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SummaryThe intensively studied Eurasian orchid genus Dactylorhiza has become a model system for exploring allopolyploid evolution, yet determining the optimal circumscriptions of, and most appropriate ranks for, its constituent taxa remain highly controversial topics. Here, novel allozyme data and detailed morphometric data for 16 Scottish marsh-orchid populations are interpreted in the context of recent DNA sequencing studies. Despite being derived from the same pair of parental species, the two allopolyploid species that currently occur in Scotland can reliably be distinguished using allozymes, haplotypes, ribotypes or sequences of nuclear genes. A modest range of diverse morphological characters are shown to distinguish the two molecularly-circumscribed species, but they have in the past been obscured by equivalent levels of infraspecific variation in characters rooted in anthocyanin pigments; these characters are better employed for distinguishing infraspecific taxa. Dactylorhiza francis-drucei (formerly D. traunsteinerioides) is confirmed as being distinct from the continental D. traunsteineri/lapponica, probably originating through allopatric isolation once the continental lineage reached Britain. All Scottish populations are attributed to the comparatively small-flowered, anthocyanin-rich subsp. francis-drucei, which includes as a variety the former D. 'ebudensis'; the less anthocyanin-rich subsp. traunsteinerioides is confined to Ireland, North Wales and northern England. In contrast with D. francis-drucei, only a minority of Scottish populations of D. purpurella are attributed to the anthocyanin-rich race, var. cambrensis. This species most likely originated through an allopolyploidy event that occurred comparatively recently within the British Isles, as it contains allozyme alleles distinctive of British rather than continental D. incarnata (its diploid pollen-parent). In contrast, the rare Scottish population of D. incarnata subsp. cruenta shares with its Irish counterparts a continental genotype, and is most likely a recent arrival in Scotland through long-distance dispersal. Among all European allotetraploid dactylorchids, D. purpurella is the species that most closely resembles D. incarnata, both molecularly and morphologically.

SummaryPrompted by concurrent completion of the latest plant atlas for Britain and Ireland, the orchid flora of these islands is reviewed in detail, focusing on 21st century progress in both systematics research and formal conservation categorisation under IUCN criteria. DNA-based phylogenies consistently circumscribe monophyletic groups that constitute obvious genera, though relationships among those genera remain less certain and resistance to phylogenetic classification persists. Comparison of species circumscription studies in critical groups such asDactylorhiza,Gymnadenia,Platanthera,EpipactisandOrchisshows that most of the recent progress has been achieved using DNA-based techniques, which have also greatly increased understanding of associated mycorrhizae. Most morphological studies remain disappointingly traditional, squandering opportunities for morphometric studies that identify discontinuities between species, allow comparison of rates of morphological and molecular evolution, and underpin accurate quantitative descriptions designed to aid identification. Possibly only one of the 53 species recognised as native is still viewed as endemic. Conventional views on the significance of both pollinator specificity and quantitative seed output as the preferred measure of fitness are questioned, and mycorrhizae are explored as possibly under-rated ecological filters. Comparison of species distributions in successive Atlases suggests diverse patterns of change that reflect a combination of habitat modification, climate change and, in a few cases, improved identification skills. Ecologically tolerant lowland species such asOphrys apiferaandDactylorhiza praetermissaare migrating northward rapidly, while the genusSerapiasappears to be mounting a cross-Channel invasion. Most formal Red-Listing categorisations, and the underlying IUCN criteria, are supported for decisions made within the British Isles, but unfortunately, they have translated poorly into the equivalent European and Global lists. Within Britain, the Data Deficient category has been employed too frequently and illogically; ironically, it is sometimes used to penalise species for having been the subjects of serious research. Far from being "complete", the present consensus view on the taxonomy and evolution of the glacially impoverished British and Irish flora is judged both parochial and complacent. Reinvigorated UK-based research programmes that integrate quantitative morphological and molecular studies of British and Irish natives in a European context are urgently needed.

SummaryThe first study of the taxonomically critical European orchid genus Dactylorhiza to use next-generation DNA sequencing generated the statistically best-supported reconstruction of its phylogeny to date. However, the two competing topologies obtained within the monophyletic Section Maculatae differed radically in the placement of the D. maculata s.s.–D. foliosa clade. Both topologies showed D. foliosa to be nested deeply within D. maculata s.s., and suggested that D. saccifera s.l. is paraphyletic, D. saccifera s.s. from south-eastern Europe and Asia Minor diverging before D. gervasiana from south-central Europe (a taxon typically viewed as a subspecies of D. saccifera or, more often, ignored completely). The poorly-sampled but character-rich morphometric comparison presented here suggests that D. saccifera s.s. and D. gervasiana cannot be distinguished with confidence using morphological characters and that, if D. gervasiana is accepted as a species, it is effectively cryptic. The diploid D. foliosa is accepted as an island endemic species, despite rendering paraphyletic the autotetraploid D. maculata s.s.; all other named taxa within the D. maculata s.s. clade are considered infraspecific. Dactylorhiza fuchsii is indisputably a bona fide species rather than a subspecies but it does not merit becoming the basis of a taxonomic section separate from D. maculata s.s. The strongly contrasting degrees of molecular vs morphological disparity reported here are representative of a more general trend evident in groups that have experienced comparatively recent speciation, as is the need to recognise occasional paraphyletic species when circumscribing species by integrating genotypic and phenotypic data categories. Explicit taxonomic criteria combined with a comparative, monographic approach are needed to achieve consistency of ranking. Disproving hypotheses of species status is judged a criminally underrated activity, actually being as important to outcomes as much-vaunted species "discovery".

Despite limited exposures, detailed field mapping and sedimentological analyses – particle size, clast petrography and fine-sand mineralogy (plus limited coarse silt and clay mineralogy) – have elucidated the ages, provenance and depositional environments of the complex and controversial Quaternary deposits covering the Ayot Paleogene Outlier. Most of the sediments were largely derived directly and/or indirectly from local facies of the Woolwich and Reading Formation. The oldest deposits are (Pre-)Pastonian proto-Thames gravels overlain by thin sands, which collectively represent crucial evidence for the earliest (Stoke Row) terrace of the proto-Thames. These materials were later thrust southwards by the Anglian ice close to its margin, which deposited a thin yet heterogeneous till at Ayot. This unit is correlated with the Ware Till, suggesting that the Anglian ice was thicker during the earlier of its two major southwestward incursions into the Vale of St Albans. An aeolian mantle was deposited on the Outlier during the Late Devensian; most of the blown material was incorporated into the upper regions of the till but some accumulated in depressions to form brickearths that resemble some Chiltern Brickearths. A colluvial apron developed during the Holocene, together with three swallow-holes. Some of these depositional events were separated by periods of poorly-preserved pedogenesis.

Circumscriptions of both the genus Gymnadenia and the 11–27 species that it contains are highly controversial. These Eurasian terrestrial orchids are nectar-rewarding and pollinated primarily by Le...

Recent phylogenetic trees of the phenotypically diverse Eurasian orchid genus Gymnadenia s.l., based on Sanger sequencing of nrITS and next-generation RAD-seq data, largely agree on species boundar...

Plastid sequences have long dominated phylogeny reconstruction at all time depths, predicated on a usually untested assumption that they accurately represent the evolutionary histories of phenotypically circumscribed species. We combined detailed in situ morphometrics (124 plants) and whole-plastome sequencing through genome skimming (71 plants) in order to better understand species-level diversity and speciation in arguably the most challenging monophyletic group within the taxonomically controversial, pseudo-copulatory bee orchid genus Ophrys. Using trees and ordinations, we interpreted the data at four nested demographic levels—macrospecies, mesospecies, microspecies, and local population—seeking the optimal level for bona fide species. Neither morphological nor molecular discontinuities are evident at any level below macrospecies, the observed overlap among taxa suggesting that both mesospecies and microspecies reflect arbitrary division of a continuum of variation. Plastomes represent geographic location more strongly than taxonomic assignment and correlate poorly with morphology, suggesting widespread plastid capture and possibly post-glacial expansion from multiple southern refugia. As they are rarely directly involved in the speciation process, plastomes depend on extinction of intermediate lineages to provide phylogenetic signal and so cannot adequately document evolutionary radiations. The popular ‘ethological’ evolutionary model recognizes as numerous ‘ecological species’ (microspecies) lineages perceived as actively diverging as a result of density-dependent selection on very few features that immediately dictate extreme pollinator specificity. However, it is assumed rather than demonstrated that the many microspecies are genuinely diverging. We conversely envisage a complex four-dimensional reticulate network of lineages, generated locally and transiently through a wide spectrum of mechanisms, but each unlikely to maintain an independent evolutionary trajectory long enough to genuinely speciate by escaping ongoing gene flow. The frequent but localized microevolution that characterizes the Ophrys sphegodes complex is often convergent and rarely leads to macroevolution. Choosing between the contrasting ‘discontinuity’ and ‘ethology’ models will require next-generation sequencing of nuclear genomes plus ordination of corresponding morphometric matrices, seeking the crucial distinction between retained ancestral polymorphism—consistent with lineage divergence—and polymorphisms reflecting gene flow through ‘hybridization’—more consistent with lineage convergence.

Premise of research. The evolutionary origin of the seed habit coincided with profound physiological and structural changes associated with underlying developmental patterns. A coenocyte is formed ...

Pulsatilla (Anemoneae, Ranunculaceae) is sister to Anemone s.s. and contains ca 40 perennial species of considerable horticultural and medical importance. We sequenced 31 of those species, plus nine subspecies, two cultivars and six outgroups, for two nuclear regions (high-copy nrITS and low-copy MLH1) and three plastid regions (rbcL, accD–psaI, trnL intron) in order to generate the first comprehensive species-level phylogeny of the genus. Phylogenetic trees were constructed using both concatenation-based (maximum likelihood and Bayesian inference) and coalescence methods. The better supported among the internal nodes were subjected to molecular clock dating and ancestral area reconstruction, and karyotypic characters identified by us using Fluorescence In Situ Hybridization were mapped across the tree. The preferred species tree from the coalescence analysis formed the basis of a new infrageneric classification based on monophyly plus degree of divergence. The earliest divergent of the three subgenera, Kostyczewianae, is represented by only a single species that is morphologically intermediate between Anemone s.s. and ‘core’ Pulsatilla. Subgenus Pulsatilla is considerably richer in species than its sister subgenus Preonanthus and contains three monophyletic sections. Species possessing nodding flowers and pectinately dissected leaves are phylogenetically derived compared with groups possessing erect flowers and palmately lobed leaves. Pulsatilla separated from Anemone s.s. at ca 25 Ma. Our results indicate a central Asian mountain origin of the genus and an initial diversification correlated with late Tertiary global cooling plus regional mountain uplift, aridification and consequent expansion of grasslands. The more rapid and extensive diversification within subgenus Pulsatilla began at ca 3 Ma and continued throughout the Quaternary, driven not only by major perturbations in global climate but also by well-documented polyploidy.

Disentangling phylogenetic relationships proves challenging for groups that have evolved recently, especially if there is ongoing reticulation. Although they are in most cases immediately isolated from diploid relatives, sets of sibling allopolyploids often hybridize with each other, thereby increasing the complexity of an already challenging situation. Dactylorhiza (Orchidaceae: Orchidinae) is a genus much affected by allopolyploid speciation and reticulate phylogenetic relationships. Here, we use genetic variation at tens of thousands of genomic positions to unravel the convoluted evolutionary history of Dactylorhiza. We first investigate circumscription and relationships of diploid species in the genus using coalescent and maximum likelihood methods, and then group 16 allotetraploids by maximum affiliation to their putative parental diploids, implementing a method based on genotype likelihoods. The direction of hybrid crosses is inferred for each allotetraploid using information from maternally inherited plastid RADseq loci. Starting from age estimates of parental taxa, the relative ages of these allotetraploid entities are inferred by quantifying their genetic similarity to the diploids and numbers of private alleles compared with sibling allotetraploids. Whereas northwestern Europe is dominated by young allotetraploids of postglacial origins, comparatively older allotetraploids are distributed further south, where climatic conditions remained relatively stable during the Pleistocene glaciations. Our bioinformatics approach should prove effective for the study of other naturally occurring, nonmodel, polyploid plant complexes.

SummaryRecent application of next-generation sequencing technology to Eurasian taxa of the primitive epidendroid orchid clade Epipactis section Epipactis has further advanced our knowledge of what has become a model system for studying the origins of numerous autogamous taxa from within arguably only one allogamous ancestral species, E. helleborine s.s. Current understanding of evolutionary pattern, species re-circumscription and speciation process within Epipactis is critically reviewed, emphasising the relevance of recent research based on next-generation sequencing and aiming to better understand taxa native to the British Isles. Conclusions include recognition that (1) the majority of named taxa currently widely viewed as species do not pass rigorous tests of species status, (2) two of the six unequivocal species native to Britain extend further east in Eurasia than was previously supposed, and none is endemic, (3) all scientifically defensible species of Epipactis have high frequencies of self-pollination (although autogamy is significantly less frequent in the ancestral species E. helleborine than in any of the remaining species derived from it), and (4) fully understanding the speciation process (including substantial increases in autogamy) will require us to further investigate not only pollinator behaviour and the diverse factors determining gynostemium morphology but also the equally diverse factors (including mycorrhizal specificity) likely to influence the invasion of novel, challenging habitat types.

In this chapter, we get acquainted with a powerful log interpretation technique that not only helped early log analysts find water saturation from a very limited set of log measurements but also set an example for later developments both in logging tool design and in formation evaluation. The pioneer, Maurice Tixier, cleverly saw that if a calculation required an unknown parameter the need for it could be sidestepped by the simple expedient of taking ratios of known or measured parameters. The principle can even be applied to find porosity from a compensated thermal neutron log by taking the ratio of the near and far count rates. The absolute counts depend on both porosity and the unknown effects of hole size and mud weight, but the ratio of the two is practically unaffected by anything but porosity. Tixier’s seminal paper was published in 1949 and has certainly stood the test of time.

This chapter covers the manual methods used with pre-digital well logs to determine connate water resistivity, Rw, and the rock matrix (zero-porosity) responses of the main “porosity” tools, namely the sonic, neutron, and density. Today these two parameters are derived from plots that are regularly run as subroutines in preinterpretation passes that automated interpretation packages offer. However, the analyst of yesteryear, working with paper prints, was obliged to build these plots manually using graph paper and laboriously reading pairs of log readings, plotting them, and then constructing the required trend lines that, hopefully, pinpointed the required parameter, which could then be used in the main calculations of formation porosity and water saturation.

Due to the complexity of finding formation porosity from the original “count rate” neutron tools that topic was covered in detail in Chapter 5. Now we turn our attention to all that followed the pioneering neutron logging tools and address the epithermal neutron tools and the compensated, (dual-detector) thermal neutron tools. In this chapter, we also provide a perspective of all the many combinations and permutations of the types of radioactive source, atomic particles propagated and detected, and the number of detectors and their spacings as used in what we may call the nuclear logging “zoo.”

Poor morphological and molecular differentiation in recently diversified lineages is a widespread phenomenon in plants. Phylogenetic relationships within such species complexes are often difficult to resolve because of the low variability in traditional molecular loci. Furthermore, biological phenomena responsible for topological incongruence such as Incomplete Lineage Sorting (ILS) and hybridisation complicate the resolution of phylogenetic relationships among closely related taxa. In this study, we employ a Genotyping-by-sequencing (GBS) approach to disentangle evolutionary relationships within a species complex belonging to the Neotropical orchid genus Cycnoches. This complex includes seven taxa distributed through Central America and the Colombian Chocó, and is nested within a clade estimated to have first diversified in the early Quaternary. Previous phylogenies inferred from few loci failed to provide support for internal relationships within the complex. Our Neighbour-net and coalescent-based analyses inferred from ca. 13,000 GBS loci obtained from 31 individuals belonging to six of the seven traditionally accepted Cycnoches taxa provided a robust phylogeny for this group. The genus Cycnoches includes three main clades that are further supported by morphological traits and geographic distributions. Similarly, a topology reconstructed through maximum likelihood (ML) inference of concatenated GBS loci produced results that are comparable with those reconstructed through coalescence and network-based methods. Our comparative phylogenetic informativeness analyses suggest that the low support evident in the ML phylogeny might be attributed to the abundance of uninformative GBS loci, which can account for up to 50% of the total number of loci recovered. The phylogenomic framework provided here, as well as morphological evidence and geographical patterns, suggest that the six entities previously thought to be different species or subspecies might actually represent only three distinct segregates. We further discuss the limited phylogenetic informativeness found in our GBS approach and its utility to disentangle relationships within recent and rapidly evolving species complexes. Our study is the first to demonstrate the utility of GBS data to reconstruct relationships within young (~2 Ma) Neotropical plant clades, opening new avenues for studies of species complexes that populate the species-rich orchid family UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Jardín Botánico Lankester (JBL)

In this chapter, the special case of running and interpreting logs recorded in empty, that is, air-drilled, holes is discussed. Practical guidelines are given for quantitative analysis using the pre-digital logs that were available at the time these analysis techniques were developed and put into practice.

Quick-look methods, by definition, allow the log analyst to find useful petrophysical formation properties without having to make any numerical calculations. This chapter summarizes several such methods as they were used in the pre-digital era. Many are based on visual comparison of analog curves on a paper log print. Some of these methods are still in use today, others have been forgotten but are worthy of being documented here because they will show up in the files of the pre-digital archives. Compatible overlays of analog curves mirror the philosophy of taking ratios to eliminate unknowns and were made use of widely in times gone by—but they still have their place today.

Background and aims Recent tissue-level observations made indirectly via flow cytometry suggest that endoreplication (duplication of the nuclear genome within the nuclear envelope in the absence of subsequent cell division) is widespread within the plant kingdom. Here, we also directly observe ploidy variation among cells within individual petals, relating size of nucleus to cell micromorphology and (more speculatively) to function. Methods We compared the labella (specialized pollinator-attracting petals) of two European orchid genera: Dactylorhiza has a known predisposition to organismal polyploidy, whereas Ophrys exhibits exceptionally complex epidermal patterning that aids pseudocopulatory pollination. Confocal microscopy using multiple staining techniques allowed us to observe directly both the sizes and the internal structures of individual nuclei across each labellum, while flow cytometry was used to test for progressively partial endoreplication. Key results In Dactylorhiza, endoreplication was comparatively infrequent, reached only low levels, and appeared randomly located across the labellum, whereas in Ophrys endoreplication was commonplace, being most frequent in large peripheral trichomes. Endoreplicated nuclei reflected both endomitosis and endocycling, the latter reaching the third round of genome doubling (16C) to generate polytene nuclei. All Ophrys individuals studied exhibited progressively partial endoreplication. Conclusions Comparison of the two genera failed to demonstrate the hypothesized pattern of frequent polyploid speciation in genera showing extensive endoreplication. Endoreplication in Ophrys appears more strongly positively correlated with cell size/complexity than with cell location or secretory role. Epigenetic control of gene overexpression by localized induction of endoreplication within individual plant organs may represent a significant component of a plant's developmental programme, contributing substantially to organ plasticity.

Although the majority of taxonomic studies of European orchids treat Pseudorchis as a monotypic genus, some observers have argued that the apparently circumboreal segregate P. straminea should also...

We describe the first reported hybrid to occur in nature between Dactylorhiza praetermissa and Gymnadenia borealis as × Dactylodenia lacerta R.M. Bateman & Tattersall. This vigorous plant was found...

Several recent palaeobotanical studies claim to have found and described pre-Cretaceous angiosperm macrofossils. With rare exceptions, these papers fail to define a flower, do not acknowledge that fossils require character-based rather than group-based classification, do not explicitly state which morphological features would unambiguously identify a fossil as angiospermous, ignore the modern conceptual framework of phylogeny reconstruction, and infer features in the fossils in question that are interpreted differently by (or even invisible to) other researchers. This unfortunate situation is compounded by the relevant fossils being highly disarticulated two-dimensional compression-impressions lacking anatomical preservation. Given current evidence, all supposed pre-Cretaceous angiosperms are assignable to other major clades among the gymnosperms sensu lato. By any workable morphological definition, flowers are not confined to, and therefore cannot delimit, the angiosperm clade. More precisely defined character states that are potentially diagnostic of angiosperms must by definition originate on the phylogenetic branch that immediately precedes the angiosperm crown group. Although the most reliable candidates for diagnostic characters (triploid endosperm reflecting double fertilization, closed carpel, bitegmic ovule, and phloem companion cells) are rarely preserved and/or difficult to detect unambiguously, similar characters have occasionally been preserved in high-quality permineralized non-angiosperm fossils. The angiosperm radiation documented by Early Cretaceous fossils involves only lineages closely similar to extant taxonomic families, lacks obvious morphological gaps, and (as agreed by both the fossil record and molecular phylogenies) was relatively rapid—all features that suggest a primary radiation. It is unlikely that ancestors of the crown group common ancestor would have fulfilled a character-based definition of (and thereby required expansion of the concept of) an angiosperm; they would instead form a new element of the non-angiosperm members of the ‘anthophyte’ grade, competing with Caytonia to be viewed as morphologically determined sister group for angiosperms. Conclusions drawn from molecular phylogenetics should not be allowed to routinely constrain palaeobotanical inferences; reciprocal illumination between different categories of data offers greater explanatory power than immediately resorting to Grand Syntheses. The Jurassic angiosperm—essentially a product of molecular phylogenetics—may have become the holy grail of palaeobotany but it appears equally mythical.

Background and Aims The terrestrial orchid genus Epipactis has become a model system for the study of speciation via transitions from allogamy to autogamy, but close phylogenetic relationships have proven difficult to resolve through Sanger sequencing. Methods We analysed with restriction site-associated sequencing (RAD-seq) 108 plants representing 29 named taxa that together span the genus, focusing on section Epipactis. Our filtered matrix of 12 543 single nucleotide polymorphisms was used to generate an unrooted network and a rooted, well-supported likelihood tree. We further inferred genetic structure through a co-ancestry heat map and admixture analysis, and estimated inbreeding coefficients per sample. Key Results The 27 named taxa of the ingroup were resolved as 11 genuine, geographically widespread species: four dominantly allogamous and seven dominantly autogamous. A single comparatively allogamous species, E. helleborine, is the direct ancestor of most of the remaining species, though one of the derived autogams has generated one further autogamous species. An assessment of shared ancestry suggested only sporadic hybridization between the re-circumscribed species. Taxa with the greatest inclination towards autogamy show less, if any, admixture, whereas the gene pools of more allogamous species contain a mixture alleles found in the autogams. Conclusions This clade is presently undergoing an evolutionary radiation driven by a wide spectrum of genotypic, phenotypic and environmental factors. Epipactis helleborine has also frequently generated many local variants showing inclinations toward autogamy (and occasionally cleistogamy), best viewed as incipient speciation from within the genetic background provided by E. helleborine, which thus becomes an example of a convincingly paraphyletic species. Autogams are often as widespread and ecologically successful as allogams.

The digitate-tubered clade (Dactylorhiza s.l. plus Gymnadenia s.l.) within subtribe Orchidinae is an important element of the North-temperate orchid flora and has become a model system for studying the genetic and epigenetic consequences of organism-wide ploidy change. Here, we integrate morphological phylogenetics with Sanger sequencing of nrITS and the plastid region trnL-F in order to explore phylogenetic relationships and phenotypic character evolution within the clade. The resulting morphological phylogenies are strongly incongruent with the molecular phylogenies, instead reconstructing through parsimony the genus-level boundaries recognised by traditional 20th Century taxonomy. They raise fresh doubts concerning whether Pseudorchis is sister to Platanthera or to Dactylorhiza plus Gymnadenia. Constraining the morphological matrix to the topology derived from ITS sequences increased tree length by 20%, adding considerably to the already exceptional level of phenotypic homoplasy. Both molecular and morphological trees agree that D. viridis and D. iberica are the earliest-diverging species within Dactylorhiza (emphasising the redundancy of the former genus Coeloglossum). Morphology and ITS both suggest that the former genus Nigritella is nested within (and thus part of) Gymnadenia, the Pyrenean endemic 'N.' gabasiana apparently forming a molecular bridge between the two radically contrasting core phenotypes. Comparatively short subtending molecular branches plus widespread (though sporadic) hybridisation indicate that Dactylorhiza and Gymnadenia approximate the minimum level of molecular divergence acceptable in sister genera. They share similar tuber morphologies and base chromosome numbers, and both genera are unusually prone to polyploid speciation. Another prominent feature of multiple speciation events within Gymnadenia is floral paedomorphosis. The 'traditional' morphological and candidate-gene approaches to phylogeny reconstruction are critically appraised.