RICERCA ITALIANA     

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

Università degli Studi di Sassari

Abstract

Marine meiofauna is a compartment of the marine biota, of particular interest for its high diversity, at all the hierarchic levels considered. Biological traits of meiobenthic organisms (such as their high specific diversity, sedentary habits, absence of larval stages, short generation times) make them ideal candidates as biological indicators for monitoring marine environmental health. However, to date, this abundant and ubiquitous assemblage of invertebrates has been largely neglected in applied sampling programmes. Disadvantages which hinder their utilisation include their small size, which requires specific competences in the handling of samples, and, eminently, the lack of supports to identification specifically addressed to non-experts taxonomists. Present project therefore aims to provide tools to facilitate the study of three groups of meiofaunal organisms, whose identification at the species level is particularly problematic, both in the grounds of traditional taxonomy and in the development of a DNA-based approach.
1) drafting of volumes of the series “Fauna d’Italia” dedicated to Gastrotricha, Platyhelminthes Proseriata and Rotifera.
Research performed under the sponsorship of the PRIN-2004 project “Contribution of meiofauna to the marine biodiversity of the italian coasts” led to the gathering of a wealth of data on the composition and distribution of the three taxa along the coasts of Italy and in general of the Mediterranean. At present, however, reference books, where the vast and disperse literature is presented and synthesized, and which can be used both by specialists and marine ecologists engaged in investigations of environmental monitoring, are lacking. The volumes of the "Fauna d’Italia", with a long tradition of prestige and authority, and easily available in every zoological library, are an ideal vehicle to make accessible to an audience of potential users the knowledge acquired only after years of experience in the taxonomy of a particular group. Contacts with the “Scientific Committee of the “Fauna d’Italia”’ (which includes the scientific coordinator of this project among its members) ensure the interest for the publication of the volumes, which will constitute a precious legacy that will facilitate, in the future, new generations of scholars.
2) contribution of experts taxonomists to a DNA-based identification system.
Because of the difficulty of sample processing and scarcity of diagnostic characters, the meiofauna is one of the groups where the use of techniques of DNA-barcoding could be more advantageous. Nevertheless, the use of this approach has so far been marginal. Present project therefore aims to the development and evaluation of the techniques of DNA-barcoding for the identification of Gastrotricha, Platyhelminthes Proseriata and Rotifera. Due to the pioneering nature of this aspect of research, in each taxon it will first be necessary to identify molecular markers capable to allow discrimination at the level of species; intra-and interspecific genetic variability will be tested in phylogenetically closely related species and in several populations within the distributional range of target species.
We believe that this approach that combines classic taxonomy with the foundations for a DNA-based taxonomy (as basis for the accession in gene bank of diagnostic sequences of the highest number possible of species) may constitute a cultural “backbone” that will allow identification of representatives of the three taxa even by non-specialists. In a scenario of “global change”, in fact, biomonitoring programmes may greatly benefit from the sound assessment of biodiversity and variation in composition and distribution of these speciose taxa that lack dispersal stages. Most of the financial request of the programme is devoted to the funding of young, mostly post-doctoral, researchers. The training of young researchers is deemed necessary to complete the proposed programme, but also to ensure that the project may play an important role in the education and transmission of scientific competences in the field of taxonomy. <<<

Principal Investigator

Marco Curini Galletti Università degli Studi di SASSARI

Research Objectives

In an era marked by phenomena of global change, the problem of the reduction of biodiversity and of the measures to tackle this threat represent an important challenge for the biological research. Sound taxonomy underpins almost all study in the environmental field. Despite this, there is currently a serious crisis in taxonomic expertise throughout the scientific community, resulting in the neglect of many highly diverse groups of organisms from ecological research. This problem is particularly acute for the taxa belonging to marine meiofauna. Biological traits of the organisms belonging to meiofauna (such as their sedentarity of adults and the absence of larval stages, high specific diversity, very short generation times) make them ideal biological indicators for investigations in the field of environmental monitoring, but their use has been so far marginal.
The project aims to contribute to the resolution of the "taxonomic impediment" present in groups of meiofauna by providing tools to facilitate identification at the species level of problematic taxa, both in the grounds of traditional taxonomy and in the development of a DNA-based approach.
1) the most basic requirement for people studying and working on aspects of biodiversity is the availability of species identification guides on a geographical basis. These are important not only as a resource for identification and stimulus for the study of little known groups, but also because they constitute a valuable cultural legacy per se. During the project PRIN-2004 “Contribution of meiofauna to the marine biodiversity of the italian coasts”, which involved all the Research Units (RRUU) partecipating to present project, a series of sampling campaigns were performed, targeted to the study of the composition and distribution of groups of marine meiofauna (Gastrotricha, Platyhelminthes Proseriata and Rotifera) along the coast of Italy and the Mediterranean. The campaigns led to the discovery of numerous new species, and the whole area ranks at present as one of the best known in the world in this respect. Therefore, it would be of particular interest to synthesize all the available data in monographic volumes, which may allow these groups to be accessible to a wider scientific audience. Given the current state of knowledge, these volumes, extended to cover all species of the Mediterranean, should not be meeting to rapid obsolescence, and could represent for years a "standard reference" for the area. The project thus aims to the production of three volumes of the series “Fauna d’Italia", dedicated to marine species belonging respectively to Gastrotricha, Platyhelminthes Proseriata and Rotifera, and conceived with the finality to result useful not only to specialists, but also to marine ecologists who may find these abundant metazoans in the course of research on interstitial benthos. To this aim, a series of identification keys will be produced, which may result particularly useful to non expert taxonomists.
2) recently, a radically different approach to traditional taxonomic search was proposed. DNA-barcoding is based on the premise that a short standardised gene sequence may allow the specific anttribution of undetermined samples, because interspecific genetic variability exceed that within species. Various regions have been emploied for species-level byosistematics (cox1, cytb, 18S-rDNA, 28S-rDNA, ITS1-rDNA, ITS2-rDNA). However, DNA-barcoding advocates the adoption of a “global standard” and a ~ 650-base fragment of the 5’ end of the mitochondrial gene cytochrome c oxidase (cox1) has gained designation as the barcode region for animals. This approach has raised great interest but also concerns in the scientific community, clearly divided between supporters and critics (especially "traditional" taxonomists). However, it is undeniable that this approach would find its ideal application in taxa of the meiofauna, characterized by the difficulties related to their study (which may require the examination of living individuals) and by the scarcity of diagnostic morphological characters. The project is therefore also aimed to the development and evaluation of techniques of DNA barcoding for the identification of Gastrotricha, Platyhelminthes Proseriata and Rotifera, on which, at present, there is no information at the regard. As expert taxonomists not new to molecular experience, we believe to be in good position to assess the advantages and avoiding the pitfalls inherent the application of DNA-barcoding in the classification of groups of organisms on which this technique has never been tested before. <<<

First Results

The research will provide, in the first place, traditional and molecular taxonomic tools useful to facilitate the study of meiofaunal groups, whose identification at the species level is seen as particularly problematical. The resolution of the taxonomic impediment will allow a wider use of these taxa whose potential for an advantageous use in the bio-environmental fields appear extremely relevant , especially in an era marked by phenomena of global changes.
More specifically, the research will allow the drafting (in English) of three volumes of the series “Fauna d’Italia” devoted respectively to Proseriata, Gastrotricha and Rotifera, which will include a total of about 500 species found throughout the Mediterranean. Since currently there are not available worldwide monographs on these groups, the three volumes will constitute a valuable, lasting legacy which can facilitate, in the future, the work of new generations of Italian and foreign scholars. The outcome of the project will benefit not only taxonomists but also the many marine ecologists engaged in investigations of the environmental impact. With regard to the latter point, our model reference are the monographs of the series “Synopses of the British Fauna” devoted to the marine nematodes (Vol. 28, 38 and 53), largely used by ecologists throughout the world, notwithstanding the fauna treated is markedly regional.
This unique opportunity will also enable the RRUU to carry out sampling campaigns focussed to the collection of rare and/or problematic species the re-description of which is necessary in order to perform taxonomic revisions essential for a more natural (phylogenetic) systematization of the taxa, that will be treated in the books.
Furthermore, the project will contribute to the development of techniques of DNA barcoding for the identification on molecular bases of Proseriata, Gastrotricha and Rotifera species. This approach to taxonomic research is radically different from the traditional one (morphological), as it uses a short DNA sequence from a standardized and agreed-upon position in the genome as a molecular diagnostic for species-level identification of biological samples of unknown identity. The possible utilization of this technique, apparently simple but not without its drawbacks, will be the subject of careful evaluation. In this respect, as morphologists with proved molecular experiences we believe to be in a good position to assess the advantages while avoiding the pitfalls inherent the application of the DNA barcode in the classification of organisms on which this technique has never been tested before. Should the technique pass the experimental verification, the scientific community will have a solid foundation on which to develop databanks of diagnostic sequences.
This part of the project will not see only the interaction between the Italian RRUU, but also the interaction between these and some prestigious foreign scientific Institution (i.e., Swedish Museum of Natural History, Stockholm and Imperial College, London). These synergies can not only solve the practical problems that might arise during the research stages, but will also bolster the cultural and scientific growth of personnel involved.
The applicants believe that such an approach that combines the classic taxonomy with the foundation of a taxonomy based on DNA may constitute a cultural inheritance that in the future will allow the identification to species of representatives of the three taxa also to non-specialists. As a consequence, these meiofaunal organisms might finally be used in a large variety of investigation spanning from topics of the basic research such as biogeography and phylogeny to more applied studies such as environmental biomonitorig.
The financial support requested will contribute substantially to the activation of scholarships and research allowances, necessary both to complete the proposed programme, but also to enable that such projects have an educational connotation while avoiding that the taxonomic skills, still present in Italian Institutions, might get lost. <<<

Timescale

24 months

National and international background

The unprecedented rate at which human activities around the world are causing widespread habitat degradation and species extinction are alarming (World Conservation Monitoring Centre, 1992). That extinctions are occurring before even half of the world’s species have been described (May 1992; Wilson 2003) is evidence of a global taxonomic crisis. The problem is particularly serious in marine environment, where the knowledge of biodiversity is still far from being adequate - even for comparatively known macrofaunal taxa (Bouchet et al., 2002) - and based upon unrepresentative data, which make assessment of its modifications during time, impossible (Sala, 2002).Present level of knowledge is even poorer for meiofaunal, “inconspicuous” organisms. The term meiofauna refers to the assemblage of marine benthic metazoa intermediate in size between macrofauna and microfauna. By definition, it includes organisms passing through a 500 µm sieve and retained on a 42 µm sieve. This size range separates a discrete group of organisms, whose morphology, physiology and life history characteristics have evolved to exploit the interstitial matrix of marine soft sediments. Meiofauna is phyletically more diverse that any other component of the marine biota, at any taxonomic level considered. In fact, all of the most recently described phyla belong to meiofauna: Gnathostomulida (1956), Loricifera (1983) and Cycliophora (1995) (Ax, 1956; Kristensen, 1983; Funch &amp; Kristensen, 1995). Application of molecular techniques has shattered present understanding of the Platyhelminthes,whose free-living representatives are, for the vast majority of species, belonging to meiofauna, resulting in the removal of Acoela and Nemertodermatida, both recently established at the phylum level, and considered of crucial phylogenetical importance as sister taxa of the remaining bilateria (Wallberg et al., 2007).
Furthermore, the suggestion of the existence of a simple relation between the number of individuals in a given size class and the number of species present in that class (May, 1975), implies that the number of species of meiofauna, which may be phenomenally abundant (up to 23 million specimens of nematods alone per square meter have been documented!) (Warwick &amp; Price, 1979), may be equally very high (Blaxter, 2003; Siemann et al., 1996).
Due to its taxonomic diversity, and species richness, meiofauna represents an important component of marine biodiversity. Therefore, it should be included in any study aimed to the individuation of high diversity areas for conservation purposes. Furthermore, recent observations suggest that meiofaunal organisms are particularly sensitive to environmental modifications (see Kennedy &amp; Jacoby, 1999, for a review). In fact, given the quick inter-generation time of meiofauna (matter of weeks (Warwick, 1981) most meiofauna produce multiple generations per year. Consequently, sublethal effects of toxicants on fecudity, growth rates, genetic expression or longevity may be detected more rapidly than in long-lived organisms belonging to the macrofauna (Coull &amp; Chandler, 1992).
Present lack of knowledge about composition and distribution of meiofauna raises concerns also in the light of the phenomena of global change, which are presently affecting the whole planet. The Earth's climate, in fact, has warmed by approximately 0.6°C over the past 100 years, with two main periods of warming, between 1910 and 1945, and from 1976 onward. The rate of warming during the latter period has been approximately double than that of the first and, thus, greater than any other time during the last 1000 years, and further warming is predicted to continue for the next 50-100 years (Parmesan et al., 1999). The anthropogenic climate change that is expected during the next century looms as an unprecedented threat to biodiversity (Stuart Chapin et al., 2000). The predicted rate of warming alone may move many species well beyond their current climate-niche ranges. Some species will find themselves in habitats that are unsuitable in many secondary ways, for example, for the lack of specific breeding microhabitats changes in seasonal resources and in the biogeography of pathogens, predators and competitors, which could trigger extinctions events (ref. in Root et al., 2003).Climatic fluctuations exert an overriding role on the marine biota. There is some evidence that Mediterranean biodiversity patterns are presently facing changes that can be related to increasing seawater temperatures (ref. in Stebbings et al., 2002). Meiofaunal organisms, characterized by short life cycles, poor dispersal, and, often, with restricted distributions, may allow documentation of the phenomenon in the sea, with a degree of confidence greater than that given by particularly vagile species (such as fishes) where it is not always easy to discriminate whether recent reports, sometimes of single specimens, outside the species' normal range may reflect an actual modification of the range or rather accidental events.
Notwithstanding all the traits of potential interest, to date, this abundant and ubiquitous assemblage of invertebrates has been largely neglected in applied sampling programmes, as well as in studied aimed to assess local levels of biodiversity. Kennedy &amp; Jacoby (1999) have exaustively examined advantages and disdvantages of the adoption of meiofauna as a tool for environmental monitoring, and they singled out taxonomic complexity, lack of specific expertise, and limited taxonomic literature accessible to non specialists, as major hindrances.
Taxonomic studies on meiofaunal groups appear indeed declinating. The so-called “taxonomy crisis”, with the progressive disappearance of experts of entire taxonomic groups, and lack of recruitment, has in fact particularly affected meiofauna research. Furthermore, the study of meiofaunal organisms, due to their particularly disperse literature, and, with very few exceptions, lacking monographies devoted to regional faunas, which may be particularly useful for identication purposes, needs a particular expertise, resulting from several years of training.
The most basic requirement for those who work in the field of biodiversity is the availability of species identification guides. Without such guides it is impractical for most of the researchers to identify individuals of a taxon, and consequently, the biology, ecology and the potential information content, at all levels, of such taxon will remain unknown. Identification guides are not only important as a resource, but also because they enable many more scientists to begin to recognise and study previously little known species or groups. Thus, plots of the accumulated rate of discovery of marine species (Costello et al., 1996) have a sigmoid shape, from little discovery in the early stages, to rapid discovery once some monography has been published, and then decreasing rates of discovery as the taxon becomes satisfactorily known, at least at the geographic scale covered by that book. To date, adequate identification guides to numerous macrofaunal taxa are available; much less is the number of monographies that concern taxa of the meiofauna. Recently, it has been stressed how their lack is particularly felt in soft-bodied meiofauna, particularly in the Mediterranean area (Costello et al., 2006). This is precisely in a time when the number of professional taxonomists is declining (Boero, 2001), and when the drafting of a monographic volume, which requires considerable commitment and time, is at variance with current policies of evaluations for careers and fundings, which rely strongly on impact factor, thereby supporting multiple publications in journals.
In order to counter the “taxonomic impediment” above, a radically different approach to traditional taxonomy was recently proposed, based on the possibility that a single gene sequence may enable to distinguish between animal species, or at least amongst their vast majority (Hebert et al., 2003). The use of a conserved portion of the mithocondrial DNA gene cytochrome c oxidase subunit I (cox1) was proposed as a global bioidentification system for animals, on the premise that genetic variation between species exceeds that within species. The sequence was likened to a “barcode”, with species being delineated by a particular sequence or by a tight cluster of very similar sequences. On these bases, a "Consortium for the barcode of Life" was developed, as outlined in the web site Barcode of Life Data Systems (www.barcodinglife.org) (Ratnasingham &amp; Hebert, 2007). The nucleotide sequence of a particular ~ 640 nucleotide region, known as “Folmer region” (Folmer et al., 1994) (the "global standard") has been taken as the identification code to facilitate the correct taxonomic identification of specimens and the discovery of new species (Moritz &amp; Cicero, 2004). It has proven particularly useful in taxa where species attribution is complicated, due to the paucity of easily obtainable diagnostic morphological features, and where cryptic diversity may be rampant (see Dericke et al., 2007, for resolution of sibling complexes in Nematoda). This technique has recently proven to be a powerful tool in the classification at the level of species of undetermined samples in numerous groups (ref. Smith at al., 2006; Kelly et al., 2007; Kerr et al., 2007).
However, not always DNA barcoding of the Folmer region has given the expected results in the resolution of closely related species. Furthermore, its amplification, within a broad supra-specific taxon, may prove problematical, due to the variability of its flanking region, which may prevent from the utilisation of the "universal" primers (Vogler &amp; Monaghan, 2006). Among recent cases of failure of cox1 as barcode, we may quote the Cypreidae (Mollusca) (Meyer &amp; Paulay, 2005) (where, however, undetected cases of cryptic speciation may be present), Porifera (Erpenbeck et al., 2006; Park et al., 2007) and Anthozoa (Shearer et al., 2002). Thus, several other markers have also been proposed as putative barcodes (cytb, 18S-rDNA, 28S-rDNA, ITS1-rDNA, ITS2-rDNA)(Markmann &amp; Tutz, 2005; Monaghan et al., 2005; Smith at al., 2006).
There are also other problems, which limit at present a widespread adoption of DNA-barcoding.
1) a DNA-based system of identification can only work if all the species have their diagnostic sequence(s) present in the data-base. An incomplete data-base will simply allow users to determine whether a given sequence is different from the others already stored. Such a result will not identify the specimen in question per se, and will not automatically imply that the specimen is a member of a new species (Dayrat, 2005). This problem is particularly true for meiofaunal taxa, where data-bases include only very few sequences (especially nematodes, cf. Dericke et al, 2007). Furthermore, for many taxa of meiofauna, even test experiments, aimed to ascertain which sequence can be used for barcoding, have not been performed. Yet, the identification of organisms of the meiofauna, due the difficulties inherent to their study, would be the ideal subject for a DNA based identification system.
1) for this approach to be effective, it must be possible to distinguish between intraspecific and interspecific mtDNA variation. Until recently, in fact, DNA-barcoding has focused on describing collections of species within geographical areas, leaving open the question of whether sympatric, closely related species might be distinguished by the technique, or which is the degree of intra-specific variability of allopatric populations, in a wide geographical area (Moritz &amp; Cicero, 2004).
Recently, the usefulness of the involvement in the research of experts taxonomists has been underlined, as the indispensable basis to identify and test the actual discriminating power of the sequences on phylogenetical bases, and on particularly problematic taxonomic cases (Kelly et al., 2007)
From what reported above, it is apparent the role of experts taxonomists, in providing tools for species identifications both on morphological and molecular bases. The fact that it was estimated that the average age of professional taxonomists, worldwide, is about 54 years (Winston, 1988), highlights the potential loss of information that we are facing in the coming decades.

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