Cryptic species diversity in a widespread neotropical tree genus: The case of Cedrela odorata.

PREMISE: Reconciling the use of taxonomy to partition morphological variation and describe genetic divergence within and among closely related species is a persistent challenge in phylogenetics. We reconstructed phylogenetic relationships among Cedrela odorata (Meliaceae) and five closely allied species to test the genetic basis for the current model of species delimitation in this economically valuable and threatened genus. METHODS: We prepared a nuclear species tree with the program SNPhylo and 16,000 single-nucleotide polymorphisms from 168 Cedrela specimens. Based on clades present and ancestral patterns ADMIXTURE, we designed nine species delimitation models and compared each model to current taxonomy with Bayes factor delimitation. Timing of major lineage divergences was estimated with the program SNAPP. RESULTS: The resulting analysis revealed that modern C. odorata evolved from two genetically distinct ancestral sources. All species delimitation models tested better fit the data than the model representing current taxonomic delimitation. Models with the greatest marginal likelihoods separated Mesoamerican C. odorata and South American C. odorata into two species and lumped C. angustifolia and C. montana as a single species. We estimated that Cedrela diversified in South America within the last 19 million years following one or more dispersal events from Mesoamerican lineages. CONCLUSIONS: Our analyses show that the present taxonomic understanding within the genus obscures divergent lineages in C. odorata due in part to morphological differentiation and taxonomic distinctions that are not predictably associated with genetic divergence. A more accurate application of taxonomy to C. odorata and related species may aid in its conservation, management, and restoration efforts.

Genomic resources for the Neotropical tree genus Cedrela (Meliaceae) and its relatives.

BACKGROUND: Tree species in the genus Cedrela P. Browne are threatened by timber overexploitation across the Neotropics. Genetic identification of processed timber can be used to supplement wood anatomy to assist in the taxonomic and source validation of protected species and populations of Cedrela. However, few genetic resources exist that enable both species and source identification of Cedrela timber products. We developed several 'omic resources including a leaf transcriptome, organelle genome (cpDNA), and diagnostic single nucleotide polymorphisms (SNPs) that may assist the classification of Cedrela specimens to species and geographic origin and enable future research on this widespread Neotropical tree genus. RESULTS: We designed hybridization capture probes to enrich for thousands of genes from both freshly preserved leaf tissue and from herbarium specimens across eight Meliaceae species. We first assembled a draft de novo transcriptome for C. odorata, and then identified putatively low-copy genes. Hybridization probes for 10,001 transcript models successfully enriched 9795 (98%) of these targets, and analysis of target capture efficiency showed that probes worked effectively for five Cedrela species, with each species showing similar mean on-target sequence yield and depth. The probes showed greater enrichment efficiency for Cedrela species relative to the other three distantly related Meliaceae species. We provide a set of candidate SNPs for species identification of four of the Cedrela species included in this analysis, and present draft chloroplast genomes for multiple individuals of eight species from four genera in the Meliaceae. CONCLUSIONS: Deforestation and illegal logging threaten forest biodiversity globally, and wood screening tools offer enforcement agencies new approaches to identify illegally harvested timber. The genomic resources described here provide the foundation required to develop genetic screening methods for Cedrela species identification and source validation. Due to their transferability across the genus and family as well as demonstrated applicability for both fresh leaves and herbarium specimens, the genomic resources described here provide additional tools for studies examining the ecology and evolutionary history of Cedrela and related species in the Meliaceae.

De novo transcriptome sequencing and SSR markers development for Cedrela balansae C.DC., a native tree species of northwest Argentina.

The endangered Cedrela balansae C.DC. (Meliaceae) is a high-value timber species with great potential for forest plantations that inhabits the tropical forests in Northwestern Argentina.Research on this species is scarce because of the limited genetic and genomic information available. Here, we explored the transcriptome of C. balansae using 454 GS FLX Titanium next-generation sequencing (NGS) technology. Following de novo assembling, we identified 27,111 non-redundant unigenes longer than 200 bp, and considered these transcripts for further downstream analysis. The functional annotation was performed searching the 27,111 unigenes against the NR-Protein and the Interproscan databases. This analysis revealed 26,977 genes with homology in at least one of the Database analyzed. Furthermore, 7,774 unigenes in 142 different active biological pathways in C. balansae were identified with the KEGG database. Moreover, after in silico analyses, we detected 2,663 simple sequence repeats (SSRs) markers. A subset of 70 SSRs related to important "stress tolerance" traits based on functional annotation evidence, were selected for wet PCR-validation in C. balansae and other Cedrela species inhabiting in northwest and northeast of Argentina (C. fissilis, C. saltensis and C. angustifolia). Successful transferability was between 77% and 93% and thanks to this study, 32 polymorphic functional SSRs for all analyzed Cedrela species are now available. The gene catalog and molecular markers obtained here represent a starting point for further research, which will assist genetic breeding programs in the Cedrela genus and will contribute to identifying key populations for its preservation.

Molecular evidence of hybrid zones of Cedrela (Meliaceae) in the Yungas of Northwestern Argentina.

In the Yungas of Northwestern Argentina, three endangered species of Cedrela (C. angustifolia, C. saltensis, and C. balansae) follow altitudinal gradients of distribution with contact zones between them. We sampled 210 individuals from 20 populations that spanned most of Cedrela's geographical range in the Yungas, and used Amplified Fragment Length Polymorphism (AFLP) markers and DNA sequences of the nuclear Internal Transcribed Spacer (ITS) to investigate hybrid zones. Data analyses employed an array of complementary methods, including principal coordinate analyses, Bayesian clustering analyses, maximum likelihood tree-building, and network techniques. Both nuclear molecular systems - AFLP and ITS - provided insights into the evolutionary history of Cedrela in the Yungas in a congruent manner. We uncovered strong support for the occurrence of natural hybridization between C. balansae and C. saltensis. Additionally, we identified hybrid zones in areas of sympatry (at both the Calilegua National Park and the San Andrés farm) and in transition zones from 820 to 1100meters above sea level (localities of Pintascayo and Acambuco). There was no evidence for hybridization of either C. balansae or C. saltensis with C. angustifolia. The role of hybrid populations in conservation and use of genetic resources in the Yungas were discussed.

Niche evolution through time and across continents: The story of Neotropical Cedrela (Meliaceae).

PREMISE OF THE STUDY: Climatic and geological changes have been considered as major drivers of biological diversification. However, it has been generally assumed that lineages retain common environmental affinities, suggesting a limited capacity to switch their climatic niche. We tested this assumption with a study of the evolution of climatic niches in the Neotropical tree genus Cedrela (Meliaceae). • METHODS: We combined distribution models of extant Cedrela with a dated molecular phylogeny based on one nuclear (ITS) and three plastid markers (psbA-trnH, trnS-G and psbB-T-N) to reconstruct the evolutionary dynamics of climatic niches. We calculated relative disparity of climatic tolerances over time to test for niche evolution within subclades or divergence between subclades and conservatism among closely related groups. Published fossil records and studies on paleosols were evaluated for the distribution and climatic conditions of extinct Cedrela. • KEY RESULTS: The fossil record of Cedrela suggested a major biome shift from paratropical conditions into warm-temperate seasonal climates in the Early Oligocene of western North America. In the Miocene, Cedrela extended from North America (John Day Formation, Oregon, USA) to southern Central America (Gatún, Panama). Diversification in the early evolutionary history was mainly driven by changes in precipitation. Temperature had an increasing impact on ecological diversification of the genus from the Miocene onwards. Sister-species comparisons revealed that recent speciation events may be related to divergence of climatic tolerances. • CONCLUSIONS: Our study highlights the complexity of climatic niche dynamics, and shows how conservatism and evolution have acted on different temporal scales and climatic parameters in Cedrela.

Tissue culture methods for the clonal propagation and genetic improvement of Spanish red cedar (Cedrela odorata).

The choice of a method to culture red cedar tissues depends on the final objectives pursued. If homogeneous clonal material is required for experimental purposes, the easiest way is to generate the lines through adventitious shoot induction from seedlings germinated from seeds. If the objective is to generate high yielding material for plantation purposes, the choice will be the same method but starting from mature vegetative tissues from selected elite plants. Most of the process are the same, but the initial steps are less efficient and much more elaborate. If the purpose is to generate lines with new genetic characteristics through somaclonal variation, mutagenesis, or genetic transformation, somatic embryogenesis will be required. No single method in its present form is suitable for all purposes. Eventually, the efficient production of somatic embryos from rejuvenated shoots collected from mature selected plants is the ideal way to culture this species, but for the time being we have to choose one or the other. In this chapter, we present a grafting procedure to rejuvenate and maintain mother plants in the greenhouse and the in vitro culture systems we have developed for the production of Cedrela odorata propagules using explants from both young seedlings and mature tissues from selected old trees. Using a modified TY17 medium and the BioMINT(®) temporary immersion system, we obtained high multiplication and ex vitro transplantation rates for efficient large-scale propagation of this species.

Molecular evidence of cryptic speciation, historical range expansion, and recent intraspecific hybridization in the Neotropical seasonal forest tree Cedrela fissilis (Meliaceae).

Molecular phylogeography can lead to a better understanding of the interaction between past climate events, large-scale vegetation shifts, and the evolutionary history of Neotropical seasonal forests. The endangered timber tree species Cedrela fissilis is associated with seasonal forests and occurs throughout South America. We sampled C. fissilis from 56 sites across the species' range in Brazil and Bolivia and obtained sequence data for nuclear and chloroplast DNA. Most specimens (149 out of 169) exhibited intraindividual polymorphism for the nuclear internal transcribed spacer (ITS). Cloning and an array of complementary sequence analyses indicated that the multiple copies of ITS were functional paralogs--concerted evolution in C. fissilis appeared to be incomplete. Independent Bayesian analyses using either ITS or cpDNA data revealed two separate phylogenetic lineages within C. fissilis that corresponded to populations located in separate geographic regions. The divergence occurred in the Early Pliocene and Late Miocene. We argue that climate-mediated events triggered dispersal events and split ancestral populations into at least two large refugial areas of seasonal forest that were located to the east and west of the present day Cerrado. Upon recent climate amelioration, formerly isolated lineages reconnected and intraspecific hybridization gave rise to intraindividual polymorphism and incomplete concerted evolution in C. fissilis.

Molecular phylogenetics of Neotropical Cedreleae (mahogany family, Meliaceae) based on nuclear and plastid DNA sequences reveal multiple origins of "Cedrela odorata".

This study focuses on the 17 Neotropical species described in Cedrela (Meliaceae; Cedreleae), in particular C. odorata, which has been shown in past population genetic studies to be more variable than other tree species. We sampled two sets of molecular markers, nuclear ribosomal spacers (nrITS), and several plastid regions (the psbB, psbN, psbT exons and the trnS-trnG spacer). Both sets of markers produced congruent results using parsimony, maximum likelihood and Bayesian methods for a set of taxa that include outgroups (outside Cedreleae) as well as other members of the related genera within Cedreleae. All results confirm current delimitation of Cedreleae and genetic distinctiveness for four new species with divergent morphologies. Our study also provides evidence that there are several genetic entities among the accessions of C. odorata that have distinct ranges or habitat preferences. These entities may constitute new species, are morphologically cryptic and require a great deal more investigation to delimit their ranges. Our results have important implications for conservation of Cedrela odorata, which is heavily harvested throughout its range in the American tropics.

The tropical cedar tree (Cedrela fissilis Vell., Meliaceae) homolog of the Arabidopsis LEAFY gene is expressed in reproductive tissues and can complement Arabidopsis leafy mutants.

A homolog of FLORICAULA/LEAFY, CfLFY (for Cedrela fissilis LFY), was isolated from tropical cedar. The main stages of the reproductive development in C. fissilis were documented by scanning electron microscopy and the expression patterns of CfLFY were studied during the differentiation of the floral meristems. Furthermore, the biological role of the CfLFY gene was assessed using transgenic Arabidopsis plants. CfLFY showed a high degree of similarity to other plant homologs of FLO/LFY. Southern analysis showed that CfLFY is a single-copy gene in the tropical cedar genome. Northern blot analysis and in situ hybridization results showed that CfLFY was expressed in the reproductive buds during the transition from vegetative to reproductive growth, as well as in floral meristems and floral organs but was excluded from the vegetative apex and leaves. Transgenic Arabidopsis lfy26 mutant lines expressing the CfLFY coding region, under the control of the LFY promoter, showed restored wild-type phenotype. Taken together, our results suggest that CfLFY is a FLO/LFY homolog probably involved in the control of tropical cedar reproductive development.

Chloroplast DNA phylogeography reveals colonization history of a Neotropical tree, Cedrela odorata L., in Mesoamerica.

Spanish Cedar (Cedrela odorata L.) is a globally important timber species which has been severely exploited in Mesoamerica for over 200 years. Using polymerase chain reaction-restriction fragment length polymorphisms, its chloroplast (cp) DNA phylogeography was studied in Mesoamerica with samples from 29 populations in six countries. Five haplotypes were characterized, phylogenetically grouped into three lineages (Northern, Central and Southern). Spatial analysis of ordered genetic distance confirmed deviation from a pattern of isolation by distance. The geographically proximate Northern and Central cpDNA lineages were genetically the most differentiated, with the Southern lineage appearing between them on a minimum spanning tree. However, populations possessing Southern lineage haplotypes occupy distinct moist habitats, in contrast to populations possessing Northern and Central lineage haplotypes which occupy drier and more seasonal habitats. Given the known colonization of the proto-Mesoamerican peninsula by South American flora and fauna prior to the formation of the Isthmus of Panama, it seems most likely that the observed population structure in C. odorata results from repeated colonization of Mesoamerica from South American source populations. Such a model would imply an ancient, pre-Isthmian colonization of a dry-adapted type (possessing the Northern lineage or a prototype thereof), with a secondary colonization via the land bridge. Following this, a more recent (possibly post-Pleistocene) expansion of moist-adapted types possessing the Southern lineage from the south fits the known vegetation history of the region.