Systematic assignment of fossil otoliths is virtually always based on studies of otolith morphology and subsequent comparisons with otoliths from collections and/or literature. Although this usually represents a practical method, comparisons and subsequent evaluation may be biased by subjective criteria used in the individual descriptions. Quantitative morphometric studies focusing on variations in the otolith morphology of extant fishes have been conducted in fisheries research, mostly based on Fourier shape analysis and related methods. However, with regard to fossil otoliths, these approaches are generally not suitable, mainly due to preservation-related problems. Here we present a new approach for quantifying otolith variation between species and populations of killifish (cyprinodontiforms) in the genera Aphanius Nardo and daggerProlebias Sauvage that can be used with both extant and fossil otoliths. Our new approach includes the definition of 10 variables from linear and angle measurements of an otolith and statistical analyses. Best results were obtained by presorting the otoliths into three groups based on sulcus shape (straight, bent, S-shaped). In this case, canonical discriminant analysis (CDA) with jackknifed cross-validation yielded an overall species classification success of 86-96%. The three groups based on sulcus shape separate according to zoogeographic patterns (i.e., Mediterranean Aphanius, Arabian Aphanius, European daggerProlebias) and probably reflect phylogenetic lineages. Application of CDA to compare otolith variation between populations resulted in an overall classification success (jackknifed) of 33-83%. High levels of variation were observed for Aphanius dispar and daggerProlebias malzi, but not for A. fasciatus and daggerP. weileri. We suggest that otolith variation between populations results predominantly from geographic separation. Combination of qualitative characters (sulcus morphology) with quantitative approaches (otolith morphometry) presents a new approach for obtaining a better understanding of the taxonomy, diversity, and zoogeography of both fossil and extant killifishes. Moreover, the method may also be suitable for assessing taxonomy and diversity in other species-rich groups like the atheriniforms and many perciforms because these groups display otolith Bauplans that are similar to those seen in killifishes.

Twenty paleogeographic maps are presented for Middle Eocene (Lutetian) to Late Pliocene times according to the stratigraphical data given in the companion paper by Berger et al. this volume. Following a first lacustrine-continental sedimentation during the Middle Eocene, two and locally three Rupelian transgressive events were identified with the first corresponding with the Early Rupelian Middle Pechelbronn beds and the second and third with the Late Rupelian ( Serie Grise ) (Fischschiefer and equivalents). During the Early Rupelian (Middle Pechelbronn beds), a connection between North Sea and URG is clearly demonstrated, but a general connection between North Sea, URG and Paratethys, via the Alpine sea, is proposed, but not proved, during the late Rupelian. Whereas in the southern URG, a major hiatus spans Early Aquitanian to Pliocene times, Early and Middle Miocene marine, brackish and freshwater facies occur in the northern URG and in the Molasse Basin (OMM, OSM); however, no marine connections between these basins could be demonstrated during this time. After the deposition of the molasse series, a very complex drainage pattern developed during the Late Miocene and Pliocene, with a clear connection to the Bresse Graben during the Piacenzian (Sundgau gravels). During the Late Miocene, Pliocene and Quaternary sedimentation persisted in the northern URG with hardly any interruptions. The present drainage pattern of the Rhine river (from Alpine area to the lower Rhine Embayment) was not established before the Early Pleistocene.

We present a general stratigraphic synthesis for the Upper Rhine Graben (URG) and the Swiss Molasse Basin (SMB) from Eocene to Pliocene times. The stratigraphic data were compiled both from literature and from research carried out by the authors during the past 6 years ; an index of the stratigraphically most important localitites is provided. We distinguish 14 geographical areas from the Helvetic domain in the South to the Hanau Basin in the North. For each geographical area, we give a synthesis of the biostratigraphy, lithofacies, and chronostratigraphic ranges. The relationships between this stratigraphic record and the global sea-level changes are generally disturbed by the geodynamic (e.g., subsidence) evolution of the basins. However, global sea-level changes probably affected the dynamic of transgression-regression in the URG (e.g., Middle Pechelbronn Beds and Serie Grise corresponding with sea-level rise between Ru1/Ru2 and Ru2/Ru3 sequences, respectively) as well as in the Molasse basin (regression of the UMM corresponding with the sea-level drop at the Ch1 sequence). The URGENT-project (Upper Rhine Graben evolution and neotectonics) provided an unique opportunity to carry out and present this synthesis. Discussions with scientists addressing sedimentology, tectonics, geophysics and geochemistry permitted the comparison of the sedimentary history and stratigraphy of the basin with processes controlling its geodynamic evolution. Data presented here back up the palaeogeographic reconstructions presented in a companion paper by the same authors (see Berger et al. in Int J Earth Sci 2005).

Among the species of Aphanius Nardo, 1827, Aphanius dispar (Rüppell, 1828) is the most common taxon and has long been viewed as representing a species group rather than a single species. This study provides comprehensive data on the phylogenetic relationships, morphology, and otoliths within the A. dispar species group, including the description of a new species. Our data demonstrate that the “true” A. dispar is restricted to the Red Sea drainages and that all other populations hitherto identified as A. dispar actually represent separate species. Four main clades are defined and named for the geographic areas in which the respective species of Aphanius occur. The oldest one is the “Red Sea clade,” it comprises A. dispar. The “Dead Sea clade” is represented by A. richardsoni (Boulenger, 1907). It is sister to both the “Hormuzgan clade” in S Iran (containing A. hormuzensis sp. nov. and A. ginaonis (Holly, 1929)) and the “Persian Gulf & Gulf of Oman clade” (comprising A. stoliczkanus (Day, 1872)). The species separation within the A. dispar group is confirmed by the distinctive otolith morphology of each species. Moreover, we present a time‐calibrated phylogeny (chronogram) for the A. dispar species group using †A. princeps (16–17 Mya) as a minimum age and the first appearance of †Prolebias (33–34 Mya) as a maximum age for the genus Aphanius. The evolution and historical biogeography routes are discussed based on the outcome of the chronogram and in the context of the geological and climatic history of the Near East in Pliocene–Pleistocene times.

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