Plastid DNA Sequences Research Articles

Integrative taxonomy of the model liverwort Riella helicophylla (Riellaceae, Sphaerocarpales) reveals its extreme rarity and an overlooked widespread new species, R. macrocarpa

AbstractRiella (Riellaceae) is a genus of rare, ephemeral, aquatic liverworts that includes some taxa specialized in growing submerged in brackish, seasonal water ponds and lagoons. One of these species, Riella helicophylla is the type of the genus. Apart from being of conservation concern, it has been used as a model organism for cytological and physiological processes. The species was described from Algeria in the mid‐nineteenth century and was not found elsewhere for almost a century, when it was discovered in central Spain. Nonetheless, those new populations were considered morphologically different and served to describe a new variety (var. macrocarpa), and a form (f. latealata) of the latter. The number of population records increased considerably in the following decades, especially in the late twentieth century; at the same time, it was discovered in several countries throughout the Mediterranean basin. Those reports, however, did not account for its infraspecific distinction. As part of a broader study of the genus, in this work, we have revisited the taxonomy of R. helicophylla based on an integrative approach considering morphological and molecular DNA sequence data. Our broadly sampled study revealed that R. helicophylla includes two morphotypes corresponding with the previously recognized varieties of this species. These varieties differ consistently in a number of morphological gametophytic and spore traits. Riella helicophylla was resolved as paraphyletic in phylogenies based on nuclear and plastid DNA sequences. The accessions clustered in two monophyletic clades in concert with morphology, supporting their recognition at the species level. Accordingly, a new combination is proposed, R. macrocarpa, which includes the neglected R. helicophylla var. macrocarpa and R. helicophylla var. macrocarpa f. latealata. Lectotypes are designated for the two latter taxa and for R. helicophylla. The two species are fully described and illustrated, and an updated identification key of the Mediterranean species is included. Updated information on the conservation status of both species is provided.

The taxonomy of Leucanthemum ircutianum (Asteraceae, Anthemideae) in the Apennine Peninsula based on AFLP fingerprinting, plastid DNA sequence variation and eco-climatological niche reconstruction

Abstract The tetraploid species Leucanthemum ircutianum (Asteraceae, Anthemideae) is represented in the Apennine Peninsula by three infraspecific taxa, L. ircutianum subsp. ircutianum, L. ircutianum subsp. asperulum and L. ircutianum subsp. leucolepis. The present study uses AFLP fingerprinting and sequencing of the petN–psbM intergenic spacer (IGS) region of the plastid genome for inferring genetic relationships among these three subspecies and aims at the elaboration of an appropriate taxonomic treatment of these taxa. Due to the amphi-Adriatic distribution of these taxa, additional samples from the Balkan Peninsula and representatives of L. ircutianum subsp. ircutianum from north-western Italy, south-eastern France, Romania and southern Germany were included. Genetic analyses reveal that L. ircutianum subsp. asperulum from southern Italy and L. ircutianum subsp. leucolepis from the Balkan Peninsula and the northern and central Apennine Peninsula are identical and should be treated as a single taxon. The observation of genetically intermediate individuals throughout the overlapping distributional ranges of L. ircutianum subspp. ircutianum and leucolepis and in a surveyed mixed stand of the two taxa indicate that these are not reproductively isolated from each other. The analysis of eco-climatological niches of the two subspecies indicates that L. ircutianum subsp. ircutianum is found in more temperate regions, whereas L. ircutianum subsp. leucolepis prefers the Mediterranean fringe of the total distributional range of the species. A hybrid-zone analysis based on AFLP ‘allele frequency’ clines along a transect from Germany to southern Italy is interpreted as supporting a scenario of secondary contact of the two entities after some diversification in allopatry. On the basis of our results, we provide a taxonomic treatment of the species L. ircutianum for Italy, including the designation of three lectotypes.

Inflorescence lignification of natural species and horticultural hybrids of Phalaenopsis orchids

Phalaenopsis is an important ornamental pot plant for the global horticultural market. The inflorescences of Phalaenopsis horticultural hybrids require support from a stick during plant cultivation because of the weight of multiple large flowers. Developing a horticultural hybrid with a sufficiently lignified inflorescence stem that does not require additional support could be a way to reduce the costs of production. This study aimed to (i) determine the orientation and degree of lignification in the inflorescence stem of different species and horticultural hybrids of Phalaenopsis and investigate whether these lignification patterns follow any (ii) topological or (iii) phylogenetic pattern of interest to further explore in genetic precision breeding. Inflorescences of comparable developmental stages of six species and 17 horticultural hybrids of flowering Phalaenopsis orchids were sampled. The orientation of the inflorescence varied from erect, sub-erect, arching, to pendant. The degree of lignification was measured with ImageJ using stained microscopic tissue sections and statistically analyzed. A molecular phylogeny of the species of Phalaenopsis was reconstructed based on plastid and nuclear ribosomal DNA sequences to analyze phylogenetic patterns. Our results show a significant difference in the degree of lignification between the different Phalaenopsis species and hybrids, between peduncle and rachis, and among the six different inflorescence positions analyzed. We found a positive correlation between inflorescence orientation and the proportion of lignified area per total stem area and the proportional thickness of the lignified fiber walls in the peduncle. We conclude that the degree of lignification is heritable, as we observed among our sample size a higher positive correlation between stem lignification variables among closely related taxa compared to more distantly related ones. However, a larger species sampling is needed to further validate our results.

Temporal diversification in the genus Onosma (Boraginaceae) based on nuclear and plastid DNA sequences

The genus Onosma is a homogeneous taxon with high morphological variation and due to the similarities among the Onosma taxa, there are many problems in their identification. Hence, systematically and taxonomically, it is considered a difficult genus. In the present study, we included a large number of Onosma species throughout their distribution range. Using nuclear (ITS) and two plastids (rpl32-trnL(UAG) and trnH-psbA) markers, we analyzed the evolutionary history, divergence time and diversification patterns of Onosma across the tropical, subtropical and temperate regions. Divergence time estimates suggest the early radiation of Onosma s.l. happened at the Oligocene-Miocene boundary. BAMM analyses indicate that the best configuration included one significant shift in diversification rates within Onosma: on the branch leading to the clade comprised of species of confined to Iran.

Exploring the tempo of species diversification in Astragalus section Incani DC. based on nuclear and plastid DNA sequences

This study uses phylogenetic relationships of the species-rich section Astragalus (Incani), to follow up on recent evidence pointing to rapid and recent plant diversification patterns in the west of Iran. Section Incani is introduced for its taxonomic complication resulting from overlapping morphological characters, but few studies have been done on this section; hence, we also lack a robust time-calibrated chronogram to address hypotheses (e.g., biogeography and diversification rates) that have implicit time assumptions. Two loci (rpl32-trnL(UAG) and nrDNA ITS) were amplified and sequenced for 87 taxa across Incani for phylogenetic reconstruction and a chronogram in BEAST. Incani is identified as the sister clade to all remaining sections with high support, and within the clade Incani, two strongly supported groups are seen: (1) Clade I includes nine species restricted to eastern Iran and Central Asia, and (2) clade II includes a bulk of the species from west and northwestern Iran, Turkey and southern Europe. Divergence time estimates suggest Incani diverged from remaining sections 3 Mya during the late Pliocene. The crown date for Incani is estimated at 1.5 Mya (Pleistocene). Biogeography showed significant improvement in the likelihood score when the ??jump dispersal? parameter was added. An eastern origin (Central Asia) is implicated as important ancestral area in all deeper nodes. BAMM analyses indicate that the best configuration included one significant shift in diversification rates within Incani: near the crown of Incani (1.5-2 Mya) including clade II. Issues with conducting diversification analyses more generally are examined in the context of scale, taxon sampling, and larger sets of phylogenetic trees.

Is Lotus zhegulensis Klok. (Fabaceae) a separate species endemic of the Middle Volga Region or just a variety of a widespread L. corniculatus L.?

We have studied several samples from Lotus corniculatus s. l. and L. zhegulensis populations with the use of SSR and sequenced nuclear (ITS) and plastid (atpB-rbcL, ndhC-trnV, rpl32-trnL(UAG), trnH-psbA, trnL-trnF) markers. We analysed 9 local populations from the Volga River valley ranging from Ulyanovsk in the north to Volgograd in the south, as well as two local populations from Moscow and Lipetsk Provinces. Analyses of SSR markers using the STRUCRURE program divided the sample into three genetic clusters. Populations from Moscow and Lipetsk Provinces, and three local populations from the ‘locus classicus’ location of L. zhegulensis in Samara Province appeared to be the most genetically differentiated while all the other populations were variously genetically admixed. Low pairwise Fst values indicate low genetic differentiation of Lotus populations and the intraspecific nature of the revealed diversity. The nuclear and plastid DNA sequences analyzed yielded little information. Substitutions and indels revealed were mostly autapomorphies characteristic of separate specimens or small groups of specimens but not of populations. Our study enables to firmly conclude that Lotus populations from the right bank of the Volga River in vicinity of Zhiguli upland described as L. zhegulensis are weakly genetically differentiated from other populations of Lotus corniculatus s. l. and, hence, cannot be regarded as a separate species.

A phylogenetic analysis of the wild Tulipa species (Liliaceae) of Kosovo based on plastid and nuclear DNA sequence

[This corrects the article DOI: 10.1002/ggn2.202100016.].

Natural hybridization among three Rhododendron species (Ericaceae) revealed by morphological and genomic evidence

BackgroundNatural hybridization can influence the adaptive response to selection and accelerate species diversification. Understanding the composition and structure of hybrid zones may elucidate patterns of hybridization processes that are important to the formation and maintenance of species, especially for taxa that have experienced rapidly adaptive radiation. Here, we used morphological traits, ddRAD-seq and plastid DNA sequence data to investigate the structure of a Rhododendron hybrid zone and uncover the hybridization patterns among three sympatric and closely related species.ResultsOur results show that the hybrid zone is complex, where bi-directional hybridization takes place among the three sympatric parental species: R. spinuliferum, R. scabrifolium, and R. spiciferum. Hybrids between R. spinuliferum and R. spiciferum (R. ×duclouxii) comprise multiple hybrid classes and a high proportion of F1 generation hybrids, while a novel hybrid taxon between R. spinuliferum and R. scabrifolium dominated the F2 generation, but no backcross individuals were detected. The hybrid zone showed basically coincident patterns of population structure between genomic and morphological data.ConclusionsNatural hybridization exists among the three Rhododendron species in the hybrid zone, although patterns of hybrid formation vary between hybrid taxa, which may result in different evolutionary outcomes. This study represents a unique opportunity to dissect the ecological and evolutionary mechanisms associated with adaptive radiation of Rhododendron species in a biodiversity hotspot.

Phylogenetic analysis ofDioscorea(Dioscoreaceae) from Japan and adjacent regions based on plastid and nuclear DNA sequences, with special reference to the taxonomic status of selected taxa

AbstractTo clarify phylogenetic relationships among Japanese Dioscorea taxa and to re-examine taxonomy based on these relationships, we conducted molecular phylogenetic analyses of four plastid regions [trnK (including matK), rbcL, atpB and trnL-F] and two nuclear regions (PHYC and 18S rDNA) for all Dioscorea taxa from Japan and selected taxa from adjacent regions. Although species are often sympatric and variation in leaf shape is relatively wide within species and not clearly demarcated between species, our results suggest that Japanese Dioscorea spp. do not hybridize. Our results also resolve several taxonomic issues surrounding Japanese species. We confirm that D. japonica, D. polystachya, D. pseudojaponica and D. tabatae, which are morphologically similar, are distinct species. Further, plants from the Daito Islands identified as D. luzonensis, which have never been observed to flower to our knowledge, are attributable to D. pseudojaponica. Dioscorea zentaroana, often treated as a synonym of D. asclepiadea, was found to be distinct from the latter. Dioscorea izuensis should be treated as D. collettii var. izuensis. In conclusion, 16 species and three varieties of native Dioscorea should be recognized in Japan.

A dated molecular phylogeny and biogeographical analysis reveals the evolutionary history of the trans-pacifically disjunct tropical tree genus Ormosia (Fabaceae)

The papilionoid legume genus Ormosia (Fabaceae) comprises about 150 species of trees and exhibits a striking disjunct geographical distribution between the New World- and Asian and Australasian wet tropics and subtropics. Modern classifications of Ormosia are not grounded on a well-substantiated phylogenetic hypothesis and have been limited to just portions of the geographical range of the genus. The lack of an evolutionarily-based foundation for systematic studies has hindered taxonomic work on the genus and prevented the testing of biogeographical hypotheses related to the origin of the Old World/New World disjunction and the individual dispersal histories within both areas. Here, we present the most comprehensively sampled molecular phylogeny of Ormosia to date, based on analysis of both nuclear (ITS) and plastid (matK and trnL–F) DNA sequences from 82 species of the genus. Phylogenetically-based divergence times and ancestral range estimations are employed to test hypotheses related to the biogeographical history of the genus. We find strong support for the monophyly of Ormosia and the grouping of all sampled Asian species of the genus into two comparably sized clades, one of which is sister to another large clade containing all sampled New World species. Within the New World clade, additional resolution supports the grouping of most species into three mutually exclusive subordinate clades. The remaining New World species form a fourth well-supported clade in the analyses of plastid sequences, but that result is contradicted by the analysis of ITS. With few exceptions the supported clades have not been previously recognized as taxonomic groups. The biogeographical analysis suggests that Ormosia originated in continental Asia and dispersed to the New World in the Oligocene or early Miocene via long-distance trans-oceanic dispersal. We reject the hypothesis that the inter-hemispheric disjunction in Ormosia resulted from fragmentation of a more continuous “Boreotropical” distribution since the dispersal post-dates Eocene climatic maxima. Both of the Old World clades appear to have originated in mainland Asia and subsequently dispersed into the Malay Archipelago and beyond, at least two lineages dispersing across Wallace’s Line as far as the Solomon Islands and northeastern Australia. In the New World, the major clades all originated in Amazonia. Dispersal from Amazonia into peripheral areas in Central America, the Caribbean, and Extra-Amazonian Brazil occurred multiple times over varying time scales, the earliest beginning in the late Miocene. In a few cases, these dispersals were followed by local diversification, but not by reverse migration back to Amazonia. Within each of the two main areas of distribution, multiple modest bouts of oceanic dispersal were required to achieve the modern distributions.

Molecular‐based phylogenetic placement and revision of Micrasepalum (Spermacoceae‐Rubiaceae)

AbstractMicrasepalum is a taxonomically and phylogenetically poorly understood genus of Rubiaceae endemic to the global biodiversity hotspot of the Caribbean islands. It has traditionally been associated with species in the Spermacoce clade, but never properly evaluated in a phylogenetic context. Also, little information about seed and fruit morphology of Micrasepalum species was provided in their original descriptions and the few subsequent taxonomic treatments. We performed a molecular phylogenetic study based on nuclear (ITS, ETS) and plastid (atpB‐rbcL, petD, rps16, trnL‐trnF) DNA sequence data to resolve the enigmatic taxonomic identity of Micrasepalum. Our analyses show a largely unresolved or poorly supported backbone in both the nuclear and plastid phylogenies of the Spermacoce clade, so that the sister relationship of Micrasepalum is still uncertain. Merging Micrasepalum with any lineage of the polyphyletic Borreria and Spermacoce or the phylogenetically well‐defined Hexasepalum, Mitracarpus or Richardia would result in a morphologically heterogeneous collection of genera difficult to recognize within the Spermacoce clade. The unresolved relationships within the species‐rich Spermacoce clade are suggestive of a rapid radiation in which Micrasepalum is one of several phylogenetically recalcitrant lineages, but that can be clearly defined morphologically. The genus is distinctive in having a caducous, 2‐lobed calyx and indehiscent, one‐seeded fruit. An updated identification key for all Caribbean genera of the Spermacoce clade and a full taxonomic revision of Micrasepalum are presented, including descriptions focused on reproductive characters, palynological analysis, a distribution map and new lectotypifications, as well as a new synonym for M. eritrichoides.

Genomics-Based Phylogenetic and Population Genetic Analysis of Global Samples Confirms Halophila johnsonii Eiseman as Halophila ovalis (R.Br.) Hook.f.

Halophila johnsonii is an endangered seagrass species that is restricted to the southeast coast of Florida, United States. Its taxonomic status has been called into question, in particular, given the close morphological and genetic similarity of H. johnsonii and the widely distributed and morphologically variable Halophila ovalis, which is largely restricted to the Indo-Pacific region. While a close relationship to H. ovalis is uncontroversial, it remains uncertain whether H. johnsonii represents a distinct lineage or is a recent introduction to the Florida region. Given the conservation status of H. johnsonii, distinguishing these alternatives has important implications for the management of the species and its habitat. Here, we develop molecular data sets for samples of H. johnsonii and H. ovalis including DNA sequences, genome-wide SNPs and microsatellites with the view to resolving the affinities of H. johnsonii with respect to the wider H. ovalis complex. Phylogenetic hypotheses based upon plastid (∼18000 bp) and low copy nuclear DNA (∼6500 bp) sequences derived from hybrid capture, along with 990 genome-wide ddRAD SNPs consistently resolved H. johnsonii within H. ovalis. Specifically, we found a close affinity between H. johnsonii and H. ovalis sampled from the east coast of Africa. In addition, Halophila specimens collected in Antigua, which are within the range of morphological variation typical for H. ovalis, are virtually identical to H. johnsonii and the East African H. ovalis samples based upon DNA sequence analyses and these group together using Bayesian clustering analyses of microsatellites and ddRAD SNPs. We conducted population genetic analyses using large number of H. johnsonii samples collected over a 17-year period. Genotypic data generated through microsatellites and ddRAD SNPs revealed genetic uniformity for all 132 H. johnsonii samples across the Indian River Lagoon, Florida, while samples of H. ovalis from Antigua shared the same genotype as H. johnsonii. We conclude that the lack of genetic diversity and the absence of sexual reproduction strongly indicates that the total range of H. johnsonii is actually one clone that is closely related to populations in Africa and Antigua and may be derived from a recent introduction from one of those regions.

Arundinella tengchongensis (Poaceae), a name at new rank and newly combined based on morphological and molecular data

To clarify the taxonomic status of Arundinella setosa var. tengchongensis and its relationships with A. setosa var. setosa and other related species, molecular phylogenetic analyses based on nuclear ITS and two plastid DNA (matK and trnL‐F) sequences, and morphological comparisons using one‐way ANOVA were carried out. Our results reveal that A. setosa var. tengchongensis phylogenetically belong in a different clade separated from the type variety. Morphologically, A. setosa var. tengchongensis is easily distinguished from A. setosa var. setosa and other varieties by its smaller spikelets, shorter awns and bearded nodes. Thus, A. setosa var. tengchongensis is here elevated to species rank and newly combined as A. tengchongensis.

Phylogeography of the East Asian grassland plant, Viola orientalis (Violaceae), inferred from plastid and nuclear restriction site-associated DNA sequencing data.

To elucidate the origin and migration history of the "Mansen elements," a group of temperate grassland plants mainly distributed in northeastern Asia, phylogeographic analyses based on chloroplast DNA markers and double-digest restriction site-associated DNA sequencing (ddRAD-seq) data were performed on Viola orientalis, one of the representative species of the group. Phylogenetic analyses using ddRAD-seq data revealed that the populations of V. orientalis were clustered into five clades, among which the continental clades made of populations from Russia and Korea diverged more than 100,000years earlier than the Japanese clades. The Japanese clade likely diverged during the last glacial period, followed by a further post-glacial divergence into the Kyushu and the Honshu subclades. Our study demonstrated that V. orientalis originated in the continental area of northeastern Asia and, during the last glacial period, has spread southward through the Korean Peninsula across the Japanese Islands. This finding supports the previously proposed evolutionary hypothesis regarding the origin and migration routes of the Mansen elements.

Descending Dysploidy and Bidirectional Changes in Genome Size Accompanied Crepis (Asteraceae) Evolution.

The evolution of the karyotype and genome size was examined in species of Crepis sensu lato. The phylogenetic relationships, inferred from the plastid and nrITS DNA sequences, were used as a framework to infer the patterns of karyotype evolution. Five different base chromosome numbers (x = 3, 4, 5, 6, and 11) were observed. A phylogenetic analysis of the evolution of the chromosome numbers allowed the inference of x = 6 as the ancestral state and the descending dysploidy as the major direction of the chromosome base number evolution. The derived base chromosome numbers (x = 5, 4, and 3) were found to have originated independently and recurrently in the different lineages of the genus. A few independent events of increases in karyotype asymmetry were inferred to have accompanied the karyotype evolution in Crepis. The genome sizes of 33 Crepis species differed seven-fold and the ancestral genome size was reconstructed to be 1C = 3.44 pg. Both decreases and increases in the genome size were inferred to have occurred within and between the lineages. The data suggest that, in addition to dysploidy, the amplification/elimination of various repetitive DNAs was likely involved in the genome and taxa differentiation in the genus.

A phylogenetic analysis of the wild Tulipa species (Liliaceae) of Kosovo based on plastid and nuclear DNA sequence.

In Kosovo, the genus Tulipa is represented by eight taxa, most of which form a species complex surrounding Tulipa scardica. To investigate the phylogenetic relationship of these Tulipa species a Bayesian analysis was undertaken using the ITS nuclear marker and trnL-trnF, rbcL and psbA-trnH plastid markers. The resulting phylogenetic trees show that Kosovarian Tulipa species consistently group into two main clades, the subgenera Eriostemones and Tulipa. Furthermore, our analyses provide some evidence that the subspecies of Tulipa sylvestris are genetically distinguishable, however not significantly enough to support their reclassification as species. In contrast, the markers provide some novel information to reassess the species concepts of the T. scardica complex. Our data provide support for the synonymisation of Tulipa luanica and Tulipa kosovarica under the species Tulipa serbica. Resolution and sampling limitations hinder any concrete conclusion about whether Tulipa albanica and T. scardica are true species, yet our data do provide some support that these are unique taxa and therefore should continue to be treated as such until further clarification. Overall, our work shows that genetic data will be important in determining species concepts in this genus, however, even with a molecular perspective pulling apart closely related taxa can be extremely challenging.

Phylogeny of Lantana, Lippia, and related genera (Lantaneae: Verbenaceae).

Lantana and Lippia (Verbenaceae) are two large Linnean genera whose classification has been based on associated fruit traits: fleshy vs. dry fruits and one vs. two seed-bearing units. We reconstruct evolutionary relationships and the evolution of the two fruit traits to test the validity of these traits for classification. Previous studies of plastid DNA sequences provided limited resolution for this group. Consequently, seven nuclear loci, including ITS, ETS, and five PPR loci, were sequenced for 88 accessions of the Lantana/Lippia clade and three outgroups. Neither Lantana nor Lippia is monophyletic. Burroughsia, Nashia, Phyla, and several Aloysia species are included within the clade comprising Lantana and Lippia. We provide a hypothesis for fruit evolution and biogeographic history in the group and their relevance for classification. Fleshy fruits evolved multiple times in the Lantana/Lippia clade and thus are not suitable taxonomic characters. Several sections of Lantana and Lippia and the small genera are monophyletic, but Lippia section Zappania is broadly paraphyletic, making circumscription of genera difficult. Lippia sect. Rhodolippia is a polyphyletic group characterized by convergence in showy bracts. Species of Lantana sect. Sarcolippia, previously transferred to Lippia, are not monophyletic. The clade originated and diversified in South America, with at least four expansions into both Central America and the Caribbean and two to Africa. The types species of Lantana and Lippia occur in small sister clades, rendering any taxonomy that retains either genus similar to its current circumscription impossible.

Hundreds of nuclear and plastid loci yield novel insights into orchid relationships.

The inference of evolutionary relationships in the species-rich family Orchidaceae has hitherto relied heavily on plastid DNA sequences and limited taxon sampling. Previous studies have provided a robust plastid phylogenetic framework, which was used to classify orchids and investigate the drivers of orchid diversification. However, the extent to which phylogenetic inference based on the plastid genome is congruent with the nuclear genome has been only poorly assessed. We inferred higher-level phylogenetic relationships of orchids based on likelihood and ASTRAL analyses of 294 low-copy nuclear genes sequenced using the Angiosperms353 universal probe set for 75 species (representing 69 genera, 16 tribes, 24 subtribes) and a concatenated analysis of 78 plastid genes for 264 species (117 genera, 18 tribes, 28 subtribes). We compared phylogenetic informativeness and support for the nuclear and plastid phylogenetic hypotheses. Phylogenetic inference using nuclear data sets provides well-supported orchid relationships that are highly congruent between analyses. Comparisons of nuclear gene trees and a plastid supermatrix tree showed that the trees are mostly congruent, but revealed instances of strongly supported phylogenetic incongruence in both shallow and deep time. The phylogenetic informativeness of individual Angiosperms353 genes is in general better than that of most plastid genes. Our study provides the first robust nuclear phylogenomic framework for Orchidaceae and an assessment of intragenomic nuclear discordance, plastid-nuclear tree incongruence, and phylogenetic informativeness across the family. Our results also demonstrate what has long been known but rarely thoroughly documented: nuclear and plastid phylogenetic trees can contain strongly supported discordances, and this incongruence must be reconciled prior to interpretation in evolutionary studies, such as taxonomy, biogeography, and character evolution.

Evidence for Glacial Refugia of the Forest Understorey Species Helleborus niger (Ranunculaceae) in the Southern as Well as in the Northern Limestone Alps.

Glacial refugia of alpine and subnival biota have been intensively studied in the European Alps but the fate of forests and their understory species in that area remains largely unclear. In order to fill this gap, we aimed at disentangling the spatiotemporal diversification of disjunctly distributed black hellebore Helleborus niger (Ranunculaceae). We applied a set of phylogeographic analyses based on restriction-site associated DNA sequencing (RADseq) data and plastid DNA sequences to a range-wide sampling of populations. These analyses were supplemented with species distribution models generated for the present and the Last Glacial Maximum (LGM). We used exploratory analyses to delimit genomically coherent groups and then employed demographic modeling to reconstruct the history of these groups. We uncovered a deep split between two major genetic groups with western and eastern distribution within the Southern Limestone Alps, likely reflecting divergent evolution since the mid-Pleistocene in two glacial refugia situated along the unglaciated southern margin of the Alps. Long-term presence in the Southern Limestone Alps is also supported by high numbers of private alleles, elevated levels of nucleotide diversity and the species’ modeled distribution at the LGM. The deep genetic divergence, however, is not reflected in leaf shape variation, suggesting that the morphological discrimination of genetically divergent entities within H. niger is questionable. At a shallower level, populations from the Northern Limestone Alps are differentiated from those in the Southern Limestone Alps in both RADseq and plastid DNA data sets, reflecting the North-South disjunction within the Eastern Alps. The underlying split was dated to ca. 0.1 mya, which is well before the LGM. In the same line, explicit tests of demographic models consistently rejected the hypothesis that the partial distribution area in the Northern Limestone Alps is the result of postglacial colonization. Taken together, our results strongly support that forest understory species such as H. niger have survived the LGM in refugia situated along the southern, but also along the northern or northeastern periphery of the Alps. Being a slow migrator, the species has likely survived repeated glacial-interglacial circles in distributional stasis while the composition of the tree canopy changed in the meanwhile.

Divergence time estimation and mapping of morphological and cytogenetical data in the southern South American geophyte genus Pterocactus (Cactaceae)

AbstractPterocactus has 10 species endemic to restricted areas of the Argentine Monte and Patagonia regions, also growing in neighbouring Chile. We analysed phylogenetic relationships between its species, based on plastid DNA sequences (psbA‐trnH, rpl16, trnH‐matK), exomorphological features (spine type and size, stem segment form, flower position, epidermis colour, areole pubescence, stem surface) and cytogenetical traits (genome size, somatic chromosome number). Traits were mapped using character mapping and ancestral state reconstruction to understand the dynamics of evolutionary changes. Pterocactus was monophyletic. Two lineages were recovered: clade A (with P. fischeri diverging early and P. reticulatus, P. valentinii, P. megliolii, P. gonjianii, and both P. tuberosus forms) and clade B (with P. neuquensis diverging first and P. araucanus and P. hickenii). Both samples examined of P. australis were paraphyletic. It was estimated that Pterocactus split from its sister group at 9.38 mya. The crown group age was estimated to be 7.69 myr. Species showed x = 11 with different ploidy levels (2n = 22, 44, 99, ca. 110, and 154); only P. tuberosus f. tuberosus was diploid. The numbers were typical of each taxon studied, and polyploidy was important in its diversification. Two 18S‐5.8S‐26S sites were detected regardless of the ploidy level, except P. fischeri with four sites and 2n = ca. 110. The number of 5S sites was 2 in P. gonjianii and the diploid P. tuberosus f. tuberosus, 4 in the tetraploid P. tuberosus f. lelongii, 10 in the probably decaploid P. fischeri and 8 in P. araucanus of unknown chromosome number. The genome size ranged from 2C = 4.94 pg in P. megliolii of unknown chromosome number to 2C = 16.68 pg in the tetradecaploid P. hickenii. The ancestor is reconstructed as a dwarf shrub with cylindrical central spines of 16.35–31.2 mm long, non‐woolly areoles, cylindrical to globose/obpyriform stem segments, non‐tuberculate stem surface, lateral flower position, green/brown to violet epidermis, probably diploid, and with a genome size between 1.10 and 1.23 pg.