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Chichvarkhin Anton, Chernyshev A.V., Chichvarkhina. O.V.

Molecular phylogeny, taxonomy, and DNA-barcoding use of five markers in the sub-order Runciniformes (Mollusca, Opisthobranchia). Chichvarkhin Anton, Chernyshev A.V., Chichvarkhina. O.V. Institute of Marine Biology, FEB RAS Vladivostok, Russia. Introduction.

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Chichvarkhin Anton, Chernyshev A.V., Chichvarkhina. O.V.

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  1. Molecular phylogeny, taxonomy, and DNA-barcoding use of five markers in the sub-order Runciniformes(Mollusca, Opisthobranchia) Chichvarkhin Anton, Chernyshev A.V., Chichvarkhina. O.V. Institute of Marine Biology, FEB RAS Vladivostok, Russia

  2. Introduction • Small group of rare tiny molluscs. About 20 species are known. Four species described by our group. • Simple morphology and variable coloration bring the difficulties for morphologists. • Uncertain phylogenetic position – traditionally as a family within Cephalaspidea. • Rarely used in recent molecular phylogenetic studies. Max. two species per study. • Taxonomy: Runcinidae and Runcinoidea – family and superfamily in traditional systematics Runcinaceae – clade proposed by recent authors Runciniformes – sub-order proposed in this study

  3. The Goals • To support or reject monophyly of Runcinacea. • To reveal existing lineages within Runcinacea. • To support or reject monophyly of ‘Runcinacea +other lineages’ • To reassess taxonomic ranks • To compare new specimens with other species. • To measure the levels of divergence between studied taxa

  4. Materials and Methods • Sanger-sequenced partial COI, 16s, H3, 18s and 28s genes. Totally ca. 4400 b.p. • Seq editor – BioEdit • Alignments – Muscle, by eye • Models, restrictions – MrModelTest • ILD test - PAUP • ML – PAUP, PhyML, GARLI (multigene) • MCMC – MrBayes (5M generations, 4 runs, multigene) • MP – PAUP • TR/TV/distance plots – DAMBE • Distances - MEGA

  5. Results • ILD tests revealed strong incongruence between each pair of genes (p=0.01) • GTR+I+Г model for each gene partition defined • Very high levels of divergence in Folmer’s COI region 11.2-27.8% within Runcinacea; 25-40% within ‘Opisthobranchia’, and 35-50%between ‘Opistobranchia’ and Neogastropod outgroup • The other four genes can also be used to distinguish the Runcinacea species at 10-30%.

  6. Topologies • Separate gene and combined dataset produce similar topologies regardless inference method used. • Maximum Parsimony method produced poorly supported topology but resolved clades are congruent with those obtained with the other methods.

  7. Tr and Tv to distance plots reveal high saturation in COI and H3 genes COI: A – all sites, B – 1st position only, C – 2nd position only, D – 3rd position only

  8. Bootstrap ML tree, all genes

  9. Uncertain phylogeny of ‘Runcinida sp. n.’

  10. Conclusions • All used molecular markers can be used to clearly distinguish studied Runcinoidea species at at east 5% level. • The genus Runcinida is not a synonym of Runcina. ‘Runcinida+Runcina’ is pararphyletic. • Runcinoidea is independent monophyletic lineage within Ethyneura. Previously proposed monophyletic lineages ‘Aplysioidea+ Runcinacea’ or ‘Cephalaspidea+ Runcinacea’ are not supported. We propose to classify them as the sub-order Runciniformes. • Four distinct monophyletic lineages within Runcinacea are revealed. The subfamilies Metaruncininae and Runcinidinae are described. • Two new species are supported with high genetic distances. Both in Metaruncininae but ‘Runcinida sp.’ is of uncertain position because of molecular data incongruence and morphology close to Runcinididae. • Recently discovered Runcinida sp. from the Sea of Japan is conspecific with R.valentinae known from Iturup island: p-distance=0.007.

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