AbstractUnsustainable wildlife trade is a major contributor to biodiversity loss; however, trade regulations have failed to prevent the decline of high‐profile species. Where wildlife is traded legally, opportunities exist to launder protected species through legal channels. The legal commercialization of olive ridley sea turtle eggs from Ostional, Costa Rica, has been criticized with some suggesting that the legal trade stimulates illegal extraction and sale of eggs. We aimed to identify whether the traceability rules, under which the Ostional project operates, were suitably robust. We surveyed markets across Costa Rica, by purchasing openly available sea turtle eggs and recording qualitative and quantitative data at the point of sale. We found that 378 (80%) of turtle eggs openly sold in the market were from olive ridley sea turtles. Green (n = 5) and leatherback (n = 6) turtle eggs were only on offer on three occasions, but no vendor referred to Ostional. Vendors frequently breached trade regulations, which appeared to be due to these regulations misaligning with consumer demand. Although the Ostional traceability rules are regularly flouted, we found no evidence that Ostional is being used as a cover to sell eggs from other turtle species.

Introduction: A recent revision of the generic classification of the Trochilidae based on DNA sequences revealed many inconsistencies with the current generic classification, largely based on plumage characters subject to homoplasy, especially in the Trochilini, the largest tribe. A thorough generic reorganization brought the classification into accord with the phylogeny, but due to lack of genetic data, two species remained unclassified. One of these was the Mangrove Hummingbird, “Amazilia” boucardi, endemic to Costa Rica and included in the IUCN red list of threatened species. Objective: To obtain molecular evidence to clarify the generic relationships of “A.” boucardi. Methods: We isolated DNA from tissues of this species and amplified 4 nuclear and 4 mitochondrial fragments and compared these with homologous fragments from 56 species in the Trochilini, constructing phylogenetic trees with maximum likelihood and Bayesian methods. Results: Our phylogenetic analyses confirmed the placement of boucardi in the Trochilini and definitely excluded it from Amazilia but placed it with high confidence in the genus Chrysuronia Bonaparte, 1850, within which its closest relative is C. coeruleogularis, which also inhabits mangroves. Conclusions: Our genetic data based on nuclear and mitochondrial regions clearly indicate the relationship of A. boucardi and L. coeruleogularis. Moreover, it is also support by their habitat distribution in the mangroves of the Pacific coast of Costa Rica and Western Panama. Therefore, we suggested to exclude A. boucardi as "incertae sedis".

Fruits of the cherimoya tree (Annona cherimola Mill.) were consumed by native cultures in Central America, from where the crop was disseminated to South America in pre-Columbian times. Despite its historical significance and cultivation in subtropical regions worldwide, cherimoya remains an underutilized crop, particularly in its area of origin where the conservation of its genetic diversity is under threat. In this study, we provide a comprehensive overview of the research performed on this fruit tree over the past two decades, shedding light on its current status in terms of commercial production and germplasm conservation efforts in the main cherimoya producing countries in the Americas and Europe.

A molecular genetic approach was used to elucidate the phylogeographic relationships of the clover grass (Halophila baillonii Asch.) from three key regions within its current distributional range. Halophila baillonii is a small seagrass that has historically been only found in a few locations in the Caribbean and Atlantic coast of Brazil. In the past few decades H. baillonii has also been observed in the Eastern Tropical Pacific (ETP). Very little is known about the relationship between the ETP populations and the Caribbean and Atlantic ones. To study their relationship, we used a hybrid capture approach targeting chloroplast loci on samples from Belize, Brazil, and the Pacific coast of Costa Rica. Phylogenetic analyses resolved H. baillonii as monophyletic and placed the samples from Belize and Costa Rica in a clade sister to the Brazilian ones. The results clearly indicate that the ETP samples are very closely related to Belize and that the most likely explanation of its occurrence in the ETP is a recent introduction, possibly as a consequence of the opening of the Panama Canal.

The Orthotospovirus genus encompasses a range of economically significant and emerging plant viruses that affect a variety of crops globally. While the prevalence and characteristics of these phytopathogenic viruses are extensively documented in North and South America, their presence in Central America remains comparatively underexplored. This study focuses on Costa Rica, strategically positioned at the nexus of North and South America, to enhance our understanding of orthotospovirus in this region. We analyzed 295 plant samples using enzyme-linked immunosorbent assay (ELISA) to test for the presence of INSV, IYSV, TSWV, and the GRSV/TCSV serogroup. Additionally, a subset (20 samples) underwent further scrutiny through reverse transcription-polymerase chain reaction (RT-PCR) employing both universal and species-specific primers. Our ELISA results indicated the absence of TSWV and the GRSV/TCSV serogroup. However, the presence of INSV in Costa Rica was substantiated through ELISA, RT-PCR, and partial sequencing, revealing its prevalence in both open-field and greenhouse environments. Despite previous diagnostic reports suggesting the presence of TSWV in Costa Rica, our study did not detect this virus. RT-PCR analysis with degenerate primers also found no evidence of other orthotospovirus species in our samples. The identification of a dominant INSV haplotype, along with three additional variants, suggests the likelihood of at least two independent virus introductions into the region. These findings underscore the necessity for more comprehensive surveys and research on orthotospoviruses in Central America to better understand their epidemiology and impact on agriculture.

Previously developed Annona cherimola specific primers based on the plant barcode gene matK were tested in 546 cherimoya accessions. Of those, 296 belong to an ex situ world reference germplasm collection maintained at the IHSM La Mayora-CSIC-UMA in Málaga (Spain) and 250 to cherimoya samples collected in situ in three Central American countries (Honduras, Guatemala and Costa Rica). Results showed the existence of two different haplotypes in the samples analyzed with geographical association. A new marker that amplified all the samples was designed based on the trnL-F locus using DNA information from seven species of the Annona genus. The information generated will be useful to analyze and conserve the extant genetic diversity present in situ and ex situ and to understand de dispersion of the crop from its center of origin in Central America.

Defining demographically independent units and understanding patterns of gene flow between them is essential for managing and conserving exploited populations. The critically endangered scalloped hammerhead shark, Sphyrna lewini, is a coastal semi-oceanic species found worldwide in tropical and subtropical waters. Pregnant females give birth in shallow coastal estuarine habitats that serve as nursery grounds for neonates and small juveniles, whereas adults move offshore and become highly migratory. We evaluated the population structure and connectivity of S. lewini in coastal areas and one oceanic island (Cocos Island) across the Eastern Tropical Pacific (ETP) using both sequences of the mitochondrial DNA control region (mtCR) and 9 nuclear-encoded microsatellite loci. The mtCR defined two genetically discrete groups: one in the Mexican Pacific and another one in the central-southern Eastern Tropical Pacific (Guatemala, Costa Rica, Panama, and Colombia). Overall, the mtCR data showed low levels of haplotype diversity ranging from 0.000 to 0.608, while nucleotide diversity ranged from 0.000 to 0.0015. More fine-grade population structure was detected using microsatellite loci where Guatemala, Costa Rica, and Panama differed significantly. Relatedness analysis revealed that individuals within nursery areas were more closely related than expected by chance, suggesting that S. lewini may exhibit reproductive philopatric behaviour within the ETP. Findings of at least two different management units, and evidence of philopatric behaviour call for intensive conservation actions for this highly threatened species in the ETP.

Plants are constantly exposed to biotic stress factors that affect their growth, development, and productivity. This interaction involves complex molecular mechanisms of resistance, tolerance, susceptibility, and sensibility. Po-tato is an important crop around the world, not only as a commodity but also as crop model, so to understand its gene regulation is particularly useful. The role of Transcription Factors (TFs) in gene regulation in biotic stress response in potato was not summarized and analyzed in any previous review. In this article, an inventory of potato TFs involved in biotic defenses was retrieved. Nine (ARF, NAC, WRYK, AP2/ERF-ERF, AP2/ERF-DREB, ZFP, TCP, bZIP, and BELL) from 67 TFs families were identified as having a role in defensive responses in potato. Activation/inactivation of such TFs triggers diverse metabolic pathways allowing plants to produce defensive proteins, metabolites, plant hormones, and/or transcriptional and post transcriptional modifications. Also, in this review, the phylogeny of StARF6, StARF17, and StWRKY8 was studied. An alignment and its comparison between amino acid sequences with related and distant species were conducted. As conclusions, our findings suggest divergence by functional approaches since we found they were related to other biotic response genes as well with constitutive genes for growth and development, being necessary to investigate more about homology and orthology to define more accurately response patterns among multiple species. The regulatory mechanisms reported include regulation at the transcriptional level through growth regulators and post-transcriptional regulation through modifications that inactivate or activate the resulting protein. The overexpression and activity depend not only on the number of copies of a gene but also on the elements present in its structure and the interaction/dimerization with other proteins. Synteny studies might be further conducted in potato because most of the evidence is focused on homology between species or emphasis on other Solanaceae. Analyses of potato TFs will help to understand the evolutionary and domestication processes, as well as provide useful knowledge for potato breeding programs aiming biotic stress resistance to increase productivity.

The aim of this study was to design a Trichoderma atroviride-specific qPCR oligo set, evaluate its specificity, and standardize a methodology that quantifies antagonism against Botrytis cinerea in blackberry fruits (Rubus adenotrichos Schltdl.). Primers and probe were designed based on the nuclear translation elongation factor 1-alpha (tef1-α) of T. atroviride. A commercial IGS-based oligo set was used to quantify B. cinerea. The specificity of the designed oligo set, along with ITS-based oligo sets, was assessed using other Trichoderma species and B. cinerea. Multiplex qPCR assays were performed using DNA from B. cinerea, T. atroviride, and blackberries inoculated with these fungi. Assays with the tef1-α oligo set showed high sensitivity and reproducibility. In inoculated fruits, T. atroviride and B. cinerea were quantified simultaneously, including in symptomless tissues. This work standardized a qPCR methodology that specifically targets a T. atroviride isolate. This newly-designed qPCR oligo set could be useful in future biological control programs.

Annona cherimola is a woody perennial species in the Annonaceae family that produces edible fruits and has economic importance in several regions of the world with subtropical climates. Together with other 10-12 species, A. cherimola belongs to the section Atta of the Annona genus with a center of origin in Central America and the Caribbean. Species of the section Atta produce soft skin ripe fruits with raised areoles bounded by recessed furrows. Annona cherimola is the only species of the section naturally found in the Andean region of South America. Currently, no information is available at the molecular level on the phylogenetic relationships of most of the species of Atta and closely related sections in Annona. In order to fill this gap, in this work a phylogenetic approach was performed using five coding and non-coding plastid DNA regions, to determine the phylogenetic relationships between A. cherimola and other related species included in Atta and other sections of the genus. The results obtained support recent studies that demonstrated the likely Mesoamerican origin of A. cherimola based on biogeographical analysis with SSR markers, rather than the previously considered South American origin hypothesis. In addition, the species belonging to the Atta section did not show monophyly. Finally, A. cherimola and A. pruinosa seem to be phylogenetically close species and additional studies are needed to discern the relations between them.

Phytoplasmas (class Mollicutes) are causal agents of plant diseases with an economic impact on crops or threatening local biodiversity. A survey was conducted from 2012 to 2016 on infected Catharanthus roseus plants that exhibited symptoms reminiscent of phytoplasma infection throughout Costa Rica. A total of 73 plants were collected exhibiting symptoms such as virescence, phyllody, axillary proliferation, little leaf, leaf malformation, chlorosis, or yellowing. All samples were tested by nested PCR using phytoplasma universal and specific primer pairs. Phytoplasma infection was detected in 52 (71.2 %) of the plants collected. Phytoplasmas of six subgroups belonging to 16Sr groups I, III, IX, XIII and XV were identified based on sequencing and in silico RFLP analyses. ´Candidatus Phytoplasma asteris´ (16SrI) was the predominant group among the positive samples (n = 30) showing variety of symptoms and wide distribution from sea level to ca. 1 400 masl in six of the seven Costa Rican provinces. Group 16SrIII was the second most abundant (14 samples); and the remaining three groups were seldom found in C. roseus (8 samples). Moreover, group 16SrXIII phytoplasma was detected for the first time in the country. To the best of our knowledge, this is the first report of natural infection of C. roseus with phytoplasma subgroups 16SrI-B, 16SrI-P, 16SrIII-F, 16SrIX-F, 16SrXIII-A, and 16SrXV-B in Costa Rica and Central America.

Comparative analysis of metabolic features of plants has a high potential for determination of quality control of active ingredients, ecological or chemotaxonomic purposes. Specifically, the development of efficient and rapid analytical tools that allow the differentiation among species, subspecies and varieties of plants is a relevant issue. Here we describe a multivariate model based on LC–MS/MS fingerprinting capable of discriminating between subspecies and varieties of the medicinal plant Chamaecrista nictitans, a rare distributed species in Costa Rica.

Knowledge on the structure and distribution of genetic diversity is a key aspect to plan and execute an efficient conservation and utilization of the genetic resources of any crop as well as for determining historical demographic inferences. In this work, a large data set of 1,765 accessions of cherimoya (Annona cherimola Mill, Annonaceae), an underutilized fruit tree crop native to the Neotropics and used as a food source by pre-Columbian cultures, was collected from six different countries across the American continent and amplified with nine highly informative microsatellite markers. The structure analyses, fine representation of the genetic diversity and an ABC approach suggest a Mesoamerican origin of the crop, contrary to previous reports, with clear implications for the dispersion of plant germplasm between Central and South America in pre-Columbian times. These results together with the potential distribution of the species in a climatic change context using two different climate models provide new insights for the history and conservation of extant genetic resources of cherimoya that can be applied to other currently underutilized woody perennial crops.

The phylogenetic relationships within Specklinia (Pleurothallidinae; Orchidaceae) and related genera are re-evaluated using Bayesian analyses of nrITS and chloroplast matK sequence data of a wide sampling of species. Specklinia is found paraphyletic in the DNA based trees, with species alternatively assigned to Muscarella proven distinct, monophyletic and easily recognizable. Specklinia as such includes about 100 morphologically highly diverse species. Their phenotypic differences have prompted the creation of up to eleven generic names within this relatively small group. Here we show not only that these morphologically divergent species are closely related, but also that they can still be recognized by certain conserved morphological traits. The genera Acostaea, Areldia, Empusella, Cucumeria, Gerardoa, Pseudoctomeria, Sarcinula, Sylphia, Tribulago and Tridelta are found embedded within Specklinia, and therefore reduced under the synonymy of the latter. Specklinia is confirmed as sister to a clade that includes Platystele, Scaphosepalum and Teagueia. Five well-supported subgenera are proposed for Specklinia and are characterized both geographically and morphologically. The species belonging to each subgenus are listed. Incaea is synonymized with Dryadella, while Rubellia is reduced under Platystele. New combinations for several species are proposed. The criteria for the generic delimitation of Specklinia and other genera in the Pleurothallidinae are discussed.

Sideroxylon capiri (“Tempisque”) is a threatened and economically important timber species in the dry forest of Costa Rica. To assess the extent of the genetic diversity between and within populations of this species, 86 samples were obtained from four sites in the northwestern part of the country from protected and non-protected areas. They were analyzed by amplified fragment length polymorphism. Five primers were used to generate 254 polymorphic bands. Molecular variance indicated a 92 % within locations with a PhiPT (φ st) of 0.083 and a He from 0.204 to 0.249. PCoA analysis showed two different groups: one formed by Palo Verde and La Cruz samples, and another group with Nicoya and Barra Honda samples. The dendrogram arranged the samples in three groups: individuals from Palo Verde and La Cruz divided in two groups, and a more compact group from Barra Honda and Nicoya. A positive correlation between pairwise linearized geographical distance and genetic differentiation among populations was detected by Mantel test (R 2 = 0.76241, P = 0.04). Results are discussed in terms of fragmentation of the continuous forests and geographical barriers. Despite these results, it is evident that a great genetic diversity exists for this species and the results can be used for conservation purposes.

The aim of this research was the genetic characterization of 218 accessions of Cucurbita moschata Duchesne, a squash, and its relationship with morphological characteristics of agronomic interest, which are part of the international collection conserved at Tropical Agricultural Research and Higher Education Center (CATIE), Costa Rica. The majority of the accessions came from Mexico and Central America: single genotypes from Cur zeta ao, Colombia, Peru and the Russian Federation were also included. The polymerase chain reaction (PCR) and single strand conformation polymorphism analysis (SSCP) were used for the analysis of the regions amplified with ITS1-ITS2 nuclear primers and tRNL-F chloroplast primers. Haplotypes were constructed according to band patterns in SSCP gels. Twenty-five haplotypes were found using the ITS1-ITS2 markers, and 24 haplotypes were found with the tRNL-F markers. Unique haplotypes were found with both markers. Two individuals of each tRNL-F haplotype were sequenced. The results indicated a high level of genetic diversity in CATIE squash collection. Using ITS1-ITS2 primers, it was found that the number of haplotypes was independent of the geographical source of the accession, and haplotypes were distributed randomly throughout the study area. Mexico had the highest values of total heterozygosity (HE), genetic diversity (H) and Shannon index (I) while Panama showed the lowest values. Sequences obtained from tRNL-F intergenic marker showed the highest diversity index values were present in the group of additional sequences and Mexico, and lower values were observed for Nicaragua. Guatemala and Panama. PCoA based on morphological data showed three groups and by ANOSIM (R) all group differences were significant. Results obtained in this study suggest that high diversity is a characteristic of C. moschata from Mesoamerica. (C) 2011 Elsevier B.V. All rights reserved.

Squash (Cucurbita moschata) is one of the most important crops in tropical countries. Geminiviruses are an important group of plant pathogens. In 2002 a new begomovirus was reported to naturally infect squash and some other crops in Costa Rica. Our objective was to compare, using molecular techniques, the extraction and further purification of DNA from squash by different extraction protocols and storage methods. A single infected sample was collected, half of the material was stored frozen at -70 degrees C, and the remainder was stored dehydrated in silica gel (SG). Total nucleic acids (TNAs) were extracted by three different protocols and were quantified by fluorometry, and the quality was analysed by electrophoresis in agarose gels, polymerase chain reaction (PCR) of the virus genome, dot blot and Southern blot hybridization. Even though the tissue stored in SG yielded a higher amount of TNAs, the genetic material exhibited lower integrity and this made it useful exclusively for the detection of geminiviral DNA by PCR amplification of short viral sequences and by hybridization with short viral probes. The Dellaporta method proved to be the most effective for the detection of geminiviral DNA in infected squash tissue. Although the cetyltrimethylammonium bromide method showed similar results, the procedure is more time-consuming. Surprisingly, the citrate method showed either similar or worse results than the other methods.

Phragmipedium longifolium is an endangered terrestrial orchid. In Costa Rica, these plants are found growing in small and isolated patches, some of them consisting of just four to six individuals. Information about the genetic variability within and among populations is very important for the conservation of this endangered species. A total of 160 samples were collected in six locations and analyzed with amplified fragment length polymorphism technique. The genetic diversity of P. longifolium in Costa Rica (Hw = 0.1711) is high and differentiation among sampled locations is moderate (φpt = 0.2013) in comparison with results of studies in some other terrestrial orchid species using the same technique. The percentage of polymorphic loci was, on average, 51.5. The analysis of molecular variance (AMOVA) indicated the main genetic variation was within sampled locations (80%), even though the variation among locations was also significant. In situ conservation is recommended because, in addition to protecting habitat and avoiding fragmentation, mycorrhizal fungi and pollinators are also protected. Close proximity between populations is required to maintain high genetic variability through a gene flow continuum. It is suggested that conservation of patches with higher genetic variability be prioritized as many are located in unprotected areas. A germplasm bank for ex situ conservation has been established in the living collection at Lankester Botanical Garden using the plants collected in this study. Finally, a search for new locations of this species is also suggested.

Maize rayado fino virus (MRFV) infects maize and appears to be restricted to, yet widespread in, the Americas. MRFV was previously unreported from Ecuador. Maize plants exhibiting symptoms of MRFV infection were collected at the Santa Catalina experiment station in Quito, Ecuador. RT-PCR reactions were performed on total RNA extracted from the symptomatic leaves using primers specific for the capsid protein (CP) gene and 3' non-translated region of MRFV and first strand cDNA as a template. Nucleotide sequence comparisons to previously sequenced MRFV isolates from other geographic regions revealed 88-91% sequence identity. Phylogenetic trees constructed using Maximum Likelihood, UPGMA, Minimal Evolution, Neighbor Joining, and Maximum Parsimony methods separated the MRFV isolates into four groups. These groups may represent geographic isolation generated by the mountainous chains of the American continent. Analysis of the sequences and the genetic distances among the different isolates suggests that MRFV may have originated in Mexico and/or Guatemala and from there it dispersed to the rest of the Americas.

The diversity of 42 Xylella fastidiosa strains from Costa Rica, São Paulo, Brazil, and the United States were analyzed using the sequence of the 16S rRNA gene by variable number of tandem repeat (VNTR) fragment analysis and by restriction fragment length polymorphisms (RFLP) of a specific polymerase chain reaction (PCR)-amplification product using enzyme CfoI. Limited variability in the sequence of the 16S rRNA gene was observed and, although the separation was not absolute, most strains from Costa Rica clustered with strains from the United States and not with strains from São Paulo. The PCR-RFLP produced different patterns of DNA bands. The same pattern was shared by strains from Costa Rica, the United States, and two coffee strains from São Paulo, but a different pattern was observed in six coffee and orange strains from Brazil. In all, 32 amplification products were scored in the VNTR fragment analysis. The total variation observed among the X. fastidiosa strains had significant (P < 0.001) contributions from both geography and host origin as inferred by Nei's values of genetic diversity and WINAMOVA statistics. The strains from Costa Rica were isolated from diseased grapevines, coffee, and sweet orange and these strains grouped together and could be distinguished from strains from grapevine from the United States or from either coffee or sweet orange from São Paulo. The strains tested from Costa Rica are most likely of local origin, although the possibility that they have been introduced along with horticultural crops cannot be excluded. In either case, they are examples of independent selection of strains of X. fastidiosa affecting coffee and sweet orange. Greater genetic similarity was observed between strains from Costa Rica and the United States than with those from São Paulo.