Click Here To Visit Malacological Society Website Click Here To Visit Malacological Society Website Click Here To Visit Malacological Society Website Click Here To Visit Malacological Society Website Click Here To Visit Malacological Society Website..Click An Image To Visit Society Website  
             
 


Dr Mikael Thollesson, the Annual Award Winner

The Nudibranchia is a taxon estimated to comprise between 1000 and 3000 genera. All are marine gastropods lacking a shell as adults, giving them their colloquial name sea slugs. The attractive colours and bizarre appearance of many species have also made them favourite photo objects for many amateur SCUBA divers and naturalists.

Working with this taxon for my Ph. D. thesis1, I used molecular methods to study some different problems. In this report I will leave out two of my favourite subjects, species concepts and phylogenetic approaches to nomenclature, but report briefly on the results from three studies on:

1. Species delimitation in Dendronotus

2. Phylogeny of Doridacea, a nudibranch subtaxon

3. Opisthobranch phylogeny and position/monophyly of Nudibranchia

One species or two?2

In almost all fields of biology, the concept of the species is paramount. In spite of (or perhaps because of) this, there is still a lot of dissention about this topic. The ost widely used species concept is the biological species concept, where species are "... groups of actually or potentially interbreeding populations, which are reproductively isolated from other such groups". Although this is not the concept I prefer, I will use it to illustrate the example here. The problem is how to put this concept to a test in a practical context.

Dendronotus frondosus (Ascanius, 1774) is a boreal nudibranch which appears in several colour variations. Some of these forms seem to be more distinct and the question is whether these are better regarded as separate species. In particular, I found some specimens on the Swedish West Coast that had an opaque white colour with dark purple-blackish blotches; the common Dendronotus here is brownish.

As I had specimens of both colorations from the same locality, the biological species concept could be applied and tested using allozyme electrophoresis. Allozyme electrophoresis makes it pssible to score the genotype as well as the alleles carried by each specimen. Under the null hypothesis that only one population is present in the sample, the genotype frequencies are expected to follow Hardy-Weinberg frequencies. That is, if we have two alleles at a particular locus with frequencies p and q (as counted from the electrophoretic analysis), the two homozygotes should be present in frequencies p2 and q2 respectively, and we should have a frequency of 2pq heterozygotes.

In the Dendronotus study, I found four loci where one allele was present in 100% of the brown specimens (i.e. all homozygotes) and another allele in 100% of the white specimens (all homozygotes for the other allele). Thus p = q = 0.5, and we expect that half of the specimens should be heterozygotes, yet none was found. As this pattern was repeated for four independent loci (Mpi, Mdh, Idh and Acp), this is strong evidence against the specimens coming from one population. Since the specimens came from dredge hauls at the same locality, they can be considered to be different species according to the biological species concept.

As this was further supported by morphological differences, I reinstated the name Dendronotus lacteus (Thompson, 1840) for the white specimens. What we should call D. frondosus remains another topic ...

Dorid Phylogeny

Doridacea is the largest of the four suborders recognized within Nudibranchia by most authors. The classification of the suborder is traditional rather than based on an explicit phylogenetic hypothesis, and thus many of the taxa are likely to be paraphyletic. Of particular interest are the Eudoridacea versus Anadoridacea (respectively possessing and lacking a pocket into which gills can be withdrawn) and Suctoria versus Non-suctoria (respectively possessing and lacking a muscular buccal pump).

For my thesis I used DNA sequences to estimate the phylogeny for a sample of these taxa. I sequenced part of the 16S gene (450 bp) and part of the COI gene (600 bp). Unfortunately, the sequences were not very suitable for the particular question asked and so, to enhance the fidelity of the cladistic analysis, I tried to use some different weighting schemes that had been proposed in the literature. The question is, what can one use as a criterion to determine what the optimal weighting scheme is? (The one that gives you the tree that you want is NOT the correct answer ...)

The fact that both of the genes are located in the mitochondrial genome gives you an option. Data sets that are linked, and thus have the same phylogenetic history, are expected to give the same tree when data sets are analysed both separately and combined. The apparent lack of recombination in the mitochondrial genome results in the mitochondrial genes being linked and inherited as a single locus, and the different genes in the mitochondrion thus share their phylogenetic history. In cases where analyses of data sets from different mitochondrial genes produce different trees, this cannot be attributed to different gene phylogenies. Thus increase in congruence between data sets can be used to indirectly evaluate increase in fidelity (signal over noise) and thus accuracy of weighting scheme.

Trying several weighting schemes, the highest congruence between the data sets was achieved using a successive weighting where each character was weighted with 1/s (s is the number of steps the character takes on a tree), and each kind of transformation was weighted with ln(Xij/X), where Xij is the number of transformations between character states i and j (the states can be A, C, G or T) and X is the total number of transformations (steps) on a tree. The best hypothesis under this optimal weighting scheme is shown in Fig. 1. Although the support for the clades relevant to the status of the aforementioned taxa were not sufficient to answer the questions I was interested in, the clades that had some bootstrap support were in general congruent with existing taxonomy. One exception is the genus Goniodoris, which is not monophyletic unless Okenia is included.

Opisthobranch relationships3

The classification of Gastropoda that was in use up to the last decade rested mainly on the authoritative work of Thiele, and used three subclasses: Prosobranchia, Opisthobranchia and Pulmonata. Although Opisthobranchia and Pulmonata usually have been treated as separate taxa, several authors have united them in one taxon, Heterobranchia or Euthyneura.

The unclear phylogeny of the Opisthobranchia and its subgroups has led many authors to discuss 'evolutionary trends' in their efforts to delimit this taxon from Prosobranchia and Pulmonata. Thus Opisthobranchia, as it has been perceived, is likely to be a paraphyletic group. Cladistic analyses of high-level gastropod relationships based on morphology, but with only a few euthyneuran taxa, indicate that Euthyneura is monophyletic while Opisthobranchia is not. Studies using molecular data have focused more on euthyneuran relationships, and also indicate a paraphyletic Opisthobranchia, with the pulmonates possibly monophyletic. These studies clearly show that phylogenetic studies of opisthobranchs and pulmonates are currently inseparable, as Pulmonata may be the sister group to a particular opisthobranch taxon.

Recently Mikkelsen provided a cladistic analysis and well supported phylogenetic hypothesis using morphological data for Cephalaspidea senso lato. She included representatives from other opisthobranch subtaxa, but omitted some taxa that may be nested within her study group. Among those are Nudibranchia and its posdsible sister group Notaspidea, as well as the pulmonates.

I used the same part of the 16S rRNA gene as above in a phylogenetic analysis of some euthyneuran taxa. There are some problems when trying to align this gene for distantly related taxa, as parts of it are highly variable and other parts are very conserved. Still, some parts of the sequences could not be aligned and had to be culled from the analysis.

The optimal tree (from an analysis using minimum evolution as a criterion and pairwise distances according to HKY with a gamma-distributed rate heterogeneity) is shown in Fig. 2; the parsimony tree is similar for the well-supported groups.

The main conclusions are that there are two main groups within the nudibranchs, Anthobranchia and Cladobranchia (which has been suggested before), but these may not form a monophyletic group unless Notaspidea is included. The Euthyneura form a clade, which is also supported by a deletion in an otherwise extremely conserved region of the 16S molecule. Acteon tornatilis, usually considered to be a basal opisthobranch, is found outside a clade with the pulmonates+opisthobranchs. The most surprising result is that the pteropod Clione limacina is found outside the Euthyneura.

1. THOLLESSON, M. 1998. Nudibranch systematics and molecular data. Ph. D. thesis, Göteborg University, Sweden. ISBN 91-628-2852-5.

2. THOLLESSON, M. 1998. Discrimination of two Dendronotus species by allozyme electrophoresis and the reinstatement of Dendronotus lacteus (Thompson, 1940) (Nudibranchia, Dendronotoidea). Zoologica Scripta, 27: 189-195.

3. THOLLESSON, M. 1999. Phylogenetic analysis of Euthyneura (Gastropoda) by means of the 16S rRNA gene: use of a 'fast' gene for 'higher-level' phylogenies. Proceedings of the Royal Society of London, Series B, 266: 75-83.

Fig. 1 The most parsimonious tree under the optimal weighting scheme for 16S and COI data for a dorid database. The numbers are bootstrap proportions.

Figure not yet available

Fig. 2 The minimum evolution tree for 16S data and euthyneuran dataset. The numbers are bootstrap proportions.

Figure not yet available

 


 

 

Join The Malacological Society of London Contact Information Mini-Reviews Bulletin Board Home