Are crocodiles really monophyletic?-Evidence for subdivisions from sequence and morphological dataIntroductionMolecular methodsBlood collection and DNA extractionSampling protocolsC-mos sequencesODC sequencesPCR amplification of nuclear genesND6-tRNAglu-cytB sequencesMitochondrial control region sequencesCycle or manual DNA sequencingData analysesMorphological methodsInstitutional abbreviationsResultsC-mosODCMitochondrial ND6-tRNAglu-cytB regionMitochondrialMitochondrial control regionConcatenated sequencesMorphological dataUnconstrained morphological analysisConstrained morphological analysisDiscussionAcknowledgmentsTaxa used in morphological analysisReferencesMolecular Phylogenetics and Evolution 39 (2006) 16–32www.elsevier.com/locate/ympev1055-7903/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.ympev.2006.01.012Are crocodiles really monophyletic?—Evidence for subdivisions from sequence and morphological dataL. Rex McAlileya,¤, Ray E. Willisa, David A. Raya,1, P. Scott Whiteb, Christopher A. Brochuc, Llewellyn D. Densmore IIIaa Department of Biological Sciences, Texas Tech University, P.O. Box 43131, Lubbock, TX 79409-313, USAb Genetic Variation Initiative, MailStopM888, Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USAc Department of Geoscience, University of Iowa, Iowa City, IA 52242, USAReceived 2 February 2005; revised 9 January 2006; accepted 10 January 2006Available online 21 February 2006AbstractRecently, the phylogenetic placement of the African slender snouted crocodile, Crocodylus cataphractus, has come under scrutiny andherein we address this issue using molecular and morphological techniques. Although it is often recognized as being a “basal” form, mor-phological studies have traditionally placed C. cataphractus within the genus Crocodylus, while molecular studies have suggested that C.cataphractus is very distinct from other Crocodylus. To address the relationship of this species to its congeners we have sequenced por-tions of two nuclear genes (C-mos 302 bp and ODC 294 bp), and two mitochondrial genes (ND6-tRNAglu-cytB 347 bp and control region457 bp). Analyses of these molecular datasets, both as individual gene sequences and as concatenated sequences, support the hypothesisthat C. cataphractus is not a member of Crocodylus or Osteolaemus. Examination of 165 morphological characters supports and strength-ens our resurrection of an historic genus, Mecistops (Gray 1844) for cataphractus.© 2006 Elsevier Inc. All rights reserved.Keywords: Crocodylus; Crocodylus cataphractus; C-mos; ODC; Mitochondrial ND6; Mitochondrial control region; Systematics; Mecistops1. IntroductionThe African slender-snouted crocodile, Crocodyluscataphractus, has long been a systematic enigma. In one ofthe earliest systematic treatments of what is now calledCrocodylia, Gmelin (1789) indicated that the habitat forLacerta gangeticus (now Gavialis gangeticus) included riv-ers in “Senegal Africae et Gangen Indiae.” Gavialis hasbeen restricted to the Indian subcontinent throughout his-torical times, but it is clear from Gmelin’s diagnosis thatC. cataphractus, the crocodylian from Senegal with an elon-gate, subcylindrical rostrum, would have fallen withinL. gangeticus, highlighting the morphological gulf betweenC. cataphractus and other Crocodylus, reinforcing the needfor further systematic analysis.Various members of the genus Crocodylus (the true croc-odiles) have been included in a number of phylogeneticstudies, but until recently, very little had been written aboutrelationships within Crocodylus. That the name “Crocody-lus” lacked a uniform meaning renders comparisons ofdiVerent scenarios virtually impossible. Neontologists werenecessarily restricted to the 12 recognized living species, butpaleontologists assigned fossils ranging throughout theCenozoic and into the Mesozoic (sometimes as old as theAlbian stage of the Cretaceous, between 99 and 112 mya) toCrocodylus (Markwick, 1998; Steel, 1973). Explicit diagno-ses not reliant on overall head shape were rarely used, andCrocodylus was often a default category that simply meanta fossil could not be unambiguously assigned to some other*Corresponding author. Fax: +1 806 742 2369.E-mail address: [email protected] (L.R. McAliley).1Present Address: Department of Biology, West Virginia University,53 Campus Dr. Morgantown, WV 26505, USA.L.R. McAliley et al. / Molecular Phylogenetics and Evolution 39 (2006) 16–32 17genus. Several authors have suggested that the African slen-der-snouted crocodile (C. cataphractus) is the sister taxonto a clade comprising all other members of this genus (Bro-chu, 1997, 2000; Densmore, 1983; Densmore and Owen,1989; Gatesy et al., 2003; Gatesy et al., 2004; White andDensmore, 2000). Most of these studies have suVered fromlimited taxon sampling and/or few representative individu-als from the species being compared. To date there havebeen few studies aimed speciWcally at the relationship ofthis species to its congeners and no molecular studies.Recently, Schmitz et al. (2003) in a study on genetic varia-tion within the Nile crocodile, C. niloticus, suggested that C.cataphractus formed a relationship outside the remainderof Crocodylus. However, this portion of their studyincluded just a single C. cataphractus sample, two dwarfAfrican crocodile samples (Osteolaemus tetraspis) and onlythree of the eleven recognized extant species of Crocodylus(C. cataphractus, C. jonstoni, and C. niloticus).Herein, using more thorough taxon sampling and muchlarger sample sizes (especially for the nuclear genesequences), we report sequence comparisons from both cod-ing and non-coding regions of two nuclear protein-codinggenes and from two diVerent regions of the mitochondrialgenome (also representing both coding and non-codingsequences) speciWcally to assess the relationship of C. cataph-ractus to other members of Crocodylus and to Osteolaemus.The two nuclear markers sequenced for this study arethe proto-oncogene C-mos and the gene that codes for orni-thine decarboxylase (ODC). C-mos is a single-copy geneslightly over 1000 bp in length, contains no introns andcodes for a protein (C-mos) involved in oocyte maturationduring meiotic metaphase II (Saint et al., 1998; Yew et al.,1993). Due to its relative high degree of conservation, thisgene provided the resolution necessary to examine genericlevel relationships within the Crocodylia.The ODC gene codes for a