Like a bat out of heaven: the phylogeny and diversity of the bat-winged slugs,

Gastropteridae
Elise Ong
Terrence M. Gosliner
Macalester College, MN
Invertebrate Zoology & Geology, California Academy of Sciences, CA
Abstract
A molecular phylogeny of philinacean cephalaspideans is presented for 22 newly
sequenced specimens of Gastropteridae. Cephalaspidea are known as bubble snails because some
representatives of these families of marine gastropods have thin bubble-like shells. Still others,
such as members of the Philinacea have a further reduced internal shell. The group consists of
nine families whose unifying character includes the presence of a large cephalic shield, an
adaptation to their burrowing habit. Cephalaspideans are considered to be the basal members of
Opisthobranchia and a major group of opisthobranch gastropods with an estimated 840 species
worldwide. The present phylogeny was estimated by analyzing the nuclear fragment 28S and two
mitochondrial fragments cytochrome c oxidase I (COI) and 16S using maximum likelihood and
Bayesian analyses. Members of the families Aglajidae and Philinidae were used as outgroups as
a close relationship has been previously suggested. Specimens from members of four genera of
the Gastropteridae which includes species of Gastropteron, Siphopteron, Enoptepteron and
Sagaminopteron. The results clearly support the monophyly of Gastropteridae and the
distinctness of each of three of the four constituent genera. Some species of Siphopteron are
transferred to Sagaminopteron to preserve monophyly. All seven of the taxa previously thought
to represent new species collected during 2014 Verde Island Passage Expedition are supported as
new to science based on the molecular evidence. The present study clearly expands on the
previous work that has demonstrated diversification in this poorly known taxon.
Introduction
Opisthobranchs are a group of marine gastropods within the group Heterobranchia. They
are defined as having their gills on the posterior end of the dorsal side of the organism.
Cephalaspidea are named after their head-shields that are used to burrow into the sand. Rather
than living on coral reefs, Gastropteridae are found in sandy areas of the water.
The majority of cephalaspideans have a shell, though it may be reduced or internal. Many
Gastropteridae do not possess a shell at all.
The 2014 Philippine Expedition brought a wide variety of new specimens. We
hypothesized 4 genera of Gastropteridae are represented and within these genera, seven new
species, based on color patterns of the species, are represented in the collection. These
hypotheses are supported by Bayesian and Maximum likelihood analyses and the resulting trees.
Methods
Sampling, DNA Extraction, sequencing and sequence alignment

We reconstructed a molecular phylogeny by analyzing the sequences of the nuclear

fragment 28S and two mitochondrial fragments, cytochrome c oxidase I (COI) and 16S for 22
taxa of the family Gastropteridae. We extracted DNA from animal tissue from specimens fixed
and store in 95% EtOH using either the DNeasyTissueKit (Qiagen Ltd.) or the
GentraPuregeneTissueKit (Qiagen Ltd.), depending on the size of the sample. We sampled the
parapodia of all specimens. If the sample was less than 2 millimeters we used the Puregene Kit
and for samples larger than 2 millimeters we used the standard DNeasy Kit.
The DNeasy Kit was modified for the small tissue samples, each sample was minced and
then added to 80l of the ATL buffer to each 1.5ml tube, pestled, and then the remaining 100l
was added to rinse the pestle. 20l of proteinaseK was added to each tube. Each tube was then
vortexed and placed in a 55C incubator in a rotator for 4 hours to overnight until all of the tissue
was digested. After the tissue was digested 200l of AL buffer was added to each tube, followed
by 200l of 100% EtOH. Each tube was vortexed and then pipetted into new, labeled 1.5ml
DNeasy Mini Spin Columns in a 2ml collection tube. The tubes and columns were centrifuged
for 1 minute at 6000 xg (8000rpm). They were transferred to new collectiontubes where 500l
AW1 Buffer was added to each column. They were centrifuged again for 1 minute at 6000 xg
(8000rpm). The spin columns were transferred to new 2 ml collection tubes and 500 l of AW2
Buffer was added to each spin column. The columns and tubes were centrifued again for 3
minutes at 20000 xg (14000 rpm). The columns were then tranferred to 2 ml screw cap tubes,
50l of AE Buffer was added to each spin column and incubated at 55C for 3 minutes. They
were then centrifuged for 1 minute at 6000 xg (8000 rpm). The elutions were then checked using
Nanodrop 2000C.
The Gentra Puregene Tissue Kit was also modifed to increase DNA yield, since tissue
samples were incredibly small. We chose the 5-10mg procedure, but further modified the
methods. The tissue was ground up after being placed in 100l of the Cell Lysis Solution in a
1.5ml tube and pestled. The pestle was rinsed with 200l of the Cell Lysis Solution. Since
maximum yield was required we added 1.5l of Puregene Proteinase K, inverted 25 times, then
placed on a rotator and incubated at 55C for 3 hours. 1.5L of the RNase A Solution was added
and inverted 25 times, then incubated at 37C for 15 minutes. It was then placed on ice for 3
minutes. 100l of the Protein Precipitation Solution was added then vortexed for 20 seconds at
high speed. The solution was then centrifuged for 3 minutes at 15550xg. 300l of isopropanol
was pipetted into 1.5ml tubes and the supernatant from the solution previously centrifuged was
aspirated into the isopropanol. If the protein pellet was dislodged during this process it was
centrifuged again and again until all of the supernatant was aspirated out. 1-2l of Glycogen
Solution was added and then inverted 50 times. The solution was again centrifuged for 5 minutes
at 15550 xg and the supernatant was discarded through aspiration, making sure the pellet
remained in the tube at all times. If the pellet became dislodged at any time, it was centrifuged
again before continuing to discard the supernatant. Once all the supernatant was removed and the
pellets remained, we added 300l of 70% EtOH and inverted 1-2 times. It was again centrifuged
for 5 minutes at 15550xg. The supernatant again was aspirated out and discarded. It air dried
until the remaining ethanol evaporated or put into the speed vacuum for 5 minutes. After the
2

ethanol had completely evaporated 30l of the DNA Hydration SOlution was added and
vortexed fro 5 seconds at medium speed to mix it. It was then incubated at 65C for 1 hour to
dissolve the DNA. The tubes were then incubated at room temperature (15-25C) overnight on a
gentle shaking device. After which DNA quality was checked on the Nanodrop 2000C
Gene amplification and sequencing was based on the following primers: the universal
COI primers (Folmer et al. 1994) and two COI primers NAFCOI and NARCOI (Anthes et al.
2008) for CASIZ186048; the universal 16S primers (Palumbi et al. 1991); 28S primers 28SC1,
28SC2F, 28SC2R and 28SD3 (Vonnemann et al. 2005). Amplification reactions were performed
in 25 l volumes.
COI PCR reactions used 1-2l of template DNA and the following reaction components:
15l of millipore water, 2.5l 10X USB Buffer, 0.85l 25mM MgCl2, 1l 10mM COI L1490,
1l 10mM COI H2198, 1l of 10mg/mL BSA. 0.5l 10mM dNTPs, 1l of 1.25u/l HotStart
Taq.
For CASIZ 186048, we used NAFCOI/NARCOI primers and a modified protocol: 2l of
template DNA, 14.75l of millipore water, 2.5l 10X USB Buffer, 1.25l 25mM MgCl2, 1l
10mM COI L1490, 1l 10mM COI H2198, 1l of 10mg/mL BSA. 0.5l 10mM dNTPs, 1l of
1.25u/l HotStart Taq.
16S PCR reactions used 1-2l of template DNA and the following reaction components:
15l of millipore water, 2.5l 10X USB Buffer, 1l 25mM MgCl2, 1l 10mM 16Sar, 1l 10mM
16Sbr, 1l of 10mg/mL BSA. 0.5l 10mM dNTPs, 1l of 1.25u/l HotStart Taq.
For CASIZ 199181, we used a modified protocol: 2l of template DNA, 8.75l of
millipore water, 2.5l 10X USB Buffer, 1.25l 25mM MgCl2, 1l 10mM 16Sar, 1l 10mM
16Sbr, 1l of 10mg/mL BSA. 0.5l 10mM dNTPs, 1l of 1.25u/l HotStart Taq.
28S PCR reactions used 2l of template DNA and the following reaction components:
9l of millipore water, 2.5l 10X USB Buffer, 0.85l 25mM MgCl2, 1l 10mM 28SC1, 1l
10mM 28SD3R, 1l of 10mg/mL BSA. 0.5l 10mM dNTPs, 5l 5M Betaine, 1l DMSO, 1l of
1.25u/l HotStart Taq.
For CASIZ 199181, we used a modified protocol: 3l of template DNA and the following
reaction components: 8.75l of millipore water, 2.5l 10X USB Buffer, 1.25l 25mM MgCl2,
1l 10mM 28SC1, 1l 10mM 28SD3R, 1l of 10mg/mL BSA. 0.5l 10mM dNTPs, 5l 5M
Betaine, 1l DMSO, 1l of 1.25u/l HotStart Taq.
For CASIZ 181575, 186051, 186048, 180393, we used a two modified protocols, one for
each set of primers. We split the external and internal primers and did each half of the 28S
sequence separately. We used 2l of template DNA and the following reaction components:
8.75l of millipore water, 2.5l 10X USB Buffer, 1.25l 25mM MgCl2, 1l 10mM 28SC1, 1l
10mM 28SC2R, 1l of 10mg/mL BSA. 0.5l 10mM dNTPs, 5l 5M Betaine, 1l DMSO, 1l of
1.25u/l HotStart Taq. We repeated that protocol using the 28SC2F and 28SD3R primers.
PCR cycling conditions were as follows: COI gene: 2 min at 94C, followed by 35 cycles
of 30 seconds at 94C, 30 seconds at 50C and 45 seconds at 72C, and a final extension period
of 10 minutes at 72C, we relaxed the protocol for the NAF/NARCOI primers: 2 min at 94C,
followed by 35 cycles of 30 seconds at 94C, 30 seconds at 48C and 45 seconds at 72C, and a
3

final extension period of 10 minutes at 72C, 16S gene: 2 min at 94C, followed by 35 cycles of
30 seconds at 94C, 30 seconds at 50C and 45 seconds at 72C, and a final extension period of
10 minutes at 72C, we relaxed the protocol for the modified PCR protocol for CASIZ 199181: 2
min at 94C, followed by 35 cycles of 30 seconds at 94C, 30 seconds at 48C and 45 seconds at
72C, and a final extension period of 10 minutes at 72C, 28S gene: 3 min at 94C, followed by
35 cycles of 30 seconds at 94C, 30 seconds at 52.5C and 2 minutes at 72C, and a final
extension period of 10 minutes at 72C, we relaxed the protocol for the modified PCR protocol
for CASIZ 199181, 181575, 186051, 186048, 180393: 3 min at 94C, followed by 35 cycles of
30 seconds at 94C, 30 seconds at 50C and 2 minutes at 72C, and a final extension period of 10
minutes at 72C.
Gel Electrophoreses were run in 1.0% agarose gels at 100V for 25 minutes to check
PCRs worked and how many bands were amplified. For those PCR reactions that failed we used
the modified protocols and for multiple bands we excised them from gels in order to purify the
PCR product. For those with one band we purified the product with ExoSAP-IT enzymes. We
1:5 diluted ExoSAP-IT and used 2:5 or 2:7 ratio of ExoSAP-IT to PCR product, placed the tubes
in the thermocycler for 30 minutes at 37C then 15 minutes at 80C. For those samples that were
excised from gels we used the Zymoclean Gel DNA Recovery Protocol, we added 3 times the
volume of the gel slice of ADB Buffer, incubated the tubes for 10 minutes at 50C to dissolve the
gel. Used Zymo-Spin colums and a collection tube and transferred the melted agarose samples
and spun them at 10000xg for 30s, we discarded the melted agarose and then added 200l if
DNA Wash Buffer to each column. The tubes were spun at 13000xg for 30 seconds, 200l of
DNA Wash Buffer was added to each colum and spun again at 13000g for 30 seconds. THe spin
columns were then transferred to new, labeled 1.5ml tubes and 12l of warm DNA Elution
Buffer was added to the Zymo filter. The samples were spun at 13000 xg for 1 minute.
After the samples of PCR product were purified we cycle sequenced the PCR product
using flourescently labeled dNTPs as well as regular dNTPs. Cycle sequencing reactions were
performed in 10 l volumes. All regular cycle sequencing reactions had the same protocol for all
primers each reaction used 2l of PCR product and the following reaction components: 5.45l
millipore water, 1.5l 5X Big Dye buffer, 0.3l 10M Primer, 0.75l Big Dye 3.1.
For those samples that required gel excision or had low PCR amplifications we used a
modified cycle sequencing protocols, they were performed in 20l volumes and were the same
for all primers required for the specific gene fragment. Each reaction used 3l of PCR product
and the following reaction components: 9.4l millipore water, 3l 5X Big Dye buffer, 0.6l
10M Primer, 4l Big Dye 3.1.
Cycle sequencing conditions were as follows, for both sets of protocols, conditions were
the same: 1 min at 96C, followed by 15 cycles of 10 seconds at 96C, 5 seconds at 50C and 75
seconds at 60C, 5 cycles of 10 seconds at 96C, 5 seconds at 50C and 90 seconds at 60C, 5
cycles of 10 seconds at 96C, 5 seconds at 50C and 120 seconds at 60C and a hold temperature
of 4C hold temperature.
After each cycle sequencing protocol the solutions were precipitated with ethanol in order
to purify the solution and remove excess dye terminators and recover and concentrate nucleic
4

acids from the liquid state for sequencing. 2.5l of 125mM di-Na EDTA, pH8.0, to the bottom of
each well, 30l of 100% EtOH was added to each well, tubes were sealed and vortexed. THe
tubes were left to sit in the dark for 15 minutes at room temperature to preciptate then spun at
3000 x g for 30 minutes. TUbes were removed and then carefully uncapped, and inverted onto
paper towels. They were again spun, but at 200 x g for 2 minutes. 60l of 70% EtOH was added
to each well and then spun at 2000 x g for 15 minutes. The tubes were removed and inverted and
sput at 200 x g for 2 minutes. The tubes were placed into a 65C incubator for 8 minutes to dry,
then resuspended in deionized formamide to prevent degradation. 10l of formamide was added
to each samped and then headed at 94-96C for 2 minutes and immediately placed on ice for 5
minutes to cool. They were then placed in a ABI sequencing cassette to be read with the ABI
3130.
Phylogenetic Tree Reconstruction

Once the sequences were read by the ABI 3130 sequencer we used the program Geneious
(look up version number) to check the quality of each sequence. We verified the sequences we
read were indeed opisthobranch DNA using the nucleotide BLAST function on ncbi.com. We
created contigs and then consensus sequences and created an alignment using Geneious with
other Gastropteridae and opisthobranch gene fragments from genbank. We We used members of
the families Aglajidae and Philinidae as the outgroups. Once we optimized the alignment as best
we could we ran Bayesian and Maximum likelihood analyses. We used Partition Finder version
1.1.1 and a 50% majority rule tree and a 25% burn-in value of .95 and above as significant.
Mr.Bayes with 20,000,000 generations, sampling every 1000 runs. Our maximum likelihood
analysis was run on RAxML with 50,000 fast bootstrapping runs and a 200 run setting for
another. We used a 70 bootstrap value for significance.

Catalog #
192431
177772
191213
182792
180300
186051
186048
182870
188586
199128
199113
199127
199130
199181
199135
199129
199132
177515

Family
Gastropteridae
Gastropteridae
Gastropteridae
Gastropteridae
Gastropteridae
Gastropteridae
Gastropteridae
Gastropteridae
Gastropteridae
Gastropteridae
Gastropteridae
Gastropteridae
Gastropteridae
Gastropteridae
Gastropteridae
Gastropteridae
Gastropteridae
Gastropteridae

Genus
Sagaminopteron
Sagaminopteron
Sagaminopteron
Sagaminopteron
Siphopteron
Gastropteron
Gastropteron
Sagaminopteron
Siphopteron
Siphopteron
Siphopteron
Siphopteron
Enotepteron
Gastropteron
Siphopteron
Siphopteron
Siphopteron
Siphopteron

Species
nigropunctatum
psychedelicum
psychedelicum
ornatum
quadrispinosum
bicornutum
nigropunctatum
michaeli
tigrinum
orange lines
green
sp.
sp. white
orange
vermillion
ochre
brunneomarginatum

COI
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x

16S
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x

28S
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x

182844
190655
180393
181575

Gastropteridae
Gastropteridae
Gastropteridae
Gastropteridae
Aglajidae

Aglajidae

Gastropteridae

Siphopteron
Siphopteron
Siphopteron
Enotepteron
Chelidonura
Odontoglaja
Philinopsis
Sagaminopteron
Siphopteron
Philine
Philine
Philine
Philine
Chelidonura
Navanax
Aglaja
Odontoglaja
Philinopsis
Philinopsis
Aglajidae
Melanochlamys
Siphopteron
1
2
3
1
2
Gastropteron
Sagaminopteron

nigromarginatum
cf. pohnpei
sp orange 2
africana
sp.
pilsbyi
psychedelicum
tigrinum
aperta1
aperta2
aperta
exigua
amoena
inermis
ticolorata
guamensis
cyanea
depicta
species1
diomedea
brunneomarginatum
pohnpei
quadrispinosum

species1
bicornutum
meckeli
ornatum
psychedelicum

x
x
x
x
DQ974654
DQ974655

x
x
x
x

DQ974667
DQ974668
JN825186 JN825128
JN825187 JN825129

AM421901
AM421877
AM421902
AM421869
AM421890
AM421892
AM421878
AM421866
AM421864
AM421858
AM421860
AM421859
AM421861
AM421862
AM421863

AM421841
AM421855
AM421854
AM421830
AM421832
AM421831
AM421826
AM421825
AM421816
AM421821
AM421819
AM421817
AM421818

AM421820
AM421822
AM421865 AM422902
AM421857 AM421814
AM421856 AM421815

x
x
x
x
DQ927216
DQ927218
AY427474
DQ927225
DQ927226

DQ279988
HQ168438
AM421962
AY427472
AM421950
AY427471
AM421951
AM421954
AM421948
AM421935
AM421939
AM421938
AM421941

AM421940
AM421936
DQ237990
AM421937
AY427478

Other Analyses

Uncorrected p values were calculated on PAUP (lookup verion number) and verified with
MEGA 6.0 and ABGD.
Discussion
The specimens collected from the 2014 Philippine Expedition thought to be
representatives of the Enoptepteron genus were misclassified. The Enoptepteron sp. will be
absorbed into the newly classified species as it is genetically identical to the Gastopteron. The
other Enoptepteron genus is more closely related to the Siphopteron genus and will be classified
as such. However, since the specimen was damaged upon collection it is difficult to determine
the color patterns and external morphology. The genetic evidence is also inconclusive to
accurately classify the specimen.
The Sagaminopteron psychedelicum specimens from Australia, Philippines and Papua
New Guinea all have 1% genetic difference and prove they are the same species despite their
distribution worldwide. The Sagaminopteron ornatum from Australia and Philippines have a
4.1% genetic distance and are therefore classified as the same species.
The Siphopteron cf. pohnpei and Siphopteron sp. orange have a 0.2% genetic difference,
both from the Philippines, however our phylogeny shows a more close relationship to the
Sagaminopteron genus and the node is highly supported. The Siphopteron cf. pohnpei
morphologically and based on the color patterns follow the description of the species and will
remain as Siphopteron pohnpei. The Siphopteron green and Siphopteron pohnpei are 2.2%
genetic difference and are of the same species. Siphopteron green is from the Philippines and
Siphopteron pohnpei are more closely related to the Sagaminopteron genus and will be renamed
as such. Siphopteron green will be renamed as a new species and Siphopteron pohnpei since it
does not follow the description of the true pohnpei will be renamed along with the Siphopteron
green.
Conclusion
Based on our molecular phylogeny, we sequenced 7 new species that we sequenced from
our collection. We also found 3 cryptic species within previously classified specimens. The
maximum likelihood bootstrap values provided show 65 and above however the cut off for
significance is 70. Bayesian analysis posterior probabilities presented are .90 and above, though
those significant are .95 and above. The specimens classified as Gastropteron sp. white,
Enoptepteron sp. and Gastropteron, Siphopteron green, Siphopteron vermillion, Siphopteron
ochre, Siphopteron orange, and Siphopteron orange lines are all new species. The
Sagaminopteron psychedelicum from Madagascar is a cryptic species, as is Sagaminopteron
nigropunctatum from Saudi Arabia, and the Siphopteron quadrispinosum from Australia.