DNA
Playing the DNA game (2)
31-08-2010 22:25
Having analyzed so far
only ITS/28S data, I’m now trying to combine
different partitions from both nuclear and
mitochondrial DNA. The first step is to concatenate
ITS/28S and histone 3. So far, so good (but after
getting around some stumble blocks). Some useful
literature in this respect is Planet (2006). For
recent reviews of general methodologies, see Blair
& Murphy (2010) and Pausas & Verdú (2010).
References:
Blair, C. & Murphy, R.W., 2010. Recent trends in molecular phylogenetic analysis: where to next? - Journal of Heridity (in press; doi:10.1093/jhered/esq092).
Pausas, J.G. & Verdú, M., 2010. The jungle of methods for evaluating phenotypic and phylogenetic structure of communities. - BioScience 60: 614-625.
Planet, P.J., 2006. Tree disagreement: measuring and testing incongruence in phylogenies. - Journal of Biomedical Informatics 39: 86-102.
References:
Blair, C. & Murphy, R.W., 2010. Recent trends in molecular phylogenetic analysis: where to next? - Journal of Heridity (in press; doi:10.1093/jhered/esq092).
Pausas, J.G. & Verdú, M., 2010. The jungle of methods for evaluating phenotypic and phylogenetic structure of communities. - BioScience 60: 614-625.
Planet, P.J., 2006. Tree disagreement: measuring and testing incongruence in phylogenies. - Journal of Biomedical Informatics 39: 86-102.
Playing the DNA game
30-08-2010 21:02
This weekend I have
been busy playing the DNA game. Just imagine the good
old Tetris game, where coloured blocks have
to be sorted. Aligning DNA fragments looks very
much the same, only you have to move them
horizontally instead of vertically.
This screenshot is part of a file in which I manually tried to align three different parts of DNA (CO1, histone 3 and ITS2/28S). It serves the continuing phylogenetic research on the relationships of the Orthalicoidea.
This screenshot is part of a file in which I manually tried to align three different parts of DNA (CO1, histone 3 and ITS2/28S). It serves the continuing phylogenetic research on the relationships of the Orthalicoidea.
New phylogenetic insights
16-07-2010 08:19
Today a
new paper
will be published on
the phylogenetics of the Orthalicoidea group. The
classification of this group has been quite
unstable during recent decades, as this table
shows.
It proved to be a challenge to collect enough suitable material of species covering the whole range of the group. Special thanks to Annet Breure, Giovanni Cuno, André Delsaerdt, Jens Hemmen, Valentín Mogollón, Grace Montalván, David Robinson, Constante Schizzi, Jan Schlögl and Corey Whisson, for sending me specimens that allowed DNA studies.
As I was especially interested in the phylogenetic relationships at the deeper levels within the group, the most suitable marker was used, viz. ITS2/28S. Applying various methods the following topology was found.
Two results are worth special mentioning. First the division of what I considered the Bulimulinae in my thesis. Although most genera (Bulimulus, Drymaeus, Bostryx, Naesiotus) group together, it is remarkable that Plekocheilus shows up very basally in the tree and needs to be considered an amphibulimid.
Secondly, the clustering of Plectostylus within a clade of Gondwanan origin corroborates the recent cladistic study of Cuezzo et al. (2010). The inclusion of Discoleus within this group is shown to be correct (unpublished data).
References:
Breure, A.S.H., Groenenberg, D.S.J. & Schilthuizen, M., 2010. New insights in the phylogenetic relations within the Orthalicoidea (Gastropoda, Stylommatophora) based on 28S sequence data. - Basteria 74: 25-31.
Cuezzo, M.G., Valdovinos, C. & Breure, A.S.H., 2010. Discoleus Breure, 1978: Is there a pre-Gondwanan ancient stock of Orthalicidae? (Mollusca, Stylommatophora). Poster, VI Southern Connection congress, 14-19 February 2010, Bariloche, Argentina.
It proved to be a challenge to collect enough suitable material of species covering the whole range of the group. Special thanks to Annet Breure, Giovanni Cuno, André Delsaerdt, Jens Hemmen, Valentín Mogollón, Grace Montalván, David Robinson, Constante Schizzi, Jan Schlögl and Corey Whisson, for sending me specimens that allowed DNA studies.
As I was especially interested in the phylogenetic relationships at the deeper levels within the group, the most suitable marker was used, viz. ITS2/28S. Applying various methods the following topology was found.
Two results are worth special mentioning. First the division of what I considered the Bulimulinae in my thesis. Although most genera (Bulimulus, Drymaeus, Bostryx, Naesiotus) group together, it is remarkable that Plekocheilus shows up very basally in the tree and needs to be considered an amphibulimid.
Secondly, the clustering of Plectostylus within a clade of Gondwanan origin corroborates the recent cladistic study of Cuezzo et al. (2010). The inclusion of Discoleus within this group is shown to be correct (unpublished data).
References:
Breure, A.S.H., Groenenberg, D.S.J. & Schilthuizen, M., 2010. New insights in the phylogenetic relations within the Orthalicoidea (Gastropoda, Stylommatophora) based on 28S sequence data. - Basteria 74: 25-31.
Cuezzo, M.G., Valdovinos, C. & Breure, A.S.H., 2010. Discoleus Breure, 1978: Is there a pre-Gondwanan ancient stock of Orthalicidae? (Mollusca, Stylommatophora). Poster, VI Southern Connection congress, 14-19 February 2010, Bariloche, Argentina.
DNA: from field to lab
29-09-2009 07:16
Getting good DNA isn’t as easy as often thought. The
wet collections of museums are often not suitable,
either because samples have been fixed in formalin or
because they may have stayed in alcohol 70% for too
long. But many times, it remains unclear why one
sample doesn’t yield any DNA while seemingly similar
ones give fair to good results.
The assumption is that preservation in the field might play a crucial role.
To test this assumption we have started at Naturalis an experiment with 32 combinations of killing and conservation methods. Most of the combinations are based on practices in the field, not only from malacologists but also entomologists. We are using two species of common snails, collected in Dutch gardens.
Hopefully, this experiment will make clear which combination results in the best DNA yield, given the limitations when in the field.
The assumption is that preservation in the field might play a crucial role.
To test this assumption we have started at Naturalis an experiment with 32 combinations of killing and conservation methods. Most of the combinations are based on practices in the field, not only from malacologists but also entomologists. We are using two species of common snails, collected in Dutch gardens.
Hopefully, this experiment will make clear which combination results in the best DNA yield, given the limitations when in the field.
DNA barcoding
25-09-2009 17:10
DNA barcoding
is thought to be a method to ease species
identification. Some even think that this method may
make conventional taxonomy obsolete once techniques
like
Lab-on-a-chip
are commonly used. Funding of barcoding projects is
usually aimed at building a reference library. Any
taxonomic problem-solving is seen as a useful
by-product at most (Rougerie,
Taxacom Discussion
List).
With the above in mind, it was interesting to see a recent publication by Davison et al. (2009) on DNA barcoding in land snails. In this paper they report on a study to test the accuracy of barcoding on stylommatophoran species. As basis of the study the CO1 sequences deposited in GenBank were used; this test set represented 129 species, including a number of Neotropical species (Orthalicidae, Polygyridae). Stylommatophorans frequently show extreme divergence of mitonchondrial DNA.
The following results are noteworthy:
- barcoding correctly identified species in 92% of cases.
- optimum threshold value is 4%, but with an error rate of more than 30%; it is therefore recommended that barcoding is backed-up by phylogenetic methods and conventional taxonomy.
- intra-specific variation in stylommatophorans is genrally much higher than in other organisms.
- relatively many (> 25) samples of each species are needed if barcoding is to be effective, due to the rather extreme divergence of CO1 in land snails.
The results point to the conclusion that plenty specimens should be sampled to study the occurrence of different haplotypes in a species. When the species occurs in a broad geographical area, care should be taken to cover this area as much as possible. This may be an important point to have in the back of your mind when doing fieldwork. At the same time it may prove to be difficult to realize when sampling range-restricted or rare species. In those cases one is often glad enough to find just one or a few specimens. Any resulting barcoding should thus carefully evaluated.
Another important conclusion of this study is the recommendation that barcoding should be part of a suit of methods, including traditional morphological taxonomy, to study the intra- and interspecific variation in land snail species.
Reference:
Davison, A., Blackie, R.L.E. & Scothern, G.P., 2009. DNA barcoding of stylommatophoran land snails: a test of existing sequences. - Molecular Ecology Resources 9: 1092-1101.
With the above in mind, it was interesting to see a recent publication by Davison et al. (2009) on DNA barcoding in land snails. In this paper they report on a study to test the accuracy of barcoding on stylommatophoran species. As basis of the study the CO1 sequences deposited in GenBank were used; this test set represented 129 species, including a number of Neotropical species (Orthalicidae, Polygyridae). Stylommatophorans frequently show extreme divergence of mitonchondrial DNA.
The following results are noteworthy:
- barcoding correctly identified species in 92% of cases.
- optimum threshold value is 4%, but with an error rate of more than 30%; it is therefore recommended that barcoding is backed-up by phylogenetic methods and conventional taxonomy.
- intra-specific variation in stylommatophorans is genrally much higher than in other organisms.
- relatively many (> 25) samples of each species are needed if barcoding is to be effective, due to the rather extreme divergence of CO1 in land snails.
The results point to the conclusion that plenty specimens should be sampled to study the occurrence of different haplotypes in a species. When the species occurs in a broad geographical area, care should be taken to cover this area as much as possible. This may be an important point to have in the back of your mind when doing fieldwork. At the same time it may prove to be difficult to realize when sampling range-restricted or rare species. In those cases one is often glad enough to find just one or a few specimens. Any resulting barcoding should thus carefully evaluated.
Another important conclusion of this study is the recommendation that barcoding should be part of a suit of methods, including traditional morphological taxonomy, to study the intra- and interspecific variation in land snail species.
Reference:
Davison, A., Blackie, R.L.E. & Scothern, G.P., 2009. DNA barcoding of stylommatophoran land snails: a test of existing sequences. - Molecular Ecology Resources 9: 1092-1101.
What to do with a living snail?
30-11-2007 20:46
Phylogenetic
relationships were always one of the major drivers
behind my scientific interest in snails. Consequently
there has been a need for animals. In the '70s I have
been hunting for snails because I wanted to study
their anatomy and histology. Nowadays scientific
frontiers have shifted and so has the methodology. I
need fresh tissue to be able to extract DNA that, in
the end, will reveal phylogenetic data.
The material that I have collected more than 30 years ago does not meet modern requirements (at least, with the facilities at my disposal). The animals were drown in water for about 24 hours to let them stretch, which procedure is detrimental for the quality of DNA. Moreover, the animals have been preserved in alcohol 70%; this is, to say the least, not optimal.
So, I'm constantly on the outlook for freshly collected animals. For which I have to rely on the kindness of other people.
Provided that you want to facilitate phylogenetic research. And suppose that you are willing to go out for hunting snails. What do you do with a living snail?
Some of you might know already. If so, please skip this section. If not, read on. I have tried to be as clear and concise as possible in this short guide, distinguishing 10 steps during three phases:
A) Preparation
1. It all starts in preparing your field trip. Take -- besides your collecting material -- your notebook, a camera and (preferably) a GPS. Nothing special for anyone who takes field work serious nowadays. And have enough stock of alcohol 96%.
B) Collecting
2. When you set out to the field, take enough wrapping paper (toilet paper will do nicely) with you.
3. Encountering a living snail, not only take notes of the locality, the GPS coordinates and the ecology (e.g. vegetation, soil type), but also take pictures of the animal. Macro (the snail in its environment) and micro (the animal from various sides).
4. Wrap the animal in enough paper, such that it will retract within its shell.
5. When you return in the evening to your (home) base, put the animal directly in alcohol 96% (so do NOT drown in water first!).
C) Preparing for shipment
6. Take cotton and soak it in alcohol 96%.
7. [Extra step if you want to save on shipping costs or are dealing with large animals, e.g. Orthalicidae or large Thaumastus, Plekocheilus, etc.] Take a sharp knife (scalpel), dip it in alcohol 96% and cut off the tail end of the foot, as large as possible.
If the animal is totally retracted within its shell, please skip this step or you might mess things up.
8. Wrap the animal in the cotton soaked during step 6, put in a plastic bag and seal it.
9. Put the bag, together with the location data and the identification label, in another plastic bag; repeat at least twice or three times.
[N.B.: If you followed step 7, ensure that the animal can be identified by either including an empty shell or a label like e.g. "Scutalus sp. (number)"].
10. Make sure the result is tightly sealed and doesn't smell and put it in a suitable box for shipment. Label with "Material for scientific study only - No commercial value" and the address you may find here. Please send me an email when you dispatch, explaining what you are sending and including all relevant data (e.g. explanation of numbers).
What will it bring you? Let me ensure you that I'll be most grateful for your kind cooperation. The least I will do is to give you a honourable mention in the 'Acknowledgements' section in one of my future papers :-) But generally I'll be happy to refund you the shipping costs, unless you waive it or I can help you in other ways.
The material that I have collected more than 30 years ago does not meet modern requirements (at least, with the facilities at my disposal). The animals were drown in water for about 24 hours to let them stretch, which procedure is detrimental for the quality of DNA. Moreover, the animals have been preserved in alcohol 70%; this is, to say the least, not optimal.
So, I'm constantly on the outlook for freshly collected animals. For which I have to rely on the kindness of other people.
Provided that you want to facilitate phylogenetic research. And suppose that you are willing to go out for hunting snails. What do you do with a living snail?
Some of you might know already. If so, please skip this section. If not, read on. I have tried to be as clear and concise as possible in this short guide, distinguishing 10 steps during three phases:
A) Preparation
1. It all starts in preparing your field trip. Take -- besides your collecting material -- your notebook, a camera and (preferably) a GPS. Nothing special for anyone who takes field work serious nowadays. And have enough stock of alcohol 96%.
B) Collecting
2. When you set out to the field, take enough wrapping paper (toilet paper will do nicely) with you.
3. Encountering a living snail, not only take notes of the locality, the GPS coordinates and the ecology (e.g. vegetation, soil type), but also take pictures of the animal. Macro (the snail in its environment) and micro (the animal from various sides).
4. Wrap the animal in enough paper, such that it will retract within its shell.
5. When you return in the evening to your (home) base, put the animal directly in alcohol 96% (so do NOT drown in water first!).
C) Preparing for shipment
6. Take cotton and soak it in alcohol 96%.
7. [Extra step if you want to save on shipping costs or are dealing with large animals, e.g. Orthalicidae or large Thaumastus, Plekocheilus, etc.] Take a sharp knife (scalpel), dip it in alcohol 96% and cut off the tail end of the foot, as large as possible.
If the animal is totally retracted within its shell, please skip this step or you might mess things up.
8. Wrap the animal in the cotton soaked during step 6, put in a plastic bag and seal it.
9. Put the bag, together with the location data and the identification label, in another plastic bag; repeat at least twice or three times.
[N.B.: If you followed step 7, ensure that the animal can be identified by either including an empty shell or a label like e.g. "Scutalus sp. (number)"].
10. Make sure the result is tightly sealed and doesn't smell and put it in a suitable box for shipment. Label with "Material for scientific study only - No commercial value" and the address you may find here. Please send me an email when you dispatch, explaining what you are sending and including all relevant data (e.g. explanation of numbers).
What will it bring you? Let me ensure you that I'll be most grateful for your kind cooperation. The least I will do is to give you a honourable mention in the 'Acknowledgements' section in one of my future papers :-) But generally I'll be happy to refund you the shipping costs, unless you waive it or I can help you in other ways.
A quick update
10-02-2007 17:37
More than a month
passed since my last posting, as my "normal" work
nearly prevented me from doing anything at all on
snails. First priorities first... Still, some
malacological work could be done.
First of all I dissected the material of Simpulopsis dominicensis. It's annoying to experience how difficult dissecting a snail can be, especially if it is rather small like this one. (Relatively speaking.... my friend Edi Gittenberger would consider this a rather big one, but as it is his "normal" work he is much more experienced in it). When I had carefully laid out the genitalia I became very doubtful about the identification. Frankly, I don't know yet, but probably this taxon belongs to the family Sagdidae.
The DNA-story slowly continues... Let's say, at snail pace :-) New material has been sent to Hamburg and some discussions over previous results. If further results corroborate the analyses, the family is probably paraphyletic.
New material has been promised by several collegues and contacts abroad. Maybe some interesting times are ahead...
First of all I dissected the material of Simpulopsis dominicensis. It's annoying to experience how difficult dissecting a snail can be, especially if it is rather small like this one. (Relatively speaking.... my friend Edi Gittenberger would consider this a rather big one, but as it is his "normal" work he is much more experienced in it). When I had carefully laid out the genitalia I became very doubtful about the identification. Frankly, I don't know yet, but probably this taxon belongs to the family Sagdidae.
The DNA-story slowly continues... Let's say, at snail pace :-) New material has been sent to Hamburg and some discussions over previous results. If further results corroborate the analyses, the family is probably paraphyletic.
New material has been promised by several collegues and contacts abroad. Maybe some interesting times are ahead...