Michael Calonje

Phylogeny of the cycads based on multiple single-copy nuclear genes: congruence of concatenated parsimony, likelihood and species tree inference methods

BACKGROUND AND AIMS Despite a recent new classification, a stable phylogeny for the cycads has been elusive, particularly regarding resolution of Bowenia, Stangeria and Dioon. In this study, five single-copy nuclear genes (SCNGs) are applied to the phylogeny of the order Cycadales. The specific aim is to evaluate several gene tree-species tree reconciliation approaches for developing an accurate phylogeny of the order, to contrast them with concatenated parsimony analysis and to resolve the erstwhile problematic phylogenetic position of these three genera. METHODS DNA sequences of five SCNGs were obtained for 20 cycad species representing all ten genera of Cycadales. These were analysed with parsimony, maximum likelihood (ML) and three Bayesian methods of gene tree-species tree reconciliation, using Cycas as the outgroup. A calibrated date estimation was developed with Bayesian methods, and biogeographic analysis was also conducted. KEY RESULTS Concatenated parsimony, ML and three species tree inference methods resolve exactly the same tree topology with high support at most nodes. Dioon and Bowenia are the first and second branches of Cycadales after Cycas, respectively, followed by an encephalartoid clade (Macrozamia-Lepidozamia-Encephalartos), which is sister to a zamioid clade, of which Ceratozamia is the first branch, and in which Stangeria is sister to Microcycas and Zamia. CONCLUSIONS A single, well-supported phylogenetic hypothesis of the generic relationships of the Cycadales is presented. However, massive extinction events inferred from the fossil record that eliminated broader ancestral distributions within Zamiaceae compromise accurate optimization of ancestral biogeographical areas for that hypothesis. While major lineages of Cycadales are ancient, crown ages of all modern genera are no older than 12 million years, supporting a recent hypothesis of mostly Miocene radiations. This phylogeny can contribute to an accurate infrafamilial classification of Zamiaceae.

Can a Botanic Garden Cycad Collection Capture the Genetic Diversity in a Wild Population

Premise of research. Conservation of plant species often requires ex situ (off-site) cultivation of living collections. Cycads constitute the most imperiled major group of plants, and ex situ collections are an important part of conservation planning for this group, given seed recalcitrance, difficulties with tissue culture, and ongoing in situ threats. Very little is known about the genetics of ex situ conservation collections of cycads. Thus, this study seeks to illuminate how well an ex situ collection of a cycad can capture the diversity in a wild population. Methodology. A model species, Zamia decumbens, was chosen on the basis of geographic isolation and detailed census knowledge, which allowed near-total sampling of in situ plants. Overall, 375 in situ plants were compared to 205 ex situ plants via 10 microsatellite markers. Pivotal results. Genetic-distance analysis shows high fidelity of the ex situ collections to their in situ source populations as well as clustering of ex situ progeny by accession and strong identity with their respective mother plants. Structured resampling of allele capture from the in situ populations by the ex situ collections shows that allele capture increases as number of ex situ plants maintained increases, but with a diminishing rate of increase. Conclusions. These data demonstrate that botanic garden collections can better conserve the genetic diversity of in situ cycad populations if four recommendations are followed: (1) use the species biology to inform the collecting strategy; (2) manage each population separately; (3) collect and maintain multiple accessions; and (4) collect over multiple years.

Cycad biodiversity in the Bahamas Archipelago and conservation genetics of the threatened Zamia lucayana (Zamiaceae)

A conservation assessment for the three cycad species native to the Bahamas Islands is presented. Results are based on field surveys on all islands where these species occur. Zamia angustifolia is native to Eleuthera, Zamia integrifolia is native to Abaco, Andros, Eleuthera, Grand Bahama and New Providence, and Zamia lucayana is endemic to Long Island. Z. angustifolia is of the highest conservation concern because of the small number of adult plants, its restricted distribution and the extensive development occurring within its habitat. Z. integrifolia also has a restricted distribution on Eleuthera and Grand Bahama and, although threatened by urban development in New Providence, it is relatively common on Abaco and Andros. Z. lucayana comprises three populations within a narrow strip of land of c. 1 km 2 ; we propose a reassignment of its current conservation status from Endangered to Critically Endangered. We assessed the genetic structure of Z. lucayana based on 15 polymorphic microsatellite DNA loci; this indicated that the three known populations should be considered a single management unit. However, the high number of private alleles suggests that genetic drift, indicative of recent fragmentation, is progressing. We propose in situ conservation strategies, and we also collected germplasm from a total of 24 populations of these three cycad species, for ex situ conservation.

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.

Conserved genetic regions across angiosperms as tools to develop single‐copy nuclear markers in gymnosperms: an example using cycads

Several individuals of the Caribbean Zamia clade and other cycad genera were used to identify single‐copy nuclear genes for phylogeographic and phylogenetic studies in Cycadales. Two strategies were employed to select target loci: (i) a tblastX search of Arabidopsis conserved ortholog sequence (COS) set and (ii) a tblastX search of Arabidopsis‐Populus‐Vitis‐Oryza Shared Single‐Copy genes (APVO SSC) against the EST Zamia databases in GenBank. From the first strategy, 30 loci were selected, and from the second, 16 loci. In both cases, the matching GenBank accessions of Zamia were used as a query for retrieving highly similar sequences from Cycas, Picea, Pinus species or Ginkgo biloba. After retrieving and aligning all the sequences in each locus, intron predictions were completed to assist in primer design. PCR was carried out in three rounds to detect paralogous loci. A total of 29 loci were successfully amplified as a single band of which 20 were likely single‐copy loci. These loci showed different diversity and divergence levels. A preliminary screening allowed us to select 8 promising loci (40S, ATG2, BG, GroES, GTP, LiSH, PEX4 and TR) for the Zamia pumila complex and 4 loci (COS26, GroES, GTP and HTS) for all other cycad genera.

Cyclone tolerance in new world arecaceae: biogeographic variation and abiotic natural selection.

BACKGROUND AND AIMS Consistent abiotic factors can affect directional selection; cyclones are abiotic phenomena with near-discrete geographic limits. The current study investigates selective pressure of cyclones on plants at the species level, testing for possible natural selection. METHODS New World Arecaceae (palms) are used as a model system, as plants with monopodial, unbranched arborescent form are most directly affected by the selective pressure of wind load. Living specimens of known provenance grown at a common site were affected by the same cyclone. Data on percentage mortality were compiled and analysed in biogeographic and phylogenetic contexts. KEY RESULTS Palms of cyclone-prone provenance exhibited a much lower (one order of magnitude) range in cyclone tolerance, and significantly lower (P < 0.001) mean percentage mortality than collections from cyclone-free areas. Palms of cyclone-free provenance had much greater variation in tolerance, and significantly greater mean percentage mortality. A test for serial independence recovered no significant phylogenetic autocorrelation of percentage mortality. CONCLUSIONS Variation in cyclone tolerance in New World Arecaceae correlates with biogeography, and is not confounded with phylogeny. These results suggest natural selection of cyclone tolerance in cyclone-prone areas.

Molecular Systematics of Threatened Seed Plant Species Endemic in the Caribbean Islands

A review of available Caribbean Island red-lists species (CR and EN categories based on the IUCN guidelines from 2001, and E category established according to the IUCN guidelines from 1980) is presented. A database of over 1,300 endemic species that are either Critically Endangered or Endangered sensu IUCN was created. There are molecular systematic studies available for 112 of them. Six of these species (in six genera) are the only members of early divergent lineages that are sister to groups composed of a large number of clades. Seven of the species (in seven genera) belong to clades that have a small number of taxa but are sister to species/genus-rich clades. Ten of the species (in six genera) are sister to taxa restricted to South America or nested in clades endemic to this region. Fifty-seven of the species (in 35 genera) are sister to Caribbean Island endemic species. Erigeron belliastroides, an Endangered (EN) Cuban endemic, is sister to the Galapagos genus Darwiniothamnus. The phylogenetic placement of four of the threatened species resulted in changes in their taxonomic placement; they belong to polyphyletic or paraphyletic genera.

A new arborescent species of Zamia from the Central Cordillera of Tolima, Colombia (Cycadales, Zamiaceae), with comments on the Z. poeppigiana species complex

Zamia tolimensis, a large arborescent species from the Central Cordillera of Tolima, Colombia, is described and illustrated. It is compared to Z. lindenii and Z. poeppigiana, the two most similar species. From these it is distinguished by leaflets with fewer teeth, sparse prickles on its petioles, beige-yellow ovulate strobili with shorter peduncles, and more microsporangia aggregated into a single group on the abaxial surface of the microsporophyll. Brief descriptions of Z. lindenii and Z. poeppigiana are also provided, as well as a key to separate the three species.

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.

A new species of Zamia from Chocó, Colombia (Cycadales, Zamiaceae)

Zamiapyrophylla, a new species from Chocó, Colombia, is described and illustrated. It is compared to Z. amazonum, Z. cunaria, and Z. ipetiensis. It is distinguished by its bright orange and red emerging leaves with leaflets that progressively turn green from the apex to the base as they mature, petioles with branched prickles and with ferruginous pubescence when immature, villous strobilar axes, and adaxial microsporangia.