The Rufford Small Grants Foundation
Final Report
Grant Recipient DetailsYour name / Oscar E. Ospina
Project title / Morphological and genetic analysis of population status and trends of the fossorial, endemic reptile Amphisbaena caeca in the Puerto Rican Archipelago
RSG reference / 12092-1
Reporting period / September 2012 – September 2013
Amount of grant / £ 5687
Your email address /
Date of this report / October 2013
------Congratulations on the completion of your project that was supported by The Rufford Small Grants Foundation.
We ask all grant recipients to complete a Final Report Form that helps us to gauge the success of our grant giving. We understand that projects often do not follow the predicted course but knowledge of your experiences is valuable to us and others who may be undertaking similar work. Please be as honest as you can in answering the questions – remember that negative experiences are just as valuable as positive ones if they help others to learn from them.
Please complete the form in English and be as clear and concise as you can. We will ask for further information if required. If you have any other materials produced by the project, particularly a few relevant photographs, please send these to us separately.
Please submit your final report to .
Thank you for your help.
Josh Cole
Grants Director
1. Please indicate the level of achievement of the project’s original objectives and include any relevant comments on factors affecting this.
Quantify genetic variation of A. caeca / X / The objectives have not been completely achieved due to complications in sampling. At the moment 27 specimens have been obtained from the northern part of Puerto Rico. Although the accomplishment of these objectives is not impossible, additional time will be necessary.
Estimate population structure degree of A. caeca / X
Measure the morphological variation in A. caeca / X
2. Please explain any unforeseen difficulties that arose during the project and how these were tackled (if relevant).
Due to the soil-dwelling habits of the Puerto Rican worm lizard (A. caeca), it has been very difficult to obtain all the proposed samples. The sampling requires enormous amounts of time and physical effort to unearth these organisms. I estimated that sampling at each locality would take no more than two visits, however most of the times I just ended up with one or two specimens per visit. Of course, with the passage of time I have obtained more experience and it has become easier to obtain samples.
At the moment, I have collected 27 specimens representing the northern populations of Puerto Rico. Sampling in the north of Puerto Rico has been easier than in the south. This has been mainly due to the dryness of the southern soils. Previous reports from the scientific literature and observations from my thesis advisor have demonstrated the presence of this species in the south of Puerto Rico. Further sampling will be made to confirm this information.
A second major challenge has been the standardisation of PCR protocols to obtain the sequences, especially for nuclear genes. Even with my experience about this topic, most of the procedures to obtain these nuclear DNA sequences have been not satisfactory. As a consequence, sending these PCR products (DNA fragments) for sequencing would be a waste of resources. I expect that by the end of this year, PCR protocols will be completely standardised as has been already don for mitochondrial genes.
3. Briefly describe the three most important outcomes of your project.
A. Inside each populations of A. caeca, there is low genetic and morphological diversity. However, if we compare each population they are extremely different. In other words, each population of the species has unique genetic and morphological features. Morphologically, the population from “Tortuguero Lake” is the most different, having more segments along their bodies (Figure 1). Genetically, the populations have DNA variants that they do not share with each other. The population from “Lares” is the most genetically different from the others (Figure 2).
B. The evident morphological and genetic differentiation previously mentioned is the product of isolation between these populations. The statistical analysis shed that there is scarce flow of individuals (and genes) between the populations, which has derived into the preservation of unique morphological and genetic variants inside each population. According to the assessed genes, I have found that there is an average of 0.16 individuals being exchanged between populations, per generation. Assuming that a generation of A. caeca equals 1 year, it would take approximately 7 years for two populations to have a complete genetic exchange. In other words, A. caeca populations are extremely isolated from each other.
C. Before this study, information for the assessment of the conservation status of this species was very scarce. Regarding the high genetic and morphological variability of A. caeca, I could say that it has an enormous potential for long-term survival. Nevertheless, the fact that populations are unique poses a challenge for the conservation of the species, meaning that one would need to protect almost all the populations. Fortunately, “Tortuguero Lake” and “Punta Salinas” populations are already under protection by the Department of Natural Resources of Puerto Rico. This is not the same scenario for the “Lares” population, which is inside an urban matrix.
4. Briefly describe the involvement of local communities and how they have benefitted from the project (if relevant).
This is probably one of the most exciting results of this project. During the sampling it was possible for me to interact with people from the countryside of Puerto Rico, and involve them in the collecting of specimens. Although many of them have been working with the soil during years, they just ignored the existence of these animals or even confused them with earthworms. This project helped them to detect the differences between worm lizards and other organisms. During the collecting of specimens, local people were able to know about the important role of soil fauna in the productivity of their crops. Sampling was also a good opportunity for them to solve questions and learn about other aspects of the soil biota.
I also had the opportunity to involve undergraduate students during the sampling and laboratory procedures. Most of them had never seen a worm lizard, or worked in the collecting of these organisms. Others had never worked with the molecular and morphological methods used in this project. In all of the previous cases, it was a good experience of learning and discussion for me and for the local people.
5. Are there any plans to continue this work?
Since the objectives of this project are not fully accomplished due to limitations in sampling, it is certainly necessary further work. Now, the most important step for this project is to collect individuals from populations in the south of Puerto Rico. Those populations may be even more differentiated from those already sampled in the north, given the presence of the main, central mountain range of the Island.
After completing the sampling, I was planning to make a better prediction of possible units (populations) for conservation. Such better planning will include an environmental modelling to detect preferences in habitat of these animals.
6. How do you plan to share the results of your work with others?
I am currently working in the development of a webpage for the project. The webpage will be available in English and Spanish, given that the majority of Puerto Rican population speaks Spanish natively. I expect the page to be available by the end of December 2013.
Collected specimens have been borrowed (and already returned to me) for educational purposes in schools. I am already arranging some talks in schools, taking advantage of the contact that I made by borrowing material. Infographic material showing the differences between amphisbaenians and other soil fauna will be placed in communal centres.
Finally, and as obvious outcome of a scientific study, at least two articles will be submitted for publication in peer-reviewed journals. By the next year I also expect to show the results of my work in a conservation-related scientific congress.
7. Timescale: Over what period was the RSG used? How does this compare to the anticipated or actual length of the project?
The total amount of the RSG has not been completely used at the moment. There still funds for the field trips. Given the difficulties with sampling, more funds have been spent than planned in visiting the places. Materials for field trips were obtained as proposed. The funds initially assigned to purchase DNA isolation and amplification reagents were spent according to the proposed schedule. There are enough of those materials to continue with the project.
The funds assigned to sequencing services have also been spent as proposed. Some individuals were re-sequenced to assure a correct result. Nevertheless, as specified in the proposal, additional funds are needed to complete all the sequencing of the specimens.
8. Budget: Please provide a breakdown of budgeted versus actual expenditure and the reasons for any differences. All figures should be in £ sterling, indicating the local exchange rate used.
Item / Budgeted Amount* / Actual Amount* / Difference* / CommentsField trips:
Transportation and
allowances / 733 / 310 / 423 / The difference will be used for the remaining field trips.
Field trips: Equipment
and material / 267 / 314 / - 47 / The additional money spent in equipment was the result of shipping costs and differences in actual prices not previously accounted for.
DNA isolation and
amplification reagents
and material / 3020 / 2601 / 419 / The remaining funds will be used for purchasing PCR-related materials.
DNA sequencing
service / 1667 / 610 / 1057 / The remaining funds are destined to complete sequencing of at least the proposed two mitochondrial DNA regions.
TOTAL / 5687 / 3835
* Local exchange rate: 1 Pound Sterling = 1.50 US Dollars.
9. Looking ahead, what do you feel are the important next steps?
After the project is finished, I think that it is necessary to send a report to the Department of Natural Resources of Puerto Rico to show them which areas should be considered critical for the conservation of the species. It will be important also to make a report to the IUCN, in order to classify the species according to their scheme. The species is not classified in the IUCN Red List due to deficiency in existing data.
Given the high diversity of the species, there is a possibility that A. caeca is actually a complex with more than one species not previously described. Then, it would be necessary to measure the diversity in the sister species to A. caeca (A. schmidti and A. bakeri). With this new information it is possible to test the hypothesis of new species inside A. caeca.
10. Did you use the RSGF logo in any materials produced in relation to this project? Did the RSGF receive any publicity during the course of your work?
The Department of Biology of the University of Puerto Rico made mention of the funds assigned to my research by RSGF in its online weekly. I also have acknowledged the support from RSGF during my field trips with rural communities. The RSGF logo will be included in the webpage mentioned in Question 6, and in every talk and infographics generated by this project.
11. Any other comments?
As previously stated, I will need an additional period of 9 months to obtain enough data and present a consistent conclusion. At the moment, the results suggest an interesting pattern of evolution by isolation of the populations. In terms of conservation, this may imply two key issues. First, the species could lose a considerable portion of its diversity due to habitat destruction, given that losing only one population means the loss of unique genetic and morphological variation. Secondly, the potential discovery of new species or subspecies is important for the conservation. Once we know which species are present in Puerto Rico, species-specific conservation measures can be put in motion.
Figure captions
Figure 1. Number of body segments in the collected A. caeca. The population from “Tortuguero Lake” (hollow triangles) tend to have more body segments than the other populations.
Figure 2. Haplotype networks for tow mitochondrial genes in A. caeca: 16S (top) and COI (bottom). The gene COI has more variability (haplotypes) than 16S. The dashed lines indicate extreme divergence. The “Lares” population is the most genetically variable, and the most different among the three populations.