Genetic diversity and connectivity remain high in Holothuria polii (Delle Chiaje 1823) across a coastal lagoon-open sea environmental gradient

Coastal lagoons represent habitats with widely heterogeneous environmental conditions, particularly as regards salinity and temperature,which fluctuate in both space and time. These characteristics suggest that physical and ecological factors could contribute to the genetic divergence among populations occurring in coastal lagoon and opencoast environments. This study investigates the genetic structure of Holothuria polii at a micro-geographic scale across theMar Menor coastal lagoon and nearbymarine areas, estimating the mitochondrial DNA variation in two gene fragments, cytochrome oxidase I (COI) and 16S rRNA (16S). Dataset of mitochondrial sequences was also used to test the influence of environmental differences between coastal lagoon andmarine waters on population genetic structure. All sampled locations exhibited high levels of haplotype diversity and low values of nucleotide diversity. Both genes showed contrasting signals of genetic differentiation (non-significant differences using COI and slight differences using 16S, which could due to different mutation rates or to differential number of exclusive haplotypes. We detected an excess of recent mutations and exclusive haplotypes, which can be generated as a result of population growth. However, selective processes can be also acting on the gene markers used; highly significant generalized additive models have been obtained considering genetic data from16S gene and independent variables such as temperature and salinity.

Genetic differentiation among Parastichopus regalis populations in the Western Mediterranean Sea: potential effects from its fishery and current connectivity

Parastichopus regalis (Cuvier, 1817) is the most expensive seafood product on the Catalonian market (NE Spain), with prices at approximately 130 €/Kg (fresh weight). Despite its ecological and economic importance, biological and genetic information on this sea cucumber species is scarce. Here, we provide both the first insight on the genetic structure of P. regalis using sequences of cytochrome oxidase I (COI) and 16S genes and a morphological description of its population. Individual sea cucumbers were collected in six locations along the Spanish Mediterranean coast, including an area under fishery pressure (Catalonia). We found high haplotype diversity and low nucleotide diversity for both genes, with higher levels of genetic diversity observed in the COI gene. The population pairwise fixation index (FST), AMOVA and correspondence analysis (CA) based on the COI gene revealed significant genetic differentiation among some locations. However, further analysis using nuclear markers (e.g., microsatellites) is necessary to corroborate these results. Moreover, the genetic and morphological data may indicate fishery effects on the Catalonian population with a decrease in the size and weight averages and lower genetic diversity compared with locations that lack fishery pressure. For the appropriate management of this species, we suggest the following: 1) accurately assessing the stock status along the Spanish coasts; 2) studying the reproductive cycle of this target species and the establishment of a closed fishery season according to the reproductive cycle; and 3) establishing protected areas (i.e., not take zones) to conserve healthy populations and favour recruitment in the nearby areas.

West versus East Mediterranean Sea: origin and genetic differentiation of the sea cucumber Holothuria polii

We studied the genetic structure of the sea cucumber Holothuria (Roweothuria) polii (Delle Chiaje 1823) by analysing the mitochondrial DNA variation in two fragments of cytochrome oxidase I (COI) and 16S genes. Individuals were collected in seven locations along the Mediterranean Sea, which cover a wide range of the species distribution. We found high haplotype diversity for COI and moderate diversity for 16S, and low nucleotide diversity for both genes. Our results for the COI gene showed many recent and exclusive haplotypes with few mutational changes, suggesting recent or ongoing population expansion. The Western and Eastern Mediterranean populations exhibited slight but significant genetic differentiation (COI gene) with higher genetic diversity in the East. The most ancient haplotype was not present in the westernmost sampling location (SE Spain). The oldest expansion time was observed in Turkey, corresponding to mid-Pleistocene. Turkey had also the highest genetic diversity (number of total and exclusive haplotypes, polymorphisms, haplotype and nucleotide diversity). This suggests that this region could be the origin of the subsequent colonizations through the Mediterranean Sea, a hypothesis that should be assessed with nuclear markers in future research.

Population genetics of Cerastoderma edule in Ria Formosa (southern Portugal): the challenge of understanding an intraspecific hotspot of genetic diversity

Coastal lagoons are highly variable environments that may act as hotspots of genetic diversity as a consequence of their ecological role as nursery habitats of marine species with both ecological and fisheries importance. The edible cockle (Cerastoderma edule) is a commercially important shellfish resource inhabiting coastal lagoons in Europe and their fisheries management urgently needs genetic studies to design appropriate strategies to promote the recovery of exploited populations. The aim of this study was to assess the C. edule genetic diversity and population structure at a small geographic scale, inside Ria Formosa coastal lagoon (southern Portugal) using mitochondrial cytochrome oxidase I sequences in six locations. Outcomes pointed to a common pattern of high haplotype diversity and non-significant genetic structuring inside the Ria Formosa lagoon. A high level of gene flow was detected between all localities and the presence of a single stock from a genetic point of view may be considered for fisheries management purposes. The existence of a high number of haplotypes and high values of haplotype diversity of C. edule in Ria Formosa lagoon could be consistent with the hypothesis that higher genetic diversity is expected in populations occurring in coastal lagoons, suggesting that lagoons could increase standing genetic variation and an adaptive potential of lagoon populations as an ecological response to a highly variable environment.

Land planarians (Platyhelminthes) as a model organism for fine-scale phylogeographic studies: understanding patterns of biodiversity in the Brazilian Atlantic Forest hotspot

The Brazilian Atlantic Forest is one of the richest biodiversity hotspots of the world. Paleoclimatic models have predicted two large stability regions in its northern and central parts, whereas southern regions might have suffered strong instability during Pleistocene glaciations. Molecular phylogeographic and endemism studies show, nevertheless, contradictory results: although some results validate these predictions, other data suggest that paleoclimatic models fail to predict stable rainforest areas in the south. Most studies, however, have surveyed species with relatively high dispersal rates whereas taxa with lower dispersion capabilities should be better predictors of habitat stability. Here, we have used two land planarian species as model organisms to analyse the patterns and levels of nucleotide diversity on a locality within the Southern Atlantic Forest. We find that both species harbour high levels of genetic variability without exhibiting the molecular footprint of recent colonization or population expansions, suggesting a long-term stability scenario. The results reflect, therefore, that paleoclimatic models may fail to detect refugia in the Southern Atlantic Forest, and that model organisms with low dispersal capability can improve the resolution of these models.

Molecular differentiation of Angiostrongylus costaricensis, A. cantonensis, and A. vasorum by polymerase chain reaction- restriction fragment length polymorphism

Angiostrongylus cantonensis, A. costaricensis, and A. vasorum are etiologic agents of human parasitic diseases. Their identification, at present, is only possible by examining the adult worm after a 40-day period following infection of vertebrate hosts with the third-stage larvae. In order to obtain a diagnostic tool to differentiate larvae and adult worm from the three referred species, polymerase chain reaction-restriction fragment length polymorphism was carried out. The rDNA second internal transcribed spacer (ITS2) and mtDNA cytochrome oxidase I regions were amplified, followed by digestion of fragments with the restriction enzymes RsaI, HapII, AluI, HaeIII, DdeI and ClaI. The enzymes RsaI and ClaI exhibited the most discriminating profiles for the differentiation of the regions COI of mtDNA and ITS2 of rDNA respectively. The methodology using such regions proved to be efficient for the specific differentiation of the three species of Angiostrongylus under study.

Morphology of the larvae, male genitalia and DNA sequences of Anopheles (Kerteszia) pholidotus (Diptera: Culicidae) from Colombia

Since 1984, Anopheles (Kerteszia) lepidotus has been considered a mosquito species that is involved in the transmission of malaria in Colombia, after having been incriminated as such with epidemiological evidence from a malaria outbreak in Cunday-Villarrica, Tolima. Subsequent morphological analyses of females captured in the same place and at the time of the outbreak showed that the species responsible for the transmission was not An. lepidotus, but rather Anopheles pholidotus. However, the associated morphological stages and DNA sequences of An. pholidotus from the foci of Cunday-Villarrica had not been analysed. Using samples that were caught recently from the outbreak region, the purpose of this study was to provide updated and additional information by analysing the morphology of female mosquitoes, the genitalia of male mosquitoes and fourth instar larvae of An. pholidotus, which was confirmed with DNA sequences of cytochrome oxidase I and rDNA internal transcribed spacer. A total of 1,596 adult females were collected in addition to 37 larval collections in bromeliads. Furthermore, 141 adult females, which were captured from the same area in the years 1981-1982, were analysed morphologically. Ninety-five DNA sequences were analysed for this study. Morphological and molecular analyses showed that the species present in this region corresponds to An. pholidotus. Given the absence of An. lepidotus, even in recent years, we consider that the species of mosquitoes that was previously incriminated as the malaria vector during the outbreak was indeed An. pholidotus, thus ending the controversy.

Dugesia sicula (Platyhelminthes, Tricladida): the colonizing success of an asexual Planarian

Background Dugesia sicula is the only species of its genus not presenting an endemic or restricted distribution within the Mediterranean area. It mostly comprises fissiparous populations (asexual reproduction by body division and regeneration), most likely sexually sterile, and characterized by an extremely low genetic diversity interpreted as the consequence of a recent anthropic expansion. However, its fissiparous reproduction can result in an apparent lack of diversity within the species, since genetic variation within individuals can be as large as between them because most individuals within a population are clones. We have estimated haplotype and nucleotide diversity of cytochrome oxidase I within and among individuals along the species distribution of a broad sample of D. sicula, including asexual and the two only sexual populations known today; and predicted its potential distribution based on climatic variables. Our aim was to determine the centre of colonisation origin, whether the populations are recent, and whether the species is expanding. Results The species presents 3 most frequent haplotypes, differing in a maximum of 11 base pairs. As expected from their fissiparous mode of reproduction, in half of all the analysed localities many individuals have multiple heteroplasmic haplotypes. The distribution of haplotypes is not geographically structured; however, the distribution of haplotypes and heteroplasmic populations shows higher diversity in the central Mediterranean region. The potential distribution predicted by climatic variables based modelling shows a preference for coastal areas and fits well with the observed data. Conclusions The distribution and frequency of the most frequent haplotypes and the presence of heteroplasmic individuals allow us to gain an understanding of the recent history of the species, together with previous knowledge on its phylogenetic relationships and age: The species most probably originated in Africa and dispersed through the central Mediterranean. After one or multiple populations became triploid and fissiparous, the species colonized the Mediterranean basin, likely both by its own means and helped by human activities. Its present distribution practically fulfils its potential distribution as modelled with climatic variables. Its prevalence in coastal regions with higher water temperatures predicts a likely future expansion to northern and more interior areas following the increase in temperatures due to climate change.

Forensic identification of the guitarfish species Rhinobatos horkelli, R-percellens and Zapteryx brevirostris using multiplex-PCR

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evolutionary dynamics of heterochromatin in the genome of Dichotomius beetles based on chromosomal analysis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mitochondrial DNA polymorphism and heteroplasmy in populations of Aedes aegypti in Brazil

The tropical mosquito Aedes aegypti (Diptera: Culicidae) is the most important domestic vector of urban yellow fever and dengue viruses. Ae. aegypti originated from Africa and was probably introduced into Brazil during the colonial period through embarkations, and dengue epidemics soon followed. Genetic analysis of 12 Ae. aegypti populations from five states in Brazil was conducted based on two mitochondrial DNA fragments: cytochrome oxidase I and NADH dehydrogenase subunit 4. Analyses comparing individual haplotypes indicated the existence of two well-defined clades, probably representing two mitochondrial lineages. Analysis of molecular variance showed significant variability in genetic structure among collections within groups. Mantel regression analysis showed a correlation between genetic and geographic distances, mainly because of northern and northeastern populations, in comparison with those in the southeast. The population from Santos, the largest port in Brazil, showed the greatest diversity, with 10 unique haplotypes, an indication of recent introductions that have not yet spread to other Brazilian cities. Different mitochondrial DNA sequences were found in three specimens, indicating the presence of heteroplasmy.

Occurrence of the Indo-Pacific freshwater prawn Macrobrachium equidens Dana 1852 (Decapoda, Palaemonidae) on the coast of Brazilian Amazonia, with notes on its reproductive biology

O camarão de água doce Macrobrachium equidens, nativo da região do Indo-Pacífico, foi registrada pela primeira vez na costa da Amazônia Brasileira. Esta espécie foi encontrada habitando o mesmo ambiente que duas espécies nativas do gênero Macrobrachium: M. amazonicum e M. acanthurus, e é morfologicamente muito similar à última. A identificação dessa espécie foi confirmada pela análise da sequencia genética do gene mitocondrial Citocromo Oxidase (COI). Uma descrição detalhada das características morfológicas e biologia reprodutiva de M. equidens neste novo ambiente que é apresentada.

Pseudoplatystoma metaense and P. orinocoense (Siluriformes: Pimelodidae) from the Orinoco basin, Venezuela: cytogenetic and molecular analyses

The genus Pseudoplatystoma includes catfish species distributed throughout the fresh waters of South America. These species are important fisheries resources and play a significant ecological role due to their piscivorous and migratory habits. The taxonomy of this genus is still debated: traditionally, only three species have been recognised, but recently, this number was raised to eight. The validity of these eight morphospecies, however, was not confirmed by two subsequent molecular phylogenetic studies, which identified either five or four main clades. In this study, we focused on the two morphospecies restricted to the Orinoco basin, P. metaense and P. orinocoense, which have been assigned to either the same or different clades in previous studies. We carried out cytogenetic analyses to describe their unknown karyotypes and to look for cytotaxonomic markers. We also analysed their mitochondrial sequences in order to assign the sampled specimens to the previously identified molecular clades. The two presumptive species show similar karyotypes (2n=56, 42 biarmed and 14 uniarmed chromosomes) and cytogenetic features in terms of the constitutive heterochromatin distribution and the number and location of minor and major ribosomal genes. Thus, no species-specific chromosome markers could be identified. The analysis of cytochrome b and cytochrome oxidase I mitochondrial genes (carried out by retrieving all the mtDNA Pseudoplatystoma sequences available in GenBank) distributed the sampled specimens into two distinct molecular clades and confirmed the need to re-evaluate, by parallel morphological and molecular analyses, the monophyly of some lineages.

Evolutionary Relationships of the Triatoma matogrossensis Subcomplex, the Endemic Triatoma in Central-Western Brazil, Based on Mitochondrial DNA Sequences

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Multiple mitochondrial genes of some sylvatic Brazilian Triatoma: Non-monophyly of the T. brasiliensis subcomplex and the need for a generic revision in the Triatomini

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)