New data on the diversity, host specificity and distribution of

New data on the diversity, host specificity and distribution of
crustacean copepods associated with stony corals
(Cnidaria: Anthozoa: Scleractinia)
of the Indo-Pacific coral reefs
V.N. Ivanenko1, Mary Wakeford2, Julian Caley2
1Department
of Invertebrate Zoology, Moscow State
University, Russia
2Australian Institute of Marine Science, Australia
Diversity of Crustacean Copepods
(insects of the Sea)
11 orders
Number of valid species 15 000+
Presumed number of species: 60 000-100 000 to 1 million
The most diverse
orders of Copepoda
Specimens
with
Barcodes
(BOLD)
Species
with
Barcodes
(BOLD)
Valid
Species
(WoRMS
database)
Genera
(WoRMS
database)
Families
(WoRMS
database)
Poecilostomatoida
86
4
2364
430+
59
Calanoida
2,268
298
3059
330+
47
Cyclopoida
476
40
2376
260+
30
Siphonostomatoida
631
49
2339
360+
40
Harpacticoida
224
17
5427
660+
59
Total
3685
408
15565
2040+
235
• ~2.5% of known copepod species are barcoded
• Most of the barcoded copepods are planktonic or fish parasites
Diversity of Symbiotic Copepoda
•
~ 2000 species of symbiotic copepods
associate with invertebrate hosts (Annelida,
Cnidaria, Crustacea, Mollusca, Porifera,
Echinodermata, Urochordata, etc)
•
This number represents only a small fraction
(~1%) of the species living with marine
invertebrates (Humes, 1994)
Diversity of Copepoda
living on/in scleractinian corals
(from original database)
•
•
•
•
•
•
900+ records of copepods from scleractinian corals
(the first record was in 1960)
275 species 89 genera, 14 families of copepods
158 species 52 genera 15 families of scleractinian
corals
Copepoda associates with most examined colonies
of scleractinian corals
Most scleractinian species have not yet been
examined for copepod associates
No barcoded copepods living on scleractinian
corals were known till our project
Common Copepoda associate with Scleractinia
Body length 0.4-3 mm
Obligate ecto- or endosymbionts
Use their coral host as a habitat and source of food
•
Poecilostomatoida:
– Lichomolgidea
• Anchimolgidae 90+ sp
• Rhynchomolgidae 30+ sp
– Xarifidae 85+ species
•
Siphonostomatoida:
– Asterocheridae 40+ sp
– Corallomyzontidae 6+ sp
•
Harpacticoida:
– Tegastidae 4 sp
Multiple association and abundance of Copepoda
(Humes, 1994)
•Copepoda are found in
multiple association with
scleractinian hosts
•Copepoda living on corals
can be abundant.
In one fragment of Acropora
hyacinthus 30 x 30 cm there
were 1250 copepods
Acropora hyacinthus
Heron Island, 2010
Material
(May – Dec 2010, 262 colonies of stony corals)
Vietnam,
October 2010
48 colonies
•
.
Lizard Island
Sep 2010
96 colonies
Vietnam-Russia
Tropical Centre,
Na Trang
Ningaloo Reef
May 2010
48 colonies
CReefs
project, AIMS
Heron Island
Nov 2010
69 colonies
•
•
•
Corals were collected by SCUBA
(70+ dives, max depth 30m)
All corals were photographed
alive underwater
Pieces of coral colonies were
isolated in plastic bags and
brought to the surface
Ningaloo Reef, May 2010
Lizard Island, Sep 2010
Heron Island, Nov 2010
Material and Methods
•
•
•
•
•
In the lab the small amount of ethanol
(~10%) were added to the plactic bag with
the coral hosts
20-30 munities later the corals were heavily
shaked in the solution which was filtered
through a fine net.
The obtained residue was sorted under the
dissecting microscope
The copepods were photographed and
preserved in 95% ethanol.
In addition DNA subsamples of the
scleractinian corals were preserved and their
skeletons were cleaned by bleaching
powder, dried and labeled skeletons of the
coral hosts were photographed and
deposited to the Museum of Tropical
Queensland
CReefs Database
(AIMS)
Field and taxonomic data
obtained from the copepods
and their hosts, including
underwater photographs of
the living corals and their
skeletons, are stored in an the
MS Access database.
The data entry interface consists
of a series of forms organised
into tabs. The three major
tabs are Sites, Samples and
Specimens. Specimens
contains four sub-formsSpecimen Details, Lot,
Images and DNA.
The database comes with a utility
called IrfanView which greatly
improves the speed at which
large image files are
processed.
DNA extraction, PCR amplification, and DNA sequencing
Sequence analysis was carried out at the
Canadian Centre for DNA Barcoding
using standard barcoding protocols
Compete specimen of morphospecies
represented by several specimens in the
sample (body length 0. 5-1.5 mm) were
used for DNA extraction.
Two methods of DNA extraction were tested:
• 1) Standard automated CCDB bind-washelute protocol (Ivanova et al. 2006) and
• 2) HotSHOT alkaline lysis (Montero-Pau
et al. 2008) in combination with voucher
recovery protocol (Porco et al. 2010).
For PCR amplification and sequencing,
LCO1490 and HCO2198 primers (Folmer,
1994) were used.
Methods of analysis
•
•
•
•
Sequence data were managed using the Barcode of Life
Data System (BOLD)
Analysis of genetic similarity was performed using MEGA
version 5.0
Trees were built using the NJ algorithm with the K2P
model.
jMOTU 1.0.7, a Java program, was used for clustering
sequences into Molecular Operational Taxonomic Units
(MOTU) (Blaxter et al., 2005).
Results
I. New collection of copepods and their coral hosts
•
In total 258 colonies of 83 species
of scleractinian corals
representing 28 genera and 15
families were collected
•
Washing of colonies discovered
many copepods on most collected
colonies (1 to 100s of specimens
of up to 10 morphospecies of
copepods on one colony).
Fig. Photos from the database: colonies of scleractinian
corals investigated for symbiotic copepods, Lizard
Island, GBR, 2010.
II Preliminary results of DNA barcoding of copepods from
scleractinian corals
•
•
DNA barcodes were generated from 119 of 190
selected for DNA barcoding specimens
25% morphospecies (=comparative units) are lost.
•
There was no significant difference in barcode
recovery between the two methods of DNA extraction
(Standard automated CCDB bind-wash-elute
protocol and HotSHOT alkaline lysis)
•
However using HotSHOT alkaline lysis in
combination with voucher recovery protocol allowed
80% (instead of 10%) vouchers to be preserved in a
condition suitable for morphological studies
.
Sequence Length Distribution - COI-5P
Siphonostomatoida
Accumulation Curves of DNA
barcoded copepods associated
with scleractinian corals of Lizard
Island shows:
-The number of DNA barcodes of
copepods increases more rapidly
for the order Poecilostomatoida
than Siphonostomatoida.
-Further investigation of copepods
associated with corals will provide
more data about molecular
diversity of copepods.
Poecilostomatoida
(obtained from BOLD)
Siphonostomatoida
Poecilostomatoida
Harpacticoida
Cyclopoida
Calanoida
Unrooted COI tree for DNA
barcoded copepods (BOLD)
including new data reconstructed
by Neighbor-Joining with Kimura-2Parameter (K-2-P) distances
66 Taxonomic Units
40
Number of MOTU
35
30
25
Ряд1
20
15
10
5
Number of specimens
sp
10
sp
9
sp
8
sp
7
sp
6
sp
5
sp
4
sp
3
sp
2
1
sp
ec
i
m
en
0
The average value of intraspecies distinction of
copepods was 1.56 %
Interspecies distinctions of all copepods
investigated during the project varies within
10.2 % to 45.9 % and is characterized by a
normal distribution.
Clustering of sequences by
jMOTU program was
conducted at cutoffs from 0 to
30 bases.
96 MOTU
The number of MOTU
dropping from 96 (0 base
difference) to 66 (at 11+ bases
or ~ 1.75+ % difference)
11+ bases or
1.75% difference
66 MOTU
The sequences included in
each MOTU correspond to the
results obtained by NeighborJoining analysis (2% threshold
values)
Acroporidae
Acropora
aculeus/carduu
s cf
Acroporidae
Acropora
divaricata cf
Acroporidae
Acropora
humilis
Acroporidae
Acropora
nasuta cf
Acroporidae
Acropora
palifera
Acroporidae
Acropora
pulchra
Acroporidae
Acropora
loripes cf
Acroporidae
Astreopora
ocellata
Fungiidae
Ctenactis
crassa
Faviidae
Echinopora
lamellosa cf
Faviidae
Echinopora
mammiformis
Fungiidae
Fungia
danai cf
Oculinidae
Galaxea
astreata
Fungiidae
Herpolitha
limax
Mussidae
Lobophyllia
flabelliformis cf
Mussidae
Lobophyllia
hemprichii
Merulinidae
Merulina
scabricula cf
4
Acroporidae
Montipora
peltiformis cf
2
Acroporidae
Montipora
sp
Pectiniidae
Pectinia
lactuca
Faviidae
Platygyra
daedalea cf
Faviidae
Platygyra
pini cf
Fungiidae
Polyphyllia
talpina
Poritidae
Porites
sp
Pocilloporidae
Seriatopora
hystrix
Pocilloporidae
Stylophora
pistillata
Dendrophylliidae
Tubastraea
micrantha
10
17
26
Host Specificity and Multiple Association of
Copepods from Corals
•
Only one species of host have all but one randomly
selected copepods (60 MOTUs, 109 specimens) from
randomly selected 26 species of corals (33 of 96 colonies
from Lizard Island) representing 17 genera and 10 families
•
Different multiple (2-5) associations of MOTUs on one
species of a coral host are found (not all potential species
of copepods was sent for the test barcoding
12
10
8
Ряд1
6
0
1 MOTU
2 MOTU
3 MOTU
4 MOTU
5 MOTU
Fig. Frequency histogram illustrating the number of host
species of corals used by on or more MOTUs
Host Specificity and Multiple Associations in Xarifidae (Poecilostomatoida)
Fig. CO1 NJ tree for 5 MOTUs of Xarifia from from 4 colonies (Xarifidae are
found in 55 colonies)
Host Specificity and Multiple Associations in Asterocheridae +
Corallomyzontidae (Siphonostomatoida)
Fig. CO1 NJ tree for 40 specimens, 11 MOTU of copepods
from 8 species of 11 colonies of corals
Our preliminary results show:
•
•
•
•
•
Effectives of DNA barcoding for investigation
of symbiotic copepods from scleractinian
corals
High diversity, species level host specificity
and multiple association of symbiotic
copepods
Necessity of DNA barcoding of the remaining
collection of copepods
Necessity of DNA barcoding of the coral hosts
Necessity of better preservation of copepods
during DNA extraction
Funding
•
•
•
•
•
The samples from Australia were collected as part of the CReefs
Australia project.
The samples from Vietnam were collected as part of a project of
Vietnam-Russia Tropical Centre (Trung tâm nhiệt đới Việt Nga) .
Molecular data were obtained at the Canadian Center for DNA
Barcoding funded by the Government of Canada through Genome
Canada and the Ontario Genomics Institute (2008-OGI-ICI-03).
Research of VNI was supported by grants from Ministry of
Education and Science of the Russian Federation, Russian
Foundation for Basic Research, and Smithsonian Institution.
The conference trip of VNI was supported by the Russian
Foundation for Basic Research.
Acknowledges
•
•
•
Chad Buxton (Museum of Tropical Queensland) as well as Mary
Wakeford (Australian Institute of Marine Science) were the great
diving buddies of VNI during sampling of corals in Australia.
Chad Walter (Smithsonian Institution) for taking part in preparation
database on Copepoda from scleractinians
Natalia Ivanova and Alex Borisenko (Canadian Center for DNA
Barcoding) helped to obtain and analyse molecular data.
Confocal microscopy of a
copepod nauplius (exuvia,
exoskeleton) in a permanent
glycerin slide.
Soft tissues were:
- removed without damaging
the external anatomy by
immersing for 10 min in a Kprotease
digestive medium from
QIAGEN
- staining
procedure with a solution of
Yellow Fuchsin.
Ivanenko et al. 2011
Lizard Island
•
The northern Great Barrier Reef, 270
km north of Cairns, Queensland.
•
Lizard Island Research Station
operated by the Australian Museum.
Creef’s team, September, 2010
Poecilostomatoida
Female
urosome
Xarifidae Humes 1960
Body length: 0.5-3 mm
Diversity: 85+ species in 5
genera
Distribution: Indian Ocean and
West Pacific
Ecology: all species live in the
coral polyps
Fig. Female of Xarifia scutipes sp.
from Goniopora tenuidens, in 3 m,
Moluccas, from A.G. Humes and M.
Dojiri, 1983
caudal ramus
egg sac
Harpacticoida
Tegastidae
Body length: 0.5 mm
Distribution: worldwide
Ecological comments:
4 species are found on scleractinians of
Indo-Pacific:
T. georgei from Stylophora sp. and
Pocillopora sp., Red Sea;
T. acroporanus from Acropora Jlorida,
Marshall Islands;
T. gemmeus from Cyphastrea ocellina
and Montipora verrucosa, Hawaii;
T. paulipes from Pocillopora verrucosa
at Moorea, Society Islands.
Tegastids are known as Red Bugs of badly
destroying colonies of cultivating
scleractinians corals
Frequency histogram of the genetic
variation
•
The average value of
intraspecies distinction of
copepods was 1.56 %
•
Interspecies distinctions
of all copepods
investigated during the
project varies within 10.2
% to 45.9 % and is
characterized by a
normal distribution.
Fig. Frequency histogram of the genetic variation of 119
specimens of 66MOTUs of copepods from Scleractinian
corals at Lizard Island, GBR, 2010). (p-dist %). The gap
between distribution of intraspecific variation and
interspecific divergence is arrowed.