Conservation genetics of coconut crab, Birgus latro in the Lutao (Green Island): Genetic connectivity to the Pacific Islands and marine protected area network


Abstract

 Lutao, also known as Green Island, is a volcanic island located in the southeastern coast of Taiwan. Sitting in the midway of the Kuroshio Current, Lutao has the well-developed fringing coral reefs in association with highly-diverse marine organisms. The coconut crab or rubber crab, Birgus latro, is the largest crustacean found around the highland reefs in Lutao. Nevertheless, coral reefs in Lutao are in a depleting status due to increasing of tourists and overharvsting of reef-associated resources. The coconut crab is also inevitably facing local extinction due to overharvesting in Lutao. Studies on the life history, including reproductive periodicity, larval development, and population biology of B. latro in Lutao suggested that effort should be implemented on this flagship species for conservation promotion in Lutao.

 Birgus latro is widely-distributed on isolated tropical islands throughout the Indo-Pacific. However, being a favor food source and loss of habitat, coconut crab in these islands also facing the same depletion or extinction as their counterparts in Lutao. The coconut crab is completely terrestrial except for a marine planktonic larval phase of 3-4 weeks. Genetic evidences of allozymes and mitochondrial restricted enzyme polymorphisms indicated that Pacific island populations diverge in a manner consistent with isolation by distances, with only the most peripheral population being significantly different. Under this scenario, the Lutao population should have the gene flow from the populations of downstream islands such as Lanyu (Orchid Island) along the Kuroshio Current and show genetic differentiation from distant islands such as Fiji or Cook Island from the equatorial currents. On the other hand, Lutao may also play a stepping-stone role for the distant populations of the Ryukyus. In contrast, recent studies indicated that marine organisms with long dispersal larvae might still tend to have the recruitment patterns of self-seeding.

 In this research project, conservation genetics of Birgus latro is proposed. By developing highly-resolute multi-loci genetic markers, genetic connectivity of B. latro will be examined by nested hierarchical design from different geographic scales. The outcomes of genetic structure and connectivity will be utilized to answer the questions of long distance dispersal or self-seeding of coconut crabs, and provide the recommendation for the marine protected area design in Lutao.


Aims

 In this research proposal, we plan to study the conservation genetics of coconut crab, Birgus latro, by examining the genetic structure, gene flow, and recruitment patterns using hypervariable molecular markers.

This project is designed in two phases:

Phase I (2005-2006):

  1. Develop highly-resolute mitochondrial DNA gene sequences and at least 10 set of microsatellite loci, and evaluate the utility of these markers in resolve the genetic structures at different geographic scales.
  2. Detect the gene flow, genetic subdivision, and connectivity of existed populations, Lutao, Lanyu, and Kenting in southern Taiwan.

Phase II (2007-2008):

  1. Extend the population sampling from distant Pacific islands, including the Ryukyus, and discuss the large scale genetic connectivity using these markers.

 Elucidate the recruitment patterns of coconut crab for conservation and marine protected area design in Lutao.


Background

 Taiwan is the biggest island located at a junction of the South China Sea and the West Pacific archipelagos, connecting the Philippines, Ryukyus, and the mainland Japan. Coral reefs and coral community are well-developed at the southern and northern tips of Taiwan, respectively. With numerous adjacent islands, Taiwan has been suggested to play a role of “stepping-stone” in maintaining the connectivity of coral reef biodiversity in the West Pacific (reviewed in Dai 1997). Lutao with 16.2 square kilometer in area, also known as the Green Island, is one of the volcanic islands located 33 kilometer off the southeastern coast of Taiwan (Fig. 1). Sitting in the midway of the Kuroshio Current, Lutao possesses the well-developed fringing coral reefs in association with highly-diverse marine organisms. Over 150 species reef-building corals, 50 species of soft corals, and 600 fishes are recorded in Lutao (reviewed in Dai 1997). This high marine diversity attracted local and international tourists to visit Lutao with increasing number of over 300,000 visitors annually. This uncontrolled tourists and overharvesting of local reef resources has severely depleted the coral reef diversity in Lutao, and cause several taxa of marine organisms locally extinct or in an endangered status (Chen et al. 2004).

Fig. 1 Map showing the Lutao

 The coconut crab or rubber crab, Birgus latro, is one of the endangered creatures in the Lutao. B. latro is the largest crustacean found around the highland reefs in Lutao. However, it is also inevitably facing local extinction due to overharvesting. Studies on the life history, including reproductive periodicity, larval development, and population biology of B. latro in Lutao suggested that effort should be implemented on this flagship species for conservation promotion in Lutao (Chen et al. 2004).

 The coconut crab lives on oceanic islets and atolls as well as on the coasts of islands in the tropical Indo-Pacific area (exception for South China Sea). As a result of overharvesting and environmental modification, Birgus latro numbers have declined over the last decades in several regions (Fletcher et al. 1990), and numerous efforts have been undertaken to manage the stocks (e.g. Fletcher et al. 1990; Schiller 1992; Schiller et al. 1991; Wolcott 1988). The planktonic larval stage has duration of between 3 and 4 weeks, and larvae remain in the surface layers of the water column, exhibiting only weak swimming behavior (Schiller et al. 1991) suggesting that they disperse passively via surface and wind-driven currents. The larval period could be sufficient for dispersal among islands, but information on genetic structure is needed to evaluate gene flow and to identify population units and the results could be applied to the conservation effort.

Fig. 2 Ocean circulation in the West Pacific. The current patterns are modified from Benzie and Williams (1997)

 There are four major ocean circulations across the Pacific island chain where are recorded populations of coconut crab, including the North Pacific Current, the North Equatorial Counter Current, the South Equatorial Counter Current, and the South Pacific Current (Fig. 3). These current systems have been demonstrated to play an important role in the genetic connectivity of marine invertebrates along the Pacific Islands (reviewed in Benzie 1997). The up-to-date understanding of genetic structure of Birgus latropopulation was consistent with dispersal by ocean currents. The high levels of gene flow of coconut crab populations among neighboring islands inferred from both allozymes (Lavery et al. 1995) and mitochondrial DNA (mtDNA) (Lavery et al. 1996) and the evidence for restricted gene flow between more distant islands separated by thousands of kilometers are consistent with information on dispersal capacity of planktonic larvae (Schiller et al. 1991). Within the Pacific, the significant difference among populations was between the most peripheral sampling locations, and no significant differences are detected among the central populations of the West Pacific. It is suggested that the ongoing gene flow across the Pacific would occur in a stepping-stone fashion. In addition, the star-like phylogeny of alleles from the Pacific Islands indicated a recent and rapid colonization of a small number of closely related haplotypes spreading widely throughout the Pacific (Lavery et al. 1996). Under this scenario, the Lutao’s coconut crabs should have the strong gene flow from the populations of downstream islands such as Lanyu (Orchid Island) along the Kuroshio current and show genetic differentiation from distant islands such as Fiji or Cook Island from the equatorial currents. In addition, Lutao may also play a stepping-stone role for the distant populations of the Ryukyus. In contrast, recent studies indicated that marine organisms with long dispersal larvae tend to have the recruitment patterns of self-seeding (reviewed in Palumbi 2003).

 In order to promote coconut crabs as the flagship species for coral reef conservation in the Lutao (Chen et al. 2004), we propose to study the genetic connectivity of Birgus latro among Lutao and other Pacific Islands, specifically on the issue of long distance dispersal vs self-seeding. For the long-distance dispersal with sea currents, reproduction and recruitment in the sea may be spatially decoupled so that recruitment in one area is highly depend upon production elsewhere (Robert 1998). This lead to the much greater need for network of protected areas in the sea than on land (Sala et al. 2002) and a regional rather than local view of conservation planning (Beck 2003). Identification of “ source” of marine larvae and “sinks”- areas dependent on the import of larvae from other locations- is critical for placement of marine reserves (Ogden 1997). Thus, both optimal distance between marine reserves and identification of upstream for connections are the key point to design marine reserves network.

 In this research project, conservation genetics of Birgus latro is proposed. By developing highly-resolute multi-loci genetic markers, genetic connectivity of B. latro will be examined by nested hierarchical design from different geographic scales. The outcomes of genetic structure and connectivity will be utitlised to answer the questions of long distance dispersal or self-seeding of coconut crab, and at the mean time, provide the recommendation for the marine protected area design in Lutao.


Material and methods

Sampling design

 

The collection of coconut crab will follow the hierarchical nested design (1500, 200 and 50 km) in the West Pacific. The coconut crab will be collected coving the substantial Oceania islands chain located at Cook Islands, Niue, Vanuatu, Soloman Islands, Papua New Guinea, Palau, Philippines, Ryukyu Arch and Taiwan with an average distance of 1500 km. This sampling strategy will cover the Kuroshio Current, Equatorial Counter Current, and South Pacific Current system. At a meso-geographical scale, collection sites along the Ryukyu Arch and Taiwan will be in a distance of 200 km. In a local scale in Taiwan, there will be three sites including Green Island, Orchid Island, and Kenting, which will be separated away 50km (fig. 1).

Fig. 3 The collection sites of coconut crab in the West Pacific. s: proposed collection site

 For the first phase (2005-2006), sampling will be targeted at the small scale which are the three sites around Taiwan (Lutao, Lanyu, and Kenting). For the second phase (2007-2008), sampling will be extended to cover the Pacific region.
Coconut crabs can be rare or abundant depending on the localities and methods we use. Thus, at least 20 male and 20 female coconut crabs from each sampling site are aimed to collect, and then tagged and released. However, this number can be variable depending on the condition. The gender sampling will be used to detect sexual-bias dispersal with microsatellite. All the muscle tissue (from the tibia of the fourth leg) of samples will be stored in 70% EtOH, in prior of DNA extraction.

Molecular marker

 Microsatellite loci will be utilized as the bi-parent nuclear markers, and the maternal mitochondrial AT- rich region will be integrated into evaluating genetic distance.

Isolation of Microsatellite and genotyping

 Approximately 10μg of genomic DNA extracted from a single coconut crab will be digested with restriction enzyme, Sau3A1. After separation on a 1.5% agarose gel, DNA fragments in the size range of 200- 800 base pairs will be excised, purified and ligated to an equal volume of plasmid vector pUC18 (Amersham-Pharmacia). The plasmids will have previously been digested with BamHI and dephosphorylated to create overhanging ends to match those resulting from the Sau3A1 digest. Recombinant plasmids will be electroporated into competent Escherichia coli cells and incubated for an hour at 37°C. Cells will be spread on to agar plates containing LB-Ampicillin and incubated overnight at 37°C to promote selective growth of transformed colonies. The recombinant colonies will be picked from plates and incubated overnight in a grid formation on new LB-Ampicillin agar plates and later stored at 4°C. Recombinant colonies will be blotted from the plates on to filter membranes (Hybond-N, Amersham). DNA from this transfer was crosslinked with the membrane, denatured and probed with oligonucleotides that had been end-labelled with [l 32P] dATP (Perkin Elmer). Cross-linked single-stranded DNA will be hybridized with the probes overnight before being exposed onto X-ray film. The positive clones that hybridized with probed repeat will be revealed. Colonies containing repeats will be thus identified and picked from the stored agar plates and cultured overnight at 37°C. Plasmid DNA will be extracted from cultures and sequenced. The clones containing the perfect repeat sequences will be amplified with PCR and gene scanner will perform the microsatellite genotyping of DNA samples from each collected sits. At least 10 loci will be excited from the library for genotyping.
Mitochondrial AT-rich region

  Total genomic DNA from muscle tissue will be extracted with Blood & Tissue Genomic DNA Extraction Miniprep System Kit (VIOGENE). The primer of AT- rich region will be designed from another species of hermit crab (Pagurus longicarpus) between tRNA Tyr (Y) and tRNA Pro (P) (Hickerson and Cunningham 2000). PCR amplification reaction should be tried for best condition and performed then. PCR products will be purified and directly sequenced on an automated DNA sequencer, and with the same primers used for the amplification.


Data analysis
(1) Estimates of population structure will be made using both IAM and SMM evolutionary models. Under the former model, Weir and Cockerham's (1984) weighted analysis-of-variance approach will be used to estimate f, the correlation of alleles within individuals, and h, the correlation of alleles within populations for each locus. The statistics f and h are minimum-bias estimators of Wright's FIS and FST, respectively (Wright 1978). Single-locus estimates of f and h will be weighted as described by Weir and Cockerham to create a combined estimate. Using the SMM, population structure will be estimated for each locus by Slatkin's (1995) RST, an analog of h that estimates the correlation of allele size within populations. The standardization approach of Goodman (1997) will be used to make a multi-locus estimate of RST. The significance of each estimate will be evaluated through permutational procedures (minimum 1000 permutations), by permuting alleles among individuals within populations (f) or individuals among populations (h and RST) to generate the null distribution of the statistics.


(2) Calculation of all estimates of population structure and their significance will be performed with the aid of ARLEQUIN 2.0 (Schneider et al. 2000) and RST CALC (Goodman 1997). Genetic distances will be assessed with (dm) 2 (genetic distance between locales) and with pairwise estimates of RST, both of which assume the SMM.


(3) Fixation indices (F-statistics) (Wright 1951) are applicable to mtDNA under the assumption of uniparental, nonrecombining inheritance to represent population difference. Hudson et al. (1992) gave an expression to estimate FST values based on sequence data: FST = 1 -Hw/Hb, where Hw is the average nucleotide variation among individuals within a subpopulation, and Hb is the average variation between subpopulations. This allows for an estimation of gene flow, Nem = 1/2 [1/ FST - 1], where Ne is the effective number of females and m is the migration rate. FST values and Nem were calculated with Arlequin 2.0 (Schneider et al. 2000).


Expected achievement

 The cococnut crab, Birgus latro, represents one of the listed most-endangered marine invertebrates in the IUCN Red List. However, according to the IUCN Red List criteria, there is not enough information on this species to make an assessment of its risk of extinction based on its distribution and/or population status. It is therefore classified as Data Deficient (DD). Thus, our efforts in studying the life history, reproductive biology, and the conservation genetics of coconut crabs in Lutao have and will contribute toward the understanding of this endangered macro crustacean. For this research project we expect to achieve the following goals:

  1. Provide highly-resolute mitochondrial DNA gene sequences and at least 10 set of microsatellite loci, and evaluate the utility of these markers in resolve the genetic structures of coconut crabs at different geographic scales.
  2. Reveal the small scale of gene flow, genetic subdivision, and connectivity of currently existed populations in Taiwan, including Lutao, Lanyu, and Kenting.
  3. Detect the large-scale genetic connectivity between Taiwan populations and distant Pacific islands; reveal the role of stepping stone of Lutao to the Ryukyus populations.

 Synthesize the conservation genetic information for the promotion of coral reef conservation and marine protected area design in Lutao.


References
1.Beck, M. 2003. The sea around: marine regional planning. In Groves, C.R. Drafting a conservation blueprint: a practitioners guide to planning for biodiversity. Island Press.

2.Benzie, J.A.H., Williams, S.T. 1997. Genetic structure of giant clam (Tridacna maxima) populations in the West Pacific is not consistent with dispersal by present-day ocean currents. Evolution 51: 768-783.

3.Chen, C.-P., Lin, S.-T., Wang, F.-L. 2004. Coconut crabs as a target for promoting the establishment of marine protected areas in the Green Island, Taiwan. 4th ISIA conference, Kimen Island, Taiwan. (Abstract).

4.Dai, C.-F. 1997. Assessment of the present health of coral reefs in Taiwan. Status of coral reefs in the Pacific. R. Grigg and C. Birkeland. Honolulu, University of Hawaii.

5. Fletcher, W.J., Brown, I.W., and Fielder, D.R. 1990. Growth of the coconut crab Birgus latro in Vanuatu. Journal of Experimental Marine Biology and Ecology 141: 63- 78.

6. Goodman S.J. 1997. Rst Calc: a collection of computer programs for calculating estimates of genetic differentiation from microsatellite data and determining their significance. Molecular Ecology 6: 881- 885.

7.Hill, A. E., 1991. Advection-diffusion-mortality solutions for investigating pelagic larval dispersal. Marine Ecology Progress Series, 70, 117-128.

8. Lavery, S., Moritz C., Fielder, D. R. 1995. Changing patterns of population structure and gene flow at different spatial scales in Birgus latro the coconut crab. Heredity. 74: 531-541.

9.Lavery S, Moritz C., Fielder D.R. (1996) Indo-Pacific population structure and evolutionary history of the coconut crab Birguslatro. Molecular Ecology, 5, 557-570.

10.Ogden, J.C. 1997. Marine manager look upstream for connections. Science, 278, 1414- 1415.

11.Palumbi S. R. 2003. Population genetics, demographic connectivity and the design of marine protected areas. Ecol. Appl. 13: S146-S158.

12.Roberts, C. M. 1998. Sources, sinks, and the design of marine reserve networks. Fisheries 23: 16-19.

13.Sala, E., O. Aburto-Oropez, G. Paredes, I. Parra, J. C. Barrera, and P. K. Dayton. 2002. A general model for designing networks of marine reserves. Science 298:1991-1993.

14.Schneider S., Roessli D., Excoffier L. 2000. ARLEQUIN ver 2.0. A software for population genetics data analysis. Geneva, Switzerland.Schiller CB (1988) Pilot stock survey of the coconut crab (Birgus latro) in Niue islands, Pacific Ocean. Consultancy report. FAO Rome Italy: 42p.

15.Schiller C.B. 1992. Assessment of the status of the coconut crab Birgus latro on Niue island with recommendations regarding an appropriate resource management strategy. Consultancy report. FAO Rome Italy: 69p.

16.Schiller, C., Fielder, D.R., Brown, I.W. 1991. Reproduction, early life-history and recruitment. Pp. 13- 34 in: Brown, I.W., and Fielder, D.R. (eds), The coconut crab: aspects of Birgus latro biology and ecology in Vanuatu. Canberra. Aciar Monographs 8.

17.Slatkin M (1995) Measure of population subdivision based on microsatellite allele frequencies. Genetics, Austin, Tex 139: 457- 462.

18.Weir B.S., Cockerham C. C. 1984. Estimating F-statistics for the analysis of population structure. Evolution 38: 1358- 1370.

19.Wolcott, T.G. 1992. Water and solute balance in the transition to land. American Zoologist 32: 428- 437.

20.Wright, S., 1951. The genetical structure of populations. Ann. Eugen. 15:323-354.

21Wright, S., 1978 Evolution and the genetics of populations. Vol. 4. Variability within and among natural populations. University of Chicago Press, Chicago.


Justification of Budget

  1. Personnel. This project will request a Msc-level research assistant with molecular training to develop the microsat library, sequencing, and identify the genotyping process. A 24-mo salary with 1.5 mo of work bonus are requested.
  2. Equipment: A genotyping apparatus, GelScan2000, will be arrived in the RCBAS under the core facility at the early January 2005. This apparatus will be installed under the supervision the program director, Allen Chen. The newly-recruited assistant will be the person in charge with the genotyping for this project.

Maintenance, materials and supplies.

  Hsieh’s lab has experience in running research programs in conservation and marine benthic ecology of marine invertebrates. The molecular work will be conducted in the program director Allen Chen’s lab under the collaboration. The requested budget consulted by A. Chen, I believe, is realistic for 24-month cost of chemical consumption, field expense, and bench fees in developing the microsat library and genotyping.


Address: Room 636, Research Center of Biodiversity, 128 Sec. 2, Academia Rd, Nankang, Taipei 115 Taiwan, R.O.C.
E-Mail: zocp@gate.sinica.edu.tw
Tel: 886-2-27899546
Fax: 886-2-27899548