Monday, March 16, 2009

In Search Of The Blue Piñon – Part 2 of 6

Note: I’m away and offline this week. I’ve set up an auto-post series of this Mexican-Tree-Adventure story in my absence.

My searching found a number of botanical research papers. One of these was a 2001 analysis* of the nutritional composition of Martinez Piñon nuts by Lauro Lopez Mata, of the Universidad Nacional Autónoma de México. Lopez Mata found the nuts to be of “outstanding dietary value compared to other pinyon pine species and commercial nuts”, exceptional in their protein content and including nine out of 10 essential amino acids.

*Regeneracion, crecimiento y dinamica poblacional del pino azul Pinus maximartinezii Rzedowski, Lauro Lopez Mata, 1998

I also found an abstract of a genetic analysis of P. maximartinezii, by F. Thomas Ledig of the University of California, Davis, published in the Journal of Botany in 1999. I tracked down Tom Ledig, emailed him, and he was kind enough to mail me a copy of his paper**.

**Evidence for an Extreme Bottleneck in a Rare Mexican Pinyon: Genetic Diversity, Disequilibrium, and the Mating System in Pinus Maximartinezii, F. Thomas Ledig, M. Thompson Conkle, Basilio Bermejo-Velazquez,Teobaldo Eguiliz-Piedra, Paul D. Hodgkiss, David R. Johnson, and William S. Dvorak, 1999

In the late 1990’s, Ledig and his colleagues examined maximartinezii chromosomes extracted from four separate seed collections. What they found was both unusual and surprising. Plants, like animals, have genes packaged into distinct chromosomes. Pines, like people, reproduce sexually and are fully diploid, meaning that they receive half their chromosomes from each parent.

allele Ledig and his team germinated a number of seeds, extracted DNA from the germinated seed radicles, or embryonic seed roots, and examined a number of loci, or specific locations along chromosomes. The first surprise was that only 30% of the loci examined were polymorphic, meaning that they displayed more than one allele, or version of the DNA coding, or genes, at that locus. On average, about 65% of pine loci are polymorphic. The second surprise was that at no polymorphic locus did they find more than two alleles. Pines usually exhibit several alleles at a given polymorphic locus. Weeping Piñon, for instance, another rare Mexican piñon suspected to be Martinez Piñon’s closest living relative, exhibits up to 6 alleles at some of its loci, and averages 3 to 3.5 across all of its polymorphic loci.

According to Ledig et al, there are only a few possible explanations for this lack of variation. First, maximartinezii could have arisen as a single mutant individual from some other pine. Second, it could have come about as a result of a unique hybridization event between two other, unknown pines. Third, and most likely, is that at some point in the recent past, the species pinus maximartinezii experienced an extreme genetic bottleneck, or exceedingly small population. Since a single seed of a chromosomally diploid species can carry no more than two alleles at any given locus, this bottleneck was possibly as small as a single tree, or even a single seed.

Linkage Disequilibrium

Ledig and his team then analyzed the frequency of linkage disequilibrium in the maximartinezii genome. Linkage disequilibrium is a measure of difference between a random distribution and the actual distribution of alleles within a population. lincoln-pennies An easy way to think of linkage disequilibrium is this: if you came across one hundred pennies lying on the ground, you’d expect roughly fifty of them to be heads up and fifty to be tails up. If in fact fifty-one were heads up and forty-nine were tails up, one could say that the coins exhibited a very low level of linkage disequilibrium. But if you found that ninety-five of the pennies were heads up, and only five were tails-up, one would say that the coins exhibited a very high level of linkage disequilibrium, and you’d probably wonder what external force or agent had caused so many pennies to be heads-up. In living things, including plants, linkage disequilibrium is indicative of non-adaptive pressures on a population such as inbreeding, or interactions between two genes where the affected trait is fitness, or an extreme population bottleneck.

Linkage disequilibrium is rarely found in pines, or conifers in general; the frequency of linkage disequilibrium in maximartinezii is the highest found in any conifer.

Linkage disequilibrium naturally dissipates very rapidly in successive generations, as the distribution of alleles tends naturally toward random. This rate of dissipation can be estimated mathematically. If maximartinezii’s past “population” consisted of one seed containing all observed alleles, the “initial” linkage disequilibrium can be determined, and compared with disequilibrium in the present population. Calculating the rate of dissipation, the number of generations between initial and present disequilibrium can be calculated.

Ledig et al used this methodology to determine that the possible single-seed bottleneck event for pinus maximartinezii occurred between four and five generations previously.

Mature maxipiñon trees produce between 900 and 1800 seeds per year. Ledig et al estimated that if only half of a tenth of a percent of the seeds produced by a tree over an average of 100 years of reproductivity germinated and survived, the resulting population would equal 2,496 after four generations – remarkably close to the 2,000 – 2,500 estimate mapped in 1985.

The oldest maximartinezii have been dated to between 200 and 220 years old, meaning that the possible bottleneck occurred no more than 800 to 1,000 years ago, and possibly much more recently (most piñons start bearing cones at around age 25.) The net of Ledig’s paper is this: Sometime between Leif Erikson and Christopher Columbus, the entire ancestral population of Pinus maximartinezii was likely one seed.

I Am Obsessed

Between Internet searches, plodding my way through Spanish research papers, waiting for Ledig’s paper, and teaching myself enough botany and genetics to understand it, it took me well over a month to gain this perspective of the Blue Piñon. Not just a rare tree with big cones, it was a unique, beautiful super-piñon, bearing giant-sized, almost impossibly nutritious nuts. Alive only through an apparently miraculous single-seed survival, hugging a single mountain in a remote Mexican range and only a forest fire away from extinction, the Blue Piñon fascinated me. I needed to do more than just read about it. I needed to see it, to smell it, to experience the feel of its needles and its bark, and, if possible, to taste its nuts.

Several years ago, I took a year-long sabbatical between jobs. One of the most rewarding things I did during that time was to take an intensive Spanish class. I took to the language rapidly, was a star pupil, and followed with regular conversations with my children’s Latina nanny. AFOClimbingSheepMtn1095 In the years since, my perpetually intermediate-level Spanish has successfully navigated me through several journeys in Spanish-speaking countries, including Mexico. I’ve lived in the Western U.S for the better part of two decades, and during this time have regularly recreated by hiking and scrambling up remote, trail-less desert peaks. Between my Spanish language faculty and back-country hiking and navigational experience, I was convinced that seeking and finding the blue piñon was an adventure almost tailor-made for me.

The Plan

IMG_1665 Oddly, for someone whose day job has nothing to do with the natural world, it was my work that provided the opportunity. Every year in February my employer rewards its top salespeople for the previous year by sending them and their spouses on an all-expense-paid vacation to a tropical destination. As head of sales for the company, I have some measure of influence over the destination choice. For 2006, we selected Zihuatenejo, which is about a day’s drive from Southern Zacatecas. (pics left and below, right)

IMG_1716 Researching the exact location of, and possible access to, Cerro Piñones took a fair amount of effort. In spite of Mexico’s exalted status in the botanical world, it was frustratingly difficult to actually find any kind of guidebook to Mexican pines. There are two such guides that have been published in the last two decades. The first, “A Field Guide to the Pines of Mexico and Central America” by Aljos Farjon, Jorge A. Perez De La Rosa and Brian Styles, is only available overseas. I ordered my copy through the London Royal Botanical Gardens. When it arrived, I was a bit disappointed. Though a useful identification guide, it lacked specific location information for rare species, including Martinez Piñon. The other guide is The Pines of Mexico and Central America by Jesse P. Perry, Jr. A 200-plus page tome, I found enough references to it on the Internet to recognize it as the de facto bible of Mexican pine hunters. Unfortunately, it has been out of print for well over a decade, and the only used copies that turned up on the Internet were selling in the $400 - $600 range.

imartinezii01Several sources, including Ledig’s paper, noted the location of Cerro Piñones as being just above a village named “Pueblo Viejo”, about twelve kilometers southwest of the town of Juchipila, Zacatecas. “Pueblo Viejo” (literally “Old Town”) is such a common name in Mexico that Internet searches were meaningless. Juchipila itself is absent from any Mexican guidebook, one of countless Mexican communities too un-noteworthy to merit either recommendations or warnings for tourists.

zacatecas_sct My Internet searching did turn up another paper* by Lopez Mata, the author of the previously mentioned nutritional analysis. This paper, which analyzed and discussed (in Spanish) among other things the impact of seed harvesting on the maximartinezii population, contained a crude color-coded map of Cerro Piñones, which showed the range of the species, and included longitude and latitude. The coordinates told me which topographical map I needed to mail-order, and when I received the map I was pleased to finally see a miniscule village marked “Pueblo Viejo” at the Eastern base of a clearly marked “Cerro Piñones” at the southern tip of a long mountain range.

* Regeneracion, crecimiento y dinamica poblacional del pino azul Pinus maximartinezii Rzedowski, Lauro Lopez Mata, 1998

Intended Hiking Route My plan was straightforward. I’d rent a car in Zihuatenejo, drive to southern Zacatecas, find my way to Pueblo Viejo, and figure out a route up the mountain by asking locals. The next day I’d spend the day hiking up, seeing the trees, hiking back down, and driving back to Guadalajara before flying home the following morning.


Tomodactylus said...

First, I wanted to tell you I really enjoy your blog. It's the first one I look for and at each day. I'm amazed at the extent of your botanical knowledge, especially since it seems to have come through self-education. That's the true spirit of education -- learning to teach yourself. More people need to have it.
Now, the unpleasant task. Your explanation and description of disequilibrium is incorrect. What you describe with the penny example is the allele frequency of a single genetic locus. Disequilibrium refers to the association of alleles at two or more loci. Continuing your coin example: suppose you found a pile of 100 pennies and 100 dimes, each penny paired with a dime. 50 of the pennies are heads up and 50 are tails up. Similarly for the dimes. If there is no association between the headness or tailness of different coins you would expect to find 25 P(H),D(H), 25 P(H),D(T), 25 P(T(,D(H), and 25 P(T),D(T) pairs. This would be an example of genetic equilibrium. If, instead you found that all the pairs of coins were either both heads or both tails (50 P(H),D(H) and 50 P(T),D(T)) then you would have complete disequilibrium. The two coins of each pair are associated in their headness or tailness.

Going from coin to genes, each coin is a genetic locus and the H or T is a different allele at that locus. Disequilibrium measures the degree of association between different genetic loci.

Don't feel bad about this. Disequilibrium is a difficult concept to understand and always gives genetics students problems.

Again, I think you do a great job with your blog in your treatment of the living world. I can't wait for tomorrows post to discover how you got up to the grove of trees. I collected frogs in Mexico fifty years ago and when I asked local people directions I usually just got an "arriba" and was left to my own devices.

Watcher said...

Tomo- thanks for the correction/explanation. (Though I was tempted to pass it off on account having it written it ~2 years ago, I doubt I would’ve been much more accurate writing it last week without your explanation!)

And I welcome corrections, input and comments from readers. (Frankly I’m always pleasantly surprised people read my blog at all...) If I got discouraged every time I made a mistake I would’ve given this project up a long time ago. Regular readers know I don’t do this for a living and am learning about this stuff as I go, so they seem to be pretty tolerant of my errors. Plus, correction-opportunities have led me to some fun posts, like the 2nd moss post, which in turn got me into the whole lichen thing.

beetlesinthebush said...

Alex - I am mad at you. I am now finding myself obsessed with blue piñon and feeling an urge to recreate this journey. I've got too many things on my plate already.

I really enjoy this blog - I think we are kindred spirits, interested in far more than what we are supposed to be and thirsty for knowledge. Plus we're both cyclists (though I have this season retired from racing).


p.s. Tomodactylus - you should really write a blog. Your explanation of disequilibrium was amazingly cogent and clear. Even I understood it, and I'm just a nerd who likes bugs ;-)

Tom Ledig said...

Hi Watcher!

Hope you are still there. My wife found Part 3 of your "In Search of the Blue Pinyon" in January 2013. I've just (August) got around to looking up and reading the whole 6 parts. Great!
Did you know that Socorro Gonzalez Elizondo reported on a second stand of Maxipinyon in Durango in 2011?
The only time I went up Cerro Pinones we walked. We had a deal with people in Pueblo Viejo for horses, but when we got there, all the horses had been taken off on an emergency repair of a reservoir. So we walked.
I especially liked your stories of the people that you met. Typical for working in Mexico. The most accommodating anywhere, even when they have been treated less than kindly in the US. And anytime one starts to go out in the field, a car must be found, tires repaired, relatives and friends visited to meet the loco gringo -- all familiar. Thanks for the Blog. Tom Ledig

Watcher said...

Hi Tom! Yes, I'm still here, and glad you found the post(s). Thanks again for all of your help- as you can tell here, you helped make possible a great adventure!

No, I was unaware until your comment of Gonzalez-Elizondo's discovery. (I just found her paper and am working my way through it) Fantastic!

I'm impressed btw that you walked up- that looked like one long, hot climb...