Lycocasine A, a Lycopodium Alkaloid from Lycopodiastrum casuarinoides and Its Acid-Sensing Ion Channel 1a Inhibitory Activity.

A novel Lycopodium alkaloid, lycocasine A (1), and seven known Lycopodium alkaloids (2-8), were isolated from Lycopodiastrum casuarinoides. Their structures were determined through NMR, HRESIMS, and X-ray diffraction analysis. Compound 1 features an unprecedented 5/6/6 tricyclic skeleton, highlighted by a 5-aza-tricyclic[6,3,1,02,6]dodecane motif. In bioactivity assays, compound 1 demonstrated weak inhibitory activity against acid-sensing ion channel 1a.

Hybridization and reticulate evolution in Diphasiastrum (flat-branched clubmosses, Lycopodiaceae) - New data from the island of Taiwan and Vietnam.

In the species groups related to Diphasiastrum multispicatum and D. veitchii, hybridization was investigated in samples from northern and southern Vietnam and the island of Taiwan, including available herbarium specimens from southeast Asia. The accessions were analyzed using flow cytometry (living material only), Sanger sequencing and multiplexed inter-simple sequence repeat genotyping by sequencing. We detected two cases of ancient hybridization involving different combinations of parental species; both led via subsequent duplication to tetraploid taxa. A cross D. multispicatum × D. veitchii from Malaysia represents D. wightianum, a tetraploid taxon according to reported DNA content measurements of dried material (genome formulas MM, VV and MMVV, respectively). The second case involves D. veitchii and an unknown diploid parent (genome formula XX). Three hybridogenous taxa (genome formulas VVX, VVXX, VVVX) were discernable by a combination of flow cytometry and molecular data. Taxon I (VVX, three clones found on Taiwan island) is apparently triploid. Taxon II represents another genetically diverse and sexual tetraploid species (VVXX) and can be assigned to D. yueshanense, described from Taiwan island but occurring as well in mainland China and Vietnam. Taxon III is as well most likely tetraploid (VVVX) and represented by at least one, more likely two, clones from Taiwan island. Taxa I and III are presumably asexual and new to science. Two independently inherited nuclear markers recombine only within, not between these hybrids, pointing towards reproductive isolation. We present an evolutionary scheme which explains the origin of the hybrids and the evolution of new and fully sexual species by hybridization and subsequent allopolyploidization in flat-branched clubmosses.

Casuattimines A-N, fourteen new Lycopodium alkaloids from Lycopodiastrum casuarinoides with Cav3.1 channel inhibitory activity.

Two new dimeric Lycopodium alkaloids, casuattimines A and B (1 and 2), along with twelve previously undescribed Lycopodium alkaloids, casuattimines C-N (3-14), and eight known Lycopodium alkaloids, were isolated from Lycopodiastrum casuarinoides. Casuattimines A and B (1 and 2) are the first two ether-linked Lycopodium alkaloid dimers. Casuattimines C and D (3 and 4) are unique Lycopodium alkaloids characterized by a long fatty acid chain. Structural elucidation was achieved through HRESIMS, NMR, and electronic circular dichroism (ECD) calculations. In addition, the absolute configurations of compounds 7, 13, and 14 were determined by single crystal X-ray diffraction. Compounds 1, 2, and 4 demonstrated notable Cav3.1 channel inhibitory activities presenting IC50 values of 10.75 ± 1.02 µM, 9.33 ± 0.79 µM, and 7.14 ± 0.86 µM, respectively. The dynamics of compound 4 against the Cav3.1 channel and preliminary structure-activity relationships of these active Lycopodium alkaloids were also discussed.

Evapotranspiration-linked silica deposition in a basal tracheophyte plant (Lycopodiaceae: Lycopodiella alopecuroides): implications for the evolutionary origins of phytoliths.

Phytoliths, microscopic deposits of hydrated silica within plants, play a myriad of functional roles in extant tracheophytes - yet their evolutionary origins and the original selective pressures leading to their deposition remain poorly understood. To gain new insights into the ancestral condition of tracheophyte phytolith production and function, phytolith content was intensively assayed in a basal, morphologically conserved tracheophyte: the foxtail clubmoss Lycopodiella alopecuroides. Wet ashing was employed to perform phytolith extractions from every major anatomical region of L. alopecuroides. Phytolith occurrence was recorded, alongside abundance, morphometric information, and morphological descriptions. Phytoliths were recovered exclusively from the microphylls, which were apicodistally silicified into multiphytolith aggregates. Phytolith aggregates were larger and more numerous in anatomical regions engaging in greater evapotranspirational activity. The tissue distribution of L. alopecuroides phytoliths is inconsistent with the expectations of proposed adaptive hypotheses of phytolith evolutionary origin. Instead, it is hypothesized that phytoliths may have arisen incidentally in the L. alopecuroides-like ancestral plant, polymerizing from intraplant silicon accumulations arising via bulk flow and 'leaky' cellular micronutrient channels. This basal, nonadaptive phytolith formation model would provide the evolutionary 'raw material' for later modification into the useful, adaptative, phytolith deposits seen in later-diverging plant clades.

Lycopodiastrum casuarinoides: An overview of their phytochemicals, biological activities, structure-activity relationship, biosynthetic pathway and 13C NMR data.

Huperzine A, a lycodine-type alkaloid, exhibits potent inhibitory activity against acetylcholinesterase (AChE) and has been utilized to treat neurodegenerative diseases' symptoms. Lycopodiastrum casuarinoides, a member of the family Lycopodiaceae, is renowned for its lycodine-type alkaloids. Some of these alkaloids show various pharmacological benefits, such as anti-cholinesterase, neuroprotective, and cytotoxic effects. To date, 113 chemical compounds, including seventy-four lycodine-type alkaloids, ten terpenoids, eleven aliphatics, and eighteen other compounds, have been isolated from this plant. In this review, we have discussed phytochemicals and biological activities of the reported compounds of L. casuarinoides. Moreover, structure-activity relationship (SAR), plausible biosynthetic pathways, and 13C nuclear magnetic resonance spectroscopy (13C NMR) data of the lycodine-type alkaloids are also summarized.

Phlegcarines A-C, three Lycopodium alkaloids from Phlegmariurus carinatus (Desv. ex Poir.) Ching.

Three undescribed Lycopodium alkaloids, phlegcarines A-C, along with nine known alkaloids, were isolated from the aerial parts of a gardening clubmoss Phlegmariurus carinatus (Desv. ex Poir.) Ching. Phlegcarine A is an undescribed Lycopodium alkaloid possessing an unprecedented 5/9/6/6 fused-tetracyclic ring system consisting of an oxa-cyclononanone, a piperidine, a methylcyclohaxane and an oxazolidine. Phlegcarine B is the first N-chloromethyl piperidinium Lycopodium alkaloid of (+)-lycoflexine. The semi-synthesis of phlegcarine B was investigated from (+)-fawcettimine. Phlegcarine C, an undescribed epimer of 12-epilycodoline, is a rare lycopodine-type alkaloid with ß-oriented H-4. Transformation of phlegcarine C and lycodoline to 12-epilycopodine N-oxide via keto-enol tautomerization was investigated using m-CPBA. The structural assignments were established through comprehensive spectroscopic techniques and chemical correlations. Phlegcarines A-C were assayed for their anti-acetylcholinesterase activity, but none of them exhibited biological activity more potent than that of huperzine A.

Fungal microbiomes associated with Lycopodiaceae during ecological succession.

Lycopodiaceae species form an early-diverging plant family, characterized by achlorophyllous and subterranean gametophytes that rely on mycorrhizal fungi for their nutrition. Lycopodiaceae often emerge after a disturbance, like in the Hochfeld reserve (Alsace, France) where seven lycopod species appeared on new ski trails following a forest cut. Here, to better understand their ecological dynamic, we conducted a germination experiment of lycopod spores following an anthropogenic disturbance and examined their associated fungi. Only 12% of the samples germinated, and all gametophytes were abundantly colonized by a specific clade of Densosporaceae (Endogonales, Mucoromycotina), which were also present in the roots of lycopod sporophytes, but absent from the ungerminated spores and the roots of surrounding herbaceous plants, suggesting high mycorrhizal specificity in Lycopodiaceae. In addition, ungerminated spores were profusely parasitized by chytrid fungi, also present in the surrounding lycopod gametophytes and sporophytes, which might explain the low spore germination rate. Altogether, the requirement of specific mycorrhizal Mucoromycotina fungi and the high prevalence of parasites may explain why Lycopodiaceae are often rare pioneer species in temperate regions, limited to the first stages of ecological succession. This illustrates the primordial roles that belowground microbes play in aboveground plant dynamics.

Club-mosses (Diphasiastrum, Lycopodiaceae) from the Far East - Introgression and possible cryptic speciation.

Hybridization occurs often in the genus Diphasiastrum (Lycopodiaceae), which corroborates reports for the two other recognized lycophyte families, Isoëtaceae and Selaginellaceae. Here we investigate the case of D. alpinum and D. sitchense from the Russian Far East (Kamchatka). Their hybrid, D. × takedae, was morphologically recognizable in 16 out of 22 accessions showing molecular signatures of hybridization; the remaining accessions displayed the morphology of either D. alpinum (3) or D. sitchense (3). We sequenced markers for chloroplast microsatellites (cp, 175 accessions from Kamchatka) and for the two nuclear markers RPB and LFY (175 and 152 accessions). A selection of 42 accessions, including all hybrid accessions, was analysed via genotyping by sequencing (GBS). We found multiple, but apparently uniparental hybridization, clearly characterized by a deviating group of haplotypes for D. sitchense and all hybrids. All accessions showing molecular signatures of hybridization in nuclear markers revealed the parental haplotype of D. sitchense, however only the LFY marker differentiated between the parent species. GBS, including 69,819 quality-filtered single nucleotid polymorphisms, unambiguously identified the hybrids and revealed introgression to occur. Most of the hybrids were F1, but three turned out to be backcrosses with D. alpinum (one) and with D. sitchense (two). These observations are in contrast to prior findings on three European species and their intermediates where all three hybrids turned out to be independent F1 crosses without evidence of recent backcrossing. In this study, backcrossing was detected, which indicates a limited fertility of the hybrid taxon D. × takedae. A comparison of accessions of Kamchatkian D. alpinum with plants from Europe indicated possible cryptic speciation. Accessions from the Far East had (i) a lower DNA content (7.0 vs. 7.5 pg/2C), (ii) different prevailing cp haplotypes, and (iii) RPB genotypes, and (iv) a clearly different SNP pattern in GBS. Diphasiastrum sitchense and the similar D. nikoënse, for the latter additional accessions from Japan were investigated, appeared as forms of one diverse species, sharing genotypes in both nuclear markers, although chloroplast haplotypes and DNA content show slight variations.

Bioactivity study and metabolic profiling of Colletotrichum alatae LCS1, an endophyte of club moss Lycopodium clavatum L.

Endophytes are silent microbial partners of green plants that ensure hosts' survival in odd conditions. They are known as the factories of multipotent metabolites with diverse bioactivities beneficial to modern pharmaceuticals industry. Endophytic fungi have been screened from a variety of plants and it is the first-time endophytes of club moss is being studied for production of antibacterial and antioxidative compounds. The present study reveals that Lycopodium clavatum L. harbors a potent niche of bioactive endophytic fungi and Colletotrichum alatae LCS1 was the prime producer of antibacterial and antioxidative compounds among them. The minimum inhibitory and bactericidal concentrations of ethyl- acetate culture extract ranged from 15.62 to 250 µg/mL against four Gram negative and three Gram positive microorganisms including methicillin resistant Staphylococcus aureus (ATCC-33591). Bio-autogram based screening followed by Gas chromatographic analysis confirmed the occurrence of 17 bioactive compounds and α-bisabolol is known to be the prime one. Alfa bisabolol is a unique and versatile bioactive essential oil and facilitates variety of functions. Killing kinetics data along with leakage of macromolecules into extracellular environment supports the cidal activity of the antibacterial principles at MBC values. Isolate C. alatae LCS1 was optimized by one variable at a time system coupled with response surface methodology for broad spectrum antibacterial production. The organism yielded maximum response (22.66±0.894 mm of zone of inhibition against MRSA) in 250 mL Erlenmeyer flask containing 50 mL potato dextrose broth supplemented with (g/L) glucose, 7.53; yeast extract concentration, 0.47; NaCl, 0.10 with medium pH 6.46; after 134 hours of incubation at 26°C. Optimized fermentation parameters enhanced antibacterial activity up-to more than 50% than the pre-optimized one (10.33±0.57 mm). Endophytic LCS1 was also efficient in free radical scavenging tested by DPPH, ABTS, H2O2 and FRAP assay with an IC50 values of 23.38±5.32 to 82.873±6.479 µg/mL.

Abietane diterpenoids with neuroprotective activities from Phlegmariurus carinatus.

Two new abietane diterpenoids, phlecarinatone A (1) and phlecarinatone B (2), along with two known analogues (3 and 4), were isolated from Phlegmariurus carinatus. The structures of 1 - 4 were unambiguously elucidated by comprehensive spectroscopic analyses. Abietane diterpenoids were isolated from the plant for the first time. All isolates were tested for their neuroprotective activities against H2O2-induced SH-SY5Y cells injury, and compound 2 showed moderate effect at the concentrations ranging from 5 ∼ 20 µM in vitro assay.

Phlegmacaritones A and B, a Pair of Serratane-Related Triterpenoid Epimers with an Unprecedented Carbon Skeleton from Phlegmariurus carinatus.

A pair of novel serratane-related triterpenoid epimers, phlegmacaritones A (1) and B (2), possessing an unprecedented 15,30-lactone-14,15-seco skeleton, six new serratane-type triterpenoids, phlegmanols G-L (3-5 and 14-16), and 16 known compounds were isolated from the whole plant of Phlegmariurus carinatus. The structures of the new metabolites were established on the basis of comprehensive spectroscopic data analysis and electronic circular dichroism calculations. A possible biosynthetic pathway for phlegmacaritones A (1) and B (2) was proposed. All compounds were submitted to cytological profiling on a cell line derived from a patient with Parkinson's disease. Phlegmacaritone B (2) induced a distinct phenotypic profile with alterations in α-tubulin, mitochondria, and autophagosomal and early endosomal features.

A permineralized Early Cretaceous lycopsid from China and the evolution of crown clubmosses.

Lycopodiaceae are one of three surviving families of lycopsids, a lineage of vascular plants with a fossil history dating to at least the Early Devonian or perhaps the Late Silurian (c. 415 Ma). Many fossils have been linked to crown Lycopodiaceae, but the lack of well-preserved material has hindered definitive recognition of this group in the paleobotanical record. New, exceptionally well-preserved permineralized lycopsid fossils from the Early Cretaceous (125.6 ± 1.0 Ma) of Inner Mongolia, China, were examined in detail using acetate peel and micro-computed tomography techniques. The anatomy of extant Lycopodiaceae was analyzed for comparison using fluorescence microscopy. Phylogenetic relationships of the new fossil to extant Lycopodiaceae were evaluated using parsimony and maximum likelihood analyses. Lycopodicaulis oellgaardii gen. et sp. nov. provides the earliest unequivocal and best-documented evidence of crown Lycopodiaceae and Lycopodioideae, based on anatomically-preserved fossil material. Recognition of Lycopodicaulis in Asia during the Early Cretaceous indicates the presence of crown Lycopodiaceae at this time, and striking similarities of stem anatomy with extant species provide a framework for the understanding of the interaction of branching and vascular anatomy in crown-group lycopsids.

Chemical constituents from Lycopodiella cernua and their anti-inflammatory and cytotoxic activities.

Phytochemical investigation of the whole plants of Lycopodiella cernua resulted in the isolation and identification of three new compounds (1-3), namely lycocernuaside E (1), lycernuic ketone F (2), and lycernuic B (3) and 12 known ones (4-15). Their chemical structures were established based on 1 D/2D NMR spectroscopic and HR-ESI-MS data analyses. Compounds 5, 12, and 13 displayed NO inhibitory effects in LPS-stimulated BV2 cells, with IC50 values of 21.2 ± 1.1, 28.5 ± 1.4, and 21.9 ± 1.1 µM, respectively. In addition, cytotoxic activity of the isolated compounds against MCF7 (breast carcinoma), HepG2 (hepatocarcinoma), and SK-Mel2 (melanoma) cancer cell lines were also reported.

Fractionation of Lycopodiaceae Alkaloids and Evaluation of Their Anticholinesterase and Cytotoxic Activities.

In view of the abundant evidence that Lycopodiaceae alkaloids, including the well-known huperzine A (HupA), are among the potent acetylcholinesterase (AChE) inhibitors, an attempt was made to search for new compounds responsible for this property. For this purpose, three plant species belonging to the Lycopodiaceae family, commonly found in the Euro-Asia region, were subjected to the isolation of bioactive compounds, their identification and subsequent evaluation of their anticholinesterase and cytotoxic activities. Methanolic extracts of two Lycopodium and one Hupezia species were obtained via optimized pressurized liquid extraction (PLE) and then pre-purified using innovative gradient vacuum liquid chromatography (gVLC). For the first time, three sorbents of different porosity packed in polypropylene cartridges and mobile phase systems of different polarity were used to elute the target compounds. This technique proved to be a rapid tool for the obtainment of alkaloid fractions and allowed one to select the appropriate process conditions to yield potent AChE inhibitors in each of the species studied. More than 100 collected fractions were analyzed via HPLC/ESI-QTOF-MS, which enabled one to detect more than 50 compounds, including several new ones previously unreported. Some of them were present in high purity fractions (60-90% of the established purity). TLC bioautography assays proved that the analyzed species are rich sources of AChE inhibitors, but H. selago showed the highest anti-AChE activity. Additionally, the modified silanized silica gel sorbent used allowed one to isolate L. clavatum alkaloids more efficiently using an aqueous reversed-phase solvent system. Furthermore, the tested extracts from the three plant extracts were found to be safe, as they did not exhibit cytotoxicity to skin fibroblasts.

Optimization of Pressurized Liquid Extraction of Lycopodiaceae Alkaloids Obtained from Two Lycopodium Species.

Alkaloids of the Lycopodiaceae family are of great interest to researchers due to their numerous properties and wide applications in medicine. They play a very important role mainly due to their potent antioxidant, antidepressant effects and a reversible ability to inhibit acetylcholinesterase (AChE) enzyme activity. This property is of immense importance due to the growing problem of an increasing number of patients with neurodegenerative diseases in developed countries and a lack of effective and efficient treatment for them. Numerous studies have shown that Lycopodiaceae alkaloids are a rich source of AChE inhibitors. In the obtaining of new therapeutic phytochemicals from plant material, the extraction process and its efficiency is crucial. Therefore, the aim of this work was to optimize the conditions of modern PLE to obtain bioactive alkaloids from two Lycopodium species: L. clavatum L. and L. annotinum L. Five different solvents of different polarity were used for prepared plant extracts in order to compare the alkaloid content in and thereby effectiveness of the entire extraction. PLE parameters were used based on multiple studies conducted that gave the highest alkaloids recovery. Crude extracts were purified using solid-phase extraction (SPE) on Oasis HLB cartridge and examined by HPLC/ESI-QTOF-MS of the highly abundant alkaloids. To the best of our knowledge, this is the first time such high recoveries have been obtained for known Lycopodiaceae alkaloids. The best extraction results of alkaloid-lycopodine were detected in the dichloromethane extract from L. clavatum, where the yield exceeded 45%. The high recovery of annotinine above 40% presented in L. annotinum was noticed in dichloromethane and ethyl acetate extracts. Moreover, chromatograms were obtained with all isolated alkaloids and the best separation and quality of the bands in methanolic extracts. Interestingly, no alkaloid amounts were detected in cyclohexane extracts belonging to the non-polar solvent. These results could be helpful for understanding and optimizing the best conditions for isolating potent AChE inhibitors.

Lycosquarrines A-R, Lycopodium Alkaloids from Phlegmariurus squarrosus.

Eighteen new Lycopodium alkaloids, lycosquarrines A-R (1-18), and eight known alkaloids were isolated from the aerial parts of Phlegmariurus squarrosus. Compounds 1-5 and 19, identified from natural sources for the first time, are uncommon lycopodine-type alkaloids with ß-oriented H-4. Pentacyclic 4 and 5 represent the first examples of 5,12- and 5,11-epoxy Lycopodium alkaloids, respectively, and an epoxide-opening cyclization reaction is suggested to be a key step in their biosynthesis. Compound 18 possesses the same carbon skeleton as carinatine A (22), which was previously reported as a unique Lycopodium alkaloid with a 5/6/6/6 ring system. X-ray crystallographic data analysis was used to determine the absolute configuration of 18, leading to the establishment of the absolute configuration of 22 by comparison of the ECD spectra. An anti-acetylcholinesterase activity assay showed that 11 and 20 exhibited inhibitory activities with IC50 values of 4.2 and 2.1 µM, respectively.

Identification and evolution of C4 photosynthetic pathway genes in plants.

BACKGROUND: NADP-malic enzyme (NAPD-ME), and pyruvate orthophosphate dikinase (PPDK) are important enzymes that participate in C4 photosynthesis. However, the evolutionary history and forces driving evolution of these genes in C4 plants are not completely understood. RESULTS: We identified 162 NADP-ME and 35 PPDK genes in 25 species and constructed respective phylogenetic trees. We classified NADP-ME genes into four branches, A1, A2, B1 and B2, whereas PPDK was classified into two branches in which monocots were in branch I and dicots were in branch II. Analyses of selective pressure on the NAPD-ME and PPDK gene families identified four positively selected sites, including 94H and 196H in the a5 branch of NADP-ME, and 95A and 559E in the e branch of PPDK at posterior probability thresholds of 95%. The positively selected sites were located in the helix and sheet regions. Quantitative RT-PCR (qRT-PCR) analyses revealed that expression levels of 6 NADP-ME and 2 PPDK genes from foxtail millet were up-regulated after exposure to light. CONCLUSION: This study revealed that positively selected sites of NADP-ME and PPDK evolution in C4 plants. It provides information on the classification and positive selection of plant NADP-ME and PPDK genes, and the results should be useful in further research on the evolutionary history of C4 plants.

Root apical meristem diversity and the origin of roots: insights from extant lycophytes.

The independent origin of roots in lycophytes and euphyllophytes has been proposed, mainly based on paleobotanical records. However, the question of how roots evolved within these lineages remains unresolved. Root apical meristem (RAM) organization in lycophytes would provide a clue toward understanding the early evolution of roots. Recently, we examined RAM organization in lycophytes (Lycopodiaceae, Isoetaceae, and Selaginellaceae) in terms of cell division activity and anatomy, comparing RAM among vascular plants. Lycophyte RAM exhibited four organization types (I, II, III, and apical); thus, RAM organization in extant lycophytes was more diverse than expected. Type I RAM contained a region with very low cell division frequency, reminiscent of the quiescent center (QC) in seed plant RAM. Although some euphyllophyte RAMs were structurally similar to types II and III and apical cell-type RAM, lycophyte RAM of types II and III had no QC-like area. These results support the paleobotanical predictions that roots evolved several times in lycophytes, as well as in euphyllophytes. In this review, we also introduce recent findings on RAM organization in extant lycophytes and discuss the origin of roots in vascular plants.

Palhinosides A-H: Flavone Glucosidic Truxinate Esters with Neuroprotective Activities from Palhinhaea cernua.

Palhinosides A-H (1-8), new flavone glucosidic truxinate esters, including ß-truxinate and µ-truxinate forms, were isolated from Palhinhaea cernua. Their structures were elucidated by extensive spectroscopic methods and chemical analyses. The flavone glucoside cyclodimers possess a unique cyclobutane ring in their carbon scaffolds. Compounds 2-7 represent three pairs of stereoisomers (2/3, 4/5, 6/7). The protective effects of 1-8 against the damage of HT-22 cells induced by l-glutamate were evaluated, and compounds 4 and 5 showed better neuroprotective effects than the positive control, Trolox.

A functional twintron, 'zombie' twintrons and a hypermobile group II intron invading itself in plant mitochondria.

The occurrence of group II introns in plant mitochondrial genomes is strikingly different between the six major land plant clades, contrasting their highly conserved counterparts in chloroplast DNA. Their present distribution likely reflects numerous ancient intron gains and losses during early plant evolution before the emergence of seed plants. As a novelty for plant organelles, we here report on five cases of twintrons, introns-within-introns, in the mitogenomes of lycophytes and hornworts. An internal group II intron interrupts an intron-borne maturase of an atp9 intron in Lycopodiaceae, whose splicing precedes splicing of the external intron. An invasive, hypermobile group II intron in cox1, has conquered nine further locations including a previously overlooked sdh3 intron and, most surprisingly, also itself. In those cases, splicing of the external introns does not depend on splicing of the internal introns. Similar cases are identified in the mtDNAs of hornworts. Although disrupting a group I intron-encoded protein in one case, we could not detect splicing of the internal group II intron in this 'mixed' group I/II twintron. We suggest the name 'zombie' twintrons (half-dead, half-alive) for such cases where splicing of external introns does not depend any more on prior splicing of fossilized internal introns.