Periods of steady climate on our planet have been punctuated by extraordinary paleoclimate events, from extreme greenhouses, with Arctic ocean temperatures soaring above 20°C 
, to the freezing conditions of snowball Earth 
. These dramatic fluctuations in temperature, and global anoxia that likely coincided with the freezing events 
, must have had a pronounced effect on the evolution of biological diversity. Each event probably acted as a powerful natural selection filter, with different events selecting for exactly opposite traits. It is possible that linking the history of paleoclimate to evolution of biodiversity may help to explain the present pattern of this biodiversity. We are interested in what biodiversity predictions can be made based on the paleoclimate dynamics, and whether such predictions can be verified. Here we examine a link between the diversity of microbial eukaryotes and paleoclimate events over the course of their evolution.
There is increasing evidence that several snowball Earth conditions occurred in the history of our planet (e.g., in the Paleoproterozoic 2.4 billion years ago 
, and more recently in the Neoproterozoic, 710 and 635 million years ago 
), as did the ultra-greenhouse temperatures that likely followed such conditions 
, and more moderate greenhouse events, the latest of which occurred in the Cenozoic 55 million years ago 
. Regardless of when and at what temperature the eukaryotes originated, they must have survived dramatic temperature and oxygen level fluctuations. Extreme cold would obviously select for extreme psychrophiles, making all other organisms less competitive. The opposite would be expected during extreme global warming periods. We postulate that one principal factor determining what kind of biodiversity would survive a cataclysmic shift is the presence or absence of a refuge. Depending on whether or not temperature refuges persisted throughout the history of eukaryotic life, we can envision four scenarios of how paleoclimate might-and in fact should–have influenced the evolution of microbial eukaryotes:
1. Refuges did not exist, and temperature extremes reached every corner of the biosphere. If so, neither psychrophiles nor thermophiles would be expected to survive an adverse temperature swing, and would have to evolve anew after each such event.
2. Only cold refuges persisted through time. If so, today's cold environments would be enriched with basal lineages with a long history of continuous evolution in the cold. Thermophilic lineages of today would be descendants of psychrophiles. Having evolved after the last global freezing event, these lineages would be expected to root within clades of psychrophilic origin.
3. Only hot refuges persisted through time. This would lead to a scenario opposite to #2: assemblages in hot environments would be enriched with basal lineages, with psychrophiles appearing as evolutionary newcomers.
4. Both types of refuges continuously existed throughout the history of eukaryotic life. In this case, today both low- and high-temperature environments would be expected to show a diversity of microeukaryotic life, each characterized by its own basal lineages. Continuous evolution of cold adaptations in thermophiles would produce evolutionarily young psychrophilic lineages within clades of thermophilic origin, and vice versa, leading to clades represented by mixes of thermo- and psychrophiles.
Two additional considerations add to the picture of predicted patterns of microeukaryotic diversity. First, since the snowball Earth was largely anoxic, the majority of survivors must have been obligate and facultative anaerobes. One would expect this to manifest today as a diversity of deeply rooted microbial eukaryotes with anaerobic life style. Second, it is reasonable to expect that older habitats would harbor larger biological richness by providing longer periods of time for this richness to evolve, especially since the diversification rate of microorganisms may have exceeded their extinction rate for billions of years 
. This richness should be distinct from that of a quick explosive origin (e.g.
, cichlid fish in Africa 
): the first should appear as a collection of lineages rooted deeply, whereas the second as a number of short lineages branching off from a distinct node of relatively recent origin.
The four scenarios above are not all equally plausible. The first is the least realistic, because hydrothermal environments have likely been a persistent feature of the ocean floor since the origin of our planet 
. Though rather ephemeral at individual sites, the hydrothermal vent environment in general would still provide a safe harbor for at least a selection of thermophilic (and perhaps some mesophilic) organisms, even at the peaks of the snowball Earth events.
In contrast, there is little evidence to support the persistence of cold refuges. The paleoceanographic record constructed from a sediment core from a recent drilling expedition in the Arctic Ocean suggests that early Cenozoic was very warm, with polar waters staying above 20°C year round, and no cold deep ocean 
. The mid-Cretaceous is also thought to have been exceptionally warm 
. It appears that cold environments might have been completely–and repeatedly-wiped out during such times, making scenarios 2 and 4 rather unlikely.
This leaves case 3 as the most plausible, at least in terms of current paleoclimate views. This scenario implies that microbial eukaryotes at elevated temperatures, such as in hydrothermal vent environments, should be uniquely rich in ancestral lineages, and exhibit a relatively large number of species, especially with anaerobic lifestyle. In contrast, protistan assemblages in cold environments would be expected to have lower species diversity and to be represented by lineages of recent evolutionary origin. The peak of the latest extreme greenhouse event was approximately 55 million years ago 
, and glaciation in the northern hemisphere did not start until 6–10 million years ago 
. Following the logic of the “center of origin” hypothesis 
, it seems reasonable to expect that protists from the polar regions, having evolved over that relatively short period of time, would form few (perhaps no) “old” unique clades, and would fall into groups characterized by deeply rooted thermophiles. Here we test these predictions by comparing the 18S rRNA gene diversity in two hydrothermal vent communities (HV1 
and HV2 
), to the diversity in an original dataset we obtained from a tidal flat in the Arctic (DI; Disko Island, Western Greenland). This sediment habitat represents one of the coldest tidal flats studied because it stays ice-free for only 3–4 months a year, with water temperature averaging −1.8°C 
, and rarely exceeding 4–6°C even on a sunny summer day. For comparative purposes, we include in our analyses the only available 18S rRNA surveys of temperate sediments (WH 
, and CA1 and CA2 
), as well as one of the best-studied (to date) protistan assemblages from an anoxic system (CB; Cariaco Basin in the Caribbean 
). We compare the results of these surveys by examining (1) their respective overall species richness; (2) the presence/absence, phylogeny, and richness of unique clades of high taxonomic order; and (3) the biology of relevant organisms.