By Henk Zwartepoorte, president ESF At the February 2008 meeting of the ESF South East Asia working group the situation regarding Cuora trifasciata DNA research possibilities on European collections (zoos and privates) was discussed. The numbers in captivity are limited and the willingness by privates to participate in the ESF studbook was and is still not overwhelming. Reason for this could be the high commercial value of the specimens kept. At present eight EAZA institutions and two privates are participating in the DNA research initiated last year and carried out at the Hamburg University by Dr. Jens Poschadel. The ESF studbook got a new boost last year by the taking over of the studbook by Torsten Blanck with former studbook keeper Elmar Meier of the Munster Zoo as co-studbook keeper. Within a year the number of private studbook participants increased to 16, holding 38 specimens.
The DNA research of 40 specimens of 8 oos and two privates resulted in the assessment of four different clades. Clade A is represented by 8.7 (15) specimens, clade B by 5.8 (13), clade C 4.4.3 (11) and clade D by 1.0 (1) specimens. The latter has been recently identified in US Zoologic collections, where a similar sampling has been processed in which 50 specimens were included, substantiating the clades identified by the European study.
One clade A male died in between at the Amsterdam Zoo.
Based on these results several recommendations for exchange are done and carried out.
The intention is that the Munster Zoo mainly concentrates on clade C and the other EAZA institutions will form pure clade breeding groups. In order to achieve this several specimens moved to other institutions (one specimen went from Munster to Chester and one from Chester to Amsterdam) and one specimen was moved from a private participant to the Chester oo. In 2008 and 2009 the Rotterdam Zoo clade B male was transferred to the Munster collection for short visits. These visits resulted in Munster into 4 hatchlings in 2008. This clade B male is a proven breeder at the Rotterdam oo where 3 animals were born in 1999, 3 in 2006 and 2 in 2008. Unfortunately the 2008 born hatchlings died.
The DNA research at the Hamburg university is so far carried out on just captive animals without a known origin. Morphological and genetic research done by Blanck and McCord resulted in the description of the new species Cuora cyclornata including the two subspecies C.c. cyclornata and C.c. meieri (Blanck, McCord, Le, 2008). But additional morphological and mtDNA studies, also in relation to the closely related species Cuora pani and C. aurocapitata, proved that further nuclear DNA research is desperately needed to understand the evolutionary origin of the different clades and their relationships.
For conservation reasons and keeping open the options of future reintroductions into the wild breeding of pure captive populations it is essential to monitor the captive populations as careful as possible. For these reasons the cooperation between the EAZA institutions and the fact that they participate in the privately managed ESF studbook are very good steps.
For more intensive nuclear DNA research a proposal is done by Poschadel, Blanck, Raffel, Meier, Plath, Tiedemann and Zwartepoorte. For this research a total of 15000 is needed.
Project Aims and Materials and Methods are:
In the first part of the proposed projects we will refine our mtDNA analyses in order to achieve a basis for an exact phylo genetic reference system for the Cuora genus.
In the second step a research of nuclear DNA poly morphismus will be carried out with help of Restrictions/Fragment/Lengths/Polymorphismus. This will bring more advantyages: (a) Based on high numbers of polymorphism in microsatelite-loci the genetic structure within ranges can be cleared., where standard sequence marker can not find variation (for instance for marking out boundries), (b) possible hybrid forms between species or subspecies can be discovered and (c) paternal lineages can be discovered in order to arrange pure breeding groups.
According blood and tissue samples for molecular analyses can be obtained without problems by the above mentioned cooperating institutions and persons making it worth to carry out the research in Germany. Summariing we propose to investigate the genetic variety of in particular Cuora trifasciata, Cuora cyclornata and Cuora aurocapitata with the following aims:
1. Clarification of the phylo genetic and bio geographic relationships between the lineages of Cuora trifasciata individual specimens of clade A, clade B as well as Cuora aurocapitata, Cuora cf. aurocapitata and Cuora cyclornata cf. meieri and Cuoracf. houi by using microsatelites.
2. Investigate of possible splitting up of the C. aurocapitata lineages by using mtDNA and nuclear micro satelite markers.
3. Rechecking of the numt theory of clade B, proposed by Spinks and Schaffer (2007) by using nuclear markers.
Materials and methods
* Identification of at least 12 Cuora nDNA microsatelite-loci and analyses of Restrictions- Fragment-Lengths-Polymorphismus of all (around 50) individuals of Cuora trifasciata and Cuora cyclornata, including in this study. Additional samples of closely related taxon make identification possible of former hybridiations and other impacts.
* Additional sequencing of four mtDNA markers (ND4, CO1, Cyt b, d-loop) on selected individuals as well as single representatives of all other known Cuora species.
* A sample of the holotype of Cuora trifasciata (Bell, 1825) will be included into the analyses of nDNA microsatelites and mtDNA.
* Up to 40 samples of selected C. trifasciata, C. cf. trifasciata, C cf. houi, C. c. cf. meieri, C.c. cyclornata, C. c. meieri will be included in those mtDNA analyses in order to establish a phylo genetic relationship system.
* At least ten samples of selected C. aurocapitata, C. cf. aurocapitata and C. pani, including the syntypes of C. aurocapitata and samples of animals with known origin will make it possible to identify potential hybridiation.
Applications for funding was done to the Turtle Conservation Fund (TCF) and the 8 EAZA institutions. Next to the donation of 1000 by private breeder Herbert Becker the two private participants also contributed in smaller amounts.
With respect to the progress of the Cuora trifasciata ESF studbook, the studbook keeper Blanck will proceed collecting photographic material of the studbook specimens and possible future specimens. Blanck and Zwartepoorte also asked the studbook participants for measurements of the specimens in order to achieve good comparison tools between DNA results and morphology.
Within ESF different DNA projects and options for other species are currently discussed.
With respect to the above mentioned Cuora research the EAZA institutions form a majority within the studbook, but this very well could change in the near future as more and more private keepers see the necessity of a genetically healthy captive population of this critically endangered freshwater species.
Additional funding is certainly very welcome. Donations can be done on the ESF bank account number 7231120 on ESF treasurer in Schalkwijk, The Netherlands.