Understanding a species: The topic of “species” is a touchy one, and there are many different definitions and approaches to understanding what exactly a species is and how to differentiate between species. The biological species concept is the standard and most broadly applied definition. To be considered a single species under the biological species concept, two populations must be capable of interbreeding and reproducing with other individuals to produce viable and fertile offspring. By extension, this means that in order to be considered an entirely different species the populations must be completely reproductively isolated from other species. 

Morphology: One way organisms become different species is being isolated from each other due to physical differences. A striking morphological difference exists in the North American population of Lentinus tigrinus. On this continent, there are two different morphological forms, or phenotypes, of fruiting bodies that are produced, differentiated as agaricoid or secotioid. The secotioid form has free visible gills that easily release spores through the air. The secotioid form has covered gills that trap spores inside and then release them gradually with no obvious signs of air dispersal . These two different forms appear to be controlled by a single locus. The agaricoid allele (a version of a gene) is dominant over the secotioid allele. This means that an agaricoid mushroom can have two copies of the agaricoid allele (aga/aga) or one copy of each allele (sec/aga), whereas the secotioid form can only exist with a sec/sec genotype. Because we have a situation where spores with one genotype are dispersed differently than spores with another genotype, we might expect this to result in assortative mating. 

Assortative mating: Assortative mating is a form of sexual selection, occurring when one phenotype mates at a higher rate with individuals with the same phenotype. In this case, if there is assortative mating between the different morphologies, we will see agaricoid forms mating with each other at an increased rate and secotioid forms mating with each other at an increased rate. This will eventually lead to population structuring, as well as increased differences between the two morphologies as heterozygotes become increasingly rare. There is already evidence that assortative mating may be occurring within the North American populations. During spore dispersal, many spores appear to fall within close proximity to the parental fruiting body. Lentinus tigrinus spores have a very fast germination rate, often within two days. This theoretically leads to increased instances of sibling-mating, which in turn can lead to increases in individuals mating with the same phenotype. 

So how do we test for assortative mating? We must first look to see if there is significant population structuring within a single population. The population chosen for this project can be found along the Ipswich River, although we plan to do the same for other populations in Massachusetts and Illinois. First, we divided the ipswich river into 100 segments of equal length. One sample was collected from each segment along the riverbank or from partially submerged logs. These samples are genotyped in the field (determined if the individual is agaricoid or secotioid) and brought back to the lab. 

Once in the lab, spore prints are taken from agaricoid samples, and these spore prints are then used to determine if the individual is a heterozygote or homozygote. This is done by creating a spore dilution for each sample, and these spores are grown out into germlings. These germlings, called single spore isolates (SSI’s), are then determined to be monokaryotic (meaning they include half of the genetic material necessary to produce fruiting bodies) and are crossed with a known tester strain. The tester strain is from a genotyped secotioid individual. This means that if the SSI contains the recessive allele, once crossed with the recessive tester strain a secotioid fruiting body is produced. If the sample the SSI’s were taken from, or the parental individual, produces only agaricoid offspring, then the sample is a homozygous individual. If some secotioid offspring are produced, this means that the parental individual is heterozygous. 

This is done for every sample taken from within that population. Once the offspring have been genotyped, the ratio of heterozygous to homozygous dominant to homozygous recessive individuals can be determined. This ratio will then be used to compare to the values that would be expected if there were no instances of assortative mating by using a Chi squared test. This will compare the observed allelic frequencies with the expected allelic frequencies. If they are significantly different, it can then be determined that assortative mating is occurring within this population. 

Differing Phenotypes and why they occur: As mutations arise, genotypes and resulting phenotypes of organisms within the same population can change. Mutations, however, are abundant and random within all genomes, many times resulting in changes so minor they are inconsequential and other times resulting in changes that are detrimental to the organism. In the case of Lentinus tigrinus, this mutation is responsible for a completely novel phenotype. It appears that at this moment, both alleles are present within the population. In order for an allele to remain and increase in frequency within a population, there must be some selection for it. 

So why do we see both phenotypes in a single population?  In order for this to occur, there must be some form of frequency dependent selection, either positive, negative, or balanced selection. Frequency dependant selection acts as a force of speciation when the fitness of a specific phenotype is dependent on the frequency of that phenotype, compared to alternative phenotypes that exist within that same population. We have two phenotypes within one population, a secotioid and an agaricoid phenotype. Usually, frequency dependent selection favors either rare phenotypes (as in the case of negative-frequency dependent selection) or common phenotypes (as in the case of positive-frequency selection). We may be seeing a selective sweep within this population towards the secotioid phenotype, meaning the secotioid form is beginning to increase to become less rare within this population. 

There may also be balancing-selection occurring. This is a selective process in which the different alleles are maintained within the same gene pool because, and both alleles occur at higher than expected frequencies. This may be true in the case of Lentinus tigrinus due to its environment of fluctuating water levels. The different phenotypes may be better fit for the same environment, just at different times or different circumstances. This is a very real possibility due to the constantly changing environments that this mushroom can be found in. Lentinus tigrinus grows along the waters edge, within the floodplain of a river, or grows on partially submerged logs within the river itself. This may lead to one form performing better while submerged and the other form performing better when they remain dry.

If you have any questions about our progress, comments, or just want to connect, please email me at sirons@clarku.edu or sofieirons17@gmail.com