5.0 Observed Instances of Speciation

The following are several examples of observations of speciation.

5.1 Speciations Involving Polyploidy, Hybridization or Hybridization Followed by Polyploidization.

5.1.1 Plants

(See also the discussion in de Wet 1971).

5.1.1.1 Evening Primrose (Oenothera gigas)

While studying the genetics of the evening primrose, Oenothera lamarckiana, de Vries (1905) found an unusual variant among his plants. O. lamarckiana has a chromosome number of 2N = 14. The variant had a chromosome number of 2N = 28. He found that he was unable to breed this variant with O. lamarckiana. He named this new species O. gigas.

5.1.1.2 Kew Primrose (Primula kewensis)

Digby (1912) crossed the primrose species Primula verticillata and P. floribunda to produce a sterile hybrid. Polyploidization occurred in a few of these plants to produce fertile offspring. The new species was named P. kewensis. Newton and Pellew (1929) note that spontaneous hybrids of P. verticillata and P. floribunda set tetraploid seed on at least three occasions. These happened in 1905, 1923 and 1926.

5.1.1.3 Tragopogon

Owenby (1950) demonstrated that two species in this genus were produced by polyploidization from hybrids. He showed that Tragopogon miscellus found in a colony in Moscow, Idaho was produced by hybridization of T. dubius and T. pratensis. He also showed that T. mirus found in a colony near Pullman, Washington was produced by hybridization of T. dubius and T. porrifolius. Evidence from chloroplast DNA suggests that T. mirus has originated independently by hybridization in eastern Washington and western Idaho at least three times (Soltis and Soltis 1989). The same study also shows multiple origins for T. micellus.

5.1.1.4 Raphanobrassica

The Russian cytologist Karpchenko (1927, 1928) crossed the radish, Raphanus sativus, with the cabbage, Brassica oleracea. Despite the fact that the plants were in different genera, he got a sterile hybrid. Some unreduced gametes were formed in the hybrids. This allowed for the production of seed. Plants grown from the seeds were interfertile with each other. They were not interfertile with either parental species. Unfortunately the new plant (genus Raphanobrassica) had the foliage of a radish and the root of a cabbage.

5.1.1.5 Hemp Nettle (Galeopsis tetrahit)

A species of hemp nettle, Galeopsis tetrahit, was hypothesized to be the result of a natural hybridization of two other species, G. pubescens and G. speciosa (Muntzing 1932). The two species were crossed. The hybrids matched G. tetrahit in both visible features and chromosome morphology.

5.1.1.6 Madia citrigracilis

Along similar lines, Clausen et al. (1945) hypothesized that Madia citrigracilis was a hexaploid hybrid of M. gracilis and M. citriodora As evidence they noted that the species have gametic chromosome numbers of n = 24, 16 and 8 respectively. Crossing M. gracilis and M. citriodora resulted in a highly sterile triploid with n = 24. The chromosomes formed almost no bivalents during meiosis. Artificially doubling the chromosome number using colchecine produced a hexaploid hybrid which closely resembled M. citrigracilis and was fertile.

5.1.1.7 Brassica

Frandsen (1943, 1947) was able to do this same sort of recreation of species in the genus Brassica (cabbage, etc.). His experiments showed that B. carinata (n = 17) may be recreated by hybridizing B. nigra (n = 8) and B. oleracea, B. juncea (n = 18) may be recreated by hybridizing B. nigra and B. campestris (n = 10), and B. napus (n = 19) may be recreated by hybridizing B. oleracea and B. campestris.

5.1.1.8 Maidenhair Fern (Adiantum pedatum)

Rabe and Haufler (1992) found a naturally occurring diploid sporophyte of maidenhair fern which produced unreduced (2N) spores. These spores resulted from a failure of the paired chromosomes to dissociate during the first division of meiosis. The spores germinated normally and grew into diploid gametophytes. These did not appear to produce antheridia. Nonetheless, a subsequent generation of tetraploid sporophytes was produced. When grown in the lab, the tetraploid sporophytes appear to be less vigorous than the normal diploid sporophytes. The 4N individuals were found near Baldwin City, Kansas.

5.1.1.9 Woodsia Fern (Woodsia abbeae)

Woodsia abbeae was described as a hybrid of W. cathcariana and W. ilvensis (Butters 1941). Plants of this hybrid normally produce abortive sporangia containing inviable spores. In 1944 Butters found a W. abbeae plant near Grand Portage, Minn. that had one fertile frond (Butters and Tryon 1948). The apical portion of this frond had fertile sporangia. Spores from this frond germinated and grew into prothallia. About six months after germination sporophytes were produced. They survived for about one year. Based on cytological evidence, Butters and Tryon concluded that the frond that produced the viable spores had gone tetraploid. They made no statement as to whether the sporophytes grown produced viable spores.

5.1.2 Animals

Speciation through hybridization and/or polyploidy has long been considered much less important in animals than in plants [[[refs.]]]. A number of reviews suggest that this view may be mistaken. (Lokki and Saura 1980; Bullini and Nascetti 1990; Vrijenhoek 1994). Bullini and Nasceti (1990) review chromosomal and genetic evidence that suggest that speciation through hybridization may occur in a number of insect species, including walking sticks, grasshoppers, blackflies and cucurlionid beetles. Lokki and Saura (1980) discuss the role of polyploidy in insect evolution. Vrijenhoek (1994) reviews the literature on parthenogenesis and hybridogenesis in fish. I will tackle this topic in greater depth in the next version of this **********

5.2 Speciations in Plant Species not Involving Hybridization or Polyploidy

5.2.1 Stephanomeira malheurensis

Gottlieb (1973) documented the speciation of Stephanomeira malheurensis. He found a single small population (< 250 plants) among a much larger population (> 25,000 plants) of S. exigua in Harney Co., Oregon. Both species are diploid and have the same number of chromosomes (N = 8). S. exigua is an obligate outcrosser exhibiting sporophytic self-incompatibility. S. malheurensis exhibits no self-incompatibility and self-pollinates. Though the two species look very similar, Gottlieb was able to document morphological differences in five characters plus chromosomal differences. F1 hybrids between the species produces only 50% of the seeds and 24% of the pollen that conspecific crosses produced. F2 hybrids showed various developmental abnormalities.

5.2.2 Maize (Zea mays)

Pasterniani (1969) produced almost complete reproductive isolation between two varieties of maize. The varieties were distinguishable by seed color, white versus yellow. Other genetic markers allowed him to identify hybrids. The two varieties were planted in a common field. Any plant's nearest neighbors were always plants of the other strain. Selection was applied against hybridization by using only those ears of corn that showed a low degree of hybridization as the source of the next years seed. Only parental type kernels from these ears were planted. The strength of selection was increased each year. In the first year, only ears with less than 30% intercrossed seed were used. In the fifth year, only ears with less than 1% intercrossed seed were used. After five years the average percentage of intercrossed matings dropped from 35.8% to 4.9% in the white strain and from 46.7% to 3.4% in the yellow strain.

5.2.3 Speciation as a Result of Selection for Tolerance to a Toxin: Yellow Monkey Flower (Mimulus guttatus)

At reasonably low concentrations, copper is toxic to many plant species. Several plants have been seen to develop a tolerance to this metal (Macnair 1981). Macnair and Christie (1983) used this to examine the genetic basis of a postmating isolating mechanism in yellow monkey flower. When they crossed plants from the copper tolerant "Copperopolis" population with plants from the nontolerant "Cerig" population, they found that many of the hybrids were inviable. During early growth, just after the four leaf stage, the leaves of many of the hybrids turned yellow and became necrotic. Death followed this. This was seen only in hybrids between the two populations. Through mapping studies, the authors were able to show that the copper tolerance gene and the gene responsible for hybrid inviability were either the same gene or were very tightly linked. These results suggest that reproductive isolation may require changes in only a small number of genes.

5.3 The Fruit Fly Literature

5.3.1 Drosophila paulistorum

Dobzhansky and Pavlovsky (1971) reported a speciation event that occurred in a laboratory culture of Drosophila paulistorum sometime between 1958 and 1963. The culture was descended from a single inseminated female that was captured in the Llanos of Colombia. In 1958 this strain produced fertile hybrids when crossed with conspecifics of different strains from Orinocan. From 1963 onward crosses with Orinocan strains produced only sterile males. Initially no assortative mating or behavioral isolation was seen between the Llanos strain and the Orinocan strains. Later on Dobzhansky produced assortative mating (Dobzhansky 1972).

5.3.2 Disruptive Selection on Drosophila melanogaster

Thoday and Gibson (1962) established a population of Drosophila melanogaster from four gravid females. They applied selection on this population for flies with the highest and lowest numbers of sternoplural chaetae (hairs). In each generation, eight flies with high numbers of chaetae were allowed to interbreed and eight flies with low numbers of chaetae were allowed to interbreed. Periodically they performed mate choice experiments on the two lines. They found that they had produced a high degree of positive assortative mating between the two groups. In the decade or so following this, eighteen labs attempted unsuccessfully to reproduce these results. References are given in Thoday and Gibson 1970.

5.3.3 Selection on Courtship Behavior in Drosophila melanogaster

Crossley (1974) was able to produce changes in mating behavior in two mutant strains of D. melanogaster. Four treatments were used. In each treatment, 55 virgin males and 55 virgin females of both ebony body mutant flies and vestigial wing mutant flies (220 flies total) were put into a jar and allowed to mate for 20 hours. The females were collected and each was put into a separate vial. The phenotypes of the offspring were recorded. Wild type offspring were hybrids between the mutants. In two of the four treatments, mating was carried out in the light. In one of these treatments all hybrid offspring were destroyed. This was repeated for 40 generations. Mating was carried out in the dark in the other two treatments. Again, in one of these all hybrids were destroyed. This was repeated for 49 generations. Crossley ran mate choice tests and observed mating behavior. Positive assortative mating was found in the treatment which had mated in the light and had been subject to strong selection against hybridization. The basis of this was changes in the courtship behaviors of both sexes. Similar experiments, without observation of mating behavior, were performed by Knight, et al. (1956).

5.3.4 Sexual Isolation as a Byproduct of Adaptation to Environmental Conditions in Drosophila melanogaster

Kilias, et al. (1980) exposed D. melanogaster populations to different temperature and humidity regimes for several years. They performed mating tests to check for reproductive isolation. They found some sterility in crosses among populations raised under different conditions. They also showed some positive assortative mating. These things were not observed in populations which were separated but raised under the same conditions. They concluded that sexual isolation was produced as a byproduct of selection.

5.3.5 Sympatric Speciation in Drosophila melanogaster

In a series of papers (Rice 1985, Rice and Salt 1988 and Rice and Salt 1990) Rice and Salt presented experimental evidence for the possibility of sympatric speciation. They started from the premise that whenever organisms sort themselves into the environment first and then mate locally, individuals with the same habitat preferences will necessarily mate assortatively. They established a stock population of D. melanogaster with flies collected in an orchard near Davis, California. Pupae from the culture were placed into a habitat maze. Newly emerged flies had to negotiate the maze to find food. The maze simulated several environmental gradients simultaneously. The flies had to make three choices of which way to go. The first was between light and dark (phototaxis). The second was between up and down (geotaxis). The last was between the scent of acetaldehyde and the scent of ethanol (chemotaxis). This divided the flies among eight habitats. The flies were further divided by the time of day of emergence. In total the flies were divided among 24 spatio-temporal habitats.
They next cultured two strains of flies that had chosen opposite habitats. One strain emerged early, flew upward and was attracted to dark and acetaldehyde. The other emerged late, flew downward and was attracted to light and ethanol. Pupae from these two strains were placed together in the maze. They were allowed to mate at the food site and were collected. Eye color differences between the strains allowed Rice and Salt to distinguish between the two strains. A selective penalty was imposed on flies that switched habitats. Females that switched habitats were destroyed. None of their gametes passed into the next generation. Males that switched habitats received no penalty. After 25 generations of this mating tests showed reproductive isolation between the two strains. Habitat specialization was also produced.
They next repeated the experiment without the penalty against habitat switching. The result was the same -- reproductive isolation was produced. They argued that a switching penalty is not necessary to produce reproductive isolation. Their results, they stated, show the possibility of sympatric speciation.

5.3.6 Isolation Produced as an Incidental Effect of Selection on several Drosophila species

In a series of experiments, del Solar (1966) derived positively and negatively geotactic and phototactic strains of D. pseudoobscura from the same population by running the flies through mazes. Flies from different strains were then introduced into mating chambers (10 males and 10 females from each strain). Matings were recorded. Statistically significant positive assortative mating was found.
In a separate series of experiments Dodd (1989) raised eight populations derived from a single population of D. Pseudoobscura on stressful media. Four populations were raised on a starch based medium, the other four were raised on a maltose based medium. The fly populations in both treatments took several months to get established, implying that they were under strong selection. Dodd found some evidence of genetic divergence between flies in the two treatments. He performed mate choice tests among experimental populations. He found statistically significant assortative mating between populations raised on different media, but no assortative mating among populations raised within the same medium regime. He argued that since there was no direct selection for reproductive isolation, the behavioral isolation results from a pleiotropic by-product to adaptation to the two media. Schluter and Nagel (1995) have argued that these results provide experimental support for the hypothesis of parallel speciation.
Less dramatic results were obtained by growing D. willistoni on media of different pH levels (de Oliveira and Cordeiro 1980). Mate choice tests after 26, 32, 52 and 69 generations of growth showed statistically significant assortative mating between some populations grown in different pH treatments. This ethological isolation did not always persist over time. They also found that some crosses made after 106 and 122 generations showed significant hybrid inferiority, but only when grown in acid medium.

5.3.7 Selection for Reinforcement in Drosophila melanogaster

Some proposed models of speciation rely on a process called reinforcement to complete the speciation process. Reinforcement occurs when to partially isolated allopatric populations come into contact. Lower relative fitness of hybrids between the two populations results in increased selection for isolating mechanisms. I should note that a recent review (Rice and Hostert 1993) argues that there is little experimental evidence to support reinforcement models. Two experiments in which the authors argue that their results provide support are discussed below.
Ehrman (1971) established strains of wild-type and mutant (black body) D. melanogaster. These flies were derived from compound autosome strains such that heterotypic matings would produce no progeny. The two strains were reared together in common fly cages. After two years, the isolation index generated from mate choice experiments had increased from 0.04 to 0.43, indicating the appearance of considerable assortative mating. After four years this index had risen to 0.64 (Ehrman 1973).
Along the same lines, Koopman (1950) was able to increase the degree of reproductive isolation between two partially isolated species, D. pseudoobscura and D. persimilis.

5.3.8 Tests of the Founder-flush Speciation Hypothesis Using Drosophila

The founder-flush (a.k.a. flush-crash) hypothesis posits that genetic drift and founder effects play a major role in speciation (Powell 1978). During a founder-flush cycle a new habitat is colonized by a small number of individuals (e.g. one inseminated female). The population rapidly expands (the flush phase). This is followed by the population crashing. During this crash period the population experiences strong genetic drift. The population undergoes another rapid expansion followed by another crash. This cycle repeats several times. Reproductive isolation is produced as a byproduct of genetic drift.
Dodd and Powell (1985) tested this hypothesis using D. pseudoobscura. A large, heterogeneous population was allowed to grow rapidly in a very large population cage. Twelve experimental populations were derived from this population from single pair matings. These populations were allowed to flush. Fourteen months later, mating tests were performed among the twelve populations. No postmating isolation was seen. One cross showed strong behavioral isolation. The populations underwent three more flush-crash cycles. Forty-four months after the start of the experiment (and fifteen months after the last flush) the populations were again tested. Once again, no postmating isolation was seen. Three populations showed behavioral isolation in the form of positive assortative mating. Later tests between 1980 and 1984 showed that the isolation persisted, though it was weaker in some cases.
Galina, et al. (1993) performed similar experiments with D. pseudoobscura. Mating tests between populations that underwent flush-crash cycles and their ancestral populations showed 8 cases of positive assortative mating out of 118 crosses. They also showed 5 cases of negative assortative mating (i.e. the flies preferred to mate with flies of the other strain). Tests among the founder-flush populations showed 36 cases of positive assortative mating out of 370 crosses. These tests also found 4 cases of negative assortative mating. Most of these mating preferences did not persist over time. Galina, et al. concluded that the founder-flush protocol yields reproductive isolation only as a rare and erratic event.
Ahearn (1980) applied the founder-flush protocol to D. silvestris. Flies from a line of this species underwent several flush-crash cycles. They were tested in mate choice experiments against flies from a continuously large population. Female flies from both strains preferred to mate with males from the large population. Females from the large population would not mate with males from the founder flush population. An asymmetric reproductive isolation was produced.
In a three year experiment, Ringo, et al. (1985) compared the effects of a founder-flush protocol to the effects of selection on various traits. A large population of D. simulans was created from flies from 69 wild caught stocks from several locations. Founder-flush lines and selection lines were derived from this population. The founder-flush lines went through six flush-crash cycles. The selection lines experienced equal intensities of selection for various traits. Mating test were performed between strains within a treatment and between treatment strains and the source population. Crosses were also checked for postmating isolation. In the selection lines, 10 out of 216 crosses showed positive assortative mating (2 crosses showed negative assortative mating). They also found that 25 out of 216 crosses showed postmating isolation. Of these, 9 cases involved crosses with the source population. In the founder-flush lines 12 out of 216 crosses showed positive assortative mating (3 crosses showed negative assortative mating). Postmating isolation was found in 15 out of 216 crosses, 11 involving the source population. They concluded that only weak isolation was found and that there was little difference between the effects of natural selection and the effects of genetic drift.
A final test of the founder-flush hypothesis will be described with the housefly cases below.

5.4 Housefly Speciation Experiments

5.4.1 A Test of the Founder-flush Hypothesis Using Houseflies

Meffert and Bryant (1991) used houseflies to test whether bottlenecks in populations can cause permanent alterations in courtship behavior that lead to premating isolation. They collected over 100 flies of each sex from a landfill near Alvin, Texas. These were used to initiate an ancestral population. From this ancestral population they established six lines. Two of these lines were started with one pair of flies, two lines were started with four pairs of flies and two lines were started with sixteen pairs of flies. These populations were flushed to about 2,000 flies each. They then went through five bottlenecks followed by flushes. This took 35 generations. Mate choice tests were performed. One case of positive assortative mating was found. One case of negative assortative mating was also found.

5.4.2 Selection for Geotaxis with and without Gene Flow

Soans, et al. (1974) used houseflies to test Pimentel's model of speciation. This model posits that speciation requires two steps. The first is the formation of races in subpopulations. This is followed by the establishment of reproductive isolation. Houseflies were subjected to intense divergent selection on the basis of positive and negative geotaxis. In some treatments no gene flow was allowed, while in others there was 30% gene flow. Selection was imposed by placing 1000 flies into the center of a 108 cm vertical tube. The first 50 flies that reached the top and the first 50 flies that reached the bottom were used to found positively and negatively geotactic populations. Four populations were established:

Population A + geotaxis,	no gene flow
Population B - geotaxis,	no gene flow
Population C + geotaxis,	30% gene flow
Population D - geotaxis,	30% gene flow

Selection was repeated within these populations each generations. After 38 generations the time to collect 50 flies had dropped from 6 hours to 2 hours in Pop A, from 4 hours to 4 minutes in Pop B, from 6 hours to 2 hours in Pop C and from 4 hours to 45 minutes in Pop D. Mate choice tests were performed. Positive assortative mating was found in all crosses. They concluded that reproductive isolation occurred under both allopatric and sympatric conditions when very strong selection was present.
Hurd and Eisenberg (1975) performed a similar experiment on houseflies using 50% gene flow and got the same results.

5.5 Speciation Through Host Race Differentiation

Recently there has been a lot of interest in whether the differentiation of an herbivorous or parasitic species into races living on different hosts can lead to sympatric speciation. It has been argued that in animals that mate on (or in) their preferred hosts, positive assortative mating is an inevitable byproduct of habitat selection (Rice 1985; Barton, et al. 1988). This would suggest that differentiated host races may represent incipient species.