Patricia Amavet

Microsatellite DNA markers applied to detection of multiple paternity in Caiman latirostris in Santa Fe, Argentina

Detecting multiple paternity in wild populations of the broad-snouted caiman (Caiman latirostris) has important implications for conservation efforts. We have applied microsatellite markers to examine genetic variation in C. latirostris and also have provided the first data concerning detection of multiple paternity in wild populations of this species. Blood samples from four nest-guarding C. latirostris females and their hatchlings were obtained from Santa Fe Province, Argentina. Amplified products were analyzed by electrophoresis on 10% polyacrylamide gels and visualized with silver staining. Four out of the eight markers tested reliably amplified and yielded useful data. Using polyacrylamide gels with silver staining provides high enough resolution to obtain individual genotypes. In order to assess the presence or absence of more than two parents in each nest, we used the single locus Minimum Method, and applied Cervus 3.0 and Gerud 2.0 software in parentage analyses. Our results indicate more than one father in at least two families. This behavior could be the consequence of high habitat variability in the area where our population was sampled. The ability to understand mating systems is important for maintaining viable populations of exploited taxa like C. latirostris.

Mating system and population analysis of the broad-snouted caiman (Caiman latirostris) using microsatellite markers

The knowledge about reproductive strategies of a species contributes to their conservation. Multiple paternity is a reproductive behavior increasing effective population size, which could increase genetic diversity particularly in populations submitted to bottlenecks events. In Argentina, wild populations of Caiman latirostris are subject of a management plan devoted to their preservation and sustainable utilization based on its commercial interest. This program started in response to the evident numeric reduction of the populations, as a consequence of hunting pressure and habitat modification; it had a remarkable success in population recovery allowing the commercial use of C. latirostris. Data on reproductive behavior of C. latirostris are limited because mating occurs in the water and the information about their genetic diversity is scarce too. Our specific aims were to study the mating system and population genetic structure applying microsatellite markers in twelve C. latirostris families. The obtained results showed highly significant difference among populations and a lack of correspondence between geographical distance and genetic differentiation suggesting that populations of C. latirostris represent unstable metapopulations. In the paternity analysis was detected more than one father in two nests, which could be explained by capacity of storage sperm, proposed in females of a related species. The behavior of multipaternity could contribute to maintain viable populations of C. latirostris, since the maintenance of genetic variability within populations could help increase their capacity to respond to selective pressure. Further studies employing genetic and behavioral framework are needed to better understand the reproductive biology of C. latirostris.

Genetic and morphometric variability in Caiman latirostris (broad-snouted caiman), reptilia, Alligatoridae.

Caiman latirostris (broad-snouted caiman) is a crocodilian species from Argentina subject of management plans. The goal of this study was estimating the distribution of genetic variability using RAPD markers and quantitative traits in wild populations of C. latirostris from Santa Fe province, Argentina. We sampled animals from four populations to obtain DNA and morphometric measurements. Eight RAPD primers were used and PCR products were analyzed on 4% polyacrylamide gels stained with silver nitrate. Eleven allometric measurements were obtained in animals within 48 hr after birth. We were able to reveal a relatively high number of variable markers in the studied populations. Our estimates of polymorphism and heterozygosity are higher than recorded values in other crocodilians using isozymes, the studied populations showed low levels of gene flow and some population subdivision. The study of quantitative traits conducted by nested analysis of variance and principal component analysis indicated higher levels of variance among nests within populations than among populations. We found that some head measurements have the highest contribution to morphological differences among populations; this fact could support the role of these traits in reproductive or feeding behavior. Estimated genetic differentiation value (F(ST)) among populations was higher than quantitative trait differentiation value (Q(ST)), suggesting a higher contribution of neutral than adaptive loci to the genetic differentiation among populations. Quantitative traits are probably more related with fitness and the differentiation among populations remained relatively lower. The high heritability estimated for some traits indicates great potential to improve them in management plans.

Analysis of the Population Structure of Broad-Snouted Caiman (Caiman latirostris) in Santa Fe, Argentina, Using the RAPD technique

Abstract Caiman latirostris (Broad-Snouted Caiman) is one of two crocodilian species that inhabit Argentina. Wild populations of C. latirostris in Santa Fe Province are under management by Proyecto Yacaré (MAGIC-MUPCN), a program based on a ranching system whose goal is the sustainable use of the species, together with its habitat conservation. We used 233 random amplified polymorphic DNA (RAPD) markers to obtain preliminary data about population structure and genetic variation in four populations of C. latirostris in Santa Fe province, which may be used to provide a genetic framework of conservation units to evaluate, and perhaps to modify, current plans for its sustainable management. Our results suggest low to intermediate levels of mean heterozygosity (H), mean number of alleles per locus (A), and percentage of loci polymorphic (P). Furthermore, the values of Nm and FST seem to show low levels of gene flow and some population subdivision. Distance genetic analysis appears to reveal high similarity between Estancia El Estero, Costa del Salado, and Estero del Paraje 114 populations, and results of analysis of molecular variance indicate that nearly all variation exists within populations rather than among them, implying that a great number of alleles is shared among populations. New samplings and new measurements in parameters such as H, A, and P, should be carried out in future years to confirm the genetic structure and level of population differentiation and to make new decisions about the management plan that has been successful in the recovery and conservation of C. latirostris.

Isolation and characterization of new microsatellite markers for application in population genetic studies of Caiman latirostrisand related species

Wild populations of Caiman latirostrisare subject to sustainable use programs in Argentina, becoming a species with important impact in the regional economy, based in their skin and meat. Genetic studies are fundamental to acquire information on important parameters for conservation and management, which may be obtained from analysis of molecular markers. Some microsatellites have been previously isolated in this species, but due to some difficulties in using them, we obtained new ones using Next Generation Sequencing approach. This study reports eight new microsatellites for C. latirostrisand tests their utility in a related species, Caiman yacare, with successful application in population genetics and mating systems studies. In addition, we shared data about a novel and fast bioinformatics tool to find microsatellites and to design their corresponding primers.

Phenotypic variability and heritability of the cephalic region of Caiman latirostris.

The study of the cephalic shape of crocodilian is relevant in the fields of ecology, systematics, evolution, and conservation. Therefore, the integration of geometric analysis within quantitative genetics allows the evaluation of the inheritable shape components. In this study, the dorsal cephalic region of 210 Caiman latirostris hatchlings was analyzed from seven populations in Santa Fe, Argentina, to detect intra‐, and inter‐population phenotypic variability, and to determine the heritability of biological shape and size, using newly available geometric morphometric tools. The principal component analysis showed two configurations of cephalic shape that could be related to sexual dimorphism. In the canonical variate analysis, Procrustes distances between groups indicated that there are differences in shape among populations. Furthermore, the method of partial least squares indicated a covariation between cephalic shape and environmental variables. Regarding to CS of the skull we found significant differences among populations, moreover the partial least squares was also significant. Estimates of the heritability of shape and size were high, indicating that the components of these features are susceptible to the selection. J. Morphol. 277:370–378, 2016.

Baseline values of immunologic parameters in the lizard Salvator merianae ( Teiidae , Squamata)

The genus Salvator is widely distributed throughout South America. In Argentina, the species most abundant widely distributed is Salvator merianae. Particularly in Santa Fe province, the area occupied by populations of these lizards overlaps with areas where agriculture was extended. With the aim of established baseline values for four immunologic biomarkers widely used, 36 tegu lizards were evaluated tacking into account different age classes and both sexes. Total leukocyte counts were not different between age classes. Of the leucocytes count, eosinophils levels were higher in neonates compared with juvenile and adults; nevertheless, the heterophils group was the most prevalent leukocyte in the peripheral blood in all age classes. Lymphocytes, monocytes, heterophils, azurophils and basophils levels did not differ with age. Natural antibodies titres were higher in the adults compared with neonates and juveniles lizards. Lastly, complement system activity was low in neonates compared with juveniles and adults. Statistical analysis within each age group showed that gender was not a factor in the outcomes. Based on the results, we concluded that S. merianae demonstrated age (but not gender) related differences in the immune parameters analyzed. Having established baseline values for these four widely-used immunologic biomarkers, ongoing studies will seek to optimize the use of the S. merianae model in future research.

Mating System of Caiman yacare (Reptilia: Alligatoridae) Described from Microsatellite Genotypes.

The yacare caiman (Caiman yacare) is a reptile from South America and 1 of the 2 crocodilian species present in Argentina. The degradation of their natural habitat and strong hunting pressure led to a sharp numerical decline of wild populations. Nowadays, C. yacare is included in Appendix II of CITES, and ranching practices in some areas in Argentina are helping hatching success. In this context, it is important to better understand the population structure and mating system of the species. To do this, we amplified 10 microsatellite markers (SSRs) in 148 individuals of 13 C. yacare nests. All of the markers were polymorphic with 2–12 alleles per locus, with allelic sizes ranging between 154 and 400 bp and medium levels of polymorphism (Ho = 0.152–0.551 and He = 0.221–0.621). We were able to determine the maternal genotype in 9 out of 13 nests. In 6 of them we found more than 1 paternal genotype, with a maximum of 3 fathers for a single nest. This study is the first to provide evidence of multiple paternity behavior. These findings will be useful to improve management and conservation strategies for the species.

FullSSR: Microsatellite Finder and Primer Designer

Microsatellites are genomic sequences comprised of tandem repeats of short nucleotide motifs widely used as molecular markers in population genetics. FullSSR is a new bioinformatic tool for microsatellite (SSR) loci detection and primer design using genomic data from NGS assay. The software was tested with 2000 sequences of Oryza sativa shotgun sequencing project from the National Center of Biotechnology Information Trace Archive and with partial genome sequencing with ROCHE 454® from Caiman latirostris, Salvator merianae, Aegla platensis, and Zilchiopsis collastinensis. FullSSR performance was compared against other similar SSR search programs. The results of the use of this kind of approach depend on the parameters set by the user. In addition, results can be affected by the analyzed sequences because of differences among the genomes. FullSSR simplifies the detection of SSRs and primer design on a big data set. The command line interface of FullSSR was intended to be used as part of genomic analysis tools pipeline; however, it can be used as a stand-alone program because the results are easily interpreted for a nonexpert user.

Analysis of morphological variability and heritability in the head of the Argentine Black and White Tegu (Salvator merianae): undisturbed vs. disturbed environments

The heterogeneity of biotic and abiotic factors influencing fitness produce selective pressures that promote local adaptation and divergence among different populations of the same species. In order for adaptations to be maintained through evolutionary time, heritable genetic variation controlling the expression of the morphological features under selection is necessary. Here we compare morphological shape variability and size of the cephalic region of Salvator merianae specimens from undisturbed environments to those of individuals from disturbed environments, and estimated heritability for shape and size using geometric morphometric and quantitative genetics tools. The results of these analyzes indicated that there are statistically significant differences in shape and size between populations from the two environments. Possibly, one of the main determinants of cephalic shape and size is adaptation to the characteristics of the environment and to the trophic niche. Individuals from disturbed environments have a cephalic region with less shape variation and also have a larger centroid size when compared to individuals from undisturbed environments. The high heritability values obtained for shape and size in dorsal view and right side view indicate that these phenotypic characters have a great capacity to respond to the selection pressures to which they are subjected. Data obtained here could be used as an important tool when establishing guidelines for plans for the sustainable use and conservation of S. merianae and other species living in disturbed areas.