Using ddRADseq to assess the genetic diversity of in-farm and gene bank cacao resources in the Baracoa region, eastern Cuba, for use and conservation purposes.

The Baracoa region, eastern Cuba, hosts around 80 % of the country cacao (Theobroma cacao L.) plantations. Cacao plants in farms are diverse in origin and propagation, with grafted and hybrid plants being the more common ones. Less frequent are plants from cuttings, TSH progeny, and traditional Cuban cacao. A national cacao gene bank is also present in Baracoa, with 282 accessions either prospected in Cuba or introduced from other countries. A breeding program associated with the gene bank started in the 1990s based on agro-morphological descriptors. The genetic diversity of cacao resources in Baracoa has been poorly described, except for traditional Cuban cacao, affecting the proper development of the breeding program and the cacao planting policies in the region. To assess the population structure and genetic diversity of cacao resources in Baracoa region, we genotyped plants from both cacao gene bank (CG) and cacao farms (CF) applying a new ddRADseq protocol for cacao. After data processing, two SNPs datasets containing 11,425 and 6,481 high-quality SNPs were generated with 238 CG and 135 CF plants, respectively. SNPs were unevenly distributed along the 10 cacao chromosomes and laid mainly in noncoding regions of the genome. Population structure analysis with these SNP datasets identified seven and four genetic groups in CG and CF samples, respectively. Clustering using UPGMA and principal component analysis mostly agree with population structure results. Amelonado was the predominant cacao ancestry, accounting for 49.22 % (CG) and 57.73 % (CF) of the total. Criollo, Contamana, Iquitos, and Nanay ancestries were detected in both CG and CF samples, while Nacional and Marañon backgrounds were only identified in CG. Genetic differentiation among CG (FST ranging from 0.071 to 0.407) was higher than among CF genetic groups (FST: 0.093-0.282). Genetic diversity parameters showed similar values for CG and CF samples. The CG and CF genetic groups with the lowest genetic diversity parameters had the highest proportion of Amelonado ancestry. These results should contribute to reinforcing the ongoing breeding program and updating the planting policies on cacao farms, with an impact on the social and economic life of the region.

High levels of population genetic differentiation in the American crocodile (Crocodylus acutus).

The American crocodile (Crocodylus acutus) is a widely distributed species across coastal and brackish areas of the Neotropical region of the Americas and the Greater Antilles. Available information on patterns of genetic differentiation in C. acutus shows a complex structuring influenced by interspecific interactions (mainly hybridization) and anthropogenic actions (mostly historical hunting, recent poaching, habitat loss and fragmentation, and unintentional translocation of individuals). In this study, we used data on mitochondrial DNA control region and 11 nuclear polymorphic microsatellite loci to assess the degree of population structure of C. acutus in South America, North America, Central America and the Greater Antilles. We used traditional genetic differentiation indices, Bayesian clustering and multivariate methods to create a more comprehensive picture of the genetic relationships within the species across its range. Analyses of mtDNA and microsatellite loci show evidence of a strong population genetic structure in the American crocodile, with unique populations in each sampling locality. Our results support previous findings showing large degrees of genetic differentiation between the continental and the Greater Antillean C. acutus. We report three new haplotypes unique to Venezuela, which are considerably less distant from the Central and North American haplotypes than to the Greater Antillean ones. Our findings reveal genetic population differentiation between Cuban and Jamaican C. acutus and offer the first evidence of strong genetic differentiation among the populations of Greater Antillean C. acutus.

Founded: Genetic Reconstruction of Lineage Diversity and Kinship Informs Ex situ Conservation of Cuban Amazon Parrots (Amazona leucocephala).

Captive breeding is a widespread conservation strategy, yet such programs rarely include empirical genetic data for assessing management assumptions and meeting conservation goals. Cuban Amazon parrots (Amazona leucocephala) are considered vulnerable, and multiple on-island captive populations have been established from wild-caught and confiscated individuals of unknown ancestry. Here, we used mitochondrial haplotypic and nuclear genotypic data at 9 microsatellite loci to quantify the extent and distribution of genetic variation within and among captive populations in Zapata Swamp and Managua, Cuba, and to estimate kinship among breeders (n = 88). Using Bayesian clustering analysis, we detected 2 distinct clusters within the Zapata population, one of which was shared with Managua. Individuals from the cluster unique to Zapata possessed mitochondrial haplotypes with affinities to Cuban subspecies (A. l. leucocephala, A. l. palmarum); the shared cluster was similar, but also included haplotypes closely related to the subspecies restricted to Cayman Brac (A. l. hesterna). Overall mean kinship was low within each captive population (-0.026 to -0.012), with 19 and 11 recommended breeding pairs in Zapata and Managua, respectively, ranked according to mean kinship and informed by molecular sexing. Our results highlight the importance of understanding population history within ex situ management programs, while providing genetic information to directly inform Cuban parrot conservation.

Comparative proteomic analysis of male and female venoms from the Cuban scorpion Rhopalurus junceus.

A complete mass spectrometry analysis of venom components from male and female scorpions of the species Rhophalurus junceus of Cuba is reported. In the order of 200 individual molecular masses were identified in both venoms, from which 63 are identical in male and females genders. It means that a significant difference of venom components exists between individuals of different sexes, but the most abundant components are present in both sexes. The relative abundance of identical components is different among the genders. Three well defined groups of different peptides were separated and identified. The first group corresponds to peptides with molecular masses of 1000-2000 Da; the second to peptides with 3500-4500 Da molecular weight, and the third with 6500-8000 Da molecular weights. A total of 86 peptides rich in disulfide bridges were found in the venoms, 27 with three disulfide bridges and 59 with four disulfide bridges. LC-MS/MS analysis allowed the identification and amino acid sequence determination of 31 novel peptides in male venom. Two new putative K(+)-channel peptides were sequences by Edman degradation. They contain 37 amino acid residues, packed by three disulfide bridges and were assigned the systematic numbers: α-KTx 1.18 and α-KTx 2.15.

Caracterización morfológica y evaluación de resistencia a Fusarium oxysporum en especies silvestres del género Solanum sección Lycopersicon / Morphological characterization and resistance evaluation to Fusarium oxysporum in wild species Solanum genera Lycopersicon section

Con el objetivo de evaluar la variación morfológica y la resistencia a Fusarium oxysporum en la colección del banco de germoplasma de la Universidad Nacional de Loja (UNL), 146 accesiones de cuatro especies silvestres fueron seleccionadas (Solanum pimpinellifolium, S. neorickii, S. habrochaites, S. lycopersicum var. cerasiforme); y, 20 accesiones de tomate cultivado (S. lycopersicum). Un total de 723 plántulas se transplantaron al campo y se evaluaron morfológicamente durante todo su ciclo biológico, con un descriptor que incluyó 20 caracteres cuantitativos y 25 cualitativos. Se estimó la variabilidad morfológica y se detectaron diferencias fenotípicas relacionadas con el fruto, características vegetativas de las plantas y los componentes del rendimiento. Las variables morfológicas mostraron que S. habrochaites es la especie más diferenciada con relación a las restantes. La podredumbre vascular causada por F. oxysporum es responsable de enormes pérdidas en la producción de tomate en el mundo; sin embargo, las especies silvestres que originaron las variedades cultivadas poseen genes de interés agronómico, utilizados por décadas para la generación de variedades resistentes. Por ello, entre 64 y 228 individuos por especie, se evaluaron por tres procedimientos de inoculación. Todas las especies fueron significativamente diferentes en los niveles de resistencia, tolerancia o susceptibilidad. S. neorickii fue más resistente y tolerante, seguida de S. pimpinellifolium y S. lycopersicum var. cerasiforme, mientras que todas las variedades cultivadas (S. lycopersicum) fueron susceptibles. Tales circunstancias permitieron determinar sin lugar a dudas, que las plantas sobrevivientes y tolerantes poseen un genotipo particular que determina esa característica.

In order to evaluate the morphological variation and Fusarium oxysporum resistance in the National University of Loja (UNL) genebank collection, 146 wild accessions of four species were selected (Solanum pimpinellifolium, S. neorickii, S. habrochaites , S. lycopersicum var. cerasiforme) and 20 accessions of cultivated tomato (S. lycopersicum). For a total of 723 seedlings were transplanted to the field and evaluated morphologically throughout its life cycle, with a descriptor that included 20 quantitative and 25 qualitative characters. Morphological variability was estimated were detected phenotypic differences related the fruit, plant vegetative characteristics and yield components. Morphological variables showed that S. habrochaites is the most differentiated with respect to the others. Vascular rot caused by F. oxysporum is responsible for huge losses in tomato production in the world, but the wild species that gave rise to the cultivated varieties have agronomic interest genes used for decades to generate resistant varieties. Therefore, between 64 and 228 individuals per species were evaluated for three inoculation procedures. All species were significantly different between levels of resistance, susceptibility or tolerance. S. neorickii was more resistant and tolerant, followed by S. pimpinellifolium and S. lycopersicum var. cerasiforme, while all cultivars (S. lycopersicum) were susceptible. Such circumstances allowed undoubtedly determine that survivors and tolerant plants have a particular genotype that determines the characteristic.

A K⁺ channel blocking peptide from the Cuban scorpion Rhopalurus garridoi.

A proteomic analysis of the venom obtained from the Cuban scorpion Rhopalurus garridoi was performed. Venom was obtained by electrical stimulation, separated by high performance liquid chromatography, and the molecular masses of their 50 protein components were identified by mass spectrometry. A peptide of 3940 Da molecular mass was obtained in pure form and its primary structure determined. It contains 37 amino acid residues, including three disulfide bridges. Electrophysiological experiments showed that this peptide is capable of blocking reversibly K(+)-channels hKv1.1 with a Kd close to 1 µM, but is not effective against hKv1.4, hERG1 and EAG currents, at the same concentration. This is the first protein component ever isolated from this species of scorpion and was assigned the systematic number α-KTx 2.14.

Nesting ecology of Chelonia mydas Testudines: Cheloniidae on the Guanahacabibes Peninsula, Cuba

The nesting colony of green sea turtles Chelonia mydas at Guanahacabibes Peninsula Biosphere Reserve and National Park is one of the largest in the Cuban archipelago; however, little information about its nesting ecology is available. Temporal and spatial variation in nesting and reproductive success as well as morphometric characteristics of gravid females were used to ecologically characterize this colony. Nine beaches of the Southernmost coast of Guanahacabibes Peninsula were monitored for 14 years 1998-2012 to determine green turtle nesting activity, from May to September peak nesting season in this area. Beach dimensions were measured to determine nest density using the length and the area. Afterward the beaches were divided in two categories, index and secondary. Females were measured and tagged to compare new tagged females 823 with returning tagged females 140. Remigration interval was also determined. Temporal variation was identified as the annual number of nesting emergences and oviposits per female, with apparent peaks in reproductive activity on a biennial cycle in the first six years followed by periods of annual increase in nest number 2003-2008 and periods of decreasing number of nests 2010-2012. We also found intra-seasonal variation with the highest nesting activity in July, particularly in the second half of the month. The peak emergence time was 22:00-02:00hr. In terms of spatial variation, smaller beaches had the highest nest density and nesting was more frequent 6-9m from the high tide line, where hatchling production was maximized although hatchling success was high on average, above 80. Morphometric analysis of females was made and newly tagged turtles were smaller on average than remigrants. Our results are only a first attempt at characterizing Guanahacabibes populations but have great value for establishing conservation priorities within the context of national management plans, and for efficient monitoring and protection of nesting beaches.

La colonia de Tortuga verde Chelonia mydas que anida en la Península de Guanahacabibeses es una de las más grandes del archipiélago cubano pero existe poca información disponible sobre la misma. Por ello utilizamos la variación espacial y temporal de la anidación así como el éxito reproductivo y las características morfométricas de las hembras grávidas para realizar la primera caracterización ecológica de esta colonia. Se estudiaron nueve playas durante 14 años 1998-2012 desde mayo hasta septiembre pico de anidación. Estas se dividieron en dos categorías: índices y secundarias. Las hembras fueron medidas y marcadas para comparar las nuevas hembras marcadas 823 con las remigrantes 140. Se detectó una variación temporal en la anidación a tres niveles: interanual, dentro de la temporada y durante la noche. Se observa un ciclo bienal en los primeros seis años, luego un incremento anual 2003-2008 seguido por un período de decrecimiento en las anidaciones 2010-2012. Dentro de la temporada se encontró un pico en el mes de julio aunque cambia de la primera quincena a la segunda en años diferentes y la hora de emergencia preferentemente de 22:00-02:00hr. En la variación espacial, las playas pequeñas tuvieron mayor densidad de anidación, especialmente entre los 6-9m hasta la línea media de marea alta, donde se maximiza la producción de neonatos, aunque el éxito de emergencia de los neonatos se mantuvo como promedio por encima del 80 en toda la playa. Morfométricamente las hembras remigrantes resultaron mayores como promedio que las marcadas por primera vez. Estos resultados son una primera aproximación para la caracterización de la colonia de Guanahacabibes pero tienen gran valor para establecer prioridades de conservación dentro del contexto de los planes de manejo nacionales así como para garantizar un monitoreo eficiente y la protección de las playas de anidación.

Nesting ecology of Chelonia mydas (Testudines: Cheloniidae) on the Guanahacabibes Peninsula, Cuba.

The nesting colony of green sea turtles (Chelonia mydas) at Guanahacabibes Peninsula Biosphere Reserve and National Park is one of the largest in the Cuban archipelago; however, little information about its nesting ecology is available. Temporal and spatial variation in nesting and reproductive success as well as morphometric characteristics of gravid females were used to ecologically characterize this colony. Nine beaches of the Southernmost coast of Guanahacabibes Peninsula were monitored for 14 years (1998-2012) to determine green turtle nesting activity, from May to September (peak nesting season in this area). Beach dimensions were measured to determine nest density using the length and the area. Afterward the beaches were divided in two categories, index and secondary. Females were measured and tagged to compare new tagged females (823) with returning tagged females (140). Remigration interval was also determined. Temporal variation was identified as the annual number of nesting emergences and oviposits per female, with apparent peaks in reproductive activity on a biennial cycle in the first six years followed by periods of annual increase in nest number (2003-2008) and periods of decreasing number of nests (2010-2012). We also found intra-seasonal variation with the highest nesting activity in July, particularly in the second half of the month. The peak emergence time was 22:00-02:00 hr. In terms of spatial variation, smaller beaches had the highest nest density and nesting was more frequent 6-9m from the high tide line, where hatchling production was maximized although hatchling success was high on average, above 80%. Morphometric analysis of females was made and newly tagged turtles were smaller on average than remigrants. Our results are only a first attempt at characterizing Guanahacabibes' populations but have great value for establishing conservation priorities within the context of national management plans, and for efficient monitoring and protection of nesting beaches.

Ovarian follicular development in the hawksbill turtle (Cheloniidae: Eretmochelys imbricata L.).

Ovarian follicular development is an essential process in the determination of maturation stages associated with size. This association acquires importance when managing populations of threatened species. We histologically processed 11 prepubescent ovaries, four pubescent ovaries, and one breeding adult ovary with vitellogenic follicles using specific staining techniques to identify the follicular stages of Eretmochelys imbricata. Follicular stages were compared with maturation stages [including straight carapace length (SCL)]. The ovary presented several germinal beds and a lacunar system less histochemically and morphologically heterogeneous than that of crocodiles. During previtellogenesis (four stages), the oocyte grows rapidly due to the strong transcriptional activity of lampbrush chromosomes and numerous nucleoli, and the strong metabolism associated with lipid synthesis. The Stage III ooplasm showed a Sudan positive band. This stage was the most frequent in all ovarian sections and it was independent of maximal follicular stage. Stage IV, more frequent in pubescent and adult ovaries, presented a lipid vacuole-rich ooplasm and a broadening of the zona pellucida and the theca. The vitellogenesis begins with the penetration and accumulation of spherical glycoprotein yolk platelets and chemically neutral lipid droplets which are observed to be mixed, but spatially and chemically segregated. Both the yolk platelets and lipid droplets increase in size, density, and proximity to the periphery of the oocyte due to their coalescence. The SCL of the immature females did not determine the maximal follicular stage nor its frequency in the ovaries. Straight carapace length turned out to be an imprecise measure in identifying the presence of follicular stages in females larger than the minimum legal size limit in Cuba. Consequently, for a national conservation program to be successful, it must emphasize the critically endangered status of E. imbricata, as well as the maturity of the most frequent hawksbill sizes encountered in the Cuban archipelago fishery.

Evolutionary history of Cuban crocodiles Crocodylus rhombifer and Crocodylus acutus inferred from multilocus markers.

Among crocodilians, Crocodylus rhombifer is one of the world's most endangered species with the smallest natural distribution. In Cuba, this endemic species coexists with the American crocodile (Crocodylus acutus). Hybridization between these two species is well known in captivity and might occur in the wild, but has never been demonstrated genetically. Here, we combined molecular data with environmental, geographic, and fossil data to infer the evolutionary history of Crocodylus in the Cuban Archipelago, and to evaluate genealogical support for species boundaries. We analyzed seven microsatellite loci plus DNA sequence data from nuclear (RAG-1) and mitochondrial (cytochrome b and cytochrome oxidase I) genes from 89 wild-caught individuals in Cuba, Grand Cayman Island, Jamaica, and Central America, and two samples from zoo collections. Microsatellites showed evidence of introgression, suggesting potential hybridization among Cuban groups. In Cuba, C. acutus contained one mitochondrial DNA (mtDNA) haplotype, whereas C. rhombifer contained two haplotypes. MtDNA data showed that C. acutus is paraphyletic with respect to C. rhombifer, revealing 1% sequence divergence between species within Cuba vs. 8% divergence between Cuban forms and mainland C. acutus. We suggest that hybridization has been a historical as well as a current phenomenon between C. acutus and C. rhombifer. These findings suggest that long-term conservation of crocodiles in Cuba will require identification of genetically pure and hybrid individuals, and a decrease in anthropogenic activities. We also recommend more extensive morphological and genetic analyses of Cuban population to establish clear boundaries of the hybrid zone between C. acutus and C. rhombifer.

Temporal variation of the population structure and genetic diversity of Farfantepenaeus notialis assessed by allozyme loci.

Population genetic studies carried out on penaeid shrimps have disclosed different patterns of population subdivision, revealing new aspects of shrimp biology as well as the effects of historical contingency molding those patterns. However, the stability of observed allele frequencies over time still remains untested. The objective of this article is to show the analysis of the temporal variation of allozymes in a shrimp species inhabiting Cuba which proves that the genetic structure of this species could significantly change in time. The study involves four populations of Farfantepenaeus notialis sampled in a period of 8 years. The significant statistics obtained from partitions observed in 1995 were not detected in 2003 (as suggested by AMOVA and F(ST)), whereas temporal genetic differentiation and heterozygosity became highly significant. The results strongly suggest that the effect of migrations could be the cause for the loss of F. notialis genetic structure in 2003. It is therefore imperative to call attention on the vulnerability of these populations when facing unstable environmental and habitat conditions.

Microsátelites del camarón blanco litopenaeus shmitti (Crustacea, Decapoda) / Microsatellites from the white shrimp litopenaeus chmitti (Crustacea, Dacapoda)

A partir de una genoteca de ADN construida con insertos entre 300 pb y 700 pb, se identificaron secuencias microsátelites de litopenaeus schmitti, especie cubana de camarón utilizada en la acuicultura. Las 910 colonias que se obtuvieron fueron analizadas en busca de repeticiones y resultaron positivos 114 clones de los cuales 106 lo fueron para los oligonucléotidos (TC)15, (TG)15 y (TAA)15 y 8 para el oligonucléotido (GTC)15...(AU)