Ireneusz J. Odrzykoski

Conocephalum salebrosum (Marchantiopsida, Conocephalaceae) - a new Holarctic liverwort species

Detailed analysis of 270 specimens of Conocephalum conicum (L.) Dumort., mainly from the POZW Herbarium revealed some diagnostic differences between two cryptic species originally detected on the basis of isozyme studies. Several diagnostic characters were found in the structure of the archegoniophore, sporophyte and sterile thallus. The most reliable diagnostic features are size and structure of stomatal apparatus of the archegoniophore ‘heads’, type of junction of the air chamber walls with epidermal cells, and the thallus surface details. On the basis of morphological and anatomical diagnostic characters, two formerly cryptic species are recognized following the rules of formal taxonomy. The lectotype of C. conicum preserved in FI was found to possess characteristics of cryptic species C. conicum- species L. Accordingly, the species previously named as C. conicum–species L must bear the name Conocephalum conicum (L.) Dumort. No published name was related to C. conicum – species S, which is therefore described as a new species: Conocephalum salebrosum Szweykowski, Buczkowska & Odrzykoski. Both species are fully described and their diagnostic characters are illustrated. Distribution maps for both species are given.

High levels of genetic variability in the haploid moss Plagiomnium ciliare

Horizontal starch‐gel electrophoresis was used to measure variability at 14 enzyme loci from 13 natural populations of the dioecious moss Plagiomnium ciliare. Overall levels of genetic polymorphism were unexpectedly high for a haploid organism. Using a 1% frequency criterion, 71% of the loci surveyed were polymorphic for the species as a whole. The number of alleles per polymorphic locus for the species as a whole was 2.82 ± 0.34 (mean ± standard error), and mean gene diversity per locus was 0.078 ± 0.035. While total gene diversity (HT = 0.178) was similar to that observed for highly outcrossed diploid plants such as pines, the variance within (HS = 0.098 ± 0.027) and among (DST = 0.080 ± 0.033) populations was more evenly distributed than that reported for populations of conifers. Genetic distances between populations ranged from 0.0002 to 0.2064, with mosses from the Piedmont region of the southeastern United States showing less differentiation among populations than did mosses from the Appalachian Mountains. Gene diversity was much reduced in populations from disturbed, secondary forests in the Piedmont (0.058 ± 0.018) relative to those from minimally disturbed, primary forests in the mountains (0.146 ± 0.048). Intensive sampling within populations revealed heterogeneity even within small (5 × 5 cm) clumps. The discovery of high levels of genetic variability in a plant with a dominant haploid life cycle challenges the traditional view of bryophytes as a genetically depauperate group. Multipleniche selection is proposed as a possible explanation for this anomaly, but the data are also consistent with the view that allozyme polymorphisms are selectively neutral.

Genetic differentiation without concordant morphological divergence in the thallose liverwort Conocephalum conicum

Allozyme variation was examined at 33 enzyme loci for 96 populations from throughout the geographical range ofConocephalum conicum (L.)Dum. (Marchantiales, Hepaticae). Variation was partitioned into five discrete groups, suggesting that this morphologically defined species is genetically heterogeneous. The degree of differentiation among these groups, as measured by genetic distance, is as large as is commonly reported between different vascular plant species, and much larger than that between conspecific populations. In Europe, two of these genetically distinct groups (S and L) occur sympatrically, but apparently do not interbreed. Geographical ranges of the other three groups (A, C, and J) are probably allopatric with the exception that the range of S, the most genetically divergent group, overlaps with group A in N. America. It is suggested that these five natural assemblages constitute different sibling species.

Isozyme Evidence of Reticulate Evolution in Mosses: Plagiomnium medium is an Allopolyploid of P. ellipticum x P. insigne

Electrophoretic data from 44 natural populations show that Plagiomnium medium is an allopolyploid, whose genome can be reconstructed by merely combining present-day variation of its extant haploid progenitors, P. ellipticum and P. insigne. Because the progenitor species are well- differentiated genetically, P. medium shows fixed heterozygosity in about 1/3 of the 23 loci assayed. High levels of polymorphism in P. ellipticum provide the opportunity to detect multiple origins of P. medium, which evidence shows must have originated from at least four separate hybridization events. We detected only one allele unique to P. medium and only one possible instance of gene silencing. This suggests a relatively recent origin of the allopolyploid. It may also suggest that natural selection does not favor silencing back to a haploid condition. Apparently, cross-fertilization is frequent in P. medium and generates a large number of genetic recombinants across loci. Thus, P. medium enjoys a favorable genetic situation in which there is fixed, intralocus heterozygosity combined with the potential for extensive interlocus recombination. We speculate that P. medium may have been launched as a species during the Pleistocene, when the ranges of the circumboreal, continental P. ellipticum and the coastal, Pacific Northwest endemic P. insigne may have overlapped broadly. Their present-day ranges include few sympatric populations and would not permit hy- bridization along a broad front. Contrary to conventional views, it appears that reticulate evolution occurs in bryophytes, just as it does in other land plants.

The hybrid origin of the polyploid liverwort Pellia borealis

Isozyme markers were used to investigate the origin of the polyploid liverwort, Pellia borealis (gametophytic n=18), which was believed to represent an autopolyploid form of Pellia epiphylla (n=9). Enzyme variation was studied in four taxa: polyploid P. borealis, two recently discovered sibling species of P. epiphylla complex, and the closely related P. neesiana (n=9). Gametophytes of the polyploid showed a complex electrophoretic phenotype for three diagnostic enzymes (DIA1, MPI1 and ACO) in contrast to simple pattern in all haploid taxa. It was postulated that the pattern found in the polyploid represents a ‘fixed heterozygous’ phenotype resulting from allopolyploidy. Alleles present in the polyploid were found (with only one exception) in the two sibling species of the P. epiphylla complex, suggesting that they are the parents of the allopolyploid. Pellia neesiana was excluded as a donor of either of the genomes. Variation in the polyploid suggests at least three separate origins of P. borealis.

Phylogeny of the European species of the genus Pellia (Hepaticae; Metzgeriales) based on the molecular data from nuclear tRNA(Leu)(CAA) intergenic sequences.

Species of the genus Pellia are similar to such an extent that their proper recognition based on morphological and anatomical features is difficult. To solve this problem isozyme methods were developed. As a result of these studies new cryptic species were recognized and new hypotheses concerning phylogenetic relationship in Pellia were formed. To examine hypotheses derived from isozyme data we decided to study species of the genus Pellia at the DNA level. Total DNA from all Polish Pellia species was isolated. The taxonomic and phylogenetic relationships in Pellia were examined using intergenic spacer sequences between nuclear tRNA(Leu) genes organised in tandem arrays. PCR (polymerase chain reaction) amplification of tRNA(Leu) gene spacers produced fragments of different sizes in all species and their restriction analysis showed species-specific patterns. PCR products were cloned and sequenced. Nucleotide sequence data were used for phylogenetic reconstruction. Sequence analysis confirmed previous results based on isozyme studies that P. endiviifolia- species A and species B as well as P. epiphylla- species S and species N (having different isozyme multilocus genotypes) are separate sibling species. Our results also confirmed the allopolyploid character of the only polyploid species in Pellia, P. borealis which was formed by hybridization of P. epiphylla- species N and P. epiphylla- species S cryptic species.

Adaptive evolution of rbcL in Conocephalum (Hepaticae, bryophytes)

An excess of nonsynonymous substitutions over synonymous ones has been regarded as an important indicator of adaptive evolution or positive selection at the molecular level. We now report such a case for rbcL sequences among cryptic species in Conocephalum (Hepaticae, Bryophytes). This finding can be regarded as evidence of adaptive evolution in several cryptic species (especially in F and JN types) within the genus. Bryophytes are small land plants with simple morphology. We can therefore expect the existence of several biologically distinct units or cryptic species within each morphological species. In our previous study, we found three rbcL types in Asian Conocephalum japonicum (Thunb.) Grolle and also found evidence strongly suggesting that the three types are reproductively isolated cryptic species. Additionally, we examined rbcL sequence variation in six cryptic species of C. conicum (L.) Dumort. previously recognized by allozyme analyses. As a result, we were able to discriminate the six cryptic species based only on their rbcL sequences. We were able to show that rbcL sequence variation is also useful in finding cryptic species of C. conicum.

Organellar inheritance in the allopolyploid moss Rhizomnium pseudopunctatum

Earlier isozyme studies have proved that the moss Rhizomnium pseudopunctatum is an allopolyploid species whose progenitors are the haploid species R. magnifolium and R. gracile. A sequence comparison of chloroplast tRNA L e u (UAA) and tRNA G l y (UCC) gene introns, as well as mitochondrial fragments of nad5 and nad4 gene introns and exons in all three species reveals that the nucleotide sequences studied are almost identical in R. magnifolium and R. pseudopunctatum but differ in R. gracile. Both chloroplasts and mitochondria of R. pseudopunctatum were therefore probably inherited from one parent: R. magnifolium. To our knowledge, this is the first report of uniparental transmission of organelles in mosses.

Identification of cryptic species within liverwort Conocephalum conicum based on the volatile components.

Cryptic speciation refers to cases where species are clearly genetically differentiated, but show little or no morphological differences. This study investigated whether volatile components can be used to distinguish the cryptic species within the Conocephalum conicum complex. Thirteen samples were included in this study representing four cryptic species (A, F, J, L) and the recently described Conocephalum salebrosum (formerly cryptic species S) detected in a worldwide collection. Results received from GC-MS analyses showed some differences between samples, indicating the existence of chemical polymorphism. It is evident from both statistical analyses (CA and PCA), that cryptic species L appears to be the most differentiated species, with the volatile components not significantly shared with the rest of the types. The most characteristic compound of this type is the sesquiterpene alcohol, conocephalenol. Distinction between other examined cryptic species were also indicated. However species F and J, as well as species A and C. salebrosum, share a great similarity in their volatile composition and are grouped together in both CA and PCA. Despite their similarities, we were able to find chemical markers which can describe each species. Cubebol is characteristic for C. salebrosum, while cryptic species A produces (E)-methylcinnamate. The presence of a large amount of the monoterpene hydrocarbon, sabinene, is characteristic for cryptic species J. In one of the analyzed specimens belonging to the cryptic species F, a large amount of cyclocolorenone was detected. Chemical differentiation was correlated with the geographical distribution of the analyzed samples.

Isozyme evidence regarding the nature of polyploidy in the moss genus Cinclidium (Mniaceae)

Abstract Genetic markers from horizontal starch gel electrophoresis show clearly that boreal-arctic C. stygium, a bisexual polyploid with n  =  14, is closely related to C. arcticum and C. latifolium, unisexual haploids with n  =  7. One locus of C. stygium shows fixed heterozygosity, suggesting an allopolyploid origin with these two haploids as progenitors. Two other loci, which are heterozygous in >90% of all plants screened, also support this view. It is also possible, however, that C. stygium is an autopolyploid, most likely of C. arcticum, in which meiosis has become regularized. Despite strong morphological differences between them, we detected only one fixed allelic difference between C. arcticum and C. latifolium. For C. subrotundum, another bisexual polyploid with n  =  14, the evidence for allopolyploidy is very strong, with seven fixed heterozygous loci out of 17 scored. One of the lineages involved is similar to that of C. arcticum and C. latifolium, but the other is highly divergent from any known extant species of Cinclidium. It is quite possible, however, that additional, highly divergent species exist or, at least, once existed, as our screening of populations from Alaska turned up a new, highly genetically distinct species that experts have previously mistakenly referred to C. latifolium.