The phylogenetic reconstruction of the Neotropical cycad genus Ceratozamia (Zamiaceae) reveals disparate patterns of niche evolution.

The cycad genus Ceratozamia comprises 40 species from Mexico, Guatemala, Belize, and Honduras, where cycads occur throughout climatically varied montane habitats. Ceratozamia has the potential to reveal the history and processes of species diversification across diverse Neotropical habitats in this region. However, the species relationships within Ceratozamia and the ecological trends during its evolution remain unclear. Here, we aimed to clarify the phylogenetic relationships, the timing of clade and species divergences, and the niche evolution throughout the phylogenetic history of Ceratozamia. Genome-wide DNA sequences were obtained with MIG-seq, and multiple data-filtering steps were used to optimize the dataset used to construct an ultrametric species tree. Divergence times among branches and ancestral niches were estimated. The niche variation among species was evaluated, summarized into two principal components, and their ancestral states were reconstructed to test whether niche shifts among branches can be explained by random processes, under a Brownian Motion model. Ceratozamia comprises three main clades, and most species relationships within the clades were resolved. Ceratozamia has diversified since the Oligocene, with major branching events occurring during the Miocene. This timing is consistent with fossil evidence, the timing estimated for other Neotropical plant groups, and the major geological events that shaped the topographic and climatic variation in Mexico. Patterns of niche evolution in the genus do not accord with the Brownian Motion model. Rather, non-random evolution with shifts towards more seasonal environments at high latitudes, or shifts towards humid or dry environments at low latitudes explain the diversification of Ceratozamia. We present a comprehensive phylogenetic reconstruction for Ceratozamia and identify for the first time the environmental factors involved in clade and species diversification within the genus. This study alleviates the controversies regarding the species relationships in the genus and provides the first evidence that latitude-associated environmental factors may influence processes of niche evolution in cycads.

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.

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.

Systematic relevance of pollen morphology in tribe Hylocereeae (Cactaceae).

Hylocereeae is one of the nine tribes in the subfamily Cactoideae (Cactaceae), for which the limits and recognition of genera have been controversial. Essentially, this group comprises epiphytic and hemiepiphytic genera with stems modified as climbing structures. The aim of this paper is to examine pollen attributes in representative species of genera of Hylocereeae, focusing on Selenicereus whose current circumscription comprises Hylocereus and three Weberocereus species, to find whether significant potentially apomorphic and/or autapomorphic systematic characters can be discovered. Utilizing SEM and light microscopy, 25 pollen characters were observed and measured. Tribe Hylocereeae is stenopalynous, with pollen grains isopolar and radially symmetrical monads, mostly tricolpate, except in Kimnachia, Pseudoripsalis and Weberocereus, whose pollen grains are pantocolpate. Seven attributes (five qualitative and two continuous) exhibited useful variation and were coded. The character of brevicolpate pollen grains was shared by Kimnachia ramulosa and Pseudorhipsalis amazonica. Convex quadrangular outline in the polar view was shared by Weberocereus tunilla and S. glaber. The absence of spinules on the exine was shared by S. minutiflorus and S. stenopterus. The largest pollen grain, found in Selenicereus megalanthus, might be correlated with polyploidy. Selenicereus is the taxon with the highest variation in pollen attributes, including species with an exine with or without spinules and variable polar area index and shape (subprolate or oblate-spheroidal).

Unlocking a high bacterial diversity in the coralloid root microbiome from the cycad genus Dioon.

Cycads are among the few plants that have developed specialized roots to host nitrogen-fixing bacteria. We describe the bacterial diversity of the coralloid roots from seven Dioon species and their surrounding rhizosphere and soil. Using 16S rRNA gene amplicon sequencing, we found that all coralloid roots are inhabited by a broad diversity of bacterial groups, including cyanobacteria and Rhizobiales among the most abundant groups. The diversity and composition of the endophytes are similar in the six Mexican species of Dioon that we evaluated, suggesting a recent divergence of Dioon populations and/or similar plant-driven restrictions in maintaining the coralloid root microbiome. Botanical garden samples and natural populations have a similar taxonomic composition, although the beta diversity differed between these populations. The rhizosphere surrounding the coralloid root serves as a reservoir and source of mostly diazotroph and plant growth-promoting groups that colonize the coralloid endosphere. In the case of cyanobacteria, the endosphere is enriched with Nostoc spp and Calothrix spp that are closely related to previously reported symbiont genera in cycads and other early divergent plants. The data reported here provide an in-depth taxonomic characterization of the bacterial community associated with coralloid root microbiome. The functional aspects of the endophytes, their biological interactions, and their evolutionary history are the next research step in this recently discovered diversity within the cycad coralloid root microbiome.

Ethnobotany of Mexican and northern Central American cycads (Zamiaceae).

BACKGROUND: This study documents cycad-human relationships in Mexico, Belize, Guatemala, El Salvador, and Honduras over the last 6000 years. The impetus was acute need for a better understanding of previously undocumented uses of cycads in this region, and the need to improve cycad conservation strategies using ethnobotanical data. We hypothesized that cycads are significant dietary items with no long-term neurological effects, are important to religious practice, and contribute to cultural identity and sense of place, but that traditional knowledge and uses are rapidly eroding. Guiding questions focused on nomenclature, food and toxicity, relationships to palms and maize, land management issues, roles in religious ceremony, and medicinal uses, among others, and contributions of these to preservation of cycads. METHODS: From 2000 to 2017, the authors conducted 411 semi-structured ethnographic interviews, engaged in participant-observation in Mexican and Honduran communities, and carried out archival research and literature surveys. RESULTS: We documented 235 terms and associated uses that 28 ethnic groups have for 57 species in 19 languages across 21 Mexican states and 4 Central American nations. Carbohydrate-rich cycads have been both famine foods and staples for at least six millennia across the region and are still consumed in Mexico and Honduras. Certain parts are eaten without removing toxins, while seed and stem starches are detoxified via several complex processes. Leaves are incorporated into syncretic Roman Catholic-Mesoamerican religious ceremonies such as pilgrimages, Easter Week, and Day of the Dead. Cycads are often perceived as ancestors and protectors of maize, revealing a close relationship between both groups. Certain beliefs and practices give cycads prominent roles in conceptions of sense of place and cultural heritage. CONCLUSIONS: Cycads are still used as foods in many places. Though they do not appear to cause long-term neurological damage, their health effects are not fully understood. They are often important to religion and contribute to cultural identity and sense of place. However, because most traditional knowledge and uses are rapidly eroding, new community-based biocultural conservation efforts are needed. These should incorporate tradition where possible and seek inspiration from existing successful cases in Honduras and Mexico.

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.

Estimation of genetic variation in closely related cycad species in Ceratozamia (Zamiaceae: Cycadales) using rapds markers / Variación genética en especies de cícadas estrechamente relacionadas en Ceratozamia (Zamiaceae: Cycadales) mediante el uso de RAPDs

AbstractThe Ceratozamianorstogii complex from Southern Mexico is made up of four closely related taxa and occurs in similar habitats (Quercus forest). All have linear-lanceolate leaflets with great similarity between them, especially in juvenile stages, but differentiate with age. There has been debate regarding delimitation of species due to character loss in herbarium specimens. The aim of this study was to determine the genetic variation, and to measure genetic similarity between the four taxa. We studied populations in Cintalapa (Chiapas) for C. alvarezii and C. norstogii; the Sierra Atravesada (Oaxaca) for C. chimalapensis, and Villa Flores (Chiapas) for C. mirandae. One population for each taxon was sampled (only one population is known for C. alvarezii) 11-15 randomly chosen adult individuals were sampled. Twenty-eight primers were tested of which five were polymorphic using the RAPD'S technique. The data were analyzed using Bayesian methods. Results revealed low genetic diversity, and a differentiation was found between species, suggesting a recent divergence. A previous morphological and anatomical study on the complex has found the taxa to be distinct. However, the results of this study have shown that the C. norstogii species complex is in a divergence process, probably through genetic drift and founder effects. Rev. Biol. Trop. 65 (1): 305-319. Epub 2017 March 01.

ResumenLos cuatro taxa que componen el complejo Ceratozamia norstogii de especies en el sur de México están estrechamente relacionados y se dan en hábitats similares (bosque de Quercus). Todos tienen folíolos linear-lanceolados con gran similitud entre ellos, sobre todo en las etapas juveniles, pero se diferencian con la edad. Ha habido un debate en relación con la delimitación de especies debido a la pérdida de caracteres en especímenes de herbario. Los objetivos de este estudio son determinar la variación genética y medir la similitud genética entre los cuatro taxones en el complejo. Las poblaciones estudiadas están en; Cintalapa, Chiapas para C. alvarezii y C. norstogii, la Sierra Atravesada, Oaxaca para C. chimalapensis y Villa Flores, Chiapas para C. mirandae. Se tomaron muestras de una población de cada taxón (sólo una población es conocida para C. alvarezii) 11-15 individuos adultos elegidos al azar fueron muestreados. Veintiocho primers fueron probados, de los cuales cinco fueron polimórficos mediante la técnica RAPD's. Los datos fueron analizados utilizando métodos bayesianos. Los resultados revelaron baja diversidad genética y la diferenciación encontrada entre las especies sugiere una divergencia reciente. Un estudio morfológico y anatómico anterior en el complejo encontró que los taxa son distintos. Sin embargo, los resultados del presente estudio han demostrado que el complejo C. norstogii aun se encuentra en un proceso de divergencia, probablemente a través de deriva genética y efectos de fundador.

Estimation of genetic variation in closely related cycad species in Ceratozamia (Zamiaceae: Cycadales) using RAPDs markers.

The Ceratozamia norstogii complex from Southern Mexico is made up of four closely related taxa and occurs in similar habitats (Quercus forest). All have linear-lanceolate leaflets with great similarity between them, especially in juvenile stages, but differentiate with age. There has been debate regarding delimitation of species due to character loss in herbarium specimens. The aim of this study was to determine the genetic variation, and to measure genetic similarity between the four taxa. We studied populations in Cintalapa (Chiapas) for C. alvarezii and C. norstogii; the Sierra Atravesada (Oaxaca) for C. chimalapensis, and Villa Flores (Chiapas) for C. mirandae. One population for each taxon was sampled (only one population is known for C. alvarezii) 11-15 randomly chosen adult individuals were sampled. Twenty-eight primers were tested of which five were polymorphic using the RAPD'S technique. The data were analyzed using Bayesian methods. Results revealed low genetic diversity, and a differentiation was found between species, suggesting a recent divergence. A previous morphological and anatomical study on the complex has found the taxa to be distinct. However, the results of this study have shown that the C. norstogii species complex is in a divergence process, probably through genetic drift and founder effects.

A character-based approach in the Mexican cycads supports diverse multigene combinations for DNA barcoding.

A DNA barcoding study was conducted to determine the optimal combination of loci needed for successful species-level molecular identification in three extant cycad genera-Ceratozamia, Dioon, and Zamia-that occur in Mexico. Based on conclusions of a previous multigene study in representative species of all genera in the Cycadales, we tested the DNA barcoding performance of seven chloroplast coding (matK, rpoB, rpoC1, and rbcL) and non-coding (atpF/H, psbK/I, and trnH-psbA) regions, plus sequences of the nuclear internal transcribed spacer. We analysed data under the assumptions of the "character attributes organization system" (CAOS), a character-based approach in which species are identified through the presence of 'DNA diagnostics'. In Ceratozamia, four chloroplast regions and one nuclear region were needed to achieve > 70% unique species identification. In contrast, the two-gene combination atpF/H + psbK/I and the four-gene combination atpF/H + psbK/I + rpoC1 + ITS2 were needed to reach 79% and 75% unique species identification in Dioon and Zamia, respectively. The combinations atpF/H + psbK/I and atpF/H + psbK/I + rpoC1 + ITS2 include loci previously considered by the international DNA barcoding community. However, none of the three combinations of potential DNA barcoding loci found to be optimal with a character-based approach in the Mexican cycads coincides with the 'core barcode' of chloroplast markers (matK + rbcL) recently proposed for universal use in the plant kingdom.

Allozyme variation in the three extant populations of the narrowly endemic cycad Dioon angustifolium Miq. (Zamiaceae) from North-eastern Mexico.

BACKGROUND AND AIMS: Dioon angustifolium was considered within D. edule. Recent morphometric and allozyme studies on D. edule have shown that D. angustifolium has originated from geographic isolation and is therefore considered to be a separate species. This cycad is endemic to north-eastern Mexico and is known only from three populations in the Sierra Madre Oriental mountain chain. Its populations are small when compared with its southern relative D. edule. In this study, genetic variation was determined within and between populations of D. angustifolium and the genetic consequences of habitat fragmentation and isolation of populations of this species were assessed. METHODS: Allozyme electrophoresis of 14 presumptive loci was used. The data were analysed with statistical approximations for estimating genetic diversity, structure, gene flow and recent genetic bottlenecks. KEY RESULTS: Means and standard deviations of genetic diversity estimators were: number of alleles per locus (A = 1.67 +/- 0.23), percentage of polymorphic loci (P = 52.4 +/- 23 %) and expected heterozygosity (H(E) = 0.218 +/- 0.093). The genetic variation attributable to differences among populations was 16.7 %. Mean gene flow between paired populations was Nm = 1.55 +/- 0.67, which is similar to that reported for endemic plant species of narrow geographical distribution and species with gravity-dispersed seed. A recent bottleneck is detected in the populations studied. CONCLUSIONS: Dioon angustifolium presents high levels of genetic diversity compared with other cycad species, in spite of small population sizes. The recent bottleneck effect did not effectively reduce the genetic variation to the extent of eliminating these populations. The distribution of D. angustifolium appears to be the result of historical biogeographical effects related to the Pleistocene glaciations. It is recommended that this species be catalogued in the IUCN Red List of Threatened Species and conservation efforts be made to preserve it.

Diversity and genetic structure of the Mexican endemic epiphyte Tillandsia achyrostachys E. Morr. ex Baker var. achyrostachys (Bromeliaceae).

BACKGROUND AND AIMS: The monoecious, bird-pollinated epiphytic Tillandsia achyrostachys E. Morr. ex Baker var. achyrostachys is an endemic bromeliad of the tropical dry forests of Mexico with clonal growth. In the Sierra de Huautla Natural Reserve this species shows a host preference for Bursera copallifera (Sessé & Moc ex. DC) Bullock. As a result of deforestation in the study area, B. copallifera has become a rare tree species in the remaining forest patches. This human-induced disturbance has directly affected the population densities of T. achyrostachys. In this study the genetic consequences of habitat fragmentation were assessed by comparing the genetic diversity, gene flow and genetic differentiation in six populations of T. achyrostachys in the Sierra de Huautla Natural Reserve, Mexico. METHODS: Allozyme electrophoresis of sixteen loci (eleven polymorphic and five monomorphic) were used. The data were analysed with standard statistical approximations for obtaining diversity, genetic structure and gene flow. KEY RESULTS: Genetic diversity and allelic richness were: HE = 0.21 +/- 0.02, A = 1.86 +/- 0.08, respectively. F-statistics revealed a deficiency of heterozygous plants in all populations (Fit = 0.65 +/- 0.02 and Fis = 0.43 +/- 0.06). Significant genetic differentiation between populations was detected (Fst = 0.39 +/- 0.07). Average gene flow between pairs of populations was relatively low and had high variation (Nm = 0.46 +/- 0.21), which denotes a pattern of isolation by distance. The genetic structure of populations of T. achyrostachys suggests that habitat fragmentation has reduced allelic richness and genetic diversity, and increased significant genetic differentiation (by approx. 40 %) between populations. CONCLUSIONS: The F-statistic values (>0) and the level of gene flow found suggest that habitat fragmentation has broken up the former population structure. In this context, it is proposed that the host trees of T. achyrostachys should be considered as a conservation priority, since they represent the limiting factor to bromeliad population growth and connectivity.