Sheldon I. Guttman

Electrophoretic evidence of reticulate evolution in the Appalachian Asplenium complex

The Appalachian Asplenium complex consists of six fertile species: three diploids and their three allotetraploid derivatives. This reticulate evolutionary pattern, originally proposed on the basis of morphological and cytological evidence, was later corroborated by analyses of flavonoid composition. Presented here are data from starch gel electrophoresis of 11 enzyme systems, coded by 15 interpretable loci, which are highly consistent with the proposed relationships in the Appalachian Asplenium complex. The diploids are strongly differentiated (genetic distance (D) = 0.67 to 1.30); each diploid possesses unique alleles at several loci. Each allotetraploid expresses the heterozygous, or less often homozygous, enzyme phenotypes expected for combinations of parental alleles at most loci. Exceptions are the expression of a novel PGI-2 allele and the loss of expression of parental IDH alleles in both allotetraploids. The importance of reticulate evolution in plants is well documented (Lewis 1980). This type of evolution involves hybridization of differentiated genomes resulting in high levels of heterozygosity. The application of the isozyme technique seems particularly relevant to gain additional insights into the evolution of polyploid complexes. This technique detects allelic variants (allozymes) at equivalent gene loci, thus facilitating analysis of genetic variation within and between species. Allopolyploids may be expected to exhibit heterozygous phenotypes at loci for which the presumed parental taxa possess electrophoretically different alleles. Indeed, early isozyme studies of plants often addressed the identification of diploid progenitors of allopolyploid crops (Cherry et al. 1972; Garber 1974; Reddy and Garber 1971; Sheen 1972). More recently Roose and Gottlieb (1976) demonstrated the utility of allozymes in confirming relationships and assessing heterozygosity in Tragopogon. However, given the preponderance of such complexes in nature and the general lack of allozyme studies on them, the technique is at present underexploited. Reticulate evolution is well known among most genera of temperate ferns (Walker 1979). The use of allozyme analysis to address problems of systematics and evolution in ferns has awaited development of techniques that allow extractions of active and electrophoretically resolvable enzymes from tissues noted for their high levels of phenolic compounds. Such techniques are now available (Soltis et al. 1980, 1983; Werth et al. 1982) and are in use in several laboratories. The evolutionary relationships among Asplenium species endemic to eastern North America were clarified by Wagner (1954) in a classic study of their morphology and meiotic chromosome behavior. Wagner showed that three diploid species, A. platyneuron, A. montanum, and A. rhizophyllum, gave rise to three allotetraploid derivatives, A. ebenoides (platyneuron x rhizophyllum), A. pinnatifidum (montanum x rhizophyllum), and A. bradleyi (montanum x platyneuron) (fig. 1). Studies on cytology (see Walker 1979 for review) and flavonoids (Smith and Levin 1963; Smith and Harborne 1971; Harborne et al. 1973) of the six fertile species as well as a number of sterile hybrids provided data consistent with the concept of relationships as originally hypothesized by Wagner (1954). The present paper reports allozymic evidence that further corroborates Wagners hypotheses, and demonstrates the potential, as well as some of the limitations, of allozyme data to elucidate parentages of allopolyploid taxa. MATERIALS AND METHODS Field collections from two or three populations of each fertile taxon as well as one sterile allodiploid A. ebenoides were obtained from isolated localities (table 1). Up to sixty individuals of each species were collected per population. However, collection of A. ebenoides from the unique tetraploid population in Alabama was limited to three juvenile sporophytes.

Recurring Origins of Allopolyploid Species in Asplenium

A large proportion of plant species has originated through allopolyploidy: interspecific hybridization followed by chromosome doubling. Heterozygosity remains fixed in allopolyploids because of nonsegregation of parental chromosomes. Two allotetraploid species of the fern genus Asplenium show allozyme polymorphisms at loci that are polymorphic in their diploid progenitors, indicating that each has originated more than once and implicating continued gene flow from diploids to tetraploids.

HIGH FIDELITY OF MITOCHONDRIAL GENOME TRANSMISSION UNDER THE DOUBLY UNIPARENTAL MODE OF INHERITANCE IN FRESHWATER MUSSELS (BIVALVIA: UNIONOIDEA)

Abstract.— Doubly uniparental inheritance (DUI) of mitochondrial DNA (mtDNA) has been demonstrated in both mytilid and unionid bivalves. Under DUI, females pass on their mtDNA to both sons and daughters, whereas males pass on their mtDNA to only sons. In mytilids, the loss of an original male (or M) mitotype, with its subsequent replacement by that lineages female (or F) mitotype, has been called a role‐reversal or, more specifically, a masculinization event. Multiple masculinization events have been inferred during the evolutionary history of mytilids but not unionids. The perceived lack of role‐reversal events in unionids may represent a significant difference in the evolutionary dynamics of DUI between the two bivalve taxa or simply a lack of sufficient taxon sampling in unionids. To evaluate these alternative hypotheses, six additional unionoidean bivalve genera were sampled for DUI including one genus from the sister taxon of the Unionidae, the Hyriidae. Phylogenetic analyses of 619 base pairs of cytochrome c oxidase I (COI) from eight genera (nine species) of unionoidean bivalves, plus the sister taxon to the Unionoida, Neotrigonia, revealed that the M and F unionoidean mitotypes were contained in gender‐specific, topologically congruent clades. This supports the hypothesis that either role‐reversal events do not occur in unionoideans or, if they do occur, their products are ephemeral in an evolutionary sense. Furthermore, the fact that the mantle‐tissue‐derived Neotrigonia mitotype is the sister mitotype to the unionoidean F mitotype clade suggests that DUI has been operating with high fidelity in unionoids for at least 200 million years. A relatively low incidence of interspecific hybridization in unionoideans and a possibly obligate role for the M mitotype in unionoidean gender determination are offered as potential explanations for the disparate evolutionary dynamics of DUI observed between mytilid and unionoidean bivalves.

AN ELECTROPHORETIC STUDY OF EVOLUTION IN CAPSICUM (SOLANACEAE)

Most recent treatments of the peppers, genus Capsicum (Solanaceae), recognize four or five domesticated taxa and 20 or more wild species (Eshbaugh, 1980a). The distribution of the species is fairly well documented with the exception of the Brazilian taxa. The genus includes a number of wide ranging species as well as several narrow endemics, e.g., C. galapagoensis, C. cardenasii, etc. All taxa are at least facultative self-fertilizers with the exception of C. cardenasii. A survey of chromosome numbers indicates n = 12 or 13 (Pickersgill, 1977), suggesting two lines of evolution which may eventually require some realignment at the generic level. Changing concepts on evolution of the genus have led from the hypothesis of C. frutescens as the single wild progenitor of all the domesticates (Davenport, 1970; Ramalingam, 1972; Jett, 1973), to four or five distinct progenitors each giving rise to a domesticate (Heiser et al., 1971) and alternatively, to three independent lines of evolution leading to domesticated C. baccatum, C. pubescens, and a complex of C. annuum, C. chinense, and C. frutescens (Eshbaugh, 1980b). Various approaches to investigating and understanding evolution in the genus have included comparative morphological investigation, karyotype analysis (Ohta, 1962; Shopova, 1966; Pickersgill, 1977) and the study of breeding behavior (Pickersgill, unpubl.; Eshbaugh, 1975). We use the technique of starch gel electrophoresis to provide data for a different perspective of evolution in Capsicum. Re-

Mantle biopsy: a technique for nondestructive tissue-sampling of freshwater mussels

Mantle biopsy is a means of obtaining tissue samples for genetic, physiological, and contaminant studies of bivalves; but the effects of this biopsy on survival have not been determined. We describe a simple technique for obtaining such samples from unionacean bivalves and how we compared survival among biopsied and control organisms in field experiments. Survival was not significantly different between treatment and control groups. Power estimates for these results were between 0.42 and 0.73. Results were similar among species and among habitats. Mantle biopsy is a technique that allows genetic, biochemical, and contaminant studies of mussel populations when destruction of individuals should be avoided.

GENETIC DIFFERENTIATION ALONG HOST PLANT LINES IN THE SYMPATRIC ENCHENOPA BINOTATA SAY COMPLEX (HOMOPTERA: MEMBRACIDAE)

Recent studies on the origin of insect host plant races and the evolution of species in certain phytophagous insect complexes have forced systematists to revise concepts concerning modes of animal speciation. Rapid establishment of new host races by stenophagous insects on introduced plants has led many biologists to accept that new host races and species may arise sympatrically (Bush, 1969, 1975). Since much of the evidence is indirect, however, other workers still regard geographical isolation as a prerequisite for speciation in most groups of sexually reproducing animals (Mayr, 1970). Parapatric and sympatric speciation are usually associated with specialized feeding habits as in some phytophagous and parasitic insects (Bush, 1975). Maynard Smith (1966) showed that disruptive selection operating on genotypes which influence habitat or host selection could potentially lead to sympatric host plant species. AIlochronic shifts in life histories (Alexander and Bigelow, 1960; Alexander, 1968) or adoption of new host plants by phytophagous insects (Bush, 1969; Huettel and Bush, 1972; Knerer and Atwood, 1973) have been implicated in the formation of host plant races and reproductive isolation. Edmunds and Alstad (1978) have demonstrated in scale insects that when these insects are associated with a stable resource such as a large tree, they adapt to the defense system of individual trees. Given limited dispersal and high extinction rates on newly colonized hosts (Simberloff, 1974), insects on a given individual host plant may represent a deme partially isolated from adjacent trees. Recently, Tauber and Tauber (1977a, 1977b) and Tauber et al. (1977) demonstrated the

Early evolution of chili peppers (Capsicum)

An hypothesis is advanced based upon geographical information and data from horizontal starch gel electrophoresis regarding the place and mode of evolution of the chili peppers (Capsicum). The hypothesis suggests a major portion of the genusCapsicum arose in a nuclear area in south central Bolivia with subsequent migration into the Andes and lowland Amazonia accompanied by radiation and speciation.

Natural Hybrids between Hyla cinerea and Hyla gratiosa: Morphology, Vocalization and Electrophoretic Analysis

Thirteen putative hybrids between Hyla cinerea and Hyla gratiosa were found near Savannah, Georgia. The ratio of head width to tibio-fibula length unequivocally distinguished the natural hybrids from samples of the parental species from the same area. Vocalizations of six male hybrids were H. gratiosa-like in their harmonic (fine-temporal) structure; vocalizations of six other male hybrids were H. cinerea-like. The spectral characteristics of hybrid calls were intermediate with respect to those of the parental forms, but the ranges of variation of hybrids and H. cinerea overlapped. Three of the natural hybrids, including a female which responded in discrimination tests, were characterized electrophoretically as F, hybrids.

Effects of Twenty Years of Hybridization in a Disturbed Habitat on Hyla cinerea and Hyla gratiosa

Field, acoustical, morphological, and electrophoretic analyses confirmed that more than 20 years after it began due to pond construction, hybridization between Hyla cinerea and H. gratiosa persists near Auburn, Alabama. Hyla gratiosa males began calling three weeks before H. cinerea or hybrids appeared at the ponds. Electrophoretic analysis demonstrated that the first back- crossed animals found in the breeding season were backcrossed towards H. gratiosa, while F, hy- brids and H. cinerea-like backcrossed animals appeared later. Morphological and electrophoretic identification of treefrogs generally coincided; however, 38% of individuals morphologically iden- tified as H. cinerea were electrophoretically H. cinerea backcrosses. The data indicate that F1 hy- brids, and possibly backcrossed individuals, may have reduced fitness.

Population Genetic Structure of Earthworms (Lumbricus rubellus) in Soils Contaminated by Heavy Metals

The genetic structure of earthworm (Lumbricus rubellus) populations from plots (N=3) contaminated by sewage sludge (containing Cd, Cu, Pb, Zn) and reference plots (N=3) was compared. Earthworms (N=30–40 per plot) were collected during May 2000 and electrophoretic analyses were used to assess genetic variation at three polymorphic loci (GPI, LAP, PGM). Allele and genotype frequencies for GPI and PGM differed significantly between sludge and reference populations. Sludge populations were characterized by a reduced frequency of the most common genotype and allele at these loci, and the presence of alleles not found in the reference population. Allele and genotype frequencies did not differ significantly among individual plots within the sludge or the reference population. These results suggest that certain alleles and genotypes may be more sensitive to the effects of heavy metals. Multi-locus heterozygosity was significantly higher in the sludge population compared to the reference population; the proportion of individuals heterozygous at two or more loci was more than 15% higher in the sludge population. Results demonstrate that measures of population genetic structure in earthworms may represent useful biological indicators for the assessment of impacted terrestrial habitats.