An 18S and 28S-based clock calibration for marine Proseriata (Platyhelminthes)

Geminate species are a powerful tool for calibrating the molecular clock in marine organisms, and their adoption
is mandatory for soft-bodied taxa, which lack fossil records. The first attempt to calibrate the molecular clock in
taxa belonging to meiofaunal microturbellaria (Platyhelminthes: Proseriata) based on geminate species is presented
here. We used two species pairs from both sides of the Isthmus of Panama: Minona gemella (Caribbean)
and Minona cf gemella (Pacific); Parotoplana sp. nov. 1 (Caribbean) and Parotoplana sp. nov. 2 (Pacific). The mutation
rates per million yearswere estimated for both geminate species pairs on two ribosomal regions, the complete
nuclear small subunit rDNA (18S) gene and the partial nuclear large subunit rDNA (28S) gene fragment
(spanning variable domains D1–D6). Similar values of mutation rates per million years were found in both species
pairs, ranging 0.12–0.16% for 18S and 0.49–0.52% for 28S. The values obtainedwere used as calibration points
at minimumage, in order to estimate the divergence timeswithin the phylogenetic tree of thewhole dataset, and
tested on three cases of trans-American (not-geminate) species fromPacific Panama and S-E Brazil, belonging to
the genera Kata, Archimonocelis and Duplominona. They consistently showed higher divergence times (ranging
9.4–17.9 Myr) than geminate, trans-isthmian pairs. These results suggest potential usefulness of our molecular
clock calibration, for future research on phylogeography and evolution of Proseriata.