Effects of initial density,nutrient, and water level regime on the seedling survival and growth of <Emphasis Type="Italic">Typha orientalis</Emphasis> Presl

Seedlings’ successful establishment is of importance in the preemption process of pioneers in wetlands. Although Typha orientalis Presl has been reported as a pioneer in Asia countries, studies on the seedling phase of T. orientalis are not available yet. A mesocosm experiment was conducted to understand the effects of biotic (initial density) and abiotic (nutrient and water level regime) factors on the seedling survival and growth of T. orientalis. Most seedlings survived under low initial density (93.8%) and eutrophic (95.5%) rather than high initial density (64.3%) and ombrotrophic (62.5%). Seedlings under low initial density, eutrophic, and flooded conditions showed relatively higher growth in shoot height. The final number of ramets showed an adverse tendency compared to the survival rate and shoot height particularly depending on the water level regime. T. orientalis compensated its biomass production with producing less but longer shoots under the flooded condition, indicating the phenotypic plasticity of T. orientalis as a deep water species. However, the compensation seemed to be guaranteed only under the condition of sufficient nutrients. Asian T. orientalis seemed not to be a pioneer but a weak-competitor not only in mature plant stage but also in juvenile seedling stage unless sufficient nutrients are guaranteed.     

Using oxygen stable isotopes to quantify ecosystem metabolism in northern lakes

In remote regions of the world, whole lake metabolic estimates are scarce, largely because long incubations, intensive sampling and deployment of monitoring equipment are impractical. The oxygen isotope (δ¹⁸O) mass balance approach represents a simple and efficient alternative to measure whole-lake gross primary production (GPP) and respiration (R) from a single point sample, yet this option has not been extensively explored in habitats such as remote northern lakes. Here, we explored the applicability of the method using a sensitivity analysis on simulated data, showing that in large, heterotrophic (i.e., R > GPP) lakes, model outputs are sensitive to input terms for isotopic fractionation and air–water gas exchange. Despite these sensitivities, field applications of the δ¹⁸O method generated promising results that were generally consistent with parallel, free-water diel DO metabolic modelling, but greater than in vitro incubation measurements. The isotopic approach captured both wide-ranging metabolic conditions in in situ experimental mesocosms, and the seasonal trends in GPP and R in a shallow, dystrophic lake. In a clearer, deeper heterotrophic lake, the isotope approach integrated a fraction of metalimnetic metabolism missed by diel DO metabolic estimates. Overall, metalimnetic contributions to surface δ¹⁸O–DO dynamics had the greatest impact on model outputs, but with accurate information on air–water gas exchange, mixing depth, and the vertical DO and light regime of a given system, these effects can be accounted for and the isotopic approach can yield well constrained, spatio-temporally integrated rates of GPP and R. The approach is clearly suitable for use in oligo- and mesotrophic lakes, especially in remote regions where sampling is logistically difficult.     

Responses of <Emphasis Type="Italic">Typha orientalis</Emphasis> Roots to Pb<Superscript>2+</Superscript> Stress

To investigate phytoremediation potential of Typha orientalis Presl in removing Pb²⁺ from polluted water, relevant experiments were conducted to evaluate responses activated by Pb²⁺ (0.25–2 mM) in T. orientalis roots over a period of ten days. Pb contents in subcellular fractions decreased in the following order: cell wall > organelle > soluble fraction. Most of Pb was located in cell wall and membrane system. Contents of K and Ca declined in T. orientalis roots under Pb²⁺ stress, but an opposite effect was noted for some mineral elements (Mg, Cu, Zn, and Fe). H₂O₂ level increased in a concentration-dependent manner, which induced oxidative stress. However, significant reduction in levels of O ₂ ^·? and malondialdehyde (MDA) were observed in all Pb²⁺ treatment groups. Findings indicated toxicity of Pb²⁺ to T. orientalis in terms of inducing oxidative stress and causing imbalance in mineral elements. However, T. orientalis also resisted Pb²⁺-induced damage through isolation mechanism, which involves cell wall and membrane systems.     

Non-congruent geographic patterns of genetic divergence across European species of <Emphasis Type="Italic">Branchinecta</Emphasis> (Anostraca: Branchinectidae)

Three of the five European species of Branchinecta have a disjunct distribution. In this study, we analyze populations of B. ferox and B. orientalis for mitochondrial (cox1) and nuclear (ITS2) molecular markers. We compare intraspecific genetic divergences between geographically distant populations of B. orientalis, from its only known Spanish population (originally described as B. cervantesi) and from a Hungarian population (assigned to B. orientalis since its discovery), with data from two relatively close Iberian populations of B. ferox. Results indicate that isolation between B. ferox and B. orientalis clades is ancient, and that the clade including the two Iberian populations of B. ferox is geographically structured. Conversely, Iberian and Hungarian populations of B. orientalis do not show geographical structure for the mitochondrial fragment. Lack of geographic structure coupled with very low genetic distances indicates that current Iberian and Hungarian populations of B. orientalis originated from a common population stock, and that the time elapsed since their separation has not been long enough to render the clades reciprocally monophyletic. We hypothesize that colonization of the Iberian Peninsula by B. orientalis is probably the consequence of a single recent dispersal event, and consequently we confirm the synonymy between B. cervantesi and B. orientalis.     

Time series forecasting model for fisheries in Chilika lagoon (a Ramsar site, 1981), Odisha,India: a case study

Chilika, a Ramsar site and the largest brackish water lagoon in Asia, is situated in East Coast of India, endowed with rich fisheries resources. In this study, SARIMAX fisheries forecasting model was developed by using seasonal ARIMA (Auto Regressive Integrated Moving Average) model with three external physicochemical factors (factor 1 was dominated by the combined effect of salinity and temperature and factor 2 and factor 3 were dominated by alkalinity and transparency) in Chilika. Monthly fish catch data and physico-chemical parameters of water from 2001–2002 to 2015–2016 was used to develop model. The results showed SARIMAX model; SARIMA (1,0,0)(2,0,0)₁₂ with factor 1, factor 2 and factor 3 was the best fitted model for the fish catch in Chilika. The factor 1 was found to be positive influence on catch at 10% level of significance (p = 0.089) while, factor 2 and factor 3 were found to be insignificant. The developed SARIMAX model was validated with actual annual fish catch for the years 2011–2015 with prediction error 3–7%. Further, the developed SARIMAX model was used to forecast fish catch for the period April 2016 to March 2018 indicating increasing 10% present catch in the lagoon. The developed SARIMAX model in the present case study is of the first time to forecast and visualise the positive influence of salinity and temperature on the fish catch in the Chilika lagoon.     

A new species of labrid fish <Emphasis Type="Italic">Oxycheilinus samurai</Emphasis> from the western Pacific Ocean

A new species of labrid fish Oxycheilinus samurai sp. nov. is described on the basis of five specimens from the Ryukyu Islands, Japan, and Panay Island, the Philippines. The new species is most similar to the Indo-West Pacific species Oxycheilinus orientalis in having a slender compressed body, 12 pectoral-fin rays, a blackish blotch around the anterior portion of the lateral-line anterior series, and a dark blotch basally on the membrane between the first and second dorsal-fin spines. However, O. samurai can be distinguished from O. orientalis by the following combination of characters: gill rakers 4 + 7–8; snout rounded with maximum circumference 49.8–63.5 % of standard length (SL); interorbital width 7.7–9.2 % of SL; caudal-peduncle depth 13.8–15.3 % of SL; posterior margin of caudal fin white; black or dark red area just before white posterior margin of caudal fin; poorly defined blackish blotches on inner surface of opercle in preserved specimens.     

Population development of the predatory mite <Emphasis Type="Italic">Amblydromalus limonicus</Emphasis> is modulated by habitat dispersion,diet and density of conspecifics

Habitat dispersion, diet and density can influence the per capita population growth of predators, and dispersed habitat can provide a spatial refuge that reduces the possibility of cannibalism among predators, thereby increasing their realized population growth rate. We tested the influence of variable habitat dispersion (dispersed patches, general patches and aggregated patches), two diets (Typha orientalis pollen and Ephestia kuehniella eggs) and initial predator density—one or two Amblydromalus limonicus (Garman and McGregor) (Acari: Phytoseiidae) founder females—on the population growth of A. limonicus in 7 days. Dispersed patches resulted in a higher total number of A. limonicus than the other two types of habitat dispersion from days 3–7 when fed on either of the diets, and started with either one or two A. limonicus females. Compared with E. kuehniella eggs, T. orientalis pollen resulted in more A. limonicus regardless of one or two founder females. Compared with two founder females, beginning with one founder female on pollen produced significantly more predatory mite females in dispersed and aggregated patches. A four-way ANOVA showed that the main effects indicated that habitat, diet, density, and time significantly influenced the number of immature and female A. limonicus. Significant interactions between habitat dispersion and diet were detected on immature and female A. limonicus. Our findings suggest that increasing the dispersion of artificial shelters on crop leaves may stimulate the control efficiency of predators in greenhouses. Furthermore, T. orientalis pollen provides a high nutritional quality supplemental diet that could enhance the ability of A. limonicus to control pests.     

Effects of flooding regime on wetland plant growth and species dominance in a mesocosm experiment

Flooding regimes are a primary influence on the wetland plant community. Human-induced disturbance often changes the duration and frequency of flooding in wetlands, and has a marked influence on wetland plant composition and viability. Comprehensive studies of the environmental thresholds of wetland plants are required for the development of proper practices for wetland management and restoration after hydrological disturbance. This study provides a quantitative assessment of the establishment, growth, and community shifts in dominance of three emergent plant species (Scirpus tabernaemontani, Typha orientalis, and Zizania latifolia) typical of South Korean wetlands, under five hydrological regimes (waterlogged, low-level standing water, high-level standing water, intensive periodic flooding, and intermittent flooding) over four growing seasons. A mesocosm experiment was conducted in the campus of Seoul National University, South Korea. The number and biomass of shoots of Z. latifolia responded positively to increased water level and flooding frequency, while that of the other plants did not. Zizania latifolia outcompeted S. tabernaemontani and T. orientalis irrespective of hydrological regime. This study suggests that Z. latifolia can outcompete the other two macrophytes in the field. This study will improve our ability to predict the dynamics of wetland vegetation and so facilitate the formulation of wetland management and restoration strategies.     

Fungal elicitors stimulate biomass and active ingredients accumulation in <Emphasis Type="Italic">Dendrobium catenatum</Emphasis> plantlets

Endophytic fungi have been widely used as biotic elicitors to stimulate the growth and production of metabolites in plant cells, tissues and organ cultures. Here, mycelium extract (ME), supernatant liquor (SL), ethanol sediment (ES) and protein-polysaccharide fraction (PPF) were prepared from four endophytic fungi, DO14 (Pestalotiopsis sp.), DO18 (Talaromyces sp.), DO19 (Xylariaceae sp.) and DO120 (Hypoxylon sp.), and applied to their host Dendrobium catenatum. After 8 weeks of co-culturing, ME, ES and PPF exhibited strong stimulation on biomass yields and contents of active ingredients. Among the three elicitors, PPF was found to be the active constituent responsible for the enhanced biomass and active ingredients in D. catenatum. Under the treatment of 240 mg/L PPF from DO14, we achieved maximum stem fresh weight (FW) and leaf FW. However, to maximize the productions of polysaccharides, naringenin and schaftoside one need only 60 mg/L of PPF from DO14. PPF from DO18, DO19 and DO120 showed different effects. Under 30 mg/L treatment, the ethanol extractives, total flavonoids and total phenols contents increased most. These results indicate that fungal elicitor PPFs can be used for industrial production of high quality D. catenatum seedlings and may be served as a broad microbial fertilizer resource for other plant growth.     

Precise feeding of probiotics in the treatment of edwardsiellosis by accurate estimation of <Emphasis Type="Italic">Edwardsiella tarda</Emphasis>

Edwardsiella tarda is one of the leading fish pathogens for the aquaculture industry. To realize efficient disease control of edwardsiellosis, a predictive model for E. tarda in seawater was developed. The modified logistic model was used to regress the growth curves of E. tarda JN at five different temperatures (range from 10 to 30 °C) and four organic nutrient concentrations (range from 5 to 40 mg l^?1 measured by chemical oxygen demand (COD)). The modeling effects of temperature and COD on the specific growth rate (μ) were developed by square-root model and saturation-growth rate model, respectively. The growth model was validated in turbot aquaculture tanks by estimating the dynamics of inoculated E. tarda. The accurate feeding of probiotic Bacillus pumilus strain H2 was calculated based on the estimation of E. tarda. Results showed that the logistic model produced a good fit to the growth curves of E. tarda JN (average R²?=?0.962). The overall predictions based on above models agreed well with the growth curve of E. tarda JN observed by plate counting in the validation tests (average Af?=?1.16; average Bf?=?1.32). The use of predicted amount of B. pumilus (5.66 log CFU ml^?1) successfully prevent the deterioration of disease for turbot with 13.3% mortality rate in a recirculating aquaculture system (RAS), while the feeding of 0 and 3.0 log CFU ml^?1 of B. pumilus resulted in 53.7 and 75.3% of turbot mortality rate, respectively. In conclusion, accurate estimation of E. tarda realized the precise feeding of probiotics, which successfully prevent the rapid progression of the edwardsiellosis.     

Chlorophyll Content as an Indicator of the Modern (2015–2016) Trophic State of Volga River Reservoirs

Data on the chlorophyll a (Chl a) content in water of the Volga River reservoirs sampled at 64–73 sampling stations in the summer period of 2015 and 2016, analyzed using the standard spectrophotometric method, are presented. Chl a content varied from a minimum of 0.9–3.0 to a maximum of 7.6–32.1 μg/L in June 2016, from 2.1–15.5 to 9.4–86 μg/L in August 2015, and from 1.1–6.7 to 15.1–62.7 μg/L in August 2016. A local increase in Chl a occurred in the near-dam areas of the reservoirs, as well as downstream of the confluence with tributaries. The highest Chl a concentrations were constantly recorded in the highly eutrophic Shoshinskii Reach in the Ivankovo Reservoir and at the Oka River mouth in the Cheboksary Reservoir. The average values of Chl a concentrations make it possible to classify the Ivankovo, Uglich, Rybinsk, and Cheboksary reservoirs as eutrophic; the Gorky and Kuibyshev reservoirs as moderately eutrophic; and the Saratov and Volgograd reservoirs as mesotrophic.     

Seasonal dynamics of ammonia-oxidizing microorganisms in freshwater aquaculture ponds

An annual investigation into the abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in fresh water aquaculture ponds was performed by quantitative PCR of the amoA gene. The results showed that AOB were the main ammonia-oxidizing microorganisms in water, and significantly higher copy numbers of the AOB amoA gene were observed in the summer (Aug 2012), while no significant differences were detected among the other three seasons. AOA showed low abundances throughout the year. The predominance of AOB in aquaculture water was suggested to be related to photoinhibition. Both the AOB and AOA amoA genes in aquaculture pond sediments showed typical seasonal patterns. The maximum density of AOB was observed in the autumn (Nov 2012) and winter (Jan 2013), while the maximum density of AOA was observed in winter. The minimum densities of both AOA and AOB occurred in the summer. The concentration of the AOA amoA gene was higher than that of the AOB amoA gene in sediments by almost one order of magnitude, which indicates that AOA are the dominant ammonia-oxidizing microorganisms in the aquaculture pond sediments. Dissolved oxygen is suggested to be the key factor determining the predominance of AOA in pond sediments.     

Evaluation and comparison of the genetic structure of <Emphasis Type="Italic">Bunias orientalis</Emphasis> populations in their native range and two non-native ranges

We studied the invasive warty cabbage Bunias orientalis (Brassicaceae) in three geographically distinct areas. Using inter-simple sequence repeat fingerprinting, we analyzed warty cabbages, including non-native populations, from the eastern Baltic and western Siberian regions and native populations from southwestern Russia. The eastern Baltic region and western Siberia represent the two opposite directions of B. orientalis spread in climatically different zones. The genetic structures of the native and non-native B. orientalis populations were assessed through analysis of molecular variance (AMOVA) and the Bayesian clustering method and by determining the main measures of genetic diversity. AMOVA revealed considerable population differentiation in both the native and invasive ranges. Our results did not indicate a decrease in genetic diversity in the non-native populations of B. orientalis. Similar measures of genetic diversity and genetic structure were determined in the invasive populations in two geographically and ecologically distinct, non-native regions located in Europe and Asia. In both of these regions, higher genetic diversity was detected in the non-native populations than in the native region populations, which may be due to multiple introductions. However, Bayesian clustering analysis revealed slightly different sources of invasive populations in the two non-native regions. Genetic diversity patterns revealed the lack of isolation by distance between populations and confirmed the influence of anthropogenic factors on the spread of B. orientalis. The significance of native populations as germplasm resources for breeding is discussed.     

Silicon Alleviates Cadmium Toxicity in Two Cypress Varieties by Strengthening the Exodermis Tissues and Stimulating Phenolic Exudation of Roots

The aim of this work was to investigate the effect of silicon (Si) on phenolic exudation of plant roots and cadmium (Cd) bioavailability in rhizospheres. For this purpose, pot experiments with two cypress varieties, Juniperus chinensis and Platycladus orientalis, each subjected to 100 mg kg^?1 Cd and/or 400 mg kg^?1 Si for 220 days, were conducted using a rhizobag technique. The results showed that P. orientalis accumulated a higher amount of Cd, hence caused higher growth inhibition on the leaves compared with J. chinensis. Si alleviated the growth inhibition induced by Cd toxicity on both varieties, but the mechanisms involved were species specific. For J. chinensis, Si did not affect the root exudation but enhanced the Cd retention of the roots by strengthening the exodermis tissues, restraining Cd translocation from the roots to the shoots. For P. orientalis, Si exposure significantly elevated the phenolic exudation (for example, ferulic acid, catechin, and gallic acid) of the roots, which caused greater Cd mobility in the rhizosphere and enhancement of Cd accumulation in the shoots compared with Cd treatment alone. These results suggest that Cd-chelating with the Si-induced phenolics in the rhizosphere is involved in the Cd detoxification in P. orientalis.     

Studies on Core-Shell Nanocapsules of Felodipine: <Emphasis Type="Italic">In Vitro</Emphasis>-<Emphasis Type="Italic">In Vivo</Emphasis> Evaluations

The present study aimed for in vitro-in vivo-in silico simulation studies of experimentally designed (3²-factorial) Capmul PG-8-cored, Eudragit RSPO-Lutrol F 127 nanocapsules to ferry felodipine using GastroPlus?. The in silico parameter sensitivity analysis for pharmacokinetic parameters was initially assessed to justify the preparation of felodipine-loaded nanocapsules (FLNs) with enhanced solubility to overcome the bioavailability issues of felodipine. The overall integrated desirability ranged between 0.8187 and 0.9488 for three optimized FLNs when analyzed for mean particle size, zeta potential, encapsulation efficiency, and in vitro dissolution parameters. The morphological evaluation (SEM, TEM, and AFM) demonstrated spherical nanoparticles (200–300 nm). Validated LC-MS/MS analysis demonstrated enhanced relative bioavailability (13.37-fold) of optimized FLN as compared to suspension. The simulated regional absorption of the FLN presented significant absorption from the cecum (26.3%) and ascending colon (20.1%) with overall absorption of 67.4% from the GIT tract. Furthermore, in vitro-in vivo correlation demonstrated the Wagner-Nelson method as the preferred model as compared to mechanistic and numerical deconvolution on the basis of least mean absolute prediction error, least standard error of prediction, least mean absolute error, and maximum correlation coefficient (r ² = 0.920). The study demonstrated enhanced oral absorption of felodipine-loaded nanocapsules, and GastroPlus? was found to be an efficient simulation tool for in vitro-in vivo-in silico simulations.     

Analyzing the occurrence of an invasive aquatic fern in wetland using data-driven and multivariate techniques

In the present study, the data-driven (classification trees and support vector machines) and multivariate techniques (principal component analysis and discriminant analysis) were applied to study the habitat preferences of an invasive aquatic fern (Azolla filiculoides) in the Selkeh Wildlife Refuge (a protected area in Anzali wetland, northern Iran). The applied database consisted of measurements from seven different sampling sites in the protected area over the study period 2007–2008. The cover percentage of the exotic fern was modelled based on various wetland characteristics. The predictive performances of the both data-driven methods were assessed based on the percentage of Correctly Classified Instances and Cohen’s kappa statistics. The results of the Paired Student’s t-test (p < 0.01) showed that SVMs outperformed the CTs and thus yielded more reliable prediction than the CTs. All data mining and multivariate techniques showed that both physical-habitat and water quality variables (in particular some nutrients) might affect the habitat requirements of A. filiculoides in the wetland.     

Phytoplankton as a monitoring tool in a tropical urban shallow reservoir (Garças Pond): the assemblage index application

This study aimed at evaluating phytoplankton as a monitoring tool for water quality assessment in an urban shallow eutrophic reservoir considering temporal and vertical scales. Garças Reservoir is located in the Parque Estadual das Fontes do Ipiranga Biological Reserve (23°38′08″S and 23°40′18″S; 46°36′48″W and 46°38′00″W) that lies in the southeastern part of the Municipality of São Paulo, southeast Brazil. Samplings were carried out monthly during 8 consecutive years (1997–2004) following the water column vertical profile (5 depths: subsurface, 1, 2, 3 m and ~20 cm from the bottom). Abiotic variables analyzed were: water temperature, electric conductivity, DO, pH, total alkalinity, free CO₂, dissolved inorganic carbon, N series, P series and SiO₄H₄. Biological variables studied were: total density, total biomass and chlorophyll a, which were integrated arithmetically. At the beginning of the 8 year series, Garças Reservoir was an eutrophic ecosystem with 20% of its surface covered by Eichhornia crassipes (phase I: January 1997–March 1998). Water hyacinth reached 70% of pond surface coverage (phase II: April 1998–August 1999), and then it was mechanically removed (phase III: September 1999–December 2004). After this intervention, drastic alteration in the limnological features was detected, leading to the conclusion that removal of the aquatic macrophyte modified nutrient dynamics drastically reduced water transparency and led to photosynthetic productivity and phytoplankton biomass increase, the latter becoming a physical barrier to light penetration. Twenty one functional groups ‘sensu’ Reynolds were identified. Cyanobacteria contribution played the main role during the drastic alterations that occurred after water hyacinth removal. Results of ecological status of reservoir using Q index showed statistical difference among the 3 limnological phases (one way ANOVA; F = 119.4; P = 0.000). Regarding Q index classification, Garças Reservoir limnological phases were characterized as follows: (1) phase I: 0 ≥ Q ≤ 2.9, medium to bad; (2) phase II: 1.4 ≥ Q ≤ 3, tolerable to medium; and (3) phase III: 0 ≥ Q ≤ 1.5, bad to tolerable ecological states.     

Nitrogen transformation under different dissolved oxygen levels by the anoxygenic phototrophic bacterium <Emphasis Type="Italic">Marichromatium gracile</Emphasis>

Marichromatium gracile: YL28 (M. gracile YL28) is an anoxygenic phototrophic bacterial strain that utilizes ammonia, nitrate, or nitrite as its sole nitrogen source during growth. In this study, we investigated the removal and transformation of ammonium, nitrate, and nitrite by M. gracile YL28 grown in a combinatorial culture system of sodium acetate-ammonium, sodium acetate-nitrate and sodium acetate-nitrite in response to different initial dissolved oxygen (DO) levels. In the sodium acetate-ammonium system under aerobic conditions (initial DO?=?7.20–7.25 mg/L), we detected a continuous accumulation of nitrate and nitrite. However, under semi-anaerobic conditions (initial DO?=?4.08–4.26 mg/L), we observed a temporary accumulation of nitrate and nitrite. Interestingly, under anaerobic conditions (initial DO?=?0.36–0.67 mg/L), there was little accumulation of nitrate and nitrite, but an increase in nitrous oxide production. In the sodium acetate-nitrite system, nitrite levels declined slightly under aerobic conditions, and nitrite was completely removed under semi-anaerobic and anaerobic conditions. In addition, M. gracile YL28 was able to grow using nitrite as the sole nitrogen source in situations when nitrogen gas produced by denitrification was eliminated. Taken together, the data indicate that M. gracile YL28 performs simultaneous heterotrophic nitrification and denitrification at low-DO levels and uses nitrite as the sole nitrogen source for growth. Our study is the first to demonstrate that anoxygenic phototrophic bacteria perform heterotrophic ammonia-oxidization and denitrification under anaerobic conditions.     

Unexpected presence of <Emphasis Type="Italic">Fagus orientalis</Emphasis> complex in Italy as inferred from 45,000-year-old DNA pollen samples from Venice lagoon

Background

Phylogeographic analyses on the Western Euroasiatic Fagus taxa (F. orientalis, F. sylvatica, F. taurica and F. moesiaca) is available, however, the subdivision of Fagus spp. is unresolved and there is no consensus on the phylogeny and on the identification (both with morphological than molecular markers) of Fagus Eurasiatic taxa.For the first time molecular analyses of ancient pollen, dated at least 45,000 years ago, were used in combination with the phylogeny analysis on current species, to identify the Fagus spp. present during the Last Interglacial period in Italy.In this work we aim at testing if the trn L-trn F chloroplast DNA (cpDNA) region, that has been previously proved efficient in discriminating different Quercus taxa, can be employed in distinguishing the Fagus species and in identifying the ancient pollen.

Results

86 populations from 4 Western Euroasistic taxa were sampled, and sequenced for the trn L-trn F region to verify the efficiency of this cpDNA region in identifying the Fagus spp.. Furthermore, Fagus crenata (2 populations), Fagus grandifolia (2 populations), Fagus japonica, Fagus hayatae, Quercus species and Castanea species were analysed to better resolve the phylogenetic inference.Our results show that this cpDNA region harbour some informative sites that allow to infer relationships among the species within the Fagaceae family. In particular, few specific and fixed mutations were able to discriminate and identify all the different Fagus species.Considering a short fragment of 176 base pairs within the trn L intron, 2 transversions were found able in distinguishing the F. orientalis complex taxa (F. orientalis, F. taurica and F. moesiaca) from the remaining Fagus spp. (F. sylvatica, F. japonica, F. hayataea, F. crenata and F. grandifolia). This permits to analyse this fragment also in ancient samples, where DNA is usually highly degraded.The sequences data indicate that the DNA recovered from ancient pollen belongs to the F. orientalis complex since it displays the informative sites characteristic of this complex.

Conclusion

The ancient DNA sequences demonstrate for the first time that, in contrast to current knowledge based on palynological and macrofossil data, the F. orientalis complex was already present during the Tyrrhenian period in what is now the Venice lagoon (Italy).This is a new and important insight considering that nowadays West Europe is not the natural area of Fagus orientalis complex, and up to now nobody has hypothesized the presence during the Last Interglacial period of F. orientalis complex in Italy.