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.

Evolution of Ceratozamia cycads: A proximate-ultimate approach.

Evidence suggests that past climatic fluctuations affected speciation of extant cycads. However, empirical genetic and morphological evidence explaining patterns and processes of species diversification are scarce. There are some explanations for the origin and evolution of the genus Ceratozamia, but with inconclusive results. To elucidate the evolution of Ceratozamia, we used genetic and phenotypic sources as empirical data, which were applied in a 'proximate-ultimate' framework (ecological and evolutionary scale, respectively). Our results suggested that the evolutionary mechanisms of speciation were shaped by deterministic (natural selection-adaptation) driven by climatic conditions associated to water stress, and probably enhanced by stochastic processes (gene drift and inbreeding). In general terms, punctuated evolution models were those that best explained the patterns of speciation throughout the phylogenetic history of the lineages encompassed in the genus Ceratozamia. Finally, we provide empirical evidence on the tempo and mode of the evolution of a 'living plant fossil'.

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.

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.

Pleistocene diversification in an ancient lineage: a role for glacial cycles in the evolutionary history of Dioon Lindl. (Zamiaceae).

PREMISE OF THE STUDY: Recent estimates of crown ages for cycad genera (Late Miocene) challenge us to consider what processes have produced the extant diversity of this ancient group in such relatively little time. Pleistocene climate change has driven major shifts in species distributions in Mexico and may have led to speciation in the genus Dioon by forcing populations to migrate up in elevation, thereby becoming separated by topography. METHODS: We inferred orthologs from transcriptomes of five species and sequenced these in 42 individuals representing all Dioon species. From these data and published plastid sequences, we inferred dated species trees and lineage-specific diversification rates. KEY RESULTS: Analyses of 84 newly sequenced nuclear orthologs and published plastid data confirm four major clades within Dioon, all of Pleistocene age. Gene tree analysis, divergence dates, and an increase in diversification rate support very recent and rapid divergence of extant taxa. CONCLUSIONS: This study confirms the Pleistocene age of Dioon species and implicates Pleistocene climate change and established topography in lineage spitting. These results add to our understanding of the cycads as evolutionarily dynamic lineages, not relicts or evolutionary dead ends. We also find that well-supported secondary calibration points can be reliable in the absence of fossils. Our hypothesis of lineage splitting mediated by habitat shifts may be applicable to other taxa that are restricted to elevation specific ecotones.

Conservation Genetics of Two Highly Endangered and Poorly Known Species of Zamia (Zamiaceae: Cycadales) in Colombia.

Genetic diversity is key in providing the variation needed to face stochastic change. Increased habitat loss alters population size and dynamics posing serious threats to the conservation of wild species. Colombia has undergone massive deforestation over the last century, but harbors extraordinary high species diversity of genus Zamia (Cycadales); however, most of the species are under threat. In this study, we targeted the largest accessible remaining populations of 2 closely related species growing as endemics in the Magdalena Valley region of Colombia. We successfully transferred the SSR loci used in previous Zamia studies to these species. In total, we amplified 13 microsatellite loci in 3 wild populations, aiming at: 1) assessing genetic diversity and 2) understanding if the structure found between the 3 populations reflected species and population boundaries due to ecological and historical genetic isolation. We found that the actual population size does not reflect population genetic diversity with a small population (Perales) harboring the highest genetic diversity. In addition, all populations are highly structured regardless of species containment, all showing signs of genetic isolation. Given the high degree of ecological threat, and the inherent biological traits of Cycads, we provide information regarding the prioritization of populations for ex situ management.

Shifting Quaternary migration patterns in the Bahamian archipelago: Evidence from the Zamia pumila complex at the northern limits of the Caribbean island biodiversity hotspot.

PREMISE OF THE STUDY: The Bahamas archipelago is formed by young, tectonically stable carbonate banks that harbor direct geological evidence of global ice-volume changes. We sought to detect signatures of major changes on gene flow patterns and reconstruct the phylogeographic history of the monophyletic Zamia pumila complex across the Bahamas. METHODS: Nuclear molecular markers with both high and low mutation rates were used to capture two different time scale signatures and test several gene flow and demographic hypotheses. KEY RESULTS: Single-copy nuclear genes unveiled apparent ancestral admixture on Andros, suggesting a significant role of this island as main hub of diversity of the archipelago. We detected demographic and spatial expansion of the Zamia pumila complex on both paleo-provinces around the Piacenzian (Pliocene)/Gelasian (Pleistocene). Populations evidenced signatures of different migration models that have occurred at two different times. Populations on Long Island (Z. lucayana) may either represent a secondary colonization of the Bahamas by Zamia or a rapid and early-divergence event of at least one population on the Bahamas. CONCLUSIONS: Despite changes in migration patterns with global climate, expected heterozygosity with both marker systems remains within the range reported for cycads, but with significant levels of increased inbreeding detected by the microsatellites. This finding is likely associated with reduced gene flow between and within paleo-provinces, accompanied by genetic drift, as rising seas enforced isolation. Our study highlights the importance of the maintenance of the predominant direction of genetic exchange and the role of overseas dispersion among the islands during climate oscillations.

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.

Ontogenetic Changes in Azoxyglycoside Levels in the Leaves of Dioon edule Lindl.

Plants have multiple strategies, including phytochemicals that protect their vulnerable tissues against pathogens and herbivores. Dioon edule, like all cycads, possess unique azoxy-type compounds, azoxyglycosides (AZGs) as a chemical defense; however, the ontogenetic variability of these compounds in this long-lived cycad is unknown. Here, we investigated the effects of plant age, sex, genotype and individual heterozygosity on AZG levels in mature leaves of wild D. edule populations from eastern Mexico. Individuals were divided into three ontogenetic stages: seedlings, juveniles and adults. We established overall leaf quality by quantifying pigments associated with photosynthesis; chlorophylla, chlorophyllb and lutein. Leaf chlorophylla levels were higher in seedlings compared to adult cycads. Plants were genotyped using 11 microsatellite markers and foliar AZG levels were quantified by HPLC. AZG levels do not correlate with plant genotype or the individual's heterozygosity. Genetic analysis identified a distinction between lowland and highland individuals; foliar AZG levels were higher in lowland adult cycads compared to highland individuals. In both populations, the highest AZG levels were found in seedlings compared to adult cycads. These young cycads are highly reliant on their few leaves since seedlings bear one or two leaves for the first years of their life and, thus, are unlikely to recover from defoliation. The results suggest that cycad leaves with a greater nutritive content and a higher value for long-term survival are better protected with higher AZG levels. Female adult cycads have higher AZG levels compared to males, suggesting that the benefits of defense could also be linked to reproductive costs.

Evolutionary dynamics of a cycad obligate pollination mutualism - Pattern and process in extant Macrozamia cycads and their specialist thrips pollinators.

Obligate pollination mutualisms are rare and few have been investigated deeply. This paper focuses on one such mutualism involving thrips in the genus Cycadothrips that pollinate cycads in the genus Macrozamia. Both represent old lineages relative to insects and plants generally, are endemic to Australia, and are mutually co-dependent. The phylogenetic analyses presented here demonstrate that the pollinator is much more diverse than previously considered, with each pollinator lineage being extremely specific to between one and three host species where these latter share part of their distribution. The new species diversity we demonstrate in Cycadothrips all presently falls under the species name C. chadwicki, and these different lineages diversified during two periods. An older divergence, beginning 7.3Mya (4.4-11.1, 95% HPD), resulted in three major lineages, and then further diversification within each of these three lineages took place at most 1.1Mya (0.6-1.8, 95% HPD). These divergence estimates correspond to times when aridification was increasing in Australia, suggesting that population fragmentation following climatic change has played a significant role in the evolutionary history of Cycadothrips and Macrozamia. This means that co-diversification of the host and pollinator in allopatry appears to be the dominant process affecting species diversity. Host switching is also clearly evident in the discrepancy between the divergence times of the C. chadwicki lineage and C. albrechti, about 10.8Mya (6.0-17.1, 95% HPD), and their hosts, at about 1.1Mya (0.2-3.4Mya, 95% HPD), in that the pollinator split pre-dates the origin of the associated host species of each. These results add to the body of evidence that the evolutionary processes important in obligate pollinator mutualisms are more varied than previously assumed.

An ancient satellite DNA has maintained repetitive units of the original structure in most species of the living fossil plant genus Zamia.

ZpS1 satellite DNA is specific to the genus Zamia and presents repetitive units organized as long arrays and also as very short arrays dispersed in the genome. We have characterized the structure of the ZpS1 repeats in 12 species representative of the whole geographic distribution of the genus. In most species, the clone most common sequences (cMCS) were so similar that a general most common sequence (GMCS) of the ZpS1 repetitive unit in the genus could be obtained. The few partial variations from the GMCS found in cMCS of some species correspond to variable positions present in most other species, as indicated by the clone consensus sequences (cCS). Two species have an additional species-specific variety of ZpS1 satellite. The dispersed repeats were found to contain more mutations than repeats from long arrays. Our results indicate that all or most species of Zamia inherited the ZpS1 satellite from a common ancestor in Miocene and have maintained repetitive units of the original structure till present. The features of ZpS1 satellite in the genus Zamia are poorly compatible with the model of concerted evolution, but they are perfectly consistent with a new model of satellite evolution based on experimental evidences indicating that a specific amplification-substitution repair mechanism maintains the homogeneity and stability of the repeats structure in each satellite DNA originally present in a species as long as the species exists.

Conservation and canalization of gene expression during angiosperm diversification accompany the origin and evolution of the flower.

The origin and rapid diversification of the angiosperms (Darwin's "Abominable Mystery") has engaged generations of researchers. Here, we examine the floral genetic programs of phylogenetically pivotal angiosperms (water lily, avocado, California poppy, and Arabidopsis) and a nonflowering seed plant (a cycad) to obtain insight into the origin and subsequent evolution of the flower. Transcriptional cascades with broadly overlapping spatial domains, resembling the hypothesized ancestral gymnosperm program, are deployed across morphologically intergrading organs in water lily and avocado flowers. In contrast, spatially discrete transcriptional programs in distinct floral organs characterize the more recently derived angiosperm lineages represented by California poppy and Arabidopsis. Deep evolutionary conservation in the genetic programs of putatively homologous floral organs traces to those operating in gymnosperm reproductive cones. Female gymnosperm cones and angiosperm carpels share conserved genetic features, which may be associated with the ovule developmental program common to both organs. However, male gymnosperm cones share genetic features with both perianth (sterile attractive and protective) organs and stamens, supporting the evolutionary origin of the floral perianth from the male genetic program of seed plants.

Zamia (Cycadales: Zamiaceae) on Puerto Rico: asymmetric genetic differentiation and the hypothesis of multiple introductions.

PREMISE OF THE STUDY: This study of Zamia in Puerto Rico is the most intensive population genetics investigation of a cycad to date in terms of number of markers, and one of few microsatellite DNA studies of plants from the highly critical Caribbean biodiversity hotspot. Three distinctive Zamia taxa occur on the island: Z. erosa on the north coast, and Z. portoricensis and Z. pumila, both in the south. Their relationships are largely unknown. We tested three hypotheses about their genetic diversity, including the possibility of multiple introductions. METHODS: We used 31 microsatellite loci across 10 populations and analyzed the data with AMOVA, Bayesian clustering, and ABC coalescent modeling. KEY RESULTS: Puerto Rican zamias exhibit an amalgam of patterns of genetic differentiation that have been reported for cycads. Overall, the taxa are slightly inbred, with high infra-populational variation and little evidence of recent bottlenecks. Zamia erosa exhibits a more than threefold greater degree of population differentiation than the other two taxa. Admixture is evident only between Z. portoricensis and Z. pumila. Zamia portoricensis is inferred to be the youngest taxon on the island, on the basis of estimates of coalescence time and effective population size. A selective sweep may be underway in a small population of Z. erosa in a saline environment. CONCLUSIONS: Zamia erosa may represent an independent introduction into Puerto Rico; Z. portoricensis and Z. pumila fit a scenario of allopatric speciation. This will be explored further in the context of genetic analysis across the entire Caribbean region.

Genetic diversity through life history of Dioon edule Lindley (Zamiaceae, Cycadales).

The distribution of genetic diversity and structure for three populations of Dioon edule Lindley (Zamiaceae) at Monte Oscuro (MO), El Farallón (EF) and Rancho del Niño (RN) in Veracruz, Mexico was studied using 20 allozyme loci, considering four life history classes: seeds, seedlings, juveniles and adults. The MO population is genetically less diverse than the EF and RN populations. Total and local inbreeding differ significantly between life history classes. An increment of inbreeding among all classes was observed, and genetic differentiation among populations was higher in seeds and seedlings than in juveniles and adults. In terms of percentage of polymorphic loci, the MO seeds showed least (80%), followed by RN (95%) and EF had the highest values (100%), probably because of a reduction in effective population size and habitat fragmentation processes. In this context, the mean effective population size was 23.2 +/- 11.3 for all populations. We conclude that seed cohorts in EF and RN represent a reservoir of genetic diversity within these two populations. Also, preservation of adult plants is an essential aspect to consider in management and conservation efforts for populations of Dioon edule in natural conditions.

Molecular relationships in Encephalartos (Zamiaceae, Cycadales) based on nucleotide sequences of nuclear ITS 1&2, rbcL, and genomic ISSR fingerprinting.

The cycad genus Encephalartos is restricted to Africa and is threatened with extinction in most of its range. Total DNA was extracted from 51, i.e., 78 %, of the described species of Encephalartos. The accessions were sampled from the furthest western occurrence of the genus in Nigeria, via Sudan and Uganda, to southern South Africa. The sequences of nuclear ribosomal internal transcribed spacer regions 1 and 2 (ITS 1&2), the chloroplast encoded rbcL gene, and ISSR genomic fingerprinting were employed to resolve the molecular history and the relationships within the genus. Sequence alignment, as well as ISSR fingerprinting, data show low genetic variation among all analysed accessions, indicating diversification within the Pliocene/Pleistocene. ITS 1&2 data agree well with morphological and geographical characters and resolved three major genetic clusters with overlapping distribution ranges in eastern South Africa. This area, that contains the largest diversity of genotypes of Encephalartos, may have served as a Pliocene/Pleistocene refugium.

An unusual satellite DNA from Zamia paucijuga (Cycadales) characterised by two different organisations of the repetitive unit in the plant genome.

We have cloned and characterised a highly repetitive DNA family that represents about 5% of the nuclear genome of Zamia paucijuga, a member of Cycadales, an ancient and rare group of seed plants. The unit repeat, cloned from EcoRV digested genomic DNA, has a length of about 323 bp, an high GC content (65-70%) and a subtelomeric localisation. The DNA of Z. paucijuga was digested with various restriction enzymes and analysed by Southern blot using the cloned unit repeat as a probe. These experiments indicated that the repeat is present in the plant genome in an unusual organisation, as tandem arrays typical of satellite DNA and as dispersed elements. The characterisation of these unusual "dispersed satellite DNA" elements required a complex series of experiments using combined Southern blot analyses, PCR, cloning and sequencing. Our results indicate that most of these dispersed elements are formed by few units of the GC-rich satellite DNA repeats arranged in tandem and flanked at both sites by one copy of a 0.6 kb AT-rich direct repeat. This unusual satellite DNA organisation of GC-rich repeats is present in many species of genus Zamia, and, thus, likely represent an ancient rearrangement of the satellite DNA repeats that spread in the genome as dispersed elements.

Detection of inter-spread repeat sequence in genomic DNA sequence.

Various types of periodic patterns in nucleotide sequences are known to be very abundant in a genomic DNA sequence, and to play important biological roles such as gene expression, genome structural stabilization, and recombination. We present a new method, named "STEPSTONE", to find a specific periodic pattern of repeat sequence, inter-spread repeat, in which the tandem repeats of the conserved and the not-conserved regions appear periodically. In our method, at first, the data on periods of short repeat sequences found in a target sequence are stored as a hash data, and then are selected by application of an auto-correlation test in time series analysis. Among the statistically selected sequences, the inter-spread repeats are obtained by usual alignment procedures through two steps. To test the performance of our method, we examined the inter-spread repeats in Mycobacterium tuberculosis and Zamia paucijuga genomic sequences. As a result, our method exactly detected the repeats in the two sequences, being useful for identifying systematically the inter-spread repeats in DNA sequence.

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.

Character-based, population-level DNA barcoding in Mexican species of Zamia L. (Zamiaceae: Cycadales).

BACKGROUND AND AIMS: With the recent proposal of matK and rbcL as core plant DNA barcoding regions by the Consortium for the Barcoding of Life Plant Working Group, the construction of reference libraries in the botanical DNA barcoding initiative has entered a new phase. However, in a recent DNA barcoding study in the three Mexican genera of the gymnosperm order Cycadales, we found that neither matK nor rbcL allow high levels of molecular identification of previously established species. MATERIALS AND METHODS: Our data analysis in that study rested on the "Characteristic Attributes Organization System" (CAOS), a character-based algorithm for the definition of "DNA diagnostics." Here, we use CAOS to analyze a population-level molecular data set in Zamia, one of the three cycad genera occurring in Mexico, whose populations display contrasting biogeographic patterns. Our population-level study, which includes all species in the region formally known as Megamexico, is restricted to the genome region, which showed the best single-locus molecular identification performance in our previous study-namely, the noncoding intergenic chloroplast spacer psbK-I. RESULTS: Our comparison of single-individual vs. population-level psbK-I datasets in Zamia indicates that CAOS analyses are sensitive to slight alignment changes, which in turn derive from the different amounts of molecular variation present in each matrix type. CONCLUSION: We, therefore, suggest that character-based studies that involve population-level data should contemplate this type of comparison between data matrices, before a set of DNA diagnostics in a given DNA barcoding reference library is considered definitive.

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.