Crocodilian phylogeny inferred from twelve mitochondrial protein-coding genes, with new complete mitochondrial genomic sequences for Crocodylus acutus and Crocodylus novaeguineae

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Abstract

We report complete mitochondrial genomic sequences for Crocodylus acutus and Crocodylus novaeguineae, whose gene orders match those of other crocodilians. Phylogenetic analyses based on the sequences of 12 mitochondrial protein-coding genes support monophyly of two crocodilian taxonomic families, Alligatoridae (genera Alligator, Caiman, and Paleosuchus) and Crocodylidae (genera Crocodylus, Gavialis, Mecistops, Osteolaemus, and Tomistoma). Our results are consistent with monophyly of all crocodilian genera. Within Alligatoridae, genus Alligator is the sister taxon of a clade comprising Caiman and Paleosuchus. Within Crocodylidae, the basal phylogenetic split separates a clade comprising Gavialis and Tomistoma from a clade comprising Crocodylus, Mecistops, and Osteolaemus. Mecistops and Osteolaemus form the sister taxon to Crocodylus. Within Crocodylus, we sampled five Indopacific species, whose phylogenetic ordering is ((C. mindorensis, C. novaeguineae), (C. porosus, (C. siamensis, C. palustris))). The African species C. niloticus and New World species C. acutus form the sister taxon to the Indopacific species, although our sampling lacks three other New World species and an Australian species of Crocodylus.

Highlights

► Two complete mt genomes were sequenced in Crocodylus acutus and C. novaeguineae. ► There were 16 crocodylian species included in the phylogenetic analysis. ► ML and BI trees were reconstructed based on 12 protein-coding genes. ► The results revealed the monophyly of Crocodylus and of the Indopacific group.

Introduction

The American crocodile (Crocodylus acutus) is a crocodylian species found primarily in Central America. It grows faster than the American alligators and is much more tolerant of salt water. The New Guinea crocodile (Crocodylus novaeguineae) is a small species of crocodile found on the island of New Guinea.

Numerous molecular datasets have been compiled to examine intergeneric phylogenetic relationships among crocodylians (Brochu and Densmore, 2001, Gatesy and Amato, 1992, Gatesy et al., 1993, Gatesy et al., 2003, Gatesy et al., 2004, Harshman et al., 2003, McAliley et al., 2006, Ray and Densmore, 2002, White and Densmore, 2001, Willis et al., 2007). The main controversies focus on the taxonomic status of the African slender-snouted crocodile (Mecistops cataphractus), the dwarf crocodile (Osteolaemus tetraspis), the false gharial (Tomistoma schlegelii) and Gavialis gangeticus. The three primary families commonly recognized within the Crocodylia were the Alligatoridae (Alligator, Caiman, Melanosuchus and Paleosuchus), the Crocodylidae (Crocodylus, Osteolaemus and Tomistoma) and the Gavialidae (including only one species, G. gangeticus). Morphological comparisons of extant and fossil samples consistently placed T. schlegelii within the Crocodylidae (Brochu, 1997, Norell, 1989, Vélez-Juarbe et al., 2007) and many previous studies placed G. gangeticus in a separate family, Gavialidae (Brochu, 1997, Dessauer and Densmore, 1983, Gatesy et al., 2004). However, every molecular analysis revealed the sister-group relationships between G. gangeticus and T. schlegelii (Densmore, 1983, Densmore and Owen, 1989, Densmore and White, 1991, Feng et al., 2010, Gatesy and Amato, 1992, Gatesy and Amato, 2008, Gatesy et al., 2003, Hass et al., 1992, Harshman et al., 2003, Janke et al., 2005, Ji et al., 2006, Li et al., 2007, Meganathan et al., 2010, Roos et al., 2007, White and Densmore, 2001, Willis et al., 2007). The African slender-snouted crocodile was alternatively placed in Crocodylus (Norell, 1989) or isolated as a new genus, Mecistops (Feng et al., 2010, Gatesy and Amato, 2008, Li et al., 2007, McAliley et al., 2006, Willis et al., 2007, Willis, 2009). Mecistops was alternatively grouped with Crocodylus (Brochu, 2000, Densmore and White, 1991, Li et al., 2007) or with the African dwarf crocodile (O. tetraspis) (Feng et al., 2010, Gatesy et al., 2003, White and Densmore, 2001, Willis, 2009).

Phylogenetic relationships among the species of traditional Crocodylus have received less attention, and many species lack complete mitochondrial genomic sequence data. Molecular phylogenetic studies within Crocodylus often reveal contrasting relationships (Feng et al., 2010, Gatesy and Amato, 2008, Ji et al., 2006, Li et al., 2007, McAliley et al., 2006, Meganathan et al., 2010, Roos et al., 2007, Willis, 2009). Here, we sequenced the complete mitochondrial genomes (mtDNA) of C. acutus and C. novaeguineae to evaluate the phylogenetic relationships in Crocodylus. Our phylogenetic analyses use 12 mitochondrial protein-coding genes for five Indopacific species (C. mindorensis, C. novaeguineae, C. porosus, C. siamensis and C. palustris), one New World species (C. acutus) and C. niloticus. We focus our discussion of Crocodylus relationships on the Indopacific group (Brochu, 2000, Meganathan et al., 2010).

Section snippets

Samples and DNA extraction

The blood samples of C. acutus and C. novaeguineae were acquired from the specimen storeroom of the laboratory in Anhui Normal University (presented by Dr. George Amato from AMNH), and were stored at −80 °C. The mtDNA was extracted following the procedure described by Arnason et al. (1991). Extracted DNA was diluted 20 times with doubly distilled water and stored at −20 °C.

PCR amplification and sequence assembling

The mtDNA sequences of Crocodylus niloticus, Crocodylus porosus, Crocodylus siamensis and M. cataphractus (Genbank accession

Genomes and gene arrangement

The length of the complete mtDNA nucleotide sequences of C. acutus and C. novaeguineae are 16,883 and 16,894 bp, respectively. As expected, the gene arrangements of the molecule conform to those of other crocodilians, differing from the typical vertebrate gene arrangement (Feng et al., 2010, Ji et al., 2006, Li et al., 2007, Zhong et al., 2005, Zhang et al., 2010). Both of them contain 13 protein-coding genes, two ribosomal RNAs (12S rRNA and 16S rRNA), 22 transfer RNAs and a putative control

Gene features

The organization of the genomes is consistent with other crocodylian mt genomes published previously (Fig. 1). In contrast to the traditional vertebrate mitochondrial genomes, such gene features as, TPF (tRNAThr–tRNAPro–tRNAPhe) and SHL (tRNASer(AGY)–tRNAHis–tRNALeu) clusters, base composition, tRNA structures, tandemly repeated sequences and the unconspicuous OL are all common in crocodilian mt genomes. The same gene order and gene rearrangement illustrate again that the mtDNA of crocodilians

Acknowledgments

We greatly appreciate the crocodile samples collected by Dr. George Amato from American Museum of Natural History. This work was supported by the National Natural Science Found of China (No. 30470244), Specialized Research Fund for the Doctoral Program of Higher Education (20070370002), the special Fund for Excellent Creative Research Team of Animal Biology in Anhui Normal University, and Provincial Key Laboratory of Biotic Environment and Ecological Safety in Anhui.

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