Trichostema laxum research update: the first interesting data?

Awhile back, I wrote about the beginnings of some research on Trichostema laxum. I've been slogging through the disappointing amount of data I gathered this field season and doing a little bit of writing. While I was, and still am, really excited about the project on T. laxum, it took a backseat to columbine and tarweed work this field season (most of which burned up). I did get some new data and perhaps gained some insight into the system. 

A normal array of plants in the site: normal purple Trichostema laxum, an individual with the common white and purple lower lip phenotype and some Zeltnera trichantha intermixed (a really cool plant)

My main question in the system is: how is this polymorphism in flower color maintained? If it were a fitness benefit, we might expect a high proportion of it. If it were deleterious, it should be lost (especially as it is at reasonably low frequency). If it is neutral, it might be drifted out. I actually suspect the answer is quite a bit more complicated. 

Once you start looking for variation, you find it! I don't know what this doubled lower lip is about (it showed up in a plant grown in the greenhouse - on most flowers). The plant was male-sterile, I believe. I'll be looking for it in the field though!

The first question is, of course, how common is the color polymorph? I censused the focal patch/population (separated by ~300 meters from others) in 2014 and 2015. In 2014, the polymorph was 2.0% (46/2278 individuals) in 2015, 3.7% (102/2757). Neither of these censuses was a complete census of the population - necessarily, I cannot assess the phenotype of any pre- or post-flowering individuals. Both were done roughly in the peak flowering time (over several days), so I do think it is close to accurate. I think its safe to conclude that the proportion stayed the same or even went slightly up in 2015. 

A rather large w/p morph individual. 

The next logical question is: do the two morphs have similar field fitness? Any "fitness" measure (e.g. reproductive success, height, etc.) of these plants is dictated mostly by microhabitat location. In this rocky, heavily serpentine site, most plants stay under 20 cm tall and never put out more than 50 flowers (mints have 4 ovaries per flower, so maximum seed set is four times flower number). In a wetter, less serpentine and rocky meadow, I once found a plant on a gopher mound (which brings up nutrients) that was nearly a meter wide and better than a half meter tall. It probably had >5000 flowers throughout the season. 

A veritable field of Trichostema! Not my field site - this site has huge plants (~500 flowers/plant) and very little flower color variation. It is a nice place to look at the insect communities on T. laxum, as it has really high densities of herbivores and predators (T. laxum gets most of the sticky plant predators)

Because of this microhabitat variation, the best comparison to make is nearest neighbors which differ in flower color. In both 2014 and 2015, I took data on 41 pairs (coincidentally!) of white/purple and purple/purple neighbors. I found no significant differences, either year, between any fitness variables (number of buds, flowers, fruit, height and, in 2014, number of leaves and herbivory [too low in 2015]).

A more normal-sized (for this population) individual. 

This, ostensibly, seems like the trait is fitness neutral (and lab growouts seem to bear this out - more data soonish). Given that this site burned in August this year (after most had flowered, but some [probably few] were still maturing seeds), I was curious about whether the morphs differed in phenology. Hindsight is 20/20 (I should have censused biweekly!), but the neighbor pairs data can be used to examine this in a roundabout way; I have data on buds, flowers and fruit, so later phenology plants should have a higher proportion of buds to flowers and fruit than earlier phenology plants. 

In both years, the white/purple plants had a lower proportion of buds than the purple plants (it is marginally significant). This suggests that they have a earlier phenology - which could be what is under selection - not the flower color itself. I am super, super, super, excited about this (the only positive result so far from anything in T. laxum) - there was possibly a big selective event (a fire) on 12-August (I think - could have burned on the 13th). From this, I'd predict that the w/p morph may have dehisced a higher proportion of their seed set by the fire. I'll be paying far more attention to the phenology, and recensusing more often this upcoming season. 

I also analyzed the pollinator data from 2014 and got no particularly useful insights (I wondered if there was some degree of isolation between the morphs). The pollinator communities using each morph were pretty similar and the only real differences were:  a bee on a purple flower was more likely to visit a w/p next than a bee starting on a w/p* and, only bees that started on w/p flowers next visited a snapdragon, Antirrhinum cornutum (but only 3% of the time). This last result is interesting as the snapdragon also has whitish purple flowers AND the T. laxum population with the w/p flowers is the only one  (out of ~15) I've found interspersed with large numbers of A. cornutum. I'll have to get MUCH better data for any hypotheses about its effect. 

Antirrhinum cornutum, grown in lab, showing the pale purple/white flowers. 

I'm working now on the "genetics" (well, inheritance, but that's as close as I ever get to ATGC) of the polymorphisms (this one and selfing). Could w/p be recessive and heterozygous in more individuals (~25% under HW assumptions)? I don't think it is (entirely) developmentally induced, as in the first grow out, I only got this polymorphism from this population (I grew individuals out from 4 populations). More soon! Do let me know if you have other ideas about the system!

Heliothis phloxiphaga was a very common herbivore on T. laxum in 2014 (this plant had two - I didn't stage this), but nearly absent in 2015 - though it was still common on columbines and tarweeds. 

*I think this is confounded, as I watched two plants during each observation - nearby plants that were similar in size. Therefore, I suspect that it was more likely that a bee on the p/p plant would encounter a w/p than one from the w/p. 

Classic Natural History I: Anna Bateson's botany

I've lately had a little bit more free time, having had my experiments burned (more burned in the Jerusalem fire after I wrote the blog post about the Rocky Fire) and having just submitted a manuscript I'd been working on. So I've decided to spend a few hours a week reading older natural history and ecology papers. I've been working through the 1800's in American Naturalist, Annals of Botany and Science Gossip - a wonderful popular magazine including observations of natural history, short articles and summaries of research. I've done this haphazardly, reading the table of contents to find interesting articles. I surely let many interesting ones through, but there is practically limitless material, so that is unavoidable. 

I'm going to try to highlight some of these papers - the astute observations, clever experimentation, often beautiful writing, and a little history, too. I probably would have left out the last piece, but several things - including Charley Eisemann's beautiful history/biography/natural history piece on Annette Braun, a forgotten but influential naturalist; Graham et al's the Essential Naturalist, and Bernd Heinrich's biography of his father - have inspired me over the past few years to think a bit more about the people and history behind old natural history.

Many also have beautiful illustrations (like this domestic hybrid pitcher plant). From M.C. Cooke's Freaks and Marvels of Plant Life (1882).

A few papers I found that seemed quite cool were those of Anna Bateson (1863-1928). She was sister to William Bateson, a famous botanist of the day. She worked at as an "assistant" in Cambridge's Balfour Biological Laboratory for woman students, closely with Darwin's son Francis and built upon Darwin's plant work herself. She helped found the Cambridge Women's Suffrage society in 1884. 

One thing I often appreciate about older papers is the lack of the standard paper formula (Intro, Methods, Results, Discussion), instead a more narrative style that presents data and observations where necessary in the story. Bateson's "The effect of cross fertilization on inconspicuous flowers" from Annals of Botany in 1888 is a really nicely laid out concise argument. She starts with Charles Darwin's observation that while many small flowers are selfing and not visited often by pollinators, it would be strange that they would still be open if they were completely selfing. 

Darwin did not do these experiments because of the "difficulty of [crossing] them". Bateson did this tedious job for three species (of three families!) and found clear outcrossing benefits in all. Her experimental prowess is obvious, as is her sense of experimental design ("it would have been a better method to have obtained the self-fertilized seeds by artificial fertilization also"). And her conclusion - that outcrossing is a benefit to even usually-selfing plants is certainly correct. 

Also from Cooke's book, where he explains the actions of plant tendrils, a phenomenon still being researched mechanistically.

Another clever experiment concerned geotropism. She and Francis Darwin hypothesized, based on prior theory, that a stem lying directly on the horizontal is the most stimulated to move. They note that this is actually harder to test than it might seem, as if you simply let a stem curl, it will be subjected to varying stimulus as it curls upward (if the hypothesis is correct). Cleverly, they came up with a method to expose plants to varying stimuli independent of their response, by pinning them down for two hours at a given angle (they use three: stem pointed 60 degrees up, 60 degrees down and horizontal). They then released all three treatments for an hour and measured the angle. They got quite clear data supporting their hypothesis. In both cabbage (n=36) and plantain (n = 148), horizontally-placed stems curved more intensely than either down-sloped or up-sloped stems. I don't know much about geotropism, so I can't actually comment on the lasting scientific value of this experiment, but it was an elegant and simple experiment (science fair? lab demonstration?). 

The last paper is more of a monograph and concerns irregular flowers (i.e. not radially symmetric). Anna and her brother William detail many examples of abnormalities in flowers. They suggest that irregular flowers must have evolved from regular ancestors (we know macroevolutionary patterns and part of the molecular basis for this now) and that the best way to study the possibly evolutionary pathway is to look at variation in flowers now, to see what variation evolution is acting on in the present.

Plate of floral mutants in Bateson and Bateson 1891.

They write very clearly about natural selection and macroevolution in the introduction, making clear difficulties with the lack of intermediate forms, determining descent, and the apparent lack of utility of intermediate forms of an organ (essentially Paley's watchmaker argument addressed by Dawkins). They even mention punctuated equilibrium versus gradualism "Supposing, then, that such a series of ancestors were before us, the matter to be determined would be the degree to which the series is continuous or discontinuous: that is to say whether the differences between any one member and its immediate successor are so small as to be imperceptible, or whether there are distinct and palpable difference between them; or whether they are sometimes small, and sometimes so great as to cause interruptions in the series and divide it into groups". They follow this with a nice metaphor of evolution as chemistry or physics. They fall into Gould's camp, clearly thinking that evolution proceeds with discontinuities ("the objections to supposing that the process of evolution of forms is discontinuous are derived, firstly, from the scarcity of observed instances of sudden and large variation,,, it is in the hope of dispelling [this] objection that the present observations are recorded") [1].

The crux of their careful observations of toadflax, speedwell, gladiolus, and Streptocarpus lie in the fact that they are characterizing mutants that show a phenotype of different regularity from the parent. Especially interesting are their findings of apparently radially symmetric speedwell (#19 in the plate) and varying symmetries in all the species examined. They conclude these detailed observations by pointing out that while gradualism may be common, mutations that fundamentally alter an essential feature of a species (regularity of the flower; or Lenski and lab's digestion of citrate by E. coli) do occur. They state "The facts now given, though few, are a contribution to such evidence and, in our judgement, are a sample of the kind of fact which is required to enable us to deal with the problems of descent". Given that we now know that during plant evolution, both to and from irregular flowers has occurred many times, it is likely that the Batesons' work was prescient, it was certainly detailed, well-written and well-reasoned (they present a section at the end detailing the many limitations of their observations that is nowadays hardly admitted in a question and answer session, let alone in the published paper!). 

I spent an enjoyable couple hours reading this papers and composing this post. I'd love to hear suggestions of other interesting papers, comments on these papers, and really anything else natural history. 

[1] This is of course, a sort of contrived analogy on my part, as they are taking a saltatory view - a mutant occurs in one generation in the Batesons' argument, though in my reading, they are taking a wider view in the introduction, even with the preceding "between one member and its immediate successor".