Linda R. Maxson

A Molecular Perspective on Lissamphibian Phylogeny

Relationships among the three extant orders of the Class Amphibia were examined using new DNA sequence data from the mitochondrial 12S ribosomal RNA gene. A total of 33 families of amphibians was compared, including representatives of nine of the 10 salamander families, four of the six caecilian families, and 20 of the 22 frog families. Approximately 380 nucleotides were sequenced in each taxon. Of the 333 sites that were aligned and compared for all taxa, 227 were variable. Phylogenetic analyses of these data support the monophyly of the Lissamphibia and the monophyly of each of the three orders, with the Caudata and the Gymnophiona as sister groups. Within the Anura, these molecular data also support the monophyly of the two suborders Archaeobatrachia and Neobatrachia. Although this study represents the broadest sampling of amphibian families at the molecular level, statistical confidence in the relationships must await additional sequence data.

Molecular insights into the relationships and biogeography of West Indian anoline lizards

Abstract The realization that the present day positions of continents and island arcs are a result of continental drift led to the proposal that the extant West Indian fauna reflect an ancient (Cretaceous) land connection between the proto-Antilles and North and South America. Movements of the Antillean islands throughout the Cenozoic are hypothesized to have further fragmented the fauna present on the proto-Antilles when it separated from the mainland 70–80 million years ago. This proposed vicariant origin of the West Indian fauna challenged the previous theory that some or most of the fauna arrived in the Antilles by overwater dispersal from mainland soruces. The virtual absence of appropriate fossils in the West Indies has hampered testing these alternative hypotheses for the origin of the present day West Indian fauna. We present evidence from two types of molecular data, one providing indirect estimates of amino acid sequence divergence for the protein serum albumin, and the other direct DNA sequence information from a mitochondrial ribosomal RNA gene. Our new data provide insights into the origin and relationships of two enigmatic lizard genera, Chamaeleolis and Chamaelinorops , and suggest they are recent derivatives within the genus Anolis . These data, along with molecular data presented elsewhere, strongly implicate an origin by overwater dispersal for most of the Antillean vertebrate fauna.

Crocodilian evolution: insights from immunological data.

The quantitative immunological technique of microcomplement fixation was used to examine serum albumin evolution among members of the order Crocodylia. The cross-reactivity of the albumin antisera and antigens employed in this study had been examined previously using the qualitative technique of immunodiffusion. The phylogenetic conclusions derived from these two data sets are highly congruent, including support of the families Alligatoridae and Crocodylidae, with the placement of Gavialis as the sister taxon of Tomistoma. Both methods provide similar information on the relative amounts of amino acid sequence divergence between albumin molecules; however, the data obtained from microcomplement fixation comparisons are more discriminating than those derived from immunodiffusion. The estimated divergence times within the Crocodylia derived from the fossil record are examined in light of divergence times predicted by the microcomplement fixation-based albumin clock. The traditional phylogenetic placement of Gavialis outside the remaining extant crocodilians is inconsistent with all molecular data sets and we suggest that a careful reexamination of both the extant and the fossil morphological data is warranted.

A phylogenetic analysis of the 18S ribosomal RNA sequence of the coelacanth Latimeria chalumnae

Approximately 98% of the sequence of the 18S ribosomal RNA (rRNA) of the coelacanth Latimeria chalumnae was determined by a combination of direct RNA sequencing and sequencing of rRNA genes amplified by the polymerase chain reaction. This sequence was compared with 18S rRNA sequences of similar length from seven other vertebrate species, representing the taxa Petromyzontiformes, Holocephali, Elasmobranchii, Actinopterygii, Dipnoi, Amphibia, and Amniota, in order to determine the most likely sister group of the coelacanth. Maximum parsimony analysis of these sequences resulted in a single most parsimonious tree containing a number of anomalous relationships among these groups. A bootstrap analysis showed that none of the relationships in this tree was significantly supported at the 95% level, however. Addition of data from 15 other vertebrates (providing multiple representatives of most of the higher taxa) resulted in similar ambiguous groupings, as did a number of methods of editing the sites compared (designed to eliminate rapidly evolving positions). These results may be due to a relatively rapid radiation of the major lineages of osteichthyans, the resolution of which will require molecular information from a larger portion of the coelacanth genome.

Divergent Lineages within the Bufo margaritifera Complex (Amphibia: Anura; Bufonidae) Revealed by Albumin Immunology'

The toads belonging to the Bufo margaritifera complex (Amphibia: Anura; Bufonidae) are widely distributed in the Neotropics. A molecular analysis, using the quantitative immunological technique of micro-complement fixation, assessed the degree of divergence in a plasma protein, serum albumin, among representative Central and South American toads currently placed in this complex. This analysis revealed a surprisingly large amount of genetic diversity. Comparisons of estimates of albumin sequence evolution in representatives of the Bufo margaritifera complex from 32 localities in one central and seven South American countries indicate that this complex indudes more than three lineages that are very distinct genetically but show relatively little morphological variation. The degree of albumin divergence implies that some of these lineages have been isolated for at least 30 million years. This study also suggests that the amount of genetic diversity within other Amazonian species or species groups could be substantial.

Taxonomy, relationships and conservation of a new genus and species of myobatrachid frog from the high rainfall region of southwestern Australia

We describe a frog, Spicospina flammocaerulea gen. et sp. nov., from the high rainfall region of southwestern Australia. Spicospina is defined by spines on the transverse processes of presacral vertebrae llI and can be recognized by the massive parotoid glands and distinctive orange and blue ventral coloration. Immunological comparisons based on albumin and karyotype suggest that this frog represents another major lineage in the subfamily Myobatrachinae, thus warranting generic status. This lineage is more closely related to Uperoleia, Pseudophryne, Myobatradhus, Arenophryne, and Metacrinia than to other Myobatrachine genera. The single species is only known from three dystrophic swamps (area of occupancy 0.05 km2, extent of occurrence 3.63 km2) in far southwestern Australia. This is the third, new, restricted frog species described in the last 10 yr from the high rainfall region of southwestern Australia and demonstrates the inadequacy of documentation of biodiversity in this region.

Immunological Insights into the Evolutionary History of Caecilians (Amphibia: Gymnophiona): Relationships of the Seychellean Caecilians and a Preliminary Report on Family-Level Relationships

The endemic amphibian fauna of the Seychelles Archipelago may have originated through a vicariance event in the early Cenozoic when the microcontinent that formed this group of islands rifted away from India. Albumin evolution in the three endemic genera of caecilians (Caeciliaidae: Grandisonia, Hypogeophis, and Praslinia) was examined using the quantitative immunological technique of micro-complement fixation (MCF). The data obtained and additional DNA sequence data from mitochondrial genes provide further evidence for the monophyly of this group and suggest a time frame for divergences within the group. Praslinia apparently diverged first, approximately 30 million years before the present (mybp), while the separation of Hypogeophis and Grandisonia, as well as speciation within Grandisonia, has occurred within the last 14 million years. Albumins of Seychellean caecilians average 106 units of immunological distance (ID) to the other members of the Caeciliaidae that were examined. Based upon the standard calibration of the albumin clock, this is consistent with the timing of the rifting of the Seychelles from India and suggests that this vicariance event may have been responsible for the isolation of these taxa. Three other members of the Caeciliaidae and one member of the Typhlonectidae were available for this study. Dermophis and Schistometopum cluster (mean ID = 59), and form the sister taxon of the Seychellean genera, while Caecilia and Typhlonectes cluster at a mean ID of 78. Albumin from representatives of two other families examined (Ichthyophiidae and Rhinatrematidae) were so different that measurements were outside the quantitative limits of MCF; these taxa appear to be distantly related to the Caeciliaidae and Typhlonectidae, as well as to each other.

Relationships among palearctic Hyla: Insights from immunology

Abstract The European or green tree frog, Hyla arborea (family Hylidae; subfamily Hylinae), was once believed to be a single widespread species ranging throughout Europe and Asia. Based on data from more detailed studies of morphology and mating call, and information from molecular techniques, H. arborea is now recognized as a superspecies group. To examine relationships among these frogs, we used the quantitative immunological technique of micro-complement fixation to estimate the degree of amino acid sequence divergence in the proetin serum albumin. We estimate the divergence time between the European H. arborea and the Middle Eastern H. savignity to be about 6 MYA, while some Asian species appear to have diverged much earlier, approximately 24 MYA. Several distinct patterns of albumin cross-reactivity to the two antisera used in this study were observed. The groups distinguished by these patterns are consistent with patterns of speciation that can be supported by biogeographical history.

Mitochondrial DNA sequence variation and genetic differentiation among colubrine snakes (Reptilia: Colubridae: Colubrinae)

Abstract Nucleotide sequence variation in a portion of the mitochondrial 16S ribosomal RNA gene was assessed to investigate systematic affinities within the snake subfamily Colubrinae. A total of 391 base pairs from each of 33 taxa were aligned, with Thamnophis as the outgroup to the Colubrinae. Data were analyzed using both distance (145 sites) and parsimony (75 sites) algorithms. Analyses were concordant in showing that although morphologically resembling a ratsnake, the Asian Gonyosoma oxycephalum is genetically more similar to the North American racer lineage that includes Coluber constrictor and Masticophis flagellum than to any ratsnake examined. All other ratsnakes sampled comprise a monophyletic group separate from the remaining colubrines. Within this ratsnake clade, the Nearctic ratsnakes can be distinguished as a monophyletic group. At least nine separate racer lineages can be identified. With three exceptions, racers from the same geographical region are genetically more similar to one another than to colubrines of other geographic origin. Finer resolution of phylogenetic affinities among taxa within this subfamily will require additional sequence data and more extensive sampling of taxa. The relative degree of sequence divergence observed between the tribes (racers and ratsnakes) sampled is consistent with other biochemical estimates of protein divergence, as well as with fossil evidence, that suggests a Miocene divergence between the ratsnakes and other colubrine lineages.

Relationships among the Eastern Plethodon: Evidence from Immunology

Relationships among North American plethodontid salamanders have been the subject of numerous molecular studies. In particular, immunological investigations of albumin evolution using the quantitative technique of micro-complement fixation have provided insights into relationships between the genera Ensatina and Plethodon, as well as within Plethodon. However, examination of relationships within eastern Plethodon has been hindered due to lack of antisera from a representative of each species group. An antiserum to albumin from P. wehrlei (wehrlei group) was produced and compared to all available Plethodon antigens. With this antiserum, all currently recognized species groups of eastern Plethodon (glutinosus, cinereus, welleri, and wehrlei) are now represented in a reciprocal matrix of im- munological distance values. Phylogenetic trees derived from these data indicate that the welleri and glutinosus groups are sister taxa, and cluster with the wehrlei group; the cinereus group is the most distant within the eastern Plethodon. This topology conflicts with that based on electrophoretic and morphological data, which suggest that the glutinosus and wehrlei groups are sister taxa. A close examination of the albumin immunological data shows that the rate of albumin evolution within eastern Plethodon has been variable, and therefore these data may be less reliable for phylogenetic reconstruction in this taxon.