Horseshoe crabs' exceptional morphological conservatism over the past 150 My has led to their reputation as "living fossils," but also has served to obscure phylogenetic relationships within the complex. Here we employ nucleotide sequences from two mitochondrial genes to assess molecular evolutionary rates and patterns among all extant horseshoe crab species. The American species Limulus polyphemus proved to be the sister taxon to a clade composed of the Asiatic species Tachypleus gigas, T. tridentatus, and Carcinoscorpius rotundicauda, whose relationships inter se were not resolved definitively. Both absolute and relative rate tests suggest a moderate slowdown in sequence evolution in horseshoe crabs. Nonetheless, dates of the lineage separations remain uncertain primarily because of reservations about molecular-clock calibrations resulting from large rate variances at examined loci across Arthropods and other animal lineages, as inferred in this and prior studies. Thus, ironically, separation dates as estimated by molecular evidence in general may remain most insecure in taxonomic groups for which such information is needed most-those lacking strong biogeographic or fossil benchmarks for internal-clock calibrations. In any event, the current results show that large numbers of molecular characters distinguish even these most morphologically conservative of organisms. Furthermore, comparisons against previously published mitochondrial sequence data in the morphologically dynamic hermit crab-king crab complex demonstrates that striking heterogeneity in levels of morphotypic differentiation can characterize Arthropod lineages at similar magnitudes of molecular divergence.

We assayed restriction site differences in mitochondrial DNA (mtDNA) within and among allopatric populations of the Mallard (Anas platyrhynchos) and the American Black Duck (A. rubripes). The observed mtDNA clones grouped into two phylogenetically distinct arrays that we estimate differ by about 0.8% in nucleotide sequence. Genotypes in one clonal array were present in both species, while genotypes in the other array were seen only in Mallards. In terms of the mtDNA "gene tree," the assayed Mallards exhibit a paraphyletic relationship with respect to Black Ducks, meaning that genealogical separations among some extant haplotypes in the Mallard predate the species separation. Evidence is advanced that this pattern probably resulted from demographically based processes of lineage sorting, rather than recent, secondary introgressive hybridization. However, haplotype frequencies were most similar among conspecific populations, so the Mallard and Black Ducks cluster separately in terms of a population phenogram. The results provide a clear example of the distinction between a gene tree and a population tree, and of the distinction between data analyses that view individuals versus populations as operational taxonomic units (OTUs). Overall, the mtDNA data indicate an extremely close evolutionary relationship between Mallards and Black Ducks, and in conjunction with the geographic distributions suggest that the Black Duck is a recent evolutionary derivative of a more broadly distributed Mallard-Black ancestor.

To address the suitability of mitochondrial DNA sequence data for higher avian systematics, cytochrome b sequences for nine species of Cuculiformes and Galliformes were gathered and compared to those of the Hoatzin, a specialized bird whose phylogenetic affinities have long puzzled ornithologists. Additional avian cytochrome b sequences from GenBank also were incorporated into the comparisons. Various phenetic and parsimony analyses applied to the molecular data coded in four formats (nucleotide sequences, purines versus pyrimidines, first and second positions of codons, and translated amino acid sequences) yielded several consistent phylogenetic arrangements, including the distinction of Galliformes from Cuculiformes, a clade composed of the Guira Cuckoo and Groove-billed Ani (Crotophagidae) to the exclusion of the other assayed cuculiforms (Cuculidae and Coccyzidae), and a clade composed of the New World quail (Odontophoridae, in the Sibley-Ahlquist classification) as a sister group to most or all Old World galliforms assayed (Phasianidae and Numididae). The latter result is of particular interest because it supports a controversial systematic realignment of the Galliformes based on DNA-DNA hybridization data. With regard to the phylogenetic position of the Hoatzin, the analysis differed in face-value outcome, although alternative clades were in all cases poorly supported by bootstrapping, and internodal distances were small relative to the range of genetic separations within either Cuculiformes or Galliformes. These uncertainties in phylogenetic resolution of the Hoatzin appear to be attributable to shallow slopes in the regressions relating cytochrome b sequence differences to evolutionary time, in conjunction with homoplasy in the data, sampling error in the estimation of genetic differences, and the relatively close temporal placements of relevant evolutionary nodes.

Horseshoe crabs' exceptional morphological conservatism over the past 150 My has led to their reputation as "living fossils," but also has served to obscure phylogenetic relationships within the complex. Here we employ nucleotide sequences from two mitochondrial genes to assess molecular evolutionary rates and patterns among all extant horseshoe crab species. The American species Limulus polyphemus proved to be the sister taxon to a clade composed of the Asiatic species Tachypleus gigas, T. tridentatus, and Carcinoscorpius rotundicauda, whose relationships inter se were not resolved definitively. Both absolute and relative rate tests suggest a moderate slowdown in sequence evolution in horseshoe crabs. Nonetheless, dates of the lineage separations remain uncertain primarily because of reservations about molecular-clock calibrations resulting from large rate variances at examined loci across Arthropods and other animal lineages, as inferred in this and prior studies. Thus, ironically, separation dates as estimated by molecular evidence in general may remain most insecure in taxonomic groups for which such information is needed most-those lacking strong biogeographic or fossil benchmarks for internal-clock calibrations. In any event, the current results show that large numbers of molecular characters distinguish even these most morphologically conservative of organisms. Furthermore, comparisons against previously published mitochondrial sequence data in the morphologically dynamic hermit crab-king crab complex demonstrates that striking heterogeneity in levels of morphotypic differentiation can characterize Arthropod lineages at similar magnitudes of molecular divergence.

Restriction analyses were conducted on mitochondrial DNA (mtDNA) amplified by long-PCR from an endangered bird, the Cape Sable seaside sparrow. The first of several successful mtDNA amplifications was accomplished using the partially digested tissue remains of a transmitter-monitored bird retrieved from the gut of a snake. As many as 91 mtDNA restriction fragments produced by 18 endonucleases were compared in this and four other Cape Sable specimens against mtDNA similarly amplified by long-PCR from other taxonomic forms in the seaside sparrow complex. Results indicate that the Cape Sable birds belong to an 'Atlantic' matrilineal clade, and are highly divergent from other seaside sparrows along the Gulf of Mexico.

Despite a great diversity of reproductive behaviors in fishes, few studies have examined the genetic consequences of alternative reproductive tactics. Here we develop and employ microsatellite markers to assess genetic paternity and maternity of progeny cohorts in a population of redbreast sunfish (Lepomis auritus), a species in which males build and tend nests. Nearly 1000 progeny from 25 nests, plus nest-attendant males and nearby adults, were genotyped at microsatellite loci that displayed more than 18 alleles each. The genetic data demonstrate that multiple females (at least two to six) spawned in each nest, their offspring were spatially dispersed across a nest, and more than 90% of the young were sired by the attendant male. However, about 40% of the nests also showed genetic evidence of low-level reproductive parasitism, and two nests were tended by males that had fathered none of the sampled offspring. Genetically deduced reproductive behaviors in this population of redbreast sunfish contrast with those reported previously in bluegill sunfish (L. macrochirus) wherein heteromorphic males specialized for parasitism or for parental care coexist in high frequency. Thus, nest-parasitic reproductive behaviors in fishes appear to be evolutionary labile.

A battery of allelic markers at highly polymorphic microsatellite loci was developed and employed to confirm genetically, the clonal nature of sibships in nine-banded armadillos. This phenomenon of consistent polyembryony, otherwise nearly unknown among the vertebrates, was capitalized upon to describe the micro-spatial distributions of numerous clonal sibships in a natural population of armadillos. Adult clone mates were significantly more dispersed than were juvenile sibs, suggesting limited opportunities for altruistic behavioural interactions among mature individuals. These results, and considerations of armadillo natural history, suggest that evolutionary explanations for polyembryony in this species may not reside in the kinds of ecological and kin selection theories relevant to some of the polyembryonic invertebrates. Rather, polyembryony in armadillos may be associated evolutionarily with other reproductive peculiarities of the species, including delayed uterine implantation of a single egg.

Breeding, male North American sunfish (Centrarchidae), are often brightly coloured and promiscuous. However, the largemouth bass (Micropterus salmoides) is sexually monomorphic in appearance and socially monogamous. Unlike some other nest-tending centrarchids in the genus Lepomis, largemouth bass have also been reported to provide biparental care to eggs and fry. Here we use microsatellite markers in order to test whether social monogamy predicts genetic monogamy in the largemouth bass. Offspring were collected from 26 nests each usually guarded by a pair of adults, many of which were also captured. Twenty-three of these progeny cohorts (88%) proved to be composed almost exclusively of full-sibs and were thus the product of monogamous matings. Cuckoldry by males was rare. The genetic data also revealed that some nests contain juveniles that were not the progeny of the guardian female, a finding that can be thought of as low-level 'female cuckoldry'. Overall, however, the data provide what may be the first genetic documentation of near-monogamy and biparental care in a vertebrate with external fertilization.

The Iceland population of Anguilla eels contains an elevated frequency of fish with vertebral numbers lower than those typical of European localities. Several distinct hypotheses have been advanced to account for these morphologically atypical fish: for example, they could represent (1) genetically "pure" American expatriates, (2) genetically "pure" European types with ontogenetic abnormalities, or (3) hybrids between American and European forms. Here we critically test these and other possibilities by examining the joint distributions of allozyme markers, mitochondrial DNA markers, and vertebral numbers in Icelandic eels. The particular patterns of association among the genetic and morphological traits demonstrate that the Iceland population includes, in low frequency, the products of hybridization between American and European eels. Approximately 2-4% of the gene pool in the Iceland eel population is derived from American eel ancestry. This hybrid zone is highly unusual in the biological world, because the mating events in catadromous eels presumably take place thousands of kilometers from where the hybrids are observed as maturing juveniles. The molecular data, in conjunction with the geographic distributions, strongly suggest that the differences in migrational behavior and morphology between American and European eels include an important additive genetic component. Evolutionary hypotheses are advanced to account for the original separation of North Atlantic eels into American and European populations, and for the presence of hybrids in Iceland.

Despite a great diversity of reproductive behaviors in fishes, few studies have examined the genetic consequences of alternative reproductive tactics. Here we develop and employ microsatellite markers to assess genetic paternity and maternity of progeny cohorts in a population of redbreast sunfish (Lepomis auritus), a species in which males build and tend nests. Nearly 1000 progeny from 25 nests, plus nest-attendant males and nearby adults, were genotyped at microsatellite loci that displayed more than 18 alleles each. The genetic data demonstrate that multiple females (at least two to six) spawned in each nest, their offspring were spatially dispersed across a nest, and more than 90% of the young were sired by the attendant male. However, about 40% of the nests also showed genetic evidence of low-level reproductive parasitism, and two nests were tended by males that had fathered none of the sampled offspring. Genetically deduced reproductive behaviors in this population of redbreast sunfish contrast with those reported previously in bluegill sunfish (L. macrochirus) wherein heteromorphic males specialized for parasitism or for parental care coexist in high frequency. Thus, nest-parasitic reproductive behaviors in fishes appear to be evolutionary labile.