Transcript Molecular phylogeny, taxonomy, and DNA

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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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
The Goals
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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
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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
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ILD tests revealed strong incongruence between each pair of
genes (p=0.01)
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GTR+I+Г model for each gene partition defined
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Very high levels of divergence in Folmer’s COI region 11.227.8% within Runcinacea; 25-40% within ‘Opisthobranchia’,
and 35-50% between ‘Opistobranchia’ and Neogastropod
outgroup
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The other four genes can also be used to distinguish the
Runcinacea species at 10-30%.
Topologies
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Separate gene and combined dataset
produce similar topologies regardless
inference method used.
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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
0.20
0.18
0.17
0.15
s
0.10
s
0.12
s and v
s and v
0.14
0.07
0.09
0.06
v
v
0.03
0.00
0,0000
0.03
0,0843
0,1687
0,2530
0,3374
0,4217
0,0733
A
GTR distance
0.15
0.34
0.13
0.28
s
0.10
0.08
0.05
0,1467
0,2200
0,2934
0,3667
0,4401
B
GTR distance
s and v
s and v
0.00
0,0000
0,5061
s
0.23
0.17
0.11
v
0.03
0.00
0,0000
v
0.06
0,0483
0,0965
0,1448
GTR distance
0,1931
0,2414
0.00
0,0000
0,2896
C
1,5000
3,0000
4,5000
GTR distance
6,0000
7,5000
9,0000
D
COI: A – all sites, B – 1st position only, C – 2nd position only, D – 3rd position only
Bootstrap ML tree, all genes
Nucella heyseana
ACTEONOIDEA
Acteon tornatilis
Ilbiidae
Ilbia ilbi
Runcina africana
95
Runcininae
Metaruncina nhatrangiensis
100
Metaruncininae subfam. nov.
Metaruncina hnatrangiensis
95
100
RUNCINACEA
‘Runcinida’ sp. n. Jeju
100
Metaruncina sp. n. Viet Nam
100
Runcinida marisae
Runcinida valentinae Kuril Islands
100
100
92
Runcinidinae subfam. nov.
Runcinida valentinae Sea of Japan
Elysia chlorotica
SACOGLOSSA
Turbonilla fusca
93
100
Aplysia parvula
100
Akera bullata
85
APLYSIOIDEA
Tylodina perversa
99
PYRAMIDELLOIDEA+EUPULMONATA
Onchidella sp.
Umbraculum umbraculm
UMBRACULOIDEA
60
Diaphana sp.
Philine aperta
100
35
CEPHALASPIDEA
Bulla striata
Berthella martensi
100
ln(L)=-21623.2
Chromodoris krohni
0.1
PLEUROBRANCHOIDEA
NUDIBRANCHIA
Uncertain phylogeny of ‘Runcinida sp. n.’
Conclusions
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All used molecular markers can be used to clearly distinguish studied
Runcinoidea species at at east 5% level.
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The genus Runcinida is not a synonym of Runcina. ‘Runcinida+Runcina’ is
pararphyletic.
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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.
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Four distinct monophyletic lineages within Runcinacea are revealed. The
subfamilies Metaruncininae and Runcinidinae are described.
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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.
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Recently discovered Runcinida sp. from the Sea of Japan is conspecific with
R.valentinae known from Iturup island: p-distance=0.007.