Natural hybridization within seed sources of shortleaf pine (Pinus echinata Mill.) and loblolly pine (Pinus taeda L.)

Shortleaf and loblolly pine trees (n = 93 and 102, respectively) from 22 seed sources of the Southwide Southern Pine Seed Source Study plantings or equivalent origin were evaluated for amplified fragment length polymorphism (AFLP) variation. These sampled trees represent shortleaf pine and loblolly pine, as they existed across their native geographic ranges before intensive forest management. Using 17 primer pairs, a total of 96 AFLPs between shortleaf pine and loblolly pine were produced and scored on the sample trees and two control-pollinated F1 interspecies hybrids and their parents. In addition, the well known isocitrate dehydrogenase (IDH) isozyme marker was scored for all trees. IDH detected two putative hybrids among the loblolly pine samples and two among the shortleaf pine samples, while either 13 or 12 putative hybrids were detected using all AFLP markers and IDH and either NewHybrids or Structure software, respectively. Results of this study show that later generation hybrids can be reliably identified using AFLP markers and confirmed that IDH is not a definitive marker for detecting hybrids; that is, at least in some seed sources, the alternative species’ IDH allele resides in the source species. Based on all the markers, hybridization frequency varied geographically, ranging from 30% in an Arkansas seed source to 0% in several other seed sources. The hybridization level was higher in populations west of the Mississippi River than in populations east of the river; the shortleaf pine hybridization rates were 16.3% and 2.4% and the loblolly pine rates were 4.5% and 3.3%, west and east of the river, respectively. The results suggest that hybridization between these two species is significant but varies by seed source and species, and the potential for the unintended creation of hybrids should be considered in forest management decisions regarding both natural and artificial regeneration.     

Microsatellite versus AFLP analyses of pre-management introgression levels in loblolly pine (<Emphasis Type="Italic">Pinus taeda</Emphasis> L.) and shortleaf pine (<Emphasis Type="Italic">P. echinata</Emphasis> Mill.)

Loblolly pine and shortleaf pine are known to naturally hybridize. In this study, we used 42 microsatellite markers and isocitrate dehydrogenase isozyme to create genetic profiles of 202 loblolly and shortleaf pine trees grown from seed collected in the 1950s for the Southwide Southern Pine Seed Source Study. Estimated Φ_PT was low in both loblolly (0.061) and shortleaf (0.080) pines, indicating that most of the diversity is accounted for within seed sources. However, both loblolly and shortleaf pines showed significant correlations between seed sources’ genetic and geographic distances, with R ² of 0.43 and 0.17, respectively. The hybridization rate was 4.0%, with more hybrids west of the Mississippi River (8.1%) than east of the river (2.1%). Additionally, about the same proportion of both species (4.5% of loblolly and 3.3% of shortleaf pine) were identified as hybrids. These results are consistent with prior studies on these two species but do contrast with the results from an amplified fragment length polymorphism (AFLP) analysis of the same samples. For example, the AFLP study concluded that 6.3% of the trees were hybrids, or 1.4 times higher than determined by this study. Of the 12 hybrids identified in the AFLP study, six were not identified as hybrids here, and of the eight hybrids identified here, only four were identified in the AFLP study. Although similar in overall results, we suggest that the microsatellite analysis is more convincing than the AFLP analysis because microsatellites provide more information per genetic locus than do AFLPs.     

Genetic diversity within and among populations of shortleaf pine (Pinus echinata Mill.) and loblolly pine (Pinus taeda L.)

Shortleaf pine (n = 93) and loblolly pine (n = 112) trees representing 22 seed sources or 16 physiographic populations were sampled from Southwide Southern Pine Seed Source Study plantings located in Oklahoma, Arkansas, and Mississippi. The sampled trees were grown from shortleaf pine and loblolly pine seeds formed in 1951 and 1952, prior to the start of intensive forest management across their native ranges. Amplification fragment length polymorphism (AFLP) markers were developed and used to study genetic diversity and its structure in these pine species. After screening 48 primer pairs, 17 and 21 pairs were selected that produced 794 and 647 AFLPs in shortleaf pine and loblolly pine, respectively. High-AFLP-based genetic diversity exists within shortleaf pine and loblolly pine, and most (84.73% in shortleaf pine; 87.69% in loblolly pine) of this diversity is maintained within physiographic populations. The high value of unbiased measures of genetic identity and low value of genetic distance for all pairwise comparisons indicates that the populations have similar genetic structures. For shortleaf pine, there was no significant correlation between geographic distance and genetic distance (r = 0.28), while for loblolly pine there was a weak but significant correlation (r = 0.51).     

Recovery from topkill of shortleaf pine × loblolly pine hybrids compared to their parent populations

Hybrids between shortleaf pine (Pinus echinata Mill.) and loblolly pine (Pinus taeda L.) have increased since the 1950s throughout the southeastern USA. Previously, greater sprouting capacity and the formation of a basal crook that lowers the height of dormant buds may have favored pure shortleaf pine populations on fire prone sites. The objective of this study was to determine how seasonal timing of topkill by both fire and topclipping affect sprouting of shortleaf × loblolly pine F1 hybrids compared to their parent open-pollinated populations during their third growing season. A factorial combination of top-clipping (hand pruners) and girdling by fire (propane torch) was conducted on November 2010, January, March, and April 2011 and sprouting response was measured after the growing season. Survival of topkilled shortleaf pine (94 %) was greatest followed by hybrid (78 %) and loblolly pine (35 %). However, species effects varied with topkill treatment and treatment date because survival was relatively lower for loblolly and hybrid pines in the burn-only as well as the November and April treatment dates while survival of shortleaf pine was consistently high. The number of sprouts was greatest for shortleaf (32.3) intermediate for hybrid (23.8) and lowest for loblolly pine (12.0). Overall, 83 % of shortleaf pine, 35 % of hybrid pine, and 5 % of loblolly pine exhibited a basal crook. The height from ground line to the lowest sprout was shortest for shortleaf (3.5 mm), intermediate for hybrids (7.7 mm), and largest for loblolly pine (21.3 mm). While the hybrid saplings exhibited intermediate performance in survival, sprouting capacity, and crooking, pure shortleaf pine were superior and are probably better suited to recover from fire.     

The genetics of shortleaf pine (<Emphasis Type="Italic">Pinus echinata</Emphasis> mill.) with implications for restoration and management

Shortleaf pine (Pinus echinata Mill.) is an important commercial timber resource and forest ecosystem component in the southeastern USA. The species occurs in mainly drier sites as an early- to mid-successional species, is fire-adapted, and it plays an important role in the fire ecology of the region. However, shortleaf pine genetics are not well-studied, especially in this era of molecular genetics and genomics. Most genetics research about the species has focused on provenance testing. Generally, shortleaf pine performs well in colder areas, when compared to loblolly pine (Pinus taeda L.), a close relative, which is faster growing and the most common plantation species in the region. Though not as advanced in genetic improvement as loblolly pine, tree breeders have improved shortleaf pine in one to two generations of selection, and diverse, genetically improved shortleaf pine materials are available to foresters and landowners throughout the southeastern USA. Researchers have also studied the genetic variation of shortleaf pine using various molecular markers and have found that shortleaf pine is generally a prolific outcrosser, a trait it shares with other non-isolated members of the family Pinaceae. In recent years, however, it has shared less genetic material across long ranges, probably because of habitat fragmentation. Various anthropogenic factors also affect shortleaf pine’s future, as recent studies show that shortleaf pine introgression with loblolly pine puts the species—and the resiliency of southeastern forests—at risk. Importantly, fire exclusion is a likely cause of the increase in introgression. Herein, we provide further details and up-to-date genetic information and resources for foresters and ecologists interested in the restoration and management of shortleaf pine.     

Paternal chloroplast inheritance patterns in pine hybrids detected with trnL-trnF intergenic region polymorphism

The inheritance patterns of the chloroplast genomes of shortleaf pine (Pinus echinata Mill.), loblolly pine (Pinus taeda L.) and slash pine (Pinus elliottii Engelm.) were investigated through the trnL-trnF intergenic spacer polymorphism analysis. The DNA sequences of this spacer differ among these three closely related Pinus species. A modified 'cold' PCR-SSCP (single-strand conformation polymorphism) analysis of this spacer shows that the artificial hybrids (F1) from the shortleaf pine (seed parent) 2 loblolly pine (pollen parent) cross, exhibit the loblolly pine profile. Additionally, nine putative hybrids between shortleaf pine and loblolly pine, previously identified by the IDH (Isocitrate dehydrogenase) allozyme marker, presented the shortleaf pine profile indicating that shortleaf pine, not loblolly pine, sired all of the putative hybrids. Nondenatured polyacrylamide-gel electrophoresis of the trnL-trnF intergenic spacer demonstrated that the artificial hybrids (F1) from the cross, slash pine (seed parent) 2 shortleaf pine (pollen parent), present the shortleaf pine profile. Those results confirmed that the chloroplast genome is paternally inherited in these three species of the genus Pinus. The significance of the trnL-trnF intergenic region polymorphism and our modified 'cold' SSCP protocol for population genetic studies is discussed.     

Frequency and direction of hybridization in sympatric populations of Pinus taeda and P. echinata (Pinaceae)

Two naturally occurring, sympatric, northern Georgia populations of Pinus taeda L. (loblolly pine) and P. echinata Mill. (shortleaf pine) were examined with respect to genetic diversity within populations and the frequency, spatial distribution, and morphology of putative hybrids. Shortleaf pine predominated at the "road" site while loblolly pine predominated at the "granite outcrop" site. Hybrid individuals were identified by their IDH allozyme genotype, the only such locus known to be fixed for different alleles in the two species. All allozymatically detectable hybrids (34 at the road site and two at the granite outcrop site) were juveniles that were distributed in open, sunny patches. A similar pattern of recruitment was seen for juveniles of the parental species. Hybrids were spatially distant from mature shortleaf pine, suggesting that shortleaf pine was not the seed parent. Discriminant analysis on needle characteristics indicated that loblolly pine was easily distinguished from shortleaf pine and the hybrids, but that shortleaf pine and the hybrids were barely distinguishable from each other. A diagnostic cpDNA restriction site marker indicated that shortleaf pine sired all the hybrids at both sites. No evidence of later generation hybridization was found.     

Micropropagation of shortleaf,Virginia and loblolly x shortleaf pine hybrids via organogenesis

Protocols were developed for the micropropagation of shortleaf pine (Pinus echinata Mill.), loblolly (P. taeda L.) x shortleaf pine hybrids, and Virginia pine (P. virginiana Mill.). For meristematic tissue induction, modified Gresshoff & Doy (GD) medium with a high concentration of benzyladenine (BA) and short pulse treatment was best for loblolly x shortleaf hybrids whereas a lower concentration of BA and longer pulse treatment was best for shortleaf and Virginia pines. Shoot growth rate for all species was generally slower on Schenk & Hildebrandt medium than on GD medium. Addition of activated charcoal improved shoot growth of shortleaf pine but not of Virginia pine or the loblolly x shortleaf hybrids. Separation of shoots was beneficial before placing in the advanced growth medium. Both GD and Litvay's media produced good advanced shoot growth, especially following the addition of 0.5% activated charcoal. Individual shoot heights of 2–3 cm and 8–12 weeks of age after separation from the cluster were best for rooting. Root induction declined rapidly thereafter. Modified GD medium with 0.5 mg 1^-1 -naphthaleneacetic acid plus 1.0 mg 1^-1 3-indolebutyric acid and 20 g 1^-1 sucrose was best for root induction for all species except shortleaf pine. Addition of activated charcoal produced better root systems. Too high a light intensity resulted in a lowered frequency of rooting. A large number of plantlets was produced.     

Hyphal growth of Phytophtora cinnamomi on pine callus tissue

A procedure was developed which demonstrates the expression of differential resistance in pine callus tissues to the fungal pathogen Phytophthora cinnamomi Rands. Callus tissues were maintained on a modified Murashige and Skoog medium with 10^–5M 2,4-D and inoculated with hyphae of P. cinnamomi at 26°C in the dark. The number of intracellular hyphae was used as an index of resistance. Loblolly and loblolly × shortleaf pine hybrids were determined to be more resistant to infection and invasion by the fungus than were shortleaf and Virginia pine.Abbreviations (AL) loblolly pine—Alabama - (PL) South Carolina - (AS) shortleaf pine—Alabama - (CS) Georgia - (AV) Virginia pine—Alabama - (H1) loblolly × shortleaf pine hybrids—14–42 × 6-I-43 - (H2) I-523 × 6-D-8     

Comparative mapping in Pinus: sugar pine (Pinus lambertiana Dougl.) and loblolly pine (Pinus taeda L.)

The majority of genomic research in conifers has been conducted in the Pinus subgenus Pinus mostly due to the high economic importance of the species within this taxon. Genetic maps have been constructed for several of these pines and comparative mapping analyses have consistently revealed notable synteny. In contrast, little genomic research has been conducted on the Pinus subgenus Strobus, even though these pines have strong ecological relevance. We report a consensus genetic linkage map for sugar pine (Pinus lambertiana Dougl.) constructed with 399 single nucleotide polymorphisms markers derived from annotated genes. The map is 1,231 cM in length and organized into 19 linkage groups. Two of the mapping populations were derived from trees that were segregating for the major gene of resistance (Cr1) to Cronartium ribicola, the fungal pathogen responsible for white pine blister rust. The third mapping population was derived from a full-sib cross segregating for partial resistance to white pine blister rust. In addition, we report the first comparative mapping study between subgenera Strobus and Pinus. Sixty mapped markers were found in common between sugar pine and the loblolly pine reference map with 56 of them (93%) showing collinearity. All 19 linkage groups of the sugar pine consensus map coaligned to the 12 linkage groups of the loblolly pine reference map. The syntenic relationship observed between these two clades of pines provides a foundation for advancing genomic research and genetic resources in subgenus Strobus.     

Genetic structure and association mapping of adaptive and selective traits in the east Texas loblolly pine (Pinus taeda L.) breeding populations

First-generation selection (FGS) and second-generation selection (SGS) breeding populations of loblolly pine from east Texas were studied to estimate the genetic diversity, population structure, linkage disequilibrium (LD), signatures of selection and association of breeding traits with a genome-wide panel of 4,264 single nucleotide polymorphisms (SNPs). Relatively high levels of observed (H _o?=?0.178–0.198) and expected (H _e?=?0.180–0.198) heterozygosities were observed in all populations. The amount of inbreeding was very low with many populations exhibiting a slight excess of heterozygotes. The population structure was weak, but F _ST indicated more pronounced differentiation in the SGS populations. As expected for outcrossing natural populations, the genome-wide LD was low, but marker density was insufficient to deduce the decay rate. Numerous associations were found between various phenotypic traits and SNPs, but only a few remained significant after false positive correction. Signatures of diversifying and balancing selection were found in markers representing important biological functions. These results present the first step in the application of marker-assisted selection (MAS) to the Western Gulf Forest Tree Improvement Program (WGFTIP) for loblolly pine and will contribute to the knowledgebase necessary for genomic selection technology.     

Genetic mapping of expressed sequence tag polymorphism (ESTP) markers in loblolly pine (Pinus taeda L.)

The development and mapping of genetic markers based upon expressed sequence tag polymorphisms (ESTPs) in loblolly pine (Pinus taeda L.) are reported. The new markers were generated by PCR-amplification of loblolly pine genomic DNAs with primers designed from sequenced cDNAs. The cDNA libraries were constructed from RNAs expressed in the needles of loblolly pine seedlings or in the xylem from young trees. DNA polymorphisms were identified by analyzing the amplified products for differences in fragment size or restriction sites, or by examining mobility differences using denaturing gradient gel electrophoresis (DGGE). DGGE revealed more DNA polymorphisms than the other two methods. Fifty six ESTPs were mapped using either of two mapping populations and positioned onto a loblolly pine consensus genetic map. Unlike many other markers commonly used in forestry, ESTPs can be used as orthologous markers for comparative mapping, to map genes of known function, or to identify candidate genes affecting important traits in loblolly pine. Received: 10 April 2000 / Accepted: 13 July 2000     

Photosynthetic capacity of loblolly pine (Pinus taeda L.) trees during the first year of carbon dioxide enrichment in a forest ecosystem

Our objective was to assess the photosynthetic responses of loblolly pine trees (Pinus taeda L.) during the first full growth season (1997) at the Brookhaven National Lab/Duke University Free Air CO₂ Enrichment (FACE) experiment. Gas exchange, fluorescence characteristics, and leaf biochemistry of ambient CO₂ (control) needles and ambient + 20 Pa CO₂ (elevated) needles were examined five times during the year. The enhancement of photosynthesis by elevated CO₂ in mature loblolly pine trees varied across the season and was influenced by abiotic and biotic factors. Photosynthetic enhancement by elevated CO₂ was strongly correlated with leaf temperature. The magnitude of photosynthetic enhancement was zero in March but was as great as 52% later in the season. In March, reduced sink demand and lower temperatures resulted in lower net photosynthesis, lower carboxylation rates and higher excess energy dissipation from the elevated CO₂ needles than from control needles. The greatest photosynthetic enhancement by CO₂ enrichment was observed in July during a period of high temperature and low precipitation, and in September during recovery from this period of low precipitation. In July, loblolly pine trees in the control rings exhibited lower net photosynthetic rates, lower maximum rates of photosynthesis at saturating CO₂ and light, lower values of carboxylation and electron transport rates (modelled from A–C_i curves), lower total Rubisco activity, and lower photochemical quenching of fluorescence in comparison to other measurement periods. During this period of low precipitation trees in the elevated CO₂ rings exhibited reduced net photosynthesis and photochemical quenching of fluorescence, but there was little effect on light- and CO₂-saturated rates of photosynthesis, modelled rates of carboxylation or electron transport, or Rubisco activity. These first-year data will be used to compare with similar measurements from subsequent years of the FACE experiment in order to determine whether photosynthetic acclimation to CO₂ occurs in these canopy loblolly pine trees growing in a forest ecosystem.     

The effects of UV-B radiation on epidermal anatomy in loblolly pine (Pinus taeda L.) and Scots pine (Pinus sylvestris L.)

The effects of enhanced UV‐B radiation on the needle anatomy of loblolly pine (Pinus taeda L.) and Scots pine (Pinus sylvestris L.) were studied in the field under supplemental UV‐B radiation supplied by a modulated irradiation system. The supplemental UV‐B levels were designed to simulate either a 16 or 25% loss of stratospheric ozone over College Park, Maryland. Enhanced UV‐B radiation caused different responses in these two species. The needles of loblolly pine had larger amounts of tannin in the lumen of epidermal cells and more wall‐bound phenolics in the outer epidermal walls of UV‐B‐treated needles, whereas the most pronounced effect on Scots pine needles was increased cutinization. In both species, the outer epidermal cell walls thickened and the needle cross‐sectional and mesophyll areas decreased (statistically significantly only in Scots pine). This suggests that more carbon may have been allocated to the protection mechanisms at the expense of photosynthetic area. The difference in response between these species suggests that the response to UV‐B radiation is not mediated by a single mechanism and that no generalization with regard to the effects of UV‐B on conifers can be made.     

Correlation between infection by ophiostomatoid fungi and the presence of subterranean termites in loblolly pine (Pinus taeda L.) roots

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Combination of multipoint maximum likelihood (MML) and regression mapping algorithms to construct a high-density genetic linkage map for loblolly pine (Pinus taeda L.)

Genetic maps have been successfully applied to assist in the dissection of complex traits, provide insight on genome structure, and estimate recombination in conjunction with physical maps. Despite an extensive list of genetic maps developed for loblolly pine (Pinus taeda L.) over the past two decades, a high-density consensus map has not yet been constructed. In this study, we used two reference three-generation outbred pedigrees, base and qtl, obtained from the North Carolina State University Cooperative Tree Improvement Program, to obtain a high-density genetic consensus map. Both populations were genotyped with ≈ 7,000 different markers (restriction fragment length polymorphisms, expressed sequence tag polymorphisms, simple sequence repeats, SNPs). The grouping, ordering, and spacing of the markers on each linkage group were performed with JoinMap® 4.1, which implements the multipoint maximum likelihood algorithm for outbred populations. The final consensus map contains 2,466 markers, with a total length of 1,476 centimorgans (cM). The average marker density across the 12 linkage groups was 0.62 cM/marker. This high-density map provides an important resource for breeders and geneticists and will enable comparative studies across species, as well as improve the loblolly pine genome sequence assembly.     

Population structure of a lodgepole pine (Pinus contorta) and jack pine (P. banksiana) complex as revealed by random amplified polymorphic DNA.

We studied the population structure of a lodgepole (Pinus contorta Dougl.) and jack pine (Pinus banksiana Lamb.) complex in west central Alberta and neighboring areas by assessing random amplified polymorphic DNA (RAPD) variability in 23 lodgepole pine, 9 jack pine, and 8 putative hybrid populations. Of 200 random primers screened, 10 that amplified 39 sharp and reproducible RAPDs were chosen for the study. None of the 39 RAPDs were unique to the parental species. RAPD diversity ranged from 0.085 to 0.190 among populations and averaged 0.143 for lodgepole pine, 0.156 for jack pine, 0.152 for hybrids, and 0.148 for all 40 populations. The estimated population differentiation based on G(ST) was 0.168 for hybrids, 0.162 for lodgepole pine, 0.155 for jack pine, and 0.247 across all 40 populations. Cluster analysis of genetic distances generally separated jack pine from lodgepole pine and hybrids, but no division could be identified that further separated lodgepole pine from hybrids. The observed weak to mild trend of "introgression by distance" in the complex and neighbouring areas was consistent with the view that introgressive hybridization between lodgepole and jack pines within and outside the hybrid zone may have been through secondary contact and primary intergradation, respectively.     

Identifying southern yellow pine cross sections from the southeastern United States using quadratic discriminant analysis on pith and second annual ring diameters

Southern yellow pine specimens collected from historical structures, stumps, and coarse woody debris in forests have been difficult to identify at the species level due to similar wood anatomy. This can be problematic for dendrochronologists when identifying the correct species used in the construction of historical structures, or reconstructing forest history on the landscape and using those specimens in the context of that history. We applied a quadratic discriminant analysis (QDA) to update a century-old method plotting pith diameters against second annual ring diameters to discern one species of southern yellow pine from others. Our analysis estimates error rates for false positive and false negative determinations when comparing longleaf pine (Pinus palustris Mill.) to shortleaf (Pinus echinata Mill.) and loblolly pine (Pinus taeda L.). The cross-validated false positive error rates for the smallest dataset (n = 46), was nearly twice (9.52%) that determined as a simple proportion by counting errant observations (4.76%). QDA of the largest dataset (n = 206) gave a flatter zero contour and false positive rate (3.13%) like the proportionally determined value (1.56%), despite one additional observation being falsely assigned to longleaf pine by QDA. An unknown, unearthed southern pine specimen from southeastern Virginia was radiocarbon dated up to 500 years prior and assigned as longleaf by our method (probability ≥ 0.9998). Thus, through a QDA, it is possible to greatly improve confidence in identifications of key unknown specimens that can provide evidence of discerning one species, longleaf pine, from other southern yellow pines.