Is genome downsizing associated with diversification in polyploid lineages of Veronica?

The study of genome size evolution in a phylogenetic context in related polyploid and diploid lineages can help us to understand the advantages and disadvantages of genome size changes and their effect on diversification. Here, we contribute 199 new DNA sequences and a nearly threefold increase in genome size estimates in polyploid and diploid Veronica (Plantaginaceae) (to 128 species, c. 30% of the genus) to provide a comprehensive baseline to explore the effect of genome size changes. We reconstructed internal transcribed spacer (ITS) and trnL‐trnL‐trnF phylogenetic trees and performed phylogenetic generalized least squares (PGLS), ancestral character state reconstruction, molecular dating and diversification analyses. Veronica 1C‐values range from 0.26 to 3.19 pg. Life history is significantly correlated with 1C‐value, whereas ploidy and chromosome number are strongly correlated with both 1C‐ and 1Cx‐values. The estimated ancestral Veronica 1Cx‐value is 0.65 pg, with significant genome downsizing in the polyploid Southern Hemisphere subgenus Pseudoveronica and two Northern Hemisphere subgenera, and significant genome upsizing in two diploid subgenera. These genomic downsizing events are accompanied by increased diversification rates, but a ‘core shift’ was only detected in the rate of subgenus Pseudoveronica. Polyploidy is important in the evolution of the genus, and a link between genome downsizing and polyploid diversification and species radiations is hypothesized. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178 , 243–266.     

Chromosome and genome size variation in Luzula (Juncaceae), a genus with holocentric chromosomes

The present study examines chromosome and genome size evolution in Luzula (woodrush; Juncaceae), a monocot genus with holocentric chromosomes. Detailed karyotypes and genome size estimates were obtained for seven Luzula spp., and these were combined with additional data from the literature to enable a comprehensive cytological analysis of the genus. So that the direction of karyotype and genome size changes could be determined, the cytological data were superimposed onto a phylogenetic tree based on the trnL‐F and internal transcribed spacer (ITS) DNA regions. Overall, Luzula shows considerable cytological variation both in terms of chromosome number (2n = 6–66) and genome size (15‐fold variation; 2C = 0.56–8.51 pg; 547.7–8322.8 Mb). In addition, there is considerable diversity in the genomic mechanisms responsible, with the range of karyotypes arising via agmatoploidy (chromosome fission), symploidy (chromosome fusion) and/or polyploidy accompanied, in some cases, by the amplification or elimination of DNA. Viewed in an evolutionary framework, no broad trend in karyotype or genome evolution was apparent across the genus; instead, different mechanisms of karyotype evolution appear to be operating in different clades. It is clear that Luzula exhibits considerable genomic flexibility and tolerance to large, genome‐scale changes. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170 , 529–541.     

Phylogenetics of the slipper orchids (Cypripedioideae, Orchidaceae): Nuclear rDNA ITS sequences

Cypripedioideae (Orchidaceae) have been the subject of numerous taxonomic treatments with conflicting interpretations of relationships among the five genera and the 150–170 species. We have produced nuclear ribosomal ITS nucleotide sequences for nearly 100 slipper orchid species and used parsimony analysis to investigate their relationships. Our results demonstrate that each genus, as currently circumscribed, is monophyletic (Mexipedium andSelenipedium being represented by a single taxon). LikerbcL data, ITS sequences placeMexipedium sister toPhragmipedium. Relationships at the sectional level inPaphiopedilum are largely as described byCribb. However, the division ofPaphiopedilum into subgg.Brachypetalum andPaphiopedilum is not supported; subg.Brachypetalum is paraphyletic to subg.Paphiopedilum. Phragmipedium species are divided into the same three major clades as in the taxonomic scheme ofMcCook. The plicate-leaved genera,Cypripedium andSelenipedium, are successive sister groups to the rest of the subfamily, confirming generally held opinions that they display plesiomorphic characters compared to the conduplicate-leaved genera. A survey of karyotypes in the context of the ITS tree reveals a general trend toward increased chromosome number, probably brought about by centric fission. These data also accord with a previously suggested biogeographic hypothesis of a widespread Northern Hemisphere distribution, followed by range fragmentation due to Miocene cooling.     

New chromosome counts in the genus Centaurea (Asteraceae) from Turkey

Twenty‐two chromosome counts are reported in 16 species, four subspecies and two varieties of the genus Centaurea. These are mostly Turkish local endemics of section Cheirolepis, a complicated group from the Eastern clade of the Jacea group. Twenty‐one reports are new. Prevalence of the basic chromosome number x = 9 among the eastern sections of the Jacea group is confirmed. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 280–286.     

Molecular phylogeny of the subgenus Holothuria (Selenkothuria) Deichmann, 1958 (Holothuroidea: Aspidochirotida)

The subgenus Selenkothuria comprises 12 species of tropical shallow water sea cucumbers that share morphological features, such as rods in the body wall and tube feet, modified tentacles for suspension feeding, and cryptic colours. The taxonomic status of this taxon has been controversial, but currently it is accepted as a subgenus of the genus Holothuria. Phylogenetic analyses of mitochondrial genes [cytochrome c oxidase subunit 1 (COI), 16S RNA] of ten species of Selenkothuria and related subgenera showed the polyphyly of this subgenus; monophyly was rejected by a likelihood ratio test. A geographical split divides the species of this subgenus into three different groups: one Indo‐West‐Pacific (IWP) group and two American groups. The IWP group is more closely related to Holothuria (Semperothuria) cinerascens and to other subgenera such as Roweothuria, Holothuria, and Vaneyothuria, whereas the two American groups are more closely related to each other and to some species of the subgenus Halodeima. These results suggest multiple parallel originations and diversification of ossicle morphology within the subgenus Selenkothuria. The current scheme of subgenera for the genus Holothuria is not supported, suggesting the need for a new classification. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 165 , 109–120.     

The role of chromosome changes in the diversification of Passiflora L. (Passifloraceae)

Passiflora L. has more than 575 species distributed especially in the Neotropics. The chromosome number variation in the genus is highly congruent with its main subgenera, but its basic chromosome number (x) and the underlying events responsible for this variation have remained controversial. Here, we provide a robust and well-resolved time-calibrated phylogeny that includes 102 taxa, and by means of phylogenetic comparative methods (PCM) we tested the relative importance of polyploidy and dysploidy events to Passiflora karyotype evolution and diversification. Passiflora arose 42.9 Mya, with subgenus diversification at the end of the Palaeogene (Eocene-Oligocene). The basic chromosome number of the genus is proposed as x?=?6, and a strong recent diversification found in the Passiflora subgenus (Miocene) correlated to genome size increase and a chromosome change from n?=?6 to n?=?9 by ascending dysploidy. Polyploidy, conversely, appeared restricted to few lineages, such as Astrophea and Deidamioides subgenera, and did not lead to diversification increases. Our findings suggest that ascending dysploidy provided a great advantage for generating long-term persistent lineages and promoting species diversification. Thus, chromosome numbers/genome size changes may have contributed to morphological/ecological traits that explain the pattern of diversification observed in the genus Passiflora.     

CHROMOSOME NUMBERS IN CUPHEA (LYTHRACEAE): NEW COUNTS AND A SUMMARY

The American genus Cuphea with ca. 260 species is extremely diverse with respect to chromosome number. Counts are now available for 78 species and/or varieties, or 29% of the genus. Included in this study are first reports for 15 taxa from Brazil, Cuba, Dominican Republic, Mexico, and Venezuela. Twenty-two different numbers are known for the genus, ranging from n = 6 to n = 54. The most common number in the primary center of species diversity in Brazil is n = 8, which is regarded as the base number of the genus. Two numbers are most common in the secondary center in Mexico, n = 10 and n = 12. Species with n = 14 or higher are considered to be of polyploid origin. Polyploids comprise 46% of the total species counted and appear in 9 of the 11 sections for which chromosome numbers have been reported. Aneuploid species comprise ca. 25% of the genus and are known from 7 of the 11 sections. The two subgenera are not characterized by different chromosome numbers or sequences of numbers. None of the 14 sections are circumscribed by a single chromosome number. Morphological and ecological variability in widespread, weedy species is correlated with differing chromosome numbers in some species whereas in others the chromosome number is stable. Summary of chromosome numbers by taxonomic section is presented. Section Euandra, centered in eastern Brazil, and the largest section of the genus, appears to be chromosomally most diverse. In section Trispermum, characterized by difficult, variable species with intermediate forms, two of the four species studied have polyploid races. Section Heterodon, endemic to Mexico and Central America and comprising most of the annual species of the genus, is best known chromosomally. Chromosome numbers have been counted for 25 of 28 species, and 12 different numbers are reported. The most advanced sections, Melvilla and Diploptychia, with numerous species occurring at higher altitudes, are characterized by high polyploids. Apomictic species occur in sect. Diploptycia. The cytoevolution of Cuphea is complex with frequent polyploid and aneuploid events apparently playing a significant role in speciation in both centers of diversity.     

Genome size and plastid trnK-matK markers give new insights into the evolutionary history of the genus Lavandula L.

The genus Lavandula L. consists of 39 species distributed from the North Atlantic Islands, across the Mediterranean Basin to India. We analysed 36 taxa of the genus Lavandula representing two of the three subgenera and six of the eight sections according to the most recent classification (Upson & Andrews 2004). We achieved a phylogenetic reconstruction from partial sequences from plastid trnK and matK genes; the genome size was estimated by flow cytometer measurements. The primary aim was to track phylogenetic patterns through the maternal inherited marker at the sectional level and identify possible genome duplications. The cpDNA tree shows the phylogenetic relationships between subgenus, sections and also elucidates for the first time the relationships between the endemic species of Macaronesia, Morocco and Arabia. The ancestral split between the two subgenera could be explained by dispersal followed by an early vicariance event. The C-value shows genome up-sizing within several phylogenetic clades and geographical areas. An ancestral genome-up sizing is characterized at the node of section Dentatae and Lavandula. The cpDNA tree suggests that the taxa L. angustifolia subsp. pyrenaica (DC.) Guinea and L. stoechas subsp. luiseiri are best treated as a distinct species.     

Molecular phylogeny of the bee genus Hoplitis (Megachilidae: Osmiini) – how does nesting biology affect biogeography?

The genus Hoplitis (Megachilidae: Osmiini) comprises about 360 described species and occurs on all continents except Australia, South America, and Antarctica. Using five genes, we inferred the phylogeny of Hoplitis including 23 out of the 27 currently recognized subgenera, applying both Bayesian and maximum likelihood methods. Compared to the current morphology‐based classification, our phylogeny resulted in three classificatory changes: first, the subgenera Alcidamea, Cyrtosmia, Dasyosmia, Megalosmia, Monumetha, and Prionohoplitis are merged into one large subgenus Alcidamea Cresson, 1864, comb. nov. ; second, the subgenera Annosmia, Bytinskia, Coloplitis, and Hoplitis are merged into one large subgenus Hoplitis Klug, 1807, comb. nov. ; third, the subgenera Acrosmia, Hoplitina, Penteriades, and Proteriades are merged into one large subgenus Proteriades Titus, 1904, comb. nov. We provide evidence that the genus Hoplitis has a Palaearctic origin and that colonization events to southern Africa and to the Nearctic, as well as recolonization events from the Nearctic to the Palaearctic occurred. The species of the genus Hoplitis exhibit an extraordinary diversity in nesting behaviour, comprising both below and above ground nesting. Parsimony mapping revealed that ground nesting in excavated burrows is the ancestral state amongst Hoplitis bees. We hypothesize that nesting biology strongly affected both range expansion and long‐distance dispersal in Hoplitis. © 2012 The Linnean Society of London     

Genome size and chromosome number in the New Zealand species of Schoenus (Cyperaceae)

Schoenus (Cyperaceae) has holocentric chromosomes. Chromosome numbers were counted and nuclear DNA amounts were measured for all the New Zealand species of the genus. Chromosome numbers ranged from 2n = 8 to c. 2n = 90. Two chromosome races, with 2n = 28 and 2n = 56, were found in S. pauciflorus. Flow cytometry using propidium iodide‐stained nuclei was used to measure genome size. A 14.8‐fold variation in 2C DNA content was found, with values ranging from 1.33 to 19.71 pg/2C nucleus. Phylogenetic trees based on sequence variation in the internal transcribed spacer (ITS) region of the 45S ribosomal DNA locus were constructed using several phylogenetic models to reveal possible evolutionary relationships among the New Zealand Schoenus spp. and a sample of Australian Schoenus spp. Analysis revealed heterogeneity of chromosome number, size and DNA C value within clades. Meiosis in four species showed only bivalent formation at metaphase I. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 169 , 555–564.     

Thoracic underreplication in Drosophila species estimates a minimum genome size and the dynamics of added DNA

Many cells in the thorax of Drosophila were found to stall during replication, a phenomenon known as underreplication. Unlike underreplication in nuclei of salivary and follicle cells, this stall occurs with less than one complete round of replication. This stall point allows precise estimations of early-replicating euchromatin and late-replicating heterochromatin regions, providing a powerful tool to investigate the dynamics of structural change across the genome. We measure underreplication in 132 species across the Drosophila genus and leverage these data to propose a model for estimating the rate at which additional DNA is accumulated as heterochromatin and euchromatin and also predict the minimum genome size for Drosophila. According to comparative phylogenetic approaches, the rates of change of heterochromatin differ strikingly between Drosophila subgenera. Although these subgenera differ in karyotype, there were no differences by chromosome number, suggesting other structural changes may influence accumulation of heterochromatin. Measurements were taken for both sexes, allowing the visualization of genome size and heterochromatin changes for the hypothetical path of XY sex chromosome differentiation. Additionally, the model presented here estimates a minimum genome size in Sophophora remarkably close to the smallest insect genome measured to date, in a species over 200 million years diverged from Drosophila.     

Island life – classification,speciation and cryptic species of Pycnandra (Sapotaceae) in New Caledonia

Pycnandra (Sapotaceae), the largest endemic genus in New Caledonia, comprises 66 species classified in six subgenera. We tested phylogenetic relationships and a proposed infrageneric classification by sampling 60 species for sequences of nuclear ribosomal DNA (ETS, ITS, RPB2) and plastid DNA (trnH–psbA) and nine morphological characters. Data were analysed with Bayesian inference, parsimony jackknifing and lineage through time. We recovered a phylogenetic tree supporting the recognition of six proposed subgenera (Achradotypus, Leptostylis, Pycnandra, Sebertia, Trouettia and Wagapensia). Because a subgeneric classification is used, the nomenclature will be stable when the members are transferred to Pycnandra. Morphological traits were optimized in the BEAST analysis, adding evidence to earlier work that morphology has limited value for successfully diagnosing groups in Sapotaceae. We confirm a previously suspected case of cryptic species that exhibit the same morphological features and require the same abiotic conditions, but are distantly related in the phylogenetic tree. We detected two possible new cases of cryptic sibling species that might warrant recognition. A slowdown in speciation rate in several genera has been suggested as evidence that New Caledonia was once submerged after rifting from Australia. Plotting lineages through time reveals two important intervals at 7.5–8.6 Ma and present to 1.5 Ma, when net molecular diversification within the genus was zero. This indicates that the genus presently has reached a dynamic equilibrium, providing additional evidence that New Caledonia is an old Darwinian island, being submerged during the Eocene and colonized after re‐emergence c. 37 Ma. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 57–77.     

Chromosome pairing affinities in interspecific hybrids reflect phylogenetic distances among lady's slipper orchids (Paphiopedilum)

Background and Aims

Lady''s slipper orchids (Paphiopedilum) are of high value in floriculture, and interspecific hybridization has long been used for breeding improved cultivars; however, information regarding the genome affinities of species and chromosome pairing behaviour of the hybrids remains almost unknown. The present work analyses the meiotic behaviour of interspecific hybrids by genomic in situ hybridization and cytologically evaluates the genomic relationships among parental species.

Methods

Eight interspecific F₁ hybrids of Paphiopedilum species in various subgenera or sections were investigated in this study. The chromosome behaviour in meiosis of these interspecific hybrids was analysed and subjected to genomic in situ hybridization and fluorescent in situ hybridization.

Key Results

Genomic in situ hybridization was demonstrated as an efficient method to differentiate between Paphiopedilum genomes and to visualize the chromosome pairing affinities in interspecific F₁ hybrids, clarifying the phylogenetic distances among these species. Comparatively regular chromosome pairing observed in the hybrids of P. delenatii × P. bellatulum, P. delenatii × P. rothschildianum and P. rothschildianum × P. bellatulum suggested high genomic affinities and close relationships between parents of each hybrid. In contrast, irregular chromosome associations, such as univalents, trivalents and quadrivalents occurred frequently in the hybrids derived from distant parents with divergent karyotypes, such as P. delenatii × P. callosum, P. delenatii × P. glaucophyllum, P. rothschildianum × P. micranthum and P. rothschildianum × P. moquetteanum. The existence of multivalents and autosyndesis demonstrated by genomic in situ hybridization in this study indicates that some micro-rearrangements and other structural alterations may also play a part in differentiating Paphiopedilum species at chromosomal level, demonstrated as different chromosome pairing affinities in interspecific hybrids.

Conclusions

The results indicate that genome homology and the interaction of genetic factors, but not chromosome number nor karyotype similarity, determine the chromosome pairing behaviour in Paphiopedilum hybrids.     

Genome size evolution in Sapindaceae at subfamily level: a case study of independence in relation to karyological and palynological traits

Sapindaceae s.l. is a moderately large family of trees, shrubs and lianas. Current knowledge on genome size and how it varies in this family is scarce. This research aims to characterize the DNA content in 39 species of Sapindaceae, mainly in tribe Paullinieae s.s., by the analysis of the variation in genome size relative to karyotypic and palynological features. Nuclear DNA amount was measured by flow cytometry, and linear regression analyses were conducted to analyse the relationship between genome size variation and various karyotypic and palynological features. Genome size varied nine‐fold among species, ranging from 1C = 0.305 pg (Lophostigma plumosum) to 2.710 pg (Cardiospermum heringeri). The low regression coefficients obtained suggest that genome size mainly varies independently of karyotypic and palynological features. With regard to karyotype evolution, the constant chromosome number but variable genome size in Houssayanthus, Paullinia and Serjania suggest that structural changes mainly caused by changes in the amounts of repetitive DNA are more important than numerical change. In contrast, in Cardiospermum and Urvillea, variation in chromosome number and genome size supports the suggestion that numerical and structural changes are important in the karyotype evolution of these genera. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174 , 589–600.     

An updated classification for the hyper-diverse genus Corydalis (Papaveraceae: Fumarioideae) based on phylogenomic and morphological evidence

The genus Corydalis, with ca. 530 species, has long been considered taxonomically challenging because of its great variability. Previous molecular analyses, based on a few molecular markers and incomplete taxonomic sampling, were clearly inadequate to delimit sections and subgenera. We have performed phylogenetic analyses of Corydalis and related taxa, using 65 shared protein-coding plastid genes from 313 accessions (including 280 samples of ca. 226 species of Corydalis) and 152 universal low-copy nuclear genes from 296 accessions (including 271 samples of Corydalis) covering all 42 previously recognized sections and five independent “series”. Phylogenetic trees were inferred using Bayesian Inference and Maximum Likelihood. Eight selected morphological characters were estimated using ancestral state reconstructions. Results include: (i) of the three subgenera of Corydalis, two are fully supported by both the plastid and nuclear data; the third, subg. Cremnocapnos, is weakly supported by plastid DNA only, whereas in the nuclear data the two included sections form successive outgroups to the rest of the genus; (ii) among all 42 sections and five “series”, 25 sections and one “series” are resolved as monophyletic in both data sets; (iii) the common ancestor of Corydalis is likely to be a perennial plant with a taproot, yellow flowers with a short saccate spur, linear fruits with recurved fruiting pedicels, and seeds with elaiosomes; (iv) we provide a new classification of Corydalis with four subgenera (of which subg. Bipapillatae is here newly described), 39 sections, 16 of which are consistent with the previous classification, 16 sections have been recircumscribed, one section has been reinstated and six new sections are established. Characters associated with lifespan, underground structures, floral spur, fruit and elaiosomes are important for the recognition of subgenera and sections. These new phylogenetic analyses combined with ancestral character reconstructions uncovered previously unrecognized relationships, and greatly improved our understanding of the evolution of the genus.     

Molecular phylogenetics,character evolution and systematics of the genus Micranthes (Saxifragaceae)

With c. 85 species, the genus Micranthes is among the larger genera of the Saxifragaceae. It is only distantly related to the morphologically similar genus Saxifraga, in which it has frequently been included as Saxifraga section Micranthes. To study the molecular evolution of Micranthes, we analysed nuclear ribosomal (internal transcribed spacer, ITS) and plastid (trnL–trnF) DNA sequences in a comprehensive set of taxa comprising c. 75% of the species. The molecular phylogenetic tree from the combined dataset revealed eight well‐supported clades of Micranthes. These clades agree in part with previously acknowledged subsections or series of Saxifraga section Micranthes. As these eight groups can also be delineated morphologically, we suggest that they should be recognized as sections of Micranthes. New relationships were also detected for some species and species groups, e.g. section Davuricae sister to sections Intermediae and Merkianae, and M. micranthidifolia as a member of section Micranthes. Species proposed to be excluded from the genus Micranthes for morphological reasons were resolved in the molecular tree in Saxifraga. Many morphological characters surveyed were homoplasious to varying extents. Micromorphological characters support comparatively well the clades in the phylogenetic tree. An updated nomenclature and a taxonomic conspectus of sections and species of Micranthes are provided. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178 , 47–66.     

Phylogenetic analysis based on structural and combined analyses of Rhus s.s. (Anacardiaceae)

Structural data were combined with trnL‐F and internal transcribed spacer sequences from other studies and with new sequences representing ten additional species to clarify the phylogenetic relationships of Rhus s.s. These data indicate that Rhus s.s and both subgenera, Rhus and Lobadium, are monophyletic. The genus Rhus is supported as monophyletic by the presence of red glandular hairs on the berries and inflorescence axis, cilia on the sepals and glands on the leaf blades. Subgenus Rhus can be identified by the presence of more than seven resin channels in the petiole, weakly percurrent tertiary veins and a type I vascular system in the mid‐vein. Subgenus Lobadium is characterized by the presence of short bracteoles and pedicels. This subgenus is divided into four sections, Lobadium, Rhoeidium, Styphonia and Terebinthifolia. Section Lobadium has trifoliate leaves; section Rhoeidium is monotypic, including only Rhus microphylla; section Styphonia is supported by five synapomorphies, including an incomplete marginal vein, fibres in the petiole, a thick cuticle, two layers of palisade parenchyma and prismatic crystals in the mesophyll; and section Terebinthifolia has gelatinous xylary fibres in the petiole. Hypotheses about the evolutionary changes of these characters are presented based on the cladograms. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 452–468.