A DNA barcode reference library for endemic Ponto-Caspian amphipods

Abstract

The Ponto-Caspian region is an endemicity hotspot that harbours several crustacean radiations, among which amphipods are the most diverse. These poorly known species are severely threatened in their native range, while at the same time they are invading European inland waters with significant ecological consequences. A proper taxonomic knowledge of this fauna is paramount for its conservation within the native region and monitoring outside of it. Here, we assemble a DNA barcode reference library for nearly 60% of all known Ponto-Caspian amphipod species. We use several methods to define molecular operational taxonomic units (MOTUs), based on two mitochondrial markers (COI and 16S), and assess their congruence with current species-level taxonomy based on morphology. Depending on the method, we find that 54-69% of species had congruent morpho-molecular boundaries. The cases of incongruence resulted from lumping distinct morphospecies into a single MOTU (7-27%), splitting a morphospecies into several MOTUs (4-28%), or both (4-11%). MOTUs defined by distance-based methods without a priori divergence thresholds showed the highest congruence with morphological taxonomy. These results indicate that DNA barcoding is valuable for clarifying the diversity of Ponto-Caspian amphipods, but reveals that extensive work is needed to resolve taxonomic uncertainties. Our study advances the DNA barcode reference library for the European aquatic biota, paving the way towards improved taxonomic knowledge needed to enhance monitoring and conservation efforts.

Figure 1

Map of sampling localities. Red dots indicate sampling points from this study while green rhombi indicate literature data. Blue dashed lines indicate the native Ponto-Caspian range. Pink dashed lines indicate areas that contain endemic Ponto-Caspian amphipods which are biogeographically separated from the mother region.

Figure 2

Maximum-likelihood COI tree and species delimitation results. Green dots at nodes indicate strongly supported clades (UFBS – ultrafast bootstrap support; SH aLRT – Shimodaira-Hasegawa approximate likelihood ratio test). The results of the species delimitation analyses are shown by the coloured columns to the right. Each colour represents a different method/parameter setting. Blue branches represent morphospecies which have been split into multiple MOTUs by at least half of the methods. Orange branches indicate morphospecies that have been lumped into one MOTU by at least half of the methods. Red branches indicate morphospecies that were both lumped and split by at least half of the methods. The fully annotated tree is provided in the supplementary Fig. S1.

Figure 3

Maximum-likelihood 16S tree and species delimitation results. For details see legend on Fig. 2. The fully annotated tree is provided in the supplementary Fig. S2.

Figure 4

Histogram of COI and 16S K2P distances between specimens. The red line indicates the threshold distance above which specimens are considered to belong to different species, according to ASAP.