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.

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.

Comparative anatomy of leaflets of Zamia acuminata and Z. pseudomonticola (Zamiaceae) in Costa Rica.

The genus Zamia is morphologically and ecologically the most diverse of the order Cycadales. Throughout its history this genus has been restricted to the New World and is presently almost entirely restricted to the Neotropics. Unusual anatomical traits of the leaflets, such as the sunken stomata and thick cuticle, are common in this and related genera. The objective of this research was to study and compare the leaflet anatomy of Zamia acuminata and Z pseudomonticola and establish possible phylogenetic relationships between the anatomical traits and the near relatives of these species. The leaf material was obtained from living plants and then processed for electron microscopy study. We found that both species are very similar to each other and to Z fairchildiana, and that they share several unusual traits with other species of the genus, such as the parenchyma morphology, the spatial distribution of tissues between the veins and the stomata morphology. The main differences between these species were seen in their fiber clusters and in the abundance oftrichome basal cells on the epidermis. The anatomical similarities between the three species could be the result of their close phylogenetic relationship and the divergences between them could be the result of recent speciation during the Pleistocene, resulting from geological changes in Southern Costa Rica.

Comparative anatomy of leaflets of Zamia acuminata and Z. pseudomonticola (Zamiaceae) in Costa Rica

The genus Zamia is morphologically and ecologically the most diverse of the order Cycadales. Throughout its history this genus has been restricted to the New World and is presently almost entirely restricted to the Neotropics. Unusual anatomical traits of the leaflets, such as the sunken stomata and thick cuticle, are common in this and related genera. The objective of this research was to study and compare the leaflet anatomy of Zamia acuminata and Z. pseudomonticola and establish possible phylogenetic relationships between the anatomical traits and the near relatives of these species. The leaf material was obtained from living plants and then processed for electron microscopy study. We found that both species are very similar to each other and to Z. fairchildiana, and that they share several unusual traits with other species of the genus, such as the parenchyma morphology, the spatial distribution of tissues between the veins and the stomata morphology. The main differences between these species were seen in their fiber clusters and in the abundance of trichome basal cells on the epidermis. The anatomical similarities between the three species could be the result of their close phylogenetic relationship and the divergences between them could be the result of recent speciation during the Pleistocene, resulting from geological changes in Southern Costa Rica.

Zamia es morfológica y ecológicamente el género más diverso del orden Cycadales. Este género siempre ha estado restringido a América, pero en la actualidad habita principalmente en la región neotropical. Características anatómicas inusuales en los foliolos como los estomas hundidos y las cutículas gruesas son comunes en Zamia y géneros afines. El objetivo de este trabajo consiste en comparar la anatomía de los foliolos de Zamia acuminata y Z. pseudomonticola y establecer posibles relaciones filogenéticas entre las características anatómicas y los parientes cercanos de esta especie. Las hojas de las especies seleccionadas fueron obtenidas de plantas vivas y luego procesadas para el estudio por microscopía electrónica. Ambas especies son muy similares entre sí y respecto a Z. fairchildiana y comparten varias características en común con otras especies del género como son la morfología del parénquima, de los estomas y la distribución espacial de tejidos alrededor de las haces vasculares. Las diferencias más notables entre especies se vieron a nivel de sus paquetes de fibras y en la abundancia de células basales de los tricomas en la epidermis. Las similitudes anatómicas entre estas tres especies pueden ser el resultado de su cercanía filogenética y las diferencias podrían ser el resultado de especiación durante el Pleistoceno, producto de los eventos geológicos y cambios sucedidos en el sur de Costa Rica en esa época.

Variation in the chemical composition of cone volatiles within the African cycad genus Encephalartos.

Volatiles play a key role in attraction of pollinators to cycad cones, but the extent to which volatile chemistry varies among cycad species is still poorly documented. Volatile composition of male and female cones of nineteen African cycad species (Encephalartos; Zamiaceae) was analysed using headspace technique and gas chromatography-mass spectrometry (GC-MS). A total of 152 compounds were identified among the species included in this study, the most common of which were monoterpenes, nitrogen-containing compounds and unsaturated hydrocarbons. Male and female cones emitted similar volatile compounds which varied in relative amounts with two unsaturated hydrocarbons (3E)-1,3-octadiene and (3E,5Z)-1,3,5-octatriene present in the volatile profile of most species. In a multivariate analysis of volatile profiles using non-metric multidimensional scaling (NMDS), a number of species clusters were identified according to shared emission of unsaturated hydrocarbons, pyrazines, benzenoids, aldehydes, alkanes and terpenoids. In comparison, terpenoids are common in Zamia and dominant in Macrozamia species (both in the family Zamiaceae) while benzenoids, esters, and alcohols are dominant in Cycas (Cycadaceae) and in Stangeria (Stangeriaceae). It is likely that volatile variation among Encephalartos species reflects both phylogeny and adaptations to specific beetle pollinators.

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.

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.

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.