability and as larger ORD can be allocated to higher latitudes, these characteristics are consequently related to higher latitudes (DYNESIUS & JANSSEN 2000). Less specialized species with wider ranges exhibit decreased speciation and number of species in these regions is lower in comparison with tropics.
Genetic drift Remarkable diversity in the tropics, forming series of species living without evident ecological and geographical barriers, could be explained by another kind of barrier, reduced population density (FEDOROV 1966). Lower population density, lower population size and restrictions in reproduction induce genetic drift, generating speciation. FEDOROV (1966) explains that "under such conditions mutant genes accumulate in populations and contribute to the relatively rapid origin of series of closely allied species, differing considerably from one another morphologically but possessing many 'indifferent' characters". On the other hand, MARTIN & TEWKSBURY (2008) argue that population bottlenecks caused by glaciations at higher latitudes could increase the effect of genetic drift also in these regions. Study of WRIGHT et al. (2006) tries to eliminate the factor of genetic drift by exploring rates of molecular evolution among temperate and tropical species with comparable population size. The analysis demonstrates that higher mutation rates in the tropics are independent of genetic drift. However, it is doubtful if the influence of genetic drift can be fully eliminated in this way (WRIGHT et al. 2006).
Biotic interactions More stable and benign physical conditions make biotic interactions more important in tropical regions (DOBZHANSKY 1950, FISCHER 1960). Biotic interactions are responsible for more intensive selective pressures. Geographic isolation generates stochastic organization of populations, extending the variability and thereby also the diversifying function of natural selection (SCHEMSKE 2002). SCHEMSKE (2002) assumed that adaptations have genetic 18 basis, but regarded the data insufficient to further investigate this subject. Especially, since he claimed that variability of diversification in the tropical populations should produce ecological divergence, which will allow coexistence of species.
Mutation rate (temperature) Generation time and mutation rate determine how fast evolution proceeds and are considered as consequences of the metabolic rate (ROHDE 1992, GILLOOLY et al. 2005, ALLEN et al. 2006). Further, metabolic rate depends upon body size and temperature (GILLOOLY et al. 2005). With increasing temperature in lower latitudes, levels of mutagenesis increase and more mutations result in greater genetic variability. Higher temperatures also accelerate growth rates and subsequently generation rates so there is more possibility of accumulation of mutations and space for natural selection. On the matter of body size, accumulation of DNA substitutions is faster in larger animals (MARTIN & PALOMBI 1993). This indicates how temperature and body size affect evolutionary rate. The close relationship between the rate of energy transformation in metabolism and the rate of nucleotide substitution was confirmed (GILLOOLY et al. 2005). ROHDE (1992) supported correlation of energy with species diversity and pointed out the hypothesis that the number of species in a region is restricted by amount of energy available (CURRIE 1991). At the same time, he questioned the part of this hypothesis which supposes that habitats are saturated by species, mentioning subdivision of niches as a possible resource of new diversity. For example, ALLEN et al. (2006) found out that in planktonic foraminifera, nucleotide substitution and speciation rates increase exponentially with increasing temperature and they specified energy flux as the major factor of evolutionary dynamics. This study also examined the rate of speciation through the rate of first occurence and, according to fossil data, investigated higher speciation rates in tropical regions. Upon these findings, they promote the idea of the tropics as a cradle. The temperature and productivity as determinants of mutation rate were supported as the reason causing higher rates of molecular evolution also in plants (WRIGHT et al. 2006). On the other hand, several works investigating this hypothesis failed to find evidence confirming this hypothesis (BROMHAM & CARDILLO 2003, DAVIES et al. 2004, THOMAS et al. 2006). BROMHAM & CARDILLO (2003) found no correlation between generation time, mutation rate and the rate of molecular evolution in birds. DAVIES et al. (2004) revealed that mutation rate directly depends on UV radiation and temperature (energy) 19 and species richness is also directly influenced by energy. The problem is caused by missing a direct connection between molecular rates and species richness. THOMAS et al. (2006) showed that there is no confirmation of body size and substitution rate relationship in their broad-scale study.
This summary describes the extent of the problem of explaining the latitudinal gradient in species richness and that there is still no consensus about the determining factor or factors that lead to the gradient. In consequence, it is unclear if latitudinal diversity gradient can be explained in a global manner for all groups of organisms. The same vagueness refers to actual taxonomy and phylogeny of the tribe Sciurini. My model group is a point in case in this instance, as I found a complex evolutionary history in tree squirrels that is not in agreement with currently accepted taxonomy of the group.
1.6 Aims
Aim of this research was to provide a comprehensive analysis of phylogenetic relationships of the tribe Sciurini and to discover the area of origin of the group. I questioned the monophyly of the genus Sciurus to resolve the ambiguity in Sciurini phylogeny. Moreover, I tried to find what hypothesis provided a suitable explanation for latitudinal diversity gradient of Sciurini squirrels. 20 2. MATERIALS AND METHODS
I worked with seven loci to reconstruct the phylogeny of the tribe Sciurini. Mitochondrial and nuclear genes were used for a complex study of this group (Tab 2). I obtained sequences from the GenBank database, the open-access database providing all publicly available DNA sequences (BENSON et al. 2010). Accession numbers of all used sequences are in Article 1 - Tab. 1. Sequences were processed and aligned in Geneious software v4.7 (DRUMMOND et al. 2009).
Tab 2. Characteristics of genes used in the study.
No. of No. of complete Genes Location sequences sequences 12S rRNA mitochondrial 7 1 16S rRNA mitochondrial 7 0 D-loop mitochondrial 5 1 mt-cyb mitochondrial 9 7 IRBP nuclear 12 0 c-myc nuclear 5 0 RAG1 nuclear 5 0
I transformed alignments from FASTA format to NEXUS format so that Bayesian inference of phylogeny can be performed. Bayesian analysis is based on estimation of posterior probabilities of reconstructed phylogenies. I used program MrBayes 3.1.2 (HUELSENBECK & RONQUIST 2001) for Bayesian analyses in this research. MrBayes works with Markov chain Monte Carlo (MCMC), a simulation technique. MCMC is a stochastic method which employs algorithms, introducing changes in parameters during analysis. These modifications of parameters are accepted or rejected according to changes in likelihood (CUMMINGS et al. 2003). After gaining stationarity, posterior probabilities are calculated as parameter frequencies (CUMMINGS et al. 2003). Standard values of posterior probability considered as significantly supported are greater or equal to 0.95, this pattern is used throughout this study. Model parameters and details about MCMC runs of my study are described in Article 1, 21 part Materials and Methods. Supertree is characterized by BININDA-EMONDS (2004) as "a phylogenetic approach in which many overlapping source trees, rather than the character data used to derive those trees, are combined to produce a single, larger supertree". In other words, resulting tree is not determined by primary source data but by source trees. The supertree approach is an important contribution to phylogeny reconstruction by the possibility to construct more complex phylogenies based on incomplete data. In my study, Bayesian gene trees were applied to six methods of supertree construction (standard matrix representation using parsimony (MRP), Purvis-MRP, SuperTriplets, MinCutSupertree, modified MinCutSupetree and veto method). Matrix representation with parsimony (BAUM 1992, RAGAN 1992) is a method of supertree contruction based on combination of variable data sets through binary coding (BININDA-EMONDS & BRYANT 1998). Nodes are assigned as matrix elements in the MRP method and the supertree is constructed as a maximum parsimony tree using the matrix representation as input data. Therefore, composite trees are influenced in different measure by source trees with low number of nodes and large source trees with many nodes and new clades can occur (BININDA-EMONDS & BRYANT 1998). Purvis-MRP, modification of MRP method by PURVIS (1995) is aimed at changes in coding the tree nodes to eliminate redundant data. The method regards size-based treatment in MRP by BAUM (1992) and RAGAN (1992) as incorrect (RONQUIST 1996). Properties of my MRP and Purvis-MRP analyses are described in Article 1, part Materials and Methods. New method, SuperTriplets (RANWEZ et al. 2010), was also used to reconstruct squirrel phylogeny. This method looks for an asymmetric triplet-based median supertree and applies polynomial algorithm to reach it (RANWEZ et al. 2010). I obtained results by means of online SuperTriplets program available at: http://www.supertriplets.univ- montp2.fr/execute.php, accessed on 11 August 2010. Polynomial time algorithm was used for another method, MinCutSupertree (SEMPLE & STEEL 2000). In this method, source trees are converted to a graph. Cut set is a set of branches that are cut from the graph according to minimum weight of branches (therefore the cut is called minimum cut) and branch weight depends upon number of appearances of branches in source trees (PAGE 2002). Finally, nodes connected by branches in graph are merged. Modified MinCutSupertree (PAGE 2002) provides small arrangements to the MinCutSupertree method, correcting limitations as large influence of size of source trees on final supertree and absence of uncontradicting nestings in the supertree. I used Supertree 22 software (PAGE 2002) to execute MinCut and modified MinCut analyses. I also worked with veto method in PhySIC_IST (SCORNAVACCA et al. 2008). In supertrees yielded by veto methods, there are not present branchings contradicting the source trees. Moreover, PhySIC_IST eliminates taxa producing regions of conflict and suggests multifurcations to resolve contradicting source trees. It contains statistical arrangement model Source Trees Correction to correct the source trees before the analysis (SCORNAVACCA et al. 2008). We used both versions, with and without the prior correction. 23 3. RESULTS
I worked with sequences of different lengths and generated alignments with length varying between 304 and 1151 base pairs. Gaps were found in c-myc and RAG1 protein-coding sequences, which can indicate errors in sequences.
Tab 3. Sequence lengths of alignments for seven genes.
Gene Alignment length 12S rRNA 801 16S rRNA 548 c-myc 934 D-loop 304 IRBP 1151 mt-cyb 527 RAG1 385
The output of the Bayesian analyses of alignments were seven phylogenetic trees. In all trees, T. hudsonicus was determined as outgroup. Results showed similar trends but slightly differed in branching and position of taxa. The trees of mitochondrial genes had higher root- to-tip distance than the trees of nuclear genes. Phylogenetic trends were explained in Article 1 and here I described the trees in detail. The 12S rRNA tree depicted an unusual position of R. macrotis and S. vulgaris further from the root, with M. flaviventer and S. niger in the basal position. 12S rRNA analysis suggested sister relationship of S. aestuans and Sy. brochus (Article 1 - Fig. 2). The 16S rRNA tree better fit the general model with R. macrotis and S. vulgaris at the base of the tree and monophyletic lineage of American species (Article 1 - Fig. 2). Nearctic species, S. carolinensis and S. niger, split first and then there was a close relationship of M. flaviventer and Sy. brochus but with lower posterior probability support. Phylogeny described in the mitochondrial gene for cytochrome b tree was similar but S. carolinensis formed an unresolved trichotomy with S. aberti and a group including two Neotropical species. Cytochrome b tree was the only tree with S. anomalus present (Article 1 24 - Fig. 2, Tab. 1) IRBP tree was the most complex one with phylogenetic relationship of 12 species described in the tree (Article 1 - Fig. 2). The analysis yielded S. vulgaris with basal position and a monophyletic clade comprising R. macrotis and American species. S. aberti was in a well-supported relationship with S. griseus and so was M. flaviventer with S. granatensis and S. aestuans with S. ignitus. With the exception of these well-supported relationships, phylogeny of other American species was poorly resolved due to polychotomy. D-loop provided support for sister relationships of S. lis with S. vulgaris and S. carolinensis with S. niger (Article 1 - Fig. 2). Trees of c-myc and RAG1 genes had the same structure (Article 1 - Fig. 2). S. carolinensis was on the more basal position while neotropic species were organized in a trichotomy that was supported in the c-myc tree, but not in the RAG1 tree. Using six algorithms of supertree reconstruction, I obtained four distinct supertrees (veto, Purvis-MRP, Supertriplets/MinCut/modified MinCut and standard MRP) and all shared similar general branching pattern, forming groups of palearctic, nearctic and neotropic species (Article 1 - Fig. 3). The veto method supertree positioned the neotropic Sy. brochus at the base of the tree, prior to branching of the palearctic species (Article 1 - Fig. 3). In the Purvis-MRP method, S. anomalus from the palearctic region was placed within the polychotomy including nearctic species (Article 1 - Fig. 3). Except these deviations and differences in branching, supertrees generated by veto method and Purvis-MRP demonstrated the proposed model of diversification. However, four species were missing in the veto supertree: M. flaviventer, S. aestuans, S. carolinensis and S. niger. Three methods, Supertriplets, MinCut and modified MinCut, yielded identical supertrees (Article 1 - Fig. 3). The first diversifying species were from Eurasia, sister taxa S. lis and S. vulgaris, followed by R. macrotis and S. anomalus. American species formed a monophyletic group where species from North America and South America were separated. North American taxa diversified after species of Eurasia, and South American species were phylogenetically the youngest group. In the American group, only two sister relationships were supported. One between S. aberti and S. griseus and another between M. alfari and S. granatensis. Standard MRP (also refered to as Baum-MRP) tree was very similar but had better resolved position of S. anomalus and three species, S. aestuans, S. ignitus and Sy. brochus, forming trichotomy in supertree generated by SuperTriplets, MinCut and modified MinCut. In standard MRP tree, S. aestuans and S. ignitus were sister taxa, closely related to Sy. brochus. 25 4. DISCUSSION
The aim of this study was to establish a comprehensive phylogenetic reconstruction of the group of tree squirrels, the tribe Sciurini. According to phylogeny estimation, I further investigated the place of origin of this tribe and contributed to complicated problematics of the latitudinal gradient in species richness of Sciurini squirrels.
4.1. Phylogenetic reconstruction
Based on relationships generated by my analyses, I was able to reconstruct the phylogeny of Sciurini squirrels. Seven trees generated by the Bayesian analysis produced comparable results. Trees of mitochondrial genes had higher root-to-tip distances than trees of nuclear genes, probably due to higher mutation rate of mitochondrial DNA in comparison with nuclear DNA (BROWN et al. 1979). Similarly, there were minor deviations between supertrees yielded by different algorithms, but clear conclusions can be made. Palearctic species were grouped on tree bases in most of the trees. Separated position of S. anomalus in the Purvis-MRP and basal position of Sy. brochus in the veto analysis seemed to be isolated analysis artefacts that did not represent stable relationships independent on used algorithm. Except for that, close relationship of all palearctic species was supported. In all supertrees, S. lis and S. vulgaris were confirmed as sister taxa, as previously suggested (OSHIDA & MASUDA 2000) and S. anomalus and R. macrotis diversified later. Four of applied methods (Baum-MRP, MinCut, modified MinCut and SuperTriplets) presented American species as a monophyletic group. Nearctic species diverged first, with succession of S. niger, S. carolinensis and sister taxa S. aberti and S. griseus. S. aberti and S. griseus figured in sister relationship in all of the generated supertrees. The same four methods supported monophyly of neotropic species. On the other hand, the neotropic clade was poorly resolved, except for close relationship of M. alfari with S. granatensis (supported by five of six supertree methods). These findings, described and explained similarly in Article 1 (part 4.1. Phylogenetic relationships), demonstrate that current taxonomy should by modified according to the latest research based on molecular phylogenetics. Classification with five genera of the tribe Sciurini is incorrect with reference to results of this work, as well as previous studies (MERCER & ROTH 2003, STEPPAN et al. 2004, HERRON et al. 2004). My work has demonstrated that the genus Sciurus is paraphyletic because species of Microsciurus, 26 Rheithrosciurus and Syntheosciurus are in a close relationship with Sciurus species and appear involved within the Sciurus lineage.
4.2. Explanation of recent zoogeography
Phylogenetic relationships described in the previous paragraph demonstrate how recent zoogeographic distribution has evolved. I briefly describe it here, as the conclusion was already explained in Article 1 (part 4.2. Biogeographic implications). With regards to the whole tribe Sciurini, its origin is probably in North America. In Sciurini group, Tamiasciurus is situated at the root (HAFNER et al. 1994, MERCER & ROTH 2003, HERRON et al. 2004, STEPPAN et al. 2004, VILLALOBOS & CERVANTES- REZA 2007, ROTH & MERCER 2008) and as Tamiasciurus is a nearctic genus, we can conclude that the whole tribe originated here. Clusters of palearctic, nearctic and neotropic species indicate the gradual expansion of the tribe Sciurini. As palearctic species of Sciurus are located at the most basal position, they evolved first and consequently these first species of Sciurus colonized North America through land connection in Beringia. New nearctic species diversified and spread across North America. South America was colonized after formation of the Isthmus of Panama and in addition, Microsciurus and Syntheosciurus diverged. This hypothesis was already proposed by OSHIDA et al. (2009) and our complex analysis provides further evidence to support it. To summarize, the tribe Sciurini diverged in North America but the genus Sciurus evolved in Eurasia. Genera (considered as genera according to taxonomic reference, WILSON & REEDER 2005) Microsciurus, Rheithrosciurus and Syntheosciurus diversified after the divergence Sciurus/Tamiasciurus (Article 1 - Fig. 1), indicating that the genus Sciurus should include also species from the former genera.
4.3. Tropical species richness
After the relatively recent colonization of regions of Mexico, Central and South America, intensive speciation produced remarkable biodiversity of Sciurini squirrels in tropical part of the world. Of the great amount of hypotheses described in Introduction, in Article 1 we rejected hypotheses of the latitudinal gradient in species richness that assume the tropics to host the oldest lineages of a group. My analyses of the tribe Sciurini presented here deny tropical origin of Sciurini squirrels. Therefore, models based on tropical origin (more time for 27 speciation in tropics, delimitation by ancestral niche, physiological constraints) are not a solution in this case. The most probable answer was proposed by ROTH & MERCER (2008). Their birth-death analysis found out that diversification rate in South American species of Sciurini is much higher than in other groups. However, they signalized that in case of recent radiations, extinction rates can be modified by "extinction lag" (MERCER & ROTH 2003). Extinction lag represents the delayed effect of extinction in young populations, where the real values of extinction rates occur later and diversification rate is temporary higher.
4.4. Conclusions and implications
This study was aimed at phylogeny reconstruction and obtained results present evident conception of phylogenetic relationships of the tribe Sciurini. Integration of all previously published data allowed this work to conclude that the tribe Sciurini needs systematic revision. Considering estimated phylogeny, tropical origin of the tribe was contradicted and origin- related hypotheses were excluded from explaining the increased diversity of Sciurini squirrels in the tropics. 28 REFERENCES
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• Article 1: PEČNEROVÁ P. & MARTÍNKOVÁ N. (2010): Evolutionary history and phylogeny of tree squirrels (Rodentia, Sciurini) based on supertrees reconstruction. Submitted on 22 March, 2011. 37 Evolutionary history and phylogeny of tree squirrels (Rodentia, Sciurini) based on supertrees reconstruction
Patrícia Pečnerováa, Natália Martínkováb, c, *
a Department of Botany and Zoology, Masaryk University, Kotlářská 2, 602 00 Brno, Czech
Republic; e-mail: [email protected] b Institute of Vertebrate Biology, Academy of Science, v.v.i., Květná 8, 603 65 Brno, Czech
Republic; e-mail: [email protected] c Institute of Biostatistics and Analyses, Masaryk University, Kamenice 3, 625 00 Brno, Czech
Republic
* Corresponding author. Tel: +420 606 124 586 38 Abstract
Tree squirrels of the tribe Sciurini represent a group where current taxonomy contradicts phylogenetic gene trees with limited sampling. We used sequences of mitochondrial genes for
D-loop, cytochrome b, 12S rRNA and 16S rRNA and nuclear c-myc, RAG1 and IRBP genes to reconstruct phylogenetic relationships within the tribe maximizing the number of analyzed species. Six methods of supertree reconstruction revealed common trends. We confirmed paraphyly of the genus Sciurus and close relation between phylogeny and zoogeography. The generic position of Microsciurus, Rheithrosciurus and Syntheosciurus is not consistent with molecular data and the species from these genera are nested within the Sciurus lineage in our results. Progressive branching from Palaearctic to Nearctic and Neotropical species indicates that the genus Sciurus originally diverged in Eurasia, colonizing the New World more recently. Rheithrosciurus diverged from the common ancestor prior to Sciurus divergence in
North America, and genera Microsciurus and Syntheosciurus form a monophyletic group with other Neotropical Sciurus species. We assume that the reason for species diversity gradients in
Sciurini is probably caused by higher diversification rate in tropical parts of the world.
Keywords: Sciurini; tree squirrel; phylogeny; supertrees
Abbreviations: Mya, million years ago; MRP, matrix representation with parsimony 39 1. Introduction
1.1. Biogeography of latitudinal species richness
The tribe Sciurini inhabits continents of Eurasia, North and South America. Most of the 37 currently recognized species of the tribe are distributed in Central America and northern part of South America, and only 4 species inhibit the large area of Eurasia with another species found in Borneo (Wilson & Reeder, 2005).
This group exhibits latitudinal gradient in species richness, which is one of the fundamental patterns described in biogeography and ecology (Wiens et al., 2006; Mittelbach et al., 2007;
Fuhrman et al., 2008). Squirrels of the tribe Sciurini largely follow the pattern with majority of species found in low latitudes of the Americas (Wilson & Reeder, 2005).
Reasons why the species richness is accumulated significantly more in the tropics than in any other region of the world remain without a solid consensus. Recently, evolutionary and historical hypotheses are gaining more importance in explaining the latitudinal gradient
(Mittelbach et al., 2007), and they can be divided into three main explanations.
First, many groups with high tropical diversity originated in the tropics and subsequently spread to environments with temperate climate with varying levels of success. The habitat in the tropical regions is older due to changes during glaciations in higher latitudes and consequently, species in equatorial parts of the world had more time for speciation (Stebbins,
1974; Stephens & Wiens, 2003).
The second explanation is based on the relation between the number of species and area size.
Until recently, the tropics occupied a much larger area. According to the area model
(Rosenzweig, 1995; Chown & Gaston, 2000), species with greater range size are less prone to extinction and have more opportunities for speciation. This means that in the tropics more 40 species would evolve due to more favorable rates of speciation versus extinction.
The third hypothesis assumes the tropical niche conservatism (Wiens & Donoghue, 2004). It is a pattern based on a determined ancestral niche, and a presumption that species tend to retain their ability to utilize their ancestral habitat, which then compromises expansion to new habitats. Seasonality and cold winter temperatures in the temperate zones would restrict distribution of the tropical species with physiology intolerant of the temperature range in higher latitudes (Fine, 2001; Wiens et al., 2006).
The Sciurini squirrels fit into the pattern of latitudinal gradient of species richness. Most species are found in Central and South America and the species abundance remarkably decreases northwards. Yet, the known palaeohistory and probable ancestral colonization pattern of the group do not easily fit the listed scenarios.
1.2. Evolutionary pathways in Sciurini diversification
Evolutionary history of the family Sciuridae begins with a well-preserved fossil species
Douglassciurus jeffersoni from North America 36 million years ago (Mya; Emry &
Thorington, 1982; Mercer & Roth, 2003). The dating of the earliest squirrel coincides with the time when area covered with tropics started to decrease (approximately 30-40 Mya;
Behrensmeyer et al., 1992). No fossils are known from South America at the time to our knowledge. The earliest fossil with evident traits of modern squirrels was Palaeosciurus goti from France (Thorington & Ferrell, 2006). Tree squirrels of the tribe Sciurini diverged approximately 13 Mya (Mercer & Roth, 2003), and the basal taxon of the group, genus
Tamiasciurus, is found in North America (Wilson & Reeder, 2005). Therefore, we might consider North America to be the place of early divergence of the tribe.
Oshida et al. (2009) suggested that Eurasia is the place of divergence of the genus Sciurus with subsequent dispersal through Beringia to North America. That would assume that 41 ancestors of Tamiasciurus first dispersed to Eurasia, diverged, and subsequently, as Sciurus, returned to North America. After separation, conditioned by opening of the Bering Strait, an intensive speciation occurred in North America. Extension of the genus proceeded to Central
America and after formation of the Isthmus of Panama, also to South America (Fig. 1). In tropical parts of Central and South America the Sciurini squirrels reach the highest levels of species richness.
This is in contrast to the above-mentioned scenarios explaining species richness in the tropics.
Based on the assumption that the genus Sciurus originated in Eurasia and reached South
America as recently as 3 Mya (Mercer & Roth, 2003), high biodiversity in the tropics is not caused by longer time available for speciation; the area occupied by Sciurini in the temperate zone is larger than in the tropics; and realized niches originate in the zones that are expected to represent a dispersal barrier.
Roth & Mercer (2008) suggested that the latitudinal gradient in species richness could be explained by variable rates of speciation and extinction in different regions. They found a high rate of diversification in South America by concentrating on the ‘birth-death’ relationship.
This explains the evolutionary history of the genera with known fossil record, Sciurus and
Tamiasciurus, but not the other currently recognized genera belonging to Sciurini.
Microsciurus, Rheithrosciurus, and Syntheosciurus are distributed in Central and South
America, and in southeast Asia (Wilson & Reeder, 2005). If Sciurini is a monophyletic group, and molecular data analyses suggest this to be true (Mercer & Roth, 2003; Herron et al., 2004;
Steppan et al., 2004), the evolutionary history described above is valid only as long as these additional genera diverged earlier than the diversification of Sciurus and Tamiasciurus.
Otherwise, the genus Sciurus would be paraphyletic and should include also species from the above-mentioned genera (Hafner et al., 1994; Mercer & Roth, 2003; Herron et al., 2004;
Steppan et al., 2004). The tropical regions are generally unsuitable for fossil preservation 42 (Tappen, 1994), and we could speculate that the ancestors of the three disputed genera were present in the region longer than expected despite the lack of fossil evidence.
Our intention is to establish phylogenetic relationships between species of the tribe Sciurini in a comprehensive way, using supertree reconstruction algorithms. We analyzed all species and loci with a sufficient number and length of available DNA sequences to maximize the number of investigated taxa and to increase robustness of the analyses. We confirmed the model of divergence and speciation within the tribe proposed by Oshida et al. (2009). In light of our multilocus phylogenetic analyses, distinguishing five genera within Sciurini is untenable, and the genus Sciurus should include species from Microsciurus, Rheithrosciurus and
Syntheosciurus.
2. Materials and Methods
We analyzed 7 loci (12S rRNA, 16S rRNA, mt-cyb, D-loop, IRBP, c-myc, RAG1), both mitochondrial and nuclear, and we obtained the DNA sequence data from the online sequence database GenBank (Wettstein et al., 1995; Matthee & Robinson, 1997; Barratt et al., 1999;
Bentz & Montegelard, 1999; DeBry & Sagel, 2001; Piaggio & Spicer, 2001; Montegelard et al., 2002; Mercer & Roth, 2003; Steppan et al., 2004; Tamura & Hayashi, 2007; Blanga-Kanfi et al., 2009; Oshida et al., 2009; Lance, S.L. et al., unpubl.; Neversov, A.A. & Volokhov, D.V., unpubl.; Sudman, P.D. & Hafner, M.S., unpubl.; Yaekashiwa, N. & Tamate, H.B., unpubl.).
We used datasets with partially overlapping species content, comprising in total of 17 species of the tribe Sciurini. Samples of all recently recognized genera of the tribe (Microsciurus,
Rheithrosciurus, Sciurus, Syntheosciurus, Tamiasciurus) were included. Accession numbers are available in Table 1.
We aligned the DNA sequences in Geneious 4.7 (Drummond et al., 2009). To estimate phylogenetic relationships for each locus separately, we calculated Bayesian inference 43 analysis for every dataset in MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001) with the GTR +
I + Γ substitution model. To optimize chain convergence, we ran five Markov chains Monte
Carlo for 2 million generations, sampling trees every 1000th generation. Chain heating parameter was 0.1 and 3 chain swaps were attempted every generation. The burn-in fraction was set to 30%. The outgroup was Tamiasciurus hudsonicus in this study.
We used the resulting trees to reconstruct supertrees that would include all taxa with available
DNA sequence data. We applied six methods: standard matrix representation with parsimony
(MRP; Baum, 1992; Ragan, 1992), Purvis matrix representation with parsimony (Purvis-
MRP; Purvis, 1995), SuperTriplets (Ranwez et al., 2010), veto supertree reconstruction
(Scornavacca et al., 2008), MinCut (Semple & Steel, 2000), modified MinCut supertrees
(Page, 2002).
MRP and Purvis-MRP are consensus methods, where taxa relationships defined in the phylogenetic trees are translated into a binary matrix. We constructed the matrix representation in r8s 1.70 (Sanderson, 2003) and analyzed it using maximum parsimony in
PAUP* 4b10 (Sinauer Associates, Inc.) with 10 heuristic search replicates and TBR branch swapping algorithm. Maximum number of the swapped trees was limited to 10000. The final tree was constructed as a 50% majority rule consensus.
The SuperTriplets (Ranwez et al., 2010) method searches for a median supertree that is based on decomposition of the source trees to the simplest trees, the triplets (Ranwez et al., 2010).
We conducted the analysis in the online program SuperTriplets (available at http://www.supertriplets.univ-montp2.fr/execute.php, accessed on 11 August 2010).
In the veto method, as implemented in PhySIC_IST (Scornavacca et al., 2008), the resulting supertree is a combination of the relationships agreed upon by all source trees (Ranwez et al.,
2007) as opposed to the relationships most favored by the source trees in the other supertree reconstruction algorithms. We used the veto method with and without the source tree correction. 44 The MinCut (Semple and Steel, 2000) and modified MinCut (Page, 2002) algorithms convert the source trees into a graph with allocated weights. The supertree is constructed from the graph after execution of a minimum cut, a cut of minimum weight. We worked with these algorithms in the Supertree software (Page, 2002).
3. Results
Seven phylogenetic trees yielded by Bayesian inference analyses revealed a similar basic pattern in the distribution of taxa with successive divergence of Palaearctic, Nearctic and
Neotropical species of Sciurini (Fig. 2). The majority of relationships were not significantly supported (Bayesian posterior probability < 0.95) or the supported groups differed between the gene trees with several notable exceptions.
Sciurus lis and S. vulgaris were confirmed as sister taxa (mt-cyb). The other Palaearctic species, Rheithrosciurus macrotis (12S rRNA, 16S rRNA and IRBP) and S. anomalus (mt- cyb), diverged early. Nearctic and Neotropical species formed supported monophyletic groups in two datasets (16S rRNA, mt-cyb). In North America, our study shows a sister relationship of S. carolinensis with S. niger (D-loop) and S. aberti with S. griseus (IRBP). Relationships among the Neotropical species are vague as the gene trees provide conflicting results and polychotomies (Fig. 2).
The diverse methods of supertree reconstruction partially coincided in the branching pattern
(Fig. 3). The MinCut, modified MinCut and SuperTriplets analyses generated identical trees.
The veto tree showed a similar organization of taxa, but four species, M. flaviventer, S. aestuans, S. carolinensis and S. niger, were excluded from the veto analysis because of their conflicting placement in the different gene trees. Compared to the other supertrees, S. ignitus and Syntheosciurus brochus were placed closer to the root in the veto supertree. The MRP and
Purvis-MRP supertrees slightly vary compared to the other trees in branching of the Eurasian 45 taxa and placing Sy. brochus as a sister species to S. aestuans and S. ignitus. In addition, the
Purvis-MRP analysis showed a basal polychotomy of North American species and peculiar positions of M. alfari, S. anomalus and S. granatensis that are not consistent with their distribution.
4. Discussion
4.1. Phylogenetic relationships
Despite differences between the supertrees generated by various algorithms, there are evident trends. The species branching patterns corresponds to their distribution in the biogeographic ecozones. First, species situated at the base of the supertrees include Palaearctic taxa (S. anomalus, S. lis, S. vulgaris) and R. macrotis from Borneo. The tree squirrels of the New
World form a monophyletic group with the Nearctic species (S. aberti, S. carolinensis, S. griseus, S. niger) splitting first. The Neotropical species (M. alfari, M. flaviventer, S. aestuans, S. granatensis, S. ignitus, S. stramineus, S. variegatoides, Sy. brochus) diverged as a monophyletic clade most recently.
Two species of Microsciurus, which we included in this study, form a close relationship with
Central and South American species of the genus Sciurus. Moreover, M. alfari and S. granatensis occur as sister taxa in the phylogenetic tree of IRBP sequences. This demonstrates that Microsciurus should be included within the genus Sciurus.
The same situation applies to the genus Syntheosciurus. Its single species, Sy. brochus, belongs to a deficiently resolved clade with Neotropical species S. aestuans and S. ignitus.
The position of Sy. brochus as a sister species to S. variegatoides (Villalobos & Cervantes-
Reza, 2007) was not confirmed by any of our analyses.
Mercer & Roth (2003) placed R. macrotis amongst Sciurini of the New World. However, our results indicate that R. macrotis diverged from a common ancestor as early as the Palaearctic 46 species, a conclusion that is consistent with the zoogeographic distribution of the species.
We found two well-supported sister relationships in the genus Sciurus: S. lis with S. vulgaris and S. aberti with S. griseus. The first pair is closely related to the Palaearctic S. anomalus, while the second further groups with the Nearctic S. carolinensis and S. niger. To resolve the phylogenetic relationships of the Neotropical species, more complex sampling and molecular analyses are needed.
Considering that the species belonging to Microsciurus, Rheithrosciurus and Syntheosciurus are nested within the Sciurus clade, these taxa cannot retain their generic status. We suggest that the taxa should be included in the genus Sciurus sensu lato.
4.2. Biogeographic implications
All supertrees, except the veto supertree, share one common pattern. Species are organized according to their distribution. Three Palaearctic species and R. macrotis are clustered at the base of the trees, while the New World squirrels form a monophyletic group. Furthermore, within the New World clade, there is a gradual transition from the Nearctic to the Neotropical species. Our multilocus analyses based on the supertrees reconstruction confirmed the relationships assumed by the latest research (Mercer & Roth, 2003; Steppan et al., 2004;
Oshida et al., 2009). Tamiasciurus is a North American genus and the species is consistently placed at the root of the tribe (Hafner et al., 1994; Mercer & Roth, 2003; Herron et al., 2004;
Steppan et al., 2004; Villalobos & Cervantes-Reza, 2007; Roth & Mercer, 2008). The ancestor of Sciurini originated in the Nearctic region, where it is currently represented by T. hudsonicus. On the other hand, the basal ingroup position of Palaearctic species indicates that the genus Sciurus diverged in Eurasia. We assume that when a connection to Eurasia formed across the Bering Strait, the squirrels spread westwards and diverged into Sciurus.
Phylogenetic position of R. macrotis within Palaearctic species group shows that this 47 monotypic genus colonized Borneo from Eurasia, rather than the Americas as previously suggested (Mercer & Roth, 2003).
Later, the genus Sciurus crossed Beringia back to North America where further speciation initiated and the taxa colonized the continent as far south as Central America. Expansion to
South America corresponds in timing with the formation of the Isthmus of Panama (Mercer &
Roth, 2003). The species of Microsciurus and Syntheosciurus originated in tropical South
America after that, and the tribe attained its highest biodiversity in the region.
4.3. Diversification rate
The fact that Microsciurus, Rheithrosciurus and Syntheosciurus are con-generic with Sciurus in our analyses shows that the latitudinal species richness gradient of the tree squirrels formed recently and the evolutionary mechanisms leading to it were different from those mentioned above. We cannot explain the latitudinal gradient in species richness of the Sciurini squirrels through the tropical origin, larger occupied area in the tropics or the tropical niche conservatism hypotheses. Our findings are not consistent with the basic principles of the hypotheses that assume the tropics to be the ancestral area of a group. The origin of Sciurini is most likely not in the tropics.
The cause of high species diversity in the tropics is probably the remarkably high diversification rate suggested by Roth & Mercer (2008). Higher rates of speciation or lower rates of extinction explain the best the question of high biodiversity of the tribe Sciurini in the tropics. However, reasons and factors conditioning and influencing the speciation/extinction rates need more investigation.
Acknowledgement
This study was funded from grant number AV0Z60930519 provided by the Academy of 48 Sciences of the Czech Republic.
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Fig. 2. Bayesian phylogenetic trees of Sciurini based on the mitochondrial and nuclear gene sequences. The numbers above edges indicate Bayesian posterior probability of respective nodes. All scale bars show 0.02 substitutions per site.
Fig. 3. Supertrees reconstructions based on the phylogenetic trees inferred from four mitochondrial and three nuclear gene sequence datasets. The numbers above edges indicate percentage of triplets that support the edge in the SuperTriplets analysis. 54 Fig. 1. 55 Fig. 2. 56 Fig. 3. 57 Tab. 1. Accession numbers of sequences of Sciurini used for the reconstruction of phylogenetic relationships.