Population genetics and hybridization
in the rare Colorado endemic Physaria bellii Mulligan

 

by Linda Kothera

Ph.D. candidate, Colorado State University

 

    Physaria bellii (Bell’s Twinpod) is an herbaceous, diploid (2N = 8), perennial member of the Brassicaceae (Mustard Family), which is endemic to north central Colorado.  It is a habitat specialist and is restricted to sloping shale and sandstone washes of the Niobrarra, Pierre, Lykins, and Fountain/Ingleside formations between elevations of 1580 and 1760 meters along the Front Range (Spackman et al. 1997, Doyle et al. 2004).  These geological formations have a patchy distribution, and as a result, P. bellii does as well.  According to the system used by the Colorado Natural Heritage Program, Bell’s Twinpod is ranked G2/S2, meaning it is imperiled because there is a small number of populations, and because this species depends on a habitat which is itself potentially threatened (Spackman et al. 1997, Doyle et al. 2004). 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Physaria bellii forms rosettes of leaves and bears several to many inflorescences, which flower from April through May and set seed around the middle of July.  Gerald Mulligan, who first described P. bellii and named it after a colleague, determined it was self- incompatible (Mulligan 1966).  The fruit is a small (4-6 mm) inflated silique consisting of two valves.  These fruit characteristics give P. bellii its common names, Twinpod and Bladderpod.

I studied P. bellii for my doctoral research at Colorado State University.  I am interested in rare plants, and P. bellii appealed to me because it is locally abundant, but at the same time vulnerable because its habitat is desired for human activities.  The patchy distribution of P. bellii indicates there may be genetic differentiation among the populations.  In other words, the populations may have detectable differences among them, and the detection and conservation of these differences is an important component to preserving the evolutionary potential of a rare species such as P. bellii.  With this in mind, one part of my research looked at estimating the amount of genetic diversity and genetic differentiation present in P. bellii populations.  In addition, anecdotal evidence suggested that P. bellii was hybridizing at the southern end of its range with the more common (but still endemic to central Colorado) P. vitulifera.  As a result, the other part of my research involved establishing whether hybridization had occurred and also characterization of any hybrids.

I collected leaf tissue from ten P. bellii populations for DNA extraction and for leaf measurements.  The DNA was used to generate genotypes of 300 P. bellii individuals, which allowed me to estimate the population genetic diversity and differentiation.  Overall, the data were consistent with a species whose populations are distributed linearly across a patchy habitat type.  There is a moderate amount of genetic diversity in P. bellii populations, and most of that diversity is within the populations, as opposed to being among them.  There does not appear to be appreciable levels of inbreeding in P. bellii populations, a condition that can reduce levels of genetic diversity in a species.  

 

There is also a significant correlation between the genetic distance between adjacent populations and the geographic distance between those populations, indicating that populations exchange genes most frequently with populations that are close by.  In addition, there is a high degree of genetic structure in P. bellii populations overall.  In other words, each population is fairly different from the others.   Even so, this work generated evidence that gene flow is occurring among the P. bellii populations around the city of Boulder, such that the ten sampled populations form eight genetic clusters.

 
I also collected leaves from 11 P. vitulifera Rydberg (sometimes called Rydberg’s Twinpod) populations as well as from two putative hybrid populations for DNA extraction and leaf measurements.  Instead of performing population genetic analyses on these data, I was interested if the genetic data could be used in a descriptive manner to differentiate among P. bellii, P. vitulifera and their putative hybrids.  Both data sets, genetic as well as morphological, discerned among the parental species and the putative hybrids such that members of each group clustered together.  The genetic data were much clearer in this regard, probably due to the nature of the data (either the marker was there or it was not).   I also found several species-diagnostic markers, which were found in over 90% of one parental species or the other. 

 

I constructed a hybrid index from these data (see Figure 1 below), that shows P. bellii individuals having scores closer to zero and P. vitulifera individuals scoring closer to six.  There are two types of hybrids on the figure.  The putative (naturally occurring) hybrids have scores that are closer to P. vitulifera and not strictly intermediate.  I also produced several first generation hybrids between the parental species (F1s) through controlled pollinations, and these individuals have more intermediate scores than the natural hybrids.  These results suggest that the putative hybrids are different in both regards, compared to the parental species, and genetically are more like P. vitulifera.

In addition, I measured leaves from the two species and the hybrids.  The most obvious difference between the leaves of P. bellii and P. vitulifera is the presence or absence of sinuses; P. bellii lacks them and P. vitulifera is supposed to have them.  I measured the depth of the sinuses and counted the number of pairs of sinuses on each leaf.  I also measured the length and width of leaves, the number of teeth on the margin of the leaves, and whether it had a tooth at the apex of the leaf (see Figure 2 below).  In the sample of leaves I examined, I found that P. bellii leaves never had sinuses, but only 69% (N = 100) of P. vitulifera leaves had ones deep enough to measure.  Interestingly, 51% (N = 58) of the hybrids had sinuses, and one might think, then, that the sinuses themselves would be intermediate in depth.  However, there was virtually no statistical difference between the sinuses of P. vitulifera and the putative hybrids.  In other words, when a hybrid has sinuses, it looks like P. vitulifera. 

 

With regard to the other characters, the two parental species differed for most of the characters, while the hybrids differed from each parental species on only one character, suggesting that overall, hybrids do have an intermediate leaf morphology between P. bellii and P. vitulifera.  The results from the genetic and morphological analyses support the idea that Jefferson County populations, which had previously been classified as P. bellii, should instead be classified as hybrids.

 
As part of the hybrid study, I also wanted to assess the degree to which P. bellii is threatened by hybridization with P. vitulifera.  Some P. vitulifera populations are diploid (eight chromosomes) like P. bellii, and others are tetraploid (16 chromosomes).  When two species have different numbers of chromosomes, it can act as a barrier to hybridization.  If they have the same number, then it is comparatively easier to form hybrids.  To that end, I ascertained the number of chromosomes from individuals in 11 P. vitulifera populations by germinating seed and performing root tip chromosome squashes.  To assess the ploidy levels of the putative hybrids, I germinated seed from one population and examined pollen mother cells from the other.

 

I found that most P. vitulifera populations are tetraploid, as are the hybrids, but the population closest to the hybrid populations was diploid.  These results suggest a possible scenario that could explain the ploidy level and location of the present-day hybrids.  Hybridization originally occurred between diploid individuals of both parent species, which were in closer proximity than they are today.  This was followed by a chromosome doubling event that resulted in present-day hybrids having a tetraploid number of chromosomes. 

 

I also performed test crosses between and among P. bellii and P. vitulifera individuals and found that inter-species crosses yielded less seed than intra-species crosses, which suggests that there are reproductive barriers in place between the parental species.

 
When my study began, it was thought that P. vitulifera occurred in southern Wyoming.  There was thus the possibility that hybridization might threaten P. bellii at both ends of its distribution.  I found one population of what had been identified as P. vitulifera near Wood’s Hole, WY, and collected leaves from most of this small population for DNA analysis.  In 2004, Bill Jennings (Jennings 2004) reclassified Wyoming P. vitulifera as P. acutifolia, as part of an effort to verify records in regional herbaria.  It thus appears that P. vitulifera does not occur in Wyoming, but the potential for P. acutifolia to hybridize with P. bellii is unknown, although the two species’ ranges currently do not overlap.  Most (63%) of the ISSR markers I found in P. bellii and P. vitulifera were not found in any P. acutifolia individual.  Also, some P. acutifolia individuals had bands that were not present in any P. bellii or P. vitulifera individuals.  A few bands were found in all three taxa.

 
My study generated evidence that the purported hybrid populations of P. bellii and P. vitulifera in Jefferson County are indeed hybrids.   It is good news that P. bellii does not appear to be threatened by hybridization with P. vitulifera at this time.  However, the results from this study indicate P. bellii is confined to just two counties in Colorado, Boulder and Larimer. Furthermore, the results from the population genetics part of my research indicate that each population makes a unique contribution to the genetic diversity of the species as a whole.  The loss of even a few populations could alter the current pattern of gene flow, which could reduce levels of genetic diversity.  

 

Physaria bellii faces the very real threat of loss of habitat from residential development and limestone mining.  Conservation efforts should thus involve monitoring populations to ensure that levels of genetic diversity remain stable, as well as ensuring a significant proportion of populations (there are less than 30) are not lost to human activities.   As P. bellii lacks formal protection, the impetus for this work will likely fall to the cities of Fort Collins and Boulder, as well as Larimer and Boulder county open space programs. 

 

 

  

 

Figure 2.  Illustrations of P. bellii (left) and P. vitulifera leaves showing leaf morphology characteristics.  1 = length; 2 = width; 3 = toptooth (here, present); 4, 5 = width of sinuses.  The P. vitulifera leaf shown has one pair of sinuses (6), which were counted for the character pairsinuses.  Leaves often had one or more teeth around the margin (numberteeth), which were left off this illustration for clarity.

 

Literature cited:

 

Jennings, W. 2004. The status of Physaria vitulifera in Wyoming. Castilleja: A Publication of the Wyoming Native Plant Society 23: 3-4. www.uwyo.edu/wyndd/wnps/wnps_home.htm

 

Doyle, G. A., S. L. Neid, and R. J. Rondeau. 2004. Survey of Critical Biological Resources, Larimer County, Colorado. Unpublished report. Colorado Natural Heritage Program.

 

Spackman, S., B. Jennings, J. Coles, C. Dawson, M. Minton, A. Kratz, and C. Spurrier. 1997. Colorado Rare Plant Field Guide. Prepared for the Bureau of Land Management, USDA Forest Service, and U.S. Fish and Wildlife Service by the Colorado Natural Heritage Program.

 

 

The Research Grants Committee thanks the many Colorado Native Plant Society members and Committee supporters for their donations to the research grants funds.  Your donation to these funds is encouraged and welcomed.  Please make your check to the Colorado Native Plant Society, designate the fund(s) to which you are donating and mail to:
Treasurer
Colorado Native Plant Society
P.O. Box 200
Fort Collins, Colorado 80522

Email Jan Loechell Turner

or phone her at (303) 458-4262 for further information.