Molecular systematics and historical biogeography of Araceae at a worldwide scale and in Southeast Asia

This dissertation addresses the biogeographic history of the Araceae family and of one of its largest genera, Alocasia. With >3300 species, Araceae are among the largest families of flowering plants. It is the monocot lineage with the deepest fossil record, reaching back to the Early Cretaceous. Araceae are distributed worldwide, but >3100 species occur in the tropical regions of the Americas, Asia, Africa, and Australia; most fossils from the Late Cretaceous and many younger ones come from the temperate zone in the northern hemisphere, implying much extinction and range expansion. Most subfamilies are pantropically distributed, and almost all genera are restricted to one continent. Alocasia comprises 113 species, many as yet undescribed, making it the 7 th -largest genus of the Araceae. Many species are ornamentals, and two species are of interest for man, either for food (giant taro) or in local cultures (Chinese taro). The origin of these species was not known. Alocasia is distributed in Southeast Asia from India to Australia, with species occurring on all islands of the Malay Archipelago. This region has a complex geologic history shaped by the collision of the Eurasian, the Pacific, and the Indo-Australian plate. The Malesian flora and fauna comprises Laurasian and Gondwanan elements, reflecting the influence of changing sea levels, uplift and submergence of islands, and other tectonic movement. In this thesis, I used molecular phylogenetics, Bayesian divergence dating, ancestral area reconstruction to understand the past distribution of the Araceae family and the Alocasia clade in the context of past continent movements and climate history. For the family analysis, existing chloroplast DNA matrices were augmented so that all Araceae genera were represented by one or more species, with a focus on covering geographic disjunctions, especially between continents. Divergence dating relied on seven confidently assigned fossil constraints, comparing uniform and gamma-shaped prior distributions on fossil ages, as well as several molecular clock models. Biogeographic analyses were performed in a model-based likelihood framework that took into account past dispersal routes based on continent connectivity and climate. I also integrated fossils into the ancestral area reconstruction, either simulating extinct or still existing ranges, and then compared results to those obtained from analyses without fossils. To study the morphology and ecology of Alocasia, fieldwork was conducted in Malaysia and herbarium work in Germany, the Netherlands, Indonesia, Malaysia, and Singapore. Maximum likelihood phylogenies were inferred based on chloroplast and nuclear loci, sequenced for 71 species of Alocasia plus 25 outgroup species from 16 genera. Bayesian divergence dating of the nuclear phylogeny relied on one fossil constraint and ancestral areas were reconstructed using parsimony- and likelihood-based methods. The Araceae diverged from the remaining Alismatales in the Early Cretaceous (ca. 135 Ma ago), and all eight subfamilies originated before the Cenozoic. The earliest lineages are inferred to have occurred in Laurasia (based on fossils and tree topology), and most lineages reached Africa, South America, Southeast Asia, and Australia during the Paleogene and Neogene. Many clades experienced extinction in the temperate regions of the northern hemisphere during the Oligocene climate cooling. Two continentally disjunct genera (Nephthytis and Philodendron) are polyphyletic and need taxonomic rearrangement. Plastid substitution rates are exceptionally high in free-floating and water-associated Araceae. Ancestral area reconstructions obtained when fossil (no longer occupied) ranges where included in the analyses were more plausible than those without fossil ranges. This is not a trivial result because only in a quantitative (computer-based) analysis is it possible for fossil ranges to influence results (here areas) at distant nodes in the phylogenetic tree. The nuclear and plastid phylogenies of Alocasia revealed the polyphyly of the two genera Alocasia and Colocasia; to achieve monophyly, two species (Alocasia hypnosa and Colocasia gigantea) have to be moved to other genera. There were strong incongruencies between phylogenies from the two partitions: The chloroplast data reflect geographical proximity, the nuclear morphological similarity. This may indicate hybridization events followed by chloroplast capture. Based on the nuclear tree, Alocasia split from its sister group by the end of the Oligocene (ca. 24 Ma) and colonized the Malay Archipelago from the Asian mainland. Borneo played a central role, with 11–13 of 18–19 inferred dispersal events originating there. The Philippines were reached from Borneo 4–5 times in the late Miocene and early Pliocene, and the Asian mainland 6–7 times during the Pliocene. The geographic origin of two domesticated species could be resolved: Giant taro originated on the Philippines and Chinese taro on the Asian mainland.Diese Doktorarbeit behandelt die historische Biogeografie der Araceen und eine ihrer artenreichsten Gattungen, Alocasia. Die Araceen sind mit über 3300 Arten unter den größten Familien der Blütenpflanzen. Sie sind weltweit verbreitet, wobei der Großteil (>3100 Arten) in den tropischen Regionen von Amerika, Asien, Afrika und Australien vorkommt. Die ältesten Fossilien der Monokotyledonen überhaupt sind Araceenfossilien aus der Unterkreide. Diese und viele der jüngeren Fossilien stammen jedoch aus gemäßigten Gebieten der nördlichen Hemisphäre, was auf ausgestorbene Abstammungslinien und Veränderungen der Verbreitungsgebiete hindeutet. Mit 113 Arten ist Alocasia die 7. größte Gattung der Araceen. Viele Arten sind Zierpflanzen, wobei zwei Arten als Nahrung (Riesen-Taro) oder aus kulturellen Gründen (chinesischer Taro) abgebaut werden, deren geografischer Ursprung jedoch nicht bekannt ist. Die Gattung Alocasia ist von Indien über ganz Südostasien bis nach Australien verbreitet und kommt auf jeder größeren Insel im Malaiischen Archipel vor. Diese Region hat eine komplexe geologische Geschichte, die von den Kollisionen der Pazifischen, Indisch-Australischen und Eurasischen Kontinentalplatten bestimmt wurde. Die Flora und Fauna in dem Malaiischen Archipel besteht aus laurasichen und gondwanischen Elementen, deren jeweilige Verbreitung durch schwankende Meeresspiegel, dem Auf- und Abtauchen von Inseln und anderen tektonischen Prozessen beeinflusst wurde. In dieser Dissertation rekonstruierte ich ehemalige Verbreitungsgebiete und Ausbreitungswege mit Hilfe von molekularer Phylogenetik und Bayes’scher Altersbestimmung, um die frühere Verbreitung der Araceen sowie von Alocasia im Kontext der Kontinentalbewegungen und der Klimageschichte zu verstehen. Für die Familiendatierung wurde eine Matrix von Chloroplasten Sequenzen derart erweitert, dass alle Gattungen mit mindestens einer Art vertreten sind und sofern eine Gattung geographisch disjunkt vorkommt (insbesondere zwischen Kontinenten) diese von einer mindestens Art pro Region vertreten ist. Die Altersbestimmung beruhte auf der Kalibrierung mit sieben Fossilien, dem Vergleich zwischen uniform- und gammaförmigen Priorverteilungen, sowie der Gegenüberstellung mehrerer Modelle molekularer Uhren. Biogeografische Analysen wurden mit Hilfe von einer Modell basierten Wahrscheinlichkeits-(likelihood") Methode durchgeführt, die frühere Ausbreitungswege aufgrund vergangener Kontinentalbewegungen und Klimaänderungen in Betracht ziehen kann. Zusätzlich habe ich Fossilien in die Rekonstruktion ehemaliger Verbreitungsgebiete eingebunden, entweder ausgestorbene oder noch lebende Arten simulierend, und die Ergebnisse mit den Analysen ohne Fossilien verglichen. Um die Morphologie und Ökologie von Alocasia zu untersuchen, habe ich Feldarbeit in Malaysia und Herbariumsarbeit in Deutschland, den Niederlanden, Indonesien, Malaysia und Singapur durchgeführt. Phylogenien, basierend auf Chloroplasten- und Kern- Sequenzen, wurden für 71 Alocasia Arten und 25 Außengruppenarten aus 16 Gattungen erstellt. Bayes’sche Altersbestimmung der nukleären Phylogenie wurde mit einem Fossil kalibriert und die Rekonstruktion ehemaliger Verbreitungsgebiete wurde mit Sparsamkeits- („parsimony”) und Wahrscheinlichkeits- („likelihood”) Methoden durchgeführt. Die Araceen haben sich zu Beginn der Unterkreide (vor ca. 135 Ma) von den übrigen Alismatales abgespalten. Alle acht Unterfamilien existierten schon vor dem Paläogen. Die Rekonstruktionen zeigen, dass die ersten Araceen in Laurasien verbreitet waren (basierend auf Fossilfunden und Stammbaumtopologie), und die meisten Abstammungslinien erreichten Afrika, Südamerika, Südostasien und Australien im Paläogen und Neogen. Viele Stammlinien sind während der Abkühlung des Klimas im Oligozän in den gemäßigten Regionen der nördlichen Hemisphäre ausgestorben. Zwei disjunkt verbreitete Gattungen (Nephthytis und Philodendron) sind polyphyletisch und benötigen taxonomische Bearbeitung. Plastidäre Substitutionsraten sind außergewöhnlich hoch in den freischwimmenden und wasserassoziierten Araceen sowie in basalen Abstammungslinien. Die Rekonstruktion ehemaliger Verbreitungsgebiete ist glaubwürdiger, wenn Fossilen in die Analysen eingebunden wurden. Dies ist kein triviales Ergebnis, denn nur in einer quantitativen Analyse ist es möglich, dass Fossilien die Rekonstruktionen entfernter Knoten in einer Phylogenie beeinflussen. Die Phylogenien von Alocasia enthüllten, dass Alocasia und Colocasia polyphyletisch sind. Um deren Monophylie wieder herzustellen, müssen zwei Arten (Alocasia hypnosa und Colocasia gigantea) in andere Gattungen verlegt werden. Es ergaben sich gut gestützte topologische Unterschiede zwischen den nukleären und plastidären Phylogenien. In der plastidären Phylogenie sind Arten nach ihrer geografischen Verbreitung gruppiert, in der nukleären Phylogenie nach morphologischer Ähnlichkeit. Dies deutet auf Hybridisierung mit anschließendem ’chloroplast capture’ hin. Basierend auf dem nukleären Stammbaum hat sich Alocasia zum Ende des Oligozäns hin (vor ca. 24 Ma) von der Schwestergruppe abgespalten und den Malaiischen Archipel vom asiatischen Festland aus besiedelt. Borneo, von wo 11–13 der 18–19 Ausbreitungen ausgingen, spielte dabei eine zentrale Rolle. Die Philippinen wurden von Borneo aus 4–5-mal im späten Miozän und frühen Paläogen besiedelt, und das asiatische Festland 6–7-mal im Pliozän. Die geografische Herkunft der zwei domestizierten Arten konnte geklärt werden: Riesen-Taro kommt von den Philippinen und Chinesischer Taro vom asiatischen Festland

Implications of Pairwise Genome Comparisons in Pyrus (Rosaceae) and Other Angiosperms for Marker Choice

Plastid genomes exhibit different levels of variability in their sequences, depending on the respective kinds of genomic regions. Genes are usually more conserved while noncoding introns and spacers evolve at a faster pace. While a set of about thirty maximum variable noncoding genomic regions has been suggested to provide universally promising phylogenetic markers throughout angiosperms, applications often require several regions to be sequenced for many individuals. Our project aims to illuminate evolutionary relationships and species-limits in the genus Pyrus (Rosaceae)—a typical case with very low genetic distances between taxa. In this study, we have sequenced the plastid genome of Pyrus spinosa and aligned it to the already available P. pyrifolia sequence. The overall p-distance of the two Pyrus genomes was 0.00145. The intergenic spacers between ndhC–trnV, trnR–atpA, ndhF–rpl32, psbM–trnD, and trnQ–rps16 were the most variable regions, also comprising the highest total numbers of substitutions, indels and inversions (potentially informative characters). Our comparative analysis of further plastid genome pairs with similar low p-distances from Oenothera (representing another rosid), Olea (asterids) and Cymbidium (monocots) showed in each case a different ranking of genomic regions in terms of variability and potentially informative characters. Only two intergenic spacers (ndhF–rpl32 and trnK–rps16) were consistently found among the 30 top-ranked regions. We have mapped the occurrence of substitutions and microstructural mutations in the four genome pairs. High AT content in specific sequence elements seems to foster frequent mutations. We conclude that the variability among the fastest evolving plastid genomic regions is lineage-specific and thus cannot be precisely predicted across angiosperms. The often lineage-specific occurrence of stem-loop elements in the sequences of introns and spacers also governs lineage-specific mutations. Sequencing whole plastid genomes to find markers for evolutionary analyses is therefore particularly useful when overall genetic distances are low

HybPhaser: a workflow for the detection and phasing of hybrids in target capture data sets

PREMISE: Hybrids contain divergent alleles that can confound phylogenetic analyses but can provide insights into reticulated evolution when identified and phased. We developed a workflow to detect hybrids in target capture data sets and phase reads into parental lineages using a similarity and phylogenetic framework. METHODS: We used Angiosperms353 target capture data for Nepenthes, including known hybrids to test the novel workflow. Reference mapping was used to assess heterozygous sites across the data set and to detect hybrid accessions and paralogous genes. Hybrid samples were phased by mapping reads to multiple references and sorting reads according to similarity. Phased accessions were included in the phylogenetic framework. RESULTS: All known Nepenthes hybrids and nine additional samples had high levels of heterozygous sites, had reads associated with multiple divergent clades, and were phased into accessions resembling divergent haplotypes. Phylogenetic analysis including phased accessions increased clade support and confirmed parental lineages of hybrids. DISCUSSION: HybPhaser provides a novel approach to detect and phase hybrids in target capture data sets, which can provide insights into reticulations by revealing origins of hybrids and reduce conflicting signal, leading to more robust phylogenetic analyses

Evolutionary Relationships and Range Evolution of Greenhood Orchids (Subtribe Pterostylidinae): Insights From Plastid Phylogenomics

Australia harbours a rich and highly endemic orchid flora with over 90% of native species found nowhere else. However, little is known about the assembly and evolution of Australia’s orchid flora. Here, we used a phylogenomic approach to infer evolutionary relationships, divergence times and range evolution in Pterostylidinae (Orchidoideae), the second largest subtribe in the Australian orchid flora, comprising the genera Pterostylis and Achlydosa. Phylogenetic analysis of 75 plastid genes provided well-resolved and supported phylogenies. Intrageneric relationships in Pterostylis were clarified and monophyly of eight of 10 sections supported. Achlydosa was found to not form part of Pterostylidinae and instead merits recognition at subtribal level, as Achlydosinae. Pterostylidinae were inferred to have originated in eastern Australia in the early Oligocene, coinciding with the complete separation of Australia from Antarctica and the onset of the Antarctic Circumpolar Current, which led to profound changes in the world’s climate. Divergence of all major lineages occurred during the Miocene, accompanied by increased aridification and seasonality of the Australian continent, resulting in strong vegetational changes from rainforest to more open sclerophyllous vegetation. The majority of extant species were inferred to have originated in the Quaternary, from the Pleistocene onwards. The rapid climatic oscillations during the Pleistocene may have acted as important driver of speciation in Pterostylidinae. The subtribe underwent lineage diversification mainly within its ancestral range, in eastern Australia. Long-distance dispersals to southwest Australia commenced from the late Miocene onwards, after the establishment of the Nullarbor Plain, which constitutes a strong edaphic barrier to mesic plants. Range expansions from the mesic into the arid zone of eastern Australia (Eremaean region) commenced from the early Pleistocene onwards. Extant distributions of Pterostylidinae in other Australasian regions, such as New Zealand and New Caledonia, are of more recent origin, resulting from long-distance dispersals from the Pliocene onwards. Temperate eastern Australia was identified as key source area for dispersals to other Australasian regions

Comparative analysis of plastid genomes in the non-photosynthetic genus Thismia reveals ongoing gene set reduction

Heterotrophic plants provide intriguing examples of reductive evolution. This is especially evident in the reduction of their plastid genomes, which can potentially proceed toward complete genome loss. Several milestones at the beginning of this path of degradation have been described; however, little is known about the latest stages of plastome reduction. Here we analyze a diversity of plastid genomes in a set of closely related non-photosynthetic plants. We demonstrate how a gradual loss of genes shapes the miniaturized plastomes of these plants. The subject of our study, the genus Thismia, represents the mycoheterotrophic monocot family Thismiaceae, a group that may have experienced a very ancient (60–80 mya) transition to heterotrophy. In all 18 species examined, the plastome is reduced to 14–18 kb and is highly AT-biased. The most complete observed gene set includes accD, seven ribosomal protein genes, three rRNA, and two tRNA genes. Different clades of Thismia have undergone further gene loss (complete absence or pseudogenization) compared to this set: in particular, we report two independent losses of rps2 and rps18

Plastid genome structure and phylogenomics of Nymphaeales: conserved gene order and new insights into relationships

The plastid genomes of early-diverging angiosperms were among the first land plant plastomes investigated. Despite their importance to understanding angiosperm evolution, no investigation has so far compared gene content or gene synteny of these plastid genomes with a focus on the Nymphaeales. Here, we report an evaluation and comparison of gene content, gene synteny and inverted repeat length for a set of 15 plastid genomes of early-diverging angiosperms. Seven plastid genomes of the Nymphaeales were newly sequenced for this investigation. We compare gene order and inverted repeat (IR) length across all genomes, review the gene annotations of previously published genomes, generate a multi-gene alignment of 77 plastid-encoded genes and reconstruct the phylogenetic relationships of the taxa under study. Our results show that gene content and synteny are highly conserved across early-diverging angiosperms: All species analyzed display complete gene synteny when accounting for expansions and contractions of the IRs. This conservation was initially obscured by ambiguous and potentially incorrect gene annotations in previously published genomes. We also report the presence of intact open reading frames across all taxa analyzed. The multi-gene phylogeny displays maximum support for the families Cabombaceae and Hydatellaceae, but no support for a clade of all Nymphaeaceae. It further indicates that the genus Victoria is embedded within Nymphaea. Plastid genomes of Trithuria were found to deviate by numerous substitutions and length changes in the IRs. Phylogenetic analyses further indicate that a previously published plastome named Nymphaea mexicana falls into a clade of N. odorata and should be re-evaluated

Australasian orchid biogeography at continental scale: molecular phylogenetic insights for the Sun Orchids (Thelymitra, Orchidaceae)

Australia harbours a rich and highly endemic orchid flora, with c. 90% of species endemic to the country. Despite that, the biogeographic history of Australasian orchid lineages is only poorly understood. Here we examined evolutionary relationships and the spatio-temporal evolution of the sun orchids (Thelymitra, 119 species), which display disjunct distribution patterns frequently found in Australasian orchid lineages. Phylogenetic analyses were conducted based on one nuclear (ITS) and three plastid markers (matK, psbJ-petA, ycf1) using Maximum Likelihood and Bayesian inference. Divergence time estimations were carried out with a relaxed molecular clock in a Bayesian framework. Ancestral ranges were estimated using the dispersal-extinction-cladogenesis model and an area coding based on major disjunctions. The phylogenetic analyses clarified intergeneric relationships within Thelymitrinae, with Epiblema being sister to Thelymitra plus Calochilus, both of which were well-supported. Within Thelymitra, eight major and several minor clades were retrieved in the nuclear and plastid phylogenetic reconstructions. Five major clades corresponded to species complexes previously recognized based on morphological characters, whereas other previously recognized species groups were found to be paraphyletic. Conflicting signals between the nuclear and plastid phylogenetic reconstructions provided support for hybridization and plastid capture events both in the deeper evolutionary history of the genus and more recently. Divergence time estimation placed the origin of Thelymitra in the late Miocene (c. 10.8 Ma) and the origin of the majority of the main clades within Thelymitra during the late Pliocene and early Pleistocene, with the majority of extant species arising during the Pleistocene. Ancestral range reconstruction revealed that the early diversification of the genus in the late Miocene and Pliocene took place predominantly in southwest Australia, where most species with highly restricted distributional ranges occur. Several long-distance dispersal events eastwards across the Nullarbor Plain were inferred, recurrently resulting in lineage divergence within the genus. The predominant eastwards direction of long-distance dispersal events in Thelymitra highlights the importance of the prevailing westerly winds in the Southern Hemisphere for the present-day distribution of the genus, giving rise to the Thelymitra floras of Tasmania, New Zealand and New Caledonia, which were inferred to be of comparatively recent origin

HybPhaser identifies hybrid evolution in Australian Thelypteridaceae

Hybridisation has been proposed to play an important role in fern evolution, but has been difficult to investigate. This study explores the utility of target sequence capture and read-to-reference phasing of putative hybrids to investigate the role of evolutionary reticulation in ferns using Australian Thelypteridaceae as a model. The bioinformatics workflow HybPhaser was used to assess divergence between alleles, phase sequence reads to references and construct accessions resembling parental haplotypes. These accessions were included in phylogenetic and network analyses to detect hybrids and infer their parentage. This approach identified two novel hybrid lineages in Thelypteridaceae, one occurring between two different genera (Abacopteris and Christella), and another as part of a complex of Christella. In addition, hybrid phasing successfully reduced conflicting data and improved overall resolution in the Thelypteridaceae phylogeny, highlighting the power of this approach for reconstructing evolutionary history in reticulated lineages

Genome skimming provides well resolved plastid and nuclear phylogenies, showing patterns of deep reticulate evolution in the tropical carnivorous plant genus Nepenthes (Caryophyllales)

Nepenthes is a genus of carnivorous plants consisting of ~160 species that are distributed in the paleotropics. Molecular systematics has so far not been able to resolve evolutionary relationships of most species because of the limited genetic divergence in previous studies. In the present study, we used a genome-skimming approach to infer phylogenetic relationships on the basis of 81 plastid genes and the highly repetitive rRNA (external transcribed spacer (ETS)–26S) for 39 accessions representing 34 species from eight sections. Maximum-likelihood analysis and Bayesian inference were performed separately for the nuclear and the plastid datasets. Divergence-time estimations were conducted on the basis of a relaxed molecular-clock model, using secondary calibration points. The phylogenetic analyses of the nuclear and plastid datasets yielded well resolved and supported phylogenies. Incongruences between the two datasets were detected, suggesting multiple hybridisation events or incomplete lineage sorting in the deeper and more recent evolutionary history of the genus. The inclusion of several known and suspected hybrids in the phylogenetic analysis provided insights into their parentage. Divergence-time estimations placed the crown diversification of Nepenthes in the early Miocene, c. 20 million years ago. This study showed that genome skimming provides well resolved nuclear and plastid phylogenies that provide valuable insights into the complex evolutionary relationships of Nepenthes

Australasian orchid diversification in time and space: molecular phylogenetic insights from the beard orchids (Calochilus, Diurideae)

Phylogenetic relationships in Calochilus (~30 species) were inferred based on a supermatrix of 81 loci including 22 species. To examine the spatio-temporal evolution of Calochilus, divergence-time estimations were conducted within a Bayesian framework using an uncorrelated relaxed molecular-clock model, followed by maximum-likelihood ancestral-range reconstructions comparing four biogeographic models. To trace the evolution of key floral and vegetative characters, maximum-likelihood ancestral-character reconstructions were carried out. The stem age of Calochilus was dated to ~12.0 million years ago in the mid-Miocene. Divergence of Calochilus into a tropical and a temperate clade was inferred to have occurred ~7.6 million years ago in the late Miocene. Northern Australia was reconstructed as the ancestral area of the tropical clade and the Euronotian region for the temperate clade. Range expansions from Australia to other Australasian regions, such as New Zealand and New Guinea, were inferred to have occurred only in recent geological times, commencing in the Pleistocene. The infrageneric classification for Calochilus was revised, erecting two subgenera, subgenus Calochilus and subgenus Tropichilus subgen. nov. Section Calochilus Szlach. was recircumscribed, and sect. Abrochilus sect. nov., and section Placochilus sect. nov. were erected. Identification keys to subgenera and sections and a taxonomic synopsis of the genus are provided