Chloroplast psbA Gene Research Articles

Herbicide-resistant plants produced by precision adenine base editing in plastid DNA.

CRISPR-free, protein-only cytosine base editors (CBEs) or adenine base editors, composed of DNA-binding proteins such as zinc finger proteins or transcription activator-like effectors (TALEs) and nucleobase cytosine or adenine deaminases, respectively, enable organellar DNA editing in cultured cells, animals and plants1-4. TALE-linked double-stranded DNA deaminase toxin A (DddAtox)-derived CBEs (DdCBEs) and TALE-linked adenine deaminases (TALEDs) install C-to-T and A-to-G single-nucleotide conversions, respectively, in mitochondria and chloroplasts5-9. Interestingly, whereas TALEDs exclusively induce A-to-G conversions without C-to-T conversions in mammalian mitochondrial DNA10, they often install unwanted C-to-T edits in addition to intended A-to-G edits in plastid DNA7,9,11,12. Here we show that uracil DNA glycosylase (UDG)-fused TALEDs (UDG-TALEDs) minimize C-to-T conversions without reducing the A-to-G editing efficiency and install a mutation in the chloroplast psbA gene that encodes a single-amino-acid substitution (S264G), which confers herbicide resistance in the resulting plants.

Cloning and functional characterization of the peptide deformylase encoding gene EuPDF1B from Eucommia ulmoides Oliv

Peptide deformylase can catalyse the removal of formyl groups from the N-terminal formyl methionine of the primary polypeptide chain. The peptide deformylase genes of a few herbaceous plants have been studied to some extent, but the peptide deformylase genes of woody plants have not been studied. In this study, we isolated EuPDF1B from Eucommia ulmoides Oliv. The full-length sequence of EuPDF1B is 1176 bp long with a poly-A tail and contains an open reading frame of 831 bp that encodes a protein of 276 amino acids. EuPDF1B was localized to the chloroplast. qRT‒PCR analysis revealed that this gene was expressed in almost all tissues tested but mainly in mature leaves. Moreover, the expression of EuPDF1B was enhanced by ABA, MeJA and GA and inhibited by shading treatment. The expression pattern of EuPDF1B was further confirmed in EuPDF1Bp: GUS transgenic tobacco plants. Among all the transgenic tobacco plants, EuPDF1Bp-3 showed the highest GUS histochemical staining and activity in different tissues. This difference may be related to the presence of enhancer elements in the region from − 891 bp to − 236 bp of the EuPDF1B promoter. In addition, the expression of the chloroplast gene psbA and the net photosynthetic rate, fresh weight and height of tobacco plants overexpressing EuPDF1B were greater than those of the wild-type tobacco plants, suggesting that EuPDF1B may promote the growth of transgenic tobacco plants. This is the first time that PDF and its promoter have been cloned from woody plants, laying a foundation for further analysis of the function of PDF and the regulation of its expression.

Taxonomic revision of Tinocladia (Ectocarpales s.l., Phaeophyceae): merger of Tinocladia with Eudesme and description of E. pseudocrassa sp. nov

ABSTRACT A molecular phylogeny of Tinocladia and Eudesme based on specimens covering a large proportion of the known species was done using mitochondrial cox1 and cox3, chloroplast atpB, psaA, psbA and rbcL genes and 5.8S rDNA and its ITS2 region sequences. The phylogeny revealed a close phylogenetic relationship between the two genera and the occurrence of a cryptic species within the generitype T. crassa. Eudesme species (E. borealis, E. shandongensis and the generitype E. virescens) showed a sister relationship with the northern hemisphere Tinocladia (T. crassa, including a cryptic sister species T. pseudocrassa sp. nov., and T. sanrikuensis), whereas southern hemisphere Tinocladia (T. australis, T. falklandica, T. novae-zelandiae, and a cryptic species from New Zealand) nested in the Eudesme clade. Morphologically, Tinocladia species have been distinguished from Eudesme by their denser medullary filaments and more extensive subcortical layer, but our molecular phylogenetic analyses suggest that these features do not correlate with genetic differences. The cox3 sequence divergence between northern hemisphere Tinocladia and the lineage that includes Eudesme and southern hemisphere Tinocladia was comparable to or smaller than those within selected ectocarpalean genera. We therefore propose to merge Tinocladia with Eudesme and resurrect Eudesme crassa (Suringar) Okamura for T. crassa. Although the two independent lineages of T. crassa do not show marked morphological differences, they are genetically isolated even in sympatric populations. We therefore propose to treat them as independent species. In this paper we neotypify Tinocladia crassa (=Eudesme crassa), describe E. pseudocrassa sp. nov., and determine that the Californian population of E. crassa is an introduction from Northeast Asia. HIGHLIGHTS •A taxonomic revision of Eudesme and Tinocladia, based on genetic and morpho-anatomy, provides evidence to merge Tinocladia with Eudesme. •The finding of cryptic species in New Zealand highlights the need for further taxonomic studies in this region. •The genetic data have revealed the occurrence of a cryptic species within T. crassa in Japan.

Oral booster vaccine antigen-Expression of full-length native SARS-CoV-2 spike protein in lettuce chloroplasts.

Oral booster vaccine antigen-Expression of full-length native SARS-CoV-2 spike protein in lettuce chloroplasts.

Diversity and Ecology of Lobophora Species Associated with Coral Reef Systems in the Western Gulf of Thailand, including the Description of Two New Species

The brown macroalgal genus Lobophora plays important ecological roles in many marine ecosystems. This group has received much attention over the past decade, and a considerable number of new species have been identified globally. However, our knowledge of the genus diversity and ecology along south-east Asian coasts are still limited. Given the growing body of research that uses a combination of molecular and morphological data to identify cryptic species, this study investigates the diversity of Lobophora in the western Gulf of Thailand using morphological and molecular data, as well as their interactions with scleractinian corals. A total of 36 Lobophora specimens were collected from 15 sites in the western Gulf of Thailand and used for molecular and morphological analyses. One mitochondrial (cox3) and two chloroplast (psbA and rbcL) genes were amplified and sequenced for molecular phylogenetic analyses. Based primarily on phylogenetic evidence, two new species were formally described, L. chumphonensis sp. nov. and L. thailandensis sp. nov. Additionally, L. lamourouxii was newly recorded from Thailand. Two new lineages of Lobophora obscura were identified, L. obscura12 and L. obscura13. Among the Lobophora species identified, three were found in interaction with corals, the most notable of which was the massive coral Porites. Lobophora chumphonensis sp. nov. only interacted with Porites by growing on bare coral skeleton between Porites colonies. Furthermore, L. obscura13 was observed under the branching coral Pocillopora. Our findings revealed that Lobophora presented both effects and absence of effects on coral. A thorough understanding of Lobophora diversity and ecology is essential for ongoing and future research on coral-macroalgal ecological relationships.

Transfer of Pilinia from Ectocarpales to Ishigeales (Phaeophyceae) with proposal of Piliniaceae fam. nov., and taxonomy of Porterinema in Ectocarpales

ABSTRACT Molecular phylogeny based on concatenated sequences of chloroplast atpB, psaA, psbA, psbC and rbcL genes indicates that the filamentous brown alga Pilinia rimosa is phylogenetically distant from the Ectocarpales, in which it has been classified (as Waerniella lucifuga). In the tree, Pilinia was sister to the clade comprised of Ishige and Petroderma (Ishigeales) supported by high statistical values. Observations of unialgal P. rimosa cultures originating from Helgoland (Germany), England (UK), Woods Hole (USA) and Newfoundland (Canada) showed a direct type of life history with reproduction by characteristic unilocular zoidangia typically forming only four zoids. Despite the description of plurilocular zoidangia in Kuckuck’s account as well as in later publications, we conclude that those observations were based on misinterpretation of the very small unilocular zoidangia formed in series, often with longitudinal walls between zoidangia. Pilinia rimosa grows in caves or crevices in rocks, and on walls or wood pilings in deep shade in the upper intertidal and spray zone, and is therefore regarded as a marine species, but it could also survive and mature in freshwater medium. We propose the establishment of a new family Piliniaceae for Pilinia and place it in Ishigeales. Molecular and morphological analysis of the culture strains, SAG 124.79 and SAG 2381, identified as the euryhaline alga Porterinema fluviatile, indicated that SAG 124.79 is in fact P. rimosa. However, SAG 2381 is true P. fluviatile, also displaying the characteristic plurilocular zoidangia. Porterinema was shown to be a member of the Chordariaceae, Ectocarpales sensu lato.

<strong>Diversity and phylogeny of the brown alga <em>Lobophora</em> (Dictyotales, Phaeophyceae) in Singapore</strong>

The brown macroalgal genus Lobophora (Phaeophyceae: Dictyotaceae) plays an ecologically significant role in many marine ecosystems, but their diversity and taxonomy remain poorly studied. Until 2012, six Lobophora species had been recognised globally based on morphological features. Yet, with more than 100 evolutionary taxonomic units characterised to date, it is now acknowledged that Lobophora comprises many cryptic species and its diversity was vastly underestimated. In light of a growing body of research integrating molecular and morphological data to delimit cryptic species, this study assessed the diversity and phylogeny of Lobophora in Singapore. A combination of molecular data and morphological observations were used to delimit and identify species from 33 specimens collected at eight sites in the southern islands of Singapore. The mitochondrial cox3 and chloroplast psbA genes were amplified and sequenced for phylogenetic analysis. Three formally described species, L. challengeriae, L. lamourouxii, L. pachyventera (comprising two morphotypes), as well as one undescribed putative species, Lobophora sp61, were recovered. These findings replace the record of the Atlantic species L. variegata in Singapore and suggest that there are more species to be discovered in the biodiverse region of Southeast Asia. Precise understanding of Lobophora diversity is critical for ongoing and future work on coral–macroalgal ecological relationships.

Nuclear-encoded synthesis of the D1 subunit of photosystem II increases photosynthetic efficiency and crop yield

In photosynthetic organisms, the photosystem II (PSII) complex is the primary target of thermal damage. Plants have evolved a repair process to prevent the accumulation of damaged PSII. The repair of PSII largely involves de novo synthesis of proteins, particularly the D1 subunit protein encoded by the chloroplast gene psbA. Here we report that the allotropic expression of the psbA complementary DNA driven by a heat-responsive promoter in the nuclear genome sufficiently protects PSII from severe loss of D1 protein and dramatically enhances survival rates of the transgenic plants of Arabidopsis, tobacco and rice under heat stress. Unexpectedly, we found that the nuclear origin supplementation of the D1 protein significantly stimulates transgenic plant growth by enhancing net CO2 assimilation rates with increases in biomass and grain yield. These findings represent a breakthrough in bioengineering plants to achieve efficient photosynthesis and increase crop productivity under normal and heat-stress conditions.

Functional Analysis of PSRP1, the Chloroplast Homolog of a Cyanobacterial Ribosome Hibernation Factor.

Bacterial ribosome hibernation factors sequester ribosomes in an inactive state during the stationary phase and in response to stress. The cyanobacterial ribosome hibernation factor LrtA has been suggested to inactivate ribosomes in the dark and to be important for post-stress survival. In this study, we addressed the hypothesis that Plastid Specific Ribosomal Protein 1 (PSRP1), the chloroplast-localized LrtA homolog in plants, contributes to the global repression of chloroplast translation that occurs when plants are shifted from light to dark. We found that the abundance of PSRP1 and its association with ribosomes were similar in the light and the dark. Maize mutants lacking PSRP1 were phenotypically normal under standard laboratory growth conditions. Furthermore, the absence of PSRP1 did not alter the distribution of chloroplast ribosomes among monosomes and polysomes in the light or in the dark, and did not affect the light-regulated synthesis of the chloroplast psbA gene product. These results suggest that PSRP1 does not play a significant role in the regulation of chloroplast translation by light. As such, the physiological driving force for the retention of PSRP1 during chloroplast evolution remains unclear.

Tinocladia sanrikuensis sp. nov. (Ectocarpales s.l., Phaeophyceae) from Japan

SUMMARYThe new species Tinocladia sanrikuensis sp. nov. H.Kawai, K.Takeuchi & T.Hanyuda (Ectocarpales s.l., Phaeophyceae) is described from the Pacific coast of the Tohoku region, northern Japan based on morphology and DNA sequences. The species is a spring–summer annual growing on lower intertidal to upper subtidal rocks and cobbles on relatively protected sites. T. sanrikuensis has a slimy, cylindrical, multiaxial erect thallus, slightly hollow when fully developed, branching once to twice, and resembles T. crassa in gross morphology. The erect thalli are composed of a dense medullary layer, long subcortical filaments, and assimilatory filaments of 11–35 cells, up to 425 μm long and curved in the upper portion. Unilocular zoidangia are formed on the basal part of assimilatory filaments. The species is genetically most closely related to T. crassa and has the same basic thallus structures but differs in having thinner and longer assimilatory filaments. DNA sequences of the mitochondrial cox1 and cox3, chloroplast atpB, psaA, psbA and rbcL genes support the distinctness of this species.

Taxonomic revision of Eudesme (Ectocarpales s.l., Phaeophyceae) proposing a new species E. borealis sp. nov.

ABSTRACTMolecular phylogeny of Eudesme specimens collected from various localities in the Northern Hemisphere using mitochondrial cox1 and cox3 and chloroplast atpB, psaA, psbA and rbcL gene sequences revealed an undescribed species in the genus. In the analyses, three distinct clades (clades 1, 2 and 3) with high statistical supports were recognised. Specimens of clade 1 corresponded to the generitype E. virescens because the morphology agreed with the reported descriptions of the species, and the distribution (subboreal Europe) included the type locality. Clade 2 had a broad distribution in the Arctic Sea and coldwater regions of the northern Pacific and northern Atlantic. Specimens of clade 2 were morphologically distinguishable from E. virescens in having broader sporophytes. Specimens of clade 3 collected from western Honshu, Japan, had cox3 sequence identical to one of an old voucher specimen of E. shandongensis (63-519) described from the Yellow Sea. We propose the establishment of a new species, E. borealis, distributed in the Arctic, northern Pacific, and northern Atlantic regions.

Proposal of a new brown algal species, Mesogloia japonica sp. nov. (Chordariaceae, Phaeophyceae), and transfer of Sauvageaugloia ikomae to Mesogloia

ABSTRACTA molecular phylogeny using mitochondrial cox1 and cox3 and chloroplast atpB, psaA, psbA and rbcL gene sequences revealed that the Japanese endemic brown alga Sauvageaugloia ikomae (Ectocarpales s.l.) is phylogenetically distant from the generitype S. divaricata described from Europe. It also suggests the occurrence of an undescribed species in the Seto Inland Sea and the Pacific coast of the Tohoku Region; whereas, S. ikomae is distributed in the Sea of Japan. The new species is genetically distinct from S. ikomae and is morphologically distinguishable in having thicker erect thalli with a more developed subcortical layer. In the phylogeny, the clade of Japanese Sauvageaugloia was sister to Mesogloia, both nesting in the clade of Cladosiphon species. Because ectocarpalean taxa are considered to be oversplit at generic rank compared to genetic divergence within the order, we refrain from describing a new genus for S. ikomae and the new species and instead suggest placing them in Mesogloia. We therefore propose the establishment of the new species Mesogloia japonica and a new combination M. ikomae. We also propose including Liebmannia leveillei in the genetically indistinguishable Mesogloia vermiculata.

Phylogenetic Analysis Reveals that the ‘Radial Centric’ Diatom Orthoseira Thwaites (Orthoseiraceae, Bacillariophyta) is a Member of a ‘Multipolar’ Diatom Lineage

The diatom genus Orthoseira Thwaites (Bacillariophyta) is a ubiquitous taxon in aerial diatom assemblages, with species found globally. Cylindrical cell shape and radial symmetry of this genus has led to its historical placement in the Coscinodiscophyceae ('radial centric' diatoms), but its systematic relationships have remained uncertain. We present a five-gene phylogeny, based on nuclear (nSSU rDNA) and chloroplast (rbcL, psbC, psbA, and psaB) genes to determine the phylogenetic placement of Orthoseira among the diatoms. The concatenated multi-gene phylogenies and nSSU-only gene tree demonstrate that Orthoseira is deeply embedded within a clade of the Mediophyceae ('multipolar centric' diatoms). Throughout all phylogenetic analyses, Orthoseira was shown to be sister to the genera Terpsinoë and Hydrosera. Through comparison of topologies reflecting competing hypotheses about the placement of Orthoseira, it was determined that the hypothesis that Orthoseira, represented here by O. dendroteres and O. roeseana, is a member of the Melosirales should be rejected. Therefore, lack of morphological similarity between Hydrosera, Orthoseira, and Terpsinoë is hypothesized to be the result of changes in habitat preferences that lead to an ancient divergence event between the Orthoseirales and the Hydrosera, Terpsinoë clade.

Differential sensitivity of Atriplex patula and Chenopodium album to sugar beet herbicides: a possible cause for the upsurge of A. patula in sugar beet fields

SummaryIn the last decade, the prevalence of Atriplex patula as a weed in the Belgian sugar beet area has increased. Possible reasons for its expansion in sugar beet fields, besides a poor implementation of the low‐dose phenmedipham/activator/soil‐acting herbicide (FAR) system, might be low sensitivity or evolved resistance to one or more herbicides used in sugar beet. Dose–response pot bioassays were conducted in the glasshouse to evaluate the effectiveness of five foliar‐applied sugar beet herbicides (metamitron, phenmedipham, desmedipham, ethofumesate and triallate) and three pre‐plant‐incorporated herbicides (metamitron, lenacil, dimethenamid‐P) for controlling five Belgian A. patula populations. Local metamitron‐susceptible and metamitron‐resistant populations of Chenopodium album were used as reference populations. Effective dosages and resistance indices were calculated. DNA sequence analysis of the photosystem II psbA gene was performed on putative resistant A. patula populations. Overall, A. patula exhibited large intraspecific variation in herbicide sensitivity. In general, A. patula populations were less susceptible to phenmedipham, desmedipham, ethofumesate and triallate relative to C. album populations. Two A. patula populations bear the leucine‐218 to valine mutation on the chloroplast psbA gene conferring low level to high level cross‐resistance to the photosystem II inhibitors phenmedipham, desmedipham, metamitron and lenacil. In order to avoid insufficient A. patula control and further spread, seedlings should preferentially be treated with FAR mixtures containing higher‐than‐standard doses of metamitron and phenmedipham/desmedipham and no later than the cotyledon stage.

Identification of a target‐site mutation conferring resistance to triazine herbicides in oriental mustard (Sisymbrium orientale L.) from Australia

In southern Australia, oriental mustard (Sisymbrium orientale) has been controlled successfully by triazine herbicides for several decades. The screening of 40 populations that were collected from the southern grain belt of Australia during 2010 and 2013 for resistance to six different herbicides (glyphosate, diflufenican, imazamox, chlorsulfuron, atrazine and 2,4‐dichlorophenoxyacetic acid) identified two oriental mustard populations as highly resistant to atrazine. Compared to the known oriental mustard‐susceptible populations (S1 and S2), these two resistant populations (P17 and P18) from near Horsham, Victoria, Australia, were 311‐ and 315‐fold resistant to atrazine, as determined by a comparison of the LD50 values. However, there was no resistance to diuron detected in these populations. Sequencing of the chloroplast psbA gene identified a missense mutation of serine 264 to glycine in both herbicide‐resistant oriental mustard populations, which is known to confer high‐level atrazine resistance in other species.

A preliminary survey of zoantharian endosymbionts shows high genetic variation over small geographic scales on Okinawa-jima Island, Japan.

Symbiotic dinoflagellates (genus Symbiodinium) shape the responses of their host reef organisms to environmental variability and climate change. To date, the biogeography of Symbiodinium has been investigated primarily through phylogenetic analyses of the ribosomal internal transcribed spacer 2 region. Although the marker can approximate species-level diversity, recent work has demonstrated that faster-evolving genes can resolve otherwise hidden species and population lineages, and that this diversity is often distributed over much finer geographical and environmental scales than previously recognized. Here, we use the noncoding region of the chloroplast psbA gene (psbAncr) to examine genetic diversity among clade C Symbiodinium associating with the common reef zoantharian Palythoa tuberculosa on Okinawa-jima Island, Japan. We identify four closely related Symbiodinium psbAncr lineages including one common generalist and two potential specialists that appear to be associated with particular microhabitats. The sea surface temperature differences that distinguish these habitats are smaller than those usually investigated, suggesting that future biogeographic surveys of Symbiodinium should incorporate fine scale environmental information as well as fine scale molecular data to accurately determine species diversity and their distributions.

Cladosiphon takenoensis sp. nov. (Ectocarpales s.l., Phaeophyceae) from Japan

SUMMARYThe new brown algal species Cladosiphon takenoensis H. Kawai (Chordariaceae, Ectocarpales s.l.) is described from Takeno, Hyogo, Japan based on morphology and DNA sequences. The species is a spring annual, growing on subtidal rocks at more or less exposed sites. It resembles C. umezakii in its gross morphology, and the two often grow together, but is distinguishable from C. umezakii in having a more hairy appearance. Cladosiphon takenoensis has a slimy, cylindrical, multiaxial and sympodial erect thallus, branching once to twice, and is provided with long assimilatory filaments (up to 1.8 mm long, composed of up to 100 cells). Unilocular zoidangia are formed on the basal part of assimilatory filaments. The species is genetically most related to C. umezakii and has the same basic thallus structures, but differs from C. umezakii and other Cladosiphon species in lacking phaeophycean hairs and plurilocular zoidangia of the assimilatory filaments. DNA sequences of the mitochondrial cox1 and cox3, chloroplast atpB, psaA, psbA and rbcL genes and the nuclear rDNA ITS2 region support the distinctness of the species. The genus Cladosiphon was paraphyletic in our analyses because the clades of C. okamuranus/C. zosterae and C. takenoensis/C. umezakii were split by Mesogloia vermiculata. However, since the genus‐level taxonomy of Chordariaceae needs considerable revision, we suspend the genus‐level taxonomy of the new species, and tentatively describe it as C. takenoensis.

Photosystem II Inhibitor Resistance in the Columbia Basin of Washington State

Weed management in potato and mint rely heavily on two Group 5 photosystem II–inhibiting herbicides, metribuzin and terbacil, respectively. Seed from weed escapes was collected in 2010 from 69 potato and mint production fields in Washington State and tested for resistance to metribuzin and terbacil. Of the seed screened, 26 of 51 Powell amaranth and redroot pigweed accessions and 8 of 23 common lambsquarters accessions tested expressed at least some degree of resistance to one or both herbicides. A Ser264Gly mutation in the chloroplastpsbA gene was present in all but three resistant accessions. Herbicides with other mechanisms of action registered in potato and mint controlled all identified photosystem II–resistant accessions when applied at typical labeled rates.

Identification of Triazine-ResistantVulpia bromoides

In Australia, triazine herbicides have routinely controlled theVulpiaspecies (Vulpia bromoides,Vulpia myuros, andVulpia fasciculata; collectively referred to as silvergrass). However, a simazine-resistant silvergrass biotype, collected from Pingelly in the Western Australian grain belt in 2014, has been confirmed. Compared to the pooled mortality of three simazine-susceptible silvergrass populations (S1, S2, and S3), the simazine-resistant Pingelly population was > 594-fold resistant at the LD50level. Dose-response screening of the simazine-selected progeny (> 800 g ai simazine ha−1) demonstrated that the simazine resistance mechanism was heritable. Sequencing of the chloroplastpsbA gene revealed the resistant population is homozygous for a serine 264 to glycine mutation, which confers a high-level triazine resistance. As expected this Ser-264-Gly mutation conferred resistance to atrazine and metribuzin, but not the phenyl-urea diuron. This is the first published report confirming field-evolved triazine resistance in aVulpiapopulation.

Sequencing of historic and modern specimens reveals cryptic diversity in Nothogenia (Scinaiaceae, Rhodophyta)

:Nothogenia fastigiata has been reported to exhibit great morphological variability and has been considered to be widely distributed in the Southern Hemisphere. To test its current circumscription, sequences from type material of N. fastigiata and other species currently synonymized with it were compared to those from recent collections of this and other species in the genus. Eight distinct species previously subsumed under the name N. fastigiata were identified. Multiple specimens from southern Chile and a single specimen from Campbell Island (subantarctic New Zealand) were conspecific with type material of N. fastigiata from the Falkland Islands. For other species, molecular analyses of recent collections using the nuclear ITS1-5.8S-ITS2 region of the ribosomal cistron, the chloroplast rbcL and psbA genes and the mitochondrial COI gene indicated a strong geographic pattern to species relationships. Other specimens identified as N. fastigiata from Chile represented up to five species, including N. chilensis and N. fragilis, based on sequences of type material; these Chilean species occurred on a monophyletic branch. We also recognized N. lingula comb. nov. from Tasmania, which is closely related to N. fastigiata, based on sequences of type material. Specimens from mainland New Zealand identified as N. fastigiata fell into a distinct clade with New Zealand N. pulvinata and represented a previously undescribed species, described here as N. neilliae sp. nov. Another New Zealand species, N. pseudosaccata, was distantly related to N. variolosa from Auckland Island and other subantarctic islands south of New Zealand. The New Zealand species were more closely related to South African N. erinacea and N. ovalis than to species of Nothogenia from Chile, including N. fastigiata, although bootstrap support for this interpretation was weak. These genetic data demonstrate that matching DNA sequences from archival Nothogenia material to modern specimens can be used to identify and define new and old cryptic species.