‐ Dissertation ‐ INTEGRATIVE SPECIES DELIMITATION IN THE ALPINE JUMPING‐BRISTLETAIL GENUS MACHILIS LATREILLE, 1832 Thomas Dejaco May 2014 Molecular Ecology Group Institute of Ecology University of Innsbruck 6020 Innsbruck, Austria 1st supervisor: Univ.‐Prof. Dr. Birgit C. Schlick‐Steiner 2nd supervisor: PD Dr. Florian Steiner i Leopold‐Franzens‐Universität Innsbruck Eidesstattliche Erklärung Ich erkläre hiermit an Eides statt durch meine eigenhändige Unterschrift, dass ich die vorliegende Arbeit selbständig verfasst und keine anderen als die angegebenen Quellen und Hilfsmittel verwendet habe. Alle Stellen, die wörtlich oder inhaltlich den angegebenen Quellen entnommen wurden, sind als solche kenntlich gemacht. Die vorliegende Arbeit wurde bisher in gleicher oder ähnlicher Form noch nicht als Magister- /Master-/Diplomarbeit/Dissertation eingereicht. 19. Mai 2014 Datum Unterschrift Machilis sp. B from Trögener Klamm (southern Carinthia, Austria). Photograph courtesy of Gernot Kunz. ii ABSTRACT Jumping‐bristletails (Archaeognatha or Microcoryphia) are certainly one of the least studied insect groups. Information on their phylogenetic relationship (both within and among hexapod sister groups) and their general biology is very scarce. Within the genus Machilis, 94 species have been described, among which numerous Alpine small‐scale endemics are found. Endemic species are treasures of biodiversity that need to be conserved in the face of the ongoing biodiversity crisis. This is especially important for the European Alps, which are poor in endemic species compared with other biodiversity hotspots like the Mediterranean region. The very basic requirement for any biodiversity assessment is knowledge about the number and taxonomic affiliation of species inhabiting the area under observation. Unfortunately, species determination in Alpine representatives of the genus Machilis is obscured by vaguely defined species limits. Therefore, a comprehensive, integrative approach is urgently needed to clarify the taxonomically problematic situation in the genus Machilis. Within this PhD project, I first developed a methodological toolbox for enhanced species delimitation in the genus Machilis. Subsequently, I sampled type localities and additional localities of as many nominal species as possible across the Eastern Alps. By integrating data from traditional morphometrics and molecular genetics, I was able to determine actual species limits for most of the 18 nominal species included. Moreover, I found a high proportion of incongruence among disciplines, indicating complex evolutionary histories including several instances of hybridization and parthenogenesis. Additionally, by using chromosome preparations and genome size measurements, I could gain first insights into the evolution of parthenogenesis and polyploidy. Overall, it became evident that the evolutionary history of Eastern‐Alpine Machilis species was strongly affected by hybridization, polyploidization, and parthenogenesis. These factors potentially have triggered speciation events that ultimately are responsible for the observed frequency of small‐scale endemics. Moreover, five new species have been discovered, mainly along the southern margin of the Eastern Alps. This indicates that Alpine species diversity in the genus Machilis is still underestimated. As a result of this comprehensive investigation, Eastern‐Alpine Machilis species are now widely accessible to researches interested in endemism and conservation, evolution of parthenogenesis, and genomic alterations like hybridization, polyploidization, and genome size variation. iii CONTENTS EIDESSTATTLICHE ERKLÄRUNG......................................................................................................................... i PHOTOGRAPH OF MACHILIS SP. B....................................................................................................................ii ABSTRACT...................................................................................................................................................iii BACKGROUND ............................................................................................................................................. 1 AIMS AND OBJECTIVES .................................................................................................................................. 4 1ST PUBLICATION: A toolbox for integrative species delimitation in Machilis jumping‐bristletails (Microcoryphia: Machilidae) .................................................... 5 2ND MANUSCRIPT: Taxonomist’s nightmare … evolutionist’s delight: hybridization and parthenogenesis challenge species delimitation in Machilis jumping‐bristletails .15 3RD MANUSCRIPT: Karyotypic variability and genome‐size variation in sexual and parthenogenetic species of the jumping‐bristletail genus Machilis (Archaeognatha)...................... .72 SYNTHESIS .............................................................................................................................................. 105 CONCLUSION........................................................................................................................................... 108 ACKNOWLEDGEMENTS.............................................................................................................................. 109 LITERATURE ............................................................................................................................................ 110 iv BACKGROUND Most subfields in natural sciences rely on the recognition of discrete categories, which usually provide a framework for hypotheses to be statistically tested. These can be artificial treatment variables or natural entities like molecules or cells. For researchers working on the level of organisms, categories will most likely be individuals, populations, or species. While the former two are easier to determine using physical and geographical properties, defining the species category has been a long‐lasting endeavour for generations of naturalists, taxonomists, and evolutionary biologists. The species problem Ever since Linnaeus' Systema Naturae (1735), organisms have been classified in an effort to systematically capture earth's biodiversity. The term 'species' was, at that time, no more than one among many categories based on morphological similarity, which could be further subdivided in varieties, races, etc. It was Charles Darwin who, by choosing the title for his monumental book On the origin of species (1859), assigned to the species category its fundamental role in his theory of natural selection. Following a century of intensive taxonomic discovery, the reconciliation of Darwinian Theory with the principles of genetics, the so‐called Modern Evolutionary Synthesis, became a landmark for the definition of species. Enlightened by the innovation of population genetic theory (Dobzhansky 1937), species were suddenly perceived as interbreeding populations, which are genetically isolated from other lineages. In this period, Ernst Mayr (1942) developed the framework for the Biological Species Concept (BSC), which was primarily based on the criterion of reproductive isolation. It is, until now, one of the most adopted species concepts among biologists. With the advent of phylogenetic systematics (Hennig 1966) and the availability of statistical methods for inferring phylogenies from morphological or molecular data, phylogenetic relationship (i.e., descent from a common ancestor) was claimed to be a superior operational criterion for defining species, leading to the Phylogenetic Species Concept (PSC; e.g., Eldrege & Cracraft 1980). Finally, in the last decades of the 20th century, several alternative species concepts have been proposed (see De Queiroz 2007; Mayden 1999). The majority of them are modifications of either the BSC or the PSC, and mostly differ in the criteria (e.g., monophyly, diagnosability, distinctiveness) used to recover clusters of individuals. Even though the primary goal of any species definition has been the search for 1 common ground among disciplines, it has become clear that, at the verge to the new millennium, taxonomists, systematists, and evolutionary biologists were more than ever divided in their view of how species should be defined. However, efforts have been made to reconcile species concepts (De Queiroz 2007; Mayden 1999), and lately, the Unified Species Concept (USC; De Queiroz 2007) is gaining popularity among systematists. Under the USC, species conceptualization and species delimitation are clearly separated. Regarding the former, the only defining property of the species category is the existence of a separately evolving metapopulation lineage, and this criterion is in fact inherent to any known species concept. Regarding the latter, different operational criteria should be taken as independent evidence for lineage separation along the speciation continuum. Consequently, the USC provides an open framework where no thresholds are defined. Instead, lineage divergence is corroborated by any additional, independent source of information. Regarding this, the USC is tightly linked to the concept of a multidisciplinary, integrative approach to taxonomy. The integrative future of taxonomy Integrative taxonomy is a multisource approach that takes advantage of complementarities among disciplines for improved species delimitation (Dayrat 2005; Padial et al. 2010; Schlick‐Steiner et al. 2010). Data can come from disciplines like molecular