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Friday, August 1, 2014

The Trouble with Seeds (2/3)

For someone interested in breeding plants, that some plants often don't produce seeds can be a major barrier. A breeder would be limited to looking for selectable mutations, rather than using the more general mixing and segregation of genetics to increase crop diversity.

Many of our common vegetables, fruits, and landscape plants are traditionally propagated by clonal divisions. In some cases (apples, pears, etc.) the complex genetic diversity of the crop means that every seedling would produce a distinct plant. The market demands for consistent production then encourage growers to clone the plants. In other cases, the clonal tradition is enforced by the plants themselves due to their inability to produce seeds. (I'll later discuss a third category of plants which have such long life-cycles that breeding projects become difficult to undertake.)

Researchers have figured out tricks to get seed and allow breeding to be done with plants that might otherwise prefer not to.

The Trouble with Seeds (1/3): Garlic, Horseradish, Potato Onion, Walking Onion, and Banana.
The Trouble with Seeds (2/3): Pineapple, Lily of the Valley, Potato, and Sweet Potato.
The Trouble with Seeds (3/3): Babington's Leek, Crosnes, and Bur Oak.

Most of this post is an accumulation of information from other sources (in colored quotes below) about how to get seeds from these crops. I've also included my thoughts and experiences where I felt something needed to be clarified or extended.


    How to get pineapple (Ananas comosus) to set seed.

    [1] "Pineapple is largely vegetatively propagated. Sexual reproduction is rare in nature because pineapple is self sterile; seeds if produced by self fertilization germinate slowly with low vigour and young seedlings are fragile due to inbreeding depression."

    "Self-sterile" generally doesn't mean there won't be any seeds, but rather that there will be very few seeds.   Plants are somewhat flexible in such things. The examination of one I recently purchased revealed three seeds.

    [2] "Pineapples are usually propagated vegetatively. Seeds are only used by commercial growers for breeding purposes. They are viable for 6 months, are unreliable and difficult to germinate. Pineapples from the store are hybrids and if you do manage to get the seed to germinate and grow into a plant and eventually fruit (2-3 years) the fruit will not resemble what you bought. It still could be a lot of fun. Nick the seeds and plant as deep as it is long in moist (not soggy) potting soil and place the pot inside a plastic baggie. Keep it warm (75-80 F). It will take a long time (up to 6 months to germinate)."

    The best method for germinating the seeds from [3] was to place the seeds in a glass jar with some water, sealed with plastic wrap and stored on a heating-mat.



    If you just want to grow another pineapple like the one you got from the store, simply cut off the leafy top and transfer into soil [4]. Give it plenty of light, keep it moist, and if you're lucky, the new plant will grow you a new fruit in a couple of years. If you're unlucky, the new plant will in time turn into a mold festival.
    1. http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/content/pineapple-3/$FILE/biologypineapple08_2.pdf
    2. http://uk.answers.yahoo.com/question/index?qid=20101129055936AAuEhEl
    3. http://www.youtube.com/watch?v=Z2xK9LigVJQ
    4. http://www.youtube.com/watch?v=vpJHgXaPzFA



    How to get Lilly of the Valley (Convallaria majalis) to set seed.

    Lily of the Valley flowers profusely, but rarely produces fruit. This leaves most propagation of the plant to be via vegetative means. The abundant small rooted bulbs are referred to as 'pips' and readily take to being transplanted.

    I've come across two methods which might trick Lily of the valley to set fruit.

    The first is two have two different clones growing close to each other, so that each will pollinate the other. This is based on the idea that the plant will prefer to cross-pollinate to maintain genetic diversity. I've come across this idea elsewhere, but I am not now able to find any references.

    The second method is to convince the plant that it is dying and thus will have no chance to reproduce except by producing fruit via self-polination. I potted up a single pip from a pink-flowered form, intending only to transfer it to a new garden bed. I placed the pot inside under some fluorescent lights, as I was in the process of moving and didn't have a garden for it, and wasn't that consistent with making sure it had water. Once I finished moving into my new place, I brought over the plants and discovered this one was growing a fruit. The conditions were not consistent with insect based cross-pollination, leading me to the conclusion that the fruit probably derived from a self-polination.

    Both these methods require more experimentation, but are consistent with evolutionary theory and long-term survival of the plant's genes.

    This plant is highly toxic, with reports of fatal poisonings due to eating only a few of its berries, so take care with how you handle and where you store your plants.


      How to get Potatoes (Solanum tuberosum) to set seed.

      Typical garden potatoes are propagated by saving tubers from year to year. A side effect of this process is the gardener is also saving potato viruses from year to year, inside the tubers. Over several years, the amount of viruses can build up and result in stunted plants which don't produce well. There are laboratory techniques to clean viruses from potato tissue clones, allowing the professionals to restore the varieties they grow to high production. Another method is to routinely grow potatoes from true seeds. This will eliminate the viruses which can interfere with production and will let you grow a diverse population of new potato types.

      To do this, you have to get seeds from the potatoes and not all potato varieties are forthcoming about flowering and producing seeds for you. One approach to get potatoes to make seeds for you relies on better living through chemistry.

      [1] "The effect of gibberellic acid containing mixtures, silver thiosulphate and extended photoperiod on flowering induction in 16 non-flowering potato genotypes and on flowering enhancement in 14 normally potato flowering genotypes was studied in sub-tropical plains of India during short-day autumn crop season of 2000-2001 and 2001-2002. Extended photoperiod alone was not successful in induction of flowering. Silver thiosulphate in combination with extended photoperiod effectively induced flowering in 16 potato genotypes studied for flower induction. Induced flowers of some genotypes were male fertile. Normal berry setting was observed on induced flowers and seeds obtained from such berries germinated normally. Gibberellic acid containing treatments were not very effective in flower induction as they induced some flowers only in few genotypes. In the normally flowering genotypes silver thiosulphate enhanced maximum flowering and duration of flowering to a great extent."

      A second approach takes a bit longer and dispenses with the chemistry. If you have varieties which don't produce berries, you eat the tubers and end those genetic lines. Before long, you will only have potato lines which do produce seeds easily and their progeny will also be likely to produce seeds easily. It will only take a few generations to get rid of the gene variations which prevent flowering/seed-production in typical potatoes.

      [2] "The easiest one to deal with is potatoes - mostly commercial varieties - that set a huge load of spuds which act as a photosynthate sink absorbing all the energy and nutrients the plant produces. This prevents berry set because there are not enough resources to go around. Azul Toro is a good example. The way to get these varieties to produce seed is to change the load balance. Plant these potatoes a few weeks later than normal and push them just barely into the surface of the soil. The resulting plants won't have room to make stolons and therefore will have extra resources to devote to producing seed. I accidentally stumbled on this method a few years ago and have used it several times since.

      This probably explains why I found seeds in berries produced by potatoes that were in my garden when I bought the place. They were growing right at the surface, slightly protruding in fact. This may also be why the plants I grew from seed have themselves set seed. They, too, are growing right at the surface."

      This third approach appears to work by playing with the physiology of the potato plant by how they are planted. The mechanism of disrupting the typical source-sink migration of sugar in the plant appears to be a generally useful idea that can apply in various ways to many species.
      1. http://link.springer.com/article/10.1007%2Fs10681-005-9050-y
      2. http://alanbishop.proboards.com/thread/8356/restoring-potato-fertility?page=1#ixzz3Vvu4F6Cb



      How to get Sweet Potatoes (Ipomea batatas) to set seed.

      Sweet potato vines will sometimes flower and set seed, depending on the variety and growing conditions. If you want to encourage flower/seed production, you have to break out the chemical weapons.

      [1] "Most sweet potato cultivers grown in Zimbabwe are poor in agronomic and quality traits and require improvement through breeding. However, most cultivars rarely flower yet the flowers are crucial in genetic improvements. The aim of this study was to determine the effects of different levels of 2,4-dichlorophenoxyacetic acid (2,4-D) on sweet potato flower induction. A 3*4 factorial experiment in a randomized complete block design with three replications was used. The first factor was landrace with three different landraces and the second factor was 2,4-D with four different concentrations (0, 100, 300, and 500 ppm). The 2,4-D was applied 50 days after planting. Sweet potato landraces that were sprayed with 2,4-D showed morphological and physiological disorders that included temporal drooping, petiole pinasty, stem splitting, shoot dieback and root swelling. Extensive morphological and physiological disorders were observed on landraces that were sprayed with the high levels of 2,4-D (300 and 500 ppm). However, within 30 days, all the landraces that were sprayed with 2,4-D managed to initiate buds and set flowers while the plants that were not sprayed did not flower at all. The Friedman's tests showed no significant differences in bud and flower number among the treatment combinations used. Therefore the lowest concentration of 2,4-D (100 ppm) used in this study is probably close to the optimum concentration for flower induction in sweet potato. Although this concentration is not the actual optimum, at the moment this concentration can be used to induce flowering in sweet potato and thus allow sweet potato breeding initiatives to be launched."

      2,4-D is a very commonly used herbicide, readily available in garden centers or from online vendors. Fortunately, there is a method that doesn't rely on toxic chemicals that you might rather not have around you.

      [2] "It was thought that the slight stimulation of flower production in the nonflowering noted in 1951-52 could be due to the limited storage of carbohydrates in the roots of the easy-floweringstock. This might result in a build-up of materials within the aerial portion of the plant and thus induce bud formation. Following this reasoning, grafts were made in 1952-53 on some closely related species commonly found in many home flower gardens. They were selected because they have no storage roots. These species are: Morning Glory (Ipomea purpurea) variety Heavenly Blue, Cardinal-climber-(Quamoclit sloteri), Moonflower (Calonyction aculeatum), and Cypress-vine (Quamoclit pennata). The method of grafting was the cleft graft. After insertion of the scion, the union was tightly wrapped with a strip of rubber made from a cut rubber band. No other treatment was given. A total of 125 such grafts were tried of which 95% were successful. Grafts were successful on all except Cypress-vine which has a stem smaller than that of the sweet potato; however, even these grafts took and grew for a short time before dying.
      On many of the plants flower buds began to appear about one month after grafting or soon after growth of the scion was resumed. These buds continued to appear, develop, and produce normal flowers. In some cases the plants were dwarfed (plant 103) but in others the growth of the scion was normal. The response of the sweet potato scions was the same regardless onto which ornamental they were grafted."

      This method can be performed easily. The best rootstock (I. tricolor) is readily available, grows quickly, and flowers prolifically. The physiological mechanism suggested is interesting, but later research suggests it is incomplete.

      [3] "Grafting has been used to induce flowering and to induce early flowering. For example, sweet potato (Ipomoea batatas) is routinely grafted to other nontuberous root forming Ipomoea species such as I. ruba, I. carnes, and I. tiliaceae to induce flowering. In this case, the presence of leaves on the scion and rootstock have had a profound influence on the flower-inducing response. Flowering was induced only when the rootstocks had expanded leaves, thus suggesting that flower-inducing substances are synthesized in the rootstock leaves and translocated through the graft union to induce flowering in the scion (Kher et al. 1953; Lam and Cordner 1955). Grafting sweet potatoes onto Ipomoea carnea ssp. fistulosa increased flower numbers, percentage of capsule set and number of seeds in all four tested cultivars…"

      Grafting will take some practice, but it appears to be a good way to start a sweet potato breeding project. I would select for lines that didn't require such heroic efforts to get further seed.
      1. http://www.academicjournals.org/article/article1387208292_Mutasa%20et%20al.pdf
      2. https://ucanr.edu/repositoryfiles/ca707p13-71853.pdf
      3. http://alanbishop.proboards.com/thread/7720/sweet-potato-breeding-project?page=6#ixzz3Q345UFcC


      I discuss this topic with respect to a different set of crops in my first post with the shared name. When I wrote the first post, I was thinking of the classic Star Trek episode, "The Trouble with Tribbles". Tribbles are fictional creatures that replicate rapidly by cloning themselves, having been born pregnant. The plants being discussed are generally clonally reproduced, so the name "The Trouble with Seeds" came to mind.

      Most of the lovely plant diagrams in this post were derived from public-domain images hosted at botanicalillustrations.org. Some other diagrams are public-domain images from the same era that I found via google. I chose the original images which depicted the plants under discussion in the way I appreciated and then subtracted out the yellowed background of the page using my favorite image editor (GIMP)

      Wednesday, July 9, 2014

      Oh, Deer.

      My new house has a big yard and a persistent deer population. Because I moved during the summer, my garden plans for this year are pretty limited to some planters I had at my last place, but I'm planning for how to garden next year with deer in mind.

      Most garden vegetables are tender, tasty plants -- yesterday, a deer ate the top off a specialty, high-beta-carotene tomato -- so they will have to be protected behind tall fences or inside greenhouses. However, there are several crops which should persevere without technical assistance.


      Some crops grow tall enough that deer aren't a problem.
      • Mulberry (Morus rubra)
        • We have a couple fruiting trees growing on the property and are planning to plant cuttings for more.
      • Apple (Malus domestica)
      • Cherry (Prunus avium)
      Some are protected by numerous spines.
      • Black raspberry (Rubus occidentals) and Red raspberry (Rubus idaeus)
        • Black raspberries are growing wild on the property and red raspberries are easy to find commercially.
      • Artichoke (Cynara cadunculus var. scolymus)
        • This may be hard to grow in the short Minnesota growing season, but I suspect there are short-season varieties around.
      • Litchi tomato (Solanum sisymbriifolium)
        • This tomato relative produces edible fruit, but the fruit is not likely to be a culinary match for the tomato.  I intend to see what I can do with it in the kitchen next year and on longer time-scales, this is one of the plants I want to explore mutation-breeding with.
      Some simply taste bad, to deer.
      • Common onion (Allium cepa)
        • I currently have three types of large-bulbing onions growing in planters for multiplication.
      • Nodding onion (Allium cernuum)
        • I gathered several plants of this native species, with permission, from the flower bed at an estate sale. I find the taste to be more savory and interesting than typical onion greens and am looking forward to having enough plants to harvest for the kitchen.
      • Onion chives (Allium schoenoprasum)
      • Garlic (Allium sativum)
      • Garlic chives (Allium tuberosum)

      I'll continue to look for more deer-resistant crop plants, as the fewer high fences I have to build, the better.

      Wednesday, May 28, 2014

      Making My Own Carrots 2

      The carrots have been planted in a window box for a few weeks. They experienced a bit of frost the night of the recent complete lunar eclipse, but have rebounded and are now throwing up flower clusters in a few different shades.

      I don't have any particular biology notes to mention here, but you can read the earlier post on the subject. I just wanted to share the recent photos.





      Part 1: The initial story.

      Part 3: Generation 2.

      Friday, May 23, 2014

      Astrobiology : Biology of Mars

      New research indicates that some methanogens, Archaea which breath hydrogen and carbon dioxide while excreting methane, can survive the temperature extremes currently found on the surface of Mars. I find this to be a very interesting piece of data.

      Various bacteria have been shown to survive the extreme conditions of vacuum, temperature, and radiation found in space. This second piece of interesting data indicates that bacteria can potentially be transferred from Earth to Mars, where they could survive.

      Meteorites are routinely found here on Earth, including a rare few which contain gases matching those found in the atmosphere of Mars. This third piece of interesting data indicates the potential transfer of rocky material from Earth to Mars.

      Together, these data indicate that living things could be found on Mars, if they had the opportunity to be transferred there at some time.

      This leads us to another piece of data, referred to as the late heavy bombardment. Approximately 4 billion years ago, large rocky fragments were crashing about the inner solar system. The crater-scarred surface of the moon is the most obvious piece of evidence to this time period. Impact craters on Earth and Mars which would have been produced at the same time have been worn away by the erosional processes of the thicker atmospheres and oceans both planets held in the times soon after the bombardment ended. Some of the impacts would have been large enough to sterilize the surface of either planet, evaporating nascent oceans and scattering rocky bits all throughout the solar system. Living cells which had formed on Earth would have been carried into local space on those ejected fragments, only to come raining down later to reseed the cooling planetary surface.

      The early environments of Earth and Mars were very much the same. Primordial reducing atmospheres with water oceans would have been found on both. Those rocky seeds which returned life to the sterilized surface of Earth would have also carried life to Mars. The same process would have carried Martian life to Earth, of course, but we have so little evidence from that time period that we really can't say which direction the transfer went.

      A few billion years after the late heavy bombardment concluded, we evolved and started asking questions.

      The core of Mars had cooled and lost its magnetic field, leading to the Martian atmosphere being stripped away by the persistent solar wind. Without a thick atmosphere, Mars cooled and dried out.  However, even now there is evidence for liquid water on Mars. There is also evidence for methane release into the atmosphere of Mars.

      Given how persistent life has been found to be on our planet, living in every place it could possibly find a way to live, I strongly suspect there is life on Mars today. That life would be the last remnants of a formerly thriving biosphere, much like our own, which was descended from the simple forms of life scattered through the solar system during the late heavy bombardment.

      Hopefully, we will get the opportunity to go and look while I'm still here to know about it.



      References:
      1. Methanogens can survive Martian conditions.
      2. Microbes living in space.
      3. Martian meteorites.
      4. Late heavy bombardment
      5. Martian ocean.
        1. http://en.wikipedia.org/wiki/Mars_ocean_hypothesis
        2. http://www.caltech.edu/content/evidence-martian-ocean
        3. http://www.astrobio.net/news-exclusive/new-evidence-for-ancient-ocean-on-mars/
      6. Mars loss of atmosphere.
        1. http://science.time.com/2013/07/23/revealed-how-mars-lost-its-atmosphere/
      7. Association of methane and water seeps on Mars.
      8. Evidence of methane release on Mars.

      Friday, May 16, 2014

      A gastropod's lesson.

      Guess what happens when you do an unusual project without taking before and after pictures? People might not believe your claims of having done the project.

      What does this have anything to do with biology or the photo at right?

      This fossil is a cast from a gastropod shell found in Ordovician-age rock found along the Mississippi river in the Twin-Cities of Minnesota. The local Ordovician-age rocks are chock full of brachiopods, but contain very few gastropods. In modern sandy ocean environments, there are very few brachiopods and gastropods are everywhere. The mix of species found within this particular ecosystem has changed dramatically over time, even though the environment is likely to be much the same now as it was back in the Ordovician.

      When I found the fossil, only the broken off tip and edge of the outer whorl were evident. My new fossil-hunting-buddy didn't see these hints for what was buried within the rock and expressed skepticism when I pointed them out. My intuition, from fossil hunting periodically over the last decades, led me to be almost certain there was a snail hiding in the rock. Yesterday it was sunny and relatively warm, so I decided to go about excavating the gastropod from its tomb. I sat on the sidewalk at the back of my house and carefully chiseled away the uninteresting (to me) rock surrounding the fossil.

      As is common for me, I didn't think to take a photo until the project was complete. You'll just have to (or not) believe my claim of having found and prepared this fossil myself.

      When I collected this specimen, I noted another gastropod hiding in a rock which was too large for me to carry. I now have some better tools and I should be able to extract it from the boulder it calls home. If I do collect and prepare it, I'll make sure to take more photos of the process.


      References



      Sunday, April 27, 2014

      Astrobiology : Follow the Water

      The more we've learned about life on our planet, the more we've come to realize the conditions that we consider livable are only a small fraction of the conditions that living things on this planet are happy to have. The two factors which remain are the need for an energy source and the presence of at least some liquid water.

      Almost any sort of energy can be utilized by living things (Light, organic chemical, inorganic chemical, ion gradient, electrical, radiation) even when present in minimal amounts. Energy isn't the limiting factor on our planet

      Water is ubiquitous on Earth, from the upper atmosphere to miles below the surface. We find life everywhere we find water, even if that water is horrendously toxic (acid/alkali/heavy-metal/etc.) to us. We find life subsisting between grains of sand subsisting on the trace of moisture provided by dew. We find life in the deepest mines we've dug. We find life living in lakes isolated from the surface for thousands of years of ice.

      On Earth, wherever there is water, there is life.



      The name of our planet is synonymous with solid ground, but most of the surface of our world is covered in water. From a distance, a glance at our home reveals it to be a water-world first. No other planet (under any definition of 'planet') in our system is anything like it. However, water is common in our part of the universe.

      During the formation of a planetary system, volatile elements and compounds are pushed out from the star until ambient space cools enough for those volatiles to condense and freeze. The place where water freezes is referred to as the ice-line and represents the start of the region where icy bodies are formed.

      The formation of planets produces a great deal of heat. The kinetic energy of rocky/icy bodies as they merge under gravitational attraction is converted to heat. If the resulting bodies are large enough, they melt and the different components form layers ranging from densest to lightest going from the core to the surface. Water is very low density and so ends up largely on the surface of such bodies. Because they formed far enough out from the sun for water to freeze, the surfaces begin to cool and freeze soon after they form. The residual heat of formation is tremendous, and combined with new heat generated through radioactive decay and tidal interactions can keep temperatures below the surface elevated above the freezing point of water for quite some time.

      Several bodies in our solar system appear to have under-ice oceans.

      Calisto : science1.nasa.gov/science-news/science-at-nasa/1998/ast22oct98_2/
      Enceledus : www.theguardian.com/science/2014/apr/03/ocean-enceladus-alien-life-water-saturn-moon
      Europa : www.space.com/23880-jupiter-moon-europa-hidden-oceans.html

      There are potentially living things on these moons, prospering in the warm water of thermal vents or creeping along in the cold waters near the ice.

      I can't wait until we go and look.



      References :
      1. Follow-the-water.
      2. Light energy for biology.
      3. Electrical energy for biology.
      4. Radiation energy for biology.
      5. Ice-line.
      6. Oxygenation of Earth.
        1. http://en.wikipedia.org/wiki/Great_Oxygenation_Event
      7. Snowball-Earth.
        1. http://en.wikipedia.org/wiki/Snowball_earth

      Friday, April 18, 2014

      Oxalis and the Red Queen

      http://sakaiweller.bio.uci.edu
      Breeding in the genus Oxalis is complicated by the presence of multiple flower types, referred to as heterosytly because of differences in the arrangement of styles (the female part of a flower). Oxalis generally have three forms, so they are specifically described as being tristylous.

      The physical differences between the flower types match hidden differences in chemistry which limit the effectiveness of pollination between like types. These differences, both physical and chemical, discourage inbreeding.

      With three forms, the odds that a vegetatively related plant will have the same form is 1/1 while the odds that any random plant will have the same form is 1/3. Failure of pollination from the same form to succeed results in a strong preference against inbreeding.

      Inbreeding reduces genetic diversity and increases the odds of revealing deleterious recessive alleles. Some plants, like Oxalis, have systems to discourage inbreeding, others don't. It isn't clear what drives some species to evolve these systems while others don't, but there is related theory that might be informative…



      Why do most plants/animals reproduce by sexual means? Any asexual progeny will get 100% of their
      DNA from their mother. Any sexual progeny will get only 50% of their DNA from their mother. Basic evolutionary theory suggests there would be a strong bias towards species reproducing by asexual means. So, why…?

      The current theory, backed by years of experimental evidence in different systems (fish, snails, nematodes) is that sexual reproduction allows a species to mix its genome up and keep ahead of parasites, which have much shorter generation times and are rapidly evolving to overwhelm the host immune system.

      This is referred to as the "Red Queen Hypothesis", in reference to Lewis Carol's character in "Through the Looking-Glass".

      "Now, here, you see, it takes all the running you can do, to keep in the same place."



      From a theoretical stance, the same pressures which lead to the prevalence of sexual reproduction are likely to be involved in why some plant species have evolved heterostyly. That is to say, a high level of parasitism over evolutionary time periods likely provided the selection pressure leading to the evolution of heterostyly and other mechanisms to minimize inbreeding. Proving this experimentally is a bit harder to do, however, and researchers are actively working on the subject.



      What got me interested in this system is that I have two Oxalis triangularis plants I got from a friend. One plant has dark purple leaves with a central lighter mark and pink flowers. The second plant has green leaves and white flowers. I opened up a flower from each plant and discovered they were structured differently.

      Plant #1
      • Purple leaves, light mark.
      • Pink flowers.
        • 1' : styles
        • 2' : stamens
        • 3' : stamens
      Plant #2
      • Green leaves, smaller.
      • White flowers.
        • 1' : stamens
        • 2' : styles
        • 3' : stamens
      If my two plants had happened to have the same flower structure, I wouldn't have looked into the topic of heterostyly. I would have tried crossing the flowers and had the cross fail. I would have assumed the plants were incompatible and not thought to look into it further.

      I lucked out and my plants have different flower forms, so my attempts to cross them will likely result in the production of seed. I still have to ponder on the subject of what I might want to breed this species for, but at least I will probably get some interesting combinations of traits in the offspring.



      References
      1. Dercole F, Ferriére R, and Rinaldi S. (2010) Chaotic Red Queen coevolution in three-species food chains. Procedings of the Royal Society of Biologists 277:2321-2330.
      2. King KC, Delph LF, Jokela J, and Lively CM. (2009) The Geographic Mosaic of Sex and the Red Queen. Current Biology 19:1438-1441.
      3. Marco DE and Arroyo MTE. (2014) The Breeding System of Oxalis squamata, a Tristylous South American Species. Botanica Acta 111:497-504.
      4. Moran LT, Schmidt OG, Gelarden IA, Parrish RCII, and Lively CM. (2011) Running with the Red Queen: Host-Parasite Coevolution Selects for Biparental Sex. Science 333:216-218.
      5. Quental TB and Marshall CR. (2013) How the Red Queen Drives Terrestrial Mammals to Extinction. Science 341:290-292.
      6. Weller SG, Dominguez CA, Molina-Freaner FE, Fornoni J, and Lebuhn G. (2007) The evolution of distyly from tristyly in populations of Oxalis alpina (Oxalidaceae) in the Sky Islands of the Sonoran Desert. American Journal of Botany 94:972-985.
      7. http://radix4roots.blogspot.com/2009/09/ocasional-update-1-to-bee-or-not-to-bee.html
      8. http://radix4roots.blogspot.com/2009/08/flurry-of-flowers.html
      9. http://wettingthebeds.cultivariable.com/2013/07/oca-maximizing-seed-yield-of-crosses.html