Pinus and Prostomis: a dendrochronological and palaeoentomological study of a study of a mid-Holocene woodland in Eastern England

Tree-ring analysis of sub-fossil Pinus sylvestris L. and Quercus sp. and their associated sub-fossil insect assemblages from tree rot holes have been used to study a prehistoric forest buried in the basal peats at Tyrham Hall Quarry, Hatfield Moors SSSI, in the Humberhead Levels, eastern England. The site provided a rare opportunity to examine the date, composition, age structure and entomological biodiversity of a mid-Holocene Pinus-dominated forest. The combined approaches of dendrochronology and palaeoentomology have enabled a detailed picture of the forest to be reconstructed, within a precise time frame. The Pinus chronology has been precisely dated to 2921- 2445 BC against the English Quercus master curve and represents the first English Pinus chronology to be dendrochronologically dated. A suite of important xylophilous (wood-loving) beetles that are today very rare and four species that no longer live within the British Isles were also recovered, their disappearance associated with the decline in woodland habitats as well as possible climate change. The sub-fossil insects indicate that the characteristic species of the site's modern-day fauna were already in place 4000 years ago. These findings have important implications in terms of maintaining long-term invertebrate biodiversity of mire sites.

Insect faunas associated with Pinus sylvestris L. from the mid-Holocene of the Humberhead Levels, Yorkshire, U.K

This paper presents the results of recent palaeoentomological research carried out in the Humberhead Levels, South Yorkshire, UK, including the discovery of fossils of five species of beetle previously unknown in the British Isles. The significance of these and other Urwaldrelikt species is discussed in relation to the fragmentation of forest habitats, particularly those associated with Pinus sylvestris L. The Holocene history of this tree and its associated taxa is examined. The importance of fire habitats and the dependence of some pinicolous taxa on these habitats suggests that the decline of fire ecosystems may have had some impact on the changing biogeography of some species.

Patterns of variation at a mitochondrial sequence-tagged-site locus provides new insights into the postglacial history of European Pinus sylvestris populations

Due to their maternal mode of inheritance, mitochondrial markers can be regarded as almost 'ideal' tools in evolutionary studies of conifer populations. In the present study, polymorphism was analysed at one mitochondrial intron (nad 1, exon B/C) in 23 native European Pinus sylvestris populations. In a preliminary screening for variation using a polymerase chain reaction-restriction fragment length polymorphism approach, two length variants were identified. By fully sequencing the 2.5 kb region, the observed length polymorphism was found to result from the insertion of a 31 bp sequence, with no other mutations observed within the intron. A set of primers was designed flanking the observed mutation, which identified a novel sequence-tagged-site mitochondrial marker for P. sylvestris. Analysis of 747 trees from the 23 populations using these primers revealed the occurrence of two distinct haplotypes in Europe. Within the Iberian Peninsula, the two haplotypes exhibited extensive population differentiation (Phi(ST) = 0.59; P less than or equal to 0.001) and a marked geographical structuring. In the populations of central and northern Europe, one haplotype largely predominated, with the second being found in only one individual of one population.

The effects of multiple metal contamination on ectomycorrhizal Scots pine (Pinus sylvestris) seedlings

Experiments were conducted to investigate the effects of single and multiple metal contamination (Cd, Pb, Zn, Sb, Cu) on Scots pine seedlings colonised by ectomycorrhizal (ECM) fungi from natural soil inoculum. Seedlings were grown in either contaminated field soil from the site of a chemical accident, soils amended with five metals contaminating the site, or in soil from an uncontaminated control site. Although contaminated and metal-amended soil significantly inhibited root and shoot growth of the Scots pine seedlings, total root tip density was not affected. Of the five metals tested in amended soils, Cd was the most toxic to ECM Scots pine. Field-contaminated soil had a toxic effect on ECM fungi associated with Scots pine seedlings and caused shifts in ECM species composition on ECM seedlings. When compared to soils amended with only one metal, soils amended with a combination of all five metals tested had lower relative toxicity and less accumulation of Pb, Zn and Sb into seedlings. This would indicate that the toxicity of multiple metal contamination cannot be predicted from the individual toxicity of the metals investigated.

The extended phenotype of Scots pine Pinus sylvestris structures the understorey assemblage

Heritable variation in plant secondary compounds in dominant species has been hypothesised to effect ecosystem function and the structure of associated assemblages of plants, microbes and animals. The functioning of this extended phenotype in relation to the understorey vegetation composition was tested within a boreal forest system dominated by Pinus sylvestris which contains a range of monoterpenes, the composition of which is largely under genetic control. A variance partitioning approach was adopted to identify the relative importance of tree chemistry, environment, spatial location and tree architecture in controlling the distribution of species in the ground flora under individual trees. The monoterpene composition of the pine needles appeared to contribute significantly to controlling understorey vegetation composition, but was less important than environmental factors, though similar to spatial factors. Thus there appears to be a link between variation in the chemical composition of the single, dominant tree species within this system and the pattern of occurrence and abundance in other species at the same trophic level.

Sensitivity to Cd or Zn of host and symbiont of ectomycorrhizal Pinus sylvestris L. (Scots pine) seedlings

Pinus sylvestris seedlings infected with either the ectomycorrhizal (ECM) fungus Paxillus involutus or Suillus variegatus were exposed to a range of Cd or Zn concentrations. This was done to investigate the relationship between the sensitivity of ECM fungi and their host plants over a wide range of concentrations. P involutus ameliorated the toxicity of Cd and Zn to P. sylvestris with respect to root length, despite significant inhibition of ECM infection levels by Cd (Cd EC₅₀ [effective concentration which inhibits ECM infection by 50%] values were: P. involutus 3.7 μg g^-1 Cd; S. variegatus 2.3 μg g^-1 Cd). ECM infection by P. involutus also decreased Cd and Zn transport to the plant shoots at potentially toxic concentrations and also influenced the proportion of Zn transported to the roots and shoots, with a higher proportion retained in the roots of the seedlings. ECM infection did increase host biomass production, but this was not affected by the presence of Cd or Zn. Root and shoot biomass production by P. sylvestris, in both the presence and absence of ECM fungi, was unaffected by Cd and Zn at all concentrations tested.

Toxic effects of cadmium and zinc on ectomycorrhizal colonization of scots pine (Pinus sylvestris L.) from soil inoculum

Scots pine seedlings colonized by ectomycorrhizal (ECM) fungi from natural soil inoculum were exposed to a range of Cd or Zn concentrations to investigate the effects of metals on ECM fungi-Scots pine associations in a realistic soil environment. Experiments focused on the relationship between the sensitivity of ECM fungi and their host plants, the influence of metals on ECM community dynamics on Scots pine roots, and the effects of metal exposure on ECM colonization from soil-borne propagules. Ectomycorrhizal colonization was inhibited by Cd and Zn, with a decrease in the proportion of ECM-colonized root tips. Shoot and root biomass, total root length, and total root-tip density, however, were unaffected by Cd or Zn. A decrease in the diversity of ECM morphotypes also occurred, which could have a negative effect on tree vigor. Overall, colonization by ECM fungi was more sensitive than seedling growth to Cd and Zn, and this could have serious implications for successful tree establishment on metal-contaminated soils.

Cross-colonization of scots pine (Pinus sylvestris) seedlings by the ectomycorrhizal fungus Paxillus involutus in the presence of inhibitory levels of Cd and Zn

The effects of Cd and Zn on cross-colonization by Paxillus involutus of Scots pine seedlings was examined by using pairs of ectomycorrhizal (ECM) and non-mycorrhizal (NM) seedlings grown in the same vessel. This was done to assess, first, the ability of P. involutus to colonize NM Scots pine seedlings by growth from colonized roots of other Scots pine seedlings in the presence of Cd or Zn, and, second whether ECM colonization of Scots pine by P. involutus provided a competitive advantage over NM seedlings. Ectomycorrhizal colonization of Scots pine was shown to be more sensitive than Scots pine itself to Cd and Zn, but prior colonization did provide a competitive advantage with respect to biomass production. This beneficial effect over NM seedlings was, however, equal in the control, Cd and Zn treatments, and was due simply to growth stimulation in the presence of ECM colonization. Cross-colonization from an ECM to a NM seedling was reduced but not prevented by Cd and Zn. Cd had a more negative effect on cross-colonization than on initial colonization of seedlings, whereas Zn had an equally inhibitory effect on both parameters. These results have important implications for plant establishment on metal-contaminated sites. If cross-colonization between plants is reduced by toxic metals, plant establishment on contaminated sites might be retarded.

Bursaphelenchus antoniae sp. n. (Nematoda: Parasitaphelenchidae) associated with Hylobius sp. from Pinus pinaster in Portugal

Bursaphelenchus antoniae sp. n. is described and illustrated. Dauer juveniles were isolated from the body of the large pine weevil, Hylobius sp., collected from maritime pine (Pinus pinaster) stumps, in Portugal. Bursaphelenchus antoniae sp. n. was reared and maintained in P. pinaster wood segments and on Petri dish cultures of the fungi Botrytis cinerea and Monilinia fructicola. The new species is characterised by a relatively small body length of ca 583 μm (females) and 578 μm (males), a lateral field with two incisures, presence of a small vulval flap and a conoid female tail with a rounded or pointed terminus. Males have stout spicules with a disc-like cucullus and seven caudal papillae arranged as a single midventral precloacal papilla, one precloacal pair and two postcloacal pairs. In the character of the lateral field, B. antoniae sp. n. comes close to B. abietinus, B. rainulfi and B. hylobianum, whilst spicule characters place it within the piniperdae-group sensu Ryss et al. Morphologically, B. antoniae sp. n. is closest to B. hylobianum; the spicules of these two species having flattened, wing-like, alae on the distal third of the lamina. Bursaphelenchus antoniae sp. n. is distinguished from B. hylobianum on the arrangement of the caudal papillae (two vs three pairs). ITS-RFLP profiles and the failure to hybridise support the separation of the two species. Phylogenetic analysis of the new species, based on the 18S rDNA sequence, supports the inclusion of this new species in the B. hylobianum-group sensu Braasch. Sequence analysis of the 28S rDNA D2/D3 domain did not place the new species in a definite group.

Bursaphelenchus Fuchs, 1937 (Nematoda: Parasitaphelenchidae) species associated with Pinus species in northern Turkey

A survey for Bursaphelenchus nematodes, associated with different conifer trees, was conducted in several forest areas in the northern regions of Turkey. Only pine trees (Pinus nigra, P. pinaster and P. sylvestris) yielded Bursaphelenchus specimens. Nematodes were identified using several morphological diagnostic characters of the genus (male spicule structure, number of lateral incisures, number and distribution of the male papillae, presence of female vulval flap), and confirmed by using RFLP analysis of the internal transcriber spacer (ITS) regions of ribosomal DNA. Three different species were identified from several sampled areas, namely B. mucronatus, B. pinophilus and B. sexdentati, representing a first report of the last two species for Turkey. The association of B. pinophilus with black pine (P. nigra) is herein reported for the first time.

A rapid staining-assisted wood sampling method for PCR-based detection of pine wood nematode Bursaphelenchus xylophilus in Pinus massoniana wood tissue

For reasons of unequal distribution of more than one nematode species in wood, and limited availability of wood samples required for the PCR-based method for detecting pinewood nematodes in wood tissue of Pinus massoniana, a rapid staining-assisted wood sampling method aiding PCR-based detection of the pine wood nematode Bursaphelenchus xylophilus (Bx) in small wood samples of P. massoniana was developed in this study. This comprised a series of new techniques: sampling, mass estimations of nematodes using staining techniques, and lowest limit Bx nematode mass determination for PCR detection. The procedure was undertaken on three adjoining 5-mg wood cross-sections, of 0.5 · 0.5 · 0.015 cm dimension, that were cut from a wood sample of 0.5 · 0.5 · 0.5 cm initially, then the larger wood sample was stained by acid fuchsin, from which two 5-mg wood cross-sections (that adjoined the three 5-mg wood cross-sections, mentioned above) were cut. Nematode-staining-spots (NSSs) in each of the two stained sections were counted under a microscope at 100· magnification. If there were eight or more NSSs present, the adjoining three sections were used for PCR assays. The B. xylophilus – specific amplicon of 403 bp (DQ855275) was generated by PCR assay from 100.00% of 5-mg wood cross-sections that contained more than eight Bx NSSs by the PCR assay. The entire sampling procedure took only 10 min indicating that it is suitable for the fast estimation of nematode numbers in the wood of P. massonina as the prelimary sample selections for other more expensive Bx-detection methods such as PCR assay.

Morphobiometric and molecular characterization of Bursaphelenchus Fuchs, 1937 (Nematoda: Aphelenchoididae) species associated with Pinus pinaster Ainton in Portugal

Abstract - The genus Bursaphelenchus comprises almost 100 species mainly from the northern hemisphere, with conifers as the most important hosts. Among the various nematode species, the pine wood nematode (PWN), Bursaphelenchus xylophilus, is the casual agent of pine wilt disease (PWD), and the most important forest pest for pines worldwide, classified as an A1 quarantine organism within the European Union. In 1999 this nematode was detected for the first time in Portugal and Europa associated with maritime pine, Pinus pinaster. Following detection, a national program denominated "Programa Nacional de Luta contra o Nemátodo da Madeira do Pinheiro" (PROLUNP) was created to, among other objectives, determine the distribution of the PWN and its associated vector(s) and host(s), and therefore intensive surveys covering the entire country were conducted with thousands of wood samples and suspected insects being analyzed. This thesis presents the listing, distribution, frequency and the insects associated with Bursaphelenchus species found associated with maritime pine in Portugal, identifying and characterizing the various species by morphological, biometrical and molecular biology (ITS-RFLP and rDNA sequencing analysis) techniques. To achieve the objectives, a total of 4813 maritime pine wood samples and 3294 insects from 22 species and six families were individually analyzed. A total of nine Bursaphelenchus species were found, namely: B. antoniae, B. hellenicus, B. leoni, B. mucronatus, B. pinasteri, B. sexdentati, B. teratospicularis, B. tusciae and B. xylophilus, all of them (with the exception of B. xylophilus) being new records for Portugal. Some of the species appear to have a widespread distribution, such as B. leoni, B. teratospicularis and B. tusciae while others were very rarely found and apparently have a localized distribution range within the country, namely B. antoniae and B. mucronatus. The majority of the species is characteristic of the Mediterranean region and can also be found in countries such as Spain, Italy and Greece, reflecting the affinity of our fauna with those locations. The association of B. hellenicus and B. tusciae with maritime pine is here reported for the first time. Six of the Bursaphelenchus species were also found associated with insects, mainly from the family Scolytidae (Coleoptera). Some of these interactions were described for the first time, namely: B. hellenicus with both Ips sexdentatus and Hylurgus ligniperda, B. sexdentati with both H. ligniperda and Orthotomicus erosus and B. tusciae with H. ligniperda. The exclusive association of B. xylophilus with the cerambycid Monochamus galloprovincialis was also confirmed. The nematode's dauer juveniles were usually found in low numbers in the insect vectors (ca 10-100 per insect), although for B. xylophilus a few thousand specimens per insect were sometimes found. The location of the dauer juveniles differed according to the species, although they were more common under the elytra and wings of the adult insects. A species new to science was detected and formally described as B. antoniae, associated with Hylobius sp. (Coleoptera; Curculionidae) beetles. Morphologically, this new species is very similar to B. hylobianum, although it's distinct ITS-RFLP molecular pattern (with only the enzyme Haelll producing comparable restriction bands) and the failure of hybridization supported the two species as distinct entities. Additional phylogenetic analysis of the 18S rDNA sequence further supported the taxonomical proximity of B. antoniae with B. hylobianum. Concerning the PWN, detailed studies on the development and morphology of B. xylophilus were conducted, and comparative measurements of field-collected and laboratory-maintained populations demonstrated that nematodes from the second group displayed larger size in all morphometric parameters, which could derive from more adequate conditions of nourishment and/or temperature. Taxonomical studies on the development stages of B. xylophilus confirmed the existence of four propagative juvenile stages (J1,J2,J3 and J4), an adult stage with both sexes and two dispersal stages (jIII e jIV), with the measurements of the gonad length allowing the separation of the propagative stages. It is hoped that the acquired knowledge will be useful on future surveys of nematodes of the Bursaphelenchus genus collected from either wood material or insect vectors, and facilitate the correct distinction and identification of the various species which are now known to occur. ### Resumo - 0 género Bursaphelenchus compreende quase 100 espécies, distribuídas sobretudo nos países do hemisfério norte do globo terrestre. Embora algumas espécies já tenham sido detectadas em plantas herbáceas, os hospedeiros vegetais mais comuns deste género são as coníferas, particularmente pinheiros. 0 nemátode da madeira do pinheiro (NMP), Bursaphelenchus xylophilus, é considerado a espécie mais importante deste género uma vez que é o agente causal da doença da murchidão dos pinheiros ("pine wilt disease"). Originário dos Estados Unidos, onde não causa grande impacte, o NMP foi introduzido em alguns países da Ásia (China, Japão, Coreia e Taiwan) e mais recentemente na Europa (Portugal). Nestas regiões é responsável pela destruição de milhares de hectares de coníferas, assumindo uma elevada importância económica. Em Portugal, depois da sua detecção em 1999, associado a Pinus pinaster, foi implementado um programa nacional "Programa Nacional de Luta contra o Nemátodo da Madeira do Pinheiro" (PROLUNP) que permitiu determinar a área afectada pela praga (a sul do rio Tejo, península de Setúbal) bem como definir e implementar estratégias de controlo e prevenção da disseminação do NMP a outras zonas de Portugal. Recentemente, em Junho de 2008, foi confirmada a presença de B. xylophilus em outras regiões de Portugal levando as autoridades oficiais a definir todo o território continental como zona afectada e de restrição. As prospecções intensivas realizadas nos últimos anos incluíram a recolha e análise de milhares de amostras de madeira de pinheiro bem como de insectos associados ao pinheiro bravo conduzindo à identificação de várias espécies de Bursaphelenchus. Assim, os estudos conduzidos neste trabalho tiveram como objectivos efectuar uma caracterização morfológica, biométrica e molecular das espécies associadas a P. pinaster em Portugal bem como a sua distribuição geográfica e abundância. Os estudos biométricos foram realizados com populações extraídas directamente do meio natural. Foi ainda realizada uma pesquisa que permitiu identificar os insectos a que estão associadas essas espécies, os seus possíveis vectores. Foram analisadas no total 4813 amostras de P. pinaster e 3294 insectos (22 espécies pertencentes a seis famílias diferentes). Foram identificadas um total de nove espécies: B. antoniae n. sp., B. hellenicus, B. leoni, B. mucronatus, B. pinasteri, B. sexdentati, B. teratospicularis, B. tusciae e B. xylophilus. Foram realizados estudos morfológicos e biométricos de todas as espécies com excepção de B. mucronatus; o reduzido número de exemplares encontrados em apenas uma amostra foram utilizados para efectuar o diagnóstico molecular desta espécie (ITS-RFLP). Apesar de ter havido, sempre que possível, a confirmação molecular, na maioria dos casos a caracterização morfológica e biométrica permitiu a correcta identificação das espécies. Contudo, foi imprescindível a análise molecular em algumas amostras, nomeadamente para a identificação de B. xylophilus e B. sexdentati; dada a grande semelhança entre B. xylophilus e B. mucronatus e tendo sido encontradas algumas populações de B. xylophilus que possuíam fêmeas com cauda mucronada, foi necessária a realização da confirmação molecular. Com excepção de B. xylophilus, todas as outras espécies foram reportadas pela primeira vez em Portugal. Juntamente com B. xylophilus, B. pinasteri foi a espécie encontrada nas amostras de madeira de pinheiro com maior frequência. Algumas destas espécies como B. leoni, B. teratospicularis e B. tusciae foram reportadas em diferentes localidades do norte, centro e sul de Portugal, apresentando uma vasta distribuição geográfica; este resultado está em consonância com a forte associação destas espécies a climas mediterrânicos tal como acontece em Espanha, França, Itália e Grécia. Em oposição, espécies como B. antoniae e B. mucronatus foram encontradas apenas numa ocasião na região centro (Leiria) e norte (Figueira da Foz) do país, respectivamente. Bursaphelenchus mucronatus é igualmente pouco frequente em Espanha onde ocorre sobretudo na região norte, na Galiza. Esta espécie preferirá climas mais frios, ocorrendo com uma maior frequência nas regiões de latitude norte; esta análise é corroborada pela presença constante em países como Alemanha, Finlândia, França, Noruega, Rússia e Suécia. A nível mundial são descritas neste trabalho pela primeira vez as associações das espécies B. hellenicus e B. tusciae ao hospedeiro vegetal P. pinaster.

Insignificant impact of prescribed fire on surface soil in a Pinus pinaster plantation, northern Portugal

Following a prescribed fire in a Pinus pinaster forest site located in the north-west Portugal, monitoring of any changes in selected soil characteristics and soil hydrology was undertaken to assess the effects of burning on the following: pH, electrical conductivity, water content, organic carbon and porosity. Thirty plots were established on a regular grid. At each sample plot before and after the fire, samples were collected (disturbed samples from depths of 0-1cm and 1-5cm; undisturbed core samples from 0-5cm). The results indicate that there was no measurable impact on the properties of the soil following this carefully conducted prescribed fire. The fire only affected the litter layer, as intended. Confirmation of this minimal impact on the soil was provided by regrowth of grasses and herbs already occurring two months after the fire. The implication is, therefore, that provided this wildfire-risk reduction strategy is carried out under existing strict guidelines, any impact on soil quality will be minimal.