DNA Fingerprinting of Mosses

[timebomb]

Hi, folks,

Chishio Hidaka, a Japanese girl pursuing her master's degree in the National University of Singapore sent me this email yesterday.

Dear Mr. Loh Kwek Leong,

Hello, this is Chishio Hidaka.
Thank you for visiting our lab the last time.

Since you know many people among aquarium hobbyists, I have a favour to ask of you. I am doing my master's research on aquarium mosses.
As you already know, the aquarium moss market is confused because of difficulties in identifying these moss species from one another. My research purpose is to distinguish these moss species with a method called AFLP, a DNA fingerprinting method, to make the identification more convincing.

To do this project, I need 10 samples each of the following mosses.
Java moss - Taxiphyllum barbieri
Weeping moss - Vesicularia ferriei
Erect moss - Vesicularia reticulata
Christmas moss - Vesicularia montagnei
Singapore moss - Vesicularia dubyana

If possible, I would like each sample from a different colony because I need to collect as many patterns of DNA as possible in each species.
I am going to try these 5 species first to make sure it works this way.
Will you help me get these samples?

Thank you.

Regards,
Chishio Hidaka


By different colony, she meant different population. If conditions were ideal, it would mean she has to go around collecting the moss from their natural habitats in 10 different places. But we have to be realistic. With almost all the mosses on her list, we don't even know where they can be found. But we know many of you grow them in your tanks. Although it could be highly possible that most of your mosses were from my tank in the first place, it would still count as a different population if they had been in your tank for a while. After all, I only gave you a little at the start and you grew the rest of it, didn't you? So please help. She needs 10 samples of each. She does not need a lot of each sample. Just what would fit into a small size plastic bag will do. Here's a picture:



Chishio is a good-looking young girl so it's dangerous for someone like her to give her address out to strangers. Yours truly, however, is an old man with little to lose so please send the mosses to me. Here's the address:

Loh Kwek Leong
Block 104 Towner Road
#08-324
Singapore 322104

I've spoken to Chishio and she is in agreement that with Vesicularia dubyana, she should look for it herself in their natural habitats in Singapore. But we need a lot of your help with the other mosses, Erect and Christmas Moss in particular. Over the years, I've sent out hundreds of envelopes of these 2 mosses. It's ironical that they are now hard to find in the local aquaria scene. With the former, they have all turned brown and died. With the latter, they have all gotten mixed up with Singapore Moss. So please, if you have these mosses in your tanks, I will be very grateful if you would send a small sample to me.

In return, I'll be happy to send you something yuo don't already have. I can send you Flame Moss, the Fern gametophyte, Fountain Moss (Fissidens fontanus), Zipper Moss (Fissidens zippelianus) or the moss tentatively known as Mini-Java. Bet you don't have this one yet . Here's a picture:



And oh, here's a picture of Chishio working at the lab. I persuaded her to let me put up her picture. I told her that in order to get a better response, it's always better to be sincere and forthcoming. After all, it's rude to be anonymous when one is asking for a favour. Right?

You will help her, won't you?



Loh K L

[ExpelledBAN]

one person doesnt need to send 10 at once right?

the total, has to be ten?

i think i could help with weeping and java, or taiwan

[timebomb]

Chishio needs at least 10 samples of each moss but of course, we don't expect one person to send all 10 samples.

Send whatever you have but please do not send Taiwan Moss as this moss isn't on her list. Do not worry if you're not sure of the identities. Chishio will sort them out before doing the DNA fingerprinting.

Loh K L

[imported_reinaldo]

Mr. Loh,

I can send you samples of all the refered mosses, less Singapore moss... also can send you some Londrina`s moss and another collected moss, here in Brazil, with round "leaves" if you want to...


Please, send me a PM, if you`re interested.

Best Regards, from Brazil...

Reinaldo

[Robert]

Hi Mr. Loh,
I asked around a bit. I asked in Germany but also in Brazil. See the reply above.

To help you even a bit more I opened a new topic in my own forum. You can find it here: http://www.shrimpnow.com/forums/showthread.php?p=18021. Hopefully it will generate a bit more interest in this topic.

You will receive my samples in a forthnight or so because I'm in my holidays in the meantime. But I will continue to look for more samples.

Kind regards

Robert

[hii]

I have all the 4 moss except Erect moss. Will send you some tomorrow..

[timebomb]

I have all the 4 moss except Erect moss. Will send you some tomorrow..

Thank you very much, Steven. Let me know if there's any moss you like and I'll try my best to get it for you.

I can send you samples of all the refered mosses, less Singapore moss... also can send you some Londrina`s moss and another collected moss, here in Brazil, with round "leaves" if you want to...

I'm afraid the *other mosses* won't be of any use to Chishio, Reinaldo. If you can, please send only the ones on her list. It's a long way from Brazil but should you need any moss from Singapore, let me know and I'll send it to you.

To help you even a bit more I opened a new topic in my own forum.

Thanks, Robert. You have been very kind. Besides you, several other hobbyists in other European countries are also helping me to round up the mosses for Chishio. A lady who lives in Finland even wrote to Tropica to ask them for help. And Tropica has agreed to send 3 of the mosses on Chishio's list to me. Hobbyists who live in Italy and Poland have also offered to help.

However, I still need a lot of help with Christmas and Erect Moss. For these 2 mosses, I have to rely on the hobbyists who live in other countries. Because here in Singapore, these 2 mosses have virtually disappeared from the aquaria scene. I thought I would get a good response from the Americans on the APD mailing list but so far, things aren't looking good. Only one of them has the Christmas Moss and he isn't even sure it's the genuine McCoy.

Loh K L

[imported_reinaldo]

Mr Loh,

Christmass moss is kind of common here, so i can send a good quantity of it... but i need an adress to send them...

Java moss and Weeping moss i also can send good quantities, too... erect moss is not that common, but i still have some, i think...

I'm afraid the *other mosses* won't be of any use to Chishio, Reinaldo. If you can, please send only the ones on her list. It's a long way from Brazil but should you need any moss from Singapore, let me know and I'll send it to you.

The other mosses i told you were not for Ms. Chishio, but maybe they can interest you and it will be usefull to me id these mosses...

From the list that you wrote that you have, i have none... All the Fissidens are kind of dream for brazilian enthusiasts...

I will pack the mosses ASAP and send you the required mosses and 3 new species (at least for us) of aquatic mosses.

Thank you so much.
Best regards.

Reinaldo

[timebomb]

Reinaldo,

Here's my address:

Loh Kwek Leong
Block 104 Towner Road
#08-324
Singapore 322104

Please don't forget to label the mosses when you send them. For the mosses that you wanted ID'ied, it's important that you let me know where you collected them. If you're looking for Fissidens(dream no more ), I can get the Fountain Moss (Fissidens fontanus) and Zipper Moss (Fissidens zippelianus) to you. Please let me have your mailing address through PM.

Loh K L

[fish newb]

Hey Loh, Been busy with school so kinda haven't been around (school sucks in America at least...)

I just sent a Huge portion of X-mass moss out to someone you should of told me you needed some!! Well I'll post this on TPT and get some moss hopefully. I think I have some Taiwan moss, that's about it on her list they're all a bit too common for me. I also have a bit of java but not nearly enough for what she's trying to do. (How much is the MINIMUM?)

Those Fissidens would be a dream. I just collected some new Willow mosses this past weekend Maybe a trade coming soon?

-Andrew

[fish newb]

Posted it on TPT, Pictures don't seem to be working though...

-Andrew

PS, I'll probably have a package for you being sent out the 20th give or take a day. Some moss for ID and some for this..

[timebomb]

Okay, Andrew, dream no more

I'll send the 2 species of Fissidens to you, since you're such a helpful chap. Thanks for posting the message to "The Planted Tank" forum but you're probably not aware that I've already done that earlier. The response wasn't that great. Tom Barr was the only one who promised to send some mosses.

Loh K L

[fish newb]

Okay, Andrew, dream no more

I'll send the 2 species of Fissidens to you, since you're such a helpful chap. Thanks for posting the message to "The Planted Tank" forum but you're probably not aware that I've already done that earlier. The response wasn't that great. Tom Barr was the only one who promised to send some mosses.

Loh K L

Yeah TPT isn't too big into moss. And no I wasn't aware. I got really really busy with school in the past months. Just got too tired to go check, even skipped on some homework Ohh well school is almost out.

I might have a little moss from people the response wasn't great but I'll pm a few. Tom doesn't surprise me though, he's got his masters in aquatic botany or something similar, microbiology maybe? Can't remember, but he is also very into mosses he has a newly collected red liverwort that he is debating weather to release it to the hobby (very invasive he says) I'm still trying to get some out of him

I can't remember if I sent you a PM or anything but I'm planning on sending some mosses the 20th of June the day after the official schools out day for me. Some for ID and some for this.

Also, How hard is this? Would it be remotely possible for me to try it? What is required? We have a "Senior Project" and I'm only a Freshman** this year but I assume if remotely possible it would take a lot of research to do this so I'd want to start early. And heck why do only 5 mosses? There are plenty to play with

So Its good to be back over here!
-Andrew

**(9th grader opposed to Sr. 12th grader, not sure if its different in Singapore, assume it isn't though.)

[timebomb]

Also, How hard is this?

I don't know but I think it's a lot harder than you think. Don't forget Chishio is doing this as her thesis for a master's degree in botany. There's also the problem with equipment. As far as I know, the equipment used for extracting DNA is in heavy demand at the university. I've heard that sometimes Chishio has to wait until the wee hours of the morning (that would be way past your bedtime, Andrew) before she gets her turn at the equipment.

I sat through a presentation by Chishio on her project and honestly, half the time I didn't understand what she was talking about. The title of her thesis is "Phylogenic analysis of tropical aquarium mosses genus Taxiphyllum and Vesicularia." I don't know what's "Phylogenic". It sounds like a big word to me. She also mentioned terms like AFLP etc which I think refers to a certain method of extracting DNA but I really don't have a clue. She showed us some diagrams which were supposed to be the DNA profiles of Java Moss but they looked like graphs to me.

The reason Chishio is focusing on only 5 mosses is because they are the ones we have positively identified. There would be little point in extracting the DNA profile of say, Spiky Moss, a moss that we know only the genus but not the species.

Loh K L

[ed seeley]

'Phylogenetic' refers to how closely gentically related organisms are.

My final year project was 'The molecular systematics of African Cichlidae'! I sequenced a section of the D-loop of the mitochondrial DNA of some Tanganyikan cichlids.

AFLP (Amplified Fragment Length Polymorphism) is a form of Polymerase Chain Reaction (PCR) that is a great way to copy up a section of DNA. I used PCR in my project. I was lucky that our PCR machines weren't too heavily used when I was there and I could set mine going, go and have a coffee and then come back after lunch to analyse the results. Life was hard!!!!

Of course it never worked properly the first time and some of the samples wouldn't give any usable DNA as the fish had degraded too much after death (I was getting samples that had died during shipping). I hope Chishio has better luck than me!

The only moss that I have is Java moss (Taxiphyllum, that is!) and I could send some if she wanted some 'British' samples!

[fish newb]

Also, How hard is this?

I don't know but I think it's a lot harder than you think. Don't forget Chishio is doing this as her thesis for a master's degree in botany. There's also the problem with equipment. As far as I know, the equipment used for extracting DNA is in heavy demand at the university. I've heard that sometimes Chishio has to wait until the wee hours of the morning (that would be way past your bedtime, Andrew) before she gets her turn at the equipment.

I sat through a presentation by Chishio on her project and honestly, half the time I didn't understand what she was talking about. The title of her thesis is "Phylogenic analysis of tropical aquarium mosses genus Taxiphyllum and Vesicularia." I don't know what's "Phylogenic". It sounds like a big word to me. She also mentioned terms like AFLP etc which I think refers to a certain method of extracting DNA but I really don't have a clue. She showed us some diagrams which were supposed to be the DNA profiles of Java Moss but they looked like graphs to me.

The reason Chishio is focusing on only 5 mosses is because they are the ones we have positively identified. There would be little point in extracting the DNA profile of say, Spiky Moss, a moss that we know only the genus but not the species.

Loh K L

Well I doubt it's harder than I think, because to me this is probably the hardest thing I would ever attempt before college IF its even possible for me to comprehend half the stuff and get lab time at a university (and being in highschool might pose a LARGE problem, but I've got Some connections...)

Well just think, If I could do masters level botany work while still in highschool... But yeah, I understand your point completely. AND to top it off, my school offers no botany classes whatsoever as far as I can search, I think they may of been cut because I remember hearing about one from a guy in boyscouts when I was much younger and how challenging and hard this class was and how good the teacher was. So I'm probably going to have to search for out of school classes somewhere

As for the last point, It doesn't make sense to me. The only answer to that would probably be she's doing it the way she is because of HER topic. Because If my knowledge is correct DNA would actually help us to ID mosses if we can get them. for example get the DNA of Spikey and Peacock and see if there is any difference. Now to do that you would need a large sample size because if they are different some people could have the two mosses but have the opposite name on them. But I have no idea how close the DNA is on these mosses we're so interested in.

It would be interesting to be able to ask Chishio more about this project, maybe you should get an account here and explain some about this project.

As for bedtime, Sleep just wastes time you can physically go 48hrs w.out sleep, I've done it before

'Phylogenetic' refers to how closely gentically related organisms are.

My final year project was 'The molecular systematics of African Cichlidae'! I sequenced a section of the D-loop of the mitochondrial DNA of some Tanganyikan cichlids.

AFLP (Amplified Fragment Length Polymorphism) is a form of Polymerase Chain Reaction (PCR) that is a great way to copy up a section of DNA. I used PCR in my project. I was lucky that our PCR machines weren't too heavily used when I was there and I could set mine going, go and have a coffee and then come back after lunch to analyse the results. Life was hard!!!!

Of course it never worked properly the first time and some of the samples wouldn't give any usable DNA as the fish had degraded too much after death (I was getting samples that had died during shipping). I hope Chishio has better luck than me!

The only moss that I have is Java moss (Taxiphyllum, that is!) and I could send some if she wanted some 'British' samples!

Hey Ed, neat project. I'm asuming this was for a masters as well? What do you think the chances of someone my age being able to comprehend all this and on top of it doing some themselves?

Yeah, I'm crazy

Now back to homework, I figured there wouldn't be much to respond to here... lol

-Andrew

[timebomb]

Thanks for the explanation, Ed. I thought about asking you what's "mitochondrial" but thought the better of it. Heck, you'll probably fire off another string of big words

But seriously, I would love to have a sample of your Java Moss. We probably will receive more samples of Java Moss than we need for Chishio's project but I intend to do a small project on the side. I want to find out which is Java Moss to the hobbyists in various countries. I think you're the only one in this forum who lives in England, Ed. So your sample is important to my project.

Loh K L

[timebomb]

Because If my knowledge is correct DNA would actually help us to ID mosses if we can get them.

You're forgetting that there has to be a database first before you can compare anything. The DNA fingerprint itself won't tell us what species is a moss, not unless there's a record of DNA fingerprints of mosses somewhere.
I'm not sure of this but Chishio's project could be the first attempt at building such a database.

If we compare the DNA samples of Spiky and Peacock Moss, the results will only tell us if they are the same moss or if they are different. It still won't tell us what moss species they belong to.

Loh K L

[ed seeley]

Andrew,
It's not that hard I think! Anything you'd like to know I'll certainly be happy to answer if I can, just ask!
As to doing it, once you've got access to the kit and some decent smaples, it's not too tricky! My project was in my third year for my BSc.

As for the last point, It doesn't make sense to me. The only answer to that would probably be she's doing it the way she is because of HER topic. Because If my knowledge is correct DNA would actually help us to ID mosses if we can get them. for example get the DNA of Spikey and Peacock and see if there is any difference. Now to do that you would need a large sample size because if they are different some people could have the two mosses but have the opposite name on them. But I have no idea how close the DNA is on these mosses we're so interested in.

It would be very difficult for anyone to tell what species something was purely by DNA, unless you had a known reference sample to compare it with, and even then this would not be guaranteed.

All most DNA analysis is really useful for is comparing one organism's DNA with another and looking for mutations. The number of differences in mutations correlates to the amount of time the two organisms (and by extension their populations) haven't been interbreeding.
You could then say that Spiky moss for example was similar to Peacock moss, but you would need a wide range of samples to see if they were monophyletic and could then possibly be one species. If all the spiky moss samples were more closely related to each other than any other species you could then say that they constitute one group, but other studies would be needed to say it was distinct enough to be a new species or subspecies etc.

Also a good number of species are more genetically diverse within their one species than a whole group of other species. This can happen because phylogenetic relationships are only a small part of determining what makes a species. Morphology (actual structural things) is far more important.

Thanks for the explanation, Ed. I thought about asking you what's "mitochondrial" but thought the better of it. Heck, you'll probably fire off another string of big words

But seriously, I would love to have a sample of your Java Moss. We probably will receive more samples of Java Moss than we need for Chishio's project but I intend to do a small project on the side. I want to find out which is Java Moss to the hobbyists in various countries. I think you're the only one in this forum who lives in England, Ed. So your sample is important to my project.

Loh K L

Sorry Loh, but I can't resist mate!

Mitochondria are small parts of each cell (that evolved from symbiotic bacteria!) that are within each cell of your body. They are involved in producing energy for the cell. As they evolved from bacteria they have a small, circular genome that is totally independant from the DNA in the nucleus. Mitochondria are passed on by your mother, via the egg, and it's good way of checking evolutionary relationships. They also mutate at a slower rate so this helps some evolution studies too.

I will definitely sort out a nicely sized sample for you and then Chishio can use some too if she likes! Pm me your address this weekend and i'll sort it to go off on Monday!

[timebomb]

Thanks, Ed. I thought I've plastered my address all over the world wide web but if you missed it, here it is again:

Loh Kwek Leong
Block 104 Towner Road
#08-324
Singapore 322104

I'm almost all out on the 2 species of Fissidens as many hobbyists have been asking for them but if you like, I can send you some Spiky Moss. It's one of the loveliest of the aquarium mosses around. But somehow or other, with new mosses appearing in the market regularly, the Spiky does not seem to get the recognition it deserves. Just say the word, Ed and it's yours.

Loh K L