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.

Beginning research: floral polymorphisms in Trichostema laxum

The first steps of any research project are, for me, the most exciting. Therefore, I’ll write a quick post on something I’ve been spending a bit of time on. Last summer, I spent most of the summer trying to wash off chemical defenses on leaves. One plant I chose was Trichostema laxum – a mint endemic to California (it may occur in extreme southern Oregon, too) that occurs pretty commonly on dry serpentine streambeds at my field site in Lake/Napa counties. I’ve already written a quick post mentioning this plant, but I know a LOT more now!

Trichostema laxum, October 2014, McLaughlin Reserve, Napa County. Notice the position of the stigma in relation to the anthers – the style (the stigma’s tube) projects well beyond the anthers. This is the “normal” morph of the plant, referenced in the literature and seen in all herbarium specimens I’ve looked at so far.

While the herbivory and exudate stuff awaits analysis (a dissertation proposal will force me to do that soon!), I discovered the aforementioned flower color polymorphism and took a bunch of baseline data on it, which may be important in the coming months. The flower color polymorphism interests me most because of one population which had a high (~3%) proportion of the white/purple morph – no others had it. Was it just a random neutral mutation that didn’t drift out? If not – how is it maintained?

The four polymorphs of flower color. All from summer 2015, McLaughlin Reserve, Napa/Lake Counties.

The first question was, do the various flower colors differ in fitness from the normal (purple) morph? Trichostema laxum along with most other annual plants in California grasslands and serpentine barrens, is extremely variable in size depending on the microclimatic conditions. Within a population, some individuals can have three orders of magnitude more flowers than others (~10 to ~10,000). I found one small bush-sized individual (probably nearly a meter square) on a gopher mound – clearly the gopher had changed the nutrients or hydrology of that specific location favorably! Therefore, I compared polymorphs to their nearest neighbor of the normal morph, in an attempt to minimize this variability. This was a coarse test (without a huge amount of power), but I found no differences, though large variability among individuals. I will – hopefully – be able to confirm this in the laboratory rather easily. I took a good amount of pollinator data – which also awaits analysis.

A pink morph just barely open (though the stigma is open, so maybe its deformed?).  McLaughlin Reserve, Lake County, CA. 

The next logical step in the investigation was to grow plants in the lab and find out whether the color polymorphisms were heritable – an important consideration in any investigation relating to population-level polymorphisms. Trichostema have a reputation for being a tough genus to grow, in fact, a professor at Davis told me a former grad student planned a project on them, but couldn’t get any germination. I’ve been more fortunate (with help from Danny Barney at the USDA) and got decent germination with a rather simple protocol – laxum may be less picky than its relatives. I grew them all fall – they flowered in November and early December.

One of the first individuals in the lab. Isn’t it cute?

When I started looking closely at the plants in the lab, I found two more polymorphisms. The first was the lower lip patterning. In normal plants, the lower lip – and sometimes the next lowest two petals, have some purple splotches on them. This is likely a nectar guide, leading pollinators to the reward (and often only visible/really cool in the UV). I knew in the field that the completely white morph lacked a nectar guide as it lacks anthocyanin, the red/purple pigment in most plants, completely, so a purple nectar guide would be precluded. But I was surprised to see a purple flowered plant lacking it.

Clean purple lower lip. December 2014, in lab. 

While interesting, this was only found in one plant (though I have seeds of it now). Another polymorphism was also obvious in the captive plants and it solved one of my summer mysteries. During the summer, I wanted to do crosses with the various colored flowers. I didn’t think it would be that hard – an older paper reported that T. laxum was non-selfing and covered plants produced no seed. So I placed pollinator exclosures over a bunch of plants and did crosses by moving pollen from one plant to another. I then covered the plants again, letting them naturally set seed and figuring that the only seed I’d get would be that of the crosses. I pollinated ~10 flowers per plant and since mints have only 4 ovaries per flower, I figured I could get about 40 seeds a plant (probably 30 since my fine motor skills aren’t all that great). When I uncaged the plants and collected the seeds in October, I got quite a surprise – large numbers of seeds. Though not a full complement from any plant (there are MANY reasons for this besides lack of pollen), I got way too many seeds to have been either 1) my pollination, or 2) occasional lapses in the pollinator exclosures. Clearly the plants were self-pollinating somehow.

And here is the solution to the mystery! Where is the stigma? Its pretty much in the middle of the anthers. This one isn’t quite mature yet, but instead of opening after growing far past the anthers (see the first picture), it will open either right in the anthers or ever so slightly beyond. McLaughlin Reserve, Lake County, CA.

Looking closely at the individuals in the lab revealed the reason for this mystery. Some plants, like the first picture in this post, had long styles, which projected the stigma far past the anthers. Others, like the one above, had short styles and the stigma was amidst, or ever so slightly past the anthers. This proximity (I think) allows the plant to self pollinating either directly, or with the slightest bit of wind or insect movement (the “self-pollinating” morph. In the lab, more than half the plants developed into the self-pollinating morph, and while I hadn’t noted it during the season, I was able to go back to the hundreds of pictures I took and found pictures of it in the field. Strangely, they are not in the same proportion – my pictures are primarily of the “normal” variety – which accords with the one paper on the plant, as well as descriptions. I then examined the specimens in the herbarium, all of which were the “normal” morph (and purple, with patterned lower lips). Whether the lab creates the right environment for this morph to develop (whether there is a genetic propensity for it, or it is somewhat environmentally-driven) is unknown now, but I am working on it. Jenny Van Wyk – another grad student at Davis and extremely knowledgeable plant reproductive biologist – have quantified the differences between these morphs and found some really interesting correlates.

Flowers of the two morphs (self-pollinating, top; normal, below), at the same scale (lower lip broken in lower photo). 

Preliminarily – and our sample size is low as of now – the self-pollinating morph has larger flowers (corolla length, display height, style length), produces more pollen (300x more!) and has more, but more dilute, nectar. There is variability within morph, but so far, each plant has fit into one of the two morphs easily. How much this is an artifact of the laboratory setting is unclear, but photos of the self-pollinating morph and the pollinator-excluded plants producing seed point to something interesting happening in the field. Right now we are focused on the laboratory aspect, but we are considering experiments and observational data to be performed/gathered this upcoming year. We’d love to hear what anyone thinks of the system and interesting questions we can ask with it!

I’ll have some photos and interesting observations from my three-week trip to Chile soon, too. Lots of interesting botany, entomology and birdwatching (condors!).

Polymorphic flowers – cool natural history observations

I’ve been spending as much time in the field (at McLaughlin reserve, which I can’t say enough good stuff about) as I can manage lately – trying to complete all the projects I’ve begun. A quick update on some interesting stuff.

This guy greeted me yesterday morning. Western rattlesnake, Crotalus viridis.
Not sure about the bunny – but maybe a juvenile jackrabbit?

The exudates of Trichostema laxum – also known as turpentineweed or bluecurls – are rather strange smelling (like herbaceous vinegar) and seem to be very deterrent to herbivorous insects in lab trials. So I have a large scale exudate removal experiment going, that while taking up a huge amount of time, seems to be going really well.

Trichostema laxum. You can even see the exudates shining on the leaves!

I am seeing an increase in herbivory (haven’t crunched the numbers, but it is noticeable) in the exudate-removed plants, and hopefully, I’ll be able to show a fitness effect. However, I wanted to be able to say that any fitness effect was due to herbivory, not differential pollination. So I’ve been spending 6 or so hours a day watching pollinators visit these plants. Which is also going well, but sitting for that long in the heat is driving me crazy. So I’ve been taking breaks and visiting other populations of T. laxum to look for herbivory.

All populations suffer some herbivory, but one population is getting annihilated by this leafhopper and
some noctuid caterpillar!

While scouting populations, I’ve come across some flower polymorphisms that apparently haven’t been described for the species. They occur in low frequency (1:1,000-100,000 depending on the population), but occur nonetheless, and therefore, are interesting. Are they selected against heavily? Or with big populations, are they just swamped out as neutral?

The top left is the “normal” morph – a purple flower which varies a small amount in shade, but not too much. The pink morph is in at least two populations in low frequency and probably exhibits a different molecular structure of anthocyanin – the pigment that gives most plants a reddish or pinkish color. The bottom right plant lacks anthocyanin entirely, it has no reddish parts, including the stem that is reddish in all other individuals of the species. The bottom left has anthocyanin – probably the same form as the normal, and has dark reddish stems – but lacks it on the flowers except on the lower lip.

I’m not sure what to make of these, but I’ve been gathering fitness data (the anthocyanin-less morph seems to be sterile) and pollination data and I’ll hopefully collect seed and do some breeding experiments.

Otherwise, life has been good!

Ctenucha sp. (rubroscapus/multifaria group, I do believe) which is all over the Stachys flowers here.