Contrasting morphology with molecular data: an approach to revision of species complexes based on the example of European Phoxinus (Cyprinidae)

Abstract

Background: Molecular taxonomy studies and barcoding projects can provide rapid means of detecting cryptic diversity. Nevertheless, the use of molecular data for species delimitation should be undertaken with caution. Especially the single-gene approaches are linked with certain pitfalls for taxonomical inference. In the present study, recent and historical species descriptions based upon morphology were used as primary species hypotheses, which were then evaluated with molecular data (including in type and historical museum material) to form secondary species hypotheses. As an example of cryptic diversity and taxonomic controversy, the European Phoxinus phoxinus species complex was used.

Results: The results of the revision showed that of the fourteen primary species hypotheses, three were rejected, namely P. ketmaieri, P. likai, and P. apollonicus. For three species (P. strandjae, P. strymonicus, P. morella), further investigation with increased data sampling was suggested, while two primary hypotheses, P. bigerri and P. colchicus, were supported as secondary species hypotheses. Finally, six of the primary species hypotheses (P. phoxinus, P. lumaireul, P. karsticus, P. septimanae, P. marsilii and P. csikii) were well supported by mitochondrial but only limitedly corroborated by nuclear data analysis.

Conclusion: The approach has proven useful for revision of species complexes, and the study can serve as an overview of the Phoxinus genus in Europe, as well as a solid basis for further work.

Keywords: Cryptic diversity; Molecular taxonomy; Phoxinus (Cyprinidae); Species delimitation.

Fig. 1

Sampling sites used in this study. Data set comprise a combination of new material and data downloaded from Genbank. For details see Additional file 2: Tables S2. Target symbols denote type localities of Phoxinus csikii, P. marsilii and P. morella. We do not have genetic data from the type localities of P. csikii and P. morella (points marked in pale red and pale violet, respectively). The arrows on the figure mark areas, where hybrids were detected. They correspond to arrows in Fig. 3b

Fig. 2

Phylogenetic reconstruction and haplotype network for revision of the genus Phoxinus. Figure 2a Phylogenetic tree constructed from the barcoding region of mitochondrial gene cytochrome oxidase I (COI). Collapsed alignment includes 139 unique haplotypes. The tree was created using Bayesian inference (BI) with BEAST 1.8.0 [26]. Branches carry posterior probabilities (PP) and bootstraps (BS) from the tree constructed with the Maximum-Likelihood (ML) method (PhyML; [29]). Weakly supported nodes are grey and only PP above 0.9 are shown for the main clades (no subclades). -, denotes lack of bootstraps originating from the difference between the BEAST and ML trees. Figure 2b Phylogenetic tree constructed with two partitions: COI and cytochrome b. Collapsed alignment includes 162 unique haplotypes. As in Fig. 2a, BI and ML were used. Because PhyML does not support partitions, GARLI v.2.01 [30, 31] was used for ML. Figure 2c An unrooted minimum-spanning network was constructed with COI using the median joining algorithm [32] implemented in Network 5.0 (www.fluxus-engineering.com) with default settings. The position of the type material in the network is denoted with TYPE PM for Phoxinus marsilii and TYPE PC for P. csikii

Fig. 3

Haplotype networks constructed with nuclear DNA. The colours represent lineages detected by mitochondrial DNA analysis. For both genes, the gametic phase of heterozygous individuals was determined using Phase 2.1 [38, 39], then an unrooted minimum-spanning networks were constructed with median-joining algorithm [32] implemented in Network 5.1 (www.fluxus-engineering.com) with default settings. Figure 3a Rhodopsin haplotype network was constructed from 782 base pair long phased alignment. Figure 3b Recombination activating gene 1 (RAG1) haplotype network was constructed using 1413 base pair long phased alignment. The arrows denote areas, where hybrids were detected and correspond to Fig. 1