The automation and evaluation of nested clade phylogeographic analysis

Nested clade phylogeographic analysis (NCPA) is a popular method for reconstructing the demographic history of spatially distributed populations from genetic data. Although some parts of the analysis are automated, there is no unique and widely followed algorithm for doing this in its entirety, beginning with the data, and ending with the inferences drawn from the data. This article describes a method that automates NCPA, thereby providing a framework for replicating analyses in an objective way. To do so, a number of decisions need to be made so that the automated implementation is representative of previous analyses. We review how the NCPA procedure has evolved since its inception and conclude that there is scope for some variability in the manual application of NCPA. We apply the automated software to three published datasets previously analyzed manually and replicate many details of the manual analyses, suggesting that the current algorithm is representative of how a typical user will perform NCPA. We simulate a large number of replicate datasets for geographically distributed, but entirely random-mating, populations. These are then analyzed using the automated NCPA algorithm. Results indicate that NCPA tends to give a high frequency of false positives. In our simulations we observe that 14% of the clades give a conclusive inference that a demographic event has occurred, and that 75% of the datasets have at least one clade that gives such an inference. This is mainly due to the generation of multiple statistics per clade, of which only one is required to be significant to apply the inference key. We survey the inferences that have been made in recent publications and show that the most commonly inferred processes (restricted gene flow with isolation by distance and contiguous range expansion) are those that are commonly inferred in our simulations. However, published datasets typically yield a richer set of inferences with NCPA than obtained in our random-mating simulations, and further testing of NCPA with models of structured populations is necessary to examine its accuracy.

The automation of nested clade phylogeographic analysis

ANeCA is a fully automated implementation of Nested Clade Phylogeographic Analysis. This was originally developed by Templeton and colleagues, and has been used to infer, from the pattern of gene sequence polymorphisms in a geographically structured population, the historical demographic processes that have shaped its evolution. Until now it has been necessary to perform large parts of the procedure manually. We provide a program that will take data in Nexus sequential format, and directly output a set of inferences. The software also includes TCS v1.18 and GeoDis v2.2 as part of automation.

Phylogeographic analysis of the chloroplast DNA variation in wild common bean (Phaseolus vulgaris L.) in the Americas

The wild common bean (Phaseolus vulgaris) is widely but discontinuously distributed from northern Mexico to northern Argentina on both sides of the Isthmus of Panama. Little is known on how the species has reached its current disjunct distribution. In this research, chloroplast DNA polymorphisms in seven non-coding regions were used to study the history of migration of wild P. vulgaris between Mesoamerica and South America. A penalized likelihood analysis was applied to previously published Leguminosae ITS data to estimate divergence times between P. vulgaris and its sister taxa from Mesoamerica, and divergence times of populations within P. vulgaris. Fourteen chloroplast haplotypes were identified by PCR-RFLP and their geographical associations were studied by means of a Nested Clade Analysis and Mantel Tests. The results suggest that the haplotypes are not randomly distributed but occupy discrete parts of the geographic range of the species. The current distribution of haplotypes may be explained by isolation by distance and by at least two migration events between Mesoamerica and South America: one from Mesoamerica to South America and another one from northern South America to Mesoamerica. Age estimates place the divergence of P. vulgaris from its sister taxa from Mesoamerica at or before 1.3 Ma, and divergence of populations from Ecuador-northern Peru at or before 0.6 Ma. As these ages are taken as minimum divergence times, the influence of past events, such as the closure of the Isthmus of Panama and the final uplift of the Andes, on the migration history and population structure of this species cannot be disregarded.

Phylogeographic Analysis of HIV-1 Subtype C Dissemination in Southern Brazil

The HIV-1 subtype C has spread efficiently in the southern states of Brazil (Rio Grande do Sul, Santa Catarina and Parana). Phylogeographic studies indicate that the subtype C epidemic in southern Brazil was initiated by the introduction of a single founder virus population at some time point between 1960 and 1980, but little is known about the spatial dynamics of viral spread. A total of 135 Brazilian HIV-1 subtype C pol sequences collected from 1992 to 2009 at the three southern state capitals (Porto Alegre, Florianopolis and Curitiba) were analyzed. Maximum-likelihood and Bayesian methods were used to explore the degree of phylogenetic mixing of subtype C sequences from different cities and to reconstruct the geographical pattern of viral spread in this country region. Phylogeographic analyses supported the monophyletic origin of the HIV-1 subtype C clade circulating in southern Brazil and placed the root of that clade in Curitiba (Parana state). This analysis further suggested that Florianopolis (Santa Catarina state) is an important staging post in the subtype C dissemination displaying high viral migration rates from and to the other cities, while viral flux between Curitiba and Porto Alegre (Rio Grande do Sul state) is very low. We found a positive correlation (r(2) = 0.64) between routine travel and viral migration rates among localities. Despite the intense viral movement, phylogenetic intermixing of subtype C sequences from different Brazilian cities is lower than expected by chance. Notably, a high proportion (67%) of subtype C sequences from Porto Alegre branched within a single local monophyletic sub-cluster. These results suggest that the HIV-1 subtype C epidemic in southern Brazil has been shaped by both frequent viral migration among states and in situ dissemination of local clades.

Comparative genomic and phylogeographic analysis of Mycobacterium leprae.

Reductive evolution and massive pseudogene formation have shaped the 3.31-Mb genome of Mycobacterium leprae, an unculturable obligate pathogen that causes leprosy in humans. The complete genome sequence of M. leprae strain Br4923 from Brazil was obtained by conventional methods (6x coverage), and Illumina resequencing technology was used to obtain the sequences of strains Thai53 (38x coverage) and NHDP63 (46x coverage) from Thailand and the United States, respectively. Whole-genome comparisons with the previously sequenced TN strain from India revealed that the four strains share 99.995% sequence identity and differ only in 215 polymorphic sites, mainly SNPs, and by 5 pseudogenes. Sixteen interrelated SNP subtypes were defined by genotyping both extant and extinct strains of M. leprae from around the world. The 16 SNP subtypes showed a strong geographical association that reflects the migration patterns of early humans and trade routes, with the Silk Road linking Europe to China having contributed to the spread of leprosy.

Molecular identification of mullet species of the Atlantic South Caribbean and South America and the phylogeographic analysis of mugil liza

Fundacao de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phylogeographic analysis in populations of Protonectarina sylveirae (Saussure, 1854) (Hymenoptera, Vespidae, Polistinae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phylogeographic analysis in populations of Protonectarina sylveirae (Saussure, 1854) (Hymenoptera, Vespidae, Polistinae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Forest-obligate Sabethes mosquitoes suggest palaeoecological perturbations

The origin of tropical forest diversity has been hotly debated for decades. Although specific mechanisms vary, many such explanations propose some vicariance in the distribution of species during glacial cycles and several have been supported by genetic evidence in Neotropical taxa. However, no consensus exists with regard to the extent or time frame of the vicariance events. Here, we analyse the cytochrome oxidase II mitochondrial gene of 250 Sabethes albiprivus B mosquitoes sampled from western Sao Paulo in Brazil. There was very low population structuring among collection sites (Phi(ST) = 0.03, P = 0.04). Historic demographic analyses and the contemporary geographic distribution of genetic diversity suggest that the populations sampled are not at demographic equilibrium. Three distinct mitochondrial clades were observed in the samples, one of which differed significantly in its geographic distribution relative to the other two within a small sampling area (similar to 70 x 35 km). This fact, supported by the inability of maximum likelihood analyses to achieve adequate fits to simple models for the population demography of the species, suggests a more complex history, possibly involving disjunct forest refugia. This hypothesis is supported by a genetic signal of recent population growth, which is expected if population sizes of this forest-obligate insect increased during the forest expansions that followed glacial periods. Although a time frame cannot be reliably inferred for the vicariance event leading to the three genetic clades, molecular clock estimates place this at similar to 1 Myr before present.

Molecular phylogeography of the Woodland Ringlet (Erebia medusa [Denis and Schiffermüller] 1775) in Europe

Phylogeography is a recent field of biological research that links phylogenetics to biogeography through deciphering the imprint that evolutionary history has left on the genetic structure of extant populations. During the cold phases of the successive ice ages, which drastically shaped species’ distributions since the Pliocene, populations of numerous species were isolated in refugia where many of them evolved into different genetic lineages. My dissertation deals with the phylogeography of the Woodland Ringlet (Erebia medusa [Denis and Schiffermüller] 1775) in Central and Eastern Europe. This Palaearctic butterfly species is currently distributed from central France and south eastern Belgium over large parts of Central Europe and southern Siberia to the Pacific. It is absent from those parts of Europe with mediterranean, oceanic and boreal climates. It was supposed to be a Siberian faunal element with a rather homogeneous population structure in Central Europe due to its postglacial expansion out of a single eastern refugium. An already existing evolutionary scenario for the Woodland Ringlet in Central and Eastern Europe is based on nuclear data (allozymes). To know if this is corroborated by organelle evolutionary history, I sequenced two mitochondrial markers (part of the cytochrome oxydase subunit one and the control region) for populations sampled over the same area. Phylogeography largely relies on the construction of networks of uniparentally inherited haplotypes that are compared to geographic haplotype distribution thanks to recent developed methods such as nested clade phylogeographic analysis (NCPA). Several ring-shaped ambiguities (loops) emerged from both haplotype networks in E. medusa. They can be attributed to recombination and homoplasy. Such loops usually avert the straightforward extraction of the phylogeographic signal contained in a gene tree. I developed several new approaches to extract phylogeographic information in the presence of loops, considering either homoplasy or recombination. This allowed me to deduce a consistent evolutionary history for the species from the mitochondrial data and also adds plausibility for the occurrence of recombination in E. medusa mitochondria. Despite the fact that the control region is assumed to have a lack of resolving power in other species, I found a considerable genetic variation of this marker in E. medusa which makes it a useful tool for phylogeographic studies. In combination with the allozyme data, the mitochondrial genome supports the following phylogeographic scenario for E. medusa in Europe: (i) a first vicariance, due to the onset of the Würm glaciation, led to the formation of several major lineages, and is mirrored in the NCPA by restricted gene flow, (ii) later on further vicariances led to the formation of two sub-lineages in the Western lineage and two sub-lineages in the Eastern lineage during the Last Glacial Maximum or Older Dryas; additionally the NCPA supports a restriction of gene flow with isolation by distance, (iii) finally, vicariance resulted in two secondary sub-lineages in the area of Germany and, maybe, to two other secondary sub-lineages in the Czech Republic. The last postglacial warming was accompanied by strong range expansions in most of the genetic lineages. The scenario expected for a presumably Siberian faunal element such as E. medusa is a continuous loss of genetic diversity during postglacial westward expansion. Hence, the pattern found in this thesis contradicts a typical Siberian origin of E. medusa. In contrast, it corroboratess the importance of multiple extra-Mediterranean refugia for European fauna as it was recently assumed for other continental species.

Statistical inferences in phylogeography

In conventional phylogeographic studies, historical demographic processes are elucidated from the geographical distribution of individuals represented on an inferred gene tree. However, the interpretation of gene trees in this context can be difficult as the same demographic/geographical process can randomly lead to multiple different genealogies. Likewise, the same gene trees can arise under different demographic models. This problem has led to the emergence of many statistical methods for making phylogeographic inferences. A popular phylogeographic approach based on nested clade analysis is challenged by the fact that a certain amount of the interpretation of the data is left to the subjective choices of the user, and it has been argued that the method performs poorly in simulation studies. More rigorous statistical methods based on coalescence theory have been developed. However, these methods may also be challenged by computational problems or poor model choice. In this review, we will describe the development of statistical methods in phylogeographic analysis, and discuss some of the challenges facing these methods.

Phylogeography of Hypostomus strigaticeps (Siluriformes: Loricariidae) inferred by mitochondrial DNA reveals its distribution in the upper Paraná River basin

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Multiple maternal origins and weak phylogeographic structure in domestic goats

Domestic animals have played a key role in human history. Despite their importance, however, the origins of most domestic species remain poorly understood. We assessed the phylogenetic history and population structure of domestic goats by sequencing a hypervariable segment (481 bp) of the mtDNA control region from 406 goats representing 88 breeds distributed across the Old World. Phylogeographic analysis revealed three highly divergent goat lineages (estimated divergence >200,000 years ago), with one lineage occurring only in eastern and southern Asia. A remarkably similar pattern exists in cattle, sheep, and pigs. These results, combined with recent archaeological findings, suggest that goats and other farm animals have multiple maternal origins with a possible center of origin in Asia, as well as in the Fertile Crescent. The pattern of goat mtDNA diversity suggests that all three lineages have undergone population expansions, but that the expansion was relatively recent for two of the lineages (including the Asian lineage). Goat populations are surprisingly less genetically structured than cattle populations. In goats only ≈10% of the mtDNA variation is partitioned among continents. In cattle the amount is ≥50%. This weak structuring suggests extensive intercontinental transportation of goats and has intriguing implications about the importance of goats in historical human migrations and commerce.

Using a Combination of Phylogenetic Comparative and Paleobotanical Methods to Elucidate Patterns of Lineage Selection in Rosales (Plantae: Anthophyta)

An increasing focus in evolutionary biology is on the interplay between mesoscale ecological and evolutionary processes such as population demographics, habitat tolerance, and especially geographic distribution, as potential drivers responsible for patterns of diversification and extinction over geologic time. However, few studies to date connect organismal processes such as survival and reproduction through mesoscale patterns to long-term macroevolutionary trends. In my dissertation, I investigate how mechanism of seed dispersal, mediated through geographic range size, influences diversification rates in the Rosales (Plantae: Anthophyta). In my first chapter, I validate the phylogenetic comparative methods that I use in my second and third chapters. Available state speciation and extinction (SSE) models assumptions about evolution known to be false through fossil data. I show, however, that as long as net diversification rates remain positive – a condition likely true for the Rosales – these violations of SSE’s assumptions do not cause significantly biased results. With SSE methods validated, my second chapter reconstructs three associations that appear to increase diversification rate for Rosalean genera: (1) herbaceous habit; (2) a three-way interaction combining animal dispersal, high within-genus species richness, and geographic range on multiple continents; (3) a four-way interaction combining woody habit with the other three characteristics of (2). I suggest that the three- and four-way interactions represent colonization ability and resulting extinction resistance in the face of late Cenozoic climate change; however, there are other possibilities as well that I hope to investigate in future research. My third chapter reconstructs the phylogeographic history of the Rosales using both non-fossil-assisted SSE methods as well as fossil-informed traditional phylogeographic analysis. Ancestral state reconstructions indicate that the Rosaceae diversified in North America while the other Rosalean families diversified elsewhere, possibly in Eurasia. SSE is able to successfully identify groups of genera that were likely to have been ancestrally widespread, but has poorer taxonomic resolution than methods that use fossil data. In conclusion, these chapters together suggest several potential causal links between organismal, mesoscale, and geologic scale processes, but further work will be needed to test the hypotheses that I raise here.