Simandou Project(Guinea)

Transcription

1 Simandou Project (Guinea) Port Component Crocodiles Complementary report Genetic analysis Soumbouya Kakossa Morebaya Kabak Forécariah Ouiné Mabala RIO TINTO Final Report January 2013

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3 Simandou Project(Guinea) Port Component Crocodiles Complementary report Genetic analysis Presented to: By: SNC-Lavalin Environment Environnement Illimité inc. January 2013

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5 WORK TEAM SNC-Lavalin Environment Project Director: Project Manager: Richard Fontaine, Engineer Chantal Roy Environnement Illimité inc. Project Director: Project Manager: Analysis and Report: Marc Gendron Patrick Lafrance Matthew H. Shirley Carto-Média and En Toutes Lettres Word Processing and Editing: GIS and Computer Graphics: Marie-Josée Allie Lise Blais Manel Besbes Daniel Cloutier Gabriel Morency-Parent Pier-Olivier Tremblay Simandou Project (Guinea) Port Component Crocodiles Complementary report Genetic analysis i

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7 TABLE OF CONTENTS WORK TEAM... I 1 INTRODUCTION OBJECTIVES METHOD RESULTS DISCUSSION REFERENCES List of Figures FIGURE 1 FIGURE 2 Example SNP Table. Here we present an example alignment of 16 bases for 7 individuals. Visible are two variable sites (position 5 and 8), fixed for a different nucleotide in individuals 1 5 and 6 8. DNA barcoding would then group these two sets of individuals as unique species or evolutionarily significant units Distance-based Relationships Amongst Crocodile Taxa. UPGMA trees illustrating distance-based, evolutionary relationships amongst Osteolaemus (top) and African Crocodylus (bottom) lineages. The tree is drawn to scale, with branch lengths (above the branches) illustrated, total divergence between taxa equal the sum of branches between them Simandou Project (Guinea) Port Component Crocodiles Complementary report Genetic analysis iii

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9 1 INTRODUCTION Two species of crocodile were recently identified as occurring in the study area of the Simandou Project deepwater port located in the Forécariah prefecture 1 (see Environnement Illimité 2013). Present governing policies (e.g., IUCN, CITES) recognize these as Crocodylus niloticus (IUCN Lower Risk/Least Concern) and Osteolaemus tetraspis (IUCN Vulnerable), but new species determinations are forthcoming. Until recently only three species of crocodile were recognized as occurring in Africa: Nile (Crocodylus niloticus), African dwarf (Osteolaemus tetraspis), and slender-snouted (Mecistops cataphractus). While the Nile crocodile may be considered one of the best known crocodilians from an ecology and management standpoint, the evolutionary history of all three species has long been the subject of investigation (see Eaton et al. 2009, Eaton 2010, Fergusson 2010, Hekkala et al for a review). Since the mid-19 th century, as many as five and seven distinct species have been described in the African dwarf and Nile crocodiles, respectively, and recent phylogenetic analyses support the resurrection of certain taxa as cryptic species lineages within each of these species (Eaton et al. 2009, Hekkala et al. 2011). In an effort to test the hypothesis that there were two different lineages divergent at the species level within Osteolaemus, Eaton et al. (2009) sampled dwarf crocodiles from the Congo, Ogooué, and Upper Guinea bioregions and sequenced individuals for three mitochondrial and two nuclear gene regions. Using a suite of standard phylogenetic analyses, they not only found support for O. osborni as a valid taxon restricted to the Congo Basin but also found support for a third, less widely discussed taxon from West Africa O. sp. nov. cf. tetraspis. While more work remains to delimit the exact distribution and taxonomy of this West African species, preliminary evidence suggests it is distributed from Mt. Cameroon westwards and that O. frontatus is the priority name of this species (Eaton et al. 2009, Eaton 2010, Shirley unpub. data). Among the more striking aspects of their results is the diagnosability of the three lineages with simple molecular tools (e.g., DNA barcoding) due to the high inter versus intra-species molecular divergence. All three taxa are incredibly homogeneous exhibiting less than 0.4% intraspecies sequence diversity (compared to 10-16% interspecies divergence) even where compared samples represented hundreds, sometimes thousands, of kilometers separation. Subsequent work from the western extent of the distribution of the newly recognized West African taxon, however, revealed the presence of a possible evolutionarily significant unit (ESU) in Senegambia divergent at around 2% from the rest of West Africa (Shirley, unpub. data). Though, the full extent of the distribution of this ESU is not yet understood due to limited sampling in the region. Unlike for the dwarf crocodile, previous molecular evidence existed possibly supporting the presence of two, highly divergent lineages within the Nile crocodile though these results were based on only a single mitochondrial gene from few individuals (Schmitz et al. 2003). To test the validity of these findings more fully, Hekkala et al. (2011) collected samples from known wild individuals distributed throughout the range of this species and sequenced them for five mitochondrial and four nuclear gene regions. Using a suite of standard phylogenetic analyses, they not only found that the Nile crocodile was comprised of two highly divergent 1 The names of some of the villages mentioned in this report may differ from the official name list established by the project. Simandou Project (Guinea) Port Component Crocodiles Complementary report Genetic analysis 1

10 lineages, but that these two lineages were paraphyletic with respect to the four New World crocodilians in other words they aren t even sister species. Further support of these results came from the two lineages having highly divergent karyotypes (n = 34 versus n = 32 chromosomes). Using ancient DNA extracted from crocodile mummies, Hekkala et al. (2011) demonstrated that the taxon for the newly recognized species is likely C. suchus described from Egyptian crocodile mummies by Geoffrey St. Hilaire (1807). While further works remain to delimit the distributional boundaries of these two species, the traditional Nile crocodile (C. niloticus) is largely distributed in East and southern Africa with small populations in coastal Central Africa (e.g., Gabon), while C. suchus is largely distributed throughout the Congo Basin and West Africa. The conservation ramifications of splitting these taxa into multiple species are quite severe. For example, by recognizing the presence of allopatrically distributed species within what was once a single, widespread taxon we are immediately reducing the population size of all species, though the impact of this population reduction is not spread evenly amongst them. For example, Nile crocodile populations throughout East and southern Africa are quite stable, even capable of sustaining commercial harvest (Fergusson 2010); however, a suite of anthropogenic threats has not only severely diminished the Nile crocodile population in West Africa, but is working to ensure that future positive growth is unlikely (Shirley et al. 2009). Similarly for the dwarf crocodiles, populations in the Ogooué and Congo Basins, while certainly experiencing declines, are also seemingly robust to the threats facing them today while rampant deforestation has decimated and highly fragmented populations throughout West Africa (Eaton 2010, Shirley et al. 2009). While none of these newly named taxa are yet officially evaluated for the IUCN Red List, it is likely that at least the two in West Africa will qualify for an elevated status beyond that of their parent taxa likely Endangered or above for O. sp. nov. cf. tetraspis (as opposed to the current listing of Vulnerable for O. tetraspis) and Vulnerable or above for C. suchus (as opposed to the current listing of LR/lc for C. niloticus). 2 Simandou Project (Guinea) Port Component Crocodiles Complementary report Genetic analysis

11 2 OBJECTIVES In light of this revised taxonomic understanding in African crocodiles, it is first necessary to identify which species of crocodile are present along the Guinea coast in order to best contextualize the results of crocodile surveys for the Simandou port project. While it would not be unreasonable to a priori assume that they are O. sp. nov. cf. tetraspis and C. suchus, the Republic of Guinea is geographically positioned to help further clarify as yet unanswered questions regarding the distribution of these species. For example, it is as yet unclear whether the Senegambia ESU of the West African dwarf crocodile is restricted to Senegambia or if it extends into, at least coastal, Guinea. Additionally, the presence of C. niloticus haplotypes in historic samples supposedly collected in Senegal suggests that this species was once further distributed in West Africa and Hekkala et al. (2011) posited that a route of coastal dispersal was most likely suggesting that coastal Crocodylus in Guinea may not actually be C. suchus. To better clarify the species status of crocodiles in Guinea, samples were opportunistically collected largely from dead or captive individuals and sequenced for comparison to the results of Eaton et al. (2009a, b) and Shirley (unpub. data). Simandou Project (Guinea) Port Component Crocodiles Complementary report Genetic analysis 3

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13 3 METHOD A full treatment of the molecular methods used is beyond the scope of this report; however, we provide a summary herein. Tissues samples were collected opportunistically from crocodiles found dead during crocodile surveys or captive individuals encountered on Kabak Island and around Conakry. Whole, genomic DNA was extracted using standard phenol:chloroform methods. A ± 600 bp fragment of the cytochrome c oxidase subunit I (COI) gene was amplified using the polymerase chain reaction (PCR) and cycle sequenced following the protocol outlined in (Eaton et al. 2009b). The COI gene is universally regarded as the most appropriate gene for vertebrate species recognition through a DNA barcoding approach (Hebert et al. 2003) and has been shown to robustly distinguish crocodilian species (Eaton et al. 2009b). Sequences were analyzed on an ABI 3700 automated capillary sequencer. Base calling was performed with Sequencher v4.1 (Genecodes Corp.). Forward and reverse sequences were assembled into contigs and base calls verified by eye. Osteolaemus sequences from Guinea were aligned with those from Eaton et al. (2009a) and Shirley (unpub. data Senegambia). Only limited COI data for the two African Crocodylus were previously published in Eaton et al. (2009b) and Hekkala et al. (2011) did not use this gene region. Therefore, in order to best diagnose the identity of these samples, we amplified COI in an extensive set of wild caught Crocodylus samples, including those individuals used by Hekkala et al., with which the Guinea individuals were aligned. A distance-based analysis was run to create a UPGMA tree showing clusters of individuals based on molecular divergence under the Kimura 2-parameter model of nucleotide substitution (Eaton et al. 2009a, Kimura 1980, Sneath and Sokal 1973, Tamura et al. 2011). Sequence alignments were searched for the presence of variable sites fixed for different nucleotides (SNP s) in each different species for diagnosis at the species, or ESU, level (Fig. 1). FIGURE 1 Example SNP Table. Here we present an example alignment of 16 bases for 7 individuals. Visible are two variable sites (position 5 and 8), fixed for a different nucleotide in individuals 1 5 and 6 8. DNA barcoding would then group these two sets of individuals as unique species or evolutionarily significant units. Simandou Project (Guinea) Port Component Crocodiles Complementary report Genetic analysis 5

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15 4 RESULTS Distance-based analysis showed an average 10.0% sequence divergence between O. tetraspis and O. sp. nov. cf. tetraspis and a further 2.5% divergence of the Senegambia ESU from the rest of West African animals (Fig. 2). Search of the alignment revealed 77 SNP s segregating O. tetraspis from O. sp. nov. cf. tetraspis and a further 23 SNP s distinguishing the Senegambian ESU within the West African clade. Osteolaemus samples collected in Guinea were nested well within expectations for O. sp. nov. cf. tetraspis and were fixed for a haplotype unique to Guinea, and Casamance of the Senegambia region, distinguished from other Senegambian haplotypes by 6 SNP s. Distance-based analysis showed an average 5.5% sequence divergence between C. niloticus from C. suchus (Fig. 2). Search of the Crocodylus alignment showed 29 SNP s segregating C. niloticus from C. suchus. Crocodylus samples collected in Guinea were nested well within the expectations for C. suchus and were fixed for a haplotype unique to individuals from the coast within the area of the Senegambia ESU identified for Osteolaemus, distinguished from other C. suchus haplotypes by a single SNP. Simandou Project (Guinea) Port Component Crocodiles Complementary report Genetic analysis 7

16 FIGURE 2 Distance-based Relationships Amongst Crocodile Taxa. UPGMA trees illustrating distance-based, evolutionary relationships amongst Osteolaemus (top) and African Crocodylus (bottom) lineages. The tree is drawn to scale, with branch lengths (above the branches) illustrated, total divergence between taxa equal the sum of branches between them. 8 Simandou Project (Guinea) Port Component Crocodiles Complementary report Genetic analysis

17 5 DISCUSSION These results are significant for several reasons. First, they confirm a priori expectations that the taxa present along the Guinea coast are O. sp. nov. cf. tetraspis and C. suchus. This may seem an obvious, if not trivial, result. However, confirmation of species identity within the study area was a critical step to verifying the relevance of our survey results to those previously published for the different crocodilian taxa in Africa. For example, comparison of our survey results with those from coastal C. niloticus or O. tetraspis populations may be somewhat informative because these taxa occupy similar ecological niches; however, they have different histories of interaction with local communities throughout their relative distributions and therefore comparisons may not allow strong inference of population trends and other lessons learned. Second, these results further enable us to delimit the extent of the Senegambian ESU within the West African dwarf crocodile. Though the exact provenance of our Guinean Osteolaemus sample is unknown (it was collected from a captive individual in Conakry), we feel confident that the individual is of Guinean origin and, most likely, from close proximity to Conakry. Therefore, the geographic distribution of the Senegambian ESU encompasses, at a minimum, the Guinea coast. Because of this, Osteolaemus populations in the study area are critical to the maintenance of genetic diversity, and therefore evolutionary potential, within the West Africa dwarf crocodiles and should be considered conservation priorities (e.g., Crandall et al. 2000, Fraser and Bernatchez 2001). Finally, the presence of C. suchus to the apparent exclusion of C. niloticus along the Guinea coast provides new evidence in the debate regarding the extent of the historic presence of the latter species in West Africa. Two individuals supposedly collected in Senegal near Dakar in the early 19 th century showed ancient DNA mitochondrial haplotypes consistent with contemporary C. niloticus leading Hekkala et al. (2011) to hypothesize routes of coastal dispersal from Central Africa as the most likely means of connection to the nearest C. niloticus populations. However, no contemporary samples analyzed to date support this hypothesis calling into question the true origin of the analyzed historic specimens. Simandou Project (Guinea) Port Component Crocodiles Complementary report Genetic analysis 9

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19 6 REFERENCES CRANDALL, K. A., O. R. P. BININDA-EMONDS, G. M. MACE, AND R. K. WAYNE Considering evolutionary processes in conservation biology. Trends in Ecology & Evolution 15: EATON, M J, A. MARTIN, J THORBJARNARSON, AND G AMATO Species-level diversification of African dwarf crocodiles (Genus Osteolaemus): A geographic and phylogenetic perspective. Molecular Phylogenetics and Evolution 50: EATON, MITCHELL J Dwarf Crocodile Osteolaemus tetraspis in S. C. Manolis and C. Stevenson (Eds). Crocodiles. Status Survey and Conservation Action Plan, Third Edit. Crocodile Specialist Group: Darwin. ENVIRONNEMENT ILLIMITE INC Baseline environmental report for crocodiles Kabak Island area (Guinea) (unpublished report). EATON, MITCHELL J., G. L. MEYERS, S.-O. KOLOKOTRONIS, M. S. LESLIE, A. P. MARTIN, AND GEORGE AMATO Barcoding bushmeat: molecular identification of Central African and South American harvested vertebrates. Conservation Genetics 11: FERGUSSON, R. A Nile crocodile Crocodylus niloticus in S. Manolis and C. Stevenson, (Eds). Crocodiles. Status Survey and Conservation Action Plan, Third Edit. Crocodile Specialist Group, Darwin. FRASER, D. J., AND L. BERNATCHEZ Adaptive evolutionary conservation: towards a unified concept for defining conservation units. Molecular Ecology 10: HEBERT, P. D. N., A. CYWINSKA, S. L. BALL, AND J. R. DE WAARD Biological identifications through DNA barcodes. Proceedings of the Royal Society B: Biological Sciences 270: HEKKALA, E., M. H. SHIRLEY, GEORGE AMATO, J. D. AUSTIN, S. CHARTER, JOHN THORBJARNARSON, K. A. VLIET, M. L. HOUCK, R. DESALLE, AND M. J. BLUM An ancient icon reveals new mysteries: mummy DNA resurrects a cryptic species within the Nile crocodile. Molecular ecology 20: KIMURA, M A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16: Simandou Project (Guinea) Port Component Crocodiles Complementary report Genetic analysis 11

20 SCHMITZ, A., P. MAUSFELD, E. HEKKALA, T. SHINE, H. NICKEL, GEORGE AMATO, AND W. BÖHME Molecular evidence for species level divergence in African Nile Crocodiles Crocodylus niloticus (Laurenti, 1786). C. R. Palevol 2: SHIRLEY, M. H., W. ODURO, AND H. Y. BEIBRO Conservation status of crocodiles in Ghana and Côte-d Ivoire, West Africa. Oryx 43:136. SNEATH, P. H. A., AND R. R. SOKAL Numerical Taxonomy. Freeman, San Francisco. TAMURA, K., D. PETERSON, N. PETERSON, G. STECHER, M. NEI, AND S. KUMAR MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: Simandou Project (Guinea) Port Component Crocodiles Complementary report Genetic analysis