Species Identification of Collembola by Means of PCR-Based Marker Systems
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Species identification of Collembola by means of PCR-based marker systems Vorgelegt dem Fachbereich 2 (Biologie/Chemie) der Universität Bremen als Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) von Ulrich Burkhardt Universität Bremen Fachbereich II Biologie/Chemie März 2005 Gutachter der Dissertation: Prof. Dr. Juliane Filser Dr. Martina Brückner Tag des öffentlichen Kolloquiums: 6.5.2005 Danksagung Viele Personen haben zum Gelingen dieser Arbeit beigetragen. Insbesondere möchte ich mich bedanken bei meiner Betreuerin, Prof. Dr. Juliane Filser, für ihre unermüdliche Begleitung durch alle Fährnisse des nicht immer einfachen Themas, allen Mitgliedern der Abteilung ‘Allgemeine und Theoretische Ökologie’ des UFT für die angenehme Arbeitsatmosphäre, die meine Arbeit sehr erleichtert hat, den technischen Assistentinnen Ute Uebers, Annemarie Kissling und Iris Burfeindt, die mich bei Laborarbeit und Materialbeschaffung unterstützt und in die Bedienung vieler Geräte eingewiesen haben, den Kollegen des EDIS-Projektverbundes, insbesondere dem Schwesterprojekt DNA-Tax in München, für die unkomplizierte Kooperation, allen Kollegen, die mich freigiebig mit Collembolen aus ihren Sammlungen und Ratschlägen aus ihrem Erfahrungsschatz versorgt haben: Michael Folger, David Russell, Stefan Scheu, Hans- Jürgen Schulz, Vibeke Simonsen und Steve Hopkin, ganz besonders der Arbeitsgruppe Frati in Siena, die mich auch ausgiebig in die Feinheiten der PCR mit kleinen Organismen eingewiesen hat, Arne Fjellberg, der großzügig Collembolen aus seiner Sammlung zur Verfügung gestellt und einige unsichere Artbestimmungen überprüft hat, den molekularbiologisch tätigen Kollegen in Bremen und umzu, die sich im IFOE-Arbeitskreis MoBiTech zusammengefunden haben, für zahlreiche Anregungen und Hinweise, insbesondere zu phylogenetischen Fragen, Erfahrungen mit spezieller Software und den Problemen des molekularen Laboralltages, Holger Mebes und Andrea Ruf für die Durchsicht des Manuskripts und kritische Anmerkungen, Holger Mebes ferner für seine geduldige Einführung in die Collembolentaxonomie, dem Bundesministerium für Bildung und Forschung BMBF, durch dessen Förderung diese Arbeit möglich wurde, der Universität Bremen für die Gewährung eines Promotionsabschlußstipendiums, und nicht zuletzt meiner Familie für all die Unterstützung, die sie mir zuteil werden ließ. Allen Beteiligten, auch den nicht aufgeführten, gilt mein herzlicher Dank. Isotoma viridis, Holzschnitt von Hermann Burkhardt. Mit freundlicher Genehmigung des Künstlers. Die Druckversion dieser Dissertation ist im Shaker Verlag Aachen erschienen (ISBN 3-8322-4498-0). Inhaltsverzeichnis Inhaltsverzeichnis / Contents............................................................................................. I 1 Introduction and outline ....................................................................................................1 2 Molekulare Taxonomie – Königsweg oder Werkzeug?..................................................15 in Reuter, H., Breckling, B. & Mittwollen, A. (Eds) GfÖ Arbeitskreis Theorie in der Ökologie 9: (2003): Gene, Bits und Ökosysteme (pp. 47-63). Frankfurt/M.: P. Lang Verlag 3 Fast and efficient discrimination of the Isotoma viridis group (Insecta: Collembola) by PCR-RFLP.......................................................................................................................33 Pedobiologia 48 (2004): 435-443 4 Molecular evidence for a fourth species within the Isotoma viridis group (Insecta, Collembola) .....................................................................................................................45 Zoologica Scripta 34 (2005): 177-185 5 Systematics of the Protaphorura armata group – a DNA-based approach ....................63 Pedobiologia, submitted 6 Species identification of Collembola by use of DNA fingerprinting..............................85 7 General discussion.........................................................................................................105 Summary.......................................................................................................................125 I 1 Introduction and outline Introduction and outline Whatever a character, it can be used in taxonomy Mikhail B. Potapov Taxonomy, in the broad sense of definition, identification and re-identification of taxa has found its actual form in the Linnean binomial classification system and the rules of application that have been erected since then. In this system, the unit on which all classification bases is the species, but the species concept itself is a subject of ongoing debate. As a result, different concepts of species have been developed (e.g. Grant 1981). The biological species concept, as the most widespread, proposes to identify taxonomic species in accordance with reasonable biological units, i.e. groups of freely interbreeding natural populations (Mayr 1970) or ”a group of populations possessing inherited differences that prevent gene exchange with other such groups” (Barton 1989). However, if we try to arrange biological entities in a classification that best represents the natural system of descent, we have to be conscious that these entities are a product of the ongoing process of evolution and thus reflect the variability that is its moving force. A species concept as defined above will encounter difficulties when regarding organisms that reproduce uniparentally or asexually (e.g. White 1978, Perkins 2000). Within the area of distribution of a species, distinct morphotypes may be found that live in sympatry (Olson et al. 2004, Reed et al. 2004). Even the potential to hybridise may vary between populations assumed to represent the same species (e.g. Fukami et al. 2004). Among organisms with limited dispersal potential, differentiated populations easily may be recognised that are not separated by sexual isolation and thus may be regarded as infraspecific taxa (Minelli 1993). Such effects have led to some 30 definitions of the term ‘species’, including biosystematic categories such as sibling species which are morphologically indistinguishable yet reproductively isolated, coenospecies or colour pattern species, as an attempt to describe taxonomic levels between species and subspecies, probably representing different stages on the way to speciation (Grant 1981, Minelli 1993). Characters are used to define boundaries between taxa for both taxonomic classification and re- identification. For that purpose, characters should be selected that not only separate one organism group from another but build up a row of homologous character states and thus may enlighten the direction of evolution within the group (Duncan & Stuessy 1984). The ability to re-identify a certain taxon provides the fundament for all further studies that might be helpful to evaluate the taxonomic status, species boundaries, reproduction and evolutionary history. These studies may lead to a re-evaluation of the original taxonomic status, which, too, may require a new character set 1 1 Introduction and outline for taxon identification. However, both disciplines, classification and re-identification, have to work together to achieve a better understanding of biological diversity and evolution. About 10-15 millions of species are estimated to exist on our planet (Hammond 1992). Thousands of taxonomists will be required not only for describing new species but even more for routine species identification (Hebert et al. 2003). Most of these new species are expected to occur among prokaryotes, fungi and invertebrates (Janssen et al. 1996, Bachmann 1997, Pace 1997, Klein et al. 1998, Brown 2000, Gravesen et al. 2000, Pryor & Gilbertson 2000, Witt et al. 2003, Pinceel et al. 2004). In most cases these new findings are small organisms bearing few morphological characters that might be suitable for species identification. Furthermore, variability on both the genetic and the phenotypic level may lead to intraspecific character variability impeding a correct identification. Further drawbacks for efficient routine identification are morphological cryptic species, which still have to be detected, and a lack of efficient morphological characters in juvenile life stages or even genders (e.g. Bretfeld 1999). In the functioning of terrestrial ecosystems, soil biodiversity is a crucial factor (Bengtsson 1998, Copley 2000). Soil communities are among the most species-rich components within terrestrial ecosystems (Jones & Bradford 2001). For studies dealing with biodiversity and ecology, there is need for species definition and species identification (Mallet & Willmott 2003), and such identification is difficult if the species studied are small-sized (e.g. Barrientos 2003, Behan- Pelletier 2003) or highly diverse in character patterns, thus usually having to be lumped in species groups (e.g. D’Haese & Weiner 1998, Stothard et al. 2000, Crochet et al. 2003, Polaszek et al. 2004). Within such species groups, both the scarcity and the variability of diagnostic differences makes the estimation of their members’ status as separate species difficult. Therefore such species groups remain undistinguishable as long as no reliable characters exist that could be used to discriminate all individuals of a presumed taxon from its sibling taxa. Among such morphologically ‘simple’ groups are some collembolan species groups (Deharveng 2004). Collembola are an ancient group of wingless arthropods that belong to the so-called ”Apterygota”. Due to new molecular