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'.

Phylogeographic structure of Canthon cyanellus (Coleoptera: Scarabaeidae), a Neotropical dung beetle in the Mexican Transition Zone: Insights on its origin and the impacts of Pleistocene climatic fluctuations on population dynamics.

Canthon cyanellus is a roller dung beetle with a wide distribution range in the tropical forests of the New World. In Mexico, it inhabits the Pacific and the Gulf coasts, the Yucatan Peninsula and the south mainly in the State of Chiapas. This species shows a wide geographical variation in cuticle color, which has been used as defining trait for subspecies. In this study we analyzed the phylogeographic and demographic history of the Mexican populations of C. cyanellus using DNA sequences of the nuclear ITS2, and the mitochondrial COI and 16S genes. We found that not all the current valid subspecies are supported by the molecular analysis. The populations are genetically and geographically structured in five lineages. The diversification events that gave origin to the main lineages within this species complex occurred during the Pleistocine in a time range of 1.63-0.91Myr. The demographic history of these lineages suggests post-glacial expansions toward the middle and the end of the Pleistocene. The combined data of mitochondrial and nuclear DNA suggest that the phylogeographic structure and demographic history of the C. cyanellus populations are the result of: the geological and volcanic activity that occurred from the end of the Pliocene to the Pleistocene; and the contraction and expansion of tropical forests due to the glacial and inter-glacial cycles during the Pleistocene. Landscape changes derived from historical events have affected the demographic history of the populations of this species. The results presented here point to the need to review the taxonomic status and delimitation of the lineages encompassed in the Canthon cyanellus complex.

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.

Transcriptome-derived microsatellite markers for Dioon (Zamiaceae) cycad species.

PREMISE OF THE STUDY: Dioon (Zamiaceae) is an endangered North American cycad genus of evolutionary and ornamental value. We designed and validated a set of microsatellite markers from D. edule that can be used for population-level and conservation studies, and that transferred successfully to D. angustifolium, D. spinulosum, and D. holmgrenii. METHODS AND RESULTS: We tested 50 primers from 80 microsatellite candidate loci in the OneKP D. edule transcriptome. Genotypes from 21 loci in 20 D. edule individuals revealed up to 14 alleles per locus and observed heterozygosity from 0.15 to 0.92; one locus was monomorphic. Seven of those 21 loci were polymorphic in D. angustifolium, D. spinulosum, and D. holmgrenii, with up to seven alleles, and an observed heterozygosity up to 0.89. CONCLUSIONS: The transcriptome-derived microsatellites generated here will serve as tools to advance population genetic studies and inform conservation strategies of Dioon, including the identification and origin of illegal plants in the cycad trade.

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.

Genetic variability of the narrow endemic tree Antirhea aromatica Castillo-Campos Lorence, (Rubiaceae, Guettardeae) in a tropical forest of Mexico.

BACKGROUND AND AIMS: Genetic structure and variability were examined in the only three extant populations of the narrow-endemic tree Antirhea aromatica (Rubiaceae, Guettardeae), an endangered species of the tropical forest of eastern Mexico. Patterns of genetic diversity within and among populations for adult plants and seedlings were obtained. METHODS: Allozyme electrophoresis of 15 loci was conducted and the data analysed with statistical approximation for obtaining genetic diversity, structure and gene flow. KEY RESULTS: The mean expected heterozygosity (He) in the adult and seedling populations was 0.18 +/- 0.08 and 0.20 +/- 0.09, respectively. The genetic variation explained by differences among populations was 51 and 35 %, for adult and seedling populations, respectively. On average, gene flow between paired adult populations was low (Nm = 0.26 +/- 0.09), compared with other trees from the tropical forest. CONCLUSIONS: The results indicated that the populations evaluated have high genetic variability, compared with other endemic and geographically narrowly distributed plant species, in areas with high levels of environmental heterogeneity (e.g. tropical forests). The conservation implications of the results are discussed, and in this regard it is proposed that A. aromatica should be considered as an indicator species with economic potential. It is suggested that sustainable management practices should be implemented and that the areas where the species is distributed should be declared a natural reserve to ensure the species conservation.

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.