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 RASVladivostok, Russia

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 authorsRunciniformes – sub-order proposed in this study

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

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

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%.

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.

Tr and Tv to distance plots reveal high saturation in COI and H3 genes

s

v

sa

ndv

GTR distance

0.00

0.03

0.06

0.09

0.12

0.15

0.18

0,0000 0,0733 0,1467 0,2200 0,2934 0,3667 0,4401

s

v

sa

nd

v

GTR distance

0.00

0.03

0.05

0.08

0.10

0.13

0.15

0,0000 0,0483 0,0965 0,1448 0,1931 0,2414 0,2896

s

v

sa

nd

v

GTR distance

0.00

0.06

0.11

0.17

0.23

0.28

0.34

0,0000 1,5000 3,0000 4,5000 6,0000 7,5000 9,0000

s

v

sa

ndv

GTR distance

0.00

0.03

0.07

0.10

0.14

0.17

0.20

0,0000 0,0843 0,1687 0,2530 0,3374 0,4217 0,5061

A B

C DCOI: A – all sites, B – 1st position only, C – 2nd position only, D – 3rd position only

Bootstrap ML tree, all genes

99

100

85

60

93

100

92

ln(L)=-21623.2

Chromodoris krohni

Berthella martensi

Bulla striata

Philine aperta

Diaphana sp.

35

Umbraculum umbraculm

Tylodina perversa

Akera bullata

Aplysia parvula

Onchidella sp.

Turbonilla fusca

Elysia chlorotica

Runcinida valentinae Sea of Japan

Runcinida valentinaeKuril Islands

Runcinida marisae

100

Metaruncina sp. n. Viet Nam

‘Runcinida’ sp. Jejun.

Metaruncina hnatrangiensis

Metaruncina nhatrangiensis100

95

100

100

Runcina africana

100

Acteon tornatilis

Nucella heyseana

0.1

100

100

100

ACTEONOIDEA

RUNCINACEA

Runcinidinae subfam. nov.

Metaruncininae subfam. nov.

Runcininae

APLYSIOIDEA

SACOGLOSSA

PYRAMIDELLOIDEA+EUPULMONATA

CEPHALASPIDEA

NUDIBRANCHIA

UMBRACULOIDEA

PLEUROBRANCHOIDEA

Ilbia ilbi

95

Ilbiidae

Uncertain phylogeny of ‘Runcinida sp. n.’

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.