MOLECULAR DIVERGENCE BETWEEN ASIAN AND NORTH AMERICAN SPECIES OF LIRIODENDRON (MAGNOLIACEAE) WITH IMPLICATIONS FOR INTERPRETATION OF FOSSIL FLORAS

Botanists have long been aware of the floristic similarities between eastern Asia and eastern North America. Most who have considered this classic disjunction pattern have suggested that it arose through range disruption of a flora that was once more widely distributed in the Northern Hemisphere. There is less agreement on the timing of this process, with suggestions ranging from the Paleocene to the Neogene. In this study, molecular markers from two different plant genomes were used to assess the degree of genetic divergence between the two interfertile, morphologically similar species of the genus Liriodendron, i.e., L. tulipifera and L. chinense. Resulting molecular divergence estimates were translated into approximate dates of separation, independent of evidence from the fossil record. Allozyme data (Nei's genetic identity = 0.434) suggested a divergence time of 10–16 million years before present, whereas sequence divergence in the plastid genomes (1.24%) led to an estimate of approximately 11–14 million years before present. A review of the paleobotanical literature indicated that the fossil floras that included, or might have included Liriodendron could not have survived in Beringia after the late Miocene and the onset of southward-migrating Arctic air masses on the North American continent. This interpretation suggests a minimum time of separation of approximately 13 million years before present. Thus, both molecular data sets and the paleobotanical evidence concur in suggesting a divergence time of 10–16 million years before present. Interspecific compatibility and relative morphological stasis must have, therefore, persisted from at least the late Miocene. We emphasize the need for similar studies in other genera, especially those that have both a reasonable Tertiary fossil history and extant species in mesic temperate refugia in Asia, Europe, and western as well as eastern North America.     

LIRIODENDRON (MAGNOLIACEAE) FROM THE MIOCENE CLARKIA FLORA OF IDAHO

Miocene Liriodendron carpels, whole fruiting structures and leaves from Clarkia and Oviatt Creek sites in northern Idaho are preserved as imprints and compressed fossils in soft lacustrine clays. The isolated carpels are indistinguishable from those described as L. hesperia Berry from the Spokane Latah flora. Fruit aggregates from the type Clarkia and Oviatt Creek localities and leaves from three Clarkia sites are considered to be within the range of variation of the single species L. hesperia. Comparisons were made regarding leaf architecture, lower leaf epidermal structures, leaf flavonoid and steroid analysis, morphological features of receptacles and carpels, and the venation pattern of carpels of the fossil material to the two extant species, L. tulipifera L. (native to southeastern United States) and L. chinense Sarg. (native to southeastern Asia). Leaf architecture features analyzed by standard statistical and canonical tests and marginal venation patterns near the base of leaves suggest that L. hesperia is more similar to L. tulipifera, whereas the size dimensions of lower epidermal cells and the common presence of two sterane compounds imply that L. hesperia is more similar to L. chinense. The fossil species, however, is a distinct taxon indicated by statistical discriminant and canonical tests, leaf base shape, often smaller epidermal cell dimensions, and the shape of round receptacle carpel scars. Both the fossil and the two living Liriodendron species are associated with comparable mixed mesophytic floras.     

REGENERATION OF LIRIODENDRON TULIPIFERA (FAMILY MAGNOLIACEAE) FROM PROTOPLAST CULTURE

Protoplasts were isolated enzymatically from suspension cultures of two embryogenic lines of yellow-poplar (Liriodendron tulipifera L.) and cultured in droplets of a regeneration medium supplemented with 1 mg/1 2,4-D and 0.25 mg/1 6BA and solidified with agarose. Suspension cultures grown in the light yielded substantial numbers of protoplasts, while insignificant numbers were isolated from cultures grown in the dark. The protoplasts reformed cell walls within three days, and 75 percent of them divided at least once within one week in culture. Embryogenic suspension or callus cultures were regenerated by placing agarose droplets containing protoplast-derived microcalli directly into liquid conditioning medium or onto plates of conditioning medium solidified with agar. Embryogenesis was obtained by transferring the callus to a hormone-free induction medium.     

INTRASPECIFIC CHLOROPLAST DNA VARIATION AND BIOGEOGRAPHY OF NORTH AMERICAN LIRIODENDRON L. (MAGNOLIACEAE)

Restriction site variation in chloroplast DNA (cpDNA) was surveyed to analyze population dynamics in Liriodendron tulipifera L., a woody angiosperm found in eastern North America. Two cpDNA haplotypes, differing by the presence or absence of five restriction site changes (nucleotide sequence divergence estimated as approximately 0.15%) are geographically structured; 61 widespread populations possess the “northern” haplotype and three isolated populations of central Florida possess the “southern” haplotype. This geographic break in cpDNA distribution corresponds to patterns of geographic distribution revealed by a previous survey of allozyme variation, with the exception that analyses of allozyme data further divided the populations containing the northern cpDNA haplotype into two groups, a widespread upland group and a coastal intermediate group. Analyses of these two independent data sets together support the hypothesis that L. tulipifera survived the glacial advances of the Pleistocene in two distinct refugia, possibly as different taxa, and the intermediate coastal group was putatively formed from recent hybridizations between these entities.     

EVIDENCE FOR VARIATION IN THE QUANTITY OF DNA AMONG PLASTIDS OF ACETABULARIA

The DNA content of individual plastids of the giant unicellular algae Acetabularia mediterranea, and Polyphysa cliftoni was studied. Four methods were used for localizing DNA: acridine orange staining, radioautography following actinomycin D-³H treatment, electron microscopy of thin tissue sections, and electron microscopy of osomotically disrupted plastids. With each method, DNA was readily detected in 20–35% of plastids, but no DNA was observed in the remaining 65–80%. The results further showed that in those plastids with detectable DNA the amount of DNA present was variable. The sensitivity and reliability of the localization methods are discussed, and the possible implications of these findings are considered.     

闽南地区TT病毒的变异及经输血传播的初步证据

TT virus(TTV)DNA was tested by nested-PCR from sera of hepatitis patients and volunteer blood donors in Minnan area. The amplified segment was a 189 base pair region in TTV ORF2. A total of six sequences were obtained from three non-A to G hepatits patients and two from volunteer blood donors. The sequences were found to be with 82.9% to 99.3% homology to TTV Japanese strain and Chinese strain. The divergence of sequence in these six segments varied from 0.7% to 17.1%, which indicated that the TTV had been existing for a long time in this area. In the serum of a non-A to G hepatitis patient who was negative for TTV DNA in the 14th day of disease course turned to be positive in the 30th day, two TTV sequences were obtained which showed 92.1% nucleotide homology. It indicated that different TTV strains can co exist in the same person. This patient's blood had been transfused ten times between the collection of his TTV negative sample and his positive serum sample. Seven of the blood donors were traced and sampled for sera, of which three were positive for TTV. For all 161 patients tested, the history of exposure to blood products was associated with an increased risk of TTV infection(relative risk, 3.0; 95% confidence intervals, 1.89～4.81).     

GENETIC EVIDENCE FOR DIPLOIDY IN EQUISETUM

Sporophytes and gametophytes of Equisetum arvense, E. laevigatum, and E. telmateia were analyzed using enzyme electrophoresis to estimate isozyme number. Despite their uniformly high chromosome numbers (2n = 216), these three species exhibited isozyme numbers typical of diploid seed plants for the enzymes AAT, ADH, ALD, GDH, [NADP]IDH, LAP, MDH, [NADP]ME, PGI, PGM, SkDH, and 6PGDH. All three species exhibited an additional isozyme for TPI. There is, therefore, no genetic evidence for low base numbers such as x = 9 and x = 12 suggested for Equisetum. Intact chloroplasts were isolated from E. arvense and the chloroplast extract compared electrophoretically to whole plant extracts. The single enzymes observed for LAP, GDH, [NADP]IDH, and [NADP]ME were absent from the chloroplast extract. Isozymes AAT-1, ALD-2, MDH-3, PGI-1, PGM-2, SkDH-2, 6PGDH-2, TPI-2, and TPI-3 were active in the chloroplast fraction; 6PGDH-1, PGI-2, PGM-1, and TPI-1 were lacking from the chloroplast fraction and were considered cytosolic. Isozymes AAT-2, MDH-1, MDH-2, MDH-4, and SkDH-1 were also lacking from the chloroplast fraction but because AAT, MDH, and SkDH have been reported from several subcellular compartments, their localization is unknown. These findings indicate that isozymes in Equisetum species are subcellularly compartmentalized as has also been demonstrated for homosporous ferns, gymnosperms, and angiosperms.     

DYNAMIC AND GENETIC CONSEQUENCES OF VARIATION IN HORIZONTAL TRANSMISSION FOR A MICROPARASITIC INFECTION

Transmission to a new host is a critical step in the life cycle of a parasite. Variation in the characteristics of the transmission process, for example, due to host demography, is assumed to select for different variants of the parasite. We have experimentally tested how variation in the time to transmission (early or late after infection) and exposure to adverse conditions outside the host (immediate or delayed contact with new host) interact to determine the success of the infection in the next host, using the trypanosome Crithidia bombi infecting its bumblebee host, Bombus terrestris. These two experimentally manageable steps mimic the processes of within- and among-host selection for the parasite. We found that early transmission led to higher infection success in the next host as did immediate contact with the new host. However, there was no interaction between the two parameters as would be expected if early-transmitted variants, resulting from rapid multiplication within the host, would be less adapted to the conditions encountered during the between-host transfer or infection of the next host. Furthermore, typing the genetic variability of the parasites with microsatellites showed that the four different transmission routes of our experiment selected for different degrees of allelic diversity of the infecting parasite populations. The results support the idea that variation in the transmission process selects for different genotypic variants of the parasite. At the same time, the relationship of allelic diversity with infection intensity suggested that the coinfection model of May and Nowak (1995) may be appropriate, where each parasite is able to infect and multiply independent of others within the same host.     

WEIGHING THE EVIDENCE FOR A SINGLE ORIGIN OF PLASTIDS1

The Journal of Phycology recently published a research paper entitled “A single origin of plastids revisited: convergent evolution in organellar genome content” ( Stiller et al. 2003 ). Also appearing in that issue was a minireview by Jeffrey D. Palmer ( Palmer 2003 ), “The symbiotic birth and spread of plastids: how many times and whodunit?” In his review, Palmer discussed evidence in support of a single endosymbiotic origin, in light of our analyses showing that similarities in plastid genome content are explained better by convergent evolution than by shared evolutionary history. Palmer raised a number of important issues that were not addressed in our paper, including the point that, in his view, no real evidence has been cited against a single plastid origin. After carefully considering Palmer's arguments, and this key point in particular, I am prompted to offer a few additional comments in the spirit of furthering a useful discussion begun in the February issue.     

GENETIC EVIDENCE FOR MULTIPLE ORIGINS OF DIOECY IN THE HAWAIIAN SHRUB WIKSTROEMIA (THYMELAEACEAE)

Dioecy is unusually common in the Hawaiian Islands, yet little is known about the evolutionary biology of this breeding system. A native shrub, Wikstroemia, has an unusually diverse array of breeding systems: two forms of dioecy, cryptic and morphological dioecy, as well as hermaphroditism (perfect flowers). The existence of two forms of dioecy is significant for three reasons: 1) the presence of cryptic unisexuals that are functionally unisexual, but retain the appearance of hermaphroditism in both sexes, is strong evidence for the ancestral status of hermaphroditism; 2) the production of nonfunctional pollen, by female cryptic unisexuals, is a new instance of a phenomenon which has previously been reported for a few other species; 3) the two forms of dioecy are morphological markers which are useful in hybridization studies for tracing the genetic basis of their inheritance. Crosses were made between cryptically unisexual individuals (C), between morphologically unisexual individuals (M), and between the two types of unisexuality. The offspring of crosses between individuals with the same sex type usually resulted in offspring with that sex type, but most of the progeny of between-sex type crosses were, unexpectedly, perfect-flowered hermaphrodites. These results show that genetic control of sex determination is not homologous in all populations, suggesting that dioecy has evolved at least twice in Hawaiian Wikstroemia. The genetic data further suggest that males are the heterozygous sex.     

PHLOEM OF PRIMITIVE ANGIOSPERMS. I. SIEVE-ELEMENT ONTOGENY IN THE PETIOLE OF LIRIODENDRON TULIPIFERA L. (MAGNOLIACEAE)

As part of a continuing study of sieve elements in primitive angiosperms, a study of this cell type was undertaken in Liriodendron tulipifera. A typical ontogenetic sequence was observed in which synthetic processes such as wall thickening are followed in time by cellular lysis of nucleus, ribosomes, microtubules, vacuoles, and dictyosomes. This lysis is selective in that certain cellular components (e.g., the plasmalemma) remain unaffected. Concomitant with lysis is the formation of sieve-area pores from plasmodesmata. Comparison of pore size on end and lateral walls indicates that the use of the term “sieve tube” rather than “sieve cell” to describe these elements is appropriate.     

GENETIC EVIDENCE FOR REPRODUCTIVE ISOLATION AND MULTIPLE ORIGINS OF SYMPATRIC TROPHIC ECOTYPES OF WHITEFISH (COREGONUS)

We assessed variation in mitochondrial DNA (mtDNA) by restriction fragment length polymorphism (RFLP) analysis and in nuclear genes by allozyme analysis among sympatric pairs of limnetic and benthic ecotypes of whitefish (Coregonus) coexisting in three lakes of southern Yukon to address three evolutionary questions regarding their origins. Are sympatric low and high gill-raker count ecotypes genetically differentiated? Are they issued from monophyletic or polyphyletic evolutionary events? If they are polyphyletic in origins, did they originate from multiple allopatric speciation events or intralacustrine radiation? Our results corroborated previous genetic and ecological studies of these ecotypes, indicating that they represent genetically distinct reproductive units, and therefore refuting the hypothesis of phenotypic polymorphism within a single population. However, the amount of gene flow between ecotypes varied among lakes, correlating with the extent of morphological differentiation and the potential for premating reproductive isolation. The results indicated a polyphyletic origin of ecotypes whereby each of them have been expressed independently more than once. In the two lakes of Squanga Creek drainage, the existence of sympatric pairs was best explained by the secondary contact of two monophyletic whitefish groups that evolved in allopatry during the last glaciation events. In Dezadeash L. of Alsek R. drainage, our results could not verify either sympatric or allopatric (or microallopatric) origin of ecotypes. Regardless of the mode of speciation involved in their origins, these sympatric whitefish populations provided further evidence that Pleistocene glaciation events created conditions favoring rapid divergence and phenotypic differentiation among northern freshwater fishes.     

EVIDENCE FOR SYMPATRIC GENETIC DIVERGENCE OF ANADROMOUS AND NONANADROMOUS MORPHS OF SOCKEYE SALMON (ONCORHYNCHUS NERKA)

Anadromous and nonanadromous morphs of the Pacific salmon Oncorhynchus nerka spawn in close physical proximity in tributaries to Takla Lake, British Columbia, yet differ in morphology, gill raker number, allozyme allele frequencies, and reproductive traits. Both morphs are semelparous typically maturing at age four, the anadromous morph (sockeye) at fork lengths of 38–65 cm and the nonanadromous morph (kokanee) at 17–22 cm. When reared together, pure and hybrid morphs also exhibited different growth rates and maturity schedules. Collectively, these large differences between the morphs confirm that sockeye and kokanee exist as reproductively isolated populations. Average gene flow (m) was estimated to be 0.1–0.8% between morphs, 1.7–3.7% among tributaries for kokanee, and 0.3–5.6% among tributaries for sockeye. We conclude that divergence has occurred in sympatry and examine potential isolating mechanisms.     

渐危植物鹅掌楸的授粉率及花粉管生长

对鹅掌楸Liriodendron chinense 3个自然居群连续3年直接统计落置柱头上的花粉量,其中 61.9％以上的柱头接受了花粉。每柱头上的平均花粉量为4.4～42.6粒,明显超过胚珠数2。授粉率是 结实率的6～8倍,鹅掌楸结实率低的主要原因可能不是由于花粉的限制。落置柱头的花粉几乎均可萌 发,少数花粉管穿过花柱道,经珠孔端进入胚囊,暗示花粉间存在着强烈的竞争。授粉率和柱头上的平 均花粉量与结实率呈正相关(R＝0.77,R=0.69)。在柱头上人工授同株和异株的花粉,花粉管生长速 率看不出明显差异,到达胚珠的时间在48～60 h之间。花开花药未裂时柱头上落置较高比例的花粉, 由于先期到达柱头的花粉最有可能受精,表明鹅掌楸有异交为主的繁育系统。人工处理包括套袋、套网、去除花被片,其结实率大大降低,而去雄后的结实率接近自然传粉结实率支持了这一观点。     

EVIDENCE FOR GENETIC HETEROZYGOSITY IN A HOMOSPOROUS FERN

Homosporous fern sporophytes from natural populations exhibited heterozygous electrophoretic patterns for several enzyme systems. Genetic tests utilizing individual gametophytes demonstrate that the observed heterozygosity is coded by alleles at single loci. This simple procedure makes it possible to distinguish segregating from fixed or phenotypic heterozygosity, previously a problem in homosporous vascular plants.     

ELECTROPHORETIC EVIDENCE FOR GENETIC DIPLOIDY IN PSILOTUM NUDUM

Psilotum nudum (2n = 104) has been considered an ancient polyploid, having resulted from repeated cycles of hybridization and allopolyploidy. However, electrophoretic analysis indicates that this species is genetically diploid despite its high chromosome number. Sixteen enzymes, encoded by 28 loci, revealed in P. nudum the number of isozymes typical of diploid seed plants. There is, therefore, no evidence of polyploid gene expression for the enzymes analyzed. These results for Psilotophyta are similar to those obtained for other lineages of homosporous pteridophytes, i.e., Arthrophyta and homosporous Microphyllophyta and Pteridophyta, all of which should be considered genetically diploid. Several hypotheses have been proposed to explain these results, most notably 1) cycles of allopolyploidy followed by massive gene silencing, and 2) initiation of these lineages with high chromosome numbers, possibly via chromosomal fission. Discrimination between these hypotheses awaits testing with molecular genetic techniques.     

GENETIC EVIDENCE FOR ZYGOTIC MEIOSIS IN PHYTOPHTHORA CAPSICI

Variation of several traits among single-oospore isolates of the heterothallic oomycete, Phytophthora capsici, was studied to determine genetically the place of meiosis in the life cycle. Low percentages of met^– offspring were recovered from two crosses of met^– X prototrophic isolates. Arg^– cultures were also recovered from the crosses indicating the possible presence of a suppressed arg^– gene in one of the parental cultures. Three arg^– X met^– crosses yielded all four of the possible classes of offspring. Thus meiosis was either zygotic or, if gametangial, the original parental cultures must have been heterozygous at both the arg and met loci. However, when these parental cultures were crossed, only prototrophic offspring were recovered. A ratio of 3 prototrophs: 1 met^– was obtained from cross R in which two prototrophic offspring from a previous arg^– X met^– cross were mated. This indicated the presence of a suppressor of met^– and may explain the low recovery of met^– offspring from earlier crosses. From backcrosses of met^– offspring from this cross to the A² prototrophic parent, ratios close to the expected 1:1 were obtained. When the A¹ prototrophic parent of cross R (K-10) was crossed to an A² prototrophic isolate, met^– offspring were again recovered, which indicated that K-10 carried a suppressed met deficiency. In two crosses of streptomycin-resistant (S^r) X sensitive (S^s) isolates, ratio of 108 S^s: 14 S^r and 84 S^s:14 S^r were obtained. Although 1:1 ratios would have been expected if meiosis were zygotic, segregation of S^s and S^r in the F₁ generation indicated that gametangial meiosis was unlikely. Segregation of the A¹ and A² mating types in all crosses also indicated that meiosis was zygotic in P. capsici. Possible control of mating type by a single pair of alleles, by plasmids, and by more complex mechanisms is discussed in relation to the mating type ratios obtained.     

GENETIC EVIDENCE FOR THE OCCURRENCE OF A NEW SPECIES OF NEOPHYLLAPHIS TAKAHASHI (HOMOPTERA: APHIDIDAE) IN AUSTRALIA

Abstract
Chromosomal and electrophoretic evidence is presented demonstrating the occurrence of a fourth species of Neophyllaphis Takahashi in Australia. The new species, N. lanata sp. n., occurs on Podocarpus spinulosus. Karyotypes of all four species of Neophyllaphis in Australia are provided.     

PLASTIDS IN LATICIFERS OF PAPAVER. I. DEVELOPMENT AND CYTOCHEMISTRY OF LATICIFER PLASTIDS IN P. SOMNIFERUM L. (PAPAVERACEAE)

Plastids were observed in all stages of laticifer differentiation in Papaver somniferum L. Plastids in laticifer initials were present as proplastids that later developed electron-dense inclusions, but never possessed the thylakoids or starch grains that characterize chloroplasts in other cells. Electron-dense inclusions in laticifer plastids were membrane-bound and appeared to arise from the accumulation of material within an invagination of the inner plastid membrane. Cytochemical studies of these plastid inclusions indicated that their matrix was not composed of crystalline protein, α-amylose, amylopectin or polysaccharide. The results suggest that the electron-dense, membrane-bound inclusions in laticifer plastids may be composed of lipoprotein.