TrnL-trnfF cpDNA polymorphism in some representatives of the genus Betula

We present preliminary results of the trnL-trnF cpDNA region analysis for some representatives of the g. Betula, out of which B. procurva can be considered the most interesting. The disjunctive range of this species is confined to the mountain system in southeast Central Asia (Pamir-Alai). The occurrence of the birch is isolated from the main range in the mountainous part of the Urals, in the boreal forests zone and in the Trans-Ural forest-steppe. Due to high interspecific hybridization and population variability of the g. Betula, doubts were raised about the correct identification of the representatives of B. procurva. The studied genetic variability and population structure of B. procurva, B. nana, B. pubescens, B. turkestanica, B. tianshanica and B. pendula species indicate active introgression and hybridization processes. Polymorphism in all groups is significantly reduced, increasing slightly when comparing the most distant groups. The matching of the B. procurva cpDNA haplotypes (GBS) with B. pendula, B. nana, and B. pubescens shows that this trnL-trnF cpDNA site cannot be used for molecular identification of birch species by barcoding as a single marker, but this marker use is possible for determination of certain B. procurva population. Based on the studied cpDNA region (trnL-F) we found a clear geographic subdivision in B. procurva populations of the Trans-Urals and Central Asia,.

A new method to quantify atmospheric Poaceae pollen DNA based on the trnT-F cpDNA region

Background Pollen, mold spores, bacteria and viruses are the main biological substances in the atmosphere causing allergic symptoms and disease. Distinguishing pollen and spores is quite time consuming and requires a trained expert. There is a different approach to identification of these substances such as microscopic analysis. However, DNA based identification of these is becoming popular recently. Objective We evaluated the correlation between the quantity of DNA, which was amplified using trnT-F cpDNA specific primers in samples obtained from a high volume air sampler (HVAS), and concentration of Poaceae pollen collected with a Burkard trap. Materials and methods Here, we present a method for identifying and quantifying airborne Poaceae pollen using a single step polymerase chain reaction (PCR) technique. Forty daily air samples were collected by HVAS. The method was optimised using two different methods (M1 and M2) and the trnT-F cpDNA region was amplified using a Poaceae specific primer pair. The correlation between the quantity of DNA and pollen concentration was tested using R statistical programming language. Results Although a significant correlation was obtained between the M1 and M2 methods (R2=0.655, p<0.01), the M2 method was more correlated with pollen concentration. The correlation between pollen and DNA content changed due to episodes that were observed during the pollen season. DNA concentrations from the PCR data were significantly correlated with pollen concentrations determined by light microscopy (R2=0.767, p<0.01) in episode II using the M2 method and during the entire season (R2=0.469, p<0.01) using M2. Conclusions The M2 method correctly identified Poaceae pollen in mixed air samples from Zonguldak Province. The non-coding trnT-F cpDNA region was used for the first time in aerobiological samples to identify Poaceae pollen. Use of this method that does not require DNA extraction may be a crucial step for real-time pollen monitoring devices to be developed in the future. The correlation strength between pollen and amplified DNA content could be improved using a sampler that has a lower absorption rate, and a more sensitive technique, such as qPCR.

A botanical mystery solved by phylogenetic analysis of botanical garden collections: the rediscovery of the presumed-extinct Dracaena umbraculifera

Extinction is the complete loss of a species, but the accuracy of that status depends on the overall information about the species. Dracaena umbraculifera was described in 1797 from a cultivated plant attributed to Mauritius, but repeated surveys failed to relocate it and it was categorized as Extinct on the IUCN Red List. However, several individuals labelled as D. umbraculifera grow in botanical gardens, suggesting that the species’ IUCN status may be inaccurate. The goal of this study was to understand (1) where D. umbraculifera originated, (2) which species are its close relatives, (3) whether it is extinct, and (4) the identity of the botanical garden accessions and whether they have conservation value. We sequenced a cpDNA region of Dracaena from Mauritius, botanical garden accessions labelled as D. umbraculifera, and individuals confirmed to be D. umbraculifera based on morphology, one of which is a living plant in a private garden. We included GenBank accessions of Dracaena from Madagascar and other locations and reconstructed the phylogeny using Bayesian and parsimony approaches. Phylogenies indicated that D. umbraculifera is more closely related to Dracaena reflexa from Madagascar than to Mauritian Dracaena. As anecdotal information indicated that the living D. umbraculifera originated from Madagascar, we conducted field expeditions there and located five wild populations; the species’ IUCN status should therefore be Critically Endangered because < 50 wild individuals remain. Although the identity of many botanical garden samples remains unresolved, this study highlights the importance of living collections for facilitating new discoveries and the importance of documenting and conserving the flora of Madagascar.

Short Communication: Strong genetic differentiation of the endemic rosin-producing tree Styrax sumatrana (Styracaceae) in North Sumatra, Indonesia

Rachmat HH, Susilowati A, Elfiati D, Hartini KS, Faradillah WN. 2017. Short Communication: Strong genetic differentiation of the endemic rosin-producing tree Styrax sumatrana (Styracaceae) in North Sumatra, Indonesia. Biodiversitas 18: 1331-1335. Styrax sumatrana is an economically important rosin-producing tree endemic to North Sumatra, Indonesia. Distribution of this species is very limited, and the high rate of forest degradation in Sumatra is increasing the necessity for conservation. To quantify genetic variation and population structuring, we collected individuals from 3 populations namely Pakpak Bharat, Humbang Hasundutan (Humbahas) and Tapanuli Utara in which each of the population was represented by 10 individuals. However, the successful rate of amplification was varied among populations and for later analysis, we only took an account those sequences showing clear electropherograms and disposed those which showed ambiguity. We sequenced trnL-trnF chloroplast DNA (cpDNA) region yielded 941 bp after alignment. The trnL-trnF assigned the species into 4 haplotypes in which Pakpak Bharat was differentiated significantly and not shared any similar haplotypes with two others populations. Humbahas and Tapanuli Utara was shared one common haplotype. Mean nucleotide diversity at silent sites ranged 0 - 3.33 x 10-3, while nucleotide diversity at non-synonymous site ranged 0 - 5.9 x 10-4. Strong genetic differentiation was also found among 3 origin populations, with the highest pairwise genetic differentiation found on Pakpak Bharat and Tapanuli Utara (FST= 0.80952). Clear and apparent genetic structuring was possibly caused by geographical barriers such as highland and mountain ranges (Bukit Barisan mountain ranges) which acted as effective barriers to gene flow among population. The findings suggest that conservation efforts should focus on every population because each of the population maintains distinct genetic identity.

PCR-RFLP analysis of cpDNA in Gigantochloa scortechinii (Poaceae: Bambuseae) in Peninsular Malaysia and implications for the use of cpDNA markers in systematic studies

Gigantochloa is a paleotropical woody bamboo genus that has been widely cultivated in SE Asia because of its usefulness. Recent studies have shown that species of this genus enter into an introgression complex with other genera of the same subtribe Bambusinae. Within G. scortechinii, a common species indigenous to Malay Peninsula and common in Peninsular Malaysia, two distinct chloroplast DNA (cpDNA) lineages, the Gombak- and Langat-type, were recovered. We report the development of a PCR-based restriction fragment length polymorphism (RFLP) marker for depicting the genetic differentiation in G. scortechinii based on cpDNA. We determined a cpDNA region and its corresponding restriction enzyme which can produce different RFLP profiles for the two cpDNA lineages. Our design was verified with empirical studies. The Gombak-type was the dominant cpDNA genotype for G. scortechinii in Peninsular Malaysia. Implications for the continued use of cpDNA markers in systematic studies are discussed.

Development of SCAR Markers for Species Identification in the Genus Shorea (Dipterocarpaceae)

The development of molecular markers unambiguously distinguishing groups at different taxonomic levels has numerous forensic applications. The identification of tropical timber is of particular interest in this context. We describe the development of SCAR (Sequence Characterized Amplified Region) markers for forensic applications taking the example of two closely related species of the tropical tree family Shorea (Dipterocarpaceae). Two AFLP (Amplified Fragment Length Polymorphism) fragments have been described earlier showing strong differentiation between S. leprosula and S. parvifolia. The AFLP markers were isolated from a gel, re-amplified, cloned and sequenced. Primer sets were designed from these sequences and AFLP fragments were converted into SCAR markers. The SCAR markers and PCR-RFLP markers of the chloroplast region trnLF digested with HinfI were used to screen in total 557 samples of S. parvifolia and S. leprosula from nineteen widely separated populations in Indonesia. Complete genetic differentiation between species was observed based on the putatively nuclear SCAR marker and the PCR-RFLP of the cpDNA region. We found a good agreement between leaf morphological variation and species identification based on both marker types and no indication for interspecific hybridization.

Spontaneous Hybridization between Pinus sylvestris L. and P. mugo Turra in Slovakia

Molecular evidence for spontaneous hybridization between Pinus sylvestris L. and P. mugo Turra in the putative hybrid swarm populations of the species in Slovakia was provided based on PCR-RFLP analysis of the cpDNA trnV-trnH region. Species-specific restriction profiles generated by Hinf I digests of the cpDNA products reliably identified P. sylvestris and P. mugo haplotypes of the embryos from open pollination. Simultaneous analysis of the respective cpDNA region in megagametophytes and embryos of individual seeds along with needles of a given maternal tree has enabled to score either the P. sylvestris or P. mugo haplotypes in the embryos illustrating hybridization patterns between the two species. Data obtained in this way indicate a relatively extensive hybridization which takes place between P. sylvestris and P. mugo. The extent of hybridization varied among populations as evidenced by the 41.1-58.7% proportion of hybrid embryos registered on the locality Habovka, and by the 8.3% and 2.7% proportions of hybrid embryos on the localities Tisovnica and Sucha Hora, respectively. The approach itself is recommended as a convenient method for monitoring the hybridization patterns in sympatric zones of the studied pine species.