Robert C. Fleischer

Predicting the global spread of H5N1 avian influenza

The spread of highly pathogenic H5N1 avian influenza into Asia, Europe, and Africa has resulted in enormous impacts on the poultry industry and presents an important threat to human health. The pathways by which the virus has and will spread between countries have been debated extensively, but have yet to be analyzed comprehensively and quantitatively. We integrated data on phylogenetic relationships of virus isolates, migratory bird movements, and trade in poultry and wild birds to determine the pathway for 52 individual introduction events into countries and predict future spread. We show that 9 of 21 of H5N1 introductions to countries in Asia were most likely through poultry, and 3 of 21 were most likely through migrating birds. In contrast, spread to most (20/23) countries in Europe was most likely through migratory birds. Spread in Africa was likely partly by poultry (2/8 introductions) and partly by migrating birds (3/8). Our analyses predict that H5N1 is more likely to be introduced into the Western Hemisphere through infected poultry and into the mainland United States by subsequent movement of migrating birds from neighboring countries, rather than from eastern Siberia. These results highlight the potential synergism between trade and wild animal movement in the emergence and pandemic spread of pathogens and demonstrate the value of predictive models for disease control.

Evolution on a volcanic conveyor belt: using phylogeographic reconstructions and K-Ar-based ages of the Hawaiian Islands to estimate molecular evolutionary rates

The Hawaiian Islands form as the Pacific Plate moves over a ‘hot spot’ in the earth’s mantle where magma extrudes through the crust to build huge shield volcanos. The islands subside and erode as the plate carries them to the north‐west, eventually to become coral atolls and seamounts. Thus islands are ordered linearly by age, with the oldest islands in the north‐west (e.g. Kauai at 5.1 Ma) and the youngest in the south‐east (e.g. Hawaii at 0.43 Ma). K–Ar estimates of the date of an island’s formation provide a maximum age for the taxa inhabiting the island. These ages can be used to calibrate rates of molecular change under the following assumptions: (i) K–Ar dates are accurate; (ii) tree topologies show that derivation of taxa parallels the timing of island formation; (iii) populations do not colonize long after island emergence; (iv) the coalescent point for sister taxa does not greatly predate the formation of the colonized younger island; (v) saturation effects and (vi) among‐lineage rate variation are minimal or correctable; and (vii) unbiased standard errors of distances and regressions can be estimated from multiple pairwise comparisons. We use the approach to obtain overall corrected rate calibrations for: (i) part of the mitochondrial cytochrome b gene in Hawaiian drepanidines (0.016 sequence divergence/Myr); (ii) the Yp1 gene in Hawaiian Drosophila (0.019/Myr Kambysellis et al. 1995); and (iii) parts of the mitochondrial 12S and 16S rRNA and tRNAval in Laupala crickets (0.024–0.102/Myr, Shaw 1996). We discuss the reliability of the estimates given the assumptions (i–vii) above and contrast the results with previous calibrations of Adh in Hawaiian Drosophila and chloroplast DNA in lobeliods.

Multilocus Resolution of Phylogeny and Timescale in the Extant Adaptive Radiation of Hawaiian Honeycreepers

Evolutionary theory has gained tremendous insight from studies of adaptive radiations. High rates of speciation, morphological divergence, and hybridization, combined with low sequence variability, however, have prevented phylogenetic reconstruction for many radiations. The Hawaiian honeycreepers are an exceptional adaptive radiation, with high phenotypic diversity and speciation that occurred within the geologically constrained setting of the Hawaiian Islands. Here we analyze a new data set of 13 nuclear loci and pyrosequencing of mitochondrial genomes that resolves the Hawaiian honeycreeper phylogeny. We show that they are a sister taxon to Eurasian rosefinches (Carpodacus) and probably came to Hawaii from Asia. We use island ages to calibrate DNA substitution rates, which vary substantially among gene regions, and calculate divergence times, showing that the radiation began roughly when the oldest of the current large Hawaiian Islands (Kauai and Niihau) formed, ~5.7 million years ago (mya). We show that most of the lineages that gave rise to distinctive morphologies diverged after Oahu emerged (4.0-3.7 mya) but before the formation of Maui and adjacent islands (2.4-1.9 mya). Thus, the formation of Oahu, and subsequent cycles of colonization and speciation between Kauai and Oahu, played key roles in generating the morphological diversity of the extant honeycreepers.

Global phylogeographic limits of Hawaii's avian malaria

The introduction of avian malaria (Plasmodium relictum) to Hawaii has provided a model system for studying the influence of exotic disease on naive host populations. Little is known, however, about the origin or the genetic variation of Hawaiis malaria and traditional classification methods have confounded attempts to place the parasite within a global ecological and evolutionary context. Using fragments of the parasite mitochondrial gene cytochrome b and the nuclear gene dihydrofolate reductase-thymidylate synthase obtained from a global survey of greater than 13 000 avian samples, we show that Hawaiis avian malaria, which can cause high mortality and is a major limiting factor for many species of native passerines, represents just one of the numerous lineages composing the morphological parasite species. The single parasite lineage detected in Hawaii exhibits a broad host distribution worldwide and is dominant on several other remote oceanic islands, including Bermuda and Moorea, French Polynesia. The rarity of this lineage in the continental New World and the restriction of closely related lineages to the Old World suggest limitations to the transmission of reproductively isolated parasite groups within the morphological species.

Prevalence and differential host‐specificity of two avian blood parasite genera in the Australo‐Papuan region

The degree to which widespread avian blood parasites in the genera Plasmodium and Haemoproteus pose a threat to novel hosts depends in part on the degree to which they are constrained to a particular host or host family. We examined the host distribution and host‐specificity of these parasites in birds from two relatively understudied and isolated locations: Australia and Papua New Guinea. Using polymerase chain reaction (PCR), we detected infection in 69 of 105 species, representing 44% of individuals surveyed (n = 428). Across host families, prevalence of Haemoproteus ranged from 13% (Acanthizidae) to 56% (Petroicidae) while prevalence of Plasmodium ranged from 3% (Petroicidae) to 47% (Ptilonorhynchidae). We recovered 78 unique mitochondrial lineages from 155 sequences. Related lineages of Haemoproteus were more likely to derive from the same host family than predicted by chance at shallow (average LogDet genetic distance = 0, n = 12, P = 0.001) and greater depths (average distance = 0.014, n = 11, P < 0.001) within the parasite phylogeny. Within two major Haemoproteus subclades identified in a maximum likelihood phylogeny, host‐specificity was evident up to parasite genetic distances of 0.029 and 0.007 based on logistic regression. We found no significant host relationship among lineages of Plasmodium by any method of analysis. These results support previous evidence of strong host‐family specificity in Haemoproteus and suggest that lineages of Plasmodium are more likely to form evolutionarily–stable associations with novel hosts.

A Multilocus Molecular Phylogeny of the Parrots (Psittaciformes): Support for a Gondwanan Origin during the Cretaceous

The question of when modern birds (Neornithes) first diversified has generated much debate among avian systematists. Fossil evidence generally supports a Tertiary diversification, whereas estimates based on molecular dating favor an earlier diversification in the Cretaceous period. In this study, we used an alternate approach, the inference of historical biogeographic patterns, to test the hypothesis that the initial radiation of the Order Psittaciformes (the parrots and cockatoos) originated on the Gondwana supercontinent during the Cretaceous. We utilized broad taxonomic sampling (representatives of 69 of the 82 extant genera and 8 outgroup taxa) and multilocus molecular character sampling (3,941 bp from mitochondrial DNA (mtDNA) genes cytochrome oxidase I and NADH dehydrogenase 2 and nuclear introns of rhodopsin intron 1, tropomyosin alpha-subunit intron 5, and transforming growth factor ss-2) to generate phylogenetic hypotheses for the Psittaciformes. Analyses of the combined character partitions using maximum parsimony, maximum likelihood, and Bayesian criteria produced well-resolved and topologically similar trees in which the New Zealand taxa Strigops and Nestor (Psittacidae) were sister to all other psittaciforms and the cockatoo clade (Cacatuidae) was sister to a clade containing all remaining parrots (Psittacidae). Within this large clade of Psittacidae, some traditionally recognized tribes and subfamilies were monophyletic (e.g., Arini, Psittacini, and Loriinae), whereas several others were polyphyletic (e.g., Cyclopsittacini, Platycercini, Psittaculini, and Psittacinae). Ancestral area reconstructions using our Bayesian phylogenetic hypothesis and current distributions of genera supported the hypothesis of an Australasian origin for the Psittaciformes. Separate analyses of the timing of parrot diversification constructed with both Bayesian relaxed-clock and penalized likelihood approaches showed better agreement between geologic and diversification events in the chronograms based on a Cretaceous dating of the basal split within parrots than the chronograms based on a Tertiary dating of this split, although these data are more equivocal. Taken together, our results support a Cretaceous origin of Psittaciformes in Gondwana after the separation of Africa and the India/Madagascar block with subsequent diversification through both vicariance and dispersal. These well-resolved molecular phylogenies will be of value for comparative studies of behavior, ecology, and life history in parrots.

Vocal dialects and their possible relation to honest status signalling in the Brown-headed Cowbird

We describe dialects in the flight whistle of the Brown-headed Cowbird and contrast previous hypotheses for the maintenance of dialects with a new one that assumes that a male’s ability to give the correct local dialect is an honest signal of relatively high male quality. The three dialects upon which we focus are part of an extensive dialect system along the eastern Sierra Nevada. The dialects are partially isolated by unsuitable habitat and are unusual because they differ via lexical rather than the less extreme phonetic differences that characterize song dialects in most other species. Because males whistle just before copulating, whistles may function in mate choice. Since flight whistles are also used over long distances, we predicted and confirmed that males within the same dialect have quantitative whistle differences of potential value for individual recognition. Other quantitative analyses indicated phonetic differences among homologous whistle elements from adjacent dialects. Recordings made between 1978 and 1980 showed no quantitative or qualitative differences from a large sample of 142 males recorded between 1983 and 1985. Such temporal stability is expected since the dialects are large, being 10 to 30 km in extent and probably contain hundreds of individuals. Historical records demonstrate that the dialect populations developed since the late 1930s. Males banded in one dialect but recorded in another made up 13.0% of our adult sample and were more likely to have foreign whistles than adults banded and recorded in the same dialect. Yearling males were significantly less likely to whistle than adults, and yearlings that did whistle were significantly more likely to have foreign whistles. Thus possession of a locally appropriate whistle is a reliable indicator of a male’s age which is a major correlate of male mating success and possibly of male quality, as nearly all copulations involve adult males. These age differences are consistent with our new “honest convergence” hypothesis and inconsistent with the local or genetic adaptation hypothesis which predicts that vocal ontogeny is closed by a male’s first breeding season. Bilingual males and those with hybrid whistles combining elements from two dialects made up 0 to 8% of the males within dialect areas. By contrast, 38% of males in one contact zone between two dialects were bilingual and 54% of males in another contact zone gave hybrid whistles. These trends are consistent with the honest convergence hypothesis and inconsistent with hypotheses that dialects are maintained by isolation or because they contribute to local adaptation among populations.

A noninvasive method for distinguishing among canid species: amplification and enzyme restriction of DNA from dung.

Endangered San Joaquin kit foxes Vulpes macrotis mutica can be sympatrically distributed with as many as four other canids: red fox, gray fox, coyote and domestic dog. Canid scats are often found during routine fieldwork, but cannot be reliably identified to species. To detect and study the endangered kit fox, we developed mitochondrial DNA markers that can be amplified from small amounts of DNA extracted from scats. We amplified a 412‐bp fragment of the mitochondrial cytochrome‐b gene from scat samples and digested it with three restriction enzymes. The resulting restriction profiles discriminated among all five canid species and correctly identified 10 ‘unknown’ fox scats to species in blind tests. We have applied our technique to identify canids species for an environmental management study and a conservation study. We envision that our protocol, and similar ones developed for other endangered species will be greatly used for conservation management in the future.

MITOCHONDRIAL-DNA VARIATION AND EVOLUTIONARY RELATIONSHIPS IN THE AMAKIHI COMPLEX

AssRAcT.-An analysis of restriction-site variation in mitochondrial DNA was conducted to examine relationships among five taxa in one group of honeycreepers-the amakihi complex (genus Hemignathus). We analyzed 35 ingroup and 3 outgroup samples. Tree topologies, based on both distance and parsimony methods, grouped taxa into two distinct lineages: the virens-wilsoni lineage; and the chloris-stejnegeri-parvus group. Inter-island sequence divergence (average d,X = 0.0368) is considerably higher than intra-island variation (mean d, = 0.0035), and is higher than average for avian species. Variability (measured as both nucleotide diversity and maximum divergence between haplotypes) differs among island populations. Molecular evolutionary rates were calibrated on the basis of maximum island age estimates; sequence divergence in this lineage is approximately 2% per million years. The relationships within the chloris-stejnegeri-parvus clade generally are consistent with the previously proposed model of double invasion. Genetic distances and the pattern of relationships among amakihi taxa indicate that species status for H. v. chloris and H. v. stejnegeri may be warranted. Received 15

Correlates of extra-pair fertilization success in hooded warblers

Abstract We examined correlates of extra-pair fertilization (EPF) success in the hooded warbler (Wilsonia citrina), a species where females pursue extra-pair matings. The good genes hypothesis predicts that females choose extra-pair mates with morphological or behavioral traits that reflect differences in male genetic quality. EPFs were common, as 35.3% (42/119) of broods contained extra-pair young (EPY) and 26.7% (95/356) of nestlings were the result of EPFs. There was a strong skew in male EPF success which increased the variance in annual male mating success 2–3 fold. However, male morphology did not predict EPF success, as extra-pair males were not older or larger than the males they cuckolded. Likewise, there were no significant correlations between the proportion of extra-pair young in a brood and male size or age. The good genes hypothesis predicts that high-quality males will be consistently preferred as genetic mates, but the number of young sired by a male with his social mate was not consistent from one year to the next. There was a significant negative correlation between female age and proportion of EPY produced, which could result if older females obtain higher-quality social mates. We found no strong evidence that females choose extra-pair mates for good genes, but females may use behavioral rather than morphological cues to assess relative male quality.