CHAPTER 5 Molecular Systetnatics of Atnphibians Jorg Plotner, Frank Kohler, Thomas Uzzell and Peter Beerli I. Introduction V. Application of Molecular Methods in Amphibian Systematics II. Molecules A. Determination and Identification of Species A. Proteins and Species Hybrids B. Nucleic Acids B. Molecular Phylogeny of Living Amphibians 1. Nuclear DNA 1. The Phylogenetic Relationships 2. Mitochondrial DNA between Gymnophiona, Urodela, and Anura III. Molecular Markers and Methods of 2. The Early History and Diversification Investigation of Amphibians A. Detection of Protein Polymorphisms 3. Molecular Phylogeny of the 1. Protein Electrophoresis Gymnophiona 2. Immunological Methods 4. Molecular Phylogeny of the Urodela B. DNA-Based Methods 5. Molecular Phylogeny of the Anura 1. Analysis of Restriction-Fragment­ Length Polymorphisms (RFLP) VI. Evaluation of Molecular Systematics and 2. DNA-Fingerprinting (Minisatellite Future Prospects Analysis) 3. peR-Based Methods VII. References 4. Sequence Analysis IV. Methods of Analysis of Molecular Data A. Phylogenetic Analysis 1. Tree-Building Methods 2. Statistical Methods for Testing the Reliability of a Tree 3. Other Methods B. Phylogenetics versus Population Genetics I. INTRODUCTION N the past 30 years the rapid development and steady improvement of molecular methods I have had and still are having a paramount impact on almost all fields of biology. Population genetics, evolutionary biology, and phylogenetic systematics are disciplines that particularly have benefited from the rise of molecular biology and have faced a tremendous evolution by themselves. Perceptions of fundamental questions in biology concerned with processes and causes of evolution and diversification of life are tightly connected with advances in molecular biology. PLCrINER ET AL.: MOLECULAR SYSTEMATICS OF AMPHIBIANS 2673 Molecules (RNA. DNA. proteins) carry important information that allows one to distinguish taxa, to reconstruct their phylogenetic relationships, to estimate divergence times, and to analyse evolutionary processes. As can be demonstrated by an ever-growing amount of molecular data, molecular approaches which supplement traditional nonmoleeular methods are a pivotal part of the toolkit of evolutionary biologists and systematists. For Amphibia, internet sequence databases (GenBank, EMBL) currently contain about 1.76 million entries of DNA and RNA sequences. DNA sequences in particular, because they bear the code of life, provide the most detailed anatomy possible for any organism - the instructions for how each working part should be assembled and operate (Page and Holmes 1998). Up to the end of the 1980s, most molecular work in systematics was conducted using proteins. Since then, the development of new DNA-manipulation techniques has promoted a shift from protein-based to DNA-based markers (ScWotterer 2004). The Polymerase Chain Reaction (PCR) (Saiki et al. 1985; Mullis et al. 1986), developed in the mid-1980s, was a significant milestone in molecular biology. For the first time it became possible to analyse any genomic region without the requirement of isolating large amounts of ultra-pure genomic DNA or to clone DNA segments. Since then many further technological innovations, for example, automated sequencers, have significantly simplified the technical procedures; DNA-sequencing is now a standard and routine method. Dramatic advances in computational technology have allowed processing large datasets and applying the complex mathematical algorithms and sophisticated statistical procedures that are needed to analyse phylogenetic relationships (e.g., Nei and Kumar 2000; Arbogast et al. 2002). Progress in molecular biology has also had a deep impact on the systematics of recent amphibians. Initial molecular investigations in the 1950s and 1960s were concerned with aspects of population genetics and genetic polymorphisms within and among species. Pioneer work was carried out by Guttman and collaborators on serum protein polymorphisms in toads of the genus Bufo (reviewed by Guttman 1973). In the following decades protein markers, mostly allozymes, were successfully applied in population genetics for the reconstruction of phylogenetic relationships of closely related species, for the estimation of divergence times in the context of historical biogeography, and for simple taxonomic purposes, e.g., to detect and differentiate species that are morphologically indistinguishable (e.g., Hotz and Uzzell 1982; Nishioka et al. 1987; Nishioka and Sumida 1992; Beerli et al. 1994, 1996). As elsewhere in biology, the study of nucleotide sequences has becoming increasingly popular in amphibian systematics since the early 1990s. As for other animal groups, systematists almost exclusively used mitochondrial (mt) DNA sequences. One of the early, more comprehensive studies is that of Hedges and Maxson (1993), who analysed the relationships among higher-ranked amphibian groups ("families") on the basis of a relatively short fragment (333 bp) of the mt 12S ribosomal RNA (rRNA) gene. Shortly thereafter, Hay et al. (1995) employed a dataset of about 1300 bp of mt ribosomal sequences from species representing 28 of the 40 recognized amphibian families. More recent studies have gone further in analysing complete mt genomes (e.g., Zardorya et al. 2003; Mueller et al. 2004; Zhang et al. 2005). The number of investigations using mt DNA sequences to infer phylogenetic relationships has continued to increase rapidly, but nowadays, nuclear genes, such as the recombination-activating gene 1 (RAG1), play an increasing role in systematic studies on amphibians (e.g., Hoegg et al. 2004; San Mauro et al. 2005). Shortly before this chapter went to print, a phylogeny of the living amphibians based on a combined analyis of mitochondrial and 2 300 bp of nuclear genes from 522 species representing all families and subfamilies within the Amphibia, except Protohynobiinae, was analysed on a parallel computer duster, which resulted in the most comprehensive hypothesis on the phylogenetic relationships within the Amphibia available to the present time (Frost et al. 2006). In the present chapter the molecular methods and statistical procedures that are commonly applied in systematics are surveyed and their advantages and disadvantages are 2674 AMPHIBIAN BIOLOGY discussed. The molecules that are most frequently used for systematic investigations are also described. In the second part of the chapter, new hypotheses on the phylogeny of m~or amphibian groups, based on molecular data, are presented. Since this chapter is primarily dedicated to amphibian systematics, the molecular methods that are considered relatively unimportant for systematics, e.g., DNA flow cytometry, are addressed only cursorily. It is far beyond the scope of this chapter to go into details concerning population genetics and evolutionary biology, fields that deserve chapters on their own. A note on the nomenclature used in this chapter is in order. The frequently used term "Lissamphibia" was coined by Haeckel (1866) for frogs and salamanders, but not for caecilians. For this reason, Dubois (2004) felt that this term might not be a proper choice to denominate all extant amphibians. Accordingly in the chapter reference is made simply to living or extant amphibians, and the formal term "Lissamphibia" not employed. This procedure has also been followed by Frost et ai. (2006) who restricted the term "Amphibia" to the group formed by caecilians, salamanders and frogs. Contrary to usage otherwise in systematic literature, including elsewhere in the Amphibian Biology series, Dubois (2004) is also followed in preferring the term Urodela Dumeril, 1806 as the correct scientific name for salamanders, and considering the name Caudata Scopoli, 1777 as not a valid amphibian taxon. II. MOLECULES A. Proteins Proteins are natural polymer molecules composed of amino acids that are joined together by covalent peptide bonds to form polypeptides (Fig. 1). There are 20 different essential amino acids in native proteins, each composed of a central carbon atom (the alpha carbon) to which are attached a hydrogen atom, a carboxyl group (COo-), an amino group (NH3+), and a unique side chain or R-group. The side chains determine the chemical properties of a protein, such as its ability to bind ligands and catalyse biochemical reactions. They also direct the folding of the polypeptide chain and stabilize its final conformation. While most side chains are neutral, those of five of the amino acids are either basic and thus positively charged (lysine, arginine, and histidine), or acidic and negatively charged (aspartic acid and glutamic acid). Charged side chains are responsible for the net charge of a protein and thus for its migration under the influence of an electrical field. This property of proteins is the basis for a molecular technique called protein electrophoresis (see Section on that topic). The genetically determined linear arrangement (sequence of amino acids in a protein) is called "primary structure". Uncharged amino acids are either non-polar and hydrophobic or polar. They can become hydrogen-bonded, resulting in a "secondary structure", which is defined as the local spatial arrangement of the main-chain atoms of a polypeptide chain segment. Three common secondary structures can be distinguished: alpha-helix, alpha­ sheet, and turns. Alpha-helices and alpha-structures are generally the most thermo­ dynamically stable conformations of regular secondary structures. Depending on the primary and