Conserv Genet DOI 10.1007/s10592-009-9883-3
SHORT COMMUNICATION
Population subdivision in hawksbill turtles nesting on Barbados, West Indies, determined from mitochondrial DNA control region sequences Darren C. Browne Æ J. A. Horrocks Æ F. A. Abreu-Grobois
Received: 18 May 2008 / Accepted: 24 February 2009 Ó Springer Science+Business Media B.V. 2009
Abstract A new mitochondrial DNA control region survey of the Barbados hawksbill nesting population was undertaken using larger sample sizes, reanalysis of previously reported samples, and new primers that increase the fragment length sequenced. This work revealed that haplotypes originally identified as endemic to Barbados were misread sequences. Genetic variants and a geographic subdivision on a finer scale than has previously been recorded for sea turtles were identified between the Barbados leeward and windward coasts, indicating the need for sampling at multiple sites to reveal comprehensive genetic variation at national scales. Using the updated haplotype profiles to re-estimate Barbados’ contribution to Caribbean hawksbill foraging grounds indicated a presence severalfold larger than previously calculated; a result congruent with the breeding population being one of the largest in the region. Keywords Mixed stock analysis Eretmochelys imbricata Caribbean Stock identification Population structure
D. C. Browne (&) J. A. Horrocks Department of Biological and Chemical Sciences, University of the West Indies, Cave Hill Campus, Barbados BB11000, West Indies e-mail:
[email protected] F. A. Abreu-Grobois Unidad Acade´mica Mazatla´n, Instituto de Ciencias del Mar y Limnologı´a, UNAM, Apartado Postal 811, 82000 Mazatla´n, Sinaloa, Mexico
Introduction Critically Endangered (IUCN 2008) hawksbill sea turtles (Eretmochelys imbricata) have a circum-tropical distribution within the Atlantic, Pacific and Indian Oceans (Witzell 1983). They are found in coastal waters throughout the Caribbean Sea feeding primarily on coral reef-associated sponges (Carrillo et al. 1999). Historical levels of exploitation (Meylan and Donnelly 1999; Solano et al. 2004) have resulted in most populations being in decline, depleted or remnants of formerly larger aggregations, with fewer than 100 females nesting annually in most Caribbean territories. Since the end of international trade in shell, hawksbill numbers have begun to recover at some locations, including Barbados and Antigua (Beggs et al. 2007; Richardson et al. 2006). Barbados (430 km2) now has one of the largest nesting populations (rookeries) in the Wider Caribbean, with 2,000? nests recorded annually in recent years (Beggs et al. 2007). Females nest on the leeward coast of the island, and on a single, reef-protected windward beach. Genetic characterization of the Barbados hawksbill rookery using mtDNA sequences derived from females (n = 15) nesting on leeward and windward beaches in 1992, was published as part of a regional study (Bass et al. 1996). The identification of a unique haplotype ‘‘signature’’ for Barbados, with the major haplotype (‘‘A’’) being shared with many Caribbean rookeries, and two endemics (‘‘D’’ and ‘‘E’’), suggested significant genetic differentiation between this and other populations of the region. However, the absence of these endemic haplotypes in regional foraging populations implied that Barbados’ contributions to Caribbean foraging grounds were minimal (Bowen et al. 1996, 2007; Dı´az-Ferna´ndez et al. 1999). Here, we report on a new analysis for the Barbados rookery that corrects
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the haplotype profile for the country, uncovers a surprising genetic subdivision within a small territory and revises the significance of this large nesting population’s contribution to regional foraging grounds.
Methods Fifty-four hawksbill tissue samples from leeward beaches and thirty from the single windward beach were analyzed. The samples were from 41 different females, or embryos from different females, nesting on leeward beaches between Speightstown and Dover in 2002 and 2004–2005 (Fig. 1) and 27 from embryos of different females nesting on the isolated windward beach (Bath; Fig. 1) in the 2005– 2006 nesting seasons (see Appendix 1). In addition, there were 13 leeward coast and three windward coast samples that were originally analyzed for the Bass et al. (1996) study. These 16 included one sample for which no results were generated in the Bass et al. study. DNA was isolated from tissues using phenol: chloroform extraction (Taggart et al. 1992). Approximately 740 bp of the mtDNA control region were amplified by PCR using primers LTEi9 (50 GAATAATCAAAAGAGAAGG 30 ) and H950 (50 GTCTCGGATTTAGGGGTTT 30 ) (AbreuGrobois et al. 2006a) in a volume of 50 ll. The thermocycler
Fig. 1 Sampling locations from the leeward (Speightstown to Dover) and windward coast (Bath) nesting sites in Barbados, within the Caribbean region. The TCS parsimony network for the regional haplotypes is overlaid on the Caribbean map to illustrate the complicated intermixing of lineages in this region. With the exception of EiA01 and EiA11, all haplotypes are placed over the nesting sites where they were reported. Rookeries where EiA01 and EiA11 were
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(Techne Genius Model No. 230) was programmed with the following profile: initial denaturation at 94°C for 1 min, followed by 35 cycles at 94°C for 30 s, 56°C for 30 s and 72°C for 90 s, and extension at 72°C for 7 min. Sequences were aligned using MULTALIN (Combet et al. 2000) and matched against published Atlantic and Caribbean hawksbill mtDNA control region sequences (Bass 1999; Bass et al. 1996; Bowen et al. 2007; Dı´azFerna´ndez et al. 1999) and unpublished new and longer sequences (Abreu-Grobois et al. 2006b). Sequences matching published haplotypes over homologous internal segments were assigned a combined code reflecting Bass et al. (1996), Dı´az-Ferna´ndez et al. (1999) nomenclatures, respectively, and the new standardized coding system for Atlantic hawksbills (Abreu-Grobois et al. 2006b), e.g. A/CU1 (EiA01). Haplotype frequencies were obtained from Dı´az-Ferna´ndez et al. (1999) for Cuba; Dı´az-Ferna´ndez et al. (1999) and Bass (1999) for Mexico and Puerto Rico; Bass et al. (1996) for Antigua, Belize and the original Barbados data; Bass et al. (1996) and Bowen et al. (2007) for the USVI; Troe¨ng et al. (2005) and Bowen et al. (2007) for Costa Rica; and Lara-Ruiz et al. (2006) for Brazil (including hybrid haplotypes). Data were combined where multiple surveys had been undertaken at single rookeries. Haplotype (h) and nucleotide diversities (p) were estimated using Arlequin 3.01 (Excoffier et al. 2005). For the analyzes that follow,
found are listed, with asterisks indicating rookeries where they occur at highest frequencies. Sizes of the haplotypes are proportional to their frequency in the regional data set. In the Barbados map inset, the haplotype diversities for the two coasts are indicated by black haplotypes within scaled down representations of the complete regional haplotype network
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with the exception of comparison between the Barbados leeward and windward rookeries where the longer 740 bp sequence could be used, sequences were truncated to the Bass et al. (1996) reading frame of 384 bp to enable comparison with all published results. TCS 1.21 was used to construct a network using the complete Caribbean dataset based on the criterion of statistical parsimony (Clement et al. 2000) and criteria in Pfenninger and Posada (2002) were used to resolve ambiguous connections between haplotypes. Arlequin 3.01 (Excoffier et al. 2005) was used to estimate pair-wise migration rates (Nm; Slatkin 1995) and genetic distances on the basis of conventional FST (haplotype frequencies; Reynolds et al. 1983; Slatkin 1995). A consensus of the genetic relationships among rookeries was obtained with the Consense procedure in PHYLIP 3.68 (Felsenstein 2005) from 1000 UPGMA trees of pairwise FST values produced by the Neighbor procedure (PHYLIP). These, in turn, were obtained with Arlequin from an equal number of bootstrapped rookery haplotype profiles. Preprocessing, formatting and bootstrapping of datasets were performed with ad hoc Microsoft Excel macros. AMOVA (Arlequin 3.01) was used to evaluate the proportion of the total genetic variation found distributed between rookeries and between the rookery clusters identified in our phylogenetic analysis. Due to the differences in the Barbados haplotype profile resulting from the new analysis, it was necessary to reestimate contributions of the Barbados leeward and windward rookeries to foraging ground stocks in the Wider Caribbean. Estimation was performed with maximum likelihood (SPAM 3.7; Debevec et al. 2000) and Bayesian procedures (BAYES; Pella and Masuda 2001). In the latter, nine 50,000-iteration Monte Carlo Markov chains of stock compositions were generated, one for each potential source population, with prior expectations of 0.95 for a particular source population, and 0.05 distributed equally among the remaining sites. The composition of mixed stocks was derived from the mean of chains after 25,000 burn-in steps. Genetic compositions of hawksbill aggregations on foraging grounds were obtained from Dı´az-Ferna´ndez et al. (1999) for three fishery zones in Cuba (A, B, and D); Bowen et al. (2007) for Texas, the Bahamas, Dominican Republic and the USVI; and both Dı´az-Ferna´ndez et al. (1999) and Bowen et al. (1996) for Puerto Rico.
Results and discussion All nesting females from the new sampling of Barbados’ leeward coast (2002–2005) exhibited A/CU1 (EiA01), contrasting with the higher haplotype variation amongst the new samples from windward coast nesting females
(h = 0.476; p = 0.003), with 19 F/PR1 (EiA11), 6 F/c (EiA09) and 2 A/Cu1 (EiA01). Our re-analysis of the 16 original samples used by Bass et al. (1996) found 13 A/CU1 (EiA01) from the leeward rookery, 2 F/PR1 (EiA11) and 1 A/Cu1 (EiA01) from the windward rookery, contrasting with what they reported as 11 A (EiA01), 1 D (EiA06) and 3 E (EiA08). The corrected results (Appendix 1) imply that haplotypes D (EiA06) and E (EiA08) were errors resulting from older sequencing technology available at the time of the initial analysis. This explains why the endemic haplotypes were neither detected in other rookeries or foraging grounds subsequently studied (Bass 1999; Bass et al. 1996; Bowen et al. 1996, 2007; Dı´az-Ferna´ndez et al. 1999; LaraRuiz et al. 2006; Troe¨ng et al. 2005), nor in our new surveys. Interestingly, the new haplotype profiles for the two coasts indicated significant inter-rookery differentiation (v2 = 71.62, FST = 0.798, P \ 0.05 for both). Although genetic structuring between adjacent nesting areas is known over distances of [400 km (Bowen et al. 1993; Schroth et al. 1996), this is the first report for rookeries separated by 30 km. Consequently, we evaluated the leeward and windward rookeries as separate populations. Our AMOVA indicated that overall divergences among the entire Wider Caribbean population set were significant (UST = 0.465, P \ 0.001). However, even further structuring was suggested from phylogenetic comparison (Appendix 2), as nesting populations partitioned into three clusters of closely related populations; the first included Cuba, the leeward Barbados rookery, Antigua and Brazil, the second USVI, the windward Barbados rookery, Belize, Costa Rica and Puerto Rico and the third comprised of Mexico. Variation among these groups accounted for 51% of total variation (UCT = 0.507, P \ 0.001) while withinpopulation variation accounted for 39% (UST = 0.609, P \ 0.001). Of the 45 possible pair-wise comparisons among the two Barbados rookeries and other rookeries in the region (Table 1), the only non-significant comparisons found were those between Barbados windward, Belize and USVI rookeries. The pair-wise comparisons suggest that the larger leeward Barbados rookery (*87.5% of all nests laid annually; Beggs et al. 2007) which is fixed for haplotype A/Cu1 (EiA01), is most similar to the Cuban rookery (FST = 0.058, P \ 0.001), while the small, more variable windward rookery is most closely related to rookeries in the western (Belize and Costa Rica) and northern Caribbean (USVI and Puerto Rico), but differentiated from the other rookeries, including Cuba. The new dataset allowed for a re-estimation of Barbados’ contribution to Caribbean foraging grounds (FGs), which in the most recent review (Bowen et al. 2007)
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Conserv Genet Table 1 Genetic differentiation between the Barbados rookeries and regional populations using haplotype sequence data truncated at the currently standard 384 bp fragment length Rookery
Barbados leeward coast
Mexico
0.937*
0.863*
0.034
0.080
Cuba Puerto Rico USVI
0.058*
0.773*
8.119
0.147
0.671*
0.282*
0.245
1.274
0.863* 0.079
Antigua Costa Rica
Barbados windward coast
20.022 ?
0.491*
0.637*
0.518
0.285
0.682*
0.129*
0.233
3.375
Belize
0.913*
0.023
Brazil
0.048 0.278*
20.797 0.465*
1.296 Barbados leeward coast Barbados windward coast
0.576 0.798* 0.126
0.798* 0.126
Conventional pair-wise FST values in bold, migration rates (Nm) based on Slatkin (1995) in italics. FST values significant at experimentwise a = 0.05 are denoted by asterisks ‘‘a’’ indicates very high migration rates between populations
appeared to be negligible. Although Antigua, Belize, Costa Rica, Cuba, Mexico, Puerto Rico and USVI and the two Barbados rookeries were included as baseline populations in our mixed stock estimations, only the contributions from the Barbados rookeries to regional foraging grounds are discussed here. Contributions from each of the Barbados coasts were markedly different—while the leeward rookery appears to contribute a larger proportion of individuals to the Dominican Republic, Bahamas, Cuba A, and USVI foraging grounds, the windward rookery contributed more to the Puerto Rico, Texas, Cuba B and Cuba D foraging grounds (Table 2). The leeward rookery appears to contribute most to the Dominican Republic (SPAM: 44.5%, Bayes: 43.8%) while the windward rookery appears to contribute most to Puerto Rico (SPAM: 37.8%, Bayes: 20.1%). The foraging ground to which the combined Barbados rookeries were estimated to make the largest contribution in the region was the Dominican Republic (SPAM: 65.6%, Bayes: 52.3%); significantly more than that previously published (SPAM: 0.14%, Bayes: 15.6%; Bowen et al. 2007). Given the large reproductive output from Barbados (Beggs et al. 2007; Mortimer and Donnelly
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2007), the extent to which Barbados contributes to regional foraging grounds was evidently substantially underestimated using the earlier haplotype profile. The genetic relationships between regional rookeries together with the geographic distributions of haplotype networks (Fig. 1) may indicate possible origins for Barbados rookeries in a re-colonization process that probably occurred after the historical hawksbill exploitation that led to steep declines or extinction of individual breeding colonies (Meylan and Donnelly 1999). At a regional scale, population reductions may have opened up opportunities for stochastic colonization or immigration events. In Barbados, the harvest was focused on the heavily settled, sheltered leeward coast and this could possibly have eroded genetic variability and facilitated the fixation of the single haplotype, EiA01. EiA01 is also the major and better distributed haplotype in the Lesser Antilles. Explaining the more diverse genetic composition at the windward rookery is more difficult, although there is an increased likelihood of colonization events from a broader set of sources as this coast is in the direct path of prevailing currents and approaching storms (see also Schroth et al. 1996). Finding haplotype EiA11 in addition to EiA01 is not surprising as these are the two most dominant haplotypes in the Eastern Caribbean (Fig. 1). More precise regional linkages may be discernable with the longer sequences since EiA11 can be split into three (F/c = EiA09, F/j = EiA10, and F/ PR1 = EiA11), and ongoing work in the Caribbean has found geographic structuring for these, for example, F/c (EiA09) and F/PR1 (EiA11) within the same rookery (Caribbean Conservation Corporation, unpublished data; Ve´lez-Zuazo et al. 2008), and F/c (EiA09) in the absence of F/PR1 (EiA11; R. LeRoux et al. unpublished data). Currently, the geographic pattern of the regional haplotype network (Fig. 1) indicates extensive crossings between sub-regions by the major lineages, too complicated to infer direct linkages and explain the Barbados genetic composition. Nonetheless, the current genetic makeup is likely the result of founder events and genetic drift in recovering populations, enhanced by the pronounced nest site fidelity exhibited by hawksbills (Spotila 2004). This study demonstrates the problematic nature of traditional sampling procedures, in which a single site may, at times, be used to characterize an entire population. Differences, similar to those between the turtle populations on the leeward and windward coasts of Barbados, may be found in studies of other species in island habitats when the range of sampling is expanded. The diversity uncovered by the use of a longer diagnostic sequence also illustrates the importance of new technologies in the definition and management of species. For some species, the protection of multiple sites may be needed to preserve the existing genetic diversity of populations. This is particularly important for conservation
Conserv Genet Table 2 Percentage contributions of the Barbados rookeries to Caribbean foraging grounds using the new haplotype profiles, estimated using both Maximum Likelihood (SPAM) and Bayesian (Bayes) methods Foraging population
Barbados estimated by Bowen et al. (2007)
Barbados leeward rookery
Barbados windward rookery
SPAM
SPAM
SPAM
Bayes SD
Estimate
Bayes
Estimate
SE
Mean
SE
Mean
SD
Texas, USA
0.00
0.00
0.30
0.81
0.00
0.00
0.26
0.79
Bahamas
0.00
0.00
1.53
4.25
31.90
6.09
20.06
15.50
Cuba A
0.04
0.02
2.76
7.37
32.81
33.29
12.57
19.51
Cuba B
0.00
0.00
0.77
2.17
0.01
0.02
2.16
Cuba D
0.00
0.00
0.88
2.32
0.00
0.00
1.26
Estimate
Bayes SE
Mean
SD
4.82
3.66
2.01
3.36
18.83
7.29
7.48
9.91
2.43
9.93
1.91
4.18
4.96
23.98
6.12
12.73
11.65
3.27
2.01
9.59
2.04
4.39
Dominican Rep.
0.14
0.02
15.59
21.27
44.50
5.48
43.84
8.82
21.07
8.19
8.46
10.70
Puerto Rico
0.00
0.00
1.70
4.34
14.80
7.91
7.64
8.90
37.79
6.37
20.08
19.37
US Virgin Islands
0.05
0.01
3.75
8.79
21.55
15.25
10.89
14.00
14.46
7.83
4.91
8.21
For comparative purposes, results from Bowen et al. (2007) for the Barbados contributions at these sites using the earlier haplotype profiles are also included. Details of locations of foraging grounds are found in Bowen et al. (2007)
of hawksbills in regions like the Caribbean, where management and conservation of sea turtles is often regulated through fisheries legislation that can vary from open to no take between neighboring territories. Acknowledgments We thank Anna Bass, Peter Dutton, Robin Leroux, and Erin LaCasella for providing the original 1992 samples, stored in the NOAA-Fisheries Marine Turtle tissue archive at the Southwest Fisheries Science Center, La Jolla California, for re-analysis; Patrick Leighton and the personnel of the Barbados Sea Turtle Project (UWI) who assisted with the collection of tissue samples; Emma Harrison of the Caribbean Conservation Corporation; John Candy of the Department of Fisheries and Oceans, Pacific Biological Station, British Columbia, Canada and Savita Shanker of the DNA Sequencing Core, University of Florida, Gainesville. The faculty and students of UWI’s Centre for Resource Management and Environmental Studies (CERMES) provided laboratory facilities and advice. Funding from the UWI Office of Research and a Pew Marine Conservation Fellowship to Julia Horrocks and grant FOSEMARNAT2004-01-353 from the Mexican National Science and Technology Commission (CONACYT) to Alberto Abreu-Grobois are gratefully acknowledged. We also wish to acknowledge use of the Maptool program (seaturtle.org) for map graphics in this paper.
Appendix 2 UPGMA consensus tree of genetic relationships (conventional pairwise FST values) between evaluated Caribbean hawksbill rookeries. The number on each branch indicates the percentage of times the partition of the populations into the two sets which are separated by that branch occurred among the 1,000 bootstrap trees
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Appendices
Appendix 1 Updated haplotype frequencies for Barbados rookeries from the leeward and windward coasts based on samples collected during the 2002, 2004–2005 nesting seasons, and the re-sequenced samples collected in 1992 and analyzed by Bass et al. (1996) Haplotype
A/Cu1 (EiA01)
Leeward coast
Total N
Re-sequencing of Bass et al. (1996) samples
2002 and 2004–2005 Nesting seasons
13
41
Windward coast Re-sequencing of Bass et al. (1996) samples
54
1
Total N
13
41
54
2005–2006 Nesting Seasons 2
3
6
6
2
19
21
3
27
30
F/c (EiA09) F/PR1 (EiA11)
Total N
Haplotype identification was carried out with the new primers and a sequence reading frame of 740 bp
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