Of elephants and errors: naming and identity in Linnaean taxonomy.

What is it to make an error in the identification of a named taxonomic group? In this article we argue that the conditions for being in error about the identity of taxonomic groups through their names have a history, and that the possibility of committing such errors is contingent on the regime of institutions and conventions governing taxonomy and nomenclature at any given point in time. More specifically, we claim that taxonomists today can be in error about the identity of taxonomic groups in a way that Carl Linnaeus (1707-1778), who is routinely cited as the "founder" of modern taxonomy and nomenclature, simply could not be. Starting from a remarkable recent study into Linnaeus's naming of Elephas maximus that led to the (putative) discovery of a (putative) nomenclatural error by him, we reconsider what it could mean to discover that Linnaeus misidentified a biological taxon in applying his taxon names. Through a further case study in Linnaean botany, we show that his practices of (re)applying names in taxonomic revisions reveal a take on determining "which taxon is which" that is strikingly different from that of contemporary taxonomists. Linnaeus, we argue, adopted a practice-based, hands-on concept of taxa as "nominal spaces" that could continue to represent the same taxon even if all its former members had been reallocated to other taxa.

Incongruence in molecular species delimitation schemes: What to do when adding more data is difficult.

Using multiple, independent approaches to molecular species delimitation is advocated to accommodate limitations and assumptions of a single approach. Incongruence in delimitation schemes is a potential by-product of employing multiple methods on the same data, and little attention has been paid to its reconciliation. Instead, a particular scheme is prioritized, and/or molecular delimitations are coupled with additional, independent lines of evidence that mitigate incongruence. We advocate that incongruence within a line of evidence should be accounted for before comparing across lines of evidence that can themselves be incongruent. Additionally, it is not uncommon for empiricists working in nonmodel systems to be data-limited, generating some concern for the adequacy of available data to address the question of interest. With conservation and management decisions often hinging on the status of species, it seems prudent to understand the capabilities of approaches we use given the data we have. Here, we apply two molecular species delimitation approaches, spedeSTEM and BPP, to the Castilleja ambigua (Orobanchaceae) species complex, a relatively young plant lineage in western North America. Upon finding incongruence in our delimitation, we employed a post hoc simulation study to examine the power of these approaches to delimit species. Given the data we collected, we find that spedeSTEM lacks the power to delimit while BPP is capable, thus allowing us to address incongruence before proceeding in delimitation. We suggest post hoc simulation studies like this compliment empirical delimitation and serve as a means of exploring conflict within a line of evidence and dealing with it appropriately.

Limited mitogenomic degradation in response to a parasitic lifestyle in Orobanchaceae.

In parasitic plants, the reduction in plastid genome (plastome) size and content is driven predominantly by the loss of photosynthetic genes. The first completed mitochondrial genomes (mitogenomes) from parasitic mistletoes also exhibit significant degradation, but the generality of this observation for other parasitic plants is unclear. We sequenced the complete mitogenome and plastome of the hemiparasite Castilleja paramensis (Orobanchaceae) and compared them with additional holoparasitic, hemiparasitic and nonparasitic species from Orobanchaceae. Comparative mitogenomic analysis revealed minimal gene loss among the seven Orobanchaceae species, indicating the retention of typical mitochondrial function among Orobanchaceae species. Phylogenetic analysis demonstrated that the mobile cox1 intron was acquired vertically from a nonparasitic ancestor, arguing against a role for Orobanchaceae parasites in the horizontal acquisition or distribution of this intron. The C. paramensis plastome has retained nearly all genes except for the recent pseudogenization of four subunits of the NAD(P)H dehydrogenase complex, indicating a very early stage of plastome degradation. These results lend support to the notion that loss of ndh gene function is the first step of plastome degradation in the transition to a parasitic lifestyle.

Complete Plastid Genome of the Recent Holoparasite Lathraea squamaria Reveals Earliest Stages of Plastome Reduction in Orobanchaceae.

Plants from the family Orobanchaceae are widely used as a model to study different aspects of parasitic lifestyle including host-parasite interactions and physiological and genomic adaptations. Among the latter, the most prominent are those that occurred due to the loss of photosynthesis; they include the reduction of the photosynthesis-related gene set in both nuclear and plastid genomes. In Orobanchaceae, the transition to non-photosynthetic lifestyle occurred several times independently, but only one lineage has been in the focus of evolutionary studies. These studies included analysis of plastid genomes and transcriptomes and allowed the inference of patterns and mechanisms of genome reduction that are thought to be general for parasitic plants. Here we report the plastid genome of Lathraea squamaria, a holoparasitic plant from Orobanchaceae, clade Rhinantheae. We found that in this plant the degree of plastome reduction is the least among non-photosynthetic plants. Like other parasites, Lathraea possess a plastome with elevated absolute rate of nucleotide substitution. The only gene lost is petL, all other genes typical for the plastid genome are present, but some of them-those encoding photosystem components (22 genes), cytochrome b6/f complex proteins (4 genes), plastid-encoded RNA polymerase subunits (2 genes), ribosomal proteins (2 genes), ccsA and cemA-are pseudogenized. Genes for cytochrome b6/f complex and photosystems I and II that do not carry nonsense or frameshift mutations have an increased ratio of non-synonymous to synonymous substitution rates, indicating the relaxation of purifying selection. Our divergence time estimates showed that transition to holoparasitism in Lathraea lineage occurred relatively recently, whereas the holoparasitic lineage Orobancheae is about two times older.

The genetic structure of wild Orobanche cumana Wallr. (Orobanchaceae) populations in eastern Bulgaria reflects introgressions from weedy populations.

Orobanche cumana is a holoparasitic plant naturally distributed from central Asia to south-eastern Europe, where it parasitizes wild Asteraceae species. It is also an important parasitic weed of sunflower crops. The objective of this research was to investigate genetic diversity, population structure, and virulence on sunflower of O. cumana populations parasitizing wild plants in eastern Bulgaria. Fresh tissue of eight O. cumana populations and mature seeds of four of them were collected in situ on wild hosts. Genetic diversity and population structure were studied with SSR markers and compared to weedy populations. Two main gene pools were identified in Bulgarian populations, with most of the populations having intermediate characteristics. Cross-inoculation experiments revealed that O. cumana populations collected on wild species possessed similar ability to parasitize sunflower to those collected on sunflower. The results were explained on the basis of an effective genetic exchange between populations parasitizing sunflower crops and those parasitizing wild species. The occurrence of bidirectional gene flow may have an impact on wild populations, as new physiological races continuously emerge in weedy populations. Also, genetic variability of wild populations may favour the ability of weedy populations to overcome sunflower resistance mechanisms.

Inferring phylogeny and introgression using RADseq data: an example from flowering plants (Pedicularis: Orobanchaceae).

Phylogenetic relationships among recently diverged species are often difficult to resolve due to insufficient phylogenetic signal in available markers and/or conflict among gene trees. Here we explore the use of reduced-representation genome sequencing, specifically in the form of restriction-site associated DNA (RAD), for phylogenetic inference and the detection of ancestral hybridization in non-model organisms. As a case study, we investigate Pedicularis section Cyathophora, a systematically recalcitrant clade of flowering plants in the broomrape family (Orobanchaceae). Two methods of phylogenetic inference, maximum likelihood and Bayesian concordance, were applied to data sets that included as many as 40,000 RAD loci. Both methods yielded similar topologies that included two major clades: a "rex-thamnophila" clade, composed of two species and several subspecies with relatively low floral diversity, and geographically widespread distributions at lower elevations, and a "superba" clade, composed of three species characterized by relatively high floral diversity and isolated geographic distributions at higher elevations. Levels of molecular divergence between subspecies in the rex-thamnophila clade are similar to those between species in the superba clade. Using Patterson's D-statistic test, including a novel extension of the method that enables finer-grained resolution of introgression among multiple candidate taxa by removing the effect of their shared ancestry, we detect significant introgression among nearly all taxa in the rex-thamnophila clade, but not between clades or among taxa within the superba clade. These results suggest an important role for geographic isolation in the emergence of species barriers, by facilitating local adaptation and differentiation in the absence of homogenizing gene flow.

The differentially regulated genes TvQR1 and TvPirin of the parasitic plant Triphysaria exhibit distinctive natural allelic diversity.

BACKGROUND: Plant parasitism represents an extraordinary interaction among flowering plants: parasitic plants use a specialized organ, the haustorium, to invade the host vascular system to deprive host plants of water and nutrients. Various compounds present in exudates of host plants trigger haustorium development. The two most effective haustorium inducing factors (HIFs) known for the parasitic plant Triphysaria versicolor (T. versicolor) are peonidin, an antioxidant flavonoid, and 2,6-dimethoxybenzoquinone (DMBQ), an oxidative stress agent. To date, two genes involved in haustorium initiation in T. versicolor have been identified: TvQR1, a quinone oxidoreductase that generates the active HIF from DMBQ, and TvPirin, a transcription co-factor that regulates several other DMBQ- responsive and -non-responsive genes. While the expression of these genes in response to DMBQ is well characterized, their expression in response to peonidin is not. In addition, the pattern of polymorphisms in these genes is unknown, even though nucleotide changes in TvQR1 and TvPirin may have contributed to the ability of T. versicolor to develop haustoria. To gain insights into these aspects, we investigated their transcriptional responses to HIFs and non-HIF and their natural nucleotide diversity. RESULTS: Here we show that TvQR1 and TvPirin are transcriptionally upregulated by both DMBQ and peonidin in T. versicolor roots. Yet, while TvQR1 also responded to juglone, a non-HIF quinone with toxicity comparable to that of DMBQ, TvPirin did not. We further demonstrate that TvPirin encodes a protein shorter than the one previously reported. In the T. versicolor natural population of Northern California, TvQR1 exhibited remarkably higher molecular diversity and more recombination events than TvPirin, with the highest non-synonymous substitution rate in the substrate recognition and catalytic domain of the TvQR1 protein. CONCLUSION: Our results suggest that TvQR1 and TvPirin have most likely evolved highly distinct roles for haustorium formation. Unlike TvPirin, TvQR1 might have been under diversifying selection to maintain a diverse collection of polymorphisms, which might be related to the recognition of an assortment of HIF and non-HIF quinones as substrates for successful haustorial establishment in a wide range of host plants.

Post-pollination barriers and their role in asymmetric hybridization in Rhinanthus (Orobanchaceae).

PREMISE OF THE STUDY: Several barriers against hybrid formation exist, and their combined action can affect the evolutionary outcomes of hybridization. To explain the asymmetrical introgression observed between two bumblebee-pollinated plant species (Rhinanthus angustifolius and R. minor), we focused on post-pollination barriers and ethological isolation of hybrids. METHODS: We studied pollen competition in conspecific and heterospecific crosses on both species and analyzed germination rates of hybrid and pure seeds. We also measured bumblebee visitation rates to hybrids relative to their parents using potted Rhinanthus placed in populations of each parental species. KEY RESULTS: In mixed pollinations, there was a conspecific siring advantage in both species, but no difference in pollen tube growth rates in either cross type. F(1) seeds with a R. angustifolius maternal plant germinated poorly, while those with R. minor as the maternal parent germinated better than pure seeds. Interestingly, bumblebees treated hybrids almost as equal to the background species and more often rejected the nonresident Rhinanthus. In a R. angustifolius background, bumblebees preferred R. angustifolius, but visited hybrids more often than R. minor. In contrast, visitation rates were similar on a R. minor background. CONCLUSIONS: Our results suggest that hybridization rates in Rhinanthus remain low because of several leaky barriers that make R. minor the maternal parent of most F(1) offspring. Preference for R. angustifolius and the equal treatment of F(1) and background species by bumblebees induce a visitation pattern that directs gene flow toward R. angustifolius when this species predominates.

The TvPirin gene is necessary for haustorium development in the parasitic plant Triphysaria versicolor.

The rhizosphere is teemed with organisms that coordinate their symbioses using chemical signals traversing between the host root and symbionts. Chemical signals also mediate interactions between roots of different plants, perhaps the most obvious being those between parasitic Orobanchaceae and their plant hosts. Parasitic plants use specific molecules provided by host roots to initiate the development of haustoria, invasive structures critical for plant parasitism. We took a transcriptomics approach to identify parasitic plant genes associated with host factor recognition and haustorium signaling and previously identified a gene, TvPirin, which is transcriptionally up-regulated in roots of the parasitic plant Triphysaria versicolor after being exposed to the haustorium-inducing molecule 2,6-dimethoxybenzoquinone (DMBQ). Because TvPirin shares homology with proteins associated with environmental signaling in some plants, we hypothesized that TvPirin may function in host factor recognition in parasitic plants. We tested the function of TvPirin in T. versicolor roots using hairpin-mediated RNA interference. Reducing TvPirin transcripts in T. versicolor roots resulted in significantly less haustoria development in response to DMBQ exposure. We determined the transcript levels of other root expressed transcripts and found that several had reduced basal levels of gene expression but were similarly regulated by quinone exposure. Phylogenic investigations showed that TvPirin homologs are present in most flowering plants, and we found no evidence of parasite-specific gene duplication or expansion. We propose that TvPirin is a generalized transcription factor associated with the expression of a number of genes, some of which are involved in haustorium development.

Development and characterization of polymorphic microsatellite markers for Conopholis americana (Orobanchaceae).

PREMISE OF THE STUDY: Conopholis americana is an obligate root parasite with highly reduced morphology. To investigate the population structure, genetic diversity, and mating system of this predominantly eastern North American species, we developed polymorphic microsatellite markers for C. americana. METHODS AND RESULTS: Using an enrichment cloning protocol, we report the isolation and characterization of 11 microsatellite markers. Product size varied from 198-370 bp. These loci show moderate levels of allelic variation (averaging 4.182 alleles per locus) and very low levels of heterozygosity (average observed heterozygosity = 0.054). CONCLUSIONS: These microsatellite markers will be useful in obtaining estimates of population-level genetic diversity and in phylogeographic studies of C. americana.

Transcriptomes of the parasitic plant family Orobanchaceae reveal surprising conservation of chlorophyll synthesis.

Parasitism in flowering plants has evolved at least 11 times [1]. Only one family, Orobanchaceae, comprises all major nutritional types of parasites: facultative, hemiparasitic (partially photosynthetic), and holoparasitic (nonphotosynthetic) [2]. Additionally, the family includes Lindenbergia, a nonparasitic genus sister to all parasitic Orobanchaceae [3-6]. Parasitic Orobanchaceae include species with severe economic impacts: Striga (witchweed), for example, affects over 50 million hectares of crops in sub-Saharan Africa, causing more than $3 billion in damage annually [7]. Although gene losses and increased substitution rates have been characterized for parasitic plant plastid genomes [5, 8-11], the nuclear genome and transcriptome remain largely unexplored. The Parasitic Plant Genome Project (PPGP; http://ppgp.huck.psu.edu/) [2] is leveraging the natural variation in Orobanchaceae to explore the evolution and genomic consequences of parasitism in plants through a massive transcriptome and gene discovery project involving Triphysaria versicolor (facultative hemiparasite), Striga hermonthica (obligate hemiparasite), and Phelipanche aegyptiaca (Orobanche [12]; holoparasite). Here we present the first set of large-scale genomic resources for parasitic plant comparative biology. Transcriptomes of above-ground tissues reveal that, in addition to the predictable loss of photosynthesis-related gene expression in P. aegyptiaca, the nonphotosynthetic parasite retains an intact, expressed, and selectively constrained chlorophyll synthesis pathway.

A sequential approach using genetic and morphological analyses to test species status: the case of United States federally endangered Agalinis acuta (Orobanchaceae).

PREMISE OF THE STUDY: Given that inaccurate taxonomy can have negative consequences for species of conservation concern and result in erroneous conclusions regarding macroecological patterns, efficient methods for resolving taxonomic uncertainty are essential. The primary objective of this study was to assess the evolutionary distinctiveness of the federally endangered plant species Agalinis acuta (Orobanchaceae) to ensure it represents a distinct taxon warranting protection under the United States Endangered Species Act. METHODS: We describe and implement a sequential approach that begins with the most restrictive criteria of genealogical exclusivity within which we first conducted a phylogenetic analysis based on six chloroplast DNA loci assayed from multiple representatives of five putative species. Because of the possibility that incomplete lineage sorting is responsible for the lack of genealogical exclusivity among A. acuta individuals, we then conducted intensive population level analyses based on 21 microsatellite loci and 61 morphological traits. KEY RESULTS: The distinctiveness of A. acuta from Agalinis decemloba and Agalinis tenella was not supported under the genealogical species concept. The results from the analyses of microsatellite loci and morphological characters evaluated under alternative species concepts also did not support the distinctiveness of A. acuta from A. decemloba . CONCLUSIONS: Through this successive approach, we found insufficient evidence to support the evolutionary distinctiveness of the listed taxon A. acuta . We recommend that it be synonymized under A. decemloba and also conclude that the taxon that would now include A. acuta is deserving of protection under the Endangered Species Act.

Molecular systematics of the parasitic genus Conopholis (Orobanchaceae) inferred from plastid and nuclear sequences.

PREMISE OF THE STUDY: Little is known of the evolutionary relationships within Conopholis, a small holoparasitic genus belonging to the broomrape family. Presently, Conopholis is described as having two species, C. americana and C. alpina. This classification is based on a combination of presence/absence of morphological characters along with a number of quantitative traits. We assessed the relationships among populations and species of this genus to determine whether the present taxonomic hypothesis is reflected in molecular phylogenies. METHODS: We conducted the first phylogenetic study of Conopholis using plastid (trnfM-E intergenic spacer and clpP gene/introns) and nuclear (PHYA intron 1) sequences from a wide taxonomic sampling covering its entire geographical range in North America. Analyses were carried out using a variety of phylogenetic inference approaches. KEY RESULTS: Reciprocal monophyly between the two traditionally accepted species has not yet been achieved. Instead, three distinct genetic clusters were recovered. Conopholis alpina is clearly paraphyletic and shows evidence of belonging to at least two distinct lineages. Specimens found in Costa Rica and Panama form a distinct group from those located in northern Mexico and the southwestern United States. The monophyly of C. americana was also not recovered; however, the possibility of it being monophyletic could not be rejected with confidence. CONCLUSIONS: These analyses recovered three distinct lineages indicating that there could be a minimum of three species within the genus. A reevaluation of morphological features within Conopholis may reveal shared features that could further corroborate our molecular findings.

Phylogenetic patterns and conservation among North American members of the genus Agalinis (Orobanchaceae).

BACKGROUND: North American Agalinis Raf. species represent a taxonomically challenging group and there have been extensive historical revisions at the species, section, and subsection levels of classification. The genus contains many rare species, including the federally listed endangered species Agalinis acuta. In addition to evaluating the degree to which historical classifications at the section and subsection levels are supported by molecular data sampled from 79 individuals representing 29 Agalinis species, we assessed the monophyly of 27 species by sampling multiple individuals representing different populations of those species. Twenty-one of these species are of conservation concern in at least some part of their range. RESULTS: Phylogenetic relationships estimated using maximum likelihood analyses of seven chloroplast DNA loci (aligned length = 11,076 base pairs (bp) and the nuclear ribosomal DNA ITS (internal transcribed spacer) locus (733 bp); indicated no support for the historically recognized sections except for Section Erectae. Our results suggest that North American members of the genus comprise six major lineages, however we were not able to resolve branching order among many of these lineages. Monophyly of 24 of the 29 sampled species was supported based on significant branch lengths of and high bootstrap support for subtending branches. However, there was no statistical support for the monophyly of A. acuta with respect to Agalinis tenella and Agalinis decemloba. Although most species were supported, deeper relationships among many species remain ambiguous. CONCLUSION: The North American Agalinis species sampled form a well supported, monophyletic group within the family Orobanchaceae relative to the outgroups sampled. Most hypotheses regarding section- and subsection-level relationships based on morphology were not supported and taxonomic revisions are warranted. Lack of support for monophyly of Agalinis acuta leaves the important question regarding its taxonomic status unanswered. Lack of resolution is potentially due to incomplete lineage sorting of ancestral polymorphisms among recently diverged species; however the gene regions examined did distinguish among almost all other species in the genus. Due to the important policy implications of this finding we are further evaluating the evolutionary distinctiveness of A. acuta using morphological data and loci with higher mutation rates.

Correlated evolution of life history and host range in the nonphotosynthetic parasitic flowering plants Orobanche and Phelipanche (Orobanchaceae).

Most members of the nonphotosynthetic parasitic genera Orobanche and Phelipanche (Orobanchaceae) have narrow host ranges, and, as they grow on perennial hosts, are (at least potentially) perennial themselves. A few species, however, have wide host ranges and grow on annual hosts, and are thus (at least facultatively) annuals themselves. Among the latter are the weedy species, which include economically important pest taxa such as Orobanche crenata or Phelipanche aegyptiaca. Using a phylogenetically based maximum likelihood approach, which takes phylogenetic and branch length uncertainty into account, we can show that the life trait host range and life history evolve in a correlated fashion. This supports the hypothesis that parasite specialization is associated with predictable resources (i.e. long-lived hosts) and generalism with unpredictable ones (i.e. short-lived hosts), a pattern often found in animal parasites. The mechanisms and temporal sequence of the life trait changes and their interrelations remain speculative.

Diversity and evolution of Ty1-copia and Ty3-gypsy retroelements in the non-photosynthetic flowering plants Orobanche and Phelipanche (Orobanchaceae).

We present the first study on the diversity and evolution of Ty1-copia and Ty3-gypsy retroelements in a group of non-photosynthetic flowering plants. To this end partial sequences of the reverse transcriptase (rt) gene were obtained from 20 clones for each retroelement type from seven and six accessions of Orobanche and Phelipanche (Orobanchaceae), respectively. Overall sequence similarity is higher in Ty3-gypsy elements than in Ty1-copia elements in agreement with the results from other angiosperm groups. Higher sequence diversity and stronger phylogenetic structure, especially of Ty1-copia sequences, in Orobanche species compared to Phelipanche species support the previously suggested hypothesis (based on karyological and cytological data) that genomes of Orobanche species are more dynamic than those of Phelipanche species. No evidence was found for intraspecific differences of retroelement diversity nor for differences between pest taxa and their putative wild relatives, e.g., O. crenata and O. owerini. The occurrence of a few sequences from Phelipanche species in clades otherwise comprising sequences from Orobanche species might be due to horizontal gene transfer, but the alternative of vertical transmission cannot be rejected unambiguously.

Section-level relationships of North American Agalinis (Orobanchaceae) based on DNA sequence analysis of three chloroplast gene regions.

BACKGROUND: The North American Agalinis are representatives of a taxonomically difficult group that has been subject to extensive taxonomic revision from species level through higher sub-generic designations (e.g., subsections and sections). Previous presentations of relationships have been ambiguous and have not conformed to modern phylogenetic standards (e.g., were not presented as phylogenetic trees). Agalinis contains a large number of putatively rare taxa that have some degree of taxonomic uncertainty. We used DNA sequence data from three chloroplast genes to examine phylogenetic relationships among sections within the genus Agalinis Raf. (=Gerardia), and between Agalinis and closely related genera within Orobanchaceae. RESULTS: Maximum likelihood analysis of sequences data from rbcL, ndhF, and matK gene regions (total aligned length 7323 bp) yielded a phylogenetic tree with high bootstrap values for most branches. Likelihood ratio tests showed that all but a few branch lengths were significantly greater than zero, and an additional likelihood ratio test rejected the molecular clock hypothesis. Comparisons of substitution rates between gene regions based on linear models of pairwise distance estimates between taxa show both ndhF and matK evolve more rapidly than rbcL, although the there is substantial rate heterogeneity within gene regions due in part to rate differences among codon positions. CONCLUSIONS: Phylogenetic analysis supports the monophyly of Agalinis, including species formerly in Tomanthera, and this group is sister to a group formed by the genera Aureolaria, Brachystigma, Dasistoma, and Seymeria. Many of the previously described sections within Agalinis are polyphyletic, although many of the subsections appear to form natural groups. The analysis reveals a single evolutionary event leading to a reduction in chromosome number from n = 14 to n = 13 based on the sister group relationship of section Erectae and section Purpureae subsection Pedunculares. Our results establish the evolutionary distinctiveness of A. tenella from the more widespread and common A. obtusifolia. However, further data are required to clearly resolve the relationship between A. acuta and A. tenella.