Phylogeography and conservation genetics of Hygrophila pogonocalyx (Acanthaceae) based on atpB–rbcL noncoding spacer cpDNA

Genetic variation in the atpB–rbcL intergenic spacer region of chloroplast DNA (cpDNA) was investigated in Hygrophila pogonocalyx Hayata (Acanthaceae), an endangered and endemic species in Taiwan. In this aquatic species, seed dispersal from capsules via elasticity is constrained by gravity and is thereby confined within populations, resulting in limited gene flow between populations. In this study, a total of 849 bp of the cpDNA atpB–rbcL spacer were sequenced from eight populations of H. pogonocalyx. Nucleotide diversity in the cpDNA is low (=0.00343±0.00041). The distribution of genetic variation among populations agrees with an isolation-by-distance model. Two geographically correlated groups, the western and eastern regions, were identified in a neighbor-joining tree and a minimum-spanning network. Phylogeographical analyses based on the cpDNA network suggest that the present-day differentiation between western and eastern groups of H. pogonocalyx resulted from past fragmentation. The differentiation between eastern and western populations may be ascribed to isolation since the formation of the Central Mountain Range about 5 million years ago, which is consistent with the rate estimates based on a molecular clock of cpDNA.Huang JC and Wang WK equally contributed to this work.     

TrnL–trnF Intergenic Spacer and trnL Intron Define Major Clades Within Luzula and Juncus (Juncaceae): Importance of Structural Mutations

Seven hundred fifty-two to one thousand ninety-seven base pairs of the trnL intron and trnL–trnF intergenic spacer of the chloroplast DNA of 55 Juncaceae taxa (Juncus, Luzula, Rostkovia, and Oxychloë) was sequenced. Seventeen structural mutations (13 indels marked A to M, 3 parts of the trnF pseudogene, and insertion o within a pseudogene) within the chloroplast trnL–trnF region were examined as possible indicators for phylogenetic relationships in Juncaceae. Juncus trifidus (section Steirochloa) was clearly separated from the other taxa by two large (>80 bp) indels. The Southern Hemisphere clade was strongly supported by a unique insertion (334 bp) in the trnL intron. The monophyly of Luzula was supported by three small (<10 bp) indels in the trnL-F spacer. They were found in all 22 examined members that represent the taxonomic and geographical diversity of the genus Luzula. A tandemly duplicated tRNA pseudogene was found in the Juncus subgenus Juncus species and is supported by four small unique indels too. The acceptor stem and D-domain-encoding regions are separated by a unique 8-bp insertion. The T-domain and acceptor stem-encoding regions were not found in the pseudogene repeats. Only the Juncus sections Ozophyllum and Iridifolii contain the 5 acceptor stem, D-domain, and anticodon domain of the tRNAF encoding DNA. The structural mutations in the trnL intron and the trnL–trnF intergenic spacer are useful for phylogenetic reconstruction in the Juncaceae.     

Complex structure of the ribosomal DNA spacer of Cucumis sativus (cucumber)

Summary The nuclear 18 S, 5.8 S and 25 S ribosomal RNA genes (rDNA) of Cucumis sativus (cucumber) occur in at least four different repeat types of 10.2, 10.5, 11.5, and 12.5 kb in length. The intergenic spacer of these repeats has been cloned and characterized with respect to sequence organization. The spacer structure is very unusual compared to those of other eukaryotes. Duplicated regions of 197 bp and 311 bp containing part of the 3 end of the 25 S rRNA coding region and approximately 470 bp of 25 S rRNA flanking sequences occur in the intergenic spacer. The data from sequence analysis suggest that these duplications originate from recombination events in which DNA sequences of the original rDNA spacer were paired with sequences of the 25 S rRNA coding region. The duplicated 3ends of the 25 S rRNA are separated from each other mostly by a tandemly repeated 30 bp element showing a high GC-content of 87.5%. In addition, another tandemly repeated sequence of 90 bp was found downstream of the 3flanking sequences of the 25 S rRNA coding region. These results suggest that rRNA coding sequences can be involved in the generation of rDNA spacer sequences by unequal crossing over.     

Nucleotide sequence and phylogenetic implication of the ATPase subunits β and ε encoded in the chloroplast genome of the brown alga Dictyota dichotoma

We have cloned and sequenced the genes atpB and atpE, coding for CF₁ subunits and , respectively, of the chloroplast genome of the brown alga Dictyota dichotoma. Although the coding site of atpE cannot be demonstrated by heterologous Southern hybridizations, a 417 bp reading frame 3 to atpB was identified as the gene atpE by sequence similarities with atpE genes from other sources. A maximum sequence identity of 30% is found between the predicted amino acid sequence of the Dictyota subunit and the corresponding cyanobacterial subunits. Including conserved amino acid replacements, the Dictyota subunit exhibits about 70% sequence similarity with the cyanobacterial and land plant subunits. As in cyanobacteria, the atpE gene does not overlap the preceding gene atpB. The deduced amino acid sequence of atpB is 74–79% identical to the corresponding cyanobacterial and chloroplast subunits. Entirely conserved are regions referred to as the catalytic and/or regulatory sites of ATP formation, including interacting regions between subunits and . A phylogram predicted from F₁/CF₁- subunits of eleven different organisms suggests a common evolutionary origin of plastids from chlorophytes and brown algae.     

The discovery and phylogenetic implications of a novel 41 bp plastid DNA deletion in wild potatoes

Insertions and deletions (indels) are common in intergenic spacer regions of plastid DNA and can provide important phylogenetic characters for closely related species. For example, a 241-bp plastid DNA deletion in the trnV-UAC/ndhC intergenic spacer region has been shown to have major phylogenetic importance in determining the origin of the cultivated potato. As part of a phylogenetic study of the wild potato Solanum series Piurana group we screened 199 accessions of 38 wild potato species in nine of the 19 tuber-bearing (Solanum section Petota) series that have not been examined before for indels in the trnV-UAC/ndhC intergenic spacer region. A novel 41 bp deletion (but no 241 bp deletion) was discovered for 30 accessions of three species: S. chiquidenum (5 of 10 accessions), S. chomatophilum (19 of 28), and S. jalcae (6 of 6). Accessions with and without this deletion are found throughout much of the north-south range of all three species in northern and central Peru, but not east of the Marañón River. Multivariate morphological analyses of these 44 accessions showed no morphological associations to the deletion. The results suggest extensive interspecific gene flow among these three species, or a common evolutionary history among species that have never been suggested to be interrelated.     

Chloroplast DNA-relationship in palaeoaustralLopidium concinnum (Hypopterygiaceae, Musci). An example of stenoevolution in mosses Studies in austral temperate rain forest bryophytes 2

Evolutionary relationship between disjunct populations of the palaeoaustral moss taxonLopidium concinnum (Hypopterygiaceae) from New Zealand and southern South America were studied using non-coding chloroplast DNA sequences. No or only slight changes could be observed within the sequences oftrnT_UGU —trnL_UAA 5exon intergenic spacer,trnL_UAA intron andtrnL_UAA 3exon —trnF_GAA intergenic spacer. This indicates nearly no genetic divergence between extant New Zealand and Chilean populations, i.e. no significant differing pathways of evolution within the 80–60 million years of disrupted areas with interrupted gene flow. Molecular data support the idea of an old Gondwanan relict species of stenoevolutionary character. Ecological data on short-range dispersal strengthen this assessment.     

Variation in Coding (NADH Dehydrogenase Subunits 2, 3, and 6) and Noncoding Intergenic Spacer Regions of the Mitochondrial Genome in Octocorallia (Cnidaria: Anthozoa)

Low rates of evolution in cnidarian mitochondrial genes such as COI and 16S rDNA have hindered molecular systematic studies in this important invertebrate group. We sequenced fragments of 3 mitochondrial protein-coding genes (NADH dehydrogenase subunits ND2, ND3 and ND6) as well as the COI-COII intergenic spacer, the longest noncoding region found in the octocoral mitochondrial genome, to determine if any of these regions contain levels of variation sufficient for reconstruction of phylogenetic relationships among genera of the anthozoan subclass Octocorallia. Within and between the soft coral families Alcyoniidae and Xeniidae, sequence divergence in the genes ND2 (539 bp), ND3 (102 bp), and ND6 (444 bp) ranged from 0.5% to 12%, with the greatest pairwise distances between the 2 families. The COI-COII intergenic spacer varied in length from 106 to 122 bp, and pairwise sequence divergence values ranged from 0% to 20.4%. Phylogenetic trees constructed using each region separately were poorly resolved. Better phylogenetic resolution was obtained in a combined analysis using all 3 protein-coding regions (1085 bp total). Although relationships among some pairs of species and genera were well supported in the combined analysis, the base of the alcyoniid family tree remained an unresolved polytomy. We conclude that variation in the NADH subunit coding regions is adequate to resolve phylogenetic relationships among families and some genera of Octocorallia, but insufficient for most species - or population-level studies. Although the COI-COII intergenic spacer exhibits greater variability than the protein-coding regions and may contain useful species-specific markers, its short length limits its phylogenetic utility.     

Inheritance and linkage relationships of glutamate oxaloacetate transaminase isoenzymes in apple

Summary Four zones of enzymatic activity for glutamate oxaloacetate transaminase (GOT) were found in apple tissue. A dimeric gene, GOT-1, determining the fastest migrating zone, was identified. Six alleles were found, including a near null allelle which produced detectable heterodimeric bands but not homodimeric bands. A marked deficit or absence of certain geno-types in all backcrosses and in some crosses between unrelated varieties was attributed to the close linkage (r=0.02±0.005) of GOT-1 with the incompatibility S locus. GOT-1 was also closely linked with the isocitrate dehydrogenase locus IDH-1 (0.03±0.01). Proposed incompatibility genotypes for four cultivars, and the linked GOT-1 alleles are Cox: S ₁ b/S ₂ d, Idared: S ₃ a/S ₄ c, Fiesta: S ₃ a/S ₂ d and Kent: S ₃ a/S ₁ b.The results reported in this paper are part of a PhD Thesis by the first author     

Transmission of yeast mitochondrial loci to progeny is reduced when nearby intergenic regions containing ori sequences are deleted

Summary Mitochondrial DNAs (mtDNA) from four stable revertant strains generated from high frequency petite forming strains of Saccharomyces cerevisiae have been shown to contain deletions which have eliminated intergenic sequences encompassing ori1, ori2 and ori7. The deleted sequences are dispensable for expression of the respiratory phenotype and mutant strains exhibit the same relative amount of mtDNA per cell as the wild-type (wt) parental strain. These deletion mutants were also used to study the influence of particular intergenic sequences on the transmission of closely linked mitochondrial loci. When the mutant strains were crossed with the parental wt strains, there was a strong bias towards the transmission into the progeny of mitochondrial genomes lacking the intergenic deletions. The deficiency in the transmission of the mutant regions was not a simple function of deletion length and varied between different loci. In crosses between mutant strains which had non-overlapping deletions, wt mtDNA molecules were formed by recombination. The wt recombinants were present at high frequencies among the progeny of such crosses, but recombinants containing both deletions were not detected at all. The results indicate that mitochondrial genomes can be selectively transmitted to progeny and that two particular intergenic regions positively influence transmission. Within these regions other sequences in addition to ori/rep affect transmission.This paper is dedicated to colleagues J. Jana, D. Tasi, I. Bortner, and F. Zavrl     

The use of the isoenzymic marker gene Got-1 in the recognition of incompatibility S alleles in apple

The S incompatibility system of apple was confirmed through the application of the gene Got-1 for glutamate oxaloacetate transaminase as a marker for the S locus. The 11S alleles proposed by Kobel et al. (1939) were confirmed through anomalous segregations for Got-1 observed in 14 semi-compatible crosses and regular segregations observed in 2 fully compatible crosses. The S allele genotypes of Idared (S ₃ S ₇), Cox (S ₅ S ₉) and Fiesta (S ₃ S ₅) were determined and found to fall within the original series. By associating parental incompatibility genotypes with the segregation of Got-1 alleles, we were able to deduce the coupling of S and Got-1 alleles in 9 varieties.     

Phylogenetic analysis of chloroplast restriction enzyme site mutations in the Saccharinae Griseb. subtribe of the Andropogoneae Dumort. tribe

Chloroplast (cp) DNA from 32 genotypes representing eight genera and 19 species from the Andropogoneae tribe was analyzed using 15 restriction enzymes and Southern hybridization with 12 cpDNA probes that span the complete rice chloroplast genome. Six of the genera, Saccharum, Miscanthus, Erianthus, Narenga, Eccoilopus, and Sclerostachya, are part of the Saccharinae subtribe, whereas the other two, Zea and Sorghum, were used as outgroups. Narenga, Miscanthus, Erianthus, and Sclerostachya are presumed to have been involved in the evolution of Saccharum officinarum (noble or high sucrose sugarcane) via S. spontaneum and S. robustum. Southern hybridization with the rice cpDNA probes surveyed approximately 3% of the S. officinarum Black Cheribon genome and yielded 62 restriction site mutations (18 informative) that were analyzed using cladistic parsimony and maximum likelihood. These site mutations placed the 32 genotypes into nine different chloroplast groups; seven from within the Saccharinae subtribe and the two outgroups (maize and Sorghum). Phylogenetic inferrence under various assumptions showed that the maternal lineages of Narenga, Miscanthus, Sclerostachya, and Saccharum formed a monophyletic group. This group displayed little variation. On the other hand, 5 of 6 Erianthus species and Eccoilopus longisetosus formed a separate group. The Old World Erianthus/Eccoilopus chloroplast was very different from that of the rest of the Saccharum complex members and was slightly more related to that of Sorghum bicolor. Placement of these Erianthus/Eccoilopus genotypes was, therefore, in conflict with analyses based on morphology. Surprisingly, Erianthus trinii, a New World species, had the same restriction sites as did one Miscanthus sinensis. One Miscanthus sp. from New Guinea that has a very high chromosome number (2n=192) had the same restriction sites as the majority of the Saccharum genus, suggesting that introgression between these genera occurs in the wild. The Saccharum genus was separated into two clades by single site mutation: one containing S. spontaneum, and the other containing all of the remaining Saccharum species and all 8 commerical hybrids (from various regions of the world). A physical map of the chloroplast of Saccharum officinarum Black Cheribon was constructed using 5 restriction enzymes.     

Structural features of the plastid ribosomal RNA operons of two red algae: Antithamnion sp. and Cyanidium caldarium

The nucleotide sequences of the plastid 16S rDNA of the multicellular red alga Antithamnion sp. and the 16S rDNA/23S rDNA intergenic spacers of the plastid DNAs of the unicellular red alga Cyanidium caldarium and of Antithamnion sp. were determined. Sequence comparisons support the idea of a polyphyletic origin of the red algal and the higher-plant chloroplasts. Both spacer regions include the unsplit tRNA^Ile (GAU) and tRNA^Ala (UGC) genes and so the plastids of both algae form a homogeneous group with those of chromophytic algae and Cyanophora paradoxa characterized by small-sized rDNA spacers in contrast to green algae and higher plants. Nevertheless, remarkable sequence differences within the rRNA and the tRNA genes give the plastids of Cyanidium caldarium a rather isolated position.     

Binary oscillations in the Kok model of oxygen evolution in oxygenic photosynthesis

The flash-induced kinetics of various characteristics of Photosystem II (PS II) in the thylakoids of oxygenic plants are modulated by a period of two, due to the function of a two-electron gate in the electron acceptor side, and by a period of four, due to the changes in the state of the oxygen-evolving complex. In the absence of inhibitors of PS II, the assignment of measured signal to the oxygen-evolving complex or to quinone acceptor side has frequently been done on the basis of the periodicity of its flash-induced oscillations, i.e. four or two. However, in some circumstances, the period four oscillatory processes of the donor side of PS II can generate period two oscillations. It is shown here that in the Kok model of oxygen evolution (equal misses and equal double hits), the sum of the concentrations of the S ₀ and S ₂ states (as well as the sum of concentrations of S ₁ and S ₃ states) oscillates with period of two: S ₀+S ₂S ₁+S ₃S ₀+S ₂S ₁+S ₃. Moreover, in the generalized Kok model (with specific miss factors and double hits for each S-state) there always exist such ₀, ₁, ₂, ₃ that the sum ₀[S₀] + ₁[S₁] + ₂[S₂] + ₃[S₃] oscillates with period of two as a function of flash number. Any other coefficients which are linearly connected with these coefficients, % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGak0dh9WrFfpC0xh9vqqj-hEeeu0xXdbba9frFj0-OqFf% ea0dXdd9vqaq-JfrVkFHe9pgea0dXdar-Jb9hs0dXdbPYxe9vr0-vr% 0-vqpWqaaeaabiGaciaacaqabeaadaqaaqaaaOqaaiqbew7aLzaaja% aaaa!3917!\[\hat \varepsilon \]_i = c_1i + c₂, also generate binary oscillations of this sum. Therefore, the decomposition of the flash-induced oscillations of some measured parameters into binary oscillations, depending only on the acceptor side of PS II, and quaternary oscillations, depending only on the donor side of PS II, becomes practically impossible when measured with techniques (such as fluorescence of chlorophyll a, delayed fluorescence, electrochromic shift, transmembrane electrical potential, changes of pH and others) that could not spectrally distinguish the donor and acceptor sides. This property of the Kok cycle puts limits on the simultaneous analysis of the donor and acceptor sides of the RC of PS II in vivo and suggests that binary oscillations are no longer a certain indicator of the origin of a signal in the acceptor side of PS II.Abbreviations PS II Photosystem II - P680 primary electron donor of reaction center of PS II - Q_A one electron acceptor plastoquinone - Q_B two electron acceptor plastoquinone - S _n redox state of the oxygen evolving complex, where n=0,1,2,3 and 4 - Chl a chlorophyll a This paper is dedicated to the memory of Alexander Kononenko.     

New Triterpene Glycosides from an Ulosa sp. Sponge

New triterpene glycosides, ulososides C, (20S,22S,23R,24S)-3,22,23-trihydroxy-3-O-(-D-glucopyranosyl)-32-nor-24-methyllanost-8(9)-ene-30-oic acid, D, (20S,22S,23R,24S)-3,22,23-trihydroxy-3-O-(-D-N-acetylglucosaminopyranosyl)-32-nor-24-methyllanost-8(9)-ene-30-oic acid, and E, (20S,22S,23R,24S)-3,22,23-trihydroxy-3-O-(-D-glucuronopyranosyl-(1 2)--D-arabinopyranosyl-32-nor-24-methyllanost-8(9)-ene-30-oic acid, were isolated from an Ulosa sp. sponge. Their structures were determined by spectral methods and chemical transformations. Specific features of their structures are discussed.     

Fumonisin B1 production and vegetative compatibility of strains from Gibberella fujikuroi mating population “A” (Fusarium moniliforme)

We examined 25 strains of Fusarium moniliforme from eight states known to be associated with equine leukoencephalomalacia, a disease caused by the mycotoxin fumonisin B₁. We determined the mating population, mating type, and vegetative compatibility group to which each of these strains belonged. All 25 strains were in the A mating population; 12 were A⁺ and 13 were A^–. Seventeen of the 25 strains were female fertile; these strains also averaged higher levels of fumonisin B₁ production than did the strains that were female sterile. Nitrate non-utilizing (nit) mutants were generated in all 25 strains and each strain was assigned to a unique vegetative compatibility group based on the inability of the derived nit mutants to form a prototrophic heterokaryon with complementary nit mutants derived from any of the other strains examined. From these data, we concluded that the production of fumonisin B₁ is a general characteristic of strains from the A mating population of Gibberella fujikuroi associated with equine leukoencephalomalacia, since all 25 of the isolates that we examined were genetically distinct individuals.     

Inhibitory activities against heterologous α-amylases and in vitro allergenic reactivity of Einkorn wheats

Salt extracts from seeds of 36 lines of Einkorn wheats were analyzed for their inhibitory activity towards two insect (Tenebrio molitor, Coleoptera, and Ephestia kuehniella, Lepidoptera) and one mammalian (human salivary) -amylases. Whereas all ten T. monococcum accessions tested were active towards the lepidopteran enzyme, they had no effect on the coleopteran or the mammalian ones. More variability was found among the 21 lines of T. boeticum analyzed, although none of them inhibited human -amylase. The five accessions of T. urartu showed even greater diversity. Among all Einkorn accessions tested, only two urartu lines affected the three -amylases. These lines displayed inhibition patterns similar to those of T. aestivum and T. turgidum cultivars. Since several breadwheat -amylase inhibitors are major allergens associated with baker's asthma, we also studied the in vitro allergenic activity of salt extracts from the Einkorn wheats under study. No significant differences in IgE-binding were found between these accessions and theT. aestivum or T. turgidum cultivars. Furthermore, putative allergens with molecular sizes in the range of 20–60 kDa were detected in these Einkorn wheats.     

Evaluation of random amplified polymorphic DNA for species identification and phylogenetic analysis inStylosanthes (Fabaceae)

Random amplified polymorphic DNA (RAPD) was assessed for its suitability as a tool to be used in the identification of taxa from the genusStylosanthes (Fabaceae, Papilionoideae, Aeschynomeneae). Five random primers were used to fingerprint accessions from seven species in the genus, and generated RAPD profiles that were species-specific. Data were used to examine evolutionary relationships between taxa, employing both clustering and ordination techniques, and the results were compared with those from a previous cladistic analysis of chloroplast DNA (cpDNA) restriction fragments. Both multivariate approaches indicated relationships that were generally similar to those obtained by RFLP analysis of cpDNA. However, while cluster analysis grouped together all accessions within species, ordination placed certain accessions ofS. humilis, S. macrocephala andS. capitata into separate groups. Experiments to test the assumed homology of comigrating RAPDs estimated 85.7% homology for accessions within species, and 53.8% homology for accessions between species. The value of RAPD data in systematics is discussed.     

Phylogenetic relationships among Cucumis species based on the ribosomal internal transcribed spacer sequence and microsatellite markers

The phylogenetic relationships within the genus Cucumis (a total of 25 accessions belonging to 17 species) were studied using the nuclear ribosomal DNA internal transcribed spacer (ITS) region. The analysis included commercially important species such as melon (C. melo L.) and cucumber (C. sativus). Two additional cucurbit species, watermelon and zucchini, were also included as outgroups. The data obtained reflected the clustering of Cucumis species in four main groups, comprising accessions from cucumber, melon, C. metuliferus and the wild African species. Some of the species clustered in different positions from those reported in classifications previously described by other authors. The data obtained clearly identify a division between the 2n=2x = 14 species (C. sativus) and the 2n = 2x = 24 ones (C. melo and wild species). Within the wild species we identified a subgroup that included C. sagittatus and C. globosus. Oreosyce africana, also classified as Cucumis membranifolius, was shown to be nested within Cucumis. Three accessions previously classified as independent species were shown to be genotypes of Cucumis melo. A set of melon and cucumber SSRs were also used to analyse the Cucumis species and the results were compared with the ITS data. The differential amplification of the SSRs among the accessions made it possible to distinguish three main groups: melon, cucumber and the wild species, though with less detail than applying ITS. Some SSRs were shown to be specific for melon, but other SSRs were useful for producing PCR fragments in all species of the genus.We are grateful to NCRPIS, IPK in Gatersleben, Semillas Fitó S.A., Michel Pitrat and Fernando Nuez for providing seeds. We would also like to thank Vanessa Alfaro, Trinidad Martínez and Núria Galofré for their excellent technical assistance. This work was financed by project AGL2000-0360 of Spains Ministerio de Ciencia y Tecnología (MCYT). AJMs work was supported by a postdoctoral contract from Spains MCYT.     

Ancient origin of the vacuolar H+-ATPase 69-kilodalton catalytic subunit superfamily

Recently, two distinct cDNA clones encoding the catalytic subunit of the vacuolar H⁺-ATPase (V-ATPase) were isolated from the allotetraploid cotton species Gossypium hirsutum L. cv Acala SJ-2 (Wilkins 1992, 1993). Differences in the nucleotide sequence of these clones were used as molecular markers to explore the organization and structure of the V-ATPase catalytic subunit genes in the A and D genomes of diploid and allotetraploid cotton species. Nucleotide sequencing of polymerase chain reaction (PCR) products amplified from G. arboreum (A₂, 2n=26), G. raimondii (D₅, 2n=26), and G. hirsutum cv Acala SJ-2 [(AD)₁, 2n=4x=52] revealed a V-ATPase catalytic subunit organization more complex than indicated hitherto in any species, including higher plants. In the genus Gossypium, the V-ATPase catalytic subunit genes are organized as a superfamily comprising two diverse but closely related multigene families, designated as vat69A and vat69B, present in both diploid and allotetraploid species. As expected, each vat69 subfamily is correspondingly more complex in the allotetraploid species due to the presence of both A and D alloalleles. Because of this, about one-half of the complex organization of V-ATPase catalytic subunit genes predates polyploidization and speciation of New World tetraploid species. Comparison of plant and fungal V-ATPase catalytic subunit gene structure indicates that introns accrued in the plant homologs following the bifurcation of plant and fungi but prior to the gene duplication event that gave rise to the vat69A and vat69B genes approximately 45 million years ago. The structural complexity of plant V-ATPase catalytic subunit genes is highly conserved, indicating the presence of at least ten introns dispersed throughout the coding region.