Optimization of 14 microsatellite loci in a Mediterranean demosponge subjected to population decimation, Ircinia fasciculata

The recovery potential of decimated populations of sponges will largely hinge on their populations’ size retrieval and their connectivity with conspecifics in unaffected locations. Here, we report on the development of microsatellite markers for estimation of the population connectivity and bottleneck and inbreeding signals in a Mediterranean sponge suffering from disease outbreaks, Ircinia fasciculata. From the 220,876 sequences obtained by genomic pyrosequencing, we isolated 14 polymorphic microsatellite loci and assessed the allelic variation of loci in 24 individuals from 2 populations in the Northwestern Mediterranean. The allele number per locus ranged from 3 to 11, observed heterozygosity from 0.68 to 0.73, and expected heterozygosity from 0.667 to 0.68. No significant linkage disequilibrium between pairs of loci was detected. The 14 markers developed here will be valuable tools for conservation strategies across the distributional range of this species allowing the detection of populations with large genetic diversity loss and high levels of inbreeding.


Introduction
Sponges play an ecologically important role given their abundance and diversity as well as their contribution to primary production and nitrification through complex symbioses in marine benthic communities (Webster 2007). In the Mediterranean, massive mortalities are drastically reducing population size and creating extensive gaps in the distribution of many species, including sponges. In particular, periodic episodes of massive die-offs have been reported for the genus Ircinia (Cebrián et al. 2011) and the environmental stress due to elevated seawater temperatures has been suggested to trigger the disease (Maldonado et al. 2010;Cebrián et al. 2011).
Molecular markers have been widely applied in conservation biology to evaluate the vulnerability of marine species but studies on population genetics using microsatellite markers are available for very few sponges (see Pérez-Portela et al. 2013). Here, we report on the optimization of microsatellite markers further estimation of genetic diversity indexes, populations' connectivity, and for detecting signals of recent bottlenecks in order to evaluate the degree of vulnerability of the sponge Ircinia fasciculata from the Mediterranean.

Methods
Sponge tissue was dissociated prior to DNA extraction and the bacterial symbionts removed by sequential centrifugations. Genomic DNA was extracted using DNeasy Tissue and Blood extraction kit (QIAGEN) to a final DNA concentration of 2 lg and distributed in two lanes of a plate. Pyrosequencing was performed on a Roche Life Science 454 GS-FLX System at the Scientific-Technical Services of  the University of Barcelona. Sequences (220,876) were searched for perfect microsatellites (di-, tri-, tetra-and penta-nucleotides) with at least eight repeats and enough priming regions with Phobos (http://www.rub.de/spezzoo/ cm/cm_phobos.htm). 40,109 sequences contained microsatellites, 61.7 % being dinucleotide, 3.4 % trinucleotide, 3.4 % tetranucleotide, and 0.7 % pentanucleotide. Thirtysix primer sets were designed with the software PRIMER 3 for 14 dinucleotide loci, 14 trinucleotide loci, 7 tetranucleotide loci, and 1 pentanucleotide locus.
Amplification success and polymorphism were tested in two populations of the NW Mediterranean (Costa Brava) collected in 2010-2012: Els Caials (42817 0 19 0 'N 3816 0 40 0 'E) and Blanes (41841 0 N 2848 0 E). Total DNA was extracted and amplified using the REDExtract-N-Amp Tissue PCR Kit (Sigma Aldrich). Forward primers were labeled with a fluorescent dye (Table 1). Samples were amplified on a PCR System 9700 (Applied Biosystems) with an initial 2 min denaturation step at 95°C; followed by 35-40 cycles of 95°C for 30 s, 52-70°C for 35 s (depending on each locus; Table 1) and 72°C for 15 s, followed by a 3 min final extension at 72°C. Amplification products were analyzed on an Applied Biosystems 3730xl Genetic Analyzer at the Scientific-Technical Services of the University of Barcelona. The length and allele scoring of PCR products was estimated relative to the internal size standard GeneScan 600LIZ using the software PEAKS-CANNER v1.0 (Applied Biosystems).

Results and discussion
Out of the 36 microsatellite loci attempted, a total of 14 polymorphic microsatellite were optimized, including a selection of different microsatellite types (see Table 1) because different microsatellite types are equally valid to assess genetic diversity and populations structure of marine invertebrates. No evidence of linkage disequilibrium was detected across all pairwise comparisons. Failed amplifications due to presence of null alleles were detected in 2 loci of the Caials population (Table 1). Five markers showed Hardy-Weinberg disequilibrium after Narum corrections but the overall populations were in HWE (Table 1). Heterozygosity deficit was observed in 5 loci (3 loci in the Caials population and two in the Blanes population; Table 1). The average gene diversity over 14 loci using the Tajima index was 0.684 ± 0.367 in Caials, and 0.668 ± 0.354 in Blanes. Using the genetic G-W index (Garza and Williamson 2001), it seems that all markers were indicating bottleneck events in both populations (Table 1), but more populations should be sequenced for further confirmation. The application of the microsatellite markers developed herein to additional I. fasciculata populations will allow to understand how the genetic diversity is distributed in this species, and to know its overall status, identifying hotspots of genetic diversity, populations affected by large genetic diversity loss and high levels of inbreeding. Information on population genetics of the species is crucial for the assessment of ecological threats and recovery potential following disease episodes and population decimation, and the developing of management strategies when necessary.