Radular Derivation of Opithsobrachia
By: Christina Miller, Noel Fong
Abstract:
Opisthobranchs are a group of highly complex gastropods that have undergone
much evolution from the archaeomollusc. Scientists have made many reconstructions and
revisions to phyletic placement of species within this group, and no certain evolutionary
trait is used in the sorting of these clades. This study presents the use of radula
morphology to determine phyletic affinities within the order of Opisthobranchs.
Morphologies compared among species in this study include numbers in rows of teeth
and numbers of teeth per row.
Introduction:
Opisthobranchs are found around the world from the shallow intertidal zones to
the greatest depths of the ocean. The Order, Opisthobranch is made up of specialized and
complex marine gastropods that include nine different sub-orders and include around
6000 different species (Gobbeler, 2011). These nine clades have been created on account
of many different evolutionary trends. These trends include the anatomy and morphology
of different organs such as the shell and the reproductive system, and also nucleotide
sequences. There have been a few studies performed to learn how their diets have been
reconstructed from the ancestral diet, however there have been even fewer studies done
using various radula morphologies as characteristics that can sort out clades.
Opisthobranchs feed on many different prey items and often are very particular in
the food item that they feed on. Different species of Opisthobranch are known to feed on
things from various species of algae, to sessile invertebrates such as hydroids and
bryozoans, to barnacles or even other Opisthobranchs. The main feeding apparatus of
many opisthobranchs is the scraping tongue known as the radula, which has rows of teeth
and each row is composed of lateral teeth that aid in manipulating food prey. The number
of rows of teeth on a radula and the number teeth per row differ between species based on
what they eat (Gosliner 1987). Given that each species is so particular in the food items
that they consume, it is reasonable to consider that evolutionarily they have adapted to
become more specialized feeders and that certain radula morphologies can be shared
derived characteristics among phylogenetic clades. In this study we will be looking at the
number of rows of teeth and the number of teeth per row as a characteristic to determine
derivation from the ancestral gastropod.
Methods:
General Approach:
We performed a comparative study between 10 different species of
Opisthobranchs in relation to their radula morphology, diet, and phylogeny. In order to
test our hypotheses, information all three variables had to be found for each species so
that a comparison could be made. Between each species’ radula, the number of rows and
the number of teeth per row was counted. After this data was collected, we researched the
diet of each species, through scientific databases, so that we could classify each one as a
generalist or a specialist. Generalists were classified as those species that consumed more
than one type of food. Specialists were classified as those that only fed on one type of
food. In order to relate diet and radula morphology to evolution, research through
scientific databases was done on the radula morphology and diet of an archaeogastropod,
or primitive gastropod. This was then supported with research of phylogenetic clades
created by previous experiments.
Species were collected during low tide in Betty’s Bay, South Africa from April
8th, 2013 through May 3rd, 2013. Weather conditions through this period were mostly
sunny or partially cloudy skies, with a few days of strong wind. Specimens were usually
collected from the rocky intertidal area within tide pools or from just outside the intertidal
tide pool area. The majority of the pools where Opisthobranchs were collected from were
fairly shallow during low tide, most being no more than a meter deep. A few species such
as Janolus capensis and Notobryon thompsini were found sub-tidally. Janolus capensis
was collected from a different site known as Miller’s Point. This site was sub-tidal with a
sand bottom and a few small reefs created by boulders. The plants and animals found at
this site were extremely similar to those found in the tide pools around Betty’s Bay. One
other collection was made at a different site than Betty’s Bay, at Kalk Bay during low
tide. The specimen found here was Phyllodesmium horridus. This site is closer to the
South Atlantic Ocean and contains a more tropical fauna than Betty’s Bay.
We chose to do this experiment here because we knew that we would be able to
find various species of Opisthobranchs in the intertidal without having to scuba. Prior to
arriving in South Africa we had read Two Oceans by Charles Griffiths and Nudibranchs
of Southern Africa by Terrence Gosliner. Each book covers the many different species of
Opisthobranchs you can find without having to go deeper than 5m and a brief description
on their diets so we also knew that we would be able to find both generalists and
specialists here:
General Hypothesis: Generalists will have different radula morphology
than will specialists.
To test this we tested the specific hypotheses:
•
Null: There will be no difference in radula morphology
between generalist species and specialist species.
•
Alternative: Generalists will have a different number of
rows of teeth than will specialists.
•
Alternative: Generalists will have a different amount of
teeth per row than will specialists.
Design:
At least two specimens of each species were collected and their radulae
counted. Only two individuals were needed for data collection because Opisthobranchs
have a radular morphology that is constant among individuals of each species without
variances within a species. Data was inputted into Systat version 13 to generate
normalized hierarchical cluster analysis of relatedness in rows of teeth and teeth per row
between each species. If two branches are generated in the cluster analysis separating
generalists from specialists with a significant p-value less than .05 then we can reject the
null hypothesis and accept both the alternatives since the cluster will be made by
comparing both row number and teeth per row.
The collection process consisted of skin-diving in tide pools, combing
through seaweeds, turning over rocks and scanning the ground beneath. Once an
Opisthobranch was found we would collect and place it in a container filled with salt
water in order to keep the specimen alive as long as possible so that it wouldn’t
deteriorate.
Once the specimens were brought back to the compound they were placed
in a tank with an airstone, once again trying to keep the specimens alive as long as
possible. If a specimen were to die before a dissection was able to be performed on it, it
would be placed in a 90% alcohol solution to prevent deterioration until we could dissect
it. Dissections were done using scalpels, micro-scalpels, forceps and a dissecting
microscope that had both 10x and 25x magnification. Cuts were made on the dorsal side
between rhinophores; if the organism lacked rhinophores then the cut was made anteriorposteriorly, in the center of the anterior mantle edge of the organism. Cuts were long
enough to reach to stomach of the organism and had to be made gently as to only cut
through one tissue layer at a time so that the buccal mass containing the radula wasn’t
accidentally damaged. The buccal mass is identified as a round mass of muscle connected
to the esophagus which trails from the stomach. The buccal mass is also easily identified
with glimmering chitin at some point along it, which is actually the radula protruding.
Muscle and tissue of the buccal mass carefully had to be removed from the radula so as
not to tear it and allowing for easier access when counting the rows of teeth and the
number of teeth per row. Radulae were then stored in 90% ethanol solution incase further
work must be done with them.
General Hypothesis: Archaeogastropod will relate to the generalists or the
specialists we collected in either its diet and/or radula morphology.
To test this we’ve researched via scientific databases, the diet and radula
morphology of the archaeogastropod in order to classify it as a generalist or specialist.
From there we compared its radula morphology to the Opisthobranchs from which we
collected data and that fell into the same classification.
Results:
We were able to collect nine different species of Opisthobranch, during our data
collection period. The most common species we found was Berthellina cirtina, it feeds
on sponges, algae and other sessile invertebrates so we classified it as a generalist
(Gosliner, 1987). We found its radular formula to be 70-108x50-76.R.50-76 (Fig. 1, A).
Another commonly found species was Aplysia parvula which feeds on various species of
red algae but since eats only one type of food it is classified as a specialist (Gosliner,
1987). We found the radular formula of Aplysia to be 36-44x11-15.R.11-15. Melibe rosea
was collected and has a hooded head, which it uses to trap small crustaceans; it is
classified as a specialist and has lost its radula (Gosliner, 1987). We collected Haminoea
alfrednesis, and it feeds on filamentous green algae (Gosliner, 1987). Haminoea is
classified as a specialist and was found to have a radular formula of 24-31x 35-38.R.3538. Janolus capensis was collected and is found to feed on bryozoans, preferring those
that stand up right, it was classified to be a specialist and its radula was so torn after
dissection that we used an experiment (Gosliner, 1981) to find that it has a radular
formula of 17-21x 26-42.R.26-42. Anteaeolidiella indica feeds on anemones, it is
A.
B.
Figure 1. Radulae from various
species. A. Taken through 25x
dissecting lens, generalist
Berhtellina. B. Scanning Electron
Micrograph of Elysia radula, taken
from previous study (Jensen,
1987).
especially known to prefer striped anemones (Gosliner, 1987). Anteaeolidiella is
classified as a specialist and was found to have a radular formula of 14x 0.R.0. We also
collected Notobryon wardi which is found to feed on hydroids, classified as a specialist
and has a radular formula of 15x17-19.0.17-19 (Gosliner, 1987). Elysia viridis feeds by
piercing the cell walls of the alga Codium and sucking out all the organelles (Gosliner,
1987). Elysia is classified as a specialist and was found to have a radular formula of 15x
0.R.0 (Fig. 1, B). Phyllodesmium horridus was collected upon the soft coral in which it
eats and was classified as a specialist, its radular formula was found to be 14-14x 0.R.0.
Since we only were able to find one generalist we found the diet and radular formula of
one more from a previous experiment in order to give us a more significant sample
number, Chromodoridella mirabilis with a radular formula of 58x 80.R.80 (Gosliner and
Griffiths, 1981).
After inputting all this data into Systat v.13 we saw that the generalists did cluster
into their own separate branch from the specialists with a p-value of .01 (Fig. 2). Species
that clustered together were as close of a distance as 368 from each other while
generalists and specialists were a distance of 10036 away from each other in relatedness.
We now see that our generalists have many more rows of teeth and teeth per row
than do our specialists, but in order to know which is the derived and which is the
ancestral form, we have to know the diet and radular morphology of the
archaeogastropod. From previous studies we know that it is likely that the ancestral
Opisthobranch was a grazing, infaunal omnivore (Gosliner, 2008). We then classified the
ancestral Opisthobranch as a generalist. The radula of an archaeogastropod functions
more like a broom by sweeping the substratum and exerting little force (Steneck, 1982).
A study that classifies different radulae by their form and function states that a radula that
is used to brush the surface of the substrate with little force and does not penetrate the
surface has many fine lateral teeth (Hawkins, 1989). We now know that since the
generalist archaeogastropod swept the substratum with little force that it probably had
many fine lateral teeth.
Discussion:
With our data from Systat v. 13 we can reject the null hypothesis and accept both
the alternatives that generalists have a different number of rows of teeth and teeth per row
than specialists. Our data shows that generalists have more rows of teeth and more teeth
per row than specialists. We also found that the archaeogastropod is a generalist and they
too have more rows of teeth and more teeth per row. Past experiments explain
evolutionary trends of the radula using form and function of various morphologies. A
study shows that radulae with a reduced number of points of contact on the substratum
and hardened teeth have superior excavating abilities (Steneck, 1982). This could explain
how the loss of teeth can be a gradual movement towards a more specialized diet. There
are a couple trends that even explain the radular derivation in one of our specialists,
Elysia. To penetrate harder substrates like resistant algae, fewer teeth that are more stout
and hardened are used (Hawkins 1989). Before Elysia was able to penetrate each
individual cell of the codium algae, its ancestors used to feed on rough, even calcified
algae (Jensen 1997). In linking the two studies it is probable that the ancestor of Elysia
had less teeth per row. Since Elysia only has one tooth per row, this shows there was a
gradual loss of teeth along with the reconstruction of diet from the ancestral form.
In looking at past experiments, monophyly of clades have been detected at the
molecular level to reveal that the suborder, Nudipluera, which contains both Berthellina
citrina and Chromodoridella mirabilis, is placed in the most basal clade in a phylogenetic
tree composed of 58 different species of Opisthobranchs that together cover all nine
major clades (Gobbeler, 2011). It also must be noted that a couple of our specialists,
Notobryon wardi, Anteaeolidiella indica and Janolus capensis fall within this basal clade.
However, this does not necessarily mean that these specialists have a less derived radula,
it is noted how this clade is an early offshoot from a common ancestor of all nine clades.
This early offshoot may have given this clade enough time to evolve enough
Figure 2. Systat v. 13 hierarchal cluster analysis of the ten total species in which we collected data from. P-value of .01 was calculated
through the program.
Berthellina citrina
Berthellina citrina
Chromodoridella
mirabilis
Berthellina citrina
Berthellina citrina
Janolus capensis
Janolus capensis
Notobryon Wardi
Notobryon Wardi
Harminoea
alfredensis
Harminoea
alfredensis
Harminoea
alfredensis
Aplysia parvula
Aplysia parvula
Aplysia parvula
Anteaeolidiella indica
Phyllodesmium horridus
Elysia viridis
Elysia viridis
Phyllodesmium horridus
differences in nucleotide sequences that it seems to be non-paraphyletic and more closely
related to Acteon, what is thought of to be the most primitive genus of Opisthobranchs
(*Gosliner, 1981). If this alternative is correct then we’d be saying that the whole clade is
more derived including the super families which include our generalists. This also isn’t
necessarily the case because characteristics that have defined these clades have been
much disputed over the past fifty years and nothing is for certain when it comes to the
detection and make-up of these clades (Gobbeler). Even though a molecular clade places
species close together, there can be other and more numerous shared derived
characteristics that can split up these species and place them into different clades.
Conclusion:
We think that numbers of rows of teeth and numbers of teeth per row are
somewhat broad characterizations to be sorting radulae into. It would be more useful to
use radulae morphology as a tool for phyletic placement if more specific characteristics
can be used along with row and teeth numbers such as, teeth shape, density and
chemistry. Teeth shape and density tell us a lot more about function and can be better
associated with dietary evolutionary trends (Lambert, 1991). Teeth chemistry can tell a
lot about environmental chemistry and if it played a role in tooth structure and
evolutionary trends through history during different water make-ups or during radiation
whilst the organism was moving through different environments. It seems plausible with
better technology than what we were equipped with, that radula characteristics can be
used to detect phylogenetic placement or at least aid in supporting already determined
clades.
References
Branch, G.M., C. Griffiths, L.E. Beckley, M.L. Branch. 2010. Two Oceans: A
Guide to Marine Life of Southern Africa.
Eliot, C.N.E. 1905. On some nudibranchs from the Pacific, including a new
genus, Chromodoridella. Proceedings of the Malacological Society in London 6: 229248.
Gobbeler, K., Klussmann-Kolb, A. 2011. Molecular phylogeny of the euthyneura
(Mollusca, gastropoda) with special focus on opisthobranchia as a framework for
reconstruction of evolution of diet. An International Journal of Marine Sciences 27 (2):
121-154.
Gosliner, T.M. 1987. Nudibranchs of Southern Africa: A Guide to Opisthobranch
Molluscs of South Africa by Terrence Gosliner.
Gosliner, T.M., R.J. Griffiths. 1981. Description and revision of some South
African aeolidacean Nudibrachia. Annals of The South African Museum 84: 105-150.
*Gosliner, T. M. 1981. .Origins and relationships of primitive members of the
Opisthobranchia (Molluscs: Gastroioda). Biological Journal of Linnean Society 16: 197223.
Gosliner, T.M. 1981. The South African Janolidae (Mollusca, Nudibrachia) with
the description of a new genus and two new species. Annals of The South African
Museum 86: 1-42.
Gosliner, T.M., Pola, M., et al. 2011. Molecular data illuminate cryptic
nudibranch species: the evolution of the Scyllaeidae (Nudibranchia: Dendronotina) with a
revision of Notobryon. Zoological Journal of the Linnean Society 165: 311-336
Hawkins, S. J., Watson, D. C., et al. 1989. A Comparison of feeding Mechanisms
in Microphagus, Herbivorous, Intertidal, Prosobranchs in Relation to Resource
Partitioning. Journal of Molluscan Studies 55: 151-165.
Jensen, K. R. 1997. Evolution of the Sacoglossa (Mollusca,Opisthobranchia) and
the ecological associations with their food plant. Evoulitionary Ecology 11:301-335
Lambert, W.J. 1991. Coexistence of hydroid eating nudibranchs: do feeding
biology and habitat use matter? Biological Bulletin 181: 248-260.
Rudman, W.B. 1981. The anatomy and biology of alcyonarian- feeding aeolid
opisthobranch molluscs their development of symbiosis with zooxanthelle. Zoological
journal of the Linnean Society 72: 219-262.
Steneck, R. S., Walting, L. 1982. Feeding capabilities and limitation of
herbivorous molluscs: A functional group approach. Marine Biology. V. 68, Issue 3: 299319.