Marco Cosimo Simeone

Prospects of barcoding the Italian wild dendroflora: oaks reveal severe limitations to tracking species identity.

DNA barcoding may be particularly important in influencing ecology, economic issues, and the fundamental crisis facing biodiversity as a standardized, species‐level identification tool for taxonomy assessment. Trees play important roles in the conservation of many land ecosystems, the wood trade, and the definition of biogeographical processes; nevertheless, peculiar biological, evolutionary and taxonomical features will probably constitute an intriguing challenge to barcoders. We examined whether four marker regions (trnh‐psba, rbcL, rpoc1, matK) proposed by the Consortium for the Barcode of Life (CBOL) matched species taxonomy in a preliminary tree biodiversity survey of Italian forested land. Our objective was to provide a test of future in situ applications of DNA barcodes by evaluating the efficacy of species discrimination under the criteria of uniformity of methods and natural co‐occurrence of the species in the main forest ecosystems. Fifty‐two species were included in a floristic study. We obtained 73% total discrimination success, with trnH‐psbA as the best performing marker and oaks as the least responsive plants to the markers used. A further taxon‐based study of Quercus (thirty specimens, 12 species) revealed that this genus is refractory to barcoding (0% discrimination success), a probable consequence of low variation rate at the plastid genome level, hybridization, and the incidence of biogeography. We conclude that some species‐rich tree genera in small geographical regions may prove exceptionally difficult to barcode. Until more efficient markers are developed, we recommend that improved and diversified sampling (multiple locations of sympatric and co‐occurring congenerics) be embraced as a timely and important goal for the precise assessment of haplotype specificity to facilitate the productive application of barcoding in practice.

Plastome data reveal multiple geographic origins of Quercus Group Ilex

Nucleotide sequences from the plastome are currently the main source for assessing taxonomic and phylogenetic relationships in flowering plants and their historical biogeography at all hierarchical levels. One major exception is the large and economically important genus Quercus (oaks). Whereas differentiation patterns of the nuclear genome are in agreement with morphology and the fossil record, diversity patterns in the plastome are at odds with established taxonomic and phylogenetic relationships. However, the extent and evolutionary implications of this incongruence has yet to be fully uncovered. The DNA sequence divergence of four Euro-Mediterranean Group Ilex oak species (Quercus ilex L., Q. coccifera L., Q. aucheri Jaub. & Spach., Q. alnifolia Poech.) was explored at three chloroplast markers (rbcL, trnK/matK, trnH-psbA). Phylogenetic relationships were reconstructed including worldwide members of additional 55 species representing all Quercus subgeneric groups. Family and order sequence data were harvested from gene banks to better frame the observed divergence in larger taxonomic contexts. We found a strong geographic sorting in the focal group and the genus in general that is entirely decoupled from species boundaries. High plastid divergence in members of Quercus Group Ilex, including haplotypes shared with related, but long isolated oak lineages, point towards multiple geographic origins of this group of oaks. The results suggest that incomplete lineage sorting and repeated phases of asymmetrical introgression among ancestral lineages of Group Ilex and two other main Groups of Eurasian oaks (Cyclobalanopsis and Cerris) caused this complex pattern. Comparison with the current phylogenetic synthesis also suggests an initial high- versus mid-latitude biogeographic split within Quercus. High plastome plasticity of Group Ilex reflects geographic area disruptions, possibly linked with high tectonic activity of past and modern distribution ranges, that did not leave imprints in the nuclear genome of modern species and infrageneric lineages.

Phylogeography and conservation perspectives of an endangered macaronesian endemic: Picconia azorica (Tutin) Knobl. (Oleaceae)

The Azores provide an excellent field test for research activities aimed at developing conservation strategies for endangered tree species. In this work, the urgency to promote Picconia azorica conservation programs addressed (a) insights into the biotaxonomy of the species (including an evaluation of its origin and relationships with the only congeneric species of P. azorica: P. excelsa from the Canary and Madeira islands) and (b) the evaluation of P. azorica genetic diversity. Plastid DNA sequence analysis and molecular markers (RFLP and SSR) were used for this purpose. Phylogenetic data suggest the monophyly of Picconia and support a late Miocene divergence of the two species. Three polymorphic cpSSR loci allowed the identification of five different haplotypes in P. azorica. Uniqueness and relictuality of lineages are presented and discussed. Picconia azorica intra-specific diversity patterns revealed low genetic diversity and a weak genetic structure, which could result from long-lasting ecological stability and efficient inter-island seed movement that have been severely affected in recent times. The species survival is at risk, and we suggest management practices focusing on ex situ and in situ conservation units based on eco-genetic data. Additional measures contributing to mild erosion of the genepool and to remove barriers to seed dispersal are indicated.

Phylogeographic structuring of plastome diversity in Mediterranean oaks (Quercus Group Ilex, Fagaceae)

Oaks of Quercus Group Ilex are emblematic components of the Mediterranean landscapes and the full extent of their diversity in a geographic context is still poorly assessed. In order to detail differentiation patterns within Group Ilex and to illuminate causes and circumstances that underlie the distribution of its lineages, we examined plastome differentiation in the four species currently representing this oak group across the Mediterranean Basin (Quercus ilex, Quercus coccifera, Quercus aucheri, Quercus alnifolia). Plastid DNA variation was explored with three markers on 124 individuals and spatial analyses were performed with phylogenetic and landscape genetics approaches. A strong geographic sorting, entirely decoupled from species boundaries, was found. Spatial coincidences with phylogenetic splits highlights the presence of four geographic regions (the Middle East, the Aegean region, the central and the western Mediterranean) characterised by different degrees of isolation. Distinct sub-regional structures were identified. Inter- and intra-lineage divergence patterns appear to reflect the complex orogeny of the Mediterranean region and Pleistocene climatic oscillations, while a few widespread haplotypes might represent the remnants of the ancestral Group Ilex range. Regional differentiation in this group is recognised to be triggered by a number of factors, including disruption of the original range, incomplete lineage sorting, repeated phases of asymmetrical introgression and isolation. The key role played by some Mediterranean regions on the range establishment and dynamics of this oak group is discussed.

Quercus macranthera Fisch. & Mey. ex Hohen. and Quercus iberica M. Bieb.: Taxonomic definition and systematic relationships with European oaks inferred from nuclear internal transcribed spacer (ITS) data

Abstract Systematic relationships of the two most widespread oaks in Armenia, Quercus iberica M. Bieb. and Quercus macranthera ssp. macranthera Fisch. & Mey. ex Hohen. (Fagaceae), were examined by means of internal transcribed spacer (ITS) sequence variation. The analysis included recently published and several newly derived ITS fragments of most common Eurasian Quercus spp. Orthology of sequences was supported by inspection of ITS secondary structure features. Analyses were implemented with parsimony and maximum likelihood criteria to check the position of all taxa. The robustness of the phylogenetic hypothesis was tested with bootstrap, decay, and Bayesian analysis. Three main clades emerged from the root, corresponding to subg. Quercus, Cerris, and Sclerophyllodrys. Q. iberica and Q. macranthera were included in the subg. Quercus clade and related to Quercus petraea and Q. frainetto-Q. pyrenaica, respectively. The taxonomical status of Q. iberica (Q. petraea ssp. iberica according to some authors) was analysed with respect to Q. petraea. Despite the strict phylogenetic relationship among these two species, the genetic distance (Kimura) was compatible with the hypothesis of maintaining the two taxa separated. A hypothesis on the differentiation of Q. iberica and Q. macranthera before the last glaciation, and their subsequent re-expansion from glacial refugia located in the southern shores of the Black Sea, is proposed. Q. petraea ssp. huguetiana (sequenced for the first time in this study) turned out to be an outgroup with respect to the clade formed by the other accessions of Q. petraea (including Q. iberica). On this basis, this taxon could also assume specific status. The phylogenetic position of Q. pyrenaica is discussed for the first time.

DNA barcoding as a complementary tool for conservation and valorisation of forest resources

Abstract Since the pre-historic era, humans have been using forests as a food, drugs and handcraft reservoir. Today, the use of botanical raw material to produce pharmaceuticals, herbal remedies, teas, spirits, cosmetics, sweets, dietary supplements, special industrial compounds and crude materials constitute an important global resource in terms of healthcare and economy. In recent years, DNA barcoding has been suggested as a useful molecular technique to complement traditional taxonomic expertise for fast species identification and biodiversity inventories. In this study, in situ application of DNA barcodes was tested on a selected group of forest tree species with the aim of contributing to the identification, conservation and trade control of these valuable plant resources. The “core barcode” for land plants (rbcL, matK, and trnH-psbA) was tested on 68 tree specimens (24 taxa). Universality of the method, ease of data retrieval and correct species assignment using sequence character states, presence of DNA barcoding gaps and GenBank discrimination assessment were evaluated. The markers showed different prospects of reliable applicability. RbcL and trnH-psbA displayed 100% amplification and sequencing success, while matK did not amplify in some plant groups. The majority of species had a single haplotype. The trnH-psbA region showed the highest genetic variability, but in most cases the high intraspecific sequence divergence revealed the absence of a clear DNA barcoding gap. We also faced an important limitation because the taxonomic coverage of the public reference database is incomplete. Overall, species identification success was 66.7%. This work illustrates current limitations in the applicability of DNA barcoding to taxonomic forest surveys. These difficulties urge for an improvement of technical protocols and an increase of the number of sequences and taxa in public databases.

Forest resources for second generation biofuel production

Abstract The gradual reduction in world oil reserves is increasing the importance of biofuel. First generation biofuels resulted in yields that are lower than general energy needs. At least in some cases, more energy was used for production than was obtained from the final fuel. Conflicts with food cultivation and high environmental impact are other possible drawbacks. Nevertheless, the multiple uses of plant lipids (fuels, food, many industrial uses) make fuel crops important cultivations. Moreover, cultivation of oil crops used for fuel only is probably economically advantageous for local needs and local economic cycles. Second generation biofuels should perform better in terms of yield and soil use. Among them, lignocellulosic biomass appears to be promising owing to its high available quantity and productivity. In particular, forest biomass (of cultivated trees) could potentially provide a large amount of biomass for biofuels. A general account on lignocellulosic biomass transformation to liquid biofuels (ethanol) is provided together with a quantitative example which calculates the potential biofuel production obtainable by cultivating forest biomass in abandoned and marginal land of Tuscany (Italy). Looking at official available data on energy demand in Tuscany, 15% of the current gasoline consumption could be replaced by such biofuel sources.

Use of innovative technology for the production of high-quality forest regeneration materials

Abstract The aim of this paper is to introduce the innovative technology created within the framework of PRE-FOREST project, funded by the European Commission under the CRAFT 6th Framework Research Programme. PRE-FOREST was an innovative research project in the field of forest planting stock material, enhancing co-operation between research foundations and small and medium-sized enterprises. Among the projects objectives were the introduction of a new technology based on precultivation (in mini-plugs) of forest regeneration material in a cost-efficient and environmental friendly production unit, not affected by outdoor climate; the development of a new, adapted to mini-plugs, grading technology and replug robot; the integration of these technologies into a functional prototype unit capable of large-scale forest regeneration material production; and the introduction of this production system at strategic locations in Europe. The system has been developed for year-round production and function in the same way, independently of climatic variations in different parts of Europe. To obtain these objectives the study focused on the interaction among production technology, mini-plug container design and rooting media, to develop cultivation protocols for forest reproductive materials of special economic and ecological importance for each participating country. One of the main benefits of the new technology was the environmentally friendly production of a large number of seedlings per square metre, leading to a cost-efficient result.

Morphological and molecular data from Madeira support the persistence of an ancient lineage of Taxus baccata L. in Macaronesia and call for immediate conservation actions

Macaronesia is an important biodiversity hotspot in the Mediterranean bioclimatic region, hosting a number of endemics, and encompassing outstanding refugia for ancient Tertiary plant lineages. We investig past occurrence and present distribution of yew (Taxus baccata L.) in the Madeiran archipelago, providing preliminary morphological and genetic descriptions and addressing conservation issues. Fifty-eight individuals presently occur in 19 micro-populations, as probable survivors of the continued yew exploitation across the centuries. Plants were characterized and compared with Euro-Mediterranean provenances by leaf morphology, anatomy, nuclear ITS (Internal Trascribed Spacer) and plastid trnS–trnQ DNA markers. The Madeiran provenance showed peculiar leaf size and morpho-anatomical characters. DNA sequences revealed a basal position of Madeiran yew in the Baccata phylogenetic clades along with the Azorean provenance. Gathered data suggest the survival of a lineage of T. baccata different from those on the continent, and with a possible closer derivation from the species’ ancestors. Such evidences provide a base for identifying a great phylo- and phytogeographical interest of the Macaronesian provenance, and confirm the role of the archipelagos to preserve relict flora and lineages. The risk of extinction of Madeiran yew also calls for conservation strategies and restoration programs for a prompt species rescue.

A tree species range in the face of climate change: cork oak as a study case for the Mediterranean biome

Species distribution models are feasible methods for projecting theoretical responses of living organisms’ occurrence under several future climate change scenarios. The major interest is focused on trees, which regulate the equilibrium within ecosystems and guarantee the survival of many life forms on the Earth. The repercussions of climatic drivers are expected to pose the strongest threats for the Mediterranean biome, an acknowledged hotspot of biodiversity. Here, we focused on cork oak (Quercus suber L.), a keystone species of many landscapes, sustaining a rich biodiversity, ecological processes and economic incomes. Results of 8 combined ecological modelling techniques and two Global Circulation Models highlight a broad contraction of the species potential range over the twenty-first century, both under intermediate and high emissions scenarios. Coupled northward and upward shifts are predicted, mostly pertaining Iberia and North Africa. The potential areas detected at Levantine will likely undergo disappearance. To exacerbate the impacts of climate change, the future of the ecosystems linked to cork oak remains uncertain, because of the expected implications on the phenotypic plasticity or evolutionary responses. A synergy among niche-based, physiological and eco-genetic investigations is strongly needed in the field of applied research, to improve the assessment of conservation and reforestation actions.