山 階 鳥 研 報(J. Yamashina Inst. Crnithol.), 25, 1-11, 1993 The Phylogenetic Relationships of the Rails, Based on DNA Comparisons Charles G. Sibley*, Jon E. Ahlquist**, and Paul DeBenedictis*** Abstract In most classifications of the past century, the rails (Rallidae) have been placed in the Gruiformes with the cranes and their allies, including the Sunbittern, bustards, Limpkin, sungrebes, trumpeters, seriemas, and the Kagu. In a few classifica- tions the rails have been assigned to an Order Ralliformes; in other classifications some taxa now usually placed in the Charadriiformes have been assigned to the Gruiformes, as relatives of the Rallidae. DNA-DNA hybridization data support other evidence that the rails are closer to the typical gruiforms than to any other group, but that they have no close relatives within the Gruiformes. Key words: rails, phylogeny, DNA hybridization, PHYLIP, neighbor joining. Introduction The ca. 34 genera and 142 species of rails, gallinules, and coots occur worldwide in temperate and tropical areas, including many insular species some of which have become flightless. Morphological characters, and comparisons with the other gruiform groups, are recorded in Sibley and Ahlquist (1990). The rails usually have been included in the Gruiformes, although some classifications have allied them to the Charadriiformes. In some classifications the gruiforms have been divided into several orders, depending on which characters were viewed as taxonomically informative. The history of the classifi- cation of the gruiforms was reviewed by Sibley and Ahlquist (1990: 427-440). Here we present a reanalysis of the DNA-DNA hybridization data on which Sibley, Ahlquist, and Monroe (1988), Sibley and Ahlquist (1990), and Sibley and Monroe (1990) based their classification of the Gruiformes. We have tried to answer the following questions: 1. Are the Gruiformes a monophyletic group? 2. Is the family Rallidae (rails, etc.) a member of the Gruiformes? 3. What other group is the closest relative of the Rallidae? Our ability to answer these questions is limited by the absence of data for the Mesitornithidae and the Rhynochetidae (Kagu). We have omitted data for Heliornis because of uncertainty about the identity of the DNA sample used in earlier studies. Methods The methods used to obtain the DNA comparisons on which this paper is based were described by Sibley and Ahlquist (1990). Briefly, DNA-DNA hybridization measures the degrees of similarity between the DNA sequences of two species. The technique takes advantage of the fact that the hydrogen bonds between the two strands of native DNA are Received 30 December 1992, Accepted 15 January 1993. * 95 Seafirth Road , Tiburon, California, 94920 USA ** Dept . of Biology, University of Louisville, Louisville, Kentucky, 40292 USA *** Educational Communications , SUNY Health Science Center, Syracuse, N. Y. 13210 USA 1 2 C. G. Sibley, J. E. Ahlquist, and P. DeBenedictis the weakest bonds in the structure and may be broken by modest degrees of heat, thus separating the two strands. Heteroduplex DNA molecules are formed by reassociating the single strands of the DNAs of two different species to form double-stranded structures. The melting temperatures of these DNA "hybrids" are compared with that of double- stranded homoduplex molecules composed of single strands from one of the species forming the heteroduplex molecules. The difference in degrees Celsius between the melting temperatures of the homoduplex and the heteroduplex DNAs is a measure of their genetic similarity. The experimental conditions are set so that only homologous sequences can form duplexes. The principal steps in the DNA-DNA hybridization technique follow (from Sibley 1991). 1. Extract and purify DNA from cell nuclei=remove proteins, RNAs, etc. 2. Shear long-chain DNA strands into fragments ca. 400-600 bases in length. 3. Remove most of the copies of repeated sequences from selected species to produce "single-copy DNA" , which includes copies of all repeated sequences. 4. "Label" the single-copy DNA with a radioactive isotope to produce a "tracer" DNA of one species=Species A. 5. Combine the single-stranded tracer DNA of Species A with the single-stranded "driver" DNA of the same species (A+A=homoduplex) , and with the single-stranded driver DNAs of other species (A+B, A+C, A+D, etc.=heteroduplexes). Each combi- nation is placed in a separate vial. 6. Incubate the vials in a waterbath at 60℃ for 120 hours to permit the formation of double-stranded hybrid molecules composed of one strand of the tracer (A) and one strand of the driver (B, C, D, etc.) to produce the hybrids: A×A, A×B, A×C, A×D, etc. 7. Place the DNA-DNA hybrids on hydroxyapatite (HAP) columns. Double-stranded DNA binds to HAP; single-stranded DNA does not bind to HAP. 8. Place the columns in a heated waterbath and raise the temperature in 2.5℃ incre- ments from 55℃ to 95℃. At each temperature, wash off (elute) the single-stranded DNA resulting from the "melting" of the hydrogen bonds between base pairs. Collect each eluted sample in a separate vial and assay the radioactivity in each vial. This is a measure of the percentage of hybrid molecules that melted at each temperature. 9. The melting temperature of a DNA-DNA hybrid is proportional to the degree of genetic similarity between the two single strands forming the hybrid molecule. Data are expressed as melting curves and as distance measures (delta values=Δ values) between the homoduplex and heteroduplex melting curves, for example, as median distances (ΔTm)), modal distances (ΔTmode), and as the temperature at 50%hybridization (ΔT50H). The percentage of reassociation (or hybridization) is sometimes used as a distance measure, but its variability limits its value. Dendrograms ("phylogenetic trees") were constructed from folded matrices of the available genetic distance values. Missing values were estimated so as to preserve the measured patterns of similarity and to minimize distortions such estimates might cause. The Phylogenetic Relationships of the Rails, Based on DNA Comparisons 3 Results The melting curves (Figs. 1-5) show the relative positions of various species compared with the homoduplex curves of Porphyrio porphyrio (Figs. 1-3), Eupodotis vigorsii (Fig. 4), and Psophia crepitans (Fig. 5). These are only examples; additional experimental sets contributed data to the analysis. The position of Turnix in Figs. 1 and 5 indicates that the buttonquails are not members of the Gruiformes, as often assumed in earlier classifications. Fig. 1. Purple Swamphen×Takahe, Baillon's Crake, Black-tailed Native-hen, Watercock, American Coot, Clapper Rail, Plains-wanderer, Horned Screamer, Wattled Brush-turkey, Helmeted Guineafowl, Small Buttonquail. Fig. 2. Purple Swamphen×Brolga, Crab Plover, Wattled Lapwing, Water Thick-knee, Ring-billed Gull, Least Sandpiper, Marbled Godwit, Common Snipe, Grey-breasted Seedsnipe, African Jacana, Plains-wanderer. 4 C. G. Sibley, J. E. Ahlquist, and P. DeBenedictis Fig. 3. Purple Swamphen×American Coot, Brolga, Lesser Frigatebird, Greater Flamingo, Northern Harrier, Masked Booby, Ring-billed Gull, Pelagic Cormorant, African Olive-Pigeon. Fig. 4. Karoo Bustard×Kori Bustard, Brolga, Limpkin, Grey-winged Trumpeter, Sunbittern, White- breasted Waterhen, Red-knobbed Coot. Figs. 6-8 were produced with the Distance Wagner method as modified by Daniel Faith (1985), with branch lengths estimated by linear least squares. The Distance Wagner trees are rooted at their midpoints. The approximately equal distances between taxa and the root suggests that there has been little variation in average evolutionary rates in these lineages. Most of the short branches in these trees are shorter than their estimated errors and such branches probably should be treated as polychotomies, rather than dichotomies. In Figs. 6-8 the vertical lines represent Hypothetical Taxonomic Units. Horizontal lines are proportional to estimated branch lengths and the gray bars represent one standard error of each estimated branch length. The tree in Fig. 6 is based on ΔT50H distance values. It reflects the positions of Turnix and Eurypyga observed in the melting curves, and the clusters composed of The Phylogenetic Relationships of the Rails, Based on DNA Comparisons 5 Fig.5. Grey-winged Trumpeter×Blue Crane, Limpkin, Red-legged Seriema, Karoo Bustard, Clapper Rail, American Coot, Sunbittern, Plains-wanderer, Comb-crested Jacana, Small Buttonquail. Fig. 6. 6 C. G. Sibley, J. E. Ahlquist, and P. DeBenedictis Fig. 7. bustards (Ardiotis, Lissotis, Eupodotis, Afotis), rails (Porphyrio, Amaurornis, Gallinula, Fulica), and Psophia, Aramus, Balearica, and Grus. Cariama is intermediate between the gruiform cluster and the charadriiform cluster (Vanellus to Thinocoridae). Note that the Plains-wanderer (Pedionomus) of Australia is most closely related to the South American seedsnipe (Thinocoridae). Fig. 7 is based on ΔTm distance values. The same clusters as in Fig. 6 are present, but Turnix and Eurypyga have moved to a central position in the tree and seem to be sister taxa. However, they are obviously not closely related as indicated by the long branches, especially that leading to Turnix. The ΔTm values are "compressed" over the range of elution temperatures and lose resolving power for genetically distant taxa. Therefore, the closely related taxa in Fig. 7 are most likely to be accurately portrayed, while the older branches appear closer together than they actually are. Fig. 8 is based on ΔTmode diStance values. These are the distances in degrees Celsius The Phylogenetic Relationships of the Rails, Based on DNA Comparisons 7 Fig. 8. between the modes of the frequency distributions of the melting curves. Modal values are difficult to calculate accurately, but essentially the same clusters are depicted in Fig. 8 as in Figs. 6 and 7, with Turnix as the most distant outgroup and the rails clustering with the traditional gruiforms. Cariama also clusters with the gruiform group. TableS 1-3 are folded matrices of average ΔT50H, ΔTm, and ΔTmode values. Figs. 9- 17 are trees based on these matrices which include only nine taxa, all of which were used as "tracers".