Filling gaps in cryptic-biodiversity research

Cryptic species are very similar ones which initially can be told apart only with great difficulty. Over the past decade evidence has emerged that all groups of organisms have many cryptic species. This research was mostly descriptive, though, and the analysis of trends in the evolution of cryptic species is a field ripe for study. We propose a three-step procedure to study the evolution of a complex of at least seven cryptic ant species. First, the rigour currently lacking in species delimitation will be achieved using a cohesive protocol for integrative taxonomy. Information from traditional morphometrics, and nuclear and mitochondrial DNA sequences will be first used separately and then integrated. If disagreement among the three disciplines emerges and cannot be resolved by finding an evolutionary explanation for it, additional disciplines that can be added are geometric morphometrics and near-infrared reflectance spectroscopy. Second, tools for routine identification will be tested, aimed at minimizing failure rates, and these tools will be made freely available in the internet. The tools will be based on the same data sources as used in the species- delimitation process and will assist in providing the basis for the third and ultimate goal of the project. Third, we will address three questions about the evolution of the cryptic-species complex: (i) Phylogenetic make-up of species complex? If the species form one exclusive crown group in the estimated species tree, stasis (retention of ancestral morphology) can be inferred as causal to the morphological similarity of the cryptic species. If the species belong to more than one crown group, an alternative approach is necessary: The morphological crypsis could have arisen by convergence or by stasis which was overcome by some (now non- cryptic) species. (ii) Relation of morphological similarity to evolutionary proximity? The cryptic ant species are morphologically similar, but not identical. This facilitates correlating the extents of morphological similarity and evolutionary proximity. This approach can make plausible convergence if the complex comprises more than one crown group. If the species form a single crown group, one of two novel insights into the maintenance of morphological crypsis could be gained: the gradual loss of similarity with decreasing evolutionary proximity or the lack of influence of evolutionary proximity on stasis. (iii) Relation of morphological to climatic-niche similarity? The forces driving the diversification of the cryptic ant species and the mechanisms allowing the maintenance of their species integrity remain unknown. Ecology could be involved and the species could, for example, differ in their climatic niches. Characterising the pattern of climatic-niche similarity in relation to morphological similarity could facilitate the cautious inference of a role of ecology in the species` evolution. The project will be relevant beyond meeting the specific project aims: Integrative taxonomy needs well- documented case studies for the fine-tuning of procedural details. The innovative 454-technology-based protocol we will use for developing nuclear sequence loci resolving at the species level will be widely applicable. The experiences from developing routine identification tools for the internet may assist in developing such tools for other cryptic species. The answers to the evolutionary questions addressed will facilitate follow-up research into further aspects of the species` evolution. More broadly speaking, the evolutionary-profile analysis could help revealing previously underappreciated characteristics often involved in the evolution of cryptic species.

Cryptic species are species morphologically so similar to each other that they can barely be told apart. Over the past decade, cryptic species were identified in all groups of organisms. This research was mostly descriptive, though. We, instead, applied a cohesive protocol of integrative taxonomy, that is, species delimitation using multiple, independent sources of evidence, to the Tetramorium caespitum ant species complex. Information from, among others, traditional morphometrics, nuclear and mitochondrial DNA sequences was first used separately and then integrated. Disagreements among disciplines were resolved by finding an evolutionary explanation for them, such as hybridization. To make species identification for the focal ant species complex available to everyone, a freely available (upon the papers publication in September 2017) online identification key for worker ants was developed at https://webapp.uibk.ac.at/ecology/tetramorium/. A fibre-optic near-infrared spectroscopy determination tool was established along the same end. Further, we aimed at understanding the evolutionary patterns leading to cryptic diversification. We found a gradual loss of morphological similarity over evolutionary time and thus that crypsis is a result of just little change over time, a phenomenon known as stasis. We found no indication that the evolution of morphological similarity in these ants was driven by environmental conditions. Our data further suggest that species occurring in higher altitudes are more difficult to tell apart, that is, more cryptic.