In preparing and presenting at the ICE 2016

It is always a challenge to prepare presentations for scientific meetings. You always want to do a good job in effectively communicating your research, in a way that you don't confuse or bore people, which becomes trickier when you are new to the group you are working with and even more defiant when your audience is composed by people that has been working on this for many years and are recognized experts in the field.

Thing is that I'm more interested and, so far, much more focused on morphology, which was only one of the three main themes I was supposed to talk about. I consider myself a lucky person in general. I've had all the support, data and information I've needed from my advisor, Dr. Andrew Short, our postdoc researcher, Dr. Emmanuel Toussaint and my labmates at the Short Lab. It was not easy to get smooth transitions between the molecular phylogeny that we have now, the different ecologies of the beetles and their morphological features.

In the end I think it went well. Here is the only photo from my presentation. It was taken by our Lab Technician Sarah Schmits. I didn't even made it to the water beetle people group picture... oh well...

The file with the presentation is posted at ResearchGate.

The amazing internal morphology

I've been doing full disarticulations of acidocerines, and so far, it has been fascinating!

For one thing, I wasn't sure about how to do it, but was pretty sure about what I wanted to see. I started trying to find a protocol for this, as I've seen this kind of work presented at national meetings, but maybe I didn't do a good job in my search, because I couldn't find one. And yes, I know the technique (mostly the KOH soft tissue dissolution) vary depending on several factors as the "age of preservation" of the specimen, its size and its hardness, but still I was looking for some kind of standard that I didn't find... at least not like that.

What I did found were several revisionary works on hydrophilids that include detailed morphology for the group in question. My favorite so far, in terms of the description of the methods, is by Minoshima, Komarek & Ôhara (2015) for the genus Megagraphydrus (currently Agraphydrus, which they synonymize in this work). Regarding morphological treatment and nomenclature of the head and its appendages, my favorite is Anton & Beutel for the genus Helophorus. Other useful resources are the revision of Afrotropical Anacaena by Komarek 2004 and the nice pictures provided by Fikáček & Vondráček (2014) on their revision of Pseudorygmodus.

My previous experiences with dissections were on broad-nosed weevils, of minimally 4mm which are harder (thicker cuticle) than the acidocerines, and my procedures involved nearly-boiling water and a quick "cooking" of about 10 minutes. The same treatment with water beetles can result in a disaster of two kinds: one, you overcook them in which case dissection is a very delicate and risky process where you end up tearing sclerites that you were not supposed to, or two, you undercook them, and then dissection is difficult and messy because there is soft tissue everywhere and you end up not being able to clearly see the structures under the microscope anyway. For my first experiments I used non-target specimens (I mean hydrophilids other than acidocerines from which we had a bunch sitting on vials on alcohol).

Then I made a patience commitment accompanied of deep breaths and performed the procedures as described in Minoshima et al (2015), which involve a temperature of around 60°C during about one hour. At the beginning I was afraid of overcooking but when I was able to relax and let it be, the results were quite good! I even tweeted it in excitement!!

I also have had to modify procedures, so now I separate head, prothorax and abdomen before cooking the specimen, also open the abdomen on one side (when possible), so the KOH goes through all the abdominal grease and tissues, and now I try to not cook the wings (something I should have done before... oh well).

So far I have 10 species fully dissected (one specimen each). There are interesting characters everywhere!: mouthparts (mandibles are particularly awesome!), metafurca, abdominal tergites and sternites, wings. And then of course, it helps a lot when you have a good microscope available to take a closer look!

For now, I will leave you with this mosaic of structures of Helochares abbreviatus. [structures not necessarily to scale regarding each other, images not edited -only cropped to fit slide-]

Helochares abbreviatus: 1. Labium; 2. Labrum; 3. Maxilla; 4. Mandible; 5. Antenna; 6. Prosternal process; 7. Abdominal tergites I-III; 8. Abdominal tergites V-VIII; 9. Abdominal sternite VII; 10. Abdominal sternite VIII; 11. Scutellum; 12. Metafurca; 13. Abdominal sternite IX; 14. Abdominal tergite IX.

Disarticulation allows you to see structures that are usually overlooked because you cannot see them without breaking the specimen, such as the prosternal process for example. And then of course, as with some specimens you can't identify the sex, "by accident" you dissect a female and also find a bunch of structures that apparently not many hydrophilid workers take a look at. Again, this is only starting! the tricky next part is to start coding these things.

References

• Anton, E., & Beutel, R. G. (2004). On the head morphology and systematic position of Helophorus (Coleoptera: Hydrophiloidea: Helophoridae). Zoologischer Anzeiger-A Journal of Comparative Zoology, 242(4), 313-346.

• Fikáček, M., & Vondráček, D. (2014). A review of Pseudorygmodus (Coleoptera: Hydrophilidae), with notes on the classification of the Anacaenini and on distribution of genera endemic to southern South America. Acta Entomologica Musei Nationalis Pragae, 54(2), 479-514.

• Komarek, A. Taxonomic revision of Anacaena Thomson, 1859 I. Afrotropical species. Koleopterologische Rundschau, 74, 303-349.

• Minoshima, Y. N., Komarek, A., & Ôhara, M. (2014). A revision of Megagraphydrus Hansen (Coleoptera, Hydrophilidae): synonymization with Agraphydrus Regimbart and description of seven new species. Zootaxa, 3930(1), 1-63.

A take on Quadriops

If there is one thing clear to me now regarding Neotropical acidocerines is that, for the most part, genera are fairly easy to recognize by their external morphology, but when it comes to species the challenge can get a lot harder.

Here I am, starting a new revision (a third one, with the previous ones in standby for different reasons) this time Quadriops, which is an exclusively Neotropical genus described in 1999 by Hansen. It has six described species:

Quadriops amazonensis García, 2000: 59 (Venezuela: Amazonas)

Quadriops dentatus Hansen, 1999: 134 (Venezuela: Bolívar)

Quadriops depressus Hansen, 1999: 136 (Perú: Loreto; Ecuador: Napo)

Quadriops politus Hansen, 1999: 135 (Perú: Loreto)

Quadriops reticulatus Hansen, 1999: 135 (Costa Rica: Puntarenas; Panama: Chiriquí)

Quadriops similaris Hansen, 1999: 136 (British Guiana: Ikuribisi; Venezuela: Bolívar)

Hansen offers a key to his species (translated to Spanish by García 2000). There is one species (Q. dentatus) in which the elytral punctures are not aligned in striae, all the others have aligned punctures (see image below).

The characters used by Hansen to separate the species with aligned punctures are mostly related to the presence or absence of shallow reticulation of the frons, clypeus and pronotum.

For me at this point, with the specimens I have seen, I would dare to say that all of the species with aligned punctures might be only one widespread species, given that:

- The species described so far are based on only a few specimens (usually one or two; seven at most)

- The males are known for four of the species and the differences on the illustrations for three of them are only slight.

Of course more observations are needed, also because I think differences on the shallow reticulations could be attributed to development for example, and not necessarily reflect interspecific differences.

References

• García, M. (2000). Una nueva especie de Quadriops Hansen, 1999 (Coleoptera: Hydrophilidae: Hydrophilinae) de Venezuela. Boletín del Centro de Investigaciones Biológicas, 34(1).

• Hansen, M. (1999). Fifteen new genera of Hydrophilidae (Coleoptera), with remarks on the generic classification of the family. Insect Systematics & Evolution, 30(2), 121-172.

More characters!!

So I've been keeping coding "new" morphological traits and including more taxa in the matrix. I stopped with the Chasmogenus species at sp. 9, so there are 12 Chasmogenus species included in the matrix so far. I've also included related genera, one species each: Agraphydrus, Globulosis, Quadriops, Radicidus, Tobochares and three undescribed genera, as well as one species of Cymbiodyta and one species of Sphaeridium, which as expected (for it being the only truly terrestrial included so far) is strikingly different.
As before, new characters and character states appeared, and some characters had to be split, so the matrix is now 230 characters and 28 taxa.
Here a small sample of variation on characters of the hind legs (and also an spoiler of my poster for the Evolution meeting 2016). You can see variation in the proportions, the size of the spines and area covered by hydrofuge pubescence. Images not at scale.

I also started my first full disarticulations to look at mouthparts, thorax (mesofurca, metafurca, tergum), wings, abdominal segments (tergites, laterosternites, terminal segments), male and female genitalia... thing is that the available specimens for that at this moment belong to the Hydrophilinae: Tropisternus, Berosus, Hydrophilus and Oocyclus. So far, having dedicated more time to careful fragmentation rather than to detailed observation (as I don't have a microscope yet), I have already seen interesting features that might be useful for phylogenetic purposes.
Here also just a sample (with not as pretty pictures, not at scale either) of the mandibles, showing very prominent differences, where the mandibles of Tropisternus are apparently the more common condition (at least for Hydrophilines); mandibles were illustrated by Hansen (1991, figs. 167-170), but it is astounding to see them in real life. It is the first time that I see brushes of setae on mandibles!!

This is only the tip of the iceberg, just a quick preview of many more structures to explore, to see, to compare, to code... this is still interesting!!


References

• Hansen, M. (1991). The hydrophiloid beetles. Phylogeny, classification and a revision of the genera (Coleoptera: Hydrophilidae). Biologiske Skrifter, 40, 1–367.

What about the molecules?

It is very likely that every person that has asked me about my research has come to a comment like "yes, I love beetles and morphology but I don't really like molecules... and I know I will have to deal with them at some point"

My experience with molecular data is very limited at this point in time. I have performed maybe one or two extractions and maybe just one full protocol composed of DNA extraction, electrophoresis and PCR, without ever knowing the final product, just for the technique and it was maybe in 2009 during my Master's at the Biology Department at UPRM, and then, when I joined the Short Lab in August 2015, I knew that working with molecules was going to happen!

The lab has been working actively with molecular data, so by December 2015 a full set of amplified samples were sent for sequencing, and by mid January this year it was my duty to take a look at the sequences and edit them when necessary in order to produce clean alignments. All the visualizations, edits and alignments were done using Geneious R-8, which I consider a friendly and efficient tool.

The process (which may be obvious for those familiar with this kind of data) starts with files to be imported into the Program. At this time we had for each gene of each specimen, two sequences: forward and reverse. Those two sequences needed to be matched to get one Assembly for each specimen. On the Assembly (composed of two strands) it is necessary to check for ambiguities, for example when one of the strands says A, but the other says G, and make a decision (based on the height of the peak and the probability on each strand) to call one chain as G or A to match the other one. You can check the image below to see what I'm trying to explain. For this edition process, we created files with a track of all the modifications made in the sequences, in case it is necessary to check for errors some steps ahead.

Screenshot of Geneious R-8: Edit of COI sequence.

After you get your editing done (and documented), it is time to convert the file into a Consensus Sequence, which contains only one strand, longer than either of the previous file, as it contains the forward and reverse tails of the amplified fragment. You need to do that for all of the specimens. Once you have all the edited Consensus sequences, you can align them (see image below) and ask the Program to highlight the base pairs that differ among sequences. You need to watch out for sequences that might be reversed, which causes them not to be properly aligned.

Screenshot of Geneious R-8: Edit of COI sequence.

At this stage you check for gaps and 'misplaced' bases, which strongly depend on which gene are you working with. Then you might return to the Assemblies in order to see if the gaps are real and if the odd bases are the product of one of the previously resolved ambiguities (here is where the notes come in handy). Finally, you can ask the Program to produce a tree with selected Consensus sequences (see image below).

Screenshot of Geneious R-8: Raw COI tree.

As far as I can tell, Geneious would produce a raw result that allows visualization. It also allows to run sequences through BLAST when you suspect contamination or you amplified something else by accident. I still don't know if you can run formal analyses and models of molecular evolution in the program... I haven't got there yet.

One of the 'extra' lessons learned from this process is that consistency (apply the same rules to everything), order and documentation are fundamental for an efficient development of this kind of work.

Morphological character's matrix

During this past couple of months most of my time at the lab has been dedicated to build a matrix of morphological characters in order to analyze the Acidocerinae. The focal group at this time the genus Chasmogenus which with 40 described species is a little bit of a mess itself.

At some point Chasmogenus was considered a subgenus of Helochares, and according to Hebauer (1992), the species of Chasmogenus can be separated in two groups (subgenera): Chasmogenus  sensu stricto (composed of the American species with 8 antennomeres and simple aedeagus) and the Chasmogenus Crephelochares (composed by the non American species with 9 antennomeres and "more differentiated" aedeagus). Regarding distributions, 17 species are Afrotropical, 5 Oriental, 3 Palaearctic and 15 Neotropical (see Hansen 1999). Hebauer also mentions affinities of Chasmogenus with Enochrus, which at this time is placed in a separate subfamily (see Short & Fikáček 2013).

As it happened with Helobata, the external morphology of Chasmogenus is very homogeneous, but not as extreme, so I started my matrix with the characters used by Short (2007) on the second chapter of his Dissertation: "Systematics and biology of the endemic water scavenger beetles of Hawaii (Coleoptera: Hydrophilidae)", which contains 55 characters, most of them associated with external morphology, knowing that it is very likely that not all the characters might be informative for my group. I also included characters from Hebauer (1992) and Clarkson & Ferreira (2014).

Then I selected specimens in order to start coding them on the matrix. The group of specimens at this time includes one species of Enochrus, Helobata striata, some Helochares, Chasmogenus abnormalis from Okinawa (Japan), Chasmogenus nitescens from New Caledonia, Chasmogenus ruidus from Costa Rica, and 26 unidentified Chasmogenus specimens (very likely different species, most of them undescribed) from Nicaragua, Costa Rica, Panama, Ecuador, Venezuela, Guiana, Suriname, French Guiana, Bolivia and Paraguay.

When I started looking at the specimens to code them, and as it usually happens while doing this, more characters and character states started to appear. Then I had to go back to the very useful drawings presented by Hansen (1991) and his list of characters, and at the same time get used to the terminology employed by some authors (e.g. mesoventrite to refer to the mesosternum). I have been also producing my own drawings in order to help me understand the variations and keep track of what I saw. Here are some of my schemes.

   

As of now, the matrix has 167 characters (86 binary). I know several are not going to be informative (either because all of the specimens have the same character state, or because they doesn't exhibit a clear signal) but also as soon as I start dissecting (mouthparts, wings, abdomen, male and female genitalia), many more characters are going to be added. I can't wait for that to happen!. Here is a screenshot of the matrix as it is now.

For now I need to keep coding the remainder taxa, as you can see I have only 3 out of the 26 specimens I selected, in part because every time I'm coding one specimen, new character states appear and I have to go back through all the other specimens that are already in the matrix in order to figure out the best way to code the character states, but at this time the matrix it's getting more stable.

References

• Clarkson, B., & Ferreira, N. (2014). Four new species and first nominal record of Chasmogenus Sharp, 1882 (Coleoptera: Hydrophilidae) from Brazil. Zootaxa, 3765(5), 481-494.
• Hansen, M. (1991). The hydrophiloid beetles. Phylogeny, classification and a revision of the genera (Coleoptera: Hydrophilidae). Biologiske Skrifter, 40, 1–367.
• Hansen, M. (1999). World catalogue of insects. Volume 2: Hydrophiloidea (s. str.)(Coleoptera). Apollo Books.
• Hebauer, F. (1992). The species of the genus Chasmogenus Sharp, 1882 (Coleoptera: Hydrophilidae). Acta Coleopterologica, 8(2), 61-92.
• Short, A. E. Z., & Fikáček, M. (2013). Molecular phylogeny, evolution and classification of the Hydrophilidae (Coleoptera). Systematic Entomology, 38(4), 723-752.
• Short, A. E. Z. (2007) Phylogeny, morphology, and biology of the hydrobiusine and hydrophiline water scavenger beetles (Coleoptera: Hydrophilidae: Hydrophilini). Ph.D. Dissertation, Cornell University. 219 pp.

EntSoc2015

Last week The Short Lab attended the Annual meeting of the Entomological Society of America. All members presented advances on their research, so I presented the information that is being compiled in this blog, along with other data regarding the intriguing genus Helobata. You can access the presentation via Research Gate or Academia.edu.