Evolution of Iris subgenus Xiphium based on chromosome numbers, FISH of nrDNA (5S, 45S) and trnL–trnF sequence analysis

The subgenus Xiphium is one of the six infrageneric divisions of the genus Iris. Chromosome numbers of six of the seven Xiphium species are known. Here the aim was to infer genetic and phylogenetic relationships based on chromosome numbers, chromosome markers and plastid sequences. Chromosomal locations of 5S and 45S rDNA loci were determined in 19 populations of the 7 species by fluorescence in situ hybridization (FISH). Additionally, the trnL–trnF plastid spacer was sequenced and a phylogenetic analysis performed. Based on chromosome markers, subgenus Xiphium species were classified into four groups that differed in the number and locations of both types of nrDNA: (1) I. tingitana (2n = 28), I. filifolia (2n = 30, 34) and I. xiphium (2n = 34), (2) I. juncea (2n = 32) and I. boissieri (2n = 36), (3) I. serotina (2n = 34) and (4) I. latifolia (2n = 42). Although the trnL–trnF phylogeny was not fully resolved, the sequence analysis showed a well-supported subgroup of I. filifolia, I. tingitana and I. xiphium, as well as I. juncea. FISH physical maps of the Iris subgenus Xiphium taxa are species dependent. I. filifolia, I. tingitana and I. xiphium are very closely related species and share cytogenetic characteristics. Disploidy appears to have been central in the evolution of this subgenus, given a series of chromosome numbers (2n = 28, 30, 32, 34, 36, 42) and our phylogenetic results. Clear differences were found among European and African populations of I. filifolia. A different taxonomic treatment of I. filifolia is supported for populations on both sides of the Strait of Gibraltar.     

From open areas to forests? The evolutionary history of Philodendron subgenus Meconostigma (Araceae) using morphological data

Due to its wide distribution covering three of the largest Neotropical biomes, Philodendron subgenus Meconostigma is an interesting model to discuss the diversification of Neotropical plants. The aim of this study was to test a previous hypothesis that the Eastern and Southern species of P. subg. Meconostigma have plesiomorphic gynoecial structure while in Amazonian species they are apomorphic. To this end, we conducted an analysis of maximum parsimony with generalized frequency coding method using a matrix with 59 morphological characters and 90% of the species of P. subg. Meconostigma. The phylogenetic reconstruction suggests that the subgenus is monophyletic and originates from open areas of Cerrado. Four morphological synapomorphies support the monophyly of the subgenus and seven synapomorphies support four minor clades within it. Our results also include characterization of three new gynoecial subtypes (A1, A2 and A3) within the subgenus. Subtype A2 (undeveloped stylar body with long stylar canals, absent central stylar dome, shallow compitum) is basal and represents the ancestral gynoecium in the group. These findings suggest that the balance between pollen uptake and accessibility of the locules were decisive to drive gynoecium evolution in the subgenus Meconostigma.     

Evolutionary trends in the chromosome numbers of swarm-founding social wasps

Chromosome number and morphology are relevant aspects of genomic organization of eukaryotes and are considered key components to comprehend evolutionary mechanisms and karyotypic differentiation. Social wasps belonging to the tribe Epiponini show complex social characteristics that make these insects interesting models for genetic and evolutionary studies. However, there is a paucity of genetic information in social wasps as a whole. Aiming to investigate the process of chromosomal evolution of the social Epiponini wasps, chromosomes of ten species of this group were analyzed using Giemsa and fluorochrome staining techniques and fluorescence in situ hybridization in two species. In this study a high variation in the chromosome number and morphology was found and the previous range of chromosome number of Epiponini was broadened from n = 8–32 to n = 5–33. We also suggest that chromosomal segments with high GC content must have had a key role in the karyotype diversification of these wasps. Moreover, based on a phylogenetic background we find evidence of a main role of chromosomal fusion in the occurrence of gradual decrease of chromosome number in Epiponini during its chromosomal evolutionary history.     

Integrative taxonomy and preliminary assessment of species limits in the Liolaemus walkeri complex (Squamata,Liolaemidae) with descriptions of?three?new species from Peru

Species delimitation studies based on integrative taxonomic approaches have received considerable attention in the last few years, and have provided the strongest hypotheses of species boundaries. We used three lines of evidence (molecular, morphological, and niche envelopes) to test for species boundaries in Peruvian populations of the Liolaemus walkeri complex. Our results show that different lines of evidence and analyses are congruent in different combinations, for unambiguous delimitation of three lineages that were “hidden” within known species, and now deserve species status. Our phylogenetic analysis shows that L. walkeri, L. tacnae and the three new species are strongly separated from other species assigned to the alticolor-bibronii group. Few conventional morphological characters distinguish the new species from closely related taxa and this highlights the need to integrate other sources of data to erect strong hypothesis of species limits. A taxonomic key for known Peruvian species of the subgenus Lioalemus is provided.     

Chromosome numbers in Pinguicula (Lentibulariaceae): survey, atlas, and taxonomic conclusions

Our study (survey, atlas of 136 microphotographs and 67 drawings) points out the actual chromosome numbers of 82 taxa of the genus Pinguicula L. They were gathered from literature and critically examined. In addition, numerous counts are published for the first time. They represent about 80% of all the taxa known. The basic chromosome numbers are x = 6, 8, 9, 11, and 14; the ploidy levels are 2n (diploid), 4n (tetraploid), 8n (octoploid) and 16n (hexadecaploid). The basic number x = 6 is a one-off, x = 8 and 11 are the most frequent in the genus; x = 14 indicates a hybridogenous differentiation process in the past. The caryological differentiation—chromosome numbers and ploidy level—is discussed with regard to distribution pattern, growth type, and infrageneric classification (at the level of sections).     

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.     

Ploidy variation in Trigonobalanus verticillata (Fagaceae)

Cytological studies were carried out on two populations of Trigonobalanus verticillata in Yinggeling, Hainan Province, China (YGL), and Fraser’s Hill, Malaysia (FH). In the two populations, the pattern of interphase nuclei was of the simple chromocentre type, the mitotic prophases were of the proximal interstitial type and chromosome numbers were 2n = 2x = 14 (YGL) and 2n = 6x = 42 (FH), representing diploid and hexaploid respectively. The basic chromosome number of the species was x = 7; this is a unique number in the Fagaceae and in the Fagales as well. The characteristic karyotype of the diploid population in China was 2n = 14 = 8m + 4sm + 2st. Based on the new finding of the chromosome number in this study, we clarify the previous dispute about the basic chromosome number and propose that the two Asian Trigonobalanus species have closer affinity.     

Atlantic moonfishes: independent pathways of karyotypic and morphological differentiation

Fish of the genus Selene, known as lookdowns or moonfish, are one of the most morphologically derived groups of the family Carangidae, whose phylogenetic relationships are still largely unknown. In this study, we discuss karyoevolutionary aspects of three representatives of this genus from the Western Atlantic: Selene brownii (2n = 48; FN = 48), Selene setapinnis (2n = 46; FN = 48), and Selene vomer (2n = 48; FN = 50). Their body patterns were also investigated and compared to one another and in relation to two other species of different genera. Two mechanisms of karyotypic evolution seem to have acted in the diversification of this genus, namely pericentric inversions and centric fusions. Mapping of rDNA sequences showed that chromosome pairs bearing 5S rDNA sites are similar, whereas those bearing 18 rDNA sites are morphologically distinct while apparently also exhibiting interspecies synteny. Although the nucleolar organizer-bearing chromosomes are extremely efficient cytotaxonomic markers among Selene species, others cytogenetic patterns of these species are relatively conserved. Hybridization with telomeric probes (TTAGGG)_n did not exhibit interstitial telomeric sites (ITS), especially in S. setapinnis, where, along with a reduction in diploid number, a large metacentric pair derived from centric fusion is present. Data obtained by geometric morphometrics enable a clear morphological distinction among the three species, as well as in relation to two other species of the genus Caranx and Oligoplites. Data obtained suggest that morphologic evolution in Selene species was primarily dissociated from visible changes that occurred at the chromosomal level.     

Chromosome numbers of the Australian Cymodoceaceae

The somatic chromosome numbers of the eleven Australian seagrass species belonging to five genera in the Family Cymodoceaceae were determined. The chromosome numbers in Amphibolis and Thalassodendron are reported for the first time. Cymodocea and Halodule species have the following chromosome numbers: Cymodocea angustata Ostenf., 2n = 14, 28; C. rotundata Ehrenb. & Hempr. ex Asch., 2n = 14; C. serrulata (R. Br.) Asch. & Magnus, 2n = 14, 28; Halodule pinifolia (Miki) den Hartog, 2n = 32; H. uninervis (Forsk.) Asch., 2n = 32; H. tridentata (Steinh.) Endl. ex Unger, 2n = 14. Halodule has the highest chromosome numbers among the seagrasses and they are the largest in sizes with a distinct bimodal type in the family. Syringodium isoetifolium (Asch.) Dandy has 2n = 20. Both endemic Amphibolis antarctica (Labill.) Sonder ex Asch. and A. griffithii (J. Black) den Hartog have 2n = ca. 36 and have the smallest chromosomes in the family appearing as small dots. Thalassodendron pachyrhizum den Hartog has 2n = 28. Chromosome numbers appear to be identical or closely related among different species in the same genus but they vary in the five genera in the Cymodoceaceae suggesting that these five genera may have evolved independently in the past.     

A new insight into Serjania Mill. (Sapindaceae, Paullinieae) infrageneric classification: a cytogenetic approach

Serjania (Sapindaceae, Paullinieae) comprises about 230 species, and currently two infrageneric classifications have been proposed but both are difficult to apply. This work tested which infrageneric classification fitted better in relation to cytogenetic traits added to the main morphological features used by the authors of the subgenus arrangements to gain an insight into the evolutionary karyotype relationships. In order to test the relationship between karyotypes and the systematics of this genus, the karyotypes of five species of Serjania belonging to five different sections (sensu Radlkofer) were described. Known karyological information on 26 species was used to complement the results. With these data, a cluster analysis was set up to test which infrageneric classification fitted better. In addition, a principal component analysis (PCA) was performed to examine the relevance of the traits in the subgenus classification. All the karyotypes analyzed (including new as well as previous records) had 2n?=?24 chromosomes, and the karyotypes were asymmetrical: submetacentric and metacentric chromosomes were common, whereas telocentric chromosomes were rare. The PCA revealed seven principal components, the first two explained 52?% of the total variation, and the last ones were related to all the karyotypic features studied. The phenogram obtained reflected a scarce fitting into both infrageneric classifications, with only 3 sections of the 12 proposed by Radlkofer and two of the six sections proposed by Acevedo-Rodríguez being represented. Finally, regarding karyotype evolution, the constancy of chromosome number and the variation in the length of the complement suggest that structural chromosome changes would have played a leading role.     

Phylogeny of the genus Peperomia (Piperaceae) inferred from the trnK/matK region (cpDNA)

The genus Peperomia is one of the largest genera of basal angiosperms, comprising about 1500-1700 pantropically distributed species. The currently accepted infrageneric classification divides Peperomia into nine subgenera and seven sections. This classification is based on some 200 species, primarily using fruit morphology. The monophyly of these infrageneric taxa has never been tested and molecular phylogenetic studies of a representative sampling within Peperomia do not exist. This paper provides the first molecular phylogeny for the genus Peperomia. Monophyletic clades within Peperomia are identified and previously used morphological characters are critically reviewed. We show that the importance of some morphological characters has been overestimated and that some of these characters presumably have evolved several times independently. Only one previously described subgenus has been confirmed to be monophyletic.     

Karyological data of 47 accessions of 28 Artemisia (Asteraceae, Anthemideae) species from Iran, with first new reports for Iranian populations and first absolute counts in three species

Somatic chromosome numbers of 47 accessions representing 28 Artemisia species are provided from Iran. Two basic chromosome numbers, x = 8, 9, each with diploid, tetraploid and hexaploid levels, were found. Different chromosome numbers, 2n = 16, 16 + 1B, 16 + 5B, 32, 48, and 2n = 18, 18 + 1B, 19, 36, 36 + 1B, 36 + 2B, 37, 49 + 2B, 49 + 3B, 51 + 6B, 54, 54 + 1B, 54 + 3B, 54 + 5B, in studied accessions were identified. Chromosome numbers are reported for the first time in three species, counts in four species are new for Iran, and other counts have been thoroughly compared to previous data. Forty percent of the studied accessions are polyploid and B-chromosome(s) are reported in 17 % of accessions. Aneuploidy and aneusomy are other relevant cytological phenomena. Some karyological parameters, total karyotype length, karyotype formula, mean value of centromeric indices, mean arm ratio, A ₁ and A ₂ indices, were estimated to characterize the karyotypes numerically. A ₁ and A ₂ indices showed that karyotypes tend to be asymmetric in polyploid and dysploid taxa. PCA analysis of all karyological parameters has shown some systematic and evolutionary implications. The consideration of all these chromosome numbers and cytogenetic mechanisms has led us to infer the different patterns of chromosomal evolution in the genus.     

Karyological features and cytotaxonomy of the tribe Vernonieae (Asteraceae)

Chromosome numbers are reported for 29 populations of 19 Vernonieae taxa collected mainly in the northeastern region of Brazil. Among them, data for five genera (Blanchetia, Rolandra, Pithecoseris, Stilpnopappus and Vanillosmopsis) are here reported for the first time, and the first chromosome counts are presented for 12 species. Chromosome numbers are quite diverse among and sometimes within genera, especially in the controversial and large subtribe Vernoniinae. The numbers varied from 2n = 18 to 2n = ~72. The main karyoevolutionary mechanism seems to be dysploidy, while polyploidy is probably associated with ancient hybridization processes generating most paleotetraploid genera. All studied species presented semi-reticulated interphase nuclei and proximal-early condensing behavior in prophase to prometaphase. In one species (Vernonia condensata with 2n = 40) fluorochrome staining with CMA/DAPI revealed five chromosome pairs bearing subterminal CMA⁺/DAPI^? heterochromatin, probably NOR-associated, revealing the existence of low amounts of satellite DNA. The role of these features in the evolution of the tribe is discussed, revealing some interesting aspects for understanding of the Vernonieae karyoevolution, especially regarding neotropical members.     

Molecular phylogeny of the neotropical genus Christensonella (Orchidaceae, Maxillariinae): species delimitation and insights into chromosome evolution

Background and Aims

Species'' boundaries applied within Christensonella have varied due to the continuous pattern of variation and mosaic distribution of diagnostic characters. The main goals of this study were to revise the species'' delimitation and propose a more stable classification for this genus. In order to achieve these aims phylogenetic relationships were inferred using DNA sequence data and cytological diversity within Christensonella was examined based on chromosome counts and heterochromatin patterns. The results presented describe sets of diagnostic morphological characters that can be used for species'' identification.

Methods

Phylogenetic studies were based on sequence data of nuclear and plastid regions, analysed using maximum parsimony and maximum likelihood criteria. Cytogenetic observations of mitotic cells were conducted using CMA and DAPI fluorochromes.

Key Results

Six of 21 currently accepted species were recovered. The results also support recognition of the ‘C. pumila’ clade as a single species. Molecular phylogenetic relationships within the ‘C. acicularis–C. madida’ and ‘C. ferdinandiana–C. neowiedii’ species'' complexes were not resolved and require further study. Deeper relationships were incongruent between plastid and nuclear trees, but with no strong bootstrap support for either, except for the position of C. vernicosa. Cytogenetic data indicated chromosome numbers of 2n = 36, 38 and 76, and with substantial variation in the presence and location of CMA/DAPI heterochromatin bands.

Conclusions

The recognition of ten species of Christensonella is proposed according to the molecular and cytogenetic patterns observed. In addition, diagnostic morphological characters are presented for each recognized species. Banding patterns and chromosome counts suggest the occurrence of centric fusion/fission events, especially for C. ferdinandiana. The results suggest that 2n = 36 karyotypes evolved from 2n = 38 through descendent dysploidy. Patterns of heterochromatin distribution and other karyotypic data proved to be a valuable source of information to understand evolutionary patterns within Maxillariinae orchids.Key words: Chromosome number, Christensonella, Cymbidieae, cytotaxonomy, fluorochrome staining, Maxillaria, Maxillariinae, molecular phylogenetics, species delimitation     

Systematics and evolutionary history of the Asterotricha group of the genus Onosma (Boraginaceae) in central and southern Europe inferred from AFLP and nrDNA ITS data

Onosma is a species-rich genus with complicated patterns of morphological and karyological variation, and controversial taxonomic treatments. In the present study we focused on the Asterotricha group, one of three major infrageneric groups, in the area of central and southern Europe. Ribosomal internal transcribed spacer (ITS) sequences and amplified fragment length polymorphism (AFLP) markers were used to assess species differentiation and relationships, and to gain insight into the evolutionary history of the group. ITS data supported the monophyly of the asterotrichous taxa. Six genetically differentiated entities were resolved by AFLP markers, corresponding to O. echioides, O. heterophylla s.l., O. thracica including O. rigida, O. malkarmayorum, O. taurica, and O. stellulata. Onosma stellulata is identified as the most differentiated lineage within the Asterotricha group, which is consistent with its unique chromosome number. Within O. heterophylla s.l., recognition of O. viridis as a species distinct from O. heterophylla s.str. is supported. On the other hand, O. tornensis appears to be conspecific with O. viridis. Inferring from the patterns of genetic diversity and divergence, the evolutionary history of O. heterophylla s.l. is discussed. Genetic variation within the polymorphic Apennine–Dalmatian O. echioides is almost continuous and shaped by isolation by distance, thus none of the previous intraspecific taxonomic concepts are valid. The assumed hybrid origin of O. malkarmayorum is supported, and its affinity to potential ancestors, O. heterophylla s.l. and O. thracica incl. O. rigida, is indicated. Future studies of asterotrichous Onosma species should focus on diversity centers in the eastern Mediterranean and Asia Minor, to build up a comprehensive taxonomic concept of the group and to clarify its evolution.     

Intraspecific phylogenetic relationships in Ixeridium dentatum (Compositae) complex based on genetic and morphological evidence

Ixeridium dentatum (I. dentatum; family Compositae) forms a complex species through intraspecific differentiation. In this study, intraspecific taxa of I. dentatum were analyzed by counting chromosome numbers, sequencing internal transcribed spacer (ITS), and detecting amplified fragment length polymorphisms (AFLP) to explore phylogenetic relationships within I. dentatum. Analysis of ploidy levels showed that I. dentatum subsp. kitayamense and subsp. nipponicum were diploid (2n = 2x = 14), I. dentatum subsp. kimuranum was tetraploid (2n = 4x = 28), and other intraspecific taxa of I. dentatum were triploid (2n = 3x = 21). The ITS sequences contained only 13 variable sites (2.7 %) among intraspecific taxa, suggesting that the I. dentatum complex originated by autoploidy. Our AFLP-based analysis separated these taxa into five groups: (1) I. dentatum subsp. kimuranum and subsp. kitayamense; (2) subsp. nipponicum and var. albiflorum; (3) two types of subsp. dentatum and f. atropurpureum; (4) f. amplifolium; (5) subsp. ozense and new taxa, which were discovered in this study. The identified clusters significantly differed from morphological classifications. For example, morphologically similar variety and forms of I. dentatum subsp. nipponicum were separated into different groups. Taken together with the ploidy data, this study proposes a possible evolutionary history generating the current taxonomic combinations of the I. dentatum complex. The proposed model suggests that diverse taxa recently emerged during migration to lowlands.     

Karyological features and banding patterns in Arachis species belonging to the Heteranthae section

The section Heteranthae of Arachis is endemic to Brazil, occurring mainly in the semi-arid northeastern region. The section is considered derived within the genus and includes only annual herbs. Most previous cytological evaluations were restricted to chromosome numbers and morphology. The present approach comprised karyomorphological evaluation in 10 accessions from five species of this section, including standard staining and fluorochrome banding [chromomycin A3 (CMA)/4′,6-diamidino-2-phenylindole (DAPI)]. All accessions presented diploid chromosome numbers (2n = 20) with a prevalence of metacentric to submetacentric chromosome morphology. Arachis dardani, Arachis pusilla, and Arachis interrupta presented karyotypic formula 18m + 4sm and satellite type 2, while Arachis sylvestris and Arachis giacomettii presented 16m + 4sm and satellite type 10. Despite the conserved morphological features, higher diversity was detected in terms of size and number of GC-rich (CMA⁺) heterochromatic blocks among the species; however, all of them were located in the pericentromeric regions. The species A. pusilla presented the highest number of GC-rich blocks, present in all chromosomes of the complement. Based on the data obtained and considering literature data, we suggest that A. dardani and A. interrupta occupy a basal position in the group due to their moderate asymmetry and satellite type. At least in A. pusilla, the constitutive heterochromatin seems to have suffered recent modifications of its constitution, in contrast to other species that present pericentromeric CMA⁺ blocks in all chromosomes. A. giacomettii and A. sylvestris are closely related to each other and also similar to the previously studied Arachis seridoensis, revealing two clear-cut subgroups within the section from the karyological point of view.     

Chromosome numbers in Tripleurospermum Sch. Bip. (Asteraceae) and closely related genera: relationships between ploidy level and stomatal length

This study includes 24 reports of chromosome counts in Tripleurospermum Sch. Bip. (20 taxa), Anthemis L. (1 species) and Matricaria L. (3 taxa) belonging to the tribe Anthemideae of the family Asteraceae from Turkey. Chromosome numbers of these taxa are 2n = 2x = 18, 3x = 27 and 4x = 36. Nine counts are new reports, one is not consistent with previous report, and the remainder confirm earlier information. Statistically significant differences depending on ploidy level, stomatal length or environmental factors such as altitude were determined in Tripleurospermum. Several systematic and evolutionary aspects of the genera are discussed in the light of chromosomal data.     

Analyses of cpDNA matK sequence data place Tillaea (Crassulaceae) within Crassula

Analysis of cpDNA matK sequences for a total of 43 members of the succulent plant family Crassulaceae, including 24 taxa of Crassula, recovered a well-supported clade comprising Crassula species that is sister to the remainder of the family. The resulting topologies do not support the monophyly of the currently recognized subgenera of Crassula, as one member of subgenus Disporocarpa (C. crenulata) is placed as sister to an otherwise monophyletic subgenus Crassula. The major synapomorphy that has been used to recognize the latter subgenus is a base chromosome number of x = 7 versus a base of x = 8 in the other subgenus. We cannot assess the utility of this feature for defining subgenus Crassula because a chromosome count of C. crenulata has yet to be published. The five accessions of the recently resurrected segregate genus Tillaea (of 24 total Crassula species) included here were placed in four separate, well-supported lineages, one of which is greatly removed from the other four accessions. This suggests that this genus is not valid and should not be recognized. An initial examination of the evolution of habit indicates that a perennial habit is ancestral and that the annual habit is a feature that has been derived at least twice in the genus.