Potentially toxic elements in soil-plant-water-animal continuum in a mining area from Northwestern Mexico: animal exposure pathways and health risks for children.

Mining increases environmental concentrations of potentially toxic elements (PTEs) accumulating in organisms and spreading in the human food chain-their presence in milk is of great human health concern. Pathways were identified by which these elements reach raw milk from farms within a mining area in Northwestern Mexico; health risks for dairy cattle and children were also evaluated. Water from river and cattle waterers, as well as, soils showed that PTE concentrations generally below the Mexican and international limits; cattle forage concentrations were above the World Health Organization limits. Al, Mg, Mo, Ni and Zn were recorded in raw milk samples from the mining area, showing that Cd, Co, Cr, Cu, Pb and V are transferred from soil to plants but not accumulated in raw milk. Zn concentrations in raw milk exceeded the permissible limit; milk from farms without mining operations (comparison site) showed the presence of Al, Cr and Cu. In cattle tail hair, PTE did not correlate with raw milk concentrations. Metal accumulation in milk was higher through water consumption than that accumulated through forage consumption. Daily intakes (DI) of Al, Mg and Zn in cows could represent a risk for their health. The observed biotransference was higher than in other parts of Mexico, and the calculated DI and hazard quotients indicate no adverse health effects for children. However, the hazard Index values indicate that exposure to multiple PTE represents a risk for children. Management measures should be performed to control the cumulative risks to protect young children's health.

Yeast diversity in open agave fermentations across Mexico.

Yeasts are a diverse group of fungal microorganisms that are widely used to produce fermented foods and beverages. In Mexico, open fermentations are used to obtain spirits from agave plants. Despite the prevalence of this traditional practice throughout the country, yeasts have only been isolated and studied from a limited number of distilleries. To systematically describe the diversity of yeast species from open agave fermentations, here we generate the YMX-1.0 culture collection by isolating 4524 strains from 68 sites with diverse climatic, geographical, and biological contexts. We used MALDI-TOF mass spectrometry for taxonomic classification and validated a subset of the strains by ITS and D1/D2 sequencing, which also revealed two potential novel species of Saccharomycetales. Overall, the composition of yeast communities was weakly associated with local variables and types of climate, yet a core set of six species was consistently isolated from most producing regions. To explore the intraspecific variation of the yeasts from agave fermentations, we sequenced the genomes of four isolates of the nonconventional yeast Kazachstania humilis. The genomes of these four strains were substantially distinct from a European isolate of the same species, suggesting that they may belong to different populations. Our work contributes to the understanding and conservation of an open fermentation system of great cultural and economic importance, providing a valuable resource to study the biology and genetic diversity of microorganisms living at the interface of natural and human-associated environments.

Geographic isolation and long-distance gene flow influence the genetic structure of the blue fan palm Brahea armata (Arecaceae).

The formation of the Baja California Peninsula (BCP) has impacted the microevolutionary dynamics of different species in ways that depend on biological traits such as dispersal capacity. Plants with relatively low levels of vagility have exhibited high genetic divergence between the BCP and Continental mainland. Brahea armata (Arecaceae) is a palm species inhabiting the northern part of the BCP and Sonora; its distribution occurs in isolated oases of vegetation. We aimed to evaluate the influence of the formation of the BCP on the genetic structure of B. armata using nuclear microsatellites and chloroplast markers (cpDNA) to compare patterns of genetic diversity and structure with previous published studies. Because gene flow through seeds is usually more limited compared to pollen flow, we expect to find stronger genetic structure at (cpDNA) than at nuclear markers. Moreover, larger genetic structure might also be explained by the smaller effective population size of cpDNA. We analyzed six microsatellite markers and two cpDNA regions. The main results indicated high levels of genetic differentiation among isolated populations located in the BCP, while low genetic differentiation was found between southern populations of the BCP and Sonora, suggesting long distance gene flow. In contrast, chloroplast markers indicated high levels of genetic structure between BCP and Sonora populations, suggesting asymmetrical gene flow between pollen (measured by nuclear microsatellites) and seed (cpDNA markers). This study provides valuable information on genetic diversity of B. armata that can be relevant for conservation and management; and develops microsatellites markers that can be transferred to other Brahea species.

Genomic Analyses of Wild and Cultivated Bacanora Agave (Agave angustifolia var. pacifica) Reveal Inbreeding, Few Signs of Cultivation History and Shallow Population Structure.

Due to the recent increase in demand for agave-based beverages, many wild agave populations have experienced rapid decline and fragmentation, whereas cultivated plants are now managed at monocultural plantations, in some cases involving clonal propagation. We examined the relative effect of migration, genetic drift, natural selection and human activities on the genetic repertoire of Agave angustifolia var. pacifica, an agave used for bacanora (an alcoholic spirit similar to tequila) production in northwestern Mexico. We sampled 34 wild and cultivated sites and used over eleven thousand genome-wide SNPs. We found shallow genetic structure among wild samples, although we detected differentiation between coastal and inland sites. Surprisingly, no differentiation was found between cultivated and wild populations. Moreover, we detected moderate inbreeding (FIS ~ 0.13) and similar levels of genomic diversity in wild and cultivated agaves. Nevertheless, the cultivated plants had almost no private alleles and presented evidence of clonality. The overall low genetic structure in A. angustifolia var. pacifica is apparently the result of high dispersibility promoted by pollinators and the possibility of clonal reproduction. Incipient cultivation history and reliance on wild seeds and plants are probably responsible for the observed patterns of high genetic connectivity and considerable diversity in cultivated samples.

Microstructure and Hydrophobicity of the External Surface of a Sonoran Desert Beetle.

We have studied the external surface (elytra) of the Sonoran Desert beetle (Eleodes eschscholtzii). Our aim was to assess whether this species has similar traits to some beetles from the Namibian Desert that are known to have hierarchical micropatterns that allow for water harvesting. We have conducted scanning electron microscopy (SEM) and apparent contact angle experiments on specimens collected at two sampling sites with different ambient humidity. The results show that the beetle's external surface microstructure is composed of a compact array of polygons with randomly scattered protuberances. The density of the polygons in the microstructure is different for individuals collected in different sites: the polygon array is denser in the more humid site and less dense in the drier site. The measured contact angles also depend on the sampling site. For individuals collected in the drier site, the average apparent contact angle is 70°, whereas for the more humid site, the average apparent contact angle is 92°. FT-IR experiments are consistent with the presence of hydrophobic wax compounds in the studied surfaces. Our investigation opens new questions that are currently being addressed by experiments that are underway. For instance, it would be interesting to know whether the observed nanopatterns could be used in biomimetic devices for water harvesting purposes.

A Change in Conservation Status of Pachyphytum caesium (Crassulaceae), a Threatened Species from Central Mexico Based on Genetic Studies.

Genetic studies in rare species are important to determine their patterns of genetic diversity among populations and implement conservation plans aimed to reduce extinction risks. Pachyphytum caesium is an endemic species with extremely small populations in central Mexico. This work analyzes the diversity and genetic structure of Pachyphytum cesium (Crassulaceae) to change the conservation status and propose conservation strategies. Twelve dominant ISSR markers were used to describe the genetic diversity of all known populations. Additionally, we carried out two simulations to explore how the loss of individuals or the local populations extinction affect the genetics parameters of the species. The average results indicated moderate levels of genetic diversity (number of alleles = 89.7 ± 3.9, expected heterozygosity = 0.212 ± 0.0, and percentage of polymorphic loci = 56.1 ± 3.9), parameters that decreased significantly when simulations were performed in P. caesium. Additionally, a genetic structure of the populations was found with low gene flow (Nm = 0.92). Genetic parameters are negatively affected by the loss of individuals and the local extinction of populations. Based on our results, we propose to reclassify the conservation status of the species in danger of extinction, hence a series of conservation strategies are provided to prevent its disappearance.

Speciation along a latitudinal gradient: The origin of the Neotropical cycad sister pair Dioon sonorense-D. vovidesii (Zamiaceae).

Latitude is correlated with environmental components that determine the distribution of biodiversity. In combination with geographic factors, latitude-associated environmental variables are expected to influence speciation, but empirical evidence on how those factors interplay is scarce. We evaluated the genetic and environmental variation among populations in the pair of sister species Dioon sonorense-D. vovidesii, two cycads distributed along a latitudinal environmental gradient in northwestern Mexico, to reveal their demographic histories and the environmental factors involved in their divergence. Using genome-wide loci data, we determined the species delimitation, estimated the gene flow, and compared multiple demographic scenarios of divergence. Also, we estimated the variation of climatic variables among populations and used ecological niche models to test niche overlap between species. The effect of geographic and environmental variables on the genetic variation among populations was evaluated using linear models. Our results suggest the existence of a widespread ancestral population that split into the two species ~829 ky ago. The geographic delimitation along the environmental gradient occurs in the absence of major geographic barriers, near the 28th parallel north, where a zonation of environmental seasonality exists. The northern species, D. vovidesii, occurs in more seasonal environments but retains the same niche of the southern species, D. sonorense. The genetic variation throughout populations cannot be solely explained by stochastic processes; the latitudinal-associated seasonality has been an additive factor that strengthened the species divergence. This study represents an example of how speciation can be achieved by the effect of the latitude-associated factors on the genetic divergence among populations.

Water resources affected by potentially toxic elements in an area under current and historical mining in northwestern Mexico.

Rio Sonora watershed and its aquifer-located in northwest Mexico-have been influenced by mining operations for 140 years, possibly causing emissions of potentially toxic elements (PTE) and affecting health of exposed populations. On the basis of available data from governmental surveys (2014-2017) and recent sampling (2018), this study constructed reliable PTE total concentration database that allowed us to report temporal/spatial variations in surface and groundwater and their associated health risks to the population living in the central part of the Rio Sonora basin. The data clearly showed that a mining spill that took place in 2014 has had an adverse impact on total PTE concentrations in surface water. They also indicated the presence of different PTE point source locations that have continued to cause contamination of surface water at levels of health concern. Data also suggested slight impacts of the spill event on groundwater possibly related to soil neutralizing potential. Two metal groups were detected for surface waters (Pb-Cd-As-Ni-Cr and of Zn-Al-Cr) and groundwaters (Cr-As-Cu-Cd and Zn-Al), which suggest that they have different sources or are being released by different processes. The potential health impacts of PTE concentrations were associated with specific age groups, dates, and areas. The results indicate that in this complex semi-arid rural system, current and historical mining activities, as well as contrasting hydrological conditions, have impacted surface and groundwater quality with important ecological and human health risks.

Metagenomics of mine tailing rhizospheric communities and its selection for plant establishment towards bioremediation.

Mining operations often generate tailing dams that contain toxic residues and are a source of contamination when left unconfined. The establishment of a plant community over the tailings has been proposed as a containment strategy known as phytostabilization. Previously, we described naturally occurring mine tailing colonizing plants such as Acacia farnesiana, Brickellia coulteri, Baccharis sarothroides, and Gnaphalium leucocephalum without finding local adaptation. We explored the rhizosphere microbes as contributors in plant establishment and described both the culturable and in situ diversity of rhizospheric bacteria using the 16S rRNA gene and metagenomic shotgun sequencing. We built a synthetic community (SC) of culturable rhizosphere bacteria from the mine tailings. The SC was then the foundation for a serial passes experiment grown in plant-derived nutrient sources, selecting for heavy metals tolerance, community cooperation, and competition. The outcome of the serial passes was named the 'final synthetic community' (FSC). Overall, diversity decreased from in situ uncultivable microbes from roots (399 bacteria genera) to the cultivated communities (291 genera), the SC (94 genera), and the lowest diversity was in the FSC (43 genera). Metagenomic diversity clustered into 94,245 protein families, where we found plant growth promotion-related genes such as the csgBAC and entCEBAH, coded in a metagenome-assembled genome named Kosakonia sp. Nacozari. Finally, we used the FSC to inoculate mine tailing colonizing plants in a greenhouse experiment. The plants with the FSC inocula observed higher relative plant growth rates in sterile substrates. The FSC presents promising features that might make it useful for phytostabilization tailored strategies.

Barriers for plant establishment in the abandoned tailings of Nacozari, Sonora, Mexico: the influence of compost addition on seedling performance and tailing properties.

Past mining activities have left a legacy of abandoned mine tailing deposits whose metal contaminants poses serious risks to ecosystems and human health. While the development of a vegetated cover in mine tailings can help in mitigating these risks, the local factors limiting plant establishment in these sites are not well understood, restricting phytostabilization efforts. Here, we explore some of the barriers that limit seedling establishment of two species (Vachellia farnesiana and Prosopis velutina) in a mine tailing deposit located in Nacozari, Sonora, Mexico, and assess whether compost addition can help in overcoming these barriers in pot and field experiments. Our field observations found 20 times more carbon and at least 4 times more nitrogen concentration in areas under vegetated patches than in non-vegetated areas, while a previous study found no difference in metal concentrations and other physicochemical parameters. This suggests that organic matter and nutrients are a major limitation for plant establishment. In agreement with this, species failed to establish without compost addition in the field experiment. Compost addition also had a positive effect on biomass accumulation, pH and microbial activity, but increased the substrate soluble concentration of As, Cu, and Zn. Nonetheless, only Cu, K, and Mo in P. velutina accumulated in tissues at levels considered toxic for animal consumption. Our study documents that compost addition facilitated plant establishment for the phytostabilization of mine tailings and help to prevent the dispersion of most metal contaminants via animal consumption. We encourage the use of complementary strategies to minimize the risk of dispersion of metal contaminants.

Stabilization of potentially toxic elements contained in mine waste: A microbiological approach for the environmental management of mine tailings.

Metals are key materials extensively employed in several industries to produce technological and daily-life products. The mining industry that produces such commodities generates Tons of waste that if not remediated can be transferred to the surrounding environment, thus representing a water, air, and soil pollution threat. In this work, we evaluated the feasibility of microbial sulfate reduction (SR) as a management strategy for this waste. Mine tailings were sampled from two abandoned mining sites located in Sonora (northwestern Mexico) and treated in anaerobic microcosms under SR conditions using anaerobic sludge as the inoculum at two different tailing:inoculum ratios (TIR). Major TIR's were found to be the triggering factor for the highest SR activities observed (73.6 ± 8.8 mg SO42- L-1 day-1). This stimulation was linked to the dissolution of sulfate bearing minerals (anglesite, jarosite, and gypsum) which provided additional sulfate for microbial activity. However, under this condition, longer lag phases for SR were observed, which was potentially due to pH inhibition at early incubation stages (pH ~3.7). Despite this, all biologically SR performing treatments presented important sulfide precipitation which was associated to changes in the mineralogy of the mine tailings. Metals of environmental concern such as As, Cd, Co, Cr and, Pb were detected to have shifted from the aqueous extractable phase to the bound to Fe and Mn oxides and residual phases. This finding was in accordance with the non-detectable concentrations of these metals in the aqueous phase by the end of the biological treatment which proved the effectiveness of this approach. This study provides insights into the promising potential of anaerobic microbes for the environmental management of mine tailings.

Mobility and accessibility of Zn, Pb, and As in abandoned mine tailings of northwestern Mexico.

Generation, storage, and management of waste coming from industrial processes are a growing worldwide problem. One of the main contributors is the mining industry, in particular tailings generated by historical mining, which are barely maintained, especially in developing countries. Assessing the impact of a mining site to surrounding soils and ecosystems can be complex, especially when determining mobility and accessibility of the contaminants is required to perform ecological and human health risk assessment. As an effort to obtain information regarding mobility and accessibility of some potentially toxic elements (Zn, Pb, and As) from an historical mining site of northwestern Mexico, the abandoned mine tailings of San Felipe de Jesús in central Sonora and adjacent agricultural soils were investigated. Mobility and accessibility were assessed by means of sequential extraction procedures and using simulated physiological media. Additionally, an assessment of accidental oral intake was calculated considering the bioaccessible fractions. Results show that higher concentrations of contaminants were found in sulfide-rich tailings (Zn = 92,540; Pb = 21,288; As = 19,740 mg kg-1) compared with oxide-rich tailings (Zn = 43,240; Pb = 14,763; As = 13,401 mg kg-1). Concentrations in agricultural soils were on average Zn = 4755, Pb = 2840, and As = 103 mg kg-1. Zinc was mainly recovered from labile fractions in oxide-rich tailings (~ 60%) and in a lower amount from sulfide-rich tailings (~ 30%). Pb and As were mainly associated with residual fractions (80-95%) in both types of tailings. The percentage of mobile fractions (sum of water-soluble, exchangeable, and bound to carbonate fractions) in agricultural soils was as follows: Zn ~ 60%, Pb ~ 15%, and As ~ 70%. Regarding the phytoaccessible fraction, the studied elements in mine tailings and agricultural soil samples exceeded the threshold limits, except for As in agricultural soils. According to data obtained, toxic effects were also calculated. As for daily oral intake for non-carcinogenic effects in adults and children, only Pb and As exceeded reference dose values, especially in children exposed to sulfide-rich tailings and agricultural soils. Regarding carcinogenic effects of Pb and As, most of the samples were above acceptable risk values.

Niche conservatism promotes speciation in cycads: the case of Dioon merolae (Zamiaceae) in Mexico.

Niche conservatism is the tendency of lineages to retain the same niche as their ancestors. It constrains biological groups and prevents ecological divergence. However, theory predicts that niche conservatism can hinder gene flow, strengthen drift and increase local adaptation: does it mean that it also can facilitate speciation? Why does this happen? We aim to answer these questions. We examined the variation of chloroplast DNA, genome-wide single nucleotide polymorphisms, morphological traits and environmental variables across the Dioon merolae cycad populations. We tested geographical structure, scenarios of demographic history, and niche conservatism between population groups. Lineage divergence is associated with the presence of a geographical barrier consisting of unsuitable habitats for cycads. There is a clear genetic and morphological distinction between the geographical groups, suggesting allopatric divergence. However, even in contrasting available environmental conditions, groups retain their ancestral niche, supporting niche conservatism. Niche conservatism is a process that can promote speciation. In D. merolae, lineage divergence occurred because unsuitable habitats represented a barrier against gene flow, incurring populations to experience isolated demographic histories and disparate environmental conditions. This study explains why cycads, despite their ancient lineage origin and biological stasis, have been able to diversify into modern ecosystems worldwide.

Environmental assessment and historic erosion calculation of abandoned mine tailings from a semi-arid zone of northwestern Mexico: insights from geochemistry and unmanned aerial vehicles.

Mining is known as one of the primary economic activities where exploitation of minerals and other materials have become essential for human development. However, this activity may represent a risk to the environment, starting from deforestation and ending with production of residues that might contain potentially toxic elements. Tailing deposits from historical mining are an example of waste that may represent an environmental concern when abandoned and exposed to environmental conditions. The town of Nacozari de Garcia, in northwestern Mexico, has three abandoned mine tailings (locally known as tailings I, II, and III) located around the urban area that represent important sources of dust and pollution. Images obtained using unmanned aerial vehicles (UAV) in conjunction with geochemical data are used to assess historic erosion calculation and pollution considering contamination and hazard indexes in tailings II and III. Digital elevation models of abandoned tailings were obtained using photogrammetry with UAV. A total of 37 surficial samples were collected from mine tailings to determine elemental concentrations (As, Cu, Pb, W, Zn) using portable X-ray fluorescence. Higher concentrations were found on samples from mine tailing II. Average concentrations followed the decreasing order of Cu > Zn > W > Pb > As for tailing II, whereas decreasing order of Cu > Zn > W > As > Pb was found for tailing III. Contamination Index (CI) values obtained from tailings II and III represent a low potential of pollution, whereas efflorescent crusts from these tailings represent a high potential of polluting soils and sediments by dust generation. Hazard Average Quotient (HAQ) values on both tailings suggest a very high potential of contamination if fluids infiltrate tailings and interact with surficial water and/or groundwater. Obtained surfaces of mine tailings II and III are 146,216 and 216,689 m2, respectively, which represent around 11% of the urbanized area. A loss mass of 321,675 tons was determined for mine tailing II, whereas 634,062 tons for tailing III, accounting for 0.96 million tons of total eroded mass. Since abandonment, calculated erosion rates of 493 t ha-1 year-1 (tailing II) and 232 t ha-1 year-1 (tailing III) are in agreement with those determined in other mining areas. CI and HAQ indexes provide good estimates of pollution associated with abandoned mine tailings from Nacozari de García. Historic erosion determined in these tailings is an environmental concern since eroded material and polluted water have been incorporated into the Moctezuma River, which feeds several villages, whose major activities include agriculture and livestock raising.

Tracking Host Trees: The Phylogeography of Endemic Psittacanthus sonorae (Loranthaceae) Mistletoe in the Sonoran Desert.

The host dependence of mistletoes suggests that they track the distributions of their hosts. However, the factors that determine the geographic distribution of mistletoes are not well understood. In this study, the phylogeography of Psittacanthus sonorae was reconstructed by sequencing one nuclear (ITS) and two plastid (trnL-F and atpB-rbcL) regions of 148 plants from populations separated by the Sea of Cortez. Divergence time and gene flow were estimated to gain insight into the historical demography and geographic structuring of genetic variation. We also described and mapped the spatial distribution of suitable habitat occupied by P. sonorae and its most common host Bursera microphylla in the Sonoran Desert, along with their responses to Quaternary climate fluctuations using environmental data and ecological niche modeling (ENM). We detected environmental and genetic differentiation between the peninsular and continental P. sonorae populations. Population divergence occurred during the Pleistocene, around the time of the Last Glacial Maximum. No signals of population growth were detected, with net gene flow moving from the continent to the peninsula. ENM models indicate decoupled responses by the mistletoe and its main host to past climate changes. For the Last Interglacial to the present, most models produce only partial areas of overlap on both the peninsula and the continent. Our results support a scenario of Late-Pleistocene isolation and divergence with asymmetrical gene flow between peninsular and continental P. sonorae populations. Continental populations migrated to the peninsula and the spatial isolation probably produced genetic differentiation under different environmental conditions.

Different patterns of colonization of Oxalis alpina in the Sky Islands of the Sonoran desert via pollen and seed flow.

Historical factors such as climatic oscillations during the Pleistocene epoch have dramatically impacted species distributions. Studies of the patterns of genetic structure in angiosperm species using molecular markers with different modes of inheritance contribute to a better understanding of potential differences in colonization and patterns of gene flow via pollen and seeds. These markers may also provide insights into the evolution of reproductive systems in plants. Oxalis alpina is a tetraploid, herbaceous species inhabiting the Sky Island region of the southwestern United States and northern Mexico. Our main objective in this study was to analyze the influence of climatic oscillations on the genetic structure of O. alpina and the impact of these oscillations on the evolutionary transition from tristylous to distylous reproductive systems. We used microsatellite markers and compared our results to a previous study using chloroplast genetic markers. The phylogeographic structure inferred by both markers was different, suggesting that intrinsic characteristics including the pollination system and seed dispersal have influenced patterns of gene flow. Microsatellites exhibited low genetic structure, showed no significant association between geographic and genetic distances, and all individual genotypes were assigned to two main groups. In contrast, chloroplast markers exhibited a strong association between geographic and genetic distance, had higher levels of genetic differentiation, and were assigned to five groups. Both types of DNA markers showed evidence of a northward expansion as a consequence of climate warming occurring in the last 10,000 years. The data from both types of markers support the hypothesis for several independent transitions from tristyly to distyly.

The role of hybridization during ecological divergence of southwestern white pine (Pinus strobiformis) and limber pine (P. flexilis).

Interactions between extrinsic factors, such as disruptive selection and intrinsic factors, such as genetic incompatibilities among loci, often contribute to the maintenance of species boundaries. The relative roles of these factors in the establishment of reproductive isolation can be examined using species pairs characterized by gene flow throughout their divergence history. We investigated the process of speciation and the maintenance of species boundaries between Pinus strobiformis and Pinus flexilis. Utilizing ecological niche modelling, demographic modelling and genomic cline analyses, we illustrated a divergence history with continuous gene flow. Our results supported an abundance of advanced generation hybrids and a lack of loci exhibiting steep transition in allele frequency across the hybrid zone. Additionally, we found evidence for climate-associated variation in the hybrid index and niche divergence between parental species and the hybrid zone. These results are consistent with extrinsic factors, such as climate, being an important isolating mechanism. A build-up of intrinsic incompatibilities and of coadapted gene complexes is also apparent, although these appear to be in the earliest stages of development. This supports previous work in coniferous species demonstrating the importance of extrinsic factors in facilitating speciation. Overall, our findings lend support to the hypothesis that varying strength and direction of selection pressures across the long lifespans of conifers, in combination with their other life history traits, delays the evolution of strong intrinsic incompatibilities.

The phylogeography of the cycad genus Dioon (Zamiaceae) clarifies its Cenozoic expansion and diversification in the Mexican transition zone.

Background and Aims: Biogeographic transition zones are promising areas to study processes of biogeographic evolution and its influence on biological groups. The Mexican transition zone originated due to the overlap of Nearctic and Neotropical biota, which promoted great biological diversification. However, since most previous studies in this area were focused on revealing the phylogeography of Nearctic plants, how historical biogeographic configuration influenced the expansion and diversification of the Neotropical flora remains almost unknown. Using the cycad genus Dioon (Zamiaceae), this study aimed to test whether the biogeographic provinciality of the Mexican transition zone reflects the history of diversification of Neotropical plants. Methods: Two chloroplast DNA (cpDNA) regions were analysed from 101 specimens of 15 Dioon species to reveal the distribution of haplogroups. In addition, genome-wide single nucleotide polymorphisms (SNPs) from 84 specimens were used to test the concordance between phylogenetic clusters and the biogeographic provinces. An ultrametric tree was constructed from the sequences containing SNPs to reconstruct the biogeographic events of vicariance and dispersal of Dioon across the Neotropical biogeographic provinces. Key Results: Four Dioon lineages with strong phylogeographic structures were recognized using both cpDNA and SNP data. The lineages correspond to two clades that originated from a common ancestor in Eastern Mexico. One clade expanded and diversified in South-east Mexico and Central America. Another clade diversified into three lineages that dispersed to North-east, South and North-west Mexico. Each lineage was biogeographically delimitated. Biogeographic provinces might have provided disparate ecological conditions that facilitated speciation in Dioon since the Miocene. Conclusions: The current genetic structure and species diversity of Dioon depict the history of expansion and diversification of the northernmost Neotropical provinces. Past biogeographic connectivities were favoured by elevated topographies, since mountain systems served as corridors for the migration of Dioon and as refugia of tropical communities that diversified during the formation of modern Neotropical forests.

Aridification as a driver of biodiversity: a case study for the cycad genus Dioon (Zamiaceae).

Background and Aims: Aridification is considered a selective pressure that might have influenced plant diversification. It is suggested that plants adapted to aridity diversified during the Miocene, an epoch of global aridification (≈15 million years ago). However, evidence supporting diversification being a direct response to aridity is scarce, and multidisciplinary evidence, besides just phylogenetic estimations, is necessary to support the idea that aridification has driven diversification. The cycad genus Dioon (Zamiaceae), a tropical group including species occurring from humid forests to arid zones, was investigated as a promising study system to understand the associations among habitat shifts, diversification times, the evolution of leaf epidermal adaptations, and aridification of Mexico. Methods: A phylogenetic tree was constructed from seven chloroplast DNA sequences and the ITS2 spacer to reveal the relationships among 14 Dioon species from habitats ranging from humid forests to deserts. Divergence times were estimated and the habitat shifts throughout Dioon phylogeny were detected. The epidermal anatomy among Dioon species was compared and correlation tests were performed to associate the epidermal variations with habitat parameters. Key Results: Events of habitat shifts towards arid zones happened exclusively in one of the two main clades of Dioon. Such habitat shifts happened during the species diversification of Dioon, mainly during the Miocene. Comparative anatomy showed epidermal differences between species from arid and mesic habitats. The variation of epidermal structures was found to be correlated with habitat parameters. Also, most of the analysed epidermal traits showed significant phylogenetic signals. Conclusions: The diversification of Dioon has been driven by the aridification of Mexico. The Miocene timing corresponds to the expansion of arid zones that embedded the ancestral Dioon populations. As response, species in arid zones evolved epidermal traits to counteract aridity stress. This case study provides a robust body of evidence supporting the idea that aridification is an important driver of biodiversity.