First insights on the mitochondrial genetic variability of Lightiella magdalenina (Crustacea), the sole Mediterranean cephalocarid species

Background Here we report the first insight into the mitochondrial (Cytochrome c Oxidase subunit I - COI and Cytochrome b - Cyt b) genetic variation of the only Mediterranean cephalocarid Lightiella magdalenina. Findings COI sequences provide a scenario of low intraspecific variability, while significant genetic divergence occurs between L. magdalenina and L. incisa. Interestingly, Cyt b sequences reveal a higher degree of intraspecific variability, with no shared haplotypes between the sites considered. Conclusions In the future, COI and Cyt b molecular markers could be used as valuable tools to shed new light into the extant species within the genus Lightiella thus providing molecular support to the taxonomical identifications carried out on a morphological basis.


Background
Cephalocarids are a class of small benthic crustaceans distributed from the intertidal zone to a depth of approximately 1550 m [1]. They have been recently considered to be phylogenetically related to the class Remipedia (Crustacea), using both nuclear [2] and mitochondrial [3] markers. Currently, only one order (Brachypoda) has been described with one family (Hutchinsoniellidae) containing twelve species belonging to five genera (Hutchinsoniella, Lightiella, Sandersiella, Chiltoniella, Hampsonellus) [4].
The genus Lightiella harbours the greatest number of species (five) and the largest distribution (for a review see [4] The five species belonging to the genus Lightiella show limited morphological differentiation [4,5]. Given the limited number of specimens used for taxonomical classification, their detailed external morphology is often incomplete e.g. [6]. In light of these considerations, Olesen et al. [4] advise that the genus needs an urgent overhaul. In particular, these authors suggest that the species richness within the whole Cephalocarida class may have been overestimated. A molecular taxonomic approach could address this problem although molecular data are so far available for only three cephalocarid species: Hutchinsoniella macracantha, L. magdalenina, and L. incisa. Mitochondrial and nuclear genes sequences (see [7][8][9][10][11][12][13][14] for details) are available for H. macracantha. Sequences of mitochondrial genes are known for L. magdalenina (see [15] for details), while mitochondrial and nuclear gene sequences are known for L. incisa (see [3] for details).
The species Lightiella magdalenina is the only cephalocarid reported in the Mediterranean and is only found so far from its type locality in La Maddalena Archipelago (Sardinia, Italy) (see [5] for details). Cladistic analysis based on external morphology [5] suggest that L. magdalenina is more closely related to the Caribbean species L. incisa and L. floridana than to the Pacific Ocean species L. serendipita (California, Pacific Ocean).
In this study, we provide the first preliminary insight into the genetic variation of the isolated Mediterranean cephalocarid L. magdalenina using sequences of the mitochondrial DNA Cytochrome c Oxidase subunit I (COI) and Cytochrome b (Cyt b) coding genes from the two close Mediterranean sites where this species is found.
We also provide new specific primers for the COI gene. Results obtained were compared with data from other species of cephalocarids and remipeds.

Methods
Eighteen specimens (10 adults and 8 larvae) of Lightiella magdalenina were collected at a depth of 15-25 m from muddy sand of the southern shore of La Maddalena Archipelago (Sardinia, Italy) ( Figure 1). Sampling was carried out, using SCUBA, during the 2007-2011 spring/ summer seasons. We analysed specimens from the type locality (Punta San Giorgio) and from an adjacent site (Isolotto Roma) (approximately 600 m farther), both characterised by bottoms with Posidonia oceanica and Caulerpa racemosa (see Figure 1 for details). No individuals of L. magdalenina were found when sampling campaigns were carried out in other eight sites from five western Mediterranean localities in northern Sardinia and southern France (see Figure 1 for details).
DNA was extracted from whole specimens by means of the QIAGEN® DNeasy Tissue kit (Hilden, Germany). DNA concentration was estimated through fluorometric measurements (average value per sample: 10 ng/μL).
Since COI universal primers for marine invertebrates [16] did not result in good quality sequences, the COI region was amplified using specific primers designed in the present study (L: 5′-gttactctggggggattcgga-3′ and H: 5′-ggccaaaata gaagaaaccccagcta-3′). Universal primers reported by Boore and Brown [17] (L: 5′-ggwtaygtwytwccwtgrggwcarat-3′ and H: 5′-gcrtawgcraawarraartaycaytcwgg-3′) were used for the Cyt b gene. Attempts to design new specific primers for this region, without critically affecting the quality of the amplified fragment, failed. Each 25 μL PCR mixture contained approximately 50 ng of total genomic DNA, 0.4 μM of each primer, and 2.5 U of EuroTaq DNA Polymerase (Euroclone®, Italy) in a reaction mix prepared according to the manufacturer's instructions. The MgCl 2 concentration was set at 2.5 mM.
PCR amplification was performed with an initial denaturation step of 2 min at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min and 30 sec at 49°C, and 1 min and 30 sec at 72°C. The final elongation step was for 5 min at 72°C with a final cooling at 4°C. In all experiments, negative controls and replicates were included. Electrophoretic runs were carried out at 4 V/cm for 20 min in 2% agarose gels prepared using 0.5 × TBE buffer. The gels were then stained with ethidium bromide (10 mg/mL).
In order to characterise genetic variation among individuals in both populations, the number of polymorphic sites (S), of haplotypes (H), the estimates of haplotype diversity (h), and nucleotide diversity (π), were computed using the software DnaSP 5.10 [20].
Genetic relationships among haplotypes were investigated by constructing a median-joining network using the software Network 4.6 [http://www.fluxus-engineering. com/]. In the absence of random recurrent polymorphisms across the whole dataset, an identical weight (10) was given to all mutations to preserve the original genetic information. We ran the Maximum Parsimony (MP) calculation post-processing option to delete all superfluous median vectors and links that were not contained in the shortest trees of the network. For the COI gene, the analysis was performed by adding also the sequences of two other cephalocarids, Lightiella incisa

COI analysis
Eighteen individuals were sequenced and a 216 base pair (bp) alignment was obtained. A previously published sequence [GenBank:EU530536] [15] belonging to an individual from Punta San Giorgio (see Table 1 for details) was added to the dataset. All 19 specimens shared the same haplotype, except one specimen from Isolotto Roma [GenBank:JX013545], bearing a single non-synonymous transversion (G to C). Indices of genetic variation are reported in Table 1.
Results obtained by the median-joining network analysis (Figure 2a) provided support for strong genetic differentiation between L. magdalenina and L. incisa. Sixteen (for L. magdalenina) and 12 (for L. incisa) point mutations represent the apomorphic polymorphisms of the two species. Twenty-seven point mutations separated H. macracantha from the group including L. magdalenina and L. incisa.

Cyt b analysis
As a possible consequence of the limited homology between the universal primers [17] and the annealing region in our specimens, scorable Cyt b sequences were obtained for only ten individuals (three from Punta San Giorgio and seven from Isolotto Roma). The already published Cyt b sequence [GenBank:EU530537] was not included in the dataset since a BLAST search revealed highest similarity with Saprolegnia ferax (a fungus belonging to the family Saprolegniaceae) (81% identity, coverage 87%) and indicated a possible fungal contamination of this sample. A 348 bp long alignment was obtained. In total, four haplotypes were detected. Values of genetic diversity were higher than those obtained for the COI gene (Table 1). No haplotypes were shared between Punta San Giorgio and Isolotto Roma samples. All of the ten polymorphic sites retrieved were biallelic with seven synonymous substitutions. Eight transversions and two transitions occurred.
The median-joining network analysis (Figure 2b) revealed a well-defined divergence, supported by 85 to 125 point mutations (this pattern contains a cycle with three median vectors separated from each other by 39 mutational steps) between L. magdalenina and H. macracantha. Within the L. magdalenina group, the most common haplotype was exclusive of individuals from Isolotto Roma, while two haplotypes belonging to Punta San Giorgio and one from Isolotto Roma diverged by two to five point mutations from the root sequence.

Discussion
Five decades after the first description of Cephalocarida [21] our knowledge of this taxon is still limited, especially for species other than Hutchinsoniella macracantha. The present work evidences that if COI universal primers [16] fail, even a 216 bp long internal portion (amplified by the new primers provided here) of the widely used 710 bp long Folmer [16] region can be used for the taxonomic identification of the species belonging to the genus Lightiella. Consistently, previous studies [22,23] showed that partial fragments of the Folmer region of about or less than two hundred bp may correctly assess phylogenetic/phylogeographic traits of the species. In such a context, our preliminary analyses provide molecular support to the morphology-based distinction reported between L. magdalenina and L. incisa [5].
Sequencing of the Cyt b gene, performed on L. magdalenina specimens, showed higher values of intraspecific genetic variability than the COI gene. Indeed, the absence of shared Cyt b haplotypes between L. magdalenina specimens from the two neighbouring sites sampled in this study is consistent with the possible occurrence of two distinct populations. In spite of the close geographical proximity of the two sites (only 600 m apart), the isolation can be explained by restricted population size [24] together with a low dispersion capacity of the species [1]. In fact, L. magdalenina, lacks a high-dispersal phase, given that both larval stages and adults show a strictly benthic lifestyle [1,25]. However, it should be taken into consideration that this pattern could be affected by the small number of samples from Punta San Giorgio (three individuals), thus preventing supported inferences. In conclusion, this study reports the first data on the genetic variation of a cephalocarid species, providing a preliminary survey on the mitochondrial variability of the only Mediterranean species, L. magdalenina.
The high level of interspecific genetic divergence reported between L. magdalenina and L. incisa, using the COI gene, offers molecular support to previous morphological analysis [5], allowing to consider this marker as a valuable tool for molecular taxonomic assays within the genus Lightiella. On the other hand, if confirmed by further analysis on a larger number of samples, the estimates of intraspecific variation based on the Cyt b gene for L. magdalenina suggest the possible use of this marker for assessing the phylogeographic patterns of the species.
These results highlight the importance of performing joint (COI and Cyt b) molecular analyses in cephalocarids for further taxonomic revision of the genus Lightiella and population genetic surveys.