Download Using `DNA Barcoding` to Determine Host Fish

Using ‘DNA Barcoding’ to Determine Host Fish Usage of
Freshwater Mussels in the St. Croix River
Purpose:
ƒTo build a library of genetic sequences for all known species
of freshwater mussel found in the St. Croix River to act as a
molecular key for identification of unknown juvenile mussels.
ƒTo determine the efficacy of DNA barcoding of the juvenile
stage of freshwater mussels for which morphological
identification can prove difficult and time consuming.
ƒTo examine which mussel species utilize which species of
fish as hosts in the wild.
Alexander A. Howe, Sarah L. Boyer,
Mark C. Hove, Daniel J. Hornbach
Macalester College – St. Paul, MN
An adult Quadrula metanevra taken from
the St. Croix River. A small tissue sample
from the mantle tissue was taken before
returning it to the water.
COI Phylogeny
Red: Juveniles and their grouping.
Green: Adults sequenced in lab
Black: Sequences from GenBank
Materials and Methods:
ƒAvailable COI and ND1 sequences for native St. Croix
mussel species were obtained from GenBank.
ƒFor species with less than two COI and ND1 sequences
available on GenBank, mantle tissue clippings from adult
mussels were obtained from the St. Croix River.
ƒJuvenile mussels from naturally infested fish were
obtained from several fish species caught in the St. Croix
River. The fish were brought back to a wet lab, sorted by
species, and put into separate tanks. The tanks were
periodically siphoned and the siphonate was carefully
examined for any juveniles. Juveniles were stored in
ethanol until subsequent extraction
ƒDNA was extracted from the mantle tissue of the adults
and from the entire juvenile with a Qiagen DNeasy Tissue
Kit and amplified for COI and ND1 using separate sets of
primers and PCR profiles.
ƒA QIAquick PCR Purification Kit was used to purify
successful PCR products.
ƒFlorescent tags were added to the amplified sequences
through cycle sequencing and cleaned with a Beckman’s
magnetic plate and beads.
ƒSequencing was preformed with a Beckman-Coulter
CEQ8000 Genetic Analysis System.
ƒSequences were edited using CodonCode Aligner and
aligned with MacClade.
ƒPhylogenetic trees were created with the program PAUP
and posterior probability values were obtained using
Bayesian Analysis with the program Mr.Bayes.
Results:
Introduction:
ƒNorth America has the most diverse assortment of freshwater
mussels in the world, yet over 70% are federally listed as
endangered, threatened, or of special concern. Mussels are
an important part of a healthy aquatic ecosystem, filtering
large quantities of organic matter from water.
ƒFreshwater mussels have a complex life history. The larvae
of most freshwater mussels, called glochidia, are obligate
parasites of fish and attach to their fins, gills, or scales for
dispersal and metamorphosis into the juvenile stage.
ƒHost specificity means that conservation efforts aimed at
endangered species of mussels must include the fish host(s)
as well.
ƒIdentification of glochidia in the wild to the species level
based on morphology alone can be very difficult and in some
cases impossible due to their small size. This makes it difficult
to identify what species of mussel are parasitizing what
species of fish.
ƒDNA Barcoding uses a short, standardized, mtDNA
sequence (~650bp) to identify an organism to the species
level. Cytochrome c oxidase subunit I (COI) is the generally
accepted barcoding gene.
ƒNADH dehydrogenase 1 (ND1) is another mitochondrial gene
that is commonly used in bivalve studies. Using both COI and
ND1 allows for a higher identification success rate than using
one gene alone would.
ƒmtDNA is used because it has a fast mutation rate allowing
for significant differences between species, but little difference
within species. Identification to the species level based on
genetic data is possible.
ƒWith a library of known adult mussel sequences it becomes
possible to identify the juveniles that fall off of naturally
infested fish.
ƒ8 of 9 larvae analyzed from spotfin shiner group with
Fusconaia flava. 1 groups with Pleurobema sintoxia.
ƒAll larvae analyzed from walleye group with both Lampsilis
higginsii and Actinonaias ligamentina, though 2 larvae
appear to be different fromthe rest.
ƒResults are consistent for larvae from spotfin shiner for both
COI and ND1. Bayesian osterior probability values (shown
in red for selected groupings) are very high for both, giving
strong support for these groupings.
Two views of a juvenile freshwater mussel which fell off of a bluntnose minnow. At
this age and size it can be very difficult to identify the species by morphology alone.
ND1 Phylogeny
Red: Juveniles and their grouping
Green: Adults sequenced in lab
Black: Sequences from GenBank
Conclusion and Future Work:
ƒThe results shown here indicate the viability of DNA
barcoding as a method for species identification of freshwater
mussel larvae as an alternative to morphological identification.
ƒThe resolution of identification that DNA barcoding provides
seems to generally be quite strong, however in the case of the
larvae from the walleye it is unable to resolve between
Lampsilis higginsii and Actinonaias ligamentina. The
resolution will likely become enhanced as more adult voucher
sequences are added to the dataset.
ƒThere is still much work to be done. Around 85 more adult
voucher specimens will be sequenced to enhance the
sequence library and will cover every species of mussel
known to the St. Croix.
ƒApproximately 65 more unknown mussel larvae coming from
4 fish will be sequenced to examine in greater detail the
efficacy of DNA barcoding on freshwater mussels.
Acknowledgments:
Thanks to Suzy, Emily, Scott, the Hornbach lab, and the
Hove lab for all their time and help.