Primate diversification inferred from phylogenies and fossils

Biodiversity arises from the balance between speciation and extinction. Fossils record the origins and disappearance of organisms, and the branching patterns of molecular phylogenies allow estimation of speciation and extinction rates, but the patterns of diversification are frequently incongruent between these two data sources. I tested two hypotheses about the diversification of primates based on ～600 fossil species and 90% complete phylogenies of living species: (1) diversification rates increased through time; (2) a significant extinction event occurred in the Oligocene. Consistent with the first hypothesis, analyses of phylogenies supported increasing speciation rates and negligible extinction rates. In contrast, fossils showed that while speciation rates increased, speciation and extinction rates tended to be nearly equal, resulting in zero net diversification. Partially supporting the second hypothesis, the fossil data recorded a clear pattern of diversity decline in the Oligocene, although diversification rates were near zero. The phylogeny supported increased extinction ～34 Ma, but also elevated extinction ～10 Ma, coinciding with diversity declines in some fossil clades. The results demonstrated that estimates of speciation and extinction ignoring fossils are insufficient to infer diversification and information on extinct lineages should be incorporated into phylogenetic analyses.     

Marine planktonic microbes survived climatic instabilities in the past

In the geological past, changes in climate and tectonic activity are thought to have spurred the tempo of evolutionary change among major taxonomic groups of plants and animals. However, the extent to which these historical contingencies increased the risk of extinction of microbial plankton species remains largely unknown. Here, I analyse fossil records of marine planktonic diatoms and calcareous nannoplankton over the past 65 million years from the world oceans and show that the probability of species' extinction is not correlated with secular changes in climatic instability. Further supporting these results, analyses of genera survivorship curves based on fossil data concurred with the predictions of a birth-death model that simulates the extinction of genera through time assuming stochastically constant rates of speciation and extinction. However, my results also show that these marine microbes responded to exceptional climatic contingencies in a manner that appears to have promoted net diversification. These results highlight the ability of marine planktonic microbes to survive climatic instabilities in the geological past, and point to different mechanisms underlying the processes of speciation and extinction in these micro-organisms.     

ESTIMATING SPECIATION AND EXTINCTION RATES FROM DIVERSITY DATA AND THE FOSSIL RECORD

Understanding the processes that underlie biodiversity requires insight into the evolutionary history of the taxa involved. Accurate estimation of speciation, extinction, and diversification rates is a prerequisite for gaining this insight. Here, we develop a stochastic birth–death model of speciation and extinction that predicts the probability distribution of both extinct and extant numbers of species in a clade. We present two estimation methods based on this model given data on the number of extinct species (from the fossil record) and extant species (from diversity assessments): a multivariate method of moments approach and a maximum-likelihood approach. We show that, except for some special cases, the two estimation methods produce very similar estimates. This is convenient, because the usually preferred maximum-likelihood approach is much more computationally demanding, so the method of moments can serve as a proxy. Furthermore, we introduce a correction for possible bias that can arise by the mere fact that we will normally only consider extant clades. We find that in some cases the bias correction affects the estimates profoundly. Finally, we show how our model can be extended to incorporate incomplete preservation. Preservation rates can, however, not be reliably estimated on the basis of numbers of extant and extinct species alone.     

Phylogenetic fields through time: temporal dynamics of geographical co-occurrence and phylogenetic structure within species ranges

Species co-occur with different sets of other species across their geographical distribution, which can be either closely or distantly related. Such co-occurrence patterns and their phylogenetic structure within individual species ranges represent what we call the species phylogenetic fields (PFs). These PFs allow investigation of the role of historical processes—speciation, extinction and dispersal—in shaping species co-occurrence patterns, in both extinct and extant species. Here, we investigate PFs of large mammalian species during the last 3 Myr, and how these correlate with trends in diversification rates. Using the fossil record, we evaluate species'' distributional and co-occurrence patterns along with their phylogenetic structure. We apply a novel Bayesian framework on fossil occurrences to estimate diversification rates through time. Our findings highlight the effect of evolutionary processes and past climatic changes on species'' distributions and co-occurrences. From the Late Pliocene to the Recent, mammal species seem to have responded in an individualistic manner to climate changes and diversification dynamics, co-occurring with different sets of species from different lineages across their geographical ranges. These findings stress the difficulty of forecasting potential effects of future climate changes on biodiversity.     

Mutualism with plants drives primate diversification

Understanding the origin of diversity is a fundamental problem in biology. Evolutionary diversification has been intensely explored during the last years due to the development of molecular tools and the comparative method. However, most studies are conducted using only information from extant species. This approach probably leads to misleading conclusions, especially because of inaccuracy in the estimation of extinction rates. It is critical to integrate the information generated by extant organisms with the information obtained from the fossil record. Unfortunately, this integrative approach has been seldom performed, and thus, our understanding of the factors fueling diversification is still deficient. Ecological interactions are a main factor shaping evolutionary diversification by influencing speciation and extinction rates. Most attention has focused on the effect of antagonistic interactions on evolutionary diversification. In contrast, the role of mutualistic interactions in shaping diversification has been much less explored. In this study, by combining phylogenetic, neontological, and paleontological information, we show that a facultative mutualistic plant-animal interaction emerging from frugivory and seed dispersal has most likely contributed to the diversification of our own lineage, the primates. We compiled diet and seed dispersal ability in 381 extant and 556 extinct primates. Using well-established molecular phylogenies, we demonstrated that mutualistic extant primates had higher speciation rates, lower extinction rates, and thereby higher diversification rates than nonmutualistic ones. Similarly, mutualistic fossil primates had higher geological durations and smaller per capita rates of extinction than nonmutualistic ones. As a mechanism underlying this pattern, we found that mutualistic extinct and extant primates have significantly larger geographic ranges, which promotes diversification by hampering extinction and increasing geographic speciation. All these outcomes together strongly suggest that the establishment of a facultative mutualism with plants has greatly benefited primate evolution and fueled its taxonomic diversification.     

The fossil record and macroevolutionary history of the beetles

Coleoptera (beetles) is the most species-rich metazoan order, with approximately 380 000 species. To understand how they came to be such a diverse group, we compile a database of global fossil beetle occurrences to study their macroevolutionary history. Our database includes 5553 beetle occurrences from 221 fossil localities. Amber and lacustrine deposits preserve most of the beetle diversity and abundance. All four extant suborders are found in the fossil record, with 69% of all beetle families and 63% of extant beetle families preserved. Considerable focus has been placed on beetle diversification overall, however, for much of their evolutionary history it is the clade Polyphaga that is most responsible for their taxonomic richness. Polyphaga had an increase in diversification rate in the Early Cretaceous, but instead of being due to the radiation of the angiosperms, this was probably due to the first occurrences of beetle-bearing amber deposits in the record. Perhaps, most significant is that polyphagan beetles had a family-level extinction rate of zero for most of their evolutionary history, including across the Cretaceous–Palaeogene boundary. Therefore, focusing on the factors that have inhibited beetle extinction, as opposed to solely studying mechanisms that may promote speciation, should be examined as important determinants of their great diversity today.     

EXTINCTION DURING EVOLUTIONARY RADIATIONS: RECONCILING THE FOSSIL RECORD WITH MOLECULAR PHYLOGENIES

Recent application of time‐varying birth–death models to molecular phylogenies suggests that a decreasing diversification rate can only be observed if there was a decreasing speciation rate coupled with extremely low or no extinction. However, from a paleontological perspective, zero extinction rates during evolutionary radiations seem unlikely. Here, with a more comprehensive set of computer simulations, we show that substantial extinction can occur without erasing the signal of decreasing diversification rate in a molecular phylogeny. We also find, in agreement with the previous work, that a decrease in diversification rate cannot be observed in a molecular phylogeny with an increasing extinction rate alone. Further, we find that the ability to observe decreasing diversification rates in molecular phylogenies is controlled (in part) by the ratio of the initial speciation rate (Lambda) to the extinction rate (Mu) at equilibrium (the LiMe ratio), and not by their absolute values. Here we show in principle, how estimates of initial speciation rates may be calculated using both the fossil record and the shape of lineage through time plots derived from molecular phylogenies. This is important because the fossil record provides more reliable estimates of equilibrium extinction rates than initial speciation rates.     

EXPLOSIVE EVOLUTIONARY RADIATIONS: DECREASING SPECIATION OR INCREASING EXTINCTION THROUGH TIME?

A common pattern in time-calibrated molecular phylogenies is a signal of rapid diversification early in the history of a radiation. Because the net rate of diversification is the difference between speciation and extinction rates, such "explosive-early" diversification could result either from temporally declining speciation rates or from increasing extinction rates through time. Distinguishing between these alternatives is challenging but important, because these processes likely result from different ecological drivers of diversification. Here we develop a method for estimating speciation and extinction rates that vary continuously through time. By applying this approach to real phylogenies with explosive-early diversification and by modeling features of lineage-accumulation curves under both declining speciation and increasing extinction scenarios, we show that a signal of explosive-early diversification in phylogenies of extant taxa cannot result from increasing extinction and can only be explained by temporally declining speciation rates. Moreover, whenever extinction rates are high, "explosive early" patterns become unobservable, because high extinction quickly erases the signature of even large declines in speciation rates. Although extinction may obscure patterns of evolutionary diversification, these results show that decreasing speciation is often distinguishable from increasing extinction in the numerous molecular phylogenies of radiations that retain a preponderance of early lineages.     

Phytogeographical Implication of Bridelia Will. (Phyllanthaceae) Fossil Leaf from the Late Oligocene of India

Background

The family Phyllanthaceae has a predominantly pantropical distribution. Of its several genera, Bridelia Willd. is of a special interest because it has disjunct equally distributed species in Africa and tropical Asia i.e. 18–20 species in Africa-Madagascar (all endemic) and 18 species in tropical Asia (some shared with Australia). On the basis of molecular phylogenetic study on Bridelia, it has been suggested that the genus evolved in Southeast Asia around 33±5 Ma, while speciation and migration to other parts of the world occurred at 10±2 Ma. Fossil records of Bridelia are equally important to support the molecular phylogenetic studies and plate tectonic models.

Results

We describe a new fossil leaf of Bridelia from the late Oligocene (Chattian, 28.4–23 Ma) sediments of Assam, India. The detailed venation pattern of the fossil suggests its affinities with the extant B. ovata, B. retusa and B. stipularis. Based on the present fossil evidence and the known fossil records of Bridelia from the Tertiary sediments of Nepal and India, we infer that the genus evolved in India during the late Oligocene (Chattian, 28.4–23 Ma) and speciation occurred during the Miocene. The stem lineage of the genus migrated to Africa via “Iranian route” and again speciosed in Africa-Madagascar during the late Neogene resulting in the emergence of African endemic clades. Similarly, the genus also migrated to Southeast Asia via Myanmar after the complete suturing of Indian and Eurasian plates. The emergence and speciation of the genus in Asia and Africa is the result of climate change during the Cenozoic.

Conclusions

On the basis of present and known fossil records of Bridelia, we have concluded that the genus evolved during the late Oligocene in northeast India. During the Neogene, the genus diversified and migrated to Southeast Asia via Myanmar and Africa via “Iranian Route”.     

Congruent phylogenetic and fossil signatures of mammalian diversification dynamics driven by Tertiary abiotic change

Computational methods for estimating diversification rates from extant species phylogenetic trees have become abundant in evolutionary research. However, little evidence exists about how their outcome compares to a complementary and direct source of information: the fossil record. Furthermore, there is virtually no direct test for the congruence of evolutionary rates based on these two sources. This task is only achievable in clades with both a well‐known fossil record and a complete phylogenetic tree. Here, we compare the evolutionary rates of ruminant mammals as estimated from their vast paleontological record—over 1200 species spanning 50 myr—and their living‐species phylogeny. Significantly, our results revealed that the ruminant's fossil record and phylogeny reflect congruent evolutionary processes. The concordance is especially strong for the last 25 myr, when living groups became a dominant part of ruminant diversity. We found empirical support for previous hypotheses based on simulations and neontological data: The pattern captured by the tree depends on how clade specific the processes are and which clades are involved. Also, we report fossil evidence for a postradiation speciation slowdown coupled with constant, moderate extinction in the Miocene. The recent deceleration in phylogenetic rates is connected to rapid extinction triggered by recent climatic fluctuations.     

Dealing with incomplete taxon sampling and diversification of a large clade of mushroom-forming fungi

The absence of an adequate fossil record can hinder understanding the process of diversification that underlies the evolutionary history of a given group. In such cases, investigators have used ultrametric trees derived from molecular data from extant taxa to gain insights into processes of speciation and extinction over time. Inadequate taxon sampling, however, impairs such inferences. In this study, we use simulations to investigate the effect of incomplete taxon sampling on the accumulation of lineages through time for a clade of mushroom-forming fungi, the Hebelomateae. To achieve complete taxon sampling, we use a new Bayesian approach that incorporates substitute lineages to estimate diversification rates. Unlike many studies of animals and plants, we find no evidence of a slowdown in speciation. This indicates the Hebelomateae has not undergone an adaptive radiation. Rather, these fungi have evolved under a relatively constant rate of diversification since their most recent common ancestor, which we date back to the Eocene. The estimated net diversification rate (0.08-0.19 spp./lineage/Ma) is comparable with that of many plants and animals. We suggest that continuous diversification in the Hebelomateae has been facilitated by climatic and vegetation changes throughout the Cenozoic. We also caution against modeling multiple genes as a single partition when performing phylogenetic dating analyses.     

The role of Quaternary environmental change in plant macroevolution: the exception or the rule?

The Quaternary has been described as an important time for genetic diversification and speciation. This is based on the premise that Quaternary climatic conditions fostered the isolation of populations and, in some instances, allopatric speciation. However, the 'Quaternary Ice-Age speciation model' rests on two key assumptions: (i) that biotic responses to climate change during the Quaternary were significantly different from those of other periods in Earth's history; and (ii) that the mechanisms of isolation during the Quaternary were sufficient in time and space for genetic diversification to foster speciation. These assumptions are addressed by examining the plant fossil record for the Quaternary (in detail) and for the past 410 Myr, which encompasses previous intervals of icehouse Earth. Our examination of the Quaternary record indicates that floristic responses to climate changes during the past 1.8 Myr were complex and that a distinction has to be made between those plants that were able to withstand the extremes of glacial conditions and those that could not. Generation times are also important as are different growth forms (e.g. herbaceous annuals and arborescent perennials), resulting in different responses in terms of genetic divergence rates during isolation. Because of these variations in the duration of isolation of populations and genomic diversification rates, no canonical statement about the predominant floristic response to climatic changes during the Quaternary (i.e. elevated rates of speciation or extinction, or stasis) is currently possible. This is especially true because of a sampling bias in terms of the fossil record of tree species over that of species with non-arborescent growth forms. Nevertheless, based on the available information, it appears that the dominant response of arborescent species during the Quaternary was extinction rather than speciation or stasis. By contrast, our examination of the fossil record of vascular plants for the past 410 Myr indicates that speciation rates often increased during long intervals of icehouse Earth (spanning up to 50 Myr). Therefore, longer periods of icehouse Earth than those occurring during the Quaternary may have isolated plant populations for sufficiently long periods of time to foster genomic diversification and allopatric speciation. Our results highlight the need for more detailed study of the fossil record in terms of finer temporal and spatial resolution than is currently available to examine the significance of intervals of icehouse Earth. It is equally clear that additional and detailed molecular studies of extant populations of Quaternary species are required in order to determine the extent to which these 'relic' species have genomically diversified across their current populations.     

Does coevolution promote species richness in parasitic cuckoos?

Why some lineages have diversified into larger numbers of species than others is a fundamental but still relatively poorly understood aspect of the evolutionary process. Coevolution has been recognized as a potentially important engine of speciation, but has rarely been tested in a comparative framework. We use a comparative approach based on a complete phylogeny of all living cuckoos to test whether parasite–host coevolution is associated with patterns of cuckoo species richness. There are no clear differences between parental and parasitic cuckoos in the number of species per genus. However, a cladogenesis test shows that brood parasitism is associated with both significantly higher speciation and extinction rates. Furthermore, subspecies diversification rate estimates were over twice as high in parasitic cuckoos as in parental cuckoos. Among parasitic cuckoos, there is marked variation in the severity of the detrimental effects on host fitness; chicks of some cuckoo species are raised alongside the young of the host and others are more virulent, with the cuckoo chick ejecting or killing the eggs/young of the host. We show that cuckoos with a more virulent parasitic strategy have more recognized subspecies. In addition, cuckoo species with more recognized subspecies have more hosts. These results hold after controlling for confounding geographical effects such as range size and isolation in archipelagos. Although the power of our analyses is limited by the fact that brood parasitism evolved independently only three times in cuckoos, our results suggest that coevolutionary arms races with hosts have contributed to higher speciation and extinction rates in parasitic cuckoos.     

When and how did the terrestrial mid-Permian mass extinction occur? Evidence from the tetrapod record of the Karoo Basin,South Africa

A mid-Permian (Guadalupian epoch) extinction event at approximately 260 Ma has been mooted for two decades. This is based primarily on invertebrate biostratigraphy of Guadalupian–Lopingian marine carbonate platforms in southern China, which are temporally constrained by correlation to the associated Emeishan Large Igneous Province (LIP). Despite attempts to identify a similar biodiversity crisis in the terrestrial realm, the low resolution of mid-Permian tetrapod biostratigraphy and a lack of robust geochronological constraints have until now hampered both the correlation and quantification of terrestrial extinctions. Here we present an extensive compilation of tetrapod-stratigraphic data analysed by the constrained optimization (CONOP) algorithm that reveals a significant extinction event among tetrapods within the lower Beaufort Group of the Karoo Basin, South Africa, in the latest Capitanian. Our fossil dataset reveals a 74–80% loss of generic richness between the upper Tapinocephalus Assemblage Zone (AZ) and the mid-Pristerognathus AZ that is temporally constrained by a U–Pb zircon date (CA-TIMS method) of 260.259 ± 0.081 Ma from a tuff near the top of the Tapinocephalus AZ. This strengthens the biochronology of the Permian Beaufort Group and supports the existence of a mid-Permian mass extinction event on land near the end of the Guadalupian. Our results permit a temporal association between the extinction of dinocephalian therapsids and the LIP volcanism at Emeishan, as well as the marine end-Guadalupian extinctions.     

Biogeography of curimatid fishes reveals multiple lowland–upland river transitions and differential diversification in the Neotropics (Teleostei,Curimatidae)

The Neotropics harbors a megadiverse ichthyofauna comprising over 6300 species with approximately 80% in just three taxonomic orders within the clade Characiphysi. This highly diverse group has evolved in tropical South America over tens to hundreds of millions of years influenced mostly by re‐arrangements of river drainages in lowland and upland systems. In this study, we investigate patterns of spatial diversification in Neotropical freshwater fishes in the family Curimatidae, a species‐rich clade of the order Characiformes. Specifically, we examined ancestral areas, dispersal events, and shifts in species richness using spatially explicit biogeographic and macroevolutionary models to determine whether lowlands–uplands serve as museums or cradles of diversification for curimatids. We used fossil information to estimate divergence times in BEAST, multiple time‐stratified models of geographic range evolution in BioGeoBEARS, and alternative models of geographic state‐dependent speciation and extinction in GeoHiSSE. Our results suggest that the most recent common ancestor of curimatids originated in the Late Cretaceous likely in lowland paleodrainages of northwestern South America. Dispersals from lowland to upland river basins of the Brazilian and Guiana shields occurred repeatedly across independently evolving lineages in the Cenozoic. Colonization of upland drainages was often coupled with increased rates of net diversification in species‐rich genera such as Cyphocharax and Steindachnerina. Our findings demonstrate that colonization of novel aquatic environments at higher elevations is associated with an increased rate of diversification, although this pattern is clade‐dependent and driven mostly by allopatric speciation. Curimatids reinforce an emerging perspective that Amazonian lowlands act as a museum by accumulating species along time, whereas the transitions to uplands stimulate higher net diversification rates and lineage diversification.     

No post-Cretaceous ecosystem depression in European forests? Rich insect-feeding damage on diverse middle Palaeocene plants,Menat, France

Insect herbivores are considered vulnerable to extinctions of their plant hosts. Previous studies of insect-damaged fossil leaves in the US Western Interior showed major plant and insect herbivore extinction at the Cretaceous–Palaeogene (K–T) boundary. Further, the regional plant–insect system remained depressed or ecologically unbalanced throughout the Palaeocene. Whereas Cretaceous floras had high plant and insect-feeding diversity, all Palaeocene assemblages to date had low richness of plants, insect feeding or both. Here, we use leaf fossils from the middle Palaeocene Menat site, France, which has the oldest well-preserved leaf assemblage from the Palaeocene of Europe, to test the generality of the observed Palaeocene US pattern. Surprisingly, Menat combines high floral diversity with high insect activity, making it the first observation of a ‘healthy’ Palaeocene plant–insect system. Furthermore, rich and abundant leaf mines across plant species indicate well-developed host specialization. The diversity and complexity of plant–insect interactions at Menat suggest that the net effects of the K–T extinction were less at this greater distance from the Chicxulub, Mexico, impact site. Along with the available data from other regions, our results show that the end-Cretaceous event did not cause a uniform, long-lasting depression of global terrestrial ecosystems. Rather, it gave rise to varying regional patterns of ecological collapse and recovery that appear to have been strongly influenced by distance from the Chicxulub structure.     

Relating traits to diversification: a simple test

We describe a simple comparative method for determining whether rates of diversification are correlated with continuous traits in species-level phylogenies. This involves comparing traits of species with net speciation rate (number of nodes linking extant species with the root divided by the root to tip evolutionary distance), using a phylogenetically corrected correlation. We use simulations to examine the power of this test. We find that the approach has acceptable power to uncover relationships between speciation and a continuous trait and is robust to background random extinction; however, the power of the approach is reduced when the rate of trait evolution is decreased. The test has low power to relate diversification to traits when extinction rate is correlated with the trait. Clearly, there are inherent limitations in using only data on extant species to infer correlates of extinction; however, this approach is potentially a powerful tool in analyzing correlates of speciation.     

Mass extinction, gradual cooling, or rapid radiation? Reconstructing the spatiotemporal evolution of the ancient angiosperm genus Hedyosmum (Chloranthaceae) using empirical and simulated approaches

Chloranthaceae is a small family of flowering plants (65 species) with an extensive fossil record extending back to the Early Cretaceous. Within Chloranthaceae, Hedyosmum is remarkable because of its disjunct distribution--1 species in the Paleotropics and 44 confined to the Neotropics--and a long "temporal gap" between its stem age (Early Cretaceous) and the beginning of the extant radiation (late Cenozoic). Is this gap real, reflecting low diversification and a recent radiation, or the signature of extinction? Here we use paleontological data, relaxed-clock molecular dating, diversification analyses, and parametric ancestral area reconstruction to investigate the timing, tempo, and mode of diversification in Hedyosmum. Our results, based on analyses of plastid and nuclear sequences for 40 species, suggest that the ancestor of Chloranthaceae and the Hedyosmum stem lineages were widespread in the Holarctic in the Late Cretaceous. High extinction rates, possibly associated with Cenozoic climatic fluctuations, may have been responsible for the low extant diversity of the family. Crown group Hedyosmum originated c. 36-43 Ma and colonized South America from the north during the Early-Middle Miocene (c. 20 Ma). This coincided with an increase in diversification rates, probably triggered by the uplift of the Northern Andes from the Mid-Miocene onward. This study illustrates the advantages of combining paleontological, phylogenetic, and biogeographic data to reconstruct the spatiotemporal evolution of an ancient lineage, for which the extant diversity is only a remnant of past radiations. It also shows the difficulties of inferring patterns of lineage diversification when incomplete taxon sampling is combined with high extinction rates.     

Extant primitively segmented spiders have recently diversified from an ancient lineage

Living fossils are lineages that have retained plesiomorphic traits through long time periods. It is expected that such lineages have both originated and diversified long ago. Such expectations have recently been challenged in some textbook examples of living fossils, notably in extant cycads and coelacanths. Using a phylogenetic approach, we tested the patterns of the origin and diversification of liphistiid spiders, a clade of spiders considered to be living fossils due to their retention of arachnid plesiomorphies and their exclusive grouping in Mesothelae, an ancient clade sister to all modern spiders. Facilitated by original sampling throughout their Asian range, we here provide the phylogenetic framework necessary for reconstructing liphistiid biogeographic history. All phylogenetic analyses support the monophyly of Liphistiidae and of eight genera. As the fossil evidence supports a Carboniferous Euramerican origin of Mesothelae, our dating analyses postulate a long eastward over-land dispersal towards the Asian origin of Liphistiidae during the Palaeogene (39–58 Ma). Contrary to expectations, diversification within extant liphistiid genera is relatively recent, in the Neogene and Late Palaeogene (4–24 Ma). While no over-water dispersal events are needed to explain their evolutionary history, the history of liphistiid spiders has the potential to play prominently in vicariant biogeographic studies.