High-resolution Genetic Markers Research Articles

Utilizing high-resolution genetic markers to track population-level exposure of migratory birds to renewable energy development

With new motivation to increase the proportion of energy demands met by zero-carbon sources, there is a greater focus on efforts to assess and mitigate the impacts of renewable energy development on sensitive ecosystems and wildlife, of which birds are of particular interest. One challenge for researchers, due in part to a lack of appropriate tools, has been estimating the effects from such development on individual breeding populations of migratory birds. To help address this, we utilize a newly developed, high-resolution genetic tagging method to rapidly identify the breeding population of origin of carcasses recovered from renewable energy facilities and combine them with maps of genetic variation across geographic space (called ‘genoscapes’) for five species of migratory birds known to be exposed to energy development, to assess the extent of population-level effects on migratory birds. We demonstrate that most avian remains collected were from the largest populations of a given species. In contrast, those remains from smaller, declining populations made up a smaller percentage of the total number of birds assayed. Results suggest that application of this genetic tagging method can successfully define population-level exposure to renewable energy development and may be a powerful tool to inform future siting and mitigation activities associated with renewable energy programs.

Genetic population structures of common scavenging species near hydrothermal vents in the Okinawa Trough

Deep-sea mining of hydrothermal deposits off the coast of Japan is currently under consideration, and environmental baseline studies of the area are required to understand possible impacts. The aim of this study is to clarify population structures of dominant benthic megafaunal species near hydrothermal vent fields in the Okinawa Trough, using a population genetics approach. We examined dominant deep-sea scavenging species including eels, several amphipods, and a decapod and performed population genetic analyses based on the mitochondrial cytochrome c oxidase subunit I region. Several sites were sampled within Okinawa Trough to examine intra-population diversity while two other locations 1400–2400 km away were chosen for inter-population comparisons. For synaphobranchid eels Simenchelys parasitica and Synaphobranchus kaupii, our results showed significant intra-population diversity but no inter-population genetic differentiation, suggesting strong genetic connectivity and/or large population sizes. In addition, single nucleotide polymorphism analysis also confirmed strong genetic connectivity for Simenchelys parasitica. Among scavenging amphipods, we detected seven putative species using molecular phylogenetic analysis. We evaluated population structures of the most abundant species of amphipods and a decapod species (Nematocarcinus lanceopes). Our results provide basic information on the genetic population structures of benthic megafaunal species near hydrothermal vent fields, which can be used to select candidate species for future connectivity analysis with high-resolution genetic markers and aid understanding of the potential population impacts of environmental disturbances.

High-throughput SNPs dataset reveal restricted population connectivity of marine gastropod within the narrow distribution range of peripheral oceanic islands

Molecular studies based on the high resolution genetic markers help us to grasp the factor shaping the genetic structure of marine organisms. Ecological factors linking to life history traits have often explained the process of genetic structuring in open and connectable oceanic environments. Besides, population genetic divergence can be affected by fragmented habitat, oceanic current, and past geographical events. In the present study, we demonstrated the genetic differentiation of marine gastropod Monodonta sp. within a narrow range of peripheral oceanic islands, the Ogasawara Islands. Genetic analyses were performed not only with a mitochondrial DNA marker but also with a high-throughput SNPs dataset obtained by ddRAD-seq. The results of the mtDNA analyses did not show genetic divergence among populations, while the SNPs dataset detected population genetic differentiation. Population demographic analyses and gene flow estimation suggested that the genetic structure was formed by sea level fluctuation associated with the past climatic change and regulated by temporal oceanographic conditions. These findings provide important insights into population genetic patterns in open and connectable environments.

Using stable isotopes to estimate migratory connectivity for a patchily distributed, wetland-associated Neotropical migrant

AbstractEstimates of migratory connectivity are needed for full annual cycle population models of migratory bird species experiencing rapid declines in abundance. One technique to determine migratory connectivity is through stable isotope analysis. This low-resolution method may be influenced by how data are calibrated between isotopes measured in precipitation and those measured in feathers, and can be informed by incorporating relative abundance into the assignment model. eBird abundance maps are a new tool combining citizen science data into a predictive species distribution model. In the Prothonotary Warbler (Protonotaria citrea), a wetland-associated songbird with a patchy breeding distribution, we sought to use stable-hydrogen isotope analysis informed by a species-specific calibration equation and eBird abundance data to determine the strength of migratory connectivity. We developed a species-specific calibration equation using known-origin samples from the breeding grounds and found that stable-hydrogen isotope values measured in precipitation explained 50% of the variation in stable-hydrogen isotope values among feathers. We found that the assignment model incorporating eBird abundance data correctly identified the true origins of 66% of individuals, and that the average assignment area (as a measure of precision) was 64% of the breeding distribution. These results represented a 7% increase in precision and a 3% decrease in accuracy when compared to a model that was not informed by abundance. Based on these models, wintering populations from 6 countries represented a mix of likely breeding origins, suggesting low migratory connectivity for Prothonotary Warblers. We found evidence that wintering latitude was related to likely breeding origin, with individuals at western wintering locations more likely to have southern breeding origins, but this relationship was weak. These results corroborate studies using archival light-level geolocators and high-resolution genetic markers, which also demonstrated weak migratory connectivity in this species. For patchily distributed species, eBird abundance data may not provide a useful increase in precision and accuracy for isotope assignments.

Worldwide Occurrence and Activity of the Reef-Building Coral Symbiont Symbiodinium in the Open Ocean

The dinoflagellate microalga Symbiodinium sustains coral reefs, one of the most diverse ecosystems of the biosphere, through mutualistic endosymbioses with a wide diversity of benthic hosts [1]. Despite its ecological and economic importance, the presence of Symbiodinium in open oceanic waters remains unknown, which represents a significant knowledge gap to fully understand the eco-evolutionary trajectory and resilience of endangered Symbiodinium-based symbioses. Here, we document the existence of Symbiodinium (i.e., now the family Symbiodiniaceae [2]) in tropical- and temperate-surface oceans using DNA and RNA metabarcoding of size-fractionated plankton samples collected at 109 stations across the globe. Symbiodinium from cladesA and C were, by far, the most prevalent and widely distributed lineages (representing 0.1% of phytoplankton reads), while other lineages (clades B, D, E, F, and G) were present but rare. Concurrent metatranscriptomics analyses using the Tara Oceans gene catalog [3] revealed that Symbiodinium cladesA and C were transcriptionally active in the open ocean and expressed core metabolic pathways (e.g., photosynthesis, carbon fixation, glycolysis, and ammonium uptake). Metabarcodes and expressed genes of clades A and C were detected in small and large plankton size fractions, suggesting the existence of a free-living population and a symbiotic lifestyle within planktonic hosts, respectively. However, high-resolution genetic markers and microscopy are required to confirm the life history of oceanic Symbiodinium. Overall, the previously unknown, metabolically active presence of Symbiodinium in oceanic waters opens up new avenues for investigating the potential of this oceanic reservoir to repopulate coral reefs following stress-induced bleaching.

Transcriptome-Derived Microsatellites Demonstrate Strong Genetic Differentiation in Pacific White Sharks.

Recent advances in genome-scale sequencing technology have allowed the development of high resolution genetic markers for the study of nonmodel taxa. In particular, transcriptome sequencing has proven to be highly useful in generating genomic markers for use in population genetic studies, allowing for insight into species connectivity, as well as local adaptive processes as many transcriptome-derived markers are found within or associated with functional genes. Herein, we developed a set of 30 microsatellite markers from a heart transcriptome for the white shark (Carcharodon carcharias), a widely distributed and globally vulnerable marine predator. Using these markers as well as 10 published anonymous genomic microsatellite loci, we provide 1) the first nuclear genetic assessment of the cross-Pacific connectivity of white sharks, and 2) a comparison of the levels of inferred differentiation across microsatellite marker sets (i.e., transcriptome vs. anonymous) to assess their respective utility to elucidate the population genetic dynamics of white sharks. Significant (FST = 0.083, P = 0.05; G″ST = 0.200; P = 0.001) genetic differentiation was found between Southwestern Pacific (n = 19) and Northeastern Pacific (n = 20) white sharks, indicating restricted, cross Pacific gene flow in this species. Transcriptome-derived microsatellite marker sets identified much higher (up to 2×) levels of genetic differentiation than anonymous genomic markers, underscoring potential utility of transcriptome markers in identifying subtle population genetic differences within highly vagile, globally distributed marine species.Subject areas: Population structure and phylogeography; Conservation genetics and biodiversity.

Elucidation of Genetic Markers for the Essential Oils Quantity in Mentha Plants F-AFLP

Medicinal plants are an essential source of therapeutic ingredients, being also the basis of traditional or original healing systems. Many medicinal plants belong to the family Lamiaceae. The Mint is the common name of about 25 perennial species of the genus Mentha. The essential oils of the Mentha species and their extracts are well documented and possess antimicrobial, antifungal, antiviral, pesticide, and antioxidant properties. The current study aimed to collect and genotype mint species from different sources using fluorescently labeled AFLP technique to asses the genetic variability and elucidate high-resolution genetic markers related to oil content and/or oil quantity. A total of 11 mint samples were analyzed using AFLP technique; among the three primer pair combinations showed number of peaks ranged between 49 to 117, with a minimum band size in base pair of 50 bp to 661 bp. The overall mean of number of bands per individual is 99, while the total number of polymorphic loci is 254. Based on the maximum likelihood hybrid index method, two samples were found misclassified or possibly hybrids. Out of the 254 polymorphic bands, 16 were found specific to the high oil-content group [A] compared to 95 bands that were specific for the low oil-content group [B]. In group A, two bands, P1.23 and P2.28, were found fixed as present in group A while absent from group B, the bands were scored from the primer pairs 1 and 2 of 297 and 140 bp, respectively; and considered as positive markers to high-oil content. However, no negative marker was found. When the two possible hybrids were inspected, the positive marker number 1 [PP1.23] was found consistent and absents in the two samples, which increase its validity as a positive marker for high-oil content, while was not the same case for the second marker. Comparing both the whole dataset and the selected markers using AMOVA and PCoA, the elucidated markers should higher genetic differentiation among the studied groups than the whole dataset. The selected markers can be applied for marker-assistant selection breeding program and early preselection of high-oil content samples for future studies related to the oil content in Mentha sp.

Dissecting the phyloepidemiology of Trypanosoma cruzi I (TcI) in Brazil by the use of high resolution genetic markers.

BackgroundTrypanosoma cruzi, the causal agent of Chagas disease, is monophyletic but genetically heterogeneous. It is currently represented by six genetic lineages (Discrete Typing Units, DTUs) designated TcI-TcVI. TcI is the most geographically widespread and genetically heterogeneous lineage, this as is evidenced by a wide range of genetic markers applied to isolates spanning a vast geographic range in Latin America.Methodology/Principal findingsIn total, 78 TcI isolated from hosts and vectors distributed in 5 different biomes of Brazil, were analyzed using 6 nuclear housekeeping genes, 25 microsatellite loci and one mitochondrial marker. Nuclear markers reveal substantial genetic diversity, significant gene flow between biomes, incongruence in phylogenies, and haplotypic analysis indicative of intra-DTU genetic exchange. Phylogenetic reconstructions based on mitochondrial and nuclear loci were incongruent, and consistent with introgression. Structure analysis of microsatellite data reveals that, amongst biomes, the Amazon is the most genetically diverse and experiences the lowest level of gene flow. Investigation of population structure based on the host species/genus, indicated that Didelphis marsupialis might play a role as the main disperser of TcI.Conclusions/SignificanceThe present work considers a large TcI sample from different hosts and vectors spanning multiple ecologically diverse biomes in Brazil. Importantly, we combine fast and slow evolving markers to contribute to the epizootiological understanding of TcI in five distinct Brazilian biomes. This constitutes the first instance in which MLST analysis was combined with the use of MLMT and maxicircle markers to evaluate the genetic diversity of TcI isolates in Brazil. Our results demonstrate the existence of substantial genetic diversity and the occurrence of introgression events. We provide evidence of genetic exchange in TcI isolates from Brazil and of the relative isolation of TcI in the Amazon biome. We observe the absence of strict associations with TcI genotypes to geographic areas and/or host species.

Regional genetic differentiation in the blue mussel from the Baltic Sea area

Connectivity plays an important role in shaping the genetic structure and in evolution of local adaptation. In the marine environment barriers to gene flow are in most cases caused by gradients in environmental factors, ocean circulation and/or larval behavior. Despite the long pelagic larval stages, with high potential for dispersal many marine organisms have been shown to have a fine scale genetic structuring. In this study, by using a combination of high-resolution genetic markers, species hybridization data and biophysical modeling we can present a comprehensive picture of the evolutionary landscape for a keystone species in the Baltic Sea, the blue mussel. We identified distinct genetic differentiation between the West Coast, Baltic Proper and Bothnian Sea regions, with lower gene diversity in the Bothnian Sea. Oceanographic connectivity together with salinity and to some extent species identity provides explanations for the genetic differentiation between the West Coast and the Baltic Sea (Baltic Proper and Bothnian Sea). The genetic differentiation between the Baltic Proper and Bothnian Sea cannot be directly explained by oceanographic connectivity, species identity or salinity, while the lower connectivity to the Bothnian Sea may explain the lower gene diversity.

Invasive blue mussels threaten regional scale genetic diversity in mainland and remote offshore locations: the need for baseline data and enhanced protection in the Southern Ocean

Human-mediated biological transfers of species have substantially modified many ecosystems with profound environmental and economic consequences. However, in many cases, invasion events are very hard to identify because of the absence of an appropriate baseline of information for receiving sites/regions. In this study, use of high-resolution genetic markers (single nucleotide polymorphisms - SNPs) highlights the threat of introduced Northern Hemisphere blue mussels (Mytilus galloprovincialis) at a regional scale to Southern Hemisphere lineages of blue mussels via hybridization and introgression. Analysis of a multispecies SNP dataset reveals hotspots of invasive Northern Hemisphere blue mussels in some mainland New Zealand locations, as well as the existence of unique native lineages of blue mussels on remote oceanic islands in the Southern Ocean that are now threatened by invasive mussels. Samples collected from an oil rig that has moved between South Africa, Australia, and New Zealand were identified as invasive Northern Hemisphere mussels, revealing the relative ease with which such non-native species may be moved from region to region. In combination, our results highlight the existence of unique lineages of mussels (and by extension, presumably of other taxa) on remote offshore islands in the Southern Ocean, the need for more baseline data to help identify bioinvasion events, the ongoing threat of hybridization and introgression posed by invasive species, and the need for greater protection of some of the world's last great remote areas.

Using dense locality sampling resolves the subtle genetic population structure of the dispersive fish species Plecoglossus altivelis.

In dispersive species with continuous distributions, genetic differentiation between local populations is often absent or subtle and thus difficult to detect. To incorporate such subtle differentiation into management plans, it may be essential to analyse many samples from many localities using adequate numbers of high-resolution genetic markers. Here, we evaluated the usefulness of dense locality sampling in resolving genetic population structure in the ayu (Plecoglossus altivelis), a dispersive fish important in Japanese inland fisheries. Genetic variability in, and differentiation between, ayu populations around the Japan-Ryukyu Archipelago were investigated in 4746 individuals collected from 120 localities by genotyping 12 microsatellite markers. These individuals represented the two subspecies of ayu, namely the Ryukyuan subspecies (Plecoglossus altivelis ryukyuensis) and both amphidromous and landlocked forms of the nominotypical subspecies (P.a.altivelis) along the archipelago. We successfully detected an absence of genetic differentiation within the landlocked form and subtle but significant differentiation and clear geographic patterns of genetic variation among populations of the amphidromous form, which had been considered genetically homogeneous. This suggests that dense locality sampling effectively resolves subtle differences in genetic population structure, reducing stochastic deviation in the detection of genetic differentiation and geographic patterns in local populations of this dispersive species. Resampling analyses based on empirical data sets clearly demonstrate the effectiveness of increasing the number of locality samples for stable and reliable estimations of genetic fixation indices. The genetic population structure observed within the amphidromous form provides useful information for identifying management or conservation units in ayu.

Analysis of genetic admixture in Uyghur using the 26 Y-STR loci system.

The Uyghur population has experienced extensive interaction with European and Eastern Asian populations historically. A set of high-resolution genetic markers could be useful to infer the genetic relationships between the Uyghur population and European and Asian populations. In this study we typed 100 unrelated Uyghur males living in southern Xinjiang at 26 Y-STR loci. Using the high-resolution 26 Y-STR loci system, we investigated genetic and phylogenetic relationship between the Uyghur population and 23 reference European or Asian populations. We found that the Uyghur population exhibited a genetic admixture of Eastern Asian and European populations, and had a slightly closer relationship with the selected European populations than the Eastern Asian populations. We also demonstrated that the 26 Y-STR loci system was potentially useful in forensic sciences because it has a large power of discrimination and rarely exhibits common haplotypes. However, ancestry inference of Uyghur samples could be challenging due to the admixed nature of the population.

Intraspecific and heteroplasmic variations, gene losses and inversions in the chloroplast genome of Astragalus membranaceus.

Astragalus membranaceus is an important medicinal plant in Asia. Several of its varieties have been used interchangeably as raw materials for commercial production. High resolution genetic markers are in urgent need to distinguish these varieties. Here, we sequenced and analyzed the chloroplast genome of A. membranaceus (Fisch.) Bunge var. mongholicus (Bunge) P.K. Hsiao using the next generation DNA sequencing technology. The genome was assembled using Abyss and then subjected to gene prediction using CPGAVAS and repeat analysis using MISA, Tandem Repeats Finder, and REPuter. Finally, the genome was subjected phylogenetic and comparative genomic analyses. The complete genome is 123,582 bp long, containing only one copy of the inverted repeat. Gene prediction revealed 110 genes encoding 76 proteins, 30 tRNAs, and four rRNAs. Five intra-specific hypermutation loci were identified, three of which are heteroplasmic. Furthermore, three gene losses and two large inversions were identified. Comparative genomic analyses demonstrated the dynamic nature of the Papilionoideae chloroplast genomes, which showed occurrence of numerous hypermutation loci, frequent gene losses, and fragment inversions. Results obtained herein elucidate the complex evolutionary history of chloroplast genomes and have laid the foundation for the identification of genetic markers to distinguish A. membranaceus varieties.

Low but significant genetic differentiation underlies biologically meaningful phenotypic divergence in a large Atlantic salmon population.

Despite decades of research assessing the genetic structure of natural populations, the biological meaning of low yet significant genetic divergence often remains unclear due to a lack of associated phenotypic and ecological information. At the same time, structured populations with low genetic divergence and overlapping boundaries can potentially provide excellent models to study adaptation and reproductive isolation in cases where high-resolution genetic markers and relevant phenotypic and life history information are available. Here, we combined single nucleotide polymorphism (SNP)-based population inference with extensive phenotypic and life history data to identify potential biological mechanisms driving fine-scale subpopulation differentiation in Atlantic salmon (Salmo salar) from the Teno River, a major salmon river in Europe. Two sympatrically occurring subpopulations had low but significant genetic differentiation (FST =0.018) and displayed marked differences in the distribution of life history strategies, including variation in juvenile growth rate, age at maturity and size within age classes. Large, late-maturing individuals were virtually absent from one of the two subpopulations, and there were significant differences in juvenile growth rates and size at age after oceanic migration between individuals in the respective subpopulations. Our findings suggest that different evolutionary processes affect each subpopulation and that hybridization and subsequent selection may maintain low genetic differentiation without hindering adaptive divergence.

Mapping migration in a songbird using high-resolution genetic markers.

Neotropic migratory birds are declining across the Western Hemisphere, but conservation efforts have been hampered by the inability to assess where migrants are most limited-the breeding grounds, migratory stopover sites or wintering areas. A major challenge has been the lack of an efficient, reliable and broadly applicable method for measuring the strength of migratory connections between populations across the annual cycle. Here, we show how high-resolution genetic markers can be used to identify genetically distinct groups of a migratory bird, the Wilson's warbler (Cardellina pusilla), at fine enough spatial scales to facilitate assessing regional drivers of demographic trends. By screening 1626 samples using 96 highly divergent single nucleotide polymorphisms selected from a large pool of candidates (~450 000), we identify novel region-specific migratory routes and timetables of migration along the Pacific Flyway. Our results illustrate that high-resolution genetic markers are more reliable, precise and amenable to high throughput screening than previously described intrinsic marking techniques, making them broadly applicable to large-scale monitoring and conservation of migratory organisms.

Next generation characterisation of cereal genomes for marker discovery.

Cereal crops form the bulk of the world’s food sources, and thus their importance cannot be understated. Crop breeding programs increasingly rely on high-resolution molecular genetic markers to accelerate the breeding process. The development of these markers is hampered by the complexity of some of the major cereal crop genomes, as well as the time and cost required. In this review, we address current and future methods available for the characterisation of cereal genomes, with an emphasis on faster and more cost effective approaches for genome sequencing and the development of markers for trait association and marker assisted selection (MAS) in crop breeding programs.

Amplified fragment length polymorphism (AFLP) analysis of closely related wild and captive tsetse fly (Glossina morsitans morsitans) populations

BackgroundTsetse flies (Diptera: Glossinidae) are vectors of trypanosomes that cause sleeping sickness in humans and nagana in livestock across sub-Saharan Africa. Tsetse control strategies rely on a detailed understanding of the epidemiology and ecology of tsetse together with genetic variation within and among populations. High-resolution nuclear genetic markers are useful tools for elucidation of the genetic basis of phenotypic traits. In this study amplified fragment length polymorphism (AFLP) markers were developed to analyze genetic variation in Glossina morsitans morsitans from laboratory and field-collected populations from Zimbabwe.ResultsA total of seven hundred and fifty one loci from laboratory and field populations of G. m. morsitans from Zimbabwe were genotyped using AFLP with seven primer combinations. Analysis identified 335 polymorphic loci. The two populations could be distinguished by cluster and principal components analysis (PCA) analysis, indicating that AFLP markers can be used to separate genetically similar populations; at the same time differences observed between laboratory and field populations were not very great. Among the techniques investigated, the use of acetone was the most reliable method of preservation of tsetse for subsequent extraction of high molecular weight DNA. An interesting finding was that AFLP also enabled robust within-population discrimination of male and female tsetse flies due to their different X chromosome DNA complements.ConclusionsAFLP represents a useful additional tool to add to the suite of techniques currently available for the genetic analysis of tsetse populations and represents a useful resource for identification of the genetic basis of important phenotypic traits.

Strategies for the conservation of endangered freshwater pearl mussels (Margaritifera margaritifera L.): a synthesis of Conservation Genetics and Ecology

Freshwater pearl mussels (Margartifera margaritifera L.) are among the most critically threatened freshwater bivalves worldwide. The pearl mussel simultaneously fulfils criteria of indicator, flagship, keystone and umbrella species and can thus be considered an ideal target species for the process conservation of aquatic ecosystem functioning. The development of conservation strategies for freshwater pearl mussels and for other bivalve species faces many challenges, including the selection of priority populations for conservation and strategic decisions on habitat restoration and/or captive breeding. This article summarises the current information about the species’ systematics and phylogeny, its distribution and status as well as about its life history strategy and genetic population structure. Based on this information, integrative conservation strategies for freshwater mollusc species which combine genetic and ecological information are discussed. Holistic conservation strategies for pearl mussels require the integration of Conservation Genetics and Conservation Ecology actions at various spatial scales, from the individual and population level to global biodiversity conservation strategies. The availability of high resolution genetic markers for the species and the knowledge of the critical stages in the life cycle, particularly of the most sensitive post-parasitic phase, are important prerequisites for conservation. Effective adaptive conservation management also requires an evaluation of previous actions and management decisions. As with other freshwater bivalves, an integrative conservation approach that identifies and sustains ecological processes and evolutionary lineages is urgently needed to protect and manage freshwater pearl mussel diversity. Such research is important for the conservation of free-living populations, as well as for artificial culturing and breeding techniques, which have recently been or which are currently being established for freshwater pearl mussels in several countries.

Patterns of Genetic Diversity in the Marine Heterotrophic Flagellate Oxyrrhis marina (Alveolata: Dinophyceae)

Oxyrrhis marina is an important model in ecological studies of free-living protists. Despite this, O. marina has rarely been studied in the environment and no explicit distributional studies exist. Further, phylogenetic data for a small number of isolates indicate that O. marina constitutes two divergent lineages. Here, we quantify phylogenetic variation between 58 globally distributed O. marina isolates using 5.8S--internal transcribed spacer 1 and 2 rDNA (5.8S ITS) and cytochrome c oxidase I (COI) partial sequences. 5.8S ITS and COI phylogenies both partitioned O. marina into four clades, which formed two lineages; mean sequence identity for 5.8S ITS and COI respectively was approximately 40 and 90% between these two lineages. Sequence identities for 5.8S ITS/ COI between clades within lineages were 66.3/99.4% (lineage 1: clade 1 vs 2) and 42.3/99.1% (lineage 2: clade 3 vs 4). rDNA mutation rates in O. marina appear to be abnormally high and were not interpreted in a species delineation context. Based on variation in COI sequence and comparisons with other protists, we suggest that O. marina lineages may constitute two species. In a geographic context, evidence of spatial restriction but also extensive overlap between O. marina clades occurred. Further, clade abundances varied considerably: clades 1 and 2 (belonging to one lineage) were abundant and widespread; in contrast, clades 3 and 4 (belonging to the second lineage) were rare and spatially restricted (occurring only in the Mediterranean or in culture collection). There is need for further phylogenetic and taxonomic studies to assess species delineation in O. marina, and for the application of high resolution genetic markers to resolve processes driving genetic diversity in this important model organism.

Isolation and characterization of microsatellite loci from the endangered Mediterranean sponge Spongia agaricina (Demospongiae: Dictyoceratida)

The abundance of the bath sponge Spongia agaricina has decreased drastically in recent years and it is now considered an endangered species under Annex 3 of Bern and Barcelona conventions. We describe eight microsatellite markers and present data on their allelic variation and utility as high resolution genetic markers. We analyzed 36 individuals from two populations and found that the number of alleles per locus ranged between 1 and 7. Observed heterozygosity ranged from 0 to 0.72. We found deviations from Hardy–Weinberg expectations for some loci. We exclusively detected null alleles for those loci that deviated from Hardy–Weinberg expectations. Also, distributions of allele frequencies differed significantly between the two populations, making them suitable for population genetic analyses.