To determine subdivision within the T. rotula morphospecies, it was first necessary to examine genetic divergence between T. rotula and T. gravida. Previous studies identified morphological plasticity in the characteristics used to define each species and argued for a single species designation
[49, 50]. Here, we found that culture collection isolates of the two species differed by at least 7% at the ITS1 and 0.8% at the 28S rDNA. This level of divergence is comparable to that observed between different species of the diatom Skeletonema (0.5% 28S divergence)
 and between Pseudo-nitzschia species (7.2% ITS1 divergence) that were confirmed using mating experiments
. ITS1 sequence variation indicated that T. rotula and T. gravida diverged approximately 3.28 Mya. Furthermore, the predicted ITS1 secondary structures of the two species differed considerably, a characteristic related to reproductive incompatibility in protists
[23–25, 69]. Although the links between reproductive isolation and ITS1 folding structure are not as well understood as the ITS2, it has been suggested that the ITS1 and ITS2 molecules co-evolve to maintain important biochemical interactions necessary for processing the mature ribosome
[26, 70]. Here, differences in the predicted T. gravida and T. rotula folding structures indicate that significant evolution has occurred at the ITS1
[24, 70]. Differences in rDNA sequences and predicted ITS1 secondary structures between culture collection isolates of T. rotula and T. gravida suggest that the original species designations are correct.
The majority of field isolates were not significantly different from T. rotula culture collection isolates at the 18S and 28S rDNA. This pool of isolates could be divided into three distinct ITS1 lineages which diverged from T. rotula culture collection isolates by 0–0.6%, an order of magnitude less than their divergence with T. gravida. This level of variation is comparable to that identified within other diatom species; for example, Pseudonitzchia pungens clades diverged by 0.5% at the ITS1
Several lines of evidence suggest that the three lineages may be able to interbreed. First, their predicted ITS1 secondary structures were identical to each other, suggesting that mutations in the ITS1 have not resulted in significant structural changes to this important molecule
. In addition, there were no compensatory base changes (CBCs) in the ITS1 stem regions of the three lineages, suggesting that this gene is conserved. Overall, the lack of CBCs and conservation of secondary structure among lineages suggest that they may retain the ability to interbreed. Second, there were no consistent differences in genome size among lineages, another indication that they may be able to interbreed. Importantly, differences in genome size were observed within and not between lineages. Within lineage 1, genome size differed by roughly two fold and within lineage 3, by 30%. Genome duplication, or polyploidization, is common in plants, and has been shown to result in rapid reproductive isolation
[30, 72–75]. In diatoms, few studies have examined changes in DNA content. Genome size differences of two-fold have been observed among ITS1 lineages of the diatom D. brightwellii, diverged by only 0.8% at the ITS1, suggesting that each lineage may instead represent a distinct species
. Furthermore, DNA content has been shown to vary among species within the genus Thalassiosira, suggesting that polyploidization may play a role in the evolution of diatoms as well as plants
. Because variations in genome size did not correlate with ITS1 lineage in T. rotula, this metric did not provide evidence for consistent barriers to interbreeding. Instead, the observation that two out of five strains differed in genome size suggests that T. rotula, and perhaps diatoms in general, may have a relatively plastic genome complement
Although several lines of evidence suggest that interbreeding could occur, additional data suggest that these lineages may not be actively interbreeding. To look for signatures of recombination, multiple copies of the ITS1 were sequenced from individuals representing each lineage. If lineages were interbreeding, one might expect to find, for example, some copies or recombinants of a lineage 1 sequence in a lineage 2 individual
. No signature of recombination could be detected among lineages suggesting that interbreeding, if it occurs, is infrequent and below the threshold of detection. Here, we used a single genetic marker to examine recombination; future analysis of recombination would be improved by surveying a greater number of genes. It is also worth noting that diatoms divide primarily asexually, and that sexual cycles have been examined for only a handful of the large number of described species
[77, 78]. Sexual recombination in the field has been observed
[79, 80], but rarely, and estimates of the incidence sexual recombination in diatoms varies widely, from once per year to once every 40 yrs
. In addition to an inability to detect recombination, it appears that gene flow between lineages has been reduced for significant time periods. A dated phylogenetic analysis indicated that lineage 3 diverged from T. gravida 3.28 Mya. Lineages 1 and 2 diverged later, at 0.68 Mya. Because divergence calculations can vary depending on outgroups, genes, or calibration points used in analysis
[12, 82], divergence times should be interpreted cautiously. Even if these estimations are off by orders of magnitude, the estimated time since last interbreeding is significant.
In the marine environment, interbreeding could cease to occur through such mechanisms as isolation by distance, physical barriers to gene flow, competitive exclusion, environmental adaptation, or genetic and phenological characteristics that prevent gametes from fusing in the field
. Here, it appears that isolation by distance was an unlikely mechanism promoting differentiation among T. rotula lineages. For example, genetic distance among lineages was not related to geographic distance. In fact, T. rotula lineage 3 had a cosmopolitan distribution ranging from the Mediterranean Sea and the N. Atlantic to the N. Pacific. This distribution is comparable to that observed in lineages of the pennate diatom P. pungens and contrasts with many terrestrial plant species, where genetic distance among lineages often correlates with geographic distance (eg.
[84, 85]). The observation that lineages can be broadly distributed in both centric and pennate diatoms suggests that dispersal likely plays a significant role in regulating gene flow. Lack of isolation by distance observed here suggests that there are no physical barriers impeding broad dispersal.
On smaller scales, physical features, such as water recirculation, may act to reduce gene flow, allowing different lineages to arise and be maintained. For example, hydrographic features have been hypothesized to drive genetic divergence in diatoms in coastal fjords of the NE Pacific where recirculating water may retain cells inside the fjord, allowing them to remain in and adapt to a particular location
. Interestingly, lineage 2 was observed within a recirculating coastal fjord in the NE Pacific, and exhibited significant divergence from lineage 1 sampled outside of the fjord, suggesting that water recirculation may influence genetic subdivision in multiple species of phytoplankton.
Competitive exclusion and environmental adaptation may be additional mechanisms initiating and supporting lineage divergence. Here, all but one location was dominated by just a single lineage. In those locations, the probability that other lineages were present but not detected was low. For example, a lineage representing 10% of the population would have a 99% probability of being detected in our 40 isolates collected from the N. Atlantic (Narragansett Bay and Martha’s Vineyard)
, suggesting that these sites were likely dominated by single lineage. This may be due to competitive exclusion of lineages not adapted to the environment in Narragansett Bay and coastal N. Atlantic. There may, however, be more complex dynamics in play. For example, all three lineages were sampled from the Queen Charlotte Islands in the NE Pacific. This location may act as a hub of intermixing between water masses and may provide an environment heterogeneous enough to support the ecological niches of all three lineages.
To explore potential signatures of environmental adaptation, we compared the physiological response of each lineage to a range of light intensities and temperatures, two important environmental variables known to affect phytoplankton growth
[87–89]. As established by Brand
, differences in acclimated growth rates can be used to identify underlying genetic variation among isolates grown in a single environmental condition. Analyzing the growth rates of isolates under different conditions allows for comparisons of genotypic versus environmental effects
. If environment alone were driving differences in growth, isolates would exhibit the same relative difference in growth rate, regardless of environment. Here, there was no correlation between ITS1 lineage and growth characteristics examined. Instead, isolates exhibited significant genotype by environment
 responses to light and temperature. The genotype by environment experiments conducted here suggest that there is additional clonal diversity within each T. rotula lineage, similar to that identified in other phytoplankton species
[86, 92, 93]. Lack of clear differentiation between lineages in their physiological response to light and temperature does not mean that environmental adaptation has not occurred among lineages since other factors such as predation and nutrient availability may be more important drivers of environmental adaptation among lineages.
The persistence of single lineages within individual locations suggests that there may be yet another type of environmental adaptation that allows diatoms to diverge into distinct lineages. For example, all isolates collected from Narragansett Bay represented a single lineage, regardless of the month of sampling (January, February, June or October). Furthermore, isolates collected from the Gulf of Naples between 1993 and 2008 represented the same lineage, suggesting that it persisted over many years in a single habitat. Similarly, identical genotypes of D. brightwellii were detected in Puget Sound over a seven-year period
. The persistent occurrence of lineages at individual locations may relate to the ability of many diatoms to create resting spores, which lie dormant in the sediment and may remain viable for decades
. T. rotula can form resting spores, although it is not a required part of its life cycle and the frequency of resting spore formation in the field and the rate of germination success are unknown
[95–98]. An intriguing hypothesis is that environmental adaptation to different phenological triggers for spore formation and germination could foster the initiation and maintenance of distinct lineages.
Finally, interbreeding could cease to occur among lineages if prezygotic barriers to gene flow no longer allow for sexual recombination among lineages. Prezygotic barriers to gene flow include changes to gametes that prevent them from recognizing each other
. In diatoms, the Sig1 gene has been hypothesized to play an important role in gamete recognition
. This gene has undergone rapid evolution among strains of the diatom T. weissflogii, including distinct protein changes that may alter the interaction between gametes in the field
. This type of prezygotic barrier to gene flow may lead to speciation via reinforcement of postzygotic differentiation
[76, 79]. Previous attempts to amplify the Sig1 gene in T. rotula have failed
, but future transcriptional or genome-wide sequencing analyses may shed light on the potential for a prezygotic barrier to gene flow between lineages observed here. A prezygotic barrier to gene flow could well explain the conflicting data we obtained regarding interbreeding among lineages, where genome size and RNA secondary structure indicated no barriers to interbreeding but active recombination may no longer occur. In this scenario, prezygotic barriers to gene flow would prevent sexual recombination among lineages but the rDNA would not yet have diverged to levels that compare with more distantly-related species.