Ploidy stability in embryogenic cultures and regenerated plantlets of tamarillo

Ploidy levels of short-term (1 and 2 years) and long-term (7 and 10 years) embryogenic cultures as well as of regenerated plantlets of tamarillo were analyzed by flow cytometry and chromosome counts. Embryogenic cultures were induced from expanding leaves cultured in the presence of Picloram or 2,4-dichlorophenoxyacetic acid (2,4-D) and monthly subcultured on the same media. Embryo development and plantlets were obtained following subculture of the embryogenic tissue in auxin free medium containing gibberellic acid (GA₃). Seedlings and rooted shoots from axillary shoot proliferation were used as controls. The results showed that in long-term embryogenic cultures the ability to develop somatic embryos and plantlets was reduced. Embryogenic tissues maintained for 10 years were mostly aneuploids of the tetraploid (2n = 4x = 48) level whereas those kept in culture for 7 years or less were also mostly aneuploids but of the diploid (2n = 2x = 24) level. The results obtained by flow cytometry were, in general, consistent with those obtained by chromosome counts. The chromosome alteration observed in the embryogenic tissues was already present after 1 year of culture and increased with culture age, hence impairing the maintenance of these tissues for long periods without affecting chromosome stability of the regenerated plantlets. However, the occurrence of triploids and tetraploids as well as aneuploids can be useful for breeding purposes. A value around 23 pg/2C was found for the genome size of tamarillo largely exceeding the value previously published (15.50 pg/2C).     

Nuclear genome size variation in fleshy-fruited Neotropical Myrtaceae

In Myrtaceae, reports regarding the nuclear DNA content are scarce. The aim of this study is to present genome size data for fleshy-fruited Myrteae, and to test their relation with chromosome number and ploidy, the available data for cytoevolutionary studies in Myrtaceae. Thirty species out of ten genera were investigated for chromosome number and genome size using flow cytometry. Twenty-eight species were diploid with 2n = 2x = 22 and two species were tetraploid with 2n = 4x = 44. All genome sizes measured are new. Among the diploid species, a gradual and small variation in 2C-values (0.486 pg in Gomidesia schaueriana to 0.636 pg in Eugenia multicostata) was observed, whereas the tetraploid genomes of Psidium acutangulum and P. cattleianum had about twice as much DNA (1.053 and 1.167 pg, respectively). The total interspecific variation of C-values was 2.45-fold. The fleshy-fruited Myrteae have smaller holoploid genomes than the capsular-fruited Eucalypteae and Melaleuceae.     

Metabolic and water loss rates of two cryptic species in the African velvet worm genus Opisthopatus (Onychophora)

Velvet worms (Onychophora) are characterised by a dearth of mechanisms to retain water, yet recently identified cryptic species are located in areas with seemingly different climates. Using flow-through respirometry, this study determined the metabolic, water loss and cuticular water loss rates of two cryptic species of Opisthopatus cinctipes s.l. from locations that differ in their current climate. When controlling for trial temperature and body mass, velvet worms from the drier and warmer site had significantly lower water loss rates than the wetter and cooler site. Mass-corrected metabolic rate and cuticular water loss did not differ significantly between the two sites. The scaling exponent for the relationship between log metabolic rate and log body mass for O. cinctipes s.l. declined with an increase in temperature from 5 to 15 °C. Females in the two cryptic Opisthopatus species had higher metabolic, water loss and cuticular water loss rates than males, which may represent the increased energetic demands of embryonic growth and development in these viviparous taxa.     

Flow cytometry and cytogenetic tools in eucalypts: genome size variation × karyotype stability

The eucalypts comprise a group of woody plants used in commercial forest plantations owing to their high growth rates, adaptability to various ecological conditions and multiple applications. Despite the enormous amount of molecular data available for eucalypts, a basic understanding of the nature of its genome still requires information regarding the DNA amount in the genus. In this work, we estimated the genome size and base composition of 25 eucalypt species. With a comparative karyotype approach, we aimed to identify possible chromosomal alterations correlated with the genome size variation. Classical cytogenetic and genomic in situ hybridization experiments were conducted for this purpose. The studied species showed genome size ranging from 2C = 0.91 (Corymbia intermedia) to 2C = 1.37 pg (Eucalyptus paniculata) and AT/CG ratios varying from AT = 61.3 (Eucalyptus urophylla) to AT = 62.85% (C. intermedia). Comparative karyotype analysis revealed no remarkable differences in chromosome number (2n = 22) or morphology among eucalypt species despite considerable differences in nuclear DNA content. The genome in situ hybridization method did not distinguish non-homologous chromosomal regions of Eucalyptus baileyana and Corymbia citriodora, despite the difference of 0.45 pg between their genome sizes. The results found in the present work corroborate the consideration of small and dispersed DNA changes as the main cause of genome size variation in eucalypts.     

The complete mitochondrial genome of the onychophoran Epiperipatus biolleyi reveals a unique transfer RNA set and provides further support for the ecdysozoa hypothesis

Onychophora (velvet worms) play a crucial role in current discussions on position of arthropods. The ongoing Articulata/Ecdysozoa debate is in need of additional ground pattern characters for Panarthropoda (Arthropoda, Tardigrada, and Onychophora). Hence, Onychophora is an important outgroup taxon in resolving the relationships among arthropods, irrespective of whether morphological or molecular data are used. To date, there has been a noticeable lack of mitochondrial genome data from onychophorans. Here, we present the first complete mitochondrial genome sequence of an onychophoran, Epiperipatus biolleyi (Peripatidae), which shows several characteristic features. Specifically, the gene order is considerably different from that in other arthropods and other bilaterians. In addition, there is a lack of 9 tRNA genes usually present in bilaterian mitochondrial genomes. All these missing tRNAs have anticodon sequences corresponding to 4-fold degenerate codons, whereas the persisting 13 tRNAs all have anticodons pairing with 2-fold degenerate codons. Sequence-based phylogenetic analysis of the mitochondrial protein-coding genes provides a robust support for a clade consisting of Onychophora, Priapulida, and Arthropoda, which confirms the Ecdysozoa hypothesis. However, resolution of the internal ecdysozoan relationships suffers from a cluster of long-branching taxa (including Nematoda and Platyhelminthes) and a lack of data from Tardigrada and further nemathelminth taxa in addition to nematodes and priapulids.     

Genome sizes of Eucomis L’Hér. (Hyacinthaceae) and a description of the new species Eucomis grimshawii G.D.Duncan & Zonneveld

Nuclear genome size, as measured by flow cytometry with propidium iodide, was used to investigate the relationships within the genus Eucomis L’Hér. (Hyacinthaceae). Most species of Eucomis have the same basic chromosome number, x = 15. However, the somatic DNA 2C-value (2C) is shown to range from 21 to 31 pg for the diploids. The largest genome contains roughly 10¹⁰ more base pairs than the smallest. Genome sizes are evaluated here in combination with available morphological and geographical data. Therefore, the taxonomy proposed here is not based on genome size alone. The genus Eucomis, as here determined, has 12 species. These can be divided into two groups: mainly dwarf diploid species and large-sized, tetraploid species. A small diploid plant, Eucomis (autumnalis subsp.) amaryllidifolia, is restored to species status, as a diploid subspecies seems incongruent with an allotetraploid Eucomis autumnalis. Moreover, as a diploid it is separated reproductively from the allotetraploid E. autumnalis. A new diploid species that has the lowest C value, E. grimshawii, is described here. On the basis of DNA content and other morphological characters, possible parents are suggested for all tetraploid species. Nuclear DNA content as measured by using flow cytometry may conveniently be used to produce systematic data. It is applicable even in dormant bulbs or sterile plants for the monitoring of the trade in bulbous species.     

Genome size in Dahlia Cav. (Asteraceae–Coreopsideae)

The genus Dahlia (Asteraceae–Coreopsideae) is monophyletic according to a recent DNA phylogeny (ETS and ITS of rDNA). Traditionally, the genus has been divided into sections, but these have been shown not to be monophyletic. We have studied variation in genome size (DNA C-values) in a sample of species to investigate the possible effects of secondary metabolites on flow cytometry and Feulgen densitometry, and to see whether genome size variation has any systematic or phylogenetic significance. Using a range of cultivars, secondary compounds from corollas were shown to have only minor effects on the Feulgen method; the floral pigments were found to be relatively inert and seemed to have been extracted on fixation with acetic methanol. Freshly expanded corollas showed apparent apoptotic DNA decay in epidermal cells, so need to be used with caution. Flow cytometric measurements with propidium iodide in some cultivars resulted in a very similar average genome size (2C = 8.62 pg) as compared with Feulgen densitometry (2C = 8.84 pg). Leaf cytosol of D. variabilis has a demonstrable inhibitory effect on propidium iodide fluorescence, which may explain some of the intraspecific variation of C-values observed. DNA 2C-values ranged from 3.30 pg in D. dissecta (2n = 34) to 9.62 pg in a D. variabilis cultivar (2n = 64). The D. variabilis cultivars had broadly similar C-values showing a 1.16-fold range between cultivars. Some of this variation probably results from technical variables and the extent of genuine variation is uncertain. The highest 2Cx-value occurred in one D. coccinea accession (2.47 pg, 2n = 32; x = 8). D. coccinea with 2n = 64 showed slightly reduced Cx-values compared to D. coccinea with 2n = 32. Artificially produced interspecific hybrids had C-values that corresponded closely with expectations from the measured values obtained from their parents.     

Persistence,dispersal and genetic evolution of recently formed <Emphasis Type="Italic">Spartina</Emphasis> homoploid hybrids and allopolyploids in Southern England

In Southampton Water, UK, the recent (c. 150 years ago) interspecific hybridisation between Spartina alterniflora (2n = 6x = 62; A-genome) and S. maritima (2n = 6x = 60; M-genome) gave rise to the homoploid hybrid (S. × townsendii, 2n = 6x = 62), and subsequently to the invasive allododecaploid species S. anglica (2n = 12x = 120–124) that has since spread worldwide. To address the question of dynamics of mixed ploidy populations involving these plants, we analysed several Spartina populations (fifty one individuals) in Southern England, UK, one of which was the presumed place of origin of the homoploid hybrid (Hythe). Using a combination of flow cytometry and ribosomal DNA (rDNA) genotyping we were able to identify the genomic composition and ploidy level of each individual analysed. The data show that the homoploid hybrid still dominates the population at Hythe (82 % of individuals collected in that locality) since its origin in the nineteenth century. We also identified S. × townsendii for the first time on Hayling Island (66 % individuals), indicating dispersal beyond its likely origin. The fertile allododecaploid S. anglica was mainly found in populations outside the initial hybridisation site, on Hayling Island and at Eling Marchwood. Quantification of the rDNA contributions from each parental genome showed that the ratios were mostly balanced in S. × townsendii. However, two (3 %) S. anglica individuals analysed have lost nearly all M-genome homeologs, indicating extensive repeat loss. Such variation indicates that despite the presumed single allopolyploid origin of S. anglica and genetic uniformity at other loci, it has undergone substantial changes at the rDNA loci following genome duplication.     

The first genome size estimates for six species of krill (Malacostraca, Euphausiidae): large genomes at the north and south poles

Krill (family Euphausiidae) represent some of the most abundant organisms in the both northern and southern oceanic environments and provide food for various animals including humans. Despite their importance, little is known about krill from a genomic standpoint, even with regard to basic properties such as total genome size. This study provides genome size estimates for six species of krill from both the North Atlantic and Southern Oceans which are the first such estimates for any species of euphausiid. Genome size estimates were obtained using both flow cytometry and Feulgen image analysis densitometry with chicken and trout blood as internal standards. Haploid genome sizes ranged from 12.77 to 48.53 pg, providing roughly fourfold variation within these six species alone. With such large estimates, sequencing of a krill genome will currently be costly and laborious, but further studies should be conducted to determine the composition of these exceptionally large genomes.     

Flow cytometric and karyological analyses of Calendula species from Iberian Peninsula

Calendula L. (Asteraceae) is a taxonomically and cytologically complex genus due to its high morphological and karyological variation. To gather consistent cytological information aiming to consolidate the existing knowledge, sustain the taxonomic revision of the genus and explore the evolutionary relationships among species, the genome size and chromosome number of the Iberian Peninsula representatives of this genus were assessed. The study included 11 taxa that occur in the Iberian Peninsula, one in Madeira and two from Morocco. Chromosome counts were made using the squash technique in root tips and flower buds, while nuclear DNA contents were assessed using propidium iodide flow cytometry. The following chromosome numbers are reported: 2n = 44 for C. arvensis, 2n = 30 for C. tripterocarpa, and 2n = 32 for the remaining Iberian taxa. The genome size of Calendula species was assessed for the first time and ranged from 1.75 pg/2C in C. maroccana to 5.41 pg/2C in C. arvensis. Within the complex formed by C. incana and C. suffruticosa, a gradient of genome size values was obtained. Intraspecific variation in genome size was detected in some taxa. The obtained genome size values and their variation are discussed in the light of the theories proposed for the speciation of the genus, with events of hybridization, genome duplication and dysploidy being hypothesized to play a major role in the evolution of this genus.     

Position and development of oocytes in velvet worms shed light on the evolution of the ovary in Onychophora and Arthropoda

Onychophora or velvet worms are of considerable importance in current reconstructions of animal phylogeny. Despite their otherwise conservative morphology, the detailed anatomy of the onychophoran ovary displays significant variation. However, the evolutionary significance of this variation is not well understood. We recognize three major ovarian types in the Onychophora: (1) the exogenous ovary; (2) the pseudoendogenous ovary; and (3) the endogenous ovary. The germ cells in all three ovarian types are intraepithelial in that they occur between the basal lamina and the epithelial cells that line the cavity of the gonad. This is the condition found in the endogenous ovary. Even in the exogenous ovary, with stalked oocytes projecting into the haemocoel, the maturating oocytes are still covered by a basal lamina. Stalked oocytes that are similar to those found in the exogenous ovary, but retain their intra‐ovarian position, characterize the pseudoendogenous ovary. This and additional observations support the assumption that the pseudoendogenous ovary is derived from an exogenous type and the similarities with the endogenous ovary are superficial. Embryological data and an outgroup comparison with arthropods suggest that the exogenous ovary is the ancestral condition in velvet worms and a synapomorphy of Onychophora and Arthropoda. The embryonic origin, growth, and position of oocytes outside the ovarian lumen in Onychophora and various groups of Arthropoda do not support the Articulata hypothesis, which proposes a sister‐group relationship of Panarthropoda and Annelida.     

Nuclear DNA C-values for biodiversity screening: Case of the Lebanese flora

The geographic position of Lebanon in the Mediterranean basin at the transition of two major landmasses, Eurasia and Africa, has contributed to its high plant diversity and makes its flora particularly interesting to study. This paper contributes to the plant DNA C-value database of native Lebanese taxa. These data should reinforce biodiversity evaluation, systematic and evolution studies involving processes of speciation such as polyploidisation. C-values have been estimated by flow cytometry using propidium iodide as intercalary fluorochrome stain. Each sample comprised at least five individuals. Where possible, several populations were measured for each species. This study presents C-values for 225 taxa belonging to 55 families and 141 genera. C-values are novel for 193 taxa including 126 plants endemic to the Eastern Mediterranean region. These are the first values for 50 genera. In this panel, genome size ranged from 1C = 0.28 pg in Hypericum thymifolium to 54.69 pg in Fritillaria alfredae. The life growth form and life cycle type are analysed according to the genome size class. Cases of polyploidy are reported for some species usually considered as only diploid. Examination of C-value variation through flow cytometry constitutes a powerful tool to screen taxonomic heterogeneity, opening further investigations.     

The first karyogram of a Bromeliaceae species: an allopolyploid genome

The Bromeliaceae family has been traditionally distributed in the subfamilies Bromelioideae, Tillandsioideae and Pitcairnioideae. However, phylogenetic studies have provided other classifications, highlighting the need for analyses in order to characterize the genome of different species from this family. In this sense, the present work aimed to determine nuclear 2C-value and base composition, characterize the chromosomes and establish the karyogram of Pitcairnia flammea. Flow cytometry yielded 2C = 1.44 pg, AT = 64.28 % and GC = 35.72 % for this species, indicating its relatively small genome size. Despite reduced length and morphological similarity of the chromosomes, P. flammea metaphases presented well-spread chromosomes, with well-defined primary constriction, without chromatin damage and cytoplasmic background. These aspects allowed morphometric chromosomal characterization and assembly of the first karyogram of a Bromeliaceae species. The karyogram displayed 2n = 50 chromosomes, of which all were submetacentric. Karyomorphological analysis revealed grouped pairs of cytogenetically identical chromosomes (2–3, 4–5, 6–9, 10–17, 18–19, 20–23 and 24–25), plus one isolated chromosome (1), not identical to any other. This result suggests an allopolyploid origin for the P. flammea genome. Thus, the present investigation contributed with karyotype data for taxonomic and evolutionary aspects of this group.     

Genome Size Evaluation in Tetraodontiform Fishes from the Neotropical Region

Smooth pufferfish of the family Tetraodontidae had become pure genomic models because of the remarkable compaction of their genome. This trait seems to be the result of DNA loss following its divergence from the sister family Diodontidae, which possess larger genomes. In this study, flow cytometry was used for estimate the genome size of four pufferfish species from the Neotropical region. Cytogenetic data and confocal microscopy were also used attempting to confirm relationships between DNA content and cytological parameters. The haploid genome size was 0.71?±?0.03 pg for Sphoeroides greeleyi, 0.34?±?0.01 pg for Sphoeroides spengleri, 0.82?±?0.03 pg for Sphoeroides testudineus (all Tetraodontidae), and 1.00?±?0.03 pg for Chilomycterus spinosus (Diodontidae). These differences are not related with ploidy level, because 46 chromosomes are considered basal for both families. The value for S. spengleri represents the smallest vertebrate genome reported to date. Since erythrocyte cell and nuclear sizes are strongly correlated with genome size, the variation in this last is considered under both adaptive and evolutionary perspectives.     

Immunolocalization of serotonin in Onychophora argues against segmental ganglia being an ancestral feature of arthropods

Background  

Onychophora (velvet worms) represent the most basal arthropod group and play a pivotal role in the current discussion on the evolution of nervous systems and segmentation in arthropods. Although there is a wealth of information on the immunolocalization of serotonin (5-hydroxytryptamine, 5-HT) in various euarthropods, as yet no comparable localization data are available for Onychophora. In order to understand how the onychophoran nervous system compares to that of other arthropods, we studied the distribution of serotonin-like immunoreactive neurons and histological characteristics of ventral nerve cords in Metaperipatus blainvillei (Onychophora, Peripatopsidae) and Epiperipatus biolleyi (Onychophora, Peripatidae).     

EST sequencing of Onychophora and phylogenomic analysis of Metazoa

Onychophora (velvet worms) represent a small animal taxon considered to be related to Euarthropoda. We have obtained 1873 5' cDNA sequences (expressed sequence tags, ESTs) from the velvet worm Epiperipatus sp., which were assembled into 833 contigs. BLAST similarity searches revealed that 51.9% of the contigs had matches in the protein databases with expectation values lower than 10(-4). Most ESTs had the best hit with proteins from either Chordata or Arthropoda (approximately 40% respectively). The ESTs included sequences of 27 ribosomal proteins. The orthologous sequences from 28 other species of a broad range of phyla were obtained from the databases, including other EST projects. A concatenated amino acid alignment comprising 5021 positions was constructed, which covers 4259 positions when problematic regions were removed. Bayesian and maximum likelihood methods place Epiperipatus within the monophyletic Ecdysozoa (Onychophora, Arthropoda, Tardigrada and Nematoda), but its exact relation to the Euarthropoda remained unresolved. The "Articulata" concept was not supported. Tardigrada and Nematoda formed a well-supported monophylum, suggesting that Tardigrada are actually Cycloneuralia. In agreement with previous studies, we have demonstrated that random sequencing of cDNAs results in sequence information suitable for phylogenomic approaches to resolve metazoan relationships.     

Longli is not a Hybrid of Longan and Lychee as Revealed by Genome Size Analysis and Trichome Morphology

Lychee, longan, longli, and rambutan are closely related, commercially important fruit trees in the Sapindaceae family. Longli fruits are morphologically similar to both lychee and longan, displaying a yellow-brown pericarp like longan, and small, sharp protuberances like lychee. These similarities have led to the hypothesis that longli is the result of intergeneric hybridization between longan and lychee. Scanning electron microscopy and flow cytometry were used to examine trichome morphology and genome size, respectively, to test this hypothesis. Longan, lychee, longli (D. confinis), and ‘Malesianus’ (D. longan sub spp malesianus) had morphologically distinct trichomes. The genome sizes for lychee (554 Mb), longan (444 Mb), ‘Malesianus’ (404 Mb), and rambutan (339 Mb) are distinctive and in a narrow range. ‘Malesianus’ has a genome 9% smaller than that of longan and 27% smaller than that of lychee. It is likely a species that evolved independently in northern Borneo island, and could be classified as a species, Dimocarpus malesianus, not a sub-species of longan as presently stated. Flow cytometry revealed a 50% variation in genome sizes among longli varieties, with genome sizes ranging from 450 to 678 Mb, beyond the range between longan and lychee. The genome size variation and distinct leaf hair morphology suggest that longli is not an intergeneric hybrid, and it is likely a separate genus evolved independently. The tested cultivars with distinctive genome sizes within D. confinis could be classified as separate species.     

Flow cytometric analysis in Lagenaria siceraria (Cucurbitaceae) indicates correlation of genome size with usage types and growing elevation

The occurrence and extent of genome size variation within species is controversially discussed and thorough analyses are rare. Given the large morphological variation in Lagenaria siceraria (bottle gourds) and its wide distribution in Africa we here analysed (1) the genome size variation within cultivars of L. siceraria, (2) the correlation between genome size and morphological traits, and (3) the geographical patterns of DNA content within the species. We measured 2C-values of 366 individuals from 117 accessions of L. siceraria (2n = 22) from Africa, America and Asia via flow cytometry with propidium iodide as DNA stain. We found that 2C-value in L. siceraria (0.683–0.776 pg/2C) is about two times lower than previously reported and varies by about 12% among all accessions. Moreover, our results indicated a clear correlation of genome size with two different seed or usage types and with growing elevations in West Africa. Within the seed types genome size varies by 6.6 and 7.5%, respectively. The genome size differences in seed types of L. siceraria might indicate differences in their evolutionary history and necessitates a re-evaluation of the phylogenetic relationships within L. siceraria while the correlation between 2C-value and the elevation of the collecting sites might indicate an adaptation of genome size to an unknown ecological parameter connected to altitude.     

Genome survey sequencing of red swamp crayfish <Emphasis Type="Italic">Procambarus clarkii</Emphasis>

Red swamp crayfish, Procambarus clarkii, presently is an important aquatic commercial species in China. The crayfish is a hot area of research focus, and its genetic improvement is quite urgent for the crayfish aquaculture in China. However, the knowledge of its genomic landscape is limited. In this study, a survey of P. clarkii genome was investigated based on Illumina’s Solexa sequencing platform. Meanwhile, its genome size was estimated using flow cytometry. Interestingly, the genome size estimated is about 8.50 Gb by flow cytometry and 1.86 Gb with genome survey sequencing. Based on the assembled genome sequences, total of 136,962 genes and 152,268 exons were predicted, and the predicted genes ranged from 150 to 12,807 bp in length. The survey sequences could help accelerate the progress of gene discovery involved in genetic diversity and evolutionary analysis, even though it could not successfully applied for estimation of P. clarkii genome size.