Research Article - (2015) Volume 18, Issue 1
To evaluate genetic variability within and among Populations of Hyalinoecia tubicola (Müller, 1776), 30 individuals from seven populations were compared using random amplified polymorphic DNA (RAPD) markers. Populations of Hyalinoecia tubicola (Müller 1776) were sampled during the period 2005-2006 from six different sites in the Cap Bon peninsula (North East coast of Tunisia). Genomic DNA of the 25 individuals studied was purified, and genetic analysis was conducted using RAPD primers. Molecular variability into the populations was analyzed using PopGene 1.32 software. One population of Aponuphis bilineata was used as an out group for the study. Results showed that the sampled populations are genetically diverse according to the high value of the gene flow (8.26) and the Nei’s genetic index for the total populations (0.2). These preliminary results have important implications for conservation policy.
Keywords: Hyalinoecia tubicola; Aponuphis bilineata; Population genetics; Polymorphic DNA; PCR; Cap Bon peninsula
Biodiversity was evaluated for long time by simple species inventories. The measures of intraspecific biodiversity were generally based on individual’s morphological analyses by comparison of phenotypic characters. However, molecular approaches based on genetic marker are nowadays extensively used, and are highly informative especially to study population structure. Techniques derived from PCR (Polymerase Chain Reaction) are highly effective in identifying species, varieties or individuals by DNA fingerprinting [1-4]. In this context, the RAPD (Random Amplified Polymorphic DNA) technique  is used as it provides numerous genetic markers useful to assess genetic variability and population structures when no DNA sequence data are known on the studied species. RAPD technique was previously used only for several studies on polychaetes, mainly those dealing with molecular phylogeny of some families or species. Struck HT, et al. studied the phylogeny of Eunicida and their presumed relationship to Dinophilidae; showed that monophyly of Clitellata is well-supported by both morphological and molecular data [2,6]. RAPD technique was also used to verify the cosmopolitan status of Hesionides arenaria and Stygocapitella subterranea , of Ctenodrilus serratus , and to assess the genetic variability of Neanthes succinea , of Pectinaria koreni , and of Alitta succinea .
To our knowledge, no genetic data were previously obtained from the polychaete Hyalinoecia tubicola (Müller 1776). This organism is an Onuphidae which has a broad geographic distribution living in oceans all over the world from shallows to 2448 m depth . Thus, H. tubicola is distributed in the western Atlantic [13,14], in the eastern Atlantic [15-17], in Indo-Pacific  and in the Mediterranean Sea [18-21]. In Tunisia, H. tubicola was reported in the North , in the Bay of Tunis [23,24] and in the Cap Bon Peninsula [25,26] H. tubicola lives in fine and muddy sediments of the circalittoral and bathylittoral zones of the Mediterranean Sea but occurred in coarse substrates of Atlantic coast . According to , H. tubicola is a detrital circalittoral species, but rarely found in the bathyal zone. In the present study, H. tubicola is among the most abundant polychaete species collected in the circalittoral bottoms of the Cap Bon peninsula (from 40 m to 60 m).
Aponuphis bilineata is also a member of the Onuphidae family. The distribution area of this species seems limited to the North Atlantic [29-32] and the Mediterranean coasts [33-36]. In Tunisia, it was reported in the Gulf of Tunis  and, as H. tubicola, in the Cap Bon Peninsula . According to , A. bilineata can live in a wide range of sediment as, it has been found in more or less muddy fine sands [40-42], compact muds, organic sand  or muddy gravel .
This paper is the first study assessing the intrapopulation genetic variability and the genetic differentiation level of six populations of Hyalinoecia tubicola from the Cap Bon Peninsula (Western Mediterranean) using the RAPD technique. Living in the same repartition area, A. bilineata was used as an out-group in this study for inter-specific comparison.
The study area (Figure 1), located on the north-east coast of Tunisia, covers 300 km of coastline and is a transition zone between the eastern and western Mediterranean basins. Tunisian marine waters belong to the western and eastern Mediterranean basins. It is in the Strait of Sicily that is the total trade between the two Mediterranean basins . Currents are complex due to its geographical position, the morphology of the seabed and wind regime .
The salinity and temperature of these waters present seasonal fluctuations and is influenced by the air temperature. The salinity of these waters can reach 38% and is often subject to the influence of the Atlantic surface water whose maximum occurs in winter and the waters of the eastern basin that seem rise to the surface in the spring and summer.
The depths of stations investigated varied between 45 m and 60 m. The particle size analysis shows that the sediment ranged from muddy sand to gravel, mainly constituted of muddy sand, fine sand, gravel and rocky substrate (Table 1).
|Populations||Sites||Depth (m)||Position||Sediment type||Individuals|
Table 1: Hyalinoecia tubicola and Aponuphis bilineata populations and main characteristics of sampling sites.
In this study, six populations of Hyalinoecia tubicola (Table 1) were collected from the Cap Bon Peninsula (north east coast of Tunisia) from six sites located in: Sidi Daoud (Mt2), Ras Ed Drek (Mt15 and Mt16), Dar Allouche (21) and Kerkouane (Mt24 and Mt27); and Aponuphis bilineata from one site (Mb37) between Menzel Temim and Menzel Horr, from September 2005 to May 2006. Aboard the R/V Hannibal, a Van Veen grab (0.1m²) was used on the sites 1-32, 15 and 16, and a Charcot dredge was used to sample the sites 22, 25, 26 and 37. Samples were washed through a 1-mm-mesh sieve and fixed in 7% sea water formaldehyde for identification. In the laboratory, samples were rinsed in fresh water and preserved in 70% ethanol. The sorted polychaetes were identified to species and counted.
DNA extraction and PCR amplification
Genomic DNA was purified from 20 mg of tissue from 5 individuals of each population except for population Mt2 (1 individual) and Mt27 (4 individuals), using the ‘DNeasy Blood & Tissue Kit’ (QUIAGEN). Seven random decamers RAPD primers  were tested and three primers, IBEA08 (5‘GAAACACCCC3’), IBEA09 (5‘TGTAGCTGGG3’) and IBEA10 (5‘AGGGCCGTCT3’), were selected based on their band reproducibility and variability. The amplification solution (25 μl) contained 50 ng genomic DNA templates, 100 μM of each dNTP (Promega), 5 pmol of each primer (MWG Biotech), Green Flexi 1X reaction buffer, 2 mM of MgCl2 and 1 unit of Taq DNA Polymerase (Promega).
PCR was performed in a Mastercycler thermocycler (Eppendorf) as follows: the initial denaturation at 94°C for 3 min was followed by 45 cycles of 30 s at 94°C, 30 s at 36°C, and 1 min at 72°C, with a final elongation step at 72°C for 10 min. Amplifications replicates were done for each samples.
PCR product migration and visualization
Fragments generated by PCR amplification were separated by size through electrophoresis on 1.4% agarose gels buffered with 0.5× TBE. Bands were visualized by illumination under UV light after staining with ethidium bromide using a video camera. RAPD fragment size was estimated by comparison with the 1 kb Plus DNA Ladder.
Genetic analysis of PCR data
After collecting all the results obtained for the same primer, the bands are numbered according to their location on different gels, and the presence (1) or absence (0) of each band is referenced in a binary matrix for each amplified sample. Data analysis was performed using POPGEN 1.32 software .
On the thirty individuals collected at the Cap Bon Peninsula and further analyzed by RAPD technique, 25 morphologically identical samples (6-30) belong to the species H. tubicola (Mt2, Mt15, Mt27, Mt24, Mt21 and Mt16). While one population (Mb37) with five sampled individuals (1-5) belongs to the species A. bilineata and are also morphologically identical.
The three primers (IBEA08, IBEA09 and IBEA10) selected for the study showed a broad affinity for all individual DNA studied and generated a total of 43 suitable and reproducible DNA bands, ranging from 200 to 1800 bp. RAPD fingerprinting profiles are shown in Figure 2. The total number of amplified bands generated per primer ranged from 13 (IBEA09) to 16 (IBEA08), with most of them (99%) being polymorphic (Table 2). RAPD fragment size was estimated by comparison with the DNA Ladder.
|Primers||Sequences (5’-3’)||NB||Size (pb)|
Table 2: RAPD primers, sizes and their sequences in the study.
Table 3 shows genetic variability parameters obtained within each population based on RAPD data. The highest number of alleles (na) was observed in the Mb37 population of A. bilineata (1.67) and the lowest in M2 population of H. tubicola (1.0). Maximum and minimum number of effective alleles (ne) was observed in Mb37 (1.30) and Mt2 (1.0), respectively. Identified loci showed a high polymorphic level over all the studied populations (99%). This result indicates the efficiency of the RAPD technique used for the genetic diversity analysis of the studied populations. The highest polymorphism level was found for the single population Mb37 of A. bilineata (67.44% of polymorphic loci or 29 loci), followed by the population Mt16 of H. tubicola (39.53% of polymorphic loci, 17 loci), whereas the population Mt2 with only one individual is monomorphic.
Table 3: Genetic variability within each population based on RAPD data (na: number of alleles; ne: number of effective allele; P: number of polymorphic markers; P (%): percentage polymorphic loci; h: gene diversity; I: Nei's genetic identity, he: mean expected heterozygosity .
The percentage of polymorphic loci (P loci%), Nei’s gene diversity (h) and Shannon’s information index (I), calculated for different populations, indicated that the genetic diversity within Mb37 (P loci=67.44%, h=0.19 and I=0.30), situated between Menzel Temim and Menzel Horr, showed the highest values, whereas the lowest values were observed for population Mt24 localized across from Kerkouane (P loci=20. 93%, h=0.06 and I=0.10). Both populations Mt15 and M16 have close Nei’s genetic diversity h values and Shannon’s information index I: 0.11, 0.10 and 0.16, 0.17, respectively. The same observation can be done for Mt24 and Mt27. At the populations level, a relatively low value of the diversity coefficient among populations Gst=0.057 was obtained whereas a high value of gene flow Nm (8.26) was revealed. The average coefficient θ (Fst) shows that 35% of the variation is expressed among populations while 65% of the variability lies within populations (Table 4). While, analysis performed separately for the six populations of H. tubicola revealed a relatively high value of Gst (0.60), low value of Nm (0.33) and low average coefficient θ (0.07) which indicate that 93% of the variation is within populations.
|Ab and Ht populations||0.20 (0.02)||0.057||0.35 (0.04)||8.26|
|Ht populations||0.19 (0.02)||0.60||0.07 (0.005)||0.33|
Table 4: Analysis of Nei genetic diversity in populations of two species H. tubicola and A. bilineata in the north-east coast of Tunisia. Ht, total genetic diversity (SD); GST, average coefficient of genetic differentiation of Nei (Nei, 1987); Theta (standard deviation) analogous to FST (Weir & Cockerham, 1984; Weir, 1990-1996) and Nm, gene flow [47-49].
Table 6 showed the Nei’s genetic identity (above diagonal) and genetic distance (below diagonal) between the different populations based on RAPD data.
The maximum value of the genetic distance was obtained between Mt2 and Mb37 (0.358); the lowest value of genetic distance was obtained between Mt24 and Mt16 (0.016) (Table 5). The matrix of Nei’s genetic was used to build an UPGMA based dendrogram (Figure 3). No clear clustering of the individuals from each studied population was observed. The number description of individuals and populations appearing in the dendrogram are shown in Table 5.
Table 5: Populations and individual’s numbers
Table 6: Nei's genetic identity (above diagonal) and genetic distance (below diagonal) between different populations based on RAPD data
This study showed for the first time that the RAPD-PCR procedure is a sensitive and accurate method applied on the polychaete H. tubicola as it requires no preliminary information about the DNA sequence of the studied individuals. Moreover, although the samples were fixed in formalin and preserved for more than one year in 70% alcohol, the amplification of the genomic DNA of both species H. tubicola and A. bilineata by this technique was highly informative and produced a large number of polymorphic markers. The analysis of the RAPD data succeeded in evaluating the genetic diversity among and within the applied H. tubicola. The 6 populations of H. tubicola (Mt2, Mt15, Mt27, Mt24, Mt21 and Mt16) collected at the Cap Bon Peninsula and further analyzed by RAPD markers presented samples (individuals 6-30) that cannot be morphologically differentiated. The population Mb37 with five sampled individuals (1-5) belonging to the species A. bilineata and also morphologically identical was used as an out group for this analysis. The level of genetic variability detected within the populations of H. tubicola was high (P=100%, He=0.17 and I=0.29), and demonstrated the high capacity of these molecular markers to reveal polymorphism . This could be also explained by the location of the study area, which is a transition zone between eastern and western Mediterranean leading to promote gene exchange between populations. The A. bilineata population shows a twofold higher level of intra-specific variability compared to H. tubicola populations.
Genetic diversity among populations
Overall, measures of genetic differentiation by the three parameters Nm, GST, and θ indicate a strong genetic divergence between the out-group of A. bilineata and H. tubicola from the north-east coast of Tunisia associated with high gene flow (8.26). This value reflects an important genes exchange between populations and is higher than that found by  (Nm=1.12) for the Nereididae species Alitta succinea from the Romanian coast of the Black Sea. Although this high value of gene flow, it does not prevent genetic differentiation among populations.
Genetic differentiation between populations is also confirmed by the values of the genetic distance ranging from 0.005 to 0.43 which shows relatively high genetic variability. High differences were detected between Mt2 of H. tubicola and other populations of the same species. On the UPGMA dendrogram, the two individuals 6 and 24 seem to cluster even if they belong to different species and different groups of sampling. These individuals, showing genetic divergence compared to the others individuals, could be cryptic species. This is responded in polychaetes species like Hediste diversicolor , Perinereis cultrifera , Arenicola defodiens and Arenicola marina , Notophyllum  and Pygospio elegans . Low genetic distances are observed between populations of the two species A. bilineata and H. tubicola. These relatively low values lead us to think that both species present a common genetic background and may belong to the same genetic group. Indeed, some authors point out that the physical, biological and oceanographic conditions such as temperature, areas of high productivity, food availability and marine currents [55,56] could influence migration and therefore the reproduction exchange between populations of both species. On the other hand, according to the geographical location of populations, type and duration of reproduction, life and also the morphology of individuals vary greatly .
In conclusion, this is the first estimation of the genetic diversity of Hyalinoecia tubicola from the Tunisian coast based on RAPD data analysis. Species determination is a difficult work that must now combined morphological and molecular analysis. Most studies to evaluate intra- and inter-specific diversity conducted on polychaetes used nuclear 18S and 28S genes  and mitochondrial COI and 16S genes (ref). However, this study demonstrate that the RAPD technique is valuable to determine the level of genetic diversity in H. tubicola populations and further work should be done using more specifically COI to extend our knowledge on the potential cryptic individual found in this study.
We would like to thank the personnel of the R/V “HANNIBAL” for their help in sampling and treating benthic samples. We are also grateful to the anonymous reviewers for their reviewing, advising, and critical comments on the manuscript.