Genetically Depauperate and Still Successful: Few Multilocus Genotypes of the Introduced Parthenogenetic Weevil Naupactus cervinus (Coleoptera: Curculionidae) Prevail in the Continental United States.

Naupactus cervinus is a parthenogenetic weevil native to South America that is currently distributed worldwide. This flightless species is polyphagous and capable of modifying gene expression regimes for responding to stressful situations. Naupactus cervinus was first reported in the continental United States in 1879 and has rapidly colonized most of the world since. Previous studies suggested that an invader genotype successfully established even in areas of unsuitable environmental conditions. In the present work, we analyze mitochondrial and nuclear sequences from 71 individuals collected in 13 localities across three states in the southern US, in order to describe the genetic diversity in this area of introduction that has not yet been previously studied. Our results suggest that 97% of the samples carry the most prevalent invader genotype already reported, while the rest shows a close mitochondrial derivative. This would support the hypothesis of a general purpose genotype, with parthenogenesis and its associated lack of recombination maintaining the linkage of genetic variants capable of coping with adverse conditions and enlarging its geographical range. However, demographic advantages related to parthenogenetic reproduction as the main driver of geographic expansion (such as the foundation of a population with a single virgin female) cannot be ruled out. Given the historical introduction records and the prevalence of the invader genotype, it is possible that the continental US may act as a secondary source of introductions to other areas. We propose that both the parthenogenesis and scarce genetic variation in places of introduction may, in fact, be an asset that allows N. cervinus to thrive across a range of environmental conditions.

City puzzles: Does urban land scape affect genetic population structure in Aedes aegypti?

Cities usually offer a suitable environment for the dengue vector Aedes aegypti, providing oviposition sites, accessibility to human hosts and nectar meals. However, large urban centres are highly heterogeneous environments, forming a patched landscape that could affect Ae. aegypti population dynamics and dispersal. Here, we performed a genome-wide analysis using Rad-seq data from 99 Ae. aegypti specimens collected in three areas within Buenos Aires city with varying levels of urbanization/land use: highly urbanized Area 1, intermediate Area 2 and poorly urbanized Area 3. We found an inverse association between urbanization levels and spatial genetic structure. Populations from highly urbanized Area 1 did not present genetic structure whereas two and three clusters were detected in Areas 2 and 3, respectively. In the case of Area 3, initial analyses showed separation in clusters was mostly due to elevated consanguinity within sites although three clusters were still detected after closely related individuals were discarded. Mosquitoes around each site displayed a high degree of isolation, evidencing a close dependence between the vector and human dwellings. Interestingly, specimens from distant boroughs (within the limits of the city) and the city's outskirts formed a single cluster with inner city sites (Area 1), highlighting the role of passive transport in shaping population structure. Genetic distances were poorly correlated with geographic distances in Buenos Aires, suggesting a stronger influence of passive than active dispersal on population structure. Only Area 2 displayed a significant isolation-by-distance pattern (p = 0.046), with males dispersing more than females (p = 0.004 and p = 0.016, respectively). Kinship analyses allowed us to detect full-siblings located 1.5 km apart in Area 1, which could be due to an extreme event of active female dispersal. Effective population size was higher in Area 2 confirming that cemeteries represent highly favourable environments for Ae. aegypti and need to be specifically targeted. Our results suggest that control programs should take into account urban landscape heterogeneity in order to improve vector control.

Stable inversion clines in a grasshopper species group despite complex geographical history.

Chromosomal inversions are known to play roles in adaptation and differentiation in many species. They involve clusters of correlated genes (i.e., loci in linkage disequilibrium, LD) possibly associated with environmental variables. The grasshopper "species complex" Trimerotropis pallidipennis comprises several genetic lineages distributed from North to South America in arid and semi-arid high-altitude environments. The southernmost lineage, Trimerotropis sp., segregates for four to seven putative inversions that display clinal variation, possibly through adaptation to temperate environments. We analysed chromosomal, mitochondrial and genome-wide single nucleotide polymorphism data in 19 Trimerotropis sp. populations mainly distributed along two altitudinal gradients (MS and Ju). Populations across Argentina comprise two main chromosomally and genetically differentiated lineages: one distributed across the southernmost border of the "Andes Centrales," adding evidence for a differentiation hotspot in this area; and the other widely distributed in Argentina. Within the latter, network analytical approaches to LD found three clusters of correlated loci (LD-clusters), with inversion karyotypes explaining >79% of the genetic variation. Outlier loci associated with environmental variables mapped to two of these LD-clusters. Furthermore, despite the complex geographical history indicated by population genetic analyses, the clines in inversion karyotypes have remained stable for more than 20 generations, implicating their role in adaptation and differentiation within this lineage. We hypothesize that these clines could be the consequence of a coupling between extrinsic postzygotic barriers and spatially varying selection along environmental gradients resulting in a hybrid zone. These results provide a framework for future investigations about candidate genes implicated in rapid adaptation to new environments.

Host-specific gene expression as a tool for introduction success in Naupactus parthenogenetic weevils.

Food resource access can mediate establishment success in invasive species, and generalist herbivorous insects are thought to rely on mechanisms of transcriptional plasticity to respond to dietary variation. While asexually reproducing invasives typically have low genetic variation, the twofold reproductive capacity of asexual organisms is a marked advantage for colonization. We studied host-related transcriptional acclimation in parthenogenetic, invasive, and polyphagous weevils: Naupactus cervinus and N. leucoloma. We analyzed patterns of gene expression in three gene categories that can mediate weevil-host plant interactions through identification of suitable host plants, short-term acclimation to host plant defenses, and long-term adaptation to host plant defenses and their pathogens. This approach employed comparative transcriptomic methods to investigate differentially expressed host detection, detoxification, immune defense genes, and pathway-level gene set enrichment. Our results show that weevil gene expression responses can be host plant-specific, and that elements of that response can be maintained in the offspring. Some host plant groups, such as legumes, appear to be more taxing as they elicit a complex gene expression response which is both strong in intensity and specific in identity. However, the weevil response to taxing host plants shares many differentially expressed genes with other stressful situations, such as host plant cultivation conditions and transition to novel host, suggesting that there is an evolutionarily favorable shared gene expression regime for responding to different types of stressful situations. Modulating gene expression in the absence of other avenues for phenotypic adaptation may be an important mechanism of successful colonization for these introduced insects.

Correction: A Species delimitation approach to uncover cryptic species in the South American fire ant decapitating flies (Diptera: Phoridae: Pseudacteon).

[This corrects the article DOI: 10.1371/journal.pone.0236086.].

Genome-Wide Screening of Aedes aegypti (Culicidae: Diptera) Populations From Northwestern Argentina: Active and Passive Dispersal Shape Genetic Structure.

Aedes aegypti is the primary vector of arboviruses of great impact on human health. Our goal was to assess the spatial genetic structure of Ae. aegypti at the regional and local levels in Northwestern Argentina, an area with high prevalence of dengue fever. We analyzed 59 Ae. aegypti individuals collected from six locations in Northwestern Argentina using nuclear genome-wide Single Nucleotide Polymorphisms (SNPs) generated with double digest Restriction-site Associated DNA Sequencing. We also performed an entomological survey in 70 households in the cities of Orán and Tartagal. An analysis at the regional level indicated that the populations of Ae. aegypti in Northwestern Argentina are spatially structured and present a significant IBD pattern. Our results suggest that passive transport of eggs/immature stages, in both northward and southward directions, plays an important role in structuring Ae. aegypti populations at a regional scale and also as a source for the introduction of novel genetic variants through migration events into established populations. At a local level, we found neither spatial genetic structure nor significant isolation by distance (IBD) in Tartagal, indicating high gene flow within the city and active dispersal. In contrast, samples from Orán formed two clusters with a significant IBD pattern, although weaker than that at a regional level. Both populations showed signs of recent bottleneck events, probably coincident with past eradication campaigns. The entomological survey revealed a high prevalence of Ae. aegypti in both cities, although significantly higher in Tartagal.

A Species delimitation approach to uncover cryptic species in the South American fire ant decapitating flies (Diptera: Phoridae: Pseudacteon).

South American fire ant decapitating flies in the genus Pseudacteon (Diptera: Phoridae) are potential biocontrol agents of the invasive fire ants Solenopsis invicta and S. richteri in the United States and other regions of the world due to their high host specificity and the direct and indirect damage to their host ants. Despite their importance and the fact that several flies have already been released in the US, little is known about the genetic variability and phylogenetic relationships of Pseudacteon flies parasitizing South American fire ants in the Solenopsis saevissima species-group. A species delimitation analysis was conducted using a distance-based method (ABGD) and two tree-based methods (GMYC and mPTP) using COI sequences of 103 specimens belonging to 20 of the 22 Pseudacteon species known from southern South America. Additionally, phylogenetic relationships between the already described and new candidate species were inferred using mitochondrial (COI) and nuclear (wingless) sequences. The species delimitation analysis suggests that species richness in these flies has been previously underestimated, due to the existence of putative cryptic species within nominal Pseudacteon obtusus, P. pradei, P. tricuspis, P. cultellatus, and P. nudicornis. Geographic distribution and host fire ant species seem to support cryptic lineages, though additional morphological data are needed to corroborate these results. All phylogenetic analyses reveal that South American fire ant decapitating flies are grouped into two main clades, with Pseudacteon convexicauda sister and well differentiated relative to these clades. Neither host nor geographic association appeared to be related to the differentiation of these two main clades within South American fire ant decapitating flies. This work provides information that will allow testing whether the putative cryptic phorid fly species show differences in their effectiveness as biocontrol agents against the highly invasive imported fire ants.

Southern expansion of the invasive ant Wasmannia auropunctata within its native range and its relation with clonality and human activity.

The little fire ant Wasmannia auropunctata, native to the Neotropics, has become a serious pest worldwide over the past 100 years. It was originally distributed from Mexico to northern Argentina and new evidence suggests a recent southern range expansion during the last 60 years reaching central Argentina. This supercolonial ant species has a polymorphic reproductive system. Some populations, mostly found in undisturbed natural environments, are characterised by a classical sexual haplodiploid reproductive system. In other populations, which mainly occur in human-modified habitats, diploid queens and haploid males are produced clonally while workers are produced sexually. Here we studied the association between the recent southern range expansion of W. auropunctata in relation to human activity and clonality. We carried out an extensive survey within the southern limit of the species' native distribution and characterised the type of habitat where populations were found. Moreover, we genetically determined the type of reproductive system in 35 populations by genotyping at 12 microsatellite loci a total of 191 reproductive individuals (i.e. queens and/or males). Clonality was the most common reproductive system, occurring in 31 out of 35 populations analysed. All the populations found in the recently colonised area in central Argentina were clonal and established in human-modified habitats, suggesting that clonality together with human activity might have facilitated the southwards expansion of W. auropunctata.

A tale of swinger insects: Signatures of past sexuality between divergent lineages of a parthenogenetic weevil revealed by ribosomal intraindividual variation.

Naupactus cervinus (Boheman) (Curculionidae, Naupactini) is a parthenogenetic weevil native to the Paranaense Forest which displays high levels of genetic variation. Two divergent clades were identified, one ranging in forest areas (Forest clade), and the other in open vegetation areas (Grassland clade). Both of them have individuals with high levels of heterozygosity in ribosomal sequences. Investigation of intraindividual variation in ITS1 sequences through cloning and posterior sequencing suggested that mating between both groups most likely occurred in the Paranaense Forest after a secondary contact, which led to fixed heterozygotes as a consequence of parthenogenesis. Otherwise, sexual segregation would have disrupted multilocus genotypes. Only a small number of heterozygous genotypes of all the possible combinations are found in nature. We propose the occurrence of a hybrid zone in the Paranaense Forest. The fact that it is one of the most important biodiversity hotspots of the world, together with its key role for investigating evolutionary processes, makes it worthy of conservation. This is the first genetic evidence of bisexuality in N. cervinus.

Sulfated Polysaccharides in the Freshwater Green Macroalga Cladophora surera Not Linked to Salinity Adaptation.

The presence of sulfated polysaccharides in cell walls of seaweeds is considered to be a consequence of the physiological adaptation to the high salinity of the marine environment. Recently, it was found that sulfated polysaccharides were present in certain freshwater Cladophora species and some vascular plants. Cladophora (Ulvophyceae, Chlorophyta) is one of the largest genera of green algae that are able to grow in both, seas and freshwater courses. Previous studies carried out on the water-soluble polysaccharides of the marine species C. falklandica established the presence of sulfated xylogalactoarabinans constituted by a backbone of 4-linked ß-L-arabinopyranose units partially sulfated mainly on C3 and also on C2 with partial glycosylation, mostly on C2, with terminal ß-D-xylopyranose or ß-D-galactofuranose units. Besides, minor amounts of 3-, 6- and/or 3,6-linked ß-D-galactan structures, with galactose in the pyranosic form were detected. In this work, the main water soluble cell wall polysaccharides from the freshwater alga Cladophora surera were characterized. It was found that this green alga biosynthesizes sulfated polysaccharides, with a structure similar to those found in marine species of this genus. Calibration of molecular clock with fossil data suggests that colonization of freshwater environments occurred during the Miocene by its ancestor. Therefore, the presence of sulfated polysaccharides in the freshwater green macroalga C. surera could be, in this case, an adaptation to transient desiccation and changes in ionic strength. Retention of sulfated polysaccharides at the cell walls may represent a snapshot of an evolutionary event, and, thus constitutes an excellent model for further studies on the mechanisms of sulfation on cell wall polysaccharides and environmental stress co-evolution.

Unraveling the diversification history of grasshoppers belonging to the "Trimerotropis pallidipennis" (Oedipodinae: Acrididae) species group: a hotspot of biodiversity in the Central Andes.

The Andean Mountain range has been recognized as one of the biodiversity hotspots of the world. The proposed mechanisms for such species diversification, among others, are due to the elevation processes occurring during the Miocene and the intensive glacial action during the Pleistocene. In this study we investigated the diversification history of the grasshopper Trimerotropis pallidipennis species complex which shows a particularly wide latitudinal and altitudinal distribution range across the northern, central and southern Andes in South America. Many genetic lineages of this complex have been so far discovered, making it an excellent model to investigate the role of the central Andes Mountains together with climatic fluctuations as drivers of speciation. Phylogenetics, biogeographic and molecular clock analyses using a multi-locus dataset revealed that in Peru there are at least two, and possibly four genetic lineages. Two different stocks originated from a common ancestor from North/Central America-would have dispersed toward southern latitudes favored by the closure of the Panama Isthmus giving rise to two lineages, the coastal and mountain lineages, which still coexist in Peru (i.e., T. pallidipennis and T. andeana). Subsequent vicariant and dispersal events continued the differentiation process, giving rise to three to six genetic lineages (i.e., clades) detected in this study, which were geographically restricted to locations dispersed over the central Andes Mountains in South America. Our results provide another interesting example of "island diversification" motored by the topography plus unstable climatic conditions during the Pleistocene, pointing out the presence of a hotspot of diversification in the Andean region of Peru.

Out of the forest: past and present range expansion of a parthenogenetic weevil pest, or how to colonize the world successfully.

Previous research revealed complex diversification patterns in the parthenogenetic weevil Naupactus cervinus. To understand the origin of clonal diversity and successful spreading of this weevil, we investigated its geographic origin and possible dispersal routes and whether parthenogens can persist in habitats under unsuitable environmental conditions. This study is based on samples taken throughout a broad area of the species' range. We used both mitochondrial and nuclear markers and applied phylogenetic and network analyses to infer possible relationships between haplotypes. Bayesian phylogeographic analyses and ecological niche modeling were used to investigate the processes that shaped genetic diversity and enabled the colonization of new geographic areas. Southeastern Brazil emerges as the original distribution area of N. cervinus. We detected two range expansions, one along natural corridors during the Pleistocene and the other in countries outside South America during recent times. Isolation due to climate shifts during the early Pleistocene led to diversification in two divergent clades, which probably survived in different refugia of the Paranaense Forest and the Paraná River delta. The origin of the clonal diversity was probably a complex process including mutational diversification, hybridization, and secondary colonization. The establishment of N. cervinus in areas outside its native range may indicate adaptation to drier and cooler conditions. Parthenogenesis would be advantageous for the colonization of new environments by preventing the breakup of successful gene combinations. As in other insect pests, the present distribution of N. cervinus results from both its evolutionary history and its recent history related to human activities.

Trichinella patagoniensis n. sp. (Nematoda), a new encapsulated species infecting carnivorous mammals in South America.

Until a few years ago, Trichinella spiralis was the only taxon of the genus Trichinella detected in both domestic and wild animals of South America. Recently, a new genotype, named Trichinella T12, was identified in cougars (Puma concolor) from Argentina, on the basis of molecular studies using mitochondrial and nuclear ribosomal markers. In the present study, cross-breeding experiments indicated that Trichinella T12 is reproductively isolated from all other encapsulated Trichinella spp. and suggested that it is biologically more similar to Trichinella britovi and Trichinella murrelli than to the other encapsulated species/genotypes. Biological assays revealed that the reproductive capacity index of Trichinella T12 was ~4 and >2000 times lower than those of T. spiralis in mice and rats, respectively. The reproductive capacity index of Trichinella T12 in domestic pigs ranged from 0.0 to 0.05. Larvae parasitising the muscles of carnivores were infective to mice after freezing at -5°C for 3 months, but they lost infectivity after freezing at -18°C for 1 week. The region within the rDNA, known as the expansion segment V, showed a unique sequence which differs from those of all other known Trichinella spp./genotypes. The biological, geographical and molecular data support the classification of the genotype Trichinella T12 as a new species widespread in the Neotropical region, for which we propose the name Trichinella patagoniensis n. sp.

Mito-nuclear genetic comparison in a Wolbachia infected weevil: insights on reproductive mode, infection age and evolutionary forces shaping genetic variation.

BACKGROUND: Maternally inherited endosymbionts like Wolbachia pipientis are in linkage disequilibrium with the mtDNA of their hosts. Therefore, they can induce selective sweeps, decreasing genetic diversity over many generations. This sex ratio distorter, that is involved in the origin of parthenogenesis and other reproductive alterations, infects the parthenogenetic weevil Naupactus cervinus, a serious pest of ornamental and fruit plants. RESULTS: Molecular evolution analyses of mitochondrial (COI) and nuclear (ITS1) sequences from 309 individuals of Naupactus cervinus sampled over a broad range of its geographical distribution were carried out. Our results demonstrate lack of recombination in the nuclear fragment, non-random association between nuclear and mitochondrial genomes and the consequent coevolution of both genomes, being an indirect evidence of apomixis. This weevil is infected by a single Wolbachia strain, which could have caused a moderate bottleneck in the invaded population which survived the initial infection. CONCLUSIONS: Clonal reproduction and Wolbachia infection induce the coevolution of bacterial, mitochondrial and nuclear genomes. The time elapsed since the Wolbachia invasion would have erased the traces of the demographic crash in the mtDNA, being the nuclear genome the only one that retained the signal of the bottleneck. The amount of genetic change accumulated in the mtDNA and the high prevalence of Wolbachia in all populations of N. cervinus agree with the hypothesis of an ancient infection. Wolbachia probably had great influence in shaping the genetic diversity of N. cervinus. However, it would have not caused the extinction of males, since sexual and asexual infected lineages coexisted until recent times.

Phylogeography of the armadillo Chaetophractus villosus (Dasypodidae Xenarthra): post-glacial range expansion from Pampas to Patagonia (Argentina).

We report a phylogeographic study of Chaetophractus villosus populations in Argentina. Control Region (CR) sequences (484 bp) were obtained for 76 C. villosus from 20 locations across the species whole distribution range. Seventeen new haplotypes were identified. The highest genetic variation and the earliest fossils were found in the Pampean Region, thus appearing as the most probable area of origin of the species. A general pattern of Contiguous Range Expansion (CRE) was revealed by Nested Clade Analysis (NCA) supported by mismatch analysis and Fu's test. The Pampean Region would have been the pre-expansion area, while Patagonia would have been the main dispersal route of contiguous expansion, possibly after the Pleistocenic glaciations.

Microsatellite variation in maize landraces from Northwestern Argentina: genetic diversity, population structure and racial affiliations.

The highland region or Northwestern Argentina (NWA) is one of the southernmost areas of native maize cultivation and constitutes an expansion of the peruvian Andes sphere of influence. To examine the genetic diversity and racial affiliations of the landraces cultivated in this area, 18 microsatellite markers were used to characterize 147 individuals from 6 maize races representative of traditional materials. For the whole data set, a total of 184 alleles were found, with an average of 10.2 alleles per locus. The average gene diversity was 0.571. The observed patterns of genetic differentiation suggest that historical association is probably the main factor in shaping population structure for the landraces studied here. In agreement with morphological and cytogenetic data, Bayesian analysis of NWA landraces revealed the occurrence of three main gene pools. Assessment of racial affiliations using a combined dataset including previous data on American landraces showed a clear relationship between one of these gene pools and typical Andean races, whereas the remaining two gene pools exhibited a closer association to Caribbean accessions and native germplasm from the United States, respectively. These results highlight the importance of integrating regional genetic studies if a deeper understanding of maize diversification and dispersal is to be achieved.

Molecular evidence for a novel encapsulated genotype of Trichinella from Patagonia, Argentina.

At present, Trichinella spiralis is the only species of this genus reported from South America. Herein, we detail a molecular analysis of a new encapsulated isolate of muscle larvae of Trichinella, found in a mountain lion (Puma concolor) coming from the Patagonia, Argentina. We studied three DNA regions previously probed to be useful for the identification of all eleven recognized Trichinella genotypes: expansion segment 5 (ES5), cytochrome c-oxidase subunit I (COI) and 5S ribosomal DNA intergenic spacer region (5S ISR). BLAST searches with these DNA sequences showed that the mitochondrial and nuclear ribosomal regions most closely resemble other Trichinella sequences available in GenBank. However, they did not exactly match any of the eleven recognized genotypes. The phylogenetic analysis from COI and 5S ISR sequences showed that the mountain lion isolate is grouped with encapsulated members, in concordance with morphological data. Furthermore, this new isolate was located at the base of the encapsulated genotypes, signifying that it is an old genotype that could have emerged earliest in this group. These data strongly suggest that this isolate from the Patagonia represents the twelfth genotype (T12) described in the genus Trichinella. Nevertheless, further studies are necessary to adequately establish this isolate as a unique genotype.

Phylogeographic studies on natural populations of the South American fruit fly, Anastrepha fraterculus (Diptera: Tephritidae).

Anastrepha fraterculus is an important pest of commercial fruits in South America. The variability observed for morphological and behavioural traits as well as genetic markers suggests that A. fraterculus represents a complex of synmorphic species rather than a single biological species. We studied the correlation between geographical distribution and genetic variation in natural populations from Argentina and south Brazil. Fragments of the mitochondrial gene COII were sequenced in 28 individuals. The matrix of aligned sequences was phylogenetically analysed by parsimony, yielding a cladogram of haplotypes. Based on Templeton's nested method, no clade showed any geographic pattern for the gene COII, indicating lack of significant association between haplotypic variability and geographic distribution. The analysis of nucleotide substitution distances by Neighbour-Joining algorithm showed that geographically distant populations exhibit low genetic distances. The corresponding trees clustered the populations without showing any geographic pattern. This result suggests that the populations studied are not reproductively isolated.

B chromosome polymorphism in maize landraces: adaptive vs. demographic hypothesis of clinal variation.

Cytogenetic analysis of maize landraces from northwestern Argentina has revealed an altitudinal cline in the mean number of B chromosomes (B's) per plant, with cultivars growing at higher altitudes exhibiting a higher number of B's. Altitudinal and longitudinal clines are frequently interpreted as evidence of selection, however, they can also be produced by the interplay between drift and spatially restricted gene flow or by admixture between previously isolated populations that have come into secondary contact. Here, we test the adaptive significance of the observed altitudinal gradient by comparing the levels of differentiation in the mean number of B's to those obtained from 18 selectively neutral loci [simple sequence repeats (SSRs)] among seven populations of the cline. The adequacy of alternative genetic-differentiation measures was determined, and associations between cytogenetic, genetic, and altitudinal distances were assessed by means of matrix- correspondence tests. No evidence for association between pairwise F(ST) and altitudinal distance or B-chromosome differentiation was found. The contrasting pattern of altitudinal divergence between the mean number of B's per plant and the genetic differentiation at SSR loci indicates that demographic processes cannot account for the observed levels of divergence in the mean number of B's.

Microsatellite typing of ancient maize: insights into the history of agriculture in southern South America.

Archaeological maize specimens from Andean sites of southern South America, dating from 400 to 1400 years before present, were tested for the presence of ancient DNA and three microsatellite loci were typed in the specimens that gave positive results. Genotypes were also obtained for 146 individuals corresponding to modern landraces currently cultivated in the same areas and for 21 plants from Argentinian lowland races. Sequence analysis of cloned ancient DNA products revealed a high incidence of substitutions appearing in only one clone, with transitions prevalent. In the archaeological specimens, there was no evidence of polymorphism at any one of the three microsatellite loci: each exhibited a single allelic variant, identical to the most frequent allele found in contemporary populations belonging to races Amarillo Chico, Amarillo Grande, Blanco and Altiplano. Affiliation between ancient specimens and a set of races from the Andean complex was further supported by assignment tests. The striking genetic uniformity displayed by the ancient specimens and their close relationship with the Andean complex suggest that the latter gene pool has predominated in the western regions of southern South America for at least the past 1400 years. The results support hypotheses suggesting that maize cultivation initially spread into South America via a highland route, rather than through the lowlands.