Evolutionary Shifts in Three Major Structural Features of the Mitochondrial Genome Among Iguanian Lizards
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J Mol Evol (1997) 44:660–674 © Springer-Verlag New York Inc. 1997 Evolutionary Shifts in Three Major Structural Features of the Mitochondrial Genome Among Iguanian Lizards J. Robert Macey,1 Allan Larson,1 Natalia B. Ananjeva,2 Theodore J. Papenfuss3 1Department of Biology, Box 1137, Washington University, St. Louis, MO 63130, USA 2Zoological Institute, Russian Academy of Sciences, St. Petersburg, Russia 3Museum of Vertebrate Zoology, University of California, Berkeley, CA 94720, USA Received: 28 May 1996 / Accepted: 27 December 1996 Abstract. A phylogenetic tree for major lineages of the origin of light-strand replication (OL), and secondary iguanian lizards is estimated from 1,488 aligned base structure of tRNAs (for review see Macey et al. 1997a,b). positions (858 informative) of newly reported mitochon- Both shifts in gene order and the alterations of tRNA drial DNA sequences representing coding regions for secondary structure have been suggested to result from eight tRNAs, ND2, and portions of ND1 and COI. Two errors in DNA replication (Macey et al. 1997a,b). Igua- well-supported groups are defined, the Acrodonta and nian lizards, a group that dates at least to the fragmen- the Iguanidae (sensu lato). This phylogenetic hypothesis tation of Gondwanaland (Estes 1983), show variation for is used to investigate evolutionary shifts in mitochondrial these three major structural features of the mitochondrial gene order, origin for light-strand replication, and sec- genome and provide an opportunity to examine the tim- ondary structure of tRNACys. These three characters shift ing and relative phylogenetic positions of shifts in these together on the branch leading to acrodont lizards. Plate characters. tectonics and the fossil record indicate that these char- Derived states of the three mitochondrial characters acters changed in the Jurassic. We propose that changes have been reported for an agamid lizard (Acrodonta: to the secondary structure of tRNACys may destroy func- Uromastix acanthinurus) (Macey et al. 1997a,b). This tion of the origin for light-strand replication which, in lizard contains a unique gene arrangement in which the turn, may facilitate shifts in gene order. tRNAIle and tRNAGln genes are switched in order rela- tive to the more typical vertebrate arrangement. The ori- Key words: Reptilia — Sauria — Iguania — Gene gin for light-strand replication (OL) is absent in Uromas- organization — Cystein transfer RNA — Mitochondrial tix from the usual vertebrate position between the DNA — Phylogenetics tRNAAsn and tRNACys genes, and tRNACys lacks a di- hydrouridine (D) stem and instead contains a D-arm re- placement loop (Macey et al. 1997a,b). These novel fea- tures were not observed in an iguanid lizard, Sceloporus Introduction occidentalis (Kumazawa and Nishida 1995). See Appen- dix 1 for taxonomy used. Structural organization of the vertebrate mitochondrial Iguanian lizards comprise eight iguanid (sensu lato) genome is evolutionarily stable except for three charac- groups (Etheridge and de Queiroz 1988) and four acro- teristics that are known to vary: gene order, position for dont groups (Frost and Etheridge 1989; Moody 1980). Systematics of iguanian lizards has been highly contro- versial, and the monophyly of the Iguanidae (sensu lato) with respect to the Acrodonta has been questioned (Frost Correspondence to: A. Larson; e-mail [email protected] and Etheridge 1989). With iguanian phylogenetics un- 661 certain, the position for the evolutionary shift of the three Table 1. Primers used in this studya mitochondrial characters remains unclear. We assessed the order of genes for one-third of the Human position Gene Sequence mitochondrial genome, located between ND1 and COI, and the presence or absence of a recognizable OL in L3002 16S 58-TACGACCTCGATGTTGGATCAGG-38 representatives of all major iguanian lineages. The evo- L3881 ND1 58-TTTGACCTAACAGAAGGAGA-38 lution of these characters and the secondary structural L4160 ND1 58-CGATTCCGATATGACCARCT-38 alternatives for tRNACys (Macey et al. 1997b) were ex- L4178a ND1 58-CARCTWATACACYTACTATGAAA-38 L4178b ND1 58-CAACTAATACACCTACTATGAAA-38 amined on a phylogenetic estimate derived from 1,488 L4221 tRNAIle 58-AAGGATTACTTTGATAGAGT-38 bases of aligned sequence corresponding to genes for H4419a tRNAMet 58-GGTATGAGCCCAATTGCTT-38 eight tRNAs, ND2, and portions of ND1 and COI. H4419b tRNAMet 58-GGTATGAGCCCGATAGCTT-38 L4437a tRNAMet 58-AAGCTTTCGGGCCCATACC-38 L4437b tRNAMet 58-AAGCAGTTGGGCCCATRCC-38 L4645 ND2 58-ACAGAAGCCGCAACAAAATA-38 Methods L4831 ND2 58-TGACTTCCAGAAGTAATACAAGG-38 H4980 ND2 58-ATTTTTCGTAGTTGGGTTTGRTT-38 L5002 ND2 58-AACCAAACCCAACTACGAAAAAT-38 Specimen Information. Museum numbers and approximate localities H5540 tRNATrp 58-TTTAGGGCTTTGAAGGC-38 for voucher specimens from which DNA was extracted and GenBank L5556 tRNATrp 58-GCCTTCAAAGCCCTAAA-38 accession numbers are presented in phylogenetic order. Acronyms L5551 tRNATrp 58-GACCAAAGGCCTTCAAAGCC-38 are CAS for California Academy of Sciences, San Francisco; MVZ for H5617a tRNAAla 58-AAAATRTCTGRGTTGCATTCAG-38 Museum of Vertebrate Zoology, University of California at Berkeley; H5617b tRNAAla 58-AAAGTGTCTGAGTTGCATTCAG-38 MZUSP for Museu de Zoologia, University of Sa˜o Paulo, Brazil; L5638a tRNAAla 58-CTGAATGCAACYCAGAYATTTT-38 TCWC for the Texas Cooperative Wildlife Collection, Texas A&M L5638b tRNAAla 58-CTGAATGCAACTCAGACACTTT-38 University; and USNM for United States National Museum, Washing- H5692 tRNAAsn 58-TTGGGTGTTTAGCTGTTAA-38 ton D.C. Acronyms followed by a dash RM represent field numbers L5706 tRNAAsn 58-TAGTTAACAGCTAAACAC-38 of the first author for uncatalogued specimens being deposited in either H5934 COI 58-AGRGTGCCAATGTCTTTGTGRTT-38 the California Academy of Sciences or the Museum of Vertebrate H5937 COI 58-GTGCCAATGTCTTTGTG-38 Zoology. Iguanidae: Anolis paternus Cuba (USNM 498070; U82679); Basiliscus plumifrons Costa Rica (MVZ 204068; U82680); Crota- a Primers are designated by their 38 ends, which correspond to the phytus collaris Texas, USA (TCWC 72206; U82681); Gambelia wis- position in the human mitochondrial genome (Anderson et al. 1981) by lizenii Nevada, USA (MVZ-RM2464; U82682); Hoplocercus spino- convention. H and L designate heavy-strand and light-strand primers, sus Mato Grosso, Brazil (MZUSP 907931; U82683); Liolaemus respectively. Positions with mixed bases are labeled with their standard tenuis Chile (MVZ 162076; U82684); Oplurus cuvieri Madagascar one-letter codes: R4GorA;Y4TorC;W4A or T. All primers are (CAS-RM10468; U82685); Phrynosoma douglassi New Mexico, USA from Macey et al. (1997a) except L4160 (Kumazawa and Nishida (MVZ 180332; U82686); Sauromalus obesus California, USA (MVZ 1993), L4437b (new to this study) and L5551 (Macey et al. 1997b) 144194; U82687). Acrodonta: Chamaeleo fischeri Tanzania (CAS 168965; U82688); Uromastix acanthinurus Morocco (MVZ 162567; U71325) (Macey et al. 1997a,b); Leiolepis belliana Thailand (MVZ 215497; U82689); Physignathus cocincinus Vietnam (MVZ 222159; encoding part of ND1, all of ND2, and part of COI were aligned by U82690); Phrynocephalus raddei Turkmenistan (CAS 179770; amino acid using MacClade (Maddison and Maddison 1992). Align- U82691). Anguidae: Elgaria panamintina California, USA (MVZ- ments of sequences encoding tRNAs were constructed manually based RM1641; U82692). on secondary structural models (Kumazawa and Nishida 1993; Macey and Verma 1997). Secondary structures of tRNAs were inferred from primary structures of the corresponding tRNA genes using these mod- Laboratory Protocols. Tissue samples were collected and frozen in els. Unalignable regions were excluded from phylogenetic analyses. liquid nitrogen. Genomic DNA was extracted from muscle or liver See Fig. 1 for alignment. using the Qiagen QIAamp tissue kit. Amplification of genomic DNA Most protein-coding regions were alignable (1,062 positions) but was conducted using a denaturation at 94°C for 35 s, annealing at some regions were excluded from phylogenetic analyses because of 45–53°C for 35 s, and extension at 70°C for 150 s with 4 s added to the questionable alignment. In ND1, sequences encoding amino acids that extension per cycle, for 30 cycles. Amplified products were purified on extended more than one amino acid beyond the sequence for Gallus 2.5% Nusieve GTG agarose gels and reamplified under similar condi- gallus (Desjardins and Morais 1990) were excluded from analyses as tions. Reamplified double-stranded products were purified on 2.5% well as stop codons, which sometimes overlap with adjacent tRNAs acrylamide gels (Maniatis et al. 1982). Template DNA was eluted from (positions 82–97). In ND2, a gap was placed in the Leiolepis sequence acrylamide passively over 3 days in which Maniatis elution buffer at the position corresponding to amino acid 249 in Gallus (positions (Maniatis et al. 1982) was replaced each day. Cycle-sequencing reac- 1076–1078). Sequences that encoded ND2 beyond amino acid 316 of tions were run using the Promega fmol DNA sequencing system with Gallus were unalignable and therefore excluded (positions 1280–1376). a denaturation at 95°C for 35 s, annealing at 45–53°C for 35 s, and In COI, sequences corresponding to the third amino acid of Gallus extension at 70°C for 1 min for 30 cycles. Sequencing reactions were appear only in Physignathus and Phrynocephalus; therefore, a gap was run on Long Ranger sequencing gels for 5–12 h at 38–40°C. placed in the other sequences (positions 1809–1811). Amplifications used primer H5934 and one of the following: Genes