Darwin's 'abominable mystery

Case StudiesCase Studies

Case Study

Darwin’s ‘abominable mystery’The origin of flowering plants

Version 1.1

Laura KellyRoyal Botanic Gardens, Kew

Dean Madden [Ed.]NCBE, University of Reading

STUDENT’S GUIDE

Copyright © Laura Kelly and Dean Madden, 2011

the origin of flowering plants

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Darwin’s ‘abominable mystery’The origin of flowering plants

The word ‘angiosperm’ is an informal term for a flowering plant. Literally, it means ‘borne in a vessel’, a reference to the fact that the seeds are enclosed within a carpel (the female reproductive organ of the flower). This contrasts with gymnosperms (such as conifers and cycads), which bear naked seeds.

The angiosperms are by far the largest group of seed-bearing plants that can be found on Earth today. There are at least 260,000 species of flowering plants — there may be up to half a million species. As many as 50,000 species of flowering plants probably remain undiscovered. The diversity of angiosperms in terms of their habitats, morphology and physiology is unrivalled elsewhere in the Plant Kingdom.

Molecular dating methods suggest that angiosperms first appeared between 180–140 million years ago, although the oldest confirmed angiosperm fossils date back to the early Cretaceous and are estimated to be around 132 million years old.

Modern angiosperms have a range of characteristics in common, which is strong evidence that they all share a single origin, tracing back to the same ancient ancestor.

Before the advent of flowering plants, and for much of the total history of the land plants, the Earth’s terrestrial habitats were dominated by spore-bearing plants such as lycopsids, ferns, mosses and non-flowering seed plants such as conifers and cycads. This modern tree fern forest is in Whirinaki Forest Park, New Zealand.

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In the 1700s, Carl Linnaeus’ classification used flower structure to infer relationships between flowering plants: today, DNA data is used.

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Why study Angiosperms?

The Angiosperms represent one of the largest branches of the ‘tree of life’. From their beginnings millions of years ago, this group of plants has risen to dominate the world’s vegetation, and is arguably the most important component of global biodiversity. Other organisms also diversified alongside the angiosperms, such as pollinating insects and seed-dispersing birds and mammals. The angiosperms are of overwhelming ecological importance, as they define most of the world’s major habitat types.

Angiosperms are also of great importance to the survival of humans, as they provide much of our food, fibre, medicines and timber. Despite this, for more than a century their origin and early evolution has been an enigma. Indeed, Charles Darwin famously called the origin of the angiosperms ‘an abominable mystery’. This origin has remained enigmatic and identifying which group of living plants was the first to diverge from the earliest angiosperms has been a major goal of botanical research: it has even been described as the ‘Holy Grail’ of botany.

Why is it so important to know what the earliest flowers may have looked like? Identifying the characteristics the earliest flowering plants will help us to understand how these features have allowed the flowering plants to diversify rapidly and rise to their current dominant position in the World’s flora. Some of the benefits that could arise from having a better understanding of the relationships of the angiosperms include: allowing a better understanding of species’ distributions and their ecological implications; facilitating the search for natural medicines and permitting more informed decision-making in relation to the conservation of biodiversity.

Laurus nobilisDrimys winteriIllicium floridanum

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Examples from flowering plant genera used in this Case Study.

Chimonanthus praecoxCanella winteranaNympaea alba

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Two competing hypotheses

Botanists working during the nineteenth century thought that catkin-bearing plants like Salix (willow) were similar to the first angiosperms. Such flowers have turned out to be highly-evolved, however. In recent years two main competing hypotheses have taken centre stage.

The ‘Woody magnoliid’ hypothesisDuring the early part of the 20th century a hypothesis was developed in which a Magnolia-like flower was thought to be the ancestral type. The earliest flowers are thought to have been large and complex, containing many spirally-arranged parts, such as those found in modern plants like Magnolia and Liriodendron. It was also suggested that these ancestral flowering plants would have been slow-growing trees or shrubs inhabiting shady tropical forests. Hence the name: Woody Magnoliids.

Annona crassifloraMagnolia grandifloraLiriodendron tulipifera

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Three modern species that are representative of the ‘Woody Magnoliids’.

Piper nigrumAsarum caudatumAristolochia arborea

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Three modern species that are representative of ‘Paleoherbs’.

The ‘Paleoherb’ hypothesis‘Paleoherbs’ are a group of several rather varied angiosperm families including waterlillies and pepper. Some botanists have also included the monocots (about 65,000 species) within the paleoherbs. This group of plants may have been the first amongst the angiosperms to have diverged, implying that the earliest flowering plants are likely to have been small and herbaceous (that is, non-woody). It has been proposed that a paleoherb-like ancestor would have had small, simple flowers and that it would have been fast-growing pioneer species that tolerated disturbed, sunny habitats.

In Darwin’s time, the first flowers were wrongly thought to have been like willow catkins.

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Solving the mysteryFossil evidence and morphology

Before the evolution of flowering plants, and for much of the total history of the land plants, the Earth’s terrestrial habitats were dominated by spore-bearing plants such as lycopsids, ferns and mosses and non-flowering seed plants such as conifers and cycads. Angiosperms appear suddenly in the fossil record, and seem to have undergone an ‘explosive radiation’ (in other words, many different species evolved very rapidly).

Fossils provide important additional information about plant diversity and can show detailed features of the anatomy of extinct flowering plants. However, there several problems associated with the use of data from fossils, including: the fact that different organs of large and complex plants are rarely found attached to each other (making it difficult to determine which organs belong to the same species); the incompleteness of the fossil record, with only a fraction of past diversity having been preserved and the tendency for certain kinds of plants to be under- or over-represented (due to features of their biology or ecology).

The fossil record has not been able to clarify which were the earliest groups to diverge among the angiosperms. Fossil evidence shows that there were several different groups already present early in the evolutionary history of the angiosperms. Whilst some of these very early flowering plants seem to belong to modern-day families of angiosperms, many do not. One of the problems with interpreting the earliest fossil angiosperms is that they may exhibit an unusual combination of characters that are no longer found in living species. For example, the fossil genus Archaefructus, which dates from about 125 million years ago, possesses features of both the Magnoliids and the paleoherbs and therefore cannot be used to help resolve which of the two hypotheses is true.

DNA sequence data

In recent years, botanists have begun to turn their attention towards the use of DNA sequence data as a way of unravelling the relationships of the earliest angiosperms. DNA sequence data from living angiosperms are relatively easy to obtain. Analysis of these data provides an independent line of evidence for investigating the evolution of angiosperms.

The aim of this Case Study is to construct an evolutionary tree using chloroplast DNA sequence data from the rbcL gene. With this tree it will be possible to test the competing hypotheses (‘Woody Magnoliid’ and

‘Paleoherb’) and judge which of these is most likely to be correct.

The Angiosperm Phylogeny Group (AGP) compared genes encoding a large subunit of the enzyme ribulose bisphosphate carboxylase/oxygenase, codenamed rbcL. The gene is found in the DNA of chloroplasts. Variations in the DNA sequence of the gene enabled the APG scientists to propose a new ‘family tree’ for flowering plants in 1998.

Drawings of fossil plants.

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Building a tree1. Double-click on the document angiosperm.geneious. This will start

up Geneious and load the data into the programme. This file contains 26 DNA sequences that will be used to construct the phylogenetic tree of angiosperms and some distantly-related species to use as an ‘outgroup’. If you examine the sequences by zooming in on them, you will see that they are already aligned to each other so that the tree can be constructed.

2. Ensure the sequences are highlighted in the top window, then click on the Translate button to see the amino acids encoded by the DNA.

Zoom buttons

Translate button



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4. A new file will appear in the top window, containing 26 amino sequences derived from the original DNA sequences. Use the magnifying glass button again to zoom in on the sequence data and check that the sequences are in fact made of amino acids (the single-letter amino acid codes are used here):

3. A box will appear, asking you to choose a version of the genetic code to use. Look at the options available, then choose Standard and click OK.

Note

Although the genetic code is often said to be ‘universal’ — the same in all living things — this is not quite true. There are some minor variations in different groups of organisms. Hence this dialogue box, which allows you to choose which version of the code you wish to use.

Amino acid codes The three-letter and single letter codes for the 20 amino acids that are found in proteins. Geneious uses the single-letter codes to show the different amino acids.

Asp D Aspartic acid Ala A AlanineGlu E Glutamic acid Gly G GlycineArg R Arginine Val V ValineLys K Lysine Leu L LeucineHis H Histidine Ile I IsoleucineAsn N Asparagine Pro P ProlineGln Q Glutamine Phe F PhenylalanineSer S Serine Met M MethionineThr T Threonine Trp W TryptophanTyr Y Tyrosine Cys C Cysteine



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5. Scan through the amino acid sequences by eye. Don’t take a long time doing this; you just need to obtain a quick impression of what the data shows.

Questions

a. Compare the lengths of the amino acid and DNA sequences (look back at the DNA sequences if you need to, by selecting the DNA sequence document, ‘Alignment of angiosperms and others’, in the uppermost Geneious window). Which sequences are longest? Why is this?

b. Is there as much variation between species in the amino acid sequence data as there is in the DNA sequence data? Explain why.

c. Which type of data (amino acid or DNA) do you think would be better for generating an evolutionary tree in this case?

6. Switch back to the DNA data by clicking on the DNA sequence document (‘Alignment of angiosperms and others’) in the top Geneious window, then click on the Tree button to create a phylogeny.

7. A panel will appear, offering some options for the tree building. Select Jukes-Cantor as the Genetic Distance Model. The Tree build Method should be Neighbor-Joining. Look at the pictures on the next page and see which species you think should be used as an ‘outgroup’. When you have decided, select the species name from the drop-down list and click the OK button.

Select ‘Jukes-Cantor’ and ‘Neighbour- Joining’ here.

Technical note The Jukes-Cantor distance model assumes that all nucleotide substitutions (mutations) happen at the same rate (1 in 4 or 25%). Other mathematical models assume that different amino acids mutate at different rates.

The Neighbor-Joining method is a quick and popular mathematical model for calculating genetic distances and drawing trees. Other methods will produce slightly different results (and take longer to do it).

Tree button

Choose your own ‘outgroup’ from this drop-down list.



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8. A tree will be produced in the lower central window. Re-size the other windows so that you can study the tree. The software will cluster similar DNA sequences (similar species) closer together.

Welwitschia mirabilisPinus sp.

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Plants that could be used as an outgroup.

Cycas sp.Ginkgo biloba

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Change the display options in this panel to obtain the tree that is easiest to interpret.



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Questions

d. Which species did you choose as an ‘outgroup’ and why? e. Where on your tree would the common ancestor of all the species be

placed?f. Which species is (or are) the first of the angiosperms to branch off

above the gymnosperm group (that is, to diverge)?g. Which of the groups listed above does this (or these) species belong to?h. Which of the two hypotheses does your tree support: the ‘Woody

Magnoliid’ hypothesis or the ‘Paleoherb’ hypothesis?i. Does your tree suggest that the ‘Paleoherbs’ and the ‘Magnoliids’ are

well-defined groups?

9. Obtain a paper print out of your tree diagram. Using the list below, mark on a printed copy which group each species belongs to.

GYMNOSPERMS

Cycas taitungensis Ginkgo biloba

ANGIOSPERMS

Paleoherbs Anemopsis californica Asarum caudigerum Cabomba caroliniana Nymphaea alba Piper cenocladum

Eudicots Buxus microphylla Dicentra eximia Mahonia bealei Platanus occidentalis

Monocots Acorus calamus

Asparagus officinalis

Magnoliids Amborella trichopoda Annona glabra Austrobaileya scandens Canella winterana Chimonanthus praecox Drimys granadensis Illicium oligandrum Laurus nobilis Liriodendron tulipifera Magnolia grandiflora Trimenia moorei

Pinus koraiensis Welwitschia mirabilis

University of California Museum of Paleontologywww.ucmp.berkeley.edu/anthophyta/anthophytafr.htmlAn introduction to angiosperm evolution.

Additional references are given in the Teacher’s Guide.

Further readingThe Tree of Life Web Projecthttp://tolweb.org/Angiosperms/20646This site includes pages on angiosperms and Magnoliids.

The Floral Genome Project www.flmnh.ufl.edu/flowerpower/default.html

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Darwin's 'abominable mystery