M. Patrick Griffith

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.

What is the Conservation Value of a Plant in a Botanic Garden? Using Indicators to Improve Management of Ex Situ Collections

Living botanic garden plant collections are a fundamental and underutilized worldwide resource for plant conservation. A common goal in managing a botanical living collection is to maintain the greatest biodiversity at the greatest economic and logistic efficiency. However to date there is no unified strategy for managing living plants within and among botanic gardens. We propose a strategy that combines three indicators of the management priority of a collection: information on species imperilment, genetic representation, and the operational costs associated to maintaining genetic representation. In combination or alone, these indicators can be used to assay effectiveness and efficiency of living collections, and to assign a numeric conservation value to an accession. We illustrate this approach using endangered palms that have been studied to varying degrees. Management decisions can be readily extended to other species based on our indicators. Thus, the conservation value of a species can be shared through existing databases with other botanic gardens and provide a list of recommendations toward a combined management strategy for living collections. Our approach is easily implemented and well suited for decision-making by gardens and organizations interested in plant conservation.ResumenLas colecciones vivas en jardines botánicos son una parte fundamental y poco utilizada en la conservación de plantas a nivel mundial. Un objetivo común en el manejo de las colecciones botánicas vivas es mantener la mayor biodiversidad al menor costo económico y logístico. Sin embargo hasta ahora no existe una estrategia unificada para el manejo de plantas vivas dentro y entre jardines botánicos. Aquí proponemos una estrategia que combina tres indicadores para establecer prioridades de manejo de una colección: información acerca del riesgo en estado silvestre de la especie, representación genética, y los costos operativos asociados a mantener la representación genética. En combinación o solos, estos indicadores pueden ser utilizados para evaluar la efectividad y eficiencia de las colecciones vivas, y para asignar un valor numérico de conservación a un espécimen. Demostramos esta estrategia con palmas que tienen diversos tipos de estudios e información disponible. Decisiones de manejo basadas en nuestros indicadores se pueden extender y aplicar fácilmente a otras especies de manera similar a como lo demostramos aquí. Además, el valor de conservación de un especie puede ser compartido con otros jardines botánicos utilizando bases de datos pre-existentes y así proveer una serie de recomendaciones hacia el manejo integrado de las colecciones vivas. Nuestra estrategia se puede implementar fácilmente y es apropiada para la toma de decisiones en jardines y organizaciones interesadas en la conservación de recursos en plantas.

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.

Sweet Drinks Are Made of This: Conservation Genetics of an Endemic Palm Species from the Dominican Republic

Pseudophoenix ekmanii is a threatened palm species endemic to the Dominican Republic. Sap from trees is extracted to make a local drink; once they are tapped the individual usually dies. Plants are also illegally harvested for the nursery trade and destroyed by poachers hunting the endemic and threatened Hispaniolan parrot. We used 7 DNA microsatellite markers to assist land managers in developing conservation strategies for this palm. We sampled 4 populations along the known distribution range of this species (3 populations from the mainland and 1 from the small island of Isla Beata), for a total sample of n = 104. We found strong evidence for genetic drift, inbreeding, and moderate gene flow (i.e., all populations had at least 4 loci that were not in Hardy-Weinberg equilibrium, at least 9 loci pairs were in linkage disequilibrium, the pairwise F(ST) values ranged from 0.069 to 0.266, and had positive F(IS) values). Data supported an isolation-by-distance model, and cluster analyses based on genetic distances resolved 2 groups that match a north-south split. The population from Isla Beata had the lowest levels of genetic diversity and was the only one in which we found pairs of individuals with identical shared multilocus genotypes.

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.

Variation in Leaflet Structure in Cycas (Cycadales: Cycadaceae): Does Anatomy Follow Phylogeny and Geography?

Premise of research. Cycas is the earliest-diverging extant lineage in the ancient order Cycadales, well separated phyletically from the other nine extant genera. Despite the ancient status of Cycas, all extant species are no more than 12 Myr old, and many extant species have become known only in recent decades. Given this context, a broad survey of variation in Cycas leaflet structure may show structural diversity that corresponds with and informs phylogeny and biogeography. Methodology. We investigated the leaflet anatomy of 48 Cycas species grown in a common garden to ascertain the variation of anatomical traits and compared this to available phylogenetic and geographic information to determine patterns in this variation. Pivotal results. We find a very strict anatomical bauplan within the genus and only limited qualitative variation in such features as epidermal cell type, stomatal structure, and extent of hypodermal specialization. Conclusions. Our findings thus sustain the well-accepted monophyly of the genus with a consistent series of synapomorphic features. The most distinctive character is the existence of pits within the outer wall of the epidermis, a feature almost unique for gymnosperms. Unlignified midrib fibers appear to be a unique synapomorphy for Australian and Papuan members of section Cycas. Encrypted stomata and epidermal cell shape confirm a close relationship between sections Asiorientales and Panzhihuaenses and suggest a distinction from section Stangerioides but do not show a clear geographic relationship. Mucilage canals are found only within section Stangerioides. No unique synapomorphy exists for section Indosinenses, but variation in epidermal cell shape appears correlated to the presence of an adaxial hypodermis in section Indosinenses.

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.