Species-level diversification of African dwarf crocodiles (Genus Osteolaemus): A geographic and phylogenetic perspective

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Abstract

The taxonomy of the African dwarf crocodile (genus Osteolaemus) has been disputed since a novel morphotype was discovered in the early 20th Century. Because this poorly-known reptile is widely hunted throughout the forests of Central and West Africa, resolving the existence and extent of taxonomic units has important management and conservation implications. Lack of molecular data from individuals of known origin and historical disagreement on diagnostic morphological characters have hindered attempts to settle one of the most important taxonomic questions in the Crocodylia. In an effort to clarify the evolutionary relationships among dwarf crocodiles, we sequenced three mitochondrial and two nuclear genes using a large sample of dwarf crocodiles from known localities across major drainage basins of forested Africa. Concordant results from Bayesian, maximum likelihood, maximum parsimony and population aggregation analytical methods support a previously recognized division of the dwarf crocodile into a Congo Basin form (O. osborni) and a West African form (Osteolaemus tetraspis), but also reveal a third diagnosable lineage from West Africa warranting recognition as an separate taxonomic unit. Corrected genetic distances between geographic regions ranged from 0.2% to 0.6% in nuclear fragments and 10.0 to 16.2% in mitochondrial COI. Population aggregation, using fixed and alternate character (nucleotide) states to cluster or divide populations, recovered 232 such molecular characters in 4286 bp of sequence data and unambiguously aggregated populations into their respective geographic clade. Several previously recognized morphological differences coincide with our molecular analysis to distinguish Congo Basin crocodiles from the Ogooué Basin and West Africa. Discrete morphological characters have not yet been documented between the latter two regions, suggesting further work is needed or molecular data may be required to recognize taxonomic divisions in cases where putative species are morphologically cryptic. This study highlights the importance of using widespread taxon sampling and a multiple evidence approach to diagnose species boundaries and reveal cryptic diversity.

Introduction

The African dwarf crocodile (Crocodylidae; Osteolaemus tetraspis ssp.) is a small, secretive crocodilian restricted to wetlands in closed-canopy tropical forests of Central and West Africa (Fig. 1). The dwarf crocodile is an important food and economic resource for rural inhabitants of Central Africa. In areas of abundant swamp and seasonally-flooded forest, dwarf crocodiles constitute as much as 25% of non-fish biomass in the ‘bushmeat’ harvest (Auzel and Wilkie, 2000, Eaton, unpublished data). Human population growth, modern hunting techniques and improved transportation infrastructure in Central Africa have resulted in increased commercial trade of dwarf crocodiles and other bushmeat species within the region and, increasingly, to satisfy expatriate markets outside of Africa (Milius, 2005). The dwarf crocodile is listed as an Appendix I species under the Convention on International Trade in Endangered Species (CITES) and vulnerable (VU A2cd) in the IUCN Red Book due to a suspected decline in population size caused by habitat loss and exploitation (Crocodile Specialist Group, 1996, Ross, 1998). Most ecological studies of the dwarf crocodile have been of limited scope or duration (Kofron and Steiner, 1994, Luiselli et al., 1999, Riley and Huchzermeyer, 1999, Riley and Huchzermeyer, 2000, Pauwels et al., 2007) and the few genetic studies to date have used small sample sizes from animals of unknown origin (Densmore and White, 1991, Brochu and Densmore, 2000, Ray et al., 2000).

The taxonomy of the dwarf crocodile has been debated since the early 1900’s (Schmidt, 1919, Chabanaud, 1920, Mertens, 1943, Inger, 1948, Wermuth and Fuchs, 1983, Densmore and White, 1991, Ray et al., 2000, Brochu, 2007). The type specimen of Osteolaemus tetraspis, described by Cope (Academy of Natural Sciences of Philadelphia, 1860), originated from the “Ogobai” (= Ogooué) River basin in Gabon (Fig. 1). Almost 60 years later a new genus and species of dwarf crocodile, Osteoblepharon osborni, was described from the Ituri forest of what is now eastern Democratic Republic of Congo (Schmidt, 1919; Fig. 1). The genus Osteoblepharon was later considered to be unwarranted by several authorities (Werner, 1933, Mertens, 1943, Inger, 1948). These authors were in agreement, however, that the eastern form should be considered a valid and separate species, Osteolaemus osborni Schmidt. Subsequently, the genus Osteolaemus was reduced to a single species, with differences in morphology rejected as ontogenetic or intraspecific, and two allopatric sub-species designated as O. t. osborni (Congo Basin) and O. t. tetraspis (West Africa) (Wermuth, 1953; summarized in Savage, 1956). Some authors have suggested that variations observed in Osteolaemus represent a cline and that the recognition of sub-species is not merited (King and Burke, 1989, Ross, 2006).

In the most comprehensive molecular examination of Osteolaemus to date, 10 samples (only two of known origin) revealed two clades with relatively high levels of genetic divergence in a 350 bp concatenated fragment of the mitochondrial ND6 and cytochrome b genes (Ray et al., 2000). All individuals used in the study were classified morphologically as O. t. tetraspis, and the authors predicted that higher levels of divergence between subspecies would eventually be discovered. Recent treatment of O. osborni as a full species is based on a morphological assessment in which differences of four cranial characters are equivalent or greater than in currently recognized crocodilian species (Brochu, 2006, McAliley et al., 2006, Brochu, 2007). McAliley et al. (2006), examining the phylogeny of the genus Crocodylus, recognized both species of Osteolaemus in a morphological assessment but were only able to sequence samples of O. tetraspis for their molecular analysis. Thus, the resurrection of O. osborni as a full species has not yet been evaluated by molecular analysis using multiple gene regions and sufficient individuals of known origin.

Confusion surrounding the diagnosibility and significance of morphological characters (e.g. Wermuth, 1953) and the lack of specimens of known origin has prevented confirmation of evolutionary distinct lineages of the dwarf crocodile. This is problematic because effective conservation and management of endangered or cryptic species depends on accurate taxonomy (Goldstein et al., 2000, Frankham et al., 2002, Coulon et al., 2006, Witt et al., 2006). Defining species boundaries is also a key to our understanding of broad-scale evolutionary patterns and resolving primary units of study in ecology, biogeography and conservation biology. An inability to differentiate between population boundaries (which delimit demographic processes) and species boundaries (defining the limit of evolutionary processes) will obscure inferences gained from studies at the population or species level by failing to resolve the basic units of evolution (Sites and Marshall, 2003).

In this study, we sequenced three mitochondrial (mtDNA) and two nuclear (nuDNA) gene fragments from numerous individuals sampled from the putative range of O. t. tetraspis and O. t. osborni to test hypotheses about the phylogeny, phylogeography and taxonomy of African dwarf crocodiles. Using molecular data and a limited morphological analysis we test a hypothesis of independent evolution of geographic lineages against a null hypothesis that the dwarf crocodile represents a single, panmictic species across forested Africa. Our a priori geographic groups include the Congo Basin (Rep. of Congo and Dem. Rep. of Congo), the greater Ogooué Basin (Gabon, Equatorial Guinea and southern Cameroon) and western Africa (west of the Cameroonian Highlands) (Fig. 1). Our results provide convincing evidence for three evolutionary divergent lineages of Osteolaemus and we propose a new hypothesis that each is distinguishable as a unique species representing the smallest diagnosable phylogenetic unit (Cracraft, 1983, Tattersall and Mowbray, 2005).

Section snippets

Sampling, DNA sequencing and alignment

Samples from wild-caught and locally hunted crocodiles were collected by MJE in Loango N.P. and Mayumba N.P. in the Republic of Gabon and from the Lac Télé Community Reserve in the Republic of Congo (LTCR) (Fig. 1). Wild-caught animals were captured, sexed, measured, marked and released as part of a broader research program on the ecology and population dynamics of Central African crocodiles (Eaton, 2006). Caudal scute clippings, used to individually mark animals, were collected for use in

Tree-based phylogenetic analysis

We sequenced 80–82 individuals for each of the three mtDNA regions and 45 and 57 individuals for RAG-1 and LDH-A nuclear fragments, respectively (Table 2). All representative haplotypes of the five gene fragments were submitted to GenBank under Accession Nos. FJ390066FJ390106. Corrected genetic distances between the Congo and Ogooué Basins varied from 0.4% for the LDH-A and RAG-1 nuclear genes, to 4.7% and 11.2% for mitochondrial 12S and COI, respectively (Table 3). Genetic distance between

Discussion

Comparing DNA sequences from organelle and independent nuclear genes is the most reliable way to avoid the ‘gene tree’ versus ‘species tree’ problem (Zhang and Hewitt, 2003). Our analysis of 4268 bp from both the nuclear and mitochondrial genomes of 82 individuals sampled from 10 localities across a large portion of the range of Osteolaemus provides convincing evidence for at least three divergent lineages of the African dwarf crocodile. These results offer resolution to a taxonomic debate that

Acknowledgments

This work was supported by the American Museum of Natural History, the National Geographic Society’s Committee for Research and Exploration, the Wildlife Conservation Society (WCS), Lincoln Park Zoo’s Conservation and Science Department, the Rufford Foundation, the Natural History Museum and the Rozella Smith Fellowship of the University of Colorado. Logistic and additional financial support in the field was provided by R. Swanborn, the Société de Conservation et Développement (SCD) and WCS’s

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