RICERCA ITALIANA     

Research program

Integrated approach to the identification of problematic taxa of the marine meiofauna: drafting of volumes of the series "Fauna d'Italia" and development and evaluation of methods of DNA-barcoding in Gastrotrichs, Proseriates and Rotifers

University Co-ordinator

Università degli Studi di MILANO - BIOLOGIA - ()

Research Unit Leader

Giulio Melone

Description

Milan Research Unit (UR), based on its long-term competence and of the experience acquired in the course of COFIN 2004 (prot.2004057239_004: Contribution of rotifers to biodiversity of marine meiofauna) proposes two aims:
1) We intend to contribute to the series ‘Fauna d'Italia' with an issue devoted to the marine rotifers, to be written also in English language to make it available to a larger audience. The support and the interest of the Scientific Committee for "Fauna d'Italia" on this project is testified by their letter (see attachment of mod. A).
2) We want to test the taxonomic status and validity of some species as test-cases, both of taxa with wide distribution, and of taxa with wide ecological tolerance, applying recently developed techniques of DNA barcoding, molecular phylogeny and phylogeography.

Schedule of the Project
1) All data on the rotifers that are present in the literature or that result from our campaigns will be used in the Rotifer issue. All available information will be collected, analysed, assembled and organized to be used. The issue will be organised into two parts, a "general" one and a "special" one.
a) The general part will illustrate anatomy, systematics, phylogeny, ecology, zoogeography, techniques for sampling, sorting out and preparing the rotifers for observation. Particular attention will be given to the sampling methods and to the procedures for sorting alive rotifers out and for studying them under light microscope. Rotifer species often can be identified only if active animals
are considered, in order to observe the traits useful for diagnosis. The diagnostic traits are, in particular, the general morphology of the body, including the foot, the shape and size of the corona, and the shape and size of the mastax. We shall also indicate how permanent slides can be prepared and kept.
Images, drawings and pictures will be obtained at white field, dark field, phase contrast and interferential phase contrast microscopy. When possible, images obtained at Scanning Electron Microscope (SEM) will also be included. A species card is here included to show how we intend to proceed. This card illustrates one of the two species of Seison.
The work to prepare the rotifer issue of “Fauna d'Italia' will last for the two years of this project.
The realization of this ambitious project necessitates the commitment of the whole research group, and also the involvement of collaborators. Two of them have already been trained during COFIN 2004 research project and are now actively collaborating. One new grant is required to contribute to the preparation of the species' cards and to the whole project. This aspect represents a relevant part of this funding request. The instruction of young people is necessary not only to perform the project but also to give the project an important role in the education and in the transmission of scientific competences that are still a richness of our country.
Ricci C. & Melone G., 2000. Key to the identification of the genera of bdelloid rotifers. Hydrobiologia, 418:73-80.

EXEMPLIFICATIVE SPECIES'CARD:
1. Seison nebaliae Grube, 1861
Saccobdella nebaliae van Beneden & Hesse, 1864: 48
Seison grubei Claus, 1876: 79
LOCUS TYPICUS: Trieste
DESCRIPTION: The body can be divided into four parts: head, neck, trunk and foot (Fig. 1). Neck and foot are segmented and can be retracted telescopically. Adult specimens are about 1 mm long, but Remane (1929-33) reported a length of 2.5 mm for an adult male; usually, males are longer than females (Ricci et al., 1993). At light microscope males are easily recognizable, because their cloacal opening is dorsal, at the anterior end of the trunk; in females, the cloaca is dorsal, but at the posterior end of the trunk. Moreover, males possess a longitudinal groove on the ventral side of their head. The head is oval and somewhat flattened laterally. The mouth opens apically in the middle of a small ciliated field, bilaterally lined by rows of ciliar tufts. Under the light microscope, immediately behind the mouth, can be observed the fulcrate mastax, the salivare glands, the oesophagus, the brain and the retrocerebral and subcerebral glands. On the head are located three sensorial organs (antennae) consisting of tuft of few short cilia: one antenna is dorsal, in the middle of the head; the other two are lateral. The fulcrate trophi, as described by Segers & Melone (1998), have a very long fulcrum with a wide dorso-ventral end; rami present long posterior extensions (alulae) parallel to the fulcrum; the apical parts of the rami are covered by epifaringeal plates; unci are small, elongated, slightly bent and provided with one anterior major tooth and an additional, posterior tooth (Fig. 2). The neck is long and is made of three pseudosegments, only the first one bears four rings. The neck containing excretory ducts, esophagous and muscles, can be withdrawn into the trunk. The trunk is oval, tapered caudally and flattened ventrally, with longitudinal folds. It contains the stomach sac, the very small intestine and a voluminous genital apparatus (Fig. 1)
Female genital apparatus consists of two sac-like ovaries in which growing eggs at different stages of maturation can be seen. The two ovaries join caudally in a short oviduct which ends in the cloaca, dorsal at the posterior end of the trunk. Male genital apparatus consists of two sac-like testes that join caudally in a complicate pear-shaped organ, containing the deferent duct and a voluminous seminal vesicle. The pear-shaped organ folds upwards, dorsal to the stomach, and reaches the cloaca, located dorsally at the beginning of the trunk. The foot is made of four pseudosegments and ends with an adhesive disc. In the foot are visible many ducts and glands of different lengths, most of these reach the adhesive terminal disc. The last pseudosegment is bent dorsally and possesses a ventral tubercle, which is the opening of a mucous gland.
BIOLOGY: S. nebaliae lives only as epibiont on leptostracan crustaceans of genus Nebalia. Illgen (1916) found more than one hundred specimens of S. nebaliae on a single host, but usually they are less numerous. S. nebaliae is unable to swim and can be regarded as a sessile epibiont which adheres firmly to its host. It maintains neck and foot withdrawn into the trunk while the host is
moving; on the contrary all the body appears fully extended when the host is still. It can be hypothesized that the transfer to a new host one can occur or from Nebalia mother to offspring, when the juveniles are in the brood chamber, or between adult crustaceans when they are crowded in hiding places during the day, being Nebalia species nocturnal animals. However, it has never been reported how Seison transfers between hosts or during the crustacean moults (Ricci
et al., 1993). The stomach content of S. nebaliae consists of detritus and algae (Illgen, 1916; Ricci et al., 1993), therefore the relationship with its host may be phoretic and/or commensal.
Seison nebaliae is reported to occur, on the same host, together with the congeneric Seison annulatus. However the former lives on the pleopods while the latter attaches to pereiopods, to the edges of the carapaces and, occasionally, to the antennae and abdomen (Claus, 1876; Illgen, 1916; d'Hondt, 1970; Ricci et al., 1993) (Fig. 3).
DISTRIBUTION: The species has been described on specimens collected near Trieste (North Adriatic Sea) (Grube, 1861; Claus, 1876; Illgen, 1916). Later on, Seison nebaliae has been collected at Marseille (Marion, 1872), Naples (Plate, 1887; 1888), Venice Lagoon (Ricci et al., 1993) and Majorca (Balearic Islands) (Segers & Melone, 1998; Ferraguti & Melone, 1999) in the
Mediterranean Sea; at Roscoff (de Beauchamp, 1909; Remane1929-33; d'Hondt, 1970; Koste, 1975; Ahlrichs, 1997; 1998) in the English Channel.
Markevich (1993) reported the finding of Seison nebalia (sic) in the sea of the Sachalin Island (North Pacific Ocean). In the literature are mentioned findings of an undetermined species of Seison, associated with Nebalia, in different places, like Monro Bay in California (Pacific Ocean) (Menzies & Mohr, 1952) and MacMurdo Sound and Magellanic Chile in the Antarctic Sea (Leung & Mohr, 1969) (Fig. 4).
Figure 1 - Seison nebaliae Grube. A) SEM image of a young male in ventral view. F, foot; H, head; N, neck; T, trunk;. B) Schematic male anatomy. C) Schematic female anatomy. cl, cloaca; ov, ovary; st, stomach; sv, seminal vesicle; t, testis; vd, deferent duct. Modified after Ricci et al. (1993)
Figure 2 - Trophi of Seison nebaliae Grube; 1: right lateral view, 2: dorsal view; a: alula, e: epipharynx, f: fulcrum, r: ramus, u: uncus. Bar: 10 µm. Modified after Segers & Melone (1998).
Figure 3 - Position of Seison nebaliae Grube (arrow) on Nebalia sp.. SEM image. Modified after Ricci et al. (1993).
Figure 4 - Finding sites of Seison nebaliae in Italy.

2) DNA barcoding, molecular phylogeny and phylogeography.
This project will involve different steps:
- obtaining animals for DNA extraction
We already preserved in ethanol most of the animals collected during previous surveys in Italy and Europe. Few samples, focussed on peculiar species will surely be needed.
- amplification of genes commonly used for DNA barcoding
Classical primers used to amplify the ‘Folmer region’ of the COI have been widely tested on rotifers, and the protocols are already optimised, both for bdelloids and for monogononts (e.g., Gómez et al., 2002; Fontaneto et al., 2007). Few monogonont species seem to have experienced substitutions in the flanking regions used as primers (Scott Mills, personal communication), but more internal primers have been already designed and worked well in every case they have been tested (Scott Mills, unpublished results). All protocols, from isolation of animals, their long-term preservation, DNA extraction, COI amplification and sequencing, have already been extensively performed in our labs, and absolutely no problems are expected for rotifers in this part of the project. Primer for other regions, e.g. ITS, 28S, 18S, 16S, cytb, have already been tested on rotifers and seems promising.
- sequence analysis
DNA barcoding accepts that species limits are established following traditional practices of taxonomy, usually based on morphology, and the DNA information is fitted into this system of predefined taxonomic groups. This approach is problematic because the correspondence of sequence variation with existing Linnean binomials is expected to be inexact. To avoid problems, a DNA-based taxonomic system should use the sequence information itself as the primary information source for establishing group membership and defining species boundaries.
Therefore, we propose to adopt a newly described method to delineate ‘independently evolving lineages’ from COI sequences themselves, proposed by Pons et al. (2006). This method involves: alignment, phylogeny reconstruction, obtaining an ultrametric tree, and finally a clustering test.
i) aligment of the COI sequences. This can be done with softwares freely available on the web, such as ClustalX (http://bips.u-strasbg.fr/fr/Documentation/ClustalX/), or Mega (http://www.megasoftware.net/);
ii) phylogeny reconstruction. Various methods exist up to now, being the most widely used and accepted (i) parsimony, (ii) maximum likelihood, and (iii) bayesian inference. Softwares are already available to reconstruct phylogenies applying all methods and to test their significance by bootstrapping, jackknifing, or by Bayesian supports. To reconstruct and test a tree based on parsimony models, PAUP (http://paup.csit.fsu.edu/) may be used. For maximum likelihood, two freely available softwares are available, TreeFinder (http://www.treefinder.de/) and PhyML (http://atgc.lirmm.fr/phyml/). For Bayesian inference, MyBayes is freely available (http://mrbayes.csit.fsu.edu/). All these phylogenetic methods assume some models of molecular evolution, which can be estimated from the data themselves with ModelTest (http://darwin.uvigo.es/software/modeltest.html), or MrModelTest (http://people.scs.fsu.edu/~nylander/mrmodeltest2/mrmodeltest2.html).
iii) ultrametric tree. An ultrametric tree is a special kind of a rooted additive tree where the terminal nodes are all equally distant from the root, and it is needed because the model developed by Pons et al. (2006) uses the waiting times between successive branching events on the ultrametric DNA tree as raw data. The ultrametric tree may be easily obtained by rate smoothing the DNA tree using penalised likelihood and cross-validation to choose the optimal smoothing parameter with another freely available software: r8s (http://loco.biosci.arizona.edu/r8s/);
iv) test for significant clustering. The protocol for detecting independently evolving clusters developed by Pons et al. (2006) under coalescent theory first tests that the entire sample does not derive from a single population obeying a single coalescent process; then, under this assumption, it optimises a threshold age, T, such that nodes before the threshold are considered to be diversification events with branching rate and scaling parameter estimated from the tree. Branches crossing the threshold define k clusters each obeying a separate coalescent process. The models can be fitted using an R script developed by Timothy G. Barraclough, and already tested on meiofana (Fontaneto et al., 2007). The script can be performed in R, a freely available statistical software (R Development Core Team 2006: http://www.r-project.org).
v) One of the main problems of DNA barcoding lies in the threshold to be chosen a priori on the differences within-groups and between-groups. Phylogenetic divergence, measured as genetic distances calculated upon the number of mutate position in relation to the reference sequence, may change among different taxonomic groups; therefore, defining a threshold value may be highly misleading, and very subjective. The method developed by Pons et al. (2006) allows to avoid this ambiguity, as it works on the tree itself, and not on thresholds on genetic distances. Such threshold may be obtained a posteriori, after the analysis, and will provide an idea of the degree of divergence in cox1, to be compared among groups by the different UURR.
The application of DNA barcoding has already been proven useful in some meiofaunal groups, and especially for rotifers, where at least 15 cryptyc species have been detected in the Brachionus plicatilis species complex worldwide (Gómez et al., 2002, 2007; Suatoni et al., 2006). Its application on bdelloid rotifers proved that these asexual organisms have been able to speciate in similar ways than ordinary sexual organisms (Fontaneto et al. 2007). Our expectation is therefore to be able to apply DNA barcoding to disentangle taxonomical problems already present in meiofauna, and of course to discover and analyse in detail cryptic taxa in other species traditionally considered as a single entity.