Sunday, April 27, 2014

Genetic Barcoding Reveals Another Species!

Parchicola "black tibia", feeding on Passiflora vitifolia
It's been a long time since my last posting - partly because I've been too busy!  Now I am home on Palomar Mountain and have time to reflect.  This posting will be wordy and not so photogenic (sorry!) but it's what I want to report.

The two groups shown here are "cryptic species"
I recently received printouts from "genetic barcoding" over 100 specimens of La Selva flea beetles.  Genetic barcoding is an analytical technique that compares a specific genetic region (in this case the Cytochrome Oxidase 1 gene) in different animals.  Members of the same species have a nearly identical sequence of base pairs (the "letters" of the genetic code), while members of different species differ substantially.  The analysis can be automated and streamlined to the point where large numbers of samples can be processed at low cost, and the technique is becoming widespread among field biologists interested in biological diversity. It is also used to identify insect larvae, which otherwise must be reared to the adult stage to determine their identity.

Carlos Garcia-Robledo of the Smithsonian Institution in Washington D.C. is carrying out a project at La Selva looking at beetle diversity along elevation gradients.  Genetic barcoding is an essential component of his study.  In his project and others, there are many instances of "cryptic species", where barcoding has revealed hidden species differences even though the animals in question are superficially the same.  Carlos kindly invited me to submit my Passiflora-feeding flea beetles for analysis using his high throughput system.  All I would have to do is to prepare the sample plates and create a spreadsheet of attribute data for the samples.  Barcoding would be useful for my project, since my species have not been formally described and there is much room for error in my assignment of temporary (morphospecies) names.  Also, I have not been successful in rearing flea beetle larvae to adulthood for many species, leaving larval identity in doubt. 

Black-legged (DF1) and yellow-legged (DF2) Parchicola
To carry out this analysis, I preserved 136 specimens in alcohol to represent the range of Passiflora feeding flea beetles at La Selva, including 10-20 individuals of most of the species.  Only the Red-black Monomacra chontalensis (0 specimens), the Ptocadica "yellow" (2 specimens) and Disonycha quinquelineata (0 specimens) were severely under-represented.  Adult beetles and larvae were collected in the field and recorded in my field notes.  Then back in the lab I froze the beetles to humanely kill them, and put them in vials of 95% ethanol.  The vials were carefully labelled, including a tiny label written in pencil placed in the vial with the specimens.  I kept these samples in the freezer.  Before leaving Costa Rica I obtained a legally required export permit to take the samples out of the country.  Then, back in California, I carefully removed a leg (using sharp forceps and tiny scissors) and placed it in a numbered sample well on a 96-well plate, keeping track of each well and the field origin of each leg.  I also kept the rest of each specimen in an individually labeled sample vial for later reevaluation or examination.  I sent the completed 96-well plates to Carlos and he ran them through his barcoding protocol.  He sent me the results a few days later.

Of the 136 samples representing 7 species, 109 gave successful, uncontaminated sequences (80%).  Six of the seven species fell into 6 clearly defined groups, indicating that they are unique, distinguishable species (as suggested by their morphology and behavior).  The seventh species, Parchicola DF1 ("black-legged yellow") included two totally distinct groups that differ at the same level as the other 6 species.  We had found a cryptic species! We also found that the 3 flea beetle larvae found eating Passiflora lobata belonged to Ptocadica "red", and the 4 similar-looking larvae found on P. auriculata were Ptocadica bifasciata.  This verified the identity of the larvae used in feeding trials in 2013, and leads us to believe that Ptocadica bifasciata is somewhat specialized to feed on P. auriculata as opposed to P. biflora.  The barcoding results also verified that Ptocadica "yellow" is a distinct species from Ptocadica "red", an important distinction since the two species appear identical in dried museum specimens.

Splitting Parchicola DF1, as required by the barcode finding, potentially creates many problems for my study.  If the difference is truly cryptic, such that the species can't be separated without genetic analysis, then it becomes necessary to collect every specimen and subject it to the barcoding process.  And what about all the previous data based on field observations and/or dried specimens?  How do you present and analyze the results?  I have faced this problem twice before, always with the yellow flea beetles.  In my original survey in 1975 I collected “yellow flea beetles” and pinned them in a collection.  When I showed my collection to taxonomists at the US National Museum they showed me that I had collected two species of yellow flea beetle that they called Strabala sp. and Monomacra sp.  Because I kept specimens I was then able to work backwards and rework my data with the new information.  Years later, taking up the flea beetle project again, I took my collection to David Furth, also of the US National Museum.  He showed me that the beetles I was calling yellow Monomacra actually belonged to two species which he had given provisional names of Parchicola  DF1 and DF2, one with yellow legs and other with black legs.  I again reworked my data based on the pinned specimens’ leg color.  However, some of the data did not have associated pinned specimens, and could not be reworked.  I handled this in my data set by setting those observations aside.  Now, with barcoding revealing yet another hidden species, I had the same problem again.

I decided to try to discover morphological or behavioral characters that could distinguish the two new species of black-legged yellow flea beetles in the field.  The barcode results divided Parchicola DF1 into two groups, a larger group of 14 specimens and a smaller group of 7.  Looking at the field notes I immediately noticed that 4 of the group of 7 were from P. vitifolia, a plant not commonly collected.  The rest were from either P. quadrangularis or P. oerstedii.  I wasn't sure which because my field notes failed to specify; I had collected several and had put them in the same container, thus mixing them up.  I then remembered from a previous field collection from P. vitifolia that  Parchicola DF1 seemed unusually large and robust, to the degree that, at first, I thought they might be Ptocadica "yellow".  That recalled another similar experience with beetles that Ron Vargas brought me from his garden P. quadrangularis.  They seemed unusually large. These field notes and recollections of mistakes suggested the possibility that the barcode "group of 7" species was larger, and fed upon P. vitifolia and P. quadrangularis.  It was clear from looking at the group of 14, that they were primarily found on P. oerstedii and P. ambigua.  This led me to go out and collect 5 Parchicola DF1 from P. vitifolia and 5 from P. oerstedii, and  measure them.  The P. vitifolia beetles averaged about 10% larger than the P. oerstedii beetles!  I felt I was on to a real difference, in behavior (host plant choice) and morphology (body length).
Note the yellow tibia (middle leg segment above)

I then examined the two groups of five newly collected beetles, looking for some other character that would separate them.  It took only a few minutes to discover that the hind tibia (the third leg segment of the hind leg) was black or dark brown ("melanized") in the larger "vitifolia" beetles, and was clear to light yellow in the smaller "oerstedii" beetles.  All the other tibia and tarsi in both species were melanized.  The hind-tibia color difference suggested new morphospecies names for the two species: Parchicola "black-tibia" and Pa. "yellow-tibia".  I then examined my small collection of dried "black-legged yellow" flea beetles, and was easily able to divide them according to hind tibia color.  Doing this I saw that both tibia colors could be found on P. oerstedii, P. ambigua, P. quadrangularis and P. auriculata.  Unfortunately I had no pinned specimens from P. vitifolia.  Even though I could not check the original barcode specimens because they were in storage in California, I felt the relationship was strong enough to begin separating Parchicola DF1 into the two species.  I also found I could see the hind tibia color if I could see the correct angle with my binoculars, and I began recording the new species in my field notes.  This far the evidence is that "black-tibia" is usually found on P. vitifolia at La Selva, and "yellow-tibia" is usually on P. ambigua and P. oerstedii.  After returning to my home in California I opened the bag of vials containing specimens used in the barcoding, and examined the color of the remaining hind tibia (usually the first one had been removed for the barcode analysis).  In 15/15 cases where the hind tibia color could be determined, the "group of 7" beetles had black hind tibia and the "group of 14" beetles had yellow hind tibia, confirming the validity of the hind tibia character as a way to distinguish the two species.
Note the black hind tibia for this flea beetle

The genetic barcoding analysis enabled me to characterize the Passiflora-feeding flea beetle community with greater precision and confidence than would otherwise have been possible.  It is doubtful anyone would have discovered the two hind-tibia Parchicola species until much later, perhaps during dissection of genitalia as part of the formal species description process.  The confirmation of species identity for the six remaining species is also a valuable result.  I had suspected that Ptocadica bifasciata might include two species groups, because the intensity of the brown color varies between individuals and possibly host plants.  However, the barcoding result for the specimens I analyzed clearly indicate only one species.  The analysis also enabled identification of flea beetle larvae, a very important tool for beetles that are difficult to rear from larvae to adult.  Interestingly, the "final" total species list for Passiflora feeding flea beetles at La Selva includes 10 species,  exactly the same as the total for Heliconius!

Does genetic barcoding replace classical taxonomy in studies of species diversity?  Not in most cases.  Perhaps it could replace the latin binomial system for labelling species, except that using it means reading and writing 300 letter words!  Unlike some other genetic techniques, barcoding is not very useful for looking at the classification and relationships between species, genera and higher groups.  Nor does it tell you anything about the natural history, ecology and biology of the living organisms.  Put simply, genetic barcoding is a tool for identifying and labeling species, without telling you anything else about them.

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