by Dr. Charles G. Sibley
The first comparisons
between molecules were made a century ago by the early serologists, but
the molecular age of evolutionary biology truly began with the determination
of the structure of DNA by Watson and Crick (1953). As the coding relationships
among DNA, RNA and proteins became clear it was apparent that these molecules
contain potential evolutionary information and methods to exploit them
were soon in use. At Cornell University in 1956 I began to use electrophoresis
to compare blood and egg-white proteins (e.g., Sibley 1960, 1970). These
studies solved few problems, but they provided experience and contacts
with persons who were developing improved procedures. I tried the "agar
column" method of DNA-DNA hybridization in 1963, without success. It was
the hydroxyapatite technique, perfected in the late 1960's, that made DNA
hybridization useful for phylogenetic research and I began to use this
method at Yale University in 1974, with the collaboration of Jon Ahlquist.
Between early-1975 and mid-1986, we made more than 26,000 DNA-DNA comparisons
among ca. 1700 bird species, representing all of the orders and 168 of
the 171 families in Wetmore's (1960) classification. A new classification
was proposed (Sibley et al. 1988). Sibley and Ahlquist (1990) provide details
of the history, methods and data. A book on the distribution and taxonomy of the bird species of the world (Sibley and Monroe 1990) used the DNA-based classification and a supplement was published in 1993. A brief update (Sibley 1991) included comments and corrections for several group names. Monroe and Sibley (1993) is a list of living species with brief distributional information and corrections to date.
The DNA-DNA hybridization technique
measures the degree of genetic similarity between complete genomes. The
comparison may be between the two DNA strands of an individual, or of different
individuals representing different levels of genetic and taxonomic divergence.
"Hybrid" double-stranded DNA molecules are formed from the single strands
of the DNAs of two species. The hybrid molecules are then dissociated ("melted")
in a thermal gradient under controlled conditions such that a measure of
the melting temperature of the hybrid duplex may be calculated. The experimental
conditions are set so that only homologous sequences can form double-stranded
structures. The melting temperature of a DNA duplex molecule is a function
of the number of correctly base-paired nucleotides, thus it is a measure
of the degree of genetic similarity between the two single strands forming
the duplex. Data are expressed as melting curves and as distances between
the midpoints of the melting curves. Dendrograms that do, and do not, assume
equal rates of genomic evolution along all branches may be constructed
to represent the branching pattern of the phylogeny indicated by the distance
values. Phylogenetic trees may be constructed from the melting temperature
data, using several methods of analysis.
The technique, data analysis, and other aspects of the procedures are described by Sibley and Ahlquist (1983, 1987, 1990).
The principal steps in the DNA-DNA hybridization technique follow:
(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 contains copies of all sequences in the genome.
(4). "Label" the single-copy DNA with a radioactive isotope to produce a "tracer" DNA of one species = Species A*. The asterisk* identifies the radio-labeled taxon.
(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 combination is placed in a separate vial.
(6). Incubate the vials in a water bath at 60 C 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* x A, A* x B, A* x C, A* x 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 water bath and raise the temperature in 2.5 C increments from 55 to 95 C (= 17 increments). 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. The percentage of the total radioactivity that elutes at each of the 17 temperatures is an index to the degree of base pairing, which is a product of genetic similarity.
(9). Use the amount of radioactivity ("counts") in each sample to construct melting curves and to calculate genetic distance values. Construct "trees" from the genetic distance values.
In the classification of Sibley et al. (1988) the boundaries of categories were based on a DNA hybridization distance measurement, DT50H, defined as the temperature in degrees Celsius between the 50% hybridization level of a homoduplex melting curve and the 50% hybridization level of a heteroduplex melting curve. For example, Orders were defined as groups that differ from one another by an average DT50H value between 20 and 22; Families differ by D9-11, etc. We have used the following categories and ranges of DT50H values: Class (31-33), Subclass (29-31), Infraclass (27-29), Parvclass (24.5-27), Superorder (22-24.5), Order (20-22), Suborder (18-20), Infraorder (15.5-18), Parvorder (13-15.5), Superfamily (11-13), Family (9-11), Subfamily (7-9), and Tribe (4.5-7).
Results and problems
At least 75% of our results agree with traditional views of the boundaries of natural clusters of species, such as the ratites, woodpeckers, gallinaceous birds, ducks and geese, parrots, pigeons, passerines, etc. This is some of the strongest evidence that DNA-DNA hybridization detects natural groups. Our work mainly concerned the higher categories; we did not attempt to measure relationships among species or genera, although such evidence emerged in a few cases. Most of the differences between our classification and traditional classifications are of three kinds. (1) Some of the groups we recognized differ from those in previous classifications, i.e., there is a disagreement about categorical ranking. (2) Some groups revealed unexpected internal genetic diversity which required recognition in the classification. (3) We found a few major subdivisions of the Class Aves which we called Parvclasses, namely: Ratitae, Galloanserae, Turnicae, Coliae, and Passerae. The most controversial subgroup in the Passerae is the Order Ciconiiformes, which includes several "traditional" Orders, namely, Charadriiformes, Falconiformes, Podicipediformes, Pelecaniformes, Ciconiiformes, Sphenisciformes, Gaviiformes, and Procellariiformes. These groups are morphologically and ecologically diverse and classifications based on morphology have viewed them as so taxonomically diverse that they "merit" recognition as Orders. Contrast this view with the Passeriformes, long recognized as an Order because passerine species are mainly arboreal, small in size, and morphologically similar to the human eye. Within our Ciconiiformes most of the Families cluster according to traditional ideas of relationships, with the exception of the Pelecaniformes, of which more below.
Previous classifications were based on morphological characters in which categorical levels were subjectively defined by the human eye. The classification of birds most widely used today is that of Alexander Wetmore who based it mainly on the work of Hans Gadow (1893). Wetmore published the first edition in 1930 and the latest of five in 1960. There were few changes during the intervening years and the classification remains essentially that of Gadow, developed from comparisons of 40 characters a century ago. Such classifications provide a taxonomy and usually associate closely-related species, but seldom reflect phylogeny. They are also prone to errors due to interpreting convergent morphological similarities as evidence of close relationship (e.g., the Australo-Papuan endemics discussed below). Classifications based on comparisons of DNAs reflect phylogeny because genomes evolve in a reasonably "clocklike" manner, i.e., the degrees of difference among the DNAs of different species are correlated with time, although the correlation is not perfect. Thus, the most objective and quantitative methods for the reconstruction of phylogeny are those that measure degrees of similarity between the DNAs of different species. Measurement is the essence of science and DNA-DNA hybridization and DNA sequencing are the best available methods; each has strengths and weaknesses, but a combination of these two techniques is our best hope for understanding the phylogeny of birds and other organisms. Sibley and Ahlquist (1990:184-245) reviewed the classification of birds.
Our phylogeny and classification have been criticized for various reasons, including the claim that our methods were imprecise and that our choice of T50H as the thermal stability index was inappropriate to resolve higher-category relationships. However, a portion of our non-passerine phylogeny has been re-examined by Bleiweiss, Kirsch and Shafi (in press) who used DNA-DNA hybridization to compare seven taxa from five non-passerine Orders. They developed a complete matrix among a duck (Anas), an owl (Bubo), two pigeons (Zenaida, Columba), a mousebird (Colius), and two galliforms (Gallus, Coturnix), with a reptile (Alligator) as the outgroup. They analyzed their data in several ways and concluded that their results "...support Sibley and Ahlquist's use of DT50H to assess ordinal patterns ...." and their data confirm a portion of our phylogeny based on the same technique.
John Kirsch and colleagues (Univ. of Wisconsin, Madison) have found that with as few as half of the cells of a matrix filled, it is possible to obtain the same tree as that based on a complete table. They also used the Sibley/Ahlquist data for the same taxa, which comprise ca. 39% of the possible comparisons, and achieved the same resolution as with their complete data set, except for the ambiguous position of Colius.
Bleiweiss, Kirsch and Matheus (1994) confirmed our subfamilial division of the hummingbirds. Bleiweiss, Kirsch and LaPointe (in press) analyzed a nearly complete matrix of DNA hybridization distance measurements among a hummingbird (Colibri), typical swift (Chaetura), crested swift (Hemiprocne), duck (Anas), woodpecker (Melanerpes), kingfisher (Megaceryle), mousebird (Colius), owl (Bubo), nightjar (Chordeiles) and a suboscine flycatcher (Myiarchus). They concluded that "Despite significant rate variation among different taxa, these results largely concur with those obtained with the same technique by Sibley and Ahlquist, who used the DT50H measure and UPGMA analysis. This agreement lends credence to some of their more controversial claims." Their data supported our conclusion that the woodpeckers represent an early branch and that passerines arose from within the non-passerine assemblage. These results, as well as the sister-group relationship of the two swift families and of both with respect to hummingbirds, were strongly supported by bootstrapping and jackknifing tests of their trees.
The detailed DNA hybridization study of the cranes by Krajewski (1989) also agreed with our more limited comparisons among cranes. Mindell and Honeycutt (1989) reported ribosomal DNA evidence that supports some aspects of our phylogeny.
Several DNA sequence studies have supported other portions of our work; they are noted in the following comments about some of the most interesting and/or controversial results from our research using DNA hybridization.
Loons and Grebes
The loons (Gaviidae) and grebes (Podicipedidae) have been associated in classifications from the earliest times to the present, sometimes in the same Order or in adjacent Orders. There have been morphological studies in the past that demonstrated many differences between them and concluded that their similarities are superficial and due to convergence. However, neither seemed to have other close relatives, so authors have continued to place them together. The DNA hybridization comparisons showed that the grebes are distant from other living groups, but the loons cluster with the penguins and tubenoses (petrels, shearwaters, albatrosses). We now have mtDNA sequence evidence that supports this arrangement (Hedges and Sibley, in press).
Our studies showed that there are three groups of ratites: Ostrich, the two rheas, and the Emu-cassowary-kiwi cluster. The tinamous are their closest living relatives. In earlier publications (e.g., Sibley and Ahlquist 1981) we assumed that the opening of the Atlantic ca. 80 MYA during the breakup of Gondwanaland separated Africa and South America and split a common ancestor that evolved into the living rheas and Ostrich. The UPGMA method of tree-building (Sibley and Ahlquist 1990:839) supported that assumption. However, the phylogenetic trees for the ratites based on the PHYLIP computer program link the rheas more closely to the Australian Emu and cassowaries (Sibley and Ahlquist 1990:810-811). This raises the possibility that the ancestor of the rheas reached South America from Australia via Antarctica ca. 35-40 MYA, as suggested by the fossil of the first land mammal found in Antarctica (Woodburne and Zinsmeister 1984). If this is correct, our calibration of the rate of DNA evolution based on the ratites must be revised to ca. DT50H 1.0 = 2.2 MY, instead of ca. 4.7 MY (Sibley and Ahlquist 1990:286). All calibrations are tentative and subject to further correction.
From comparisons of the tongue apparatus, Bock and Bühler (1990) proposed that the Ostrich and the elephant-birds (Aepyornis) should be associated in a suborder Struthioni, that the other ratites and the tinamous should be placed in the suborder Tinami, and that there is no evidence for dispersal between Africa and South America that would support a closer relationship between the Ostrich and rheas. I agree that the rheas are probably closer to the Australian-New Zealand ratites than to the Ostrich, but do not agree that the tinamous are members of the clade that includes the Ostrich, rheas, emu, cassowaries and kiwis. Our DNA hybridization data consistently place the tinamous outside the ratite clade.
Most classifications have assigned the New World quail to the Phasianidae (exceptions noted in Sibley and Ahlquist 1990). We found that the New World quail clade is the sister group of the phasianid-numidid clade (Parvorder Phasianida), so we placed the New World quail in an adjacent Parvorder Odontophorida, Family Odontophoridae. This has been supported by mitochondrial DNA sequence evidence by Kornegay et al. (1993), who also found that the cracids (chachalacas, guans, etc.) are the sister group of the typical galliforms plus the New World quail. Avise et al. (unpubl.) also confirmed our placement of the New World quail. We assigned the cracids and megapodes to the Order Craciformes.
The Turnicidae have been assigned to the Gruiformes in most classifications. We found that Turnix is not a gruiform and has no close living relatives, hence we placed it in its own Order Turniciformes. Our data also showed that the Plains-wanderer (Pedionomus) of Australia is not related to Turnix, but is closest to the seedsnipe (Thinocoridae) of South America, thus supporting Olson and Steadman (1981). DNA of the Lark Buttonquail (Ortyxelos) was not available and its relationships remain uncertain.
Barbets and toucans
The New World and Old World barbets have been placed in the Capitonidae; the toucans in the Ramphastidae. We found that the New World barbets are more closely related to the toucans than to the Old World barbets. This has been supported by mtDNA sequence data (Lanyon and Hall, 1994). We place the toucans and New World barbets in the Ramphastidae, superfamily Ramphastoidea. The Asian barbets (Megalaimidae) and African barbets (Lybiidae) are sufficiently distinct to be placed in separate superfamilies.
The South American Hoatzin Opisthocomus hoazin has been assigned to the Galliformes, Cuculiformes, or to a monotypic Order. Comparisons of the electrophoretic patterns of the egg-white proteins of the Hoatzin, several cuckoos, galliforms and species in other groups indicated that the Hoatzin is most closely related to the anis (Crotophaga) and the Guira Cuckoo G. guira (Sibley and Ahlquist 1973). The Hoatzin shares several behavioral and plumage characters with the anis and the Guira Cuckoo and DNA hybridization comparisons also indicated a cuculiform alliance. Bock (1992) disagreed because the Hoatzin has anisodactyl toes and the cuckoos have the zygodactyl arrangement. Our DNA hybridization data indicate that the cuckoos are genetically diverse and Berger (1960) found comparable diversity in morphology. The relationships of the Hoatzin remain unclear, but I believe that it is most closely related to the Guira Cuckoo, the anis and the roadrunners.
Sibley et al. (1988) and Sibley and Ahlquist (1990) placed the Limpkin (Aramus) and the Sungrebe (Heliornis) as subfamilies in the Heliornithidae. This may have been an error and it is being re-examined by DNA sequencing in the laboratory of Carey Krajewski at Southern Illinois Univ. Our DNA hybridization data for the relationships among the rails, the other gruiforms and the charadriiforms were re-examined by Sibley et al. (1993). We concluded that these three groups are close relatives.
The members of the traditional Order Pelecaniformes share many morphological characters, but our DNA comparisons suggested that (1) the tropicbirds (Phaethon) are not closely related to the other taxa; (2) the frigatebirds (Fregata) are most closely related to the petrels, penguins and loons; (3) the boobies, gannets, anhingas and cormorants form a monophyletic cluster, and (4) the pelicans are most closely related to the Shoebill Balaeniceps rex. Most of these suggestions are opposed by other evidence and by many avian systematists. I questioned all but the tropicbird position and the monophyly of the booby/anhinga/cormorant clade. Our proposal that the traditional Order Pelecaniformes is polyphyletic "may be the most controversial conclusion of our entire study and we expect it to be disbelieved." (Sibley and Ahlquist 1990:527). However, Blair Hedges (Penn. State Univ.) has completed an mtDNA sequence study of 16 species of birds that supports pelecaniform polyphyly, including the pelican-Shoebill alliance (Hedges and Sibley, in press). John Kirsch is using DNA-DNA hybridization to re-examine the "pelecaniform problem".
Storks and New World vultures
Morphological evidence that the New World vultures (Cathartinae) are more closely related to the storks (Ciconiinae) than to the Old World vultures (Accipitridae) was proposed by Garrod (1873) and supported by Ligon (1967), but ignored by avian systematists until DNA hybridization also suggested this relationship (Sibley and Ahlquist 1990).
Loons and grebes
The loons (Gaviidae) and grebes (Podicipedidae) often have been placed in the same, or adjacent, Orders. Their morphological similarities have been interpreted as due to common ancestry or to convergent evolution. Our DNA comparisons indicate that the grebes have no close living relatives and that the loons are members of the radiation that includes the petrels (Procellariidae), penguins (Spheniscidae) and frigatebirds (Fregatidae), all placed in the Superfamily Procellarioidea. The association of penguins and petrels has long been accepted, but the assignment of loons and frigatebirds to this cluster is certain to be controversial.
That the New World suboscine groups radiated in South America during the long isolation of that continent is apparent, but their morphological diversity has made it difficult to arrive at a consensus about their classification. The DNA comparisons seem to have solved some of the problems, but others remain. We defined a subgroup of the tyrannids, the Pipromorphinae (= "Mionectinae"), that may or may not be supported by other evidence. The Broad-billed Sapayoa Sapayoa aenigma remains an enigma.
Starlings and mockingbirds
It was surprising to discover that the Old World starlings and the New World mockingbirds and thrashers are closest living relatives (Sibley and Ahlquist 1980, 1984, 1990). This has been supported by mtDNA sequencing in John Avise's laboratory at the Univ. of Georgia (Prinsloo et al. unpubl.).
The Australo-Papuan endemic radiation
Our most important discovery may be the evidence that the old endemic passerine groups of Australia and New Guinea are the results of adaptive radiation within that area, not the products of a series of invasions from Asia. This is a complex situation; for details see Sibley and Ahlquist (1985) and (1990). Our data showed that the birds-of-paradise are more closely related to the corvoid cluster (corvines, artamines, etc.) than to the bowerbirds, with which they usually have been associated. This conclusion has been supported by mtDNA sequence data (Helm-Bychowski and Cracraft 1993). We also found that the bowerbirds are closest to the lyrebirds (Menura), but Cracraft (pers. comm.) reports mtDNA evidence that the bowerbirds and lyrebirds are not as closely related to one another as the lyrebirds are to the meliphagoid cluster (honeyeaters, e.g.). This fits one of our data analyses using the FITCH routine of the PHYLIP program (Sibley and Ahlquist 1990:831) and may be correct, but our UPGMA analysis (p. 859) allies the bowerbirds with the lyrebirds. I have doubts about our placement of the Australo-Papuan treecreepers (Climacteridae), which we viewed as the sister group of the lyrebird-bowerbird clade. Baverstock et al. (1991), using microcomplement fixation, supported most of our conclusions about the origin and relationships of the Australo-Papuan passerines, but they concluded that the climacterids have no close living relatives although they may be closest to the honeyeaters (Meliphagidae). They may be right.
The old endemic Australo-Papuan groups are those noted above, plus the fairywrens (Maluridae), pardalotes, bristlebirds, scrubwrens, thornbills and allies (Pardalotidae), Australian robins (Petroicidae), log-runners (Orthonychidae), Australian babblers (Pomatostomidae), true shrikes (Laniidae), vireos and allies (Vireonidae) and the members of an enlarged family Corvidae, the members of which are separated by unexpectedly small DNA hybridization distances. The ancestors of the crows, jays and magpies of the world originated in Australia and our Corvidae also includes the quail-thrushes, Apostlebird, White-winged Chough, sittellas, whistlers, currawongs, woodswallows, birds-of-paradise, Old World orioles, cuckooshrikes, fantails, drongos, monarchs, magpie-larks, bush-shrikes, helmet-shrikes, and vangas. The latter three groups occur in Africa and Madagascar. Some of these groups had been included in Eurasian families, such as the Muscicapidae and Sylviidae. Eurasian or African groups represented in Australia include the larks, thrushes, swallows, white-eyes, sunbirds and estrildines. Presumably, these are recent arrivals that entered Australia after it had drifted close to southeastern Asia.
Conclusions and comments
There are many problems of avian relationships that deserve study and their results will modify or verify our conclusions. As noted above, several DNA sequence studies and DNA-DNA hybridization studies have supported our conclusions. I am aware of no extensive studies that have falsified them. Our book (Sibley and Ahlquist 1990) was reviewed about 30 times in nine languages with a wide range of opinions, but I have never seen a critical review of Wetmore's classification, except in the cited book. Alex Wetmore was a dear friend and I treasure the memory of our friendship during the last 37 years of his long life. He was convinced that his classification was as close to perfection as it was possible to achieve. On one occasion, many years ago, he said to me, "With just a little more tinkering I think we'll have it about right." I wonder what he would think about the present "tinkering". Will we ever "have it about right"? I think we will.
I expect that more of our results will be verified and that some will be found to be incorrect. I believe that our phylogeny and classification represent improvements over past arrangements, but that further improvements will be made.
A frequent criticism of our work has been that our phylogeny is not based on a "complete matrix" of distance values. There are, at least, three answers: (1). Reciprocity in DNA-DNA comparisons is usually good enough so that it is not necessary to make two-way comparisons in all cases. (2). As noted above, John Kirsch and colleagues at the University of Wisconsin in Madison have shown that the same tree obtained from a complete matrix can also be obtained from as little as 50% of the cells in that matrix. (3). At the average rate of data production we achieved from 1975 to 1986 it would take ca. 1251 years to produce a complete matrix for the 1700 species used in our experiments.
At least two methods will be used to correct or confirm our results. DNA-DNA hybridization will be most useful for the resolution of the older branches because it averages the entire genome. DNA sequence studies have confirmed some of our conclusions and will become even more important when nuclear sequences of 10,000 to 50,000 bases become routine because the level of confidence will be improved and older branches can be resolved. Methods are now available to obtain phylogenies that reflect the history of life on Earth.
Acknowledgments - For their help I thank Jon Ahlquist, John Avise, Robert Bleiweiss, Joel Cracraft, Paul DeBenedictis, Blair Hedges, John Kirsch, Janet Kornegay, Carey Krajewski, Scott Lanyon, Burt Monroe and Paulette Prinsloo.
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