Evolutionary origins of the eastern North American–Mesoamerican floristic disjunction: Current status and future prospects

Biogeographic disjunction patterns, where multiple taxa are shared between isolated geographic areas, represent excellent systems for investigating the historical assembly of modern biotas and fundamental biological processes such as speciation, diversification, niche evolution, and evolutionary responses to climate change. Studies on plant genera disjunct across the northern hemisphere, particularly between eastern North America (ENA) and eastern Asia (EAS), have yielded tremendous insight on the geologic history and assembly of rich temperate floras. However, one of the most prevalent disjunction patterns involving ENA forests has been largely overlooked: that of taxa disjunct between ENA and cloud forests of Mesoamerica (MAM), with examples including Acer saccharum, Liquidambar styraciflua, Cercis canadensis, Fagus grandifolia, and Epifagus virginiana. Despite the remarkable nature of this disjunction pattern, which has been recognized for over 75 years, there have been few recent efforts to empirically examine its evolutionary and ecological origins. Here I synthesize previous systematic, paleobotanical, phylogenetic, and phylogeographic studies to establish what is known about this disjunction pattern to provide a roadmap for future research. I argue that this disjunction pattern, and the evolution and fossil record of the Mexican flora more broadly, represents a key missing piece in the broader puzzle of northern hemisphere biogeography. I also suggest that the ENA–MAM disjunction represents an excellent system for examining fundamental questions about how traits and life history strategies mediate plant evolutionary responses to climate change and for predicting how broadleaf temperate forests will respond to the ongoing climatic pressures of the Anthropocene.     

Phylogenomics,biogeography, and evolution of the blue‐ or white‐fruited dogwoods (Cornus)—Insights into morphological and ecological niche divergence following intercontinental geographic isolation

The eastern Asian (EA)–eastern North American (ENA) floristic disjunction represents a major pattern of phytogeography of the Northern Hemisphere. Despite 20 years of studies dedicated to identification of taxa that display this disjunct pattern, its origin and evolution remain an open question, especially regarding post‐isolation evolution. The blue‐ or white‐fruited dogwoods (BW) are the most species‐rich among the four major clades of Cornus L., consisting of ~35 species divided into three subgenera (subg. Yinquania, subg. Mesomora, and subg. Kraniopsis). The BW group provides an excellent example of the EA–ENA floristic disjunction for biogeographic study due to its diversity distribution centered in eastern Asia and eastern North America, yet its species relationships and delineation have remained poorly understood. In this study, we combined genome‐wide markers from RAD‐seq, morphology, fossils, and climate data to understand species relationships, biogeographic history, and ecological niche and morphological evolution. Our phylogenomic analyses with RAxML and MrBayes recovered a strongly supported and well‐resolved phylogeny of the BW group with three intercontinental disjunct clades in EA and ENA or Eurasia and North America, of which two are newly identified within subg. Kraniopsis. These analyses also recovered a potential new species but failed to resolve relationships within the C. hemsleyi–C. schindleri complex. In an effort to develop an approach to reduce computation time, analysis of different nodal age settings in treePL suggests setting a node's minimum age constraint to the lower bound of a fossil's age range to obtain similar ages to that of BEAST. Divergence time analyses with BEAST and treePL dated the BW stem back to the very Late Cretaceous and the divergence of the three subgenera in the Paleogene. By integrating fossil ages and morphology, a total evidence‐based dating approach was used in conjunction with time‐slice probabilities of dispersal under a DEC model to resolve ancestral ranges of each disjunct in the Miocene: Eurasia and ENA (disjunct 1), EA and western North America (disjunct 2), and EA (disjunct 3). The dated biogeographic history supports dispersal via the North Atlantic Land Bridge in the late Paleogene in disjunct 1 and dispersal via the Bering Land Bridge in the Miocene for disjuncts 2 and 3. Character mapping with a stochastic model in phytools and comparison of ecological niche, morphospace, and rate of evolution indicated differential divergence patterns in morphology, ecological niche, and molecules between disjunct sisters. Although morphological stasis was observed in most of the characters, evolutionary changes in growth habit and some features of leaf, flower, and fruit morphology occurred in one or both sister clades. A significant differentiation of ecological habitats in temperature, precipitation, and elevation between disjunct sisters was observed, suggesting a role of niche divergence in morphological evolution post‐isolation. The patterns of evolutionary rate between morphology and molecules varied among disjunct clades and were not always congruent between morphology and molecules, suggesting cases of non‐neutral morphological evolution driven by ecological selection. Our phylogenetic evidence and comparisons of evolutionary rate among disjunct lineages lend new insights into the formation of the diversity anomaly between EA and ENA, with particular support of an early diversification in EA. These findings, in conjunction with previous studies, again suggest that the EA–ENA disjunct floras are an assembly of lineages descended from the Mesophytic Forests that evolved from the early Paleogene “boreotropical flora” through varied evolutionary pathways across lineages.     

Phylogenomics,biogeography, and evolution of morphology and ecological niche of the eastern Asian–eastern North American Nyssa (Nyssaceae)

Nyssa (Nyssaceae, Cornales) represents a classical example of the well‐known eastern Asian–eastern North American floristic disjunction. The genus consists of three species in eastern Asia, four species in eastern North America, and one species in Central America. Species of the genus are ecologically important trees in eastern North American and eastern Asian forests. The distribution of living species and a rich fossil record of the genus make it an excellent model for understanding the origin and evolution of the eastern Asian–eastern North American floristic disjunction. However, despite the small number of species, relationships within the genus have remained unclear and have not been elucidated using a molecular approach. Here, we integrate data from 48 nuclear genes, fossils, morphology, and ecological niche to resolve species relationships, elucidate its biogeographical history, and investigate the evolution of morphology and ecological niches, aiming at a better understanding of the well‐known EA–ENA floristic disjunction. Results showed that the Central American (CAM) Nyssa talamancana was sister to the remaining species, which were divided among three, rapidly diversified subclades. Estimated divergence times and biogeographical history suggested that Nyssa had an ancestral range in Eurasia and western North America in the late Paleocene. The rapid diversification occurred in the early Eocene, followed by multiple dispersals between and within the Erasian and North American continents. The genus experienced two major episodes of extinction in the early Oligocene and end of Neogene, respectively. The Central American N. talamancana represents a relic lineage of the boreotropical flora in the Paleocene/Eocene boundary that once diversified in western North America. The results supported the importance of both the North Atlantic land bridge and the Bering land bridge (BLB) for the Paleogene dispersals of Nyssa and the Neogene dispersals, respectively, as well as the role of Central America as refugia of the Paleogene flora. The total‐evidence‐based dated phylogeny suggested that the pattern of macroevolution of Nyssa coincided with paleoclimatic changes. We found a number of evolutionary changes in morphology (including wood anatomy and leaf traits) and ecological niches (precipitation and temperature) between the EA–ENA disjunct, supporting the ecological selection driving trait evolutions after geographic isolation. We also demonstrated challenges in phylogenomic studies of lineages with rapid diversification histories. The concatenation of gene data can lead to inference of strongly supported relationships incongruent with the species tree. However, conflicts in gene genealogies did not seem to impose a strong effect on divergence time dating in our case. Furthermore, we demonstrated that rapid diversification events may not be recovered in the divergence time dating analysis using BEAST if critical fossil constraints of the relevant nodes are not available. Our study provides an example of complex bidirectional exchanges of plants between Eurasia and North America in the Paleogene, but “out of Asia” migrations in the Neogene, to explain the present disjunct distribution of Nyssa in EA and ENA.     

Timing the Eastern Asian–Eastern North American Floristic Disjunction: Molecular Clock Corroborates Paleontological Estimates

Sequence data of the chloroplast gene rbcL were used to estimate the time of the well-known eastern Asian–eastern North American floristic disjunction. Sequence divergence of rbcL was examined for 22 species of 11 genera (Campsis, Caulophyllum, Cornus, Decumaria, Liriodendron, Menispermum, Mitchella, Pachysandra, Penthorum, Podophyllum, and Phryma) representing a diverse array of flowering plants occurring disjunctly in eastern Asia and eastern North America. Divergence times of putative disjunct species pairs were estimated from synonymous substitutions, using rbcL molecular clocks calibrated for Cornus. Relative rate tests were performed to assess rate constancy of rbcL evolution among lineages. Corrections of estimates of divergence times for each species pair were made based on rate differences of rbcL between Cornus and other species pairs. Results of these analyses indicate that the time of divergence of species pairs examined ranges from 12.56 ± 4.30 million years to recent (<0.31 million years), with most within the last 10 million years (in the late Miocene and Pliocene). These results suggest that the isolation of most morphologically similar disjunct species in eastern Asia and eastern North America occurred during the global climatic cooling period that took place throughout the late Tertiary and Quaternary. This estimate is closely correlated with paleontological evidence and in agreement with the hypothesis that considers the eastern Asian–eastern North American floristic disjunction to be the result of the range restriction of a once more or less continuously distributed mixed mesophytic forest of the Northern Hemisphere that occurred during the late Tertiary and Quaternary. This implies that in most taxa the disjunction may have resulted from vicariance events. However, long-distance dispersal may explain the disjunct distribution of taxa with low divergence, such as Menispermum.     

A dated phylogeny of Lardizabalaceae reveals an unusual long-distance dispersal across the Pacific Ocean and the rapid rise of East Asian subtropical evergreen broadleaved forests in the late Miocene

Temperate South American–Asian disjunct distributions are the most unusual in organisms, and challenging to explain. Here, we address the origin of this unusual disjunction in Lardizabalaceae using explicit models and molecular data. The family (c.40 species distributed in ten genera) also provides an opportunity to explore the historical assembly of East Asian subtropical evergreen broadleaved forests, a typical and luxuriant vegetation in East Asia. DNA sequences of five plastid loci of 42 accessions representing 23 species of Lardizabalaceae (c. 57.5% of estimated species diversity), and 19 species from the six other families of Ranunculales, were used to perform phylogenetic analyses. By dating the branching events and reconstructing ancestral ranges, we infer that extant Lardizabalaceae dated to the Upper Cretaceous of East Asia and that the temperate South American lineage might have split from its East Asian sister group at c. 24.4 Ma. A trans-Pacific dispersal possibly by birds from East Asia to South America is plausible to explain the establishment of the temperate South American–East Asian disjunction in Lardizabalaceae. Diversification rate analyses indicate that net diversification rates of Lardizabalaceae experienced a significant increase around c. 7.5 Ma. Our findings suggest that the rapid rise of East Asian subtropical evergreen broadleaved forests occurred in the late Miocene, associated with the uplift of the Tibetan Plateau and the intensified East Asian monsoon, as well as the higher winter temperature and atmospheric CO₂ levels.     

Early evolutionary history of the flowering plant family Annonaceae: steady diversification and boreotropical geodispersal

Aim Rain forest‐restricted plant families show disjunct distributions between the three major tropical regions: South America, Africa and Asia. Explaining these disjunctions has become an important challenge in biogeography. The pantropical plant family Annonaceae is used to test hypotheses that might explain diversification and distribution patterns in tropical biota: the museum hypothesis (low extinction leading to steady accumulation of species); and dispersal between Africa and Asia via Indian rafting versus boreotropical geodispersal. Location Tropics and boreotropics. Methods Molecular age estimates were calculated using a Bayesian approach based on 83% generic sampling representing all major lineages within the family, seven chloroplast markers and two fossil calibrations. An analysis of diversification was carried out, which included lineage‐through‐time (LTT) plots and the calculation of diversification rates for genera and major clades. Ancestral areas were reconstructed using a maximum likelihood approach that implements the dispersal–extinction–cladogenesis model. Results The LTT plots indicated a constant overall rate of diversification with low extinction rates for the family during the first 80 Ma of its existence. The highest diversification rates were inferred for several young genera such as Desmopsis, Uvariopsis and Unonopsis. A boreotropical migration route was supported over Indian rafting as the best fitting hypothesis to explain present‐day distribution patterns within the family. Main conclusions Early diversification within Annonaceae fits the hypothesis of a museum model of tropical diversification, with an overall steady increase in lineages possibly due to low extinction rates. The present‐day distribution of species within the two largest clades of Annonaceae is the result of two contrasting biogeographic histories. The ‘long‐branch clade’ has been diversifying since the beginning of the Cenozoic and underwent numerous geodispersals via the boreotropics and several more recent long‐distance dispersal events. In contrast, the ‘short‐branch clade’ dispersed once into Asia via the boreotropics during the Early Miocene and further dispersal was limited.     

Regional differences in rates of plant speciation and molecular evolution: a comparison between eastern Asia and eastern North America

The eastern Asian (EAS)-eastern North American (ENA) floristic disjunction is one of the best-known biogeographic patterns in the Northern Hemisphere. Recent paleontological and molecular analyses have illuminated the origins of the biogeographic pattern, but subsequent diversification and evolution of the disjunct floras in each of the two continents after isolation remains poorly understood. Although similar in climate and floristic composition, EAS has twice as many species as ENA in genera occurring in both regions. Explaining such differences in species diversity between regions with similar environmental conditions (diversity anomalies) is an important goal of the study of the global patterns of biodiversity. We used a phylogenetic approach to compare rates of net speciation and molecular evolution between the two regions. We first identified EAS-ENA disjunct sister clades from ten genera (Asarum, Buckleya, Carpinus, Carya, Cornus, Hamamelis, Illicium, Panax, Stewartia, and Styrax) that represent diverse angiosperm lineages using phylogenetic analyses of ITS (internal transcribed spacer of nuclear ribosomal DNA) sequence data. Species richness and substitution rate of ITS between sister clades were compared. The results revealed a pattern of greater species diversity in the EAS counterparts. A positive relationship between species diversity and ITS substitution rate was also documented. These results suggest greater net speciation and accelerated molecular evolution in EAS. The data support the idea that a regional difference in net speciation rate related to topographic heterogeneity contributes to the diversity anomaly between EAS and ENA. The close relationship between rates of ITS evolution and species richness further suggests that species production may be directly linked to rate of nucleotide substitution.     

Molecular phylogeny and biogeography of Astilbe (Saxifragaceae) in Asia and eastern North America

Phylogenetic analyses were conducted for Astilbe (Saxifragaceae), an Asian/eastern North American disjunct genus, using sequences of nuclear ribosomal internal transcribed spacer (ITS) and plastid matK, trnL‐trnF and psbA‐trnH regions. The monophyly of Astilbe is well supported by both ITS and plastid sequences. Topological incongruence was detected between the plastid and the ITS trees, particularly concerning the placement of the single North American species, A. biternata, which may be most probably explained by its origin involving hybridization and/or allopolyploidy with plastid capture. In Astilbe, all species with hermaphroditic flowers constitute a well‐supported clade; dioecious species form a basal grade to the hermaphroditic clade. Astilbe was estimated to have split with Saxifragopsis from western North America at 20.69 Ma (95% HPD: 12.14–30.22 Ma) in the early Miocene. This intercontinental disjunction between Astilbe and Saxifragopsis most likely occurred via the Bering land bridge. The major clade of Astilbe (all species of the genus excluding A. platyphylla) was inferred to have a continental Asian origin. At least three subsequent migrations or dispersals were hypothesized to explain the expansion of Astilbe into North America, Japan and tropical Asian islands. The intercontinental disjunct lineage in Astilbe invokes a hybridization event either in eastern Asia or in North America. This disjunction in Astilbe may be explained by a Beringian migration around 3.54 Ma (95% high posterior density: 1.29–6.18 Ma) in the late Tertiary, although long‐distance dispersal from eastern Asia to North America is also likely. The biogeographical connection between continental Asia, Taiwan, the Philippines and other tropical Asian islands in Astilbe provides evidence for the close floristic affinity between temperate or alpine south‐western China and tropical Asia. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, ●● , ●●–●●.     

Phylogenetics of early branching eudicots:Comparing phylogenetic signal across plastid introns,spacers,and genes

<正>Recent phylogenetic analyses revealed a grade with Ranunculales,Sabiales,Proteales,Trochodendrales, and Buxales as first branching eudicots,with the respective positions of Proteales and Sabiales still lacking statistical confidence.As previous analyses of conserved plastid genes remain inconclusive,we aimed to use and evaluate a representative set of plastid introns(group I:trnL;group II:petD,rpll6,trnK) and intergenic spacers(trnL-F,petB-petD, atpB-rbcL,rps3-rpl16) in comparison to the rapidly evolving matK and slowly evolving atpB and rbcL genes. Overall patterns of micro structural mutations converged across genomic regions,underscoring the existence of a general mutational pattern throughout the plastid genome.Phylogenetic signal differed strongly between functionally and structurally different genomic regions and was highest in matK,followed by spacers,then group II and group I introns.The more conserved atpB and rbcL coding regions showed distinctly lower phylogenetic information content.Parsimony,maximum likelihood,and Bayesian phylogenetic analyses based on the combined dataset of non-coding and rapidly evolving regions(14 000 aligned characters) converged to a backbone topology of eudicots with Ranunculales branching first,a Proteales-Sabiales clade second,followed by Trochodendrales and Buxales. Gunnerales generally appeared as sister to all remaining core eudicots with maximum support.Our results show that a small number of intron and spacer sequences allow similar insights into phylogenetic relationships of eudicots compared to datasets of many combined genes.The non-coding proportion of the plastid genome thus can be considered an important information source for plastid phylogenomics.     

Molecular phylogenetics and biogeography of the eastern Asian–eastern North American disjunct Mitchella and its close relative Damnacanthus (Rubiaceae,Mitchelleae)

Mitchella is a small genus of the Rubiaceae with only two species. It is the only herbaceous semishrub of the family showing a disjunct distribution in eastern Asia and eastern North America, extending to Central America. Its phylogeny and biogeographical diversification remain poorly understood. In this study, we conducted phylogenetic and biogeographical analyses for Mitchella and its close relative Damnacanthus based on sequences of the nuclear internal transcribed spacer (ITS) and four plastid markers (rbcL, atpB‐rbcL, rps16 and trnL‐F). Mitchella is monophyletic, consisting of an eastern Asian M. undulata clade and a New World M. repens clade. Our results also support Michella as the closest relative to the eastern Asian Damnacanthus. The divergence time between the two intercontinental disjunct Mitchella species was dated to 7.73 Mya, with a 95% highest posterior density (HPD) of 3.14?12.53 Mya, using the Bayesian relaxed clock estimation. Ancestral area reconstructions suggest that the genus originated in eastern Asia. The semishrub Mitchella appears to have arisen from its woody ancestor in eastern Asia and then migrated to North America via the Bering land bridge in the late Miocene. © 2013 The Linnean Society of London     

Phylogenetic analysis of Chloraeinae (Orchidaceae) based on plastid and nuclear DNA sequences

The phylogenetic relationships of subtribe Chloraeinae, a group of terrestrial orchids endemic to southern South America, have not been satisfactorily investigated. A previous molecular phylogenetic analysis based on plastid DNA supported the monophyly of Chloraeinae and Gavilea, but showed that Chloraea is non‐monophyletic and that the sole species of Bipinnula analysed is sister to Geoblasta. However, that analysis included only 18 of the 73 species belonging to this subtribe. Here, the phylogenetic relationships of Chloraeinae were assessed by analysing aproximately 7500 bp of nucleotide sequences from nuclear ribosomal internal transcribed spacer (ITS) and plastid DNA (rbcL, matK, trnL‐trnF, rpoB‐trnC) for 42 species representing all four currently accepted genera of Chloraeinae and appropriate outgroups. Nuclear and plastid data were analysed separately and in combination using two different methods, namely parsimony and Bayesian inference. Our analyses support the monophyly of Chloraeinae and their inclusion in an expanded concept of Cranichideae, but none of the genera of Chloraeinae that includes more than one species is monophyletic. Gavilea and Bipinnula are paraphyletic, with Chloraea chica nested in Gavilea and Geoblasta penicillata in Bipinnula. As currently delimited, Chloraea is polyphyletic. The taxonomic changes proposed recently are for the most part not justifiable on phylogenetic grounds, except for recognition of the monotypic genus Correorchis. The lack of resolution for the relationships among species of ‘core’Chloraea suggests a relatively recent diversification of this group. The current generic classification is in need or revision, but additional study is advisable before carrying out further taxonomic changes. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 168 , 258–277.     

Phylogenetic biogeography of the leafy liverwort Herbertus (Jungermanniales, Herbertaceae) based on nuclear and chloroplast DNA sequence data: correlation between genetic variation and geographical distribution

Aim The cosmopolitan genus Herbertus is notorious for having a difficult taxonomy and for the fact that there is limited knowledge of species ranges and relationships. Topologies generated from variable molecular markers are used to discuss biogeographical patterns in Herbertus and to compare them with the geological history of continents and outcomes reported for other land plants. Location Africa, Asia, Azores, Europe, southern South America, northern South America, North America, New Zealand. Methods Phylogenetic analyses of nuclear ribosomal internal transcribed spacer and chloroplast (cp) trnL–trnF sequences of 66 accessions of Herbertus and the outgroup species Triandrophyllum subtrifidum and Mastigophora diclados were used to investigate biogeographical patterns in Herbertus. Areas of putative endemism were defined based on the distribution of species included in the analyses. Maximum parsimony analyses were undertaken to reconstruct ancestral areas and intraspecies migration routes. Results The analyses reveal species‐level cladograms with a correlation between genetic variation and the geographical distribution of the related accessions. The southern South American Herbertus runcinatus is sister to the remainder of the genus, which is split into two main clades. One contains the Neotropical–African Herbertus juniperoideus and the New Zealand/Tasmanian Herbertus oldfieldianus. An African accession of H. juniperoideus is nested within Neotropical accessions. The second main clade includes species that inhabit Asia, the Holarctic, Africa, and northern South America. Maximum parsimony analyses indicate that this clade arose in Asia. Herbertus sendtneri originated in Asia and subsequently colonized the Holarctic and northern South America. An Asian origin and colonization into Africa is indicated for H. dicranus. Main conclusions The current distribution of Herbertus cannot be explained by Gondwanan vicariance. A more feasible explanation of the range is a combination of short‐distance dispersal, rare long‐distance dispersal events (especially into regions that faced floral displacements as a result of climatic changes) extinction, recolonization, and diversification. The African Herbertus flora is a mixture of Asian and Neotropical elements. Southern South America harbours an isolated species. The molecular data indicate partial decoupling of molecular and morphological variation in Herbertus. Biogeographical patterns in Herbertus are not dissimilar to those of other groups of bryophytes, but elucidation of the geographical ranges requires a molecular approach. Some patterns could be the result of maintenance of Herbertus in the inner Tropics during glacial maxima, and dispersal into temperate regions in warm phases.     

Biogeography and diversification of colletid bees (Hymenoptera: Colletidae): emerging patterns from the southern end of the world

Aim The evolutionary history of bees is presumed to extend back in time to the Early Cretaceous. Among all major clades of bees, Colletidae has been a prime example of an ancient group whose Gondwanan origin probably precedes the complete break‐up of Africa, Antarctica, Australia and South America, because modern lineages of this family occur primarily in southern continents. In this paper, we aim to study the temporal and spatial diversification of colletid bees to better understand the processes that have resulted in the present southern disjunctions. Location Southern continents. Methods We assembled a dataset comprising four nuclear genes of a broad sample of Colletidae. We used Bayesian inference analyses to estimate the phylogenetic tree topology and divergence times. Biogeographical relationships were investigated using event‐based analytical methods: a Bayesian approach to dispersal–vicariance analysis, a likelihood‐based dispersal–extinction–cladogenesis model and a Bayesian model. We also used lineage through time analyses to explore the tempo of radiations of Colletidae and their context in the biogeographical history of these bees. Results Initial diversification of Colletidae took place at the Late Cretaceous (≥ 70 Ma). Several (6–14) lineage exchanges between Australia and South America via Antarctica during the Late Cretaceous and Eocene epochs could explain the disjunctions observed between colletid lineages today. All biogeographical methods consistently indicated that there were multiple lineage exchanges between South America and Australia, and these approaches were valuable in exploring the degree of uncertainty inherent in the ancestral reconstructions. Biogeographical and dating results preclude an explanation of Scrapterinae in Africa as a result of vicariance, so one dispersal event is assumed to explain the disjunction in relation to Euryglossinae. The net diversification rate was found to be highest in the recent history of colletid evolution. Main conclusions The biogeography and macroevolutionary history of colletid bees can be explained by a combination of Cenozoic vicariance and palaeoclimatic changes during the Neogene. The austral connection and posterior break‐up of South America, Antarctica and Australia resulted in a pattern of disjunct sister lineages. Increased biome aridification coupled with floristic diversification in the southern continents during the Neogene may have contributed to the high rates of cladogenesis in these bees in the last 25–30 million years.     

DNA barcoding of Gaultheria L. in China (Ericaceae: Vaccinioideae)

Abstract Four DNA barcoding loci, chloroplast loci rbcL, matK, trnH‐psbA, and nuclear locus internal transcribed spacer (ITS), were tested for the accurate discrimination of the Chinese species of Gaultheria by using intraspecific and interspecific pairwise P‐distance, Wilcoxon signed rank test, and tree‐based analyses. This study included 186 individuals from 89 populations representing 30 species. For all individuals, single locus markers showed high levels of sequencing universality but were ineffective for species resolvability. Polymerase chain reaction amplification and sequencing were successful for all four loci. Both ITS and matK showed significantly higher levels of interspecific species delimitation than rbcL and trnH‐psbA. A combination of matK and ITS was the most efficient DNA barcode among all studied regions, however, they do not represent an appropriate candidate barcode for Chinese Gaultheria, by which only 11 out of 30 species can be separated. Loci rbcL, matK, and trnH‐psbA, which were recently proposed as universal plant barcodes, have a very poor capacity for species separation for Chinese Gaultheria. DNA barcodes may be reliable tools to identify the evolutionary units of this group, so further studies are needed to develop more efficient DNA barcodes for Gaultheria and other genera with complicated evolutionary histories.     

DNA barcoding herbaceous and woody plant species at a subalpine forest dynamics plot in Southwest China

Although DNA barcoding has been widely used to identify plant species composition in temperate and tropical ecosystems, relatively few studies have used DNA barcodes to document both herbaceous and woody components of forest plot. A total of 201 species (72 woody species and 129 herbaceous species) representing 135 genera distributed across 64 families of seed plants were collected in a 25 ha CForBio subalpine forest dynamics plot. In total, 491 specimens were screened for three DNA regions of the chloroplast genome (rbcL, matK, and trnH‐psbA) as well as the internal transcribed spacers (ITS) of nuclear ribosomal DNA. We quantified species resolution for each barcode separately or in combination using a ML tree‐based method. Amplification and sequencing success were highest for rbcL, followed by trnH‐psbA, which performed better than ITS and matK. The rbcL + ITS barcode had slightly higher species resolution rates (88.60%) compared with rbcL + matK (86.60%) and rbcL + trnH‐psbA (86.01%). The addition of trnH‐psbA or ITS to the rbcL + matK barcode only marginally increased species resolution rates, although in combination the four barcodes had the highest discriminatory power (90.21%). The situations where DNA barcodes did not discriminate among species were typically associated with higher numbers of co‐occurring con‐generic species. In addition, herbaceous species were much better resolved than woody species. Our study represents one of the first applications of DNA barcodes in a subalpine forest dynamics plot and contributes to our understanding of patterns of genetic divergence among woody and herbaceous plant species.     

'Andean-centred' genera in the short-branch clade of Annonaceae: testing biogeographical hypotheses using phylogeny reconstruction and molecular dating

Aim We test biogeographical hypotheses regarding the origin of Andean‐centred plant groups by reconstructing phylogeny in the short‐branch clade (SBC) of Annonaceae, and estimating the timing of diversifications in four apparently Andean‐centred genera: Cremastosperma R.E.Fr., Klarobelia Chatrou, Malmea R.E.Fr. and Mosannona Chatrou. The SBC includes species distributed in both the Old and New World tropics. A number of the Neotropical genera display ‘Andean‐centred’ distribution patterns, with high species richness on both sides of the Andes mountain range. In particular, we test whether these groups could have originated on the South American continent during the time frame of the Andean orogeny [from c. 23 Ma (Miocene) to the present]. Methods Chloroplast DNA sequences were used to reconstruct phylogeny in related Annonaceae taxa plus outgroups, under maximum parsimony and Bayesian inference. The markers rbcL, trnL‐trnF and psbA‐trnH were sampled for 96 accessions to test the monophyly of each of the genera, and thus whether they might be para‐ or polyphyletic with respect to related groups distributed across Amazonia. To determine the sister groups of the four genera, the additional markers matK, ndhF, trnT‐trnL, trnS‐trnG and atpB‐rbcL were sampled for 23 of the 96 accessions. Molecular dating techniques (nonparametric rate‐smoothing; penalized likelihood; Bayesian inference) were then applied to estimate the age of the crown group of each genus and the age of their sister groups. Results Monophyly was confirmed in Cremastosperma, Malmea and Mosannona. The monotypic genus Pseudephedranthus Aristeg. was found to be nested within Klarobelia, the species of which otherwise formed a monophyletic group, and a South American‐centred (SAC) clade was identified. The SAC clade comprises all the SBC genera distributed in South America and generally to a limited extent into Central America, but not those endemic to Africa, Asia and Central America. Age estimations for clades within the SBC were no older than around 60 Myr; those for the crown groups of Cremastosperma, Klarobelia, Malmea and Mosannona fell largely within the last 10–20 Myr. Main conclusions The distribution patterns of Cremastosperma, Klarobelia, Malmea and Mosannona are not the arbitrary result of the definition of para‐ or polyphyletic groups. We infer the presence of a common ancestor of the four genera in South America, but not by vicariance of an ancestral population on Gondwana. The age estimations, instead, may suggest that the SAC clade originated in South America by dispersal across the Boreotropics. Although the strength of this test was limited by imprecision in the molecular dating results, the ages of crown groups of the four genera suggest that diversifications occurred within the time frame of the orogeny of the Northern Andes.     

Long-distance colonization of the Western Mediterranean by Cistus ladanifer (Cistaceae) despite the absence of special dispersal mechanisms

Aim There are few biogeographical and evolutionary studies that address plant colonization and lineage origins in the Mediterranean. Cistus serves as an excellent model with which to study diaspore dispersal and distribution patterns of plants exhibiting no special long‐distance dispersal mechanisms. Here we analyse the pattern of genetic variation and divergence times to infer whether the African–European disjunction of C. ladanifer L. is the result of long‐distance dispersal or of vicariance events. Location Principally the Western Mediterranean region, with a focus on the Strait of Gibraltar. Methods We used DNA sequence phylogenetic approaches, based on plastid (rbcL/trnK‐matK) and nuclear (ITS) sequence data sets, and the penalized likelihood method, to date the diversification of the 21 species of Cistus. Phylogenetic relationships and phylogeographical patterns in 47 populations of C. ladanifer were also analysed using two plastid DNA regions (trnS‐trnG, trnK‐matK). These sequence data were analysed using maximum parsimony, Bayesian inference and statistical parsimony. Results Dating estimates indicated divergence dates of the C. ladanifer lineage in the Pleistocene. Eight nucleotide‐substitution haplotypes distributed on the European (four haplotypes) and African (five haplotypes) sides of the Strait of Gibraltar were revealed from C. ladanifer sequences. Both the haplotype network and the phylogenetic analyses depicted two main Cistus lineages distributed in both Europe and North Africa. An Iberian haplotype forms part of the North African lineage, and another haplotype distributed on both continents is related to the European lineage. Haplotype relationships with respect to outgroup sequences supported the hypothesis that the centre of genetic diversity is in northern Africa. Main conclusions Based on lineage divergence‐time estimates and disassociation between geographical and lineage haplotype distributions, we inferred at least two intercontinental colonization events of C. ladanifer post‐dating the opening of the Strait of Gibraltar (c. 5 Ma). This result supports a hypothesis of long‐distance dispersal rather than a hypothesis of vicariance. We argue that, despite limited dispersal abilities, preference for disturbed habitats was integral to historical colonization after the advent of the Mediterranean climate (c. 3.2 Ma), when Cistus species diverged and became established as a dominant element in the Mediterranean scrub.     

Out of Africa: molecular phylogenetics and biogeography of Wolffiella (Lemnaceae)

The monophyletic genus Wolffiella (Lemnaceae) comprises 10 species divided taxonomically into three sections. Relative to other genera of Lemnaceae, Wolffiella has a restricted range, with species distributed in warm temperate to tropical areas of Africa and the Americas, with only one species occurring in both areas. Sequence data from coding (rbcL and matK) and non‐coding (trnK and rpl16 introns) regions of cpDNA were analyzed phylogenetically to resolve relationships within Wolffiella, and these results were compared to earlier allozyme and morphological studies. Allozymes, cpDNA and morphology all supported the recognition of three sections. Relationships among species were similar in most respects between the allozyme and cpDNA trees, as well as among the different plastid partitions. In Wolffiella, both non‐synonymous and synonymous substitutions were greater in matK than in rbcL, as observed in other taxa. The synonymous substitution rate in matK was similar to the substitution rate of the non‐coding regions. All partitions, including coding regions, exhibited some homoplasy. Biogeographical reconstructions from a combination of cpDNA partitions indicated that Wolffiella originated in Africa with early movement to and radiation in the Americas. The one species found in both Africa and the Americas, W. welwitschii, likely originated in the Americas and subsequently dispersed to Africa. Using the SOWH test, the cpDNA data could reject two alternative biogeographical hypotheses suggested from analyses of morphological and allozyme data. The present distribution of Wolffiella can be explained by two major dispersal events and this contrasts with the more complex species distributions in other Lemnaceae genera. Limited dispersal in Wolffiella relative to other Lemnaceae genera may be due to more recent origins of species, lower dispersibility and poorer colonizing ability. © 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 79 , 565–576.     

Assessing the potential of candidate DNA barcodes for identifying non‐flowering seed plants

In plants, matK and rbcL have been selected as core barcodes by the Consortium for the Barcode of Life (CBOL) Plant Working Group (PWG), and ITS/ITS2 and psbA‐trnH were suggested as supplementary loci. Yet, research on DNA barcoding of non‐flowering seed plants has been less extensive, and the evaluation of DNA barcodes in this division has been limited thus far. Here, we evaluated seven markers (psbA‐trnH, matK, rbcL, rpoB, rpoC1, ITS and ITS2) from non‐flowering seed plants. The usefulness of each region was assessed using four criteria: the success rate of PCR amplification, the differential intra‐ and inter‐specific divergences, the DNA barcoding gap and the ability to discriminate species. Among the seven loci tested, ITS2 produced the best results in the barcoding of non‐flowering seed plants. In addition, we compared the abilities of the five most‐recommended markers (psbA‐trnH, matK, rbcL, ITS and ITS2) to identify additional species using a large database of gymnosperms from GenBank. ITS2 remained effective for species identification in a wide range of non‐flowering seed plants: for the 1531 samples from 608 species of 80 diverse genera, ITS2 correctly authenticated 66% of them at the species level. In conclusion, the ITS2 region can serve as a useful barcode to discriminate non‐flowering seed plants, and this study will contribute valuable information for the barcoding of plant species.     

Testing DNA barcodes in closely related species of Curcuma (Zingiberaceae) from Myanmar and China

The genus Curcuma L. is commonly used as spices, medicines, dyes and ornamentals. Owing to its economic significance and lack of clear‐cut morphological differences between species, this genus is an ideal case for developing DNA barcodes. In this study, four chloroplast DNA regions (matK, rbcL, trnH‐psbA and trnL‐F) and one nuclear region (ITS2) were generated for 44 Curcuma species and five species from closely related genera, represented by 96 samples. PCR amplification success rate, intra‐ and inter‐specific genetic distance variation and the correct identification percentage were taken into account to assess candidate barcode regions. PCR and sequence success rate were high in matK (89.7%), rbcL (100%), trnH‐psbA (100%), trnL‐F (95.7%) and ITS2 (82.6%) regions. The results further showed that four candidate chloroplast barcoding regions (matK, rbcL, trnH‐psbA and trnL‐F) yield no barcode gaps, indicating that the genus Curcuma represents a challenging group for DNA barcoding. The ITS2 region presented large interspecific variation and provided the highest correct identification rates (46.7%) based on BLASTClust method among the five regions. However, the ITS2 only provided 7.9% based on NJ tree method. An increase in discriminatory power needs the development of more variable markers.