Large-scale patterns in of microbial from the abyssal sea floor

Frank Scheckenbacha,1, Klaus Hausmannb, Claudia Wylezicha,c, Markus Weiterea, and Hartmut Arndta

aDepartment of General and Limnology, Institute for Zoology, Biocenter, University of Cologne, Cologne, Germany; bDivision of Protozoology, Institute of Biology/Zoology, Free University of Berlin, Berlin, Germany; and cDepartment of Biological Oceanography, Leibniz Institute for Baltic Sea Research, Warnemünde, Germany

Edited by David M. Karl, University of Hawaii, Honolulu, HI, and approved November 4, 2009 (received for review August 6, 2009) Eukaryotic microbial life at abyssal depths remains “uncharted ter- role of microbial eukaryotic communities is essential to under- ritory” in eukaryotic microbiology. No phylogenetic surveys have standing global biogeochemical cycles (18). focused on the largest benthic environment on this planet, the The perceived homogeneity of abyssal environments, with abyssal plains. Moreover, knowledge of the spatial patterns of little environmental variation, has led to the assumption that deep-sea community structure is scanty, and what little is known species have broad distribution ranges. This is in fact supported originates primarily from morphology-based studies of foramini- by studies of foraminiferans, which in some cases have ranges ferans. Here we report on the great phylogenetic diversity of mi- encompassing entire abyssal plains (19). But environmental crobial eukaryotic communities of all 3 abyssal plains of the gradients do shape the deep-sea community structure, especially southeastern Atlantic Ocean—the Angola, Cape, and Guinea Abys- in benthic environments (20). Except for the studies by Count- sal Plains—from depths of 5,000 m. A high percentage of retrieved way et al. (13), molecular studies of microbial com- clones had no close representatives in genetic databases. Many munities in the deep sea have all been carried out on a local clones were affiliated with parasitic species. Furthermore, differ- scale. Local-scale studies of benthic deep-sea microbial com- ences between the communities of the Cape Abyssal Plain and the munities have reported differences in the community structures other 2 abyssal plains point to environmental gradients apparently between adjacent sampling sites (11, 21). Although currently

shaping community structure at the landscape level. On a regional scant, knowledge of large-scale patterns of deep-sea community ECOLOGY scale, local species diversity showed much less variation. Our study structures is necessary to allow the assessment of the forces provides insight into the community composition of microbial eu- driving biodiversity and biogeography in the deep. karyotes on larger scales from the wide abyssal sea floor realm In the present study, we used environmental cloning and and marks a direction for more detailed future studies aimed at sequencing techniques to investigate microbial eukaryotes in the improving our understanding of deep-sea microbes at the com- three abyssal plains of the southern Atlantic Ocean: the Angola, munity and ecosystem levels, as well as the ecological principles Cape, and Guinea Abyssal Plains (Fig. 1). We addressed and at play. compared the phylogenetic diversity and community structure of these abyssal plains using a multiple PCR primer approach, in- benthic | clone library | deep sea | | protozoa cluding general eukaryotic and group-specific (Heterokonta, Cercozoa, and Kinetoplastea) primers. The data reveal a high he abyssal sea floor (3,000–6,000 m depth) represents the phylogenetic diversity and demonstrate that large-scale patterns fl Tmost common benthic environment on this planet, covering of eukaryotic microbial biodiversity exist at the abyssal sea oor. ’ 54% of the Earth s surface (1). Studies of the abyss have been Results impeded by the considerable technical difficulties involved in Sampling Efficiency and Estimation. We were able to retrieve a total accessing this remote environment. Almost all studies to date of 763 protistan clones with an average length of 594 bp, 442 of have focused on prokaryotes and metazoans. Whereas diversity them with general eukaryotic primers and 331 of them with estimations of metazoans of meiofaunal size have a long and rich group-specific primers (Heterokonta, n = 205; Cercozoa, n = 59; history (2), microbial eukaryotes (protists) have received much Kinetoplastea, n = 57). In addition, 26 clones were identified by less attention, and studies have concentrated mainly on a single RDP Chimera Check, Pintail, or our manual procedure as po- taxonomic group, the foraminiferans (3). There remains a sig- tential chimeric sequences and consequently were excluded from nificant lack of information on the community structure of other the analysis. All clones were grouped into operational taxonomic deep-sea microbial eukaryotes (4, 5). Environmental molecular units (OTUs), following mean (5.21%; OTU5.21) and median surveys have revolutionized our understanding of microbial sys- (0.80%; OTU0.80) values for OTU delineation estimated with the tems (6). Phylogenetic surveys also have been applied success- SILVA data set (22) (Fig. S1), resulting in 180 (for OTU5.21) and fully to study the community composition of eukaryotic microbes 387 (for OTU0.80) different OTUs. Rank-abundance curves of the at several deep-sea sites (7–16); however, to date, no study has entire data set (deducible from the bar charts of Figs. 2 and 3; addressed the phylogenetic diversity of eukaryotic microbial life rank-abundance curves for each region are shown in Fig. S2) ’ showed high percentages of singletons (59% for OTU5.21 and 73% of one of the largest habitats, covering over half of the Earth s 0.80 surface: the abyssal plains. for OTU ). Rarefaction curves calculated with both the general Abyssal plains are among the Earth’s flattest and smoothest regions and are covered with muddy soft sediments. At one time, Author contributions: F.S., C.W., and H.A. designed research; F.S. performed research; F.S. these plains were assumed to be vast, desert-like, contiguous analyzed data; and F.S., K.H., C.W., M.W., and H.A. wrote the paper. habitats with relatively constant physical and chemical parame- The authors declare no conflict of interest. ters. Recent studies of prokaryotes have shown that even the This article is a PNAS Direct Submission. deepest parts of the Earth teem with a wide variety of life (17); Data deposition: The sequences reported in this paper have been deposited in the however, information on the diversity and distribution of mi- GenBank database (accession nos. GU218701–GU219463). crobial eukaryotes at abyssal depths and beyond remains scarce, 1To whom correspondence should be addressed. E-mail: [email protected]. ’ fl despite these eukarocytes important role in the material ux in This article contains supporting information online at www.pnas.org/cgi/content/full/ other aquatic ecosystems of the biosphere. Knowledge of the 0908816106/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0908816106 PNAS Early Edition | 1of6 Downloaded by guest on October 4, 2021 Community Comparisons of Deep-Sea Abyssal Plains. Clone libraries constructed with general eukaryotic primers were statistically compared. Rank-abundance curves for each sampling region (deducible from Figs. 2 and 3 and Fig. S2) show that the nu- merically dominant OTUs generally were present in all 4 sam- pling regions, highlighting the similarity of the underlying communities based on relative clone abundances. However, OTU-based similarity indices showed significant dissimilarity between the communities of the Cape Abyssal Plain and those of the Angola Abyssal Plain and the eastern and western Guinea 5.21 Abyssal Plain (0.20–0.26 θ YC), but considerably greater sim- ilarity of the communities of the Angola Abyssal Plain and the Fig. 1. Map of the southeastern Atlantic with sampling locations: western 5.21 Guinea Abyssal Plain (0.72–0.87 θ YC). FST (Table S1) and Guinea Abyssal Plain (1), eastern Guinea Abyssal Plain (2), Angola Abyssal fi Plain (3), and Cape Abyssal Plain (4). Unifrac (Table S2) tests revealed signi cant differences among the communities of the Cape Abyssal Plain and the other abyssal plains (Fig. 4). Differences in the community structures of the eukaryotic and the group-specific primers always leveled off, but Angola and the Guinea Abyssal Plain turned out to be statisti- did not reach saturation (Fig. S3). The total number of OTUs was cally nonsignificant; however, the P-test (Table S2) was non- 5.21 fi estimated at 408 for S ACE [95% confidence interval (CI), 328– signi cant only between the communities of the Angola and the 0.80 535] and 1,240 for S ACE (95% CI, 1006–1562). western Guinea Abyssal Plains. According to SIMPROF-tests (OTU5.21- and OTU0.80-based Bray–Curtis similarity indices), Large Amount of Novel Phylotypes. Comparisons of all clones with communities of the Cape Abyssal Plain differed from those of published sequences resulted in a very high percentage of clones the Angola and the 2 regions of the Guinea Abyssal Plain (P > having neither a nearest named neighbor (73%) nor any nearest .01). The communities of the Angola and the Guinea Abyssal neighbor in GenBANK (63%). We defined the nearest neighbor PlainsR could not be distinguished with the present data set (P < as the Basic Local Alignment Search Tool (BLAST) hit with the .01). -LIBSHUFF tests (Table S3) also could not distinguish highest score and no more than 5.21% p-distance to the re- between communities of the Angola Abyssal Plain and the 2 spective clone. The nearest named neighbor was accordingly regions of the Guinea Abyssal Plain (P > .05); communities of defined as the highest scoring BLAST hit with no more than both of these abyssal plains were mostly significantly different 5.21% p-distance to the respective clone and representing a se- from communities of the Cape Abyssal Plain (P < .01). quence with full species name annotated. The mean genetic p- Tests with group-specific clone libraries showed that com- distances between clones and their first BLAST hit (i.e., the munities from the same abyssal plain (the eastern and western BLAST hit with the highest score), as well as first named BLAST Guinea Abyssal Plain) were very similar (Table S4). This high- hit (i.e., the BLAST hit with the highest score representing a lights the remarkable similarity of communities retrieved from sequence with full species name annotated), were accordingly the same abyssal plain. Sampling regions from different abyssal high, with mean values of 11% and 13%, respectively (Fig. S4). plains were generally significantly different from each other. This was mainly the result of novel euglenozoan clones. The first All clone libraries were significantly different (P < .01; Fig. 4) BLAST hit within this group was nearly always a highly divergent from other published deep-sea clone libraries. named sequence and no environmental sequence, indicating that these clones rarely appeared in other clone libraries. Removing Discussion euglenozoans from the analysis resulted in mean genetic p-dis- High Diversity and High Numbers of Novel Phylotypes. The most tances between clones and published sequences of 6.5% (first obvious feature of microbial eukaryotes from abyssal plains is the 5.21 BLAST hit) and 12.4% (first named BLAST hit). Calculating substantial species richness of their communities (408 S ACE; 0.80 mean genetic p-distances between clones and published se- 1,240 S ACE), which is as high as that at proclaimed diversity hot quences without clones from group-specific clone libraries raised spots, such as hydrothermal vents, anoxic and hypersaline envi- the mean values to 15.9%–16.0% (Fig. S4), due to the fact that ronments, and methane seeps (8–10, 12, 14–16) with mean esti- 5.21 0.80 most euglenozoan clones (all diplonemids and 78% of all ki- mates ranging from 410 (S ACE) to 489 (S ACE). Furthermore, netoplastids) were present in clone libraries retrieved with gen- the mean genetic p-distances between all clones and both their first eral eukaryotic primers. BLAST hits (11.0%) and first named BLAST hits (13.0%) are higher than those reported from other deep-sea environments High Phylogenetic Diversity with Many Parasites. All retrieved (2.5% and 10.6%, respectively; Fig. S4). These values point to a clones belonged to well-established high-rank taxonomic groups. specific assemblage of microbial eukaryotes at benthic abyssal sites All phylogenetic groups generally found in deep-sea molecular and are supported by high FST values between our clone libraries surveys were present in our clone libraries (Figs. 2 and 3). The most and other published clone libraries (Fig. 4). To some extent this is abundant phylogenetic groups in clone libraries constructed with the result of a single taxonomic group: the (i.e., general eukaryotic primers were Alveolata (n = 168), Euglenozoa Diplonemea and Kinetoplastea). Although group-specific primers (n = 161), Heterokonta (n = 39) and Rhizaria (n = 26). This re- have proven useful in assessing the phylogenetic diversity of some sembles the phylogenetic composition of other deep-sea clone li- groups, such as the Cercozoa (23) and Diplonemea (24), only one- braries. Well-known major phylogenetic clades, such as the third of the kinetoplastid clones were retrieved from group- uncultured marine (UMA/MALV/NA) or marine specific clone libraries, whereas two-thirds were retrieved with stramenopiles (MAST), were abundant. Lineages and clades with general eukaryotic primers. Thus, the high representation of eu- no close representatives in GenBANK (5.21% threshold) were glenozoans in our clone libraries is the result of their high occur- retrieved for all major phylogenetic groups, even within well- rence in these environments, rather than due to a bias resulting studied groups, such as the Ciliophora (red long-dashed lines in from group-specific primers able to detect lineages that otherwise Figs. 2 and 3). Furthermore, numerous clones had parasites as may not appear in general eukaryotic clone libraries. nearest neighbors, including Amoebophrya, Cryptocaryon, Cy- The first BLAST hit within the euglenozoans was mostly a tauxzoon, , Haramonas, Ichthyobodo, Miamiensis, highly genetically divergent sequence, indicating that these deep- Paradinium, , Pirsonia, and Rhizidomyces. sea euglenozoans rarely appear in published clone libraries and

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.0908816106 Scheckenbach et al. Downloaded by guest on October 4, 2021 0.1 Color ranges (phylogeny): Legend (bar and pie charts):

Rhizaria (Radiolaria) Cape Abyssal Plain Rhizaria (Cercozoa Haplosporidia Gromia) Angola Abyssal Plain Heterokonta Eastern Guinea Abyssal Plain Haptophyta Western Guinea Abyssal Plain Dinozoa 10 clones (bar charts) Ciliophora Choanoflagellida Cryptophyceae

Telonema 9118 19.18/22.39 9049 20.47/22.39

9128 12.76/17.90

9104 10.79/11.97

Reticulamoeba Cryothecomonas9426 8.69/11.79

9109 -/13.08 9418 3.39/9.09

8901 2.16/7.80 8767 11.35/11.518897 7.41/17.14

Gromia 9116 4.94/6.50 Massisteria

8927 3.51/9.79 PseudocubusCollozoum

8935 11.61/13.79 8706 7.42/9.19 9100 18.27/19.63 Paradinium 8707 15.76/18.20 8710 -/18.92 8756 10.59/11.11 8982 18.57/19.289422 -/15.24 Amphibelone 8731 0.25/4.99 Bicosoeca 9171 4.92/11.21 Caecitellus 8932 0.00/12.22 8916 -/0.36 8819 11.39/12.78 Cafeteria 9388 -/0.17 Thraustochytrium Aplanochytrium 8951 3.78/7.40 9191 3.05/10.06 8963 0.36/3.61 9149 8.16/12.38 8723 8.39/9.82 9224 2.13/2.93 8971 6.95/7.47 8736 -/5.51 Larcopyle Developayella Spongodiscus 8998 0.00/10.27 8949 0.49/7.85 9142 3.81/7.62 8764 12.25/13.40 8735 -/4.56 9138 -/7.05 8904 8.70/10.87 Rhizidiomyces 9358 0.50/11.97 8738 4.66/4.97 8712 9.72/11.02 Pirsonia 9452 -/2.00 9347 -/2.97 Haramonas 9011 0.00/0.00 Pelagomonas Pedinella 9000 0.66/2.65 9139 0.16/4.13 Pseudo-nitzschia Telonema 8704 2.33/2.50 9008 0.31/1.10 Bolidomonas

9088 -/4.76 9136 -/13.59 ECOLOGY Chroomonas Paraphysomonas Diaphanoeca 8915 3.88/4.37 8776 3.10/4.99 Ochromonas 9069 -/4.90 9368 -/5.99 9050 0.82/0.98 8960 -/0.33 Spumella Strombidium 8730 -/8.82 Hemigastrostyla 9145 -/12.22 8917 2.78/19.91 8893 -/0.49 9051 -/8.89 Gonostomum 8913 -/11.16 Paruroleptus 9003 -/10.63 9362 -/4.91 Chrysochromulina 8749 0.50/3.17 Parastrombidinopsis 8775 -/14.33 8947 2.09/7.89 8709 7.10/7.42 9458 2.91/3.21 9119 14.13/16.67 Holosticha Perkinsus 9445 1.80/2.00 8997 -/7.02 9395 -/1.42 8945 7.27/8.55 Diophrys 8884 7.96/10.43 9450 -/6.00 9115 5.86/8.71 8990 10.54/11.64 8774 -/5.92 9394 5.49/8.89 8759 -/8.70 9376 6.03/8.41 9097 -/7.32 8750 -/16.01 8975 -/16.61 9022 -/14.77 Arcuospathidium 8943 17.73/18.63 87698718 8.22/8.79 -/8.66 8931 13.55/14.05 9120 12.29/12.64 9392 -/14.33 8986 7.00/7.00 8988 -/14.65 9122Miamiensis 3.08/4.97 8925 16.80/18.01 9117 24.39/27.58 9425 -/13.94 Cytauxzoon 8777 -/17.76 Dexitrichides 8896 5.33/5.49 Scrippsiella Pentapharsodinium 9441 10.24/13.56 8980 3.92/3.92 8970 -/14.33 8705 6.21/13.09 9437 0.00/0.63 9382 -/4.85 9168 1.43/8.47 8772 4.76/5.91 8962 0.32/11.66 9370 3.14/9.95 8937 5.96/12.72 8966 0.00/12.68 Cryptocaryon Duboscquella 8928 0.69/9.23 8899 0.81/11.97 Ichthyodinium 9459 4.84/12.64 9121 8.73/9.76 Bryometopus Ephelota 8956 0.64/8.08 8930 0.32/6.76 8931 0.70/17.17

8942 8.90/14.03 Amoebophrya [1] 8758 0.49/1.63

9169 91646.92/17.60 3.12/8.63

8885 1.78/2.67 Orthodonella

9107 1.84/11.609127 0.17/9.98 8957 1.01/7.98 8987 2.28/8.30 8771 3.06/5.54 9078 0.00/6.81 9232 5.79/7.28

8941 6.11/8.36 8952 0.80/4.01 8989 3.31/10.72 8961 1.97/4.30

8944 7.78/10.16

Amoebophrya [2]

Fig. 2. Midpoint rooted maximum likelihood tree of all clones (general eukaryotic and group-specific primers). Because of their very long branches, the euglenozoans were removed and are displayed separately (Fig. 3). Their branching position is marked by an arrow. Bootstrap support values ≥ 70 are highlighted by black circles. Branches in red are clones; branches in black are named sequences retrieved from GenBANK. For clarity, clones were grouped into OTUs (5.21% threshold; Fig. S1), and only 1 representative clone from each OTU was included in the phylogenetic tree. These clones are identified by the last 4 numbers of their GenBANK accession numbers (GU218701-GU219463) followed by the lowest p-distance to the first BLAST hit and, after the slash, the lowest p-distance to the first named BLAST hit within each OTU. When the first BLAST hit was already a named sequence, the first value was omitted. Bar charts represent the number of clones per abyssal plain obtained for each OTU; pie charts represent the relative number of clones obtained from each abyssal plain for the major phylogenetic groups. Sequences in bold represent the nearest named neighbor for each OTU. Phylogenetic branches are extended toward their labels by gray dotted lines and by red dotted lines if the lowest p-distance to the first named BLAST hit within the respective OTU was > 5.21%. These branches are also highlighted by long dashes if the lowest p-distance to the first BLAST hit was already > 5.21%. Species names and GenBANK accession numbers of the reference sequences are listed in Table S6.

Scheckenbach et al. PNAS Early Edition | 3of6 Downloaded by guest on October 4, 2021 0.1 FST distance Legend (bar and pie charts): 0.05 0.10 0.15 0.20 0.25 0.30 0 Cape Abyssal Plain Angola Abyssal Plain Drake Passage (López-García et al. 2001) Eastern Guinea Abyssal Plain Sargasso Sea (Not et al. 2007) * Western Guinea Abyssal Plain 10 clones (bar charts) Beaufort Sea (Terrado et al. 2009)§ Guaymas Basin (Edgcomb et al. 2007 [corer A]) * Color ranges (phylogeny): Caraico Basin (Stoeck et al. 2003) Kinetoplastea * Diplonemea Guaymas Basin (Edgcomb et al. 2007 [corer C]) * Guaymas Basin (Edgcomb et al. 2007 [surface of Corer C]) P *

8855 28.03/29.32

9089 -/27.72 8860 -/22.19 Diplonema [1] 8886 5.57/14.75 8929 6.51/17.12 Rhynchopus Diplonema [2] 9284 -/10.74 9282 -/8.86 Cape Abyssal Plain 9276 -/11.32 9202 -/8.199230 -/15.09 9280 13.90/14.26 8880 4.53/15.51 8906 1.42/16.22 Lost City hydrothermal field (López-García et al. 2007) 8948 6.75/14.93 * 8892 3.58/12.09 8981 5.46/15.70 Rainbow hydrothermal field (López-García et al. 2007) 8965 4.20/11.17 8977 2.69/12.00 * 9131 6.77/16.29 Sagami Through subduction zone (Takishita et al. 2007) 9187 26.49/29.80 (F) * 9114 12.09/25.89 9081 19.33/22.77 Western Greenland (Stoeck et al. 2007)§ 9144 4.64/15.46 Ichthyobodo [1] Ichthyobodo [2] Angola Abyssal Plain 9158 5.53/9.73 9156 5.30/11.11 8702 14.92/17.93 Western Guinea Abyssal Plain F/U/(P) 8784 18.43/23.22 Rhynchobodo Eastern Guinea Abyssal Plain 9160 -/12.94 8719 18.25/18.68 * 9167 -/18.38 Sargasso Sea (Countway et al. 2007 [station 1]) Rhynchomonas 9381 -/0.00 Sargasso Sea (Countway et al. 2007 [station 2]) (U)/(P) 8701 8.48/8.75 Neobodo [1] Sargasso Sea (Countway et al. 2007 [station 3]) 8703 9.85/11.36 9165 16.35/17.31 8715 7.86/8.81 Fig. 4. Dendrogram of pairwise FST values. Clone libraries of microbial eu- Neobodo [2] karyotes from depths ≥ 750 m constructed using general eukaryotic primers 9233 -/26.08 Neobodo [3] were compared. Clone libraries from this study are highlighted in bold. For 8721 12.53/16.17 comparision, two clone libraries from non–deep-sea environments were in- 8716 5.40/21.44 9159 6.02/21.62 cluded in the analysis (denoted by §). Significant P values for FST, UniFrac, and P-tests are indicated by an asterisk (P < .01). Nonsignificant P values are Fig. 3. Maximum likelihood tree of Euglenozoa. For more details, see Fig. 2. indicated by bold lines, and the respective tests are denoted by F (FST test), U (UniFrac test), and P (P-test). When only some pairwise tests within a cluster were insignificant, the respective tests are given in parentheses. have not yet been taxonomically studied. This was an unexpected finding for the euglenozoan group Kinetoplastea, because this group has been studied in detail. This supports the idea of a evaluating this missed diversity. This applies particularly to specific benthic deep-sea community of euglenozoans and is in phylogenetic groups that have been underreported in environ- agreement with the fact that most nonparasitic diplonemids and mental sequencing surveys (e.g., amoebozoans and foraminifer- kinetoplastids have been described as typical benthic species, and ans). At present, we cannot provide any reliable estimates of this some diplonemid lineages have been hypothesized to preferen- missing diversity, however (31). tially inhabit the deep oceans (24). Removing euglenozoans from the analysis did lower the mean High Diversity of Parasites. The presence of many lineages and p-distance between all clones and their first BLAST hits, al- clades associated with parasitic species or groups, such as the though the mean values (Fig. S4) were still higher than those Apicomplexa, emphasizes the importance of parasitism in reported from most other deep-sea habitats. This contradicts the aquatic (in particular, marine and deep-sea) environments assumption that the abyss is simply a “sink habitat” (25), as has reported by many recent molecular surveys (e.g., 32). Never- been suggested for some metazoans, and indicates that the di- theless, the putative parasitic diversity (in both phylogenetic di- versity of benthic, deep-sea microbial eukaryotic communities versity and relative clone abundance) reported herein is can be attributed only partially to the sedimentation of organ- noteworthy. Although inferring the trophic role of an organism isms from the pelagial. by its sequence is difficult, parasitism appears to be a major force shaping microbial community structure at abyssal depths, a force “Rare Biosphere.” Most of the OTUs obtained in this study that has been underestimated in current conceptualizations of occurred at low abundance, giving the distribution of clone rel- the marine carbon cycle. Lafferty et al. (33) have concluded that ative abundances a very long right-hand tail, with rare species up to 75% of all food web links include parasites. present as singletons. This “rare biosphere” (26, 27) is a common Studies of deep-sea metazoan parasites have reported declines phenomenon in microbiology and has traditionally been thought in parasitic abundance and diversity with depth but increases to indicate the presence of a seed bank of potential new colo- close to the sea floor (34, 35). This phenomeon has been linked nizers, according to the “everything-is-everywhere” hypothesis to the increased biomass at benthic sites and thus to an increased (28). Interestingly, this is consistent with observations of rare availability of potential intermediate hosts, resulting in higher species of metazoans present in the deep sea that led to the idea parasite abundance and diversity. This also may explain the high of a high deep-sea diversity (29). These rare species challenge occurrence of microbial eukaryotic parasites at the abyssal sea the traditional concept of diversity and are likewise hypothesized floor. One possible explanation for the paradox of a species-rich to form a pool of transient immigrants (30). Several other ex- deep-sea floor, according to Snelgrove et al. (36), was accorded planations have been given to explain this rare biosphere in to the underestimation of positive species interactions in the eukaryotic microbiology (27). deep sea due to parasites (37). The diversity of microbial eukaryotes in the deep sea seems to be undersampled, as indicated by rarefaction curves. Thus, Large-Scale Patterns in Biodiversity. Communities of the Angola future studies may reveal many additional new phylotypes. and the Guinea Abyssal Plains were relatively similar and dif- Group-specific clone libraries seem to be especially useful for fered from the community of the Cape Abyssal Plain. The

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.0908816106 Scheckenbach et al. Downloaded by guest on October 4, 2021 community structure of microbial eukaryotes showed little geo- The high similarity of communities from the Guinea and the graphic variation on large spatial scales encompassing different Angola Abyssal Plains assume that some abyssal plains form a abyssal plains located thousands of kilometers apart. Although vast interconnected environment. Thus, communities of micro- protistan biogeography is controversial (e.g., refs. 28 and 38) and bial eukaryotes might not be shaped primarily by geographical no consensus has been reached, it is well known that at least distance. The differences in the ecological conditions between some deep-sea microbial eukaryotes, including several foramin- our sampling regions suggest that ecological parameters might be iferan species (19), exist with large distribution ranges at benthic the decisive factors in shaping deep-sea communities of micro- abyssal sites, similar to some deep-sea metazoans. Allen and bial eukaryotes on larger scales. It must be noted, however, that Sanders (39) noted that 50% of North Atlantic bivalves have we sampled only 4 regions, and further sampling might challenge geographic ranges encompassing entire abyssal plains, a finding our findings. echoed by Rex et al. (25) for gastropods. A recent study (13) reported similar community compositions of microbial eukar- Conclusion yotes on a regional scale in the abyssopelagial zone of the Our findings challenge the concept of lower biodiversity on the northwestern Atlantic Ocean. At abyssal depths, the perceived abyssal sea floor than in other marine environments. The high homogeneity of sea floor habitats thus appears to be paralleled percentage of lineages with no close representatives in genetic by the homogeneity of their communities. However, by pooling databases points to rich, and to some extent specific, commun- the DNA from each region, we were not able to capture the local ities of microbial eukaryotes at the abyssal sea floor with a heterogeneity possibly present at each sampling region as re- potentially high percentage of parasites. ported by other deep-sea studies (10, 21). The abyssal sea floor appears to be a contiguous habitat for In the present study, the numerically dominant OTUs were microbial eukaryotes on regional scales. Nevertheless, the sig- generally present at all 4 sampling regions, in contrast to many nificant differences between the Cape Abyssal Plain and the rare OTUs (deducible from bar charts of Figs. 2 and 3 and Fig. other 2 abyssal plains studied suggest that ecological parameters S2). It is generally assumed that the dominant taxa contribute the might be the decisive factors in shaping microbial eukaryote most to an ecosystem’s function and consequently best describe distribution patterns and zonation on large spatial scales at fl that ecosystem. According to this assumption, different envi- abyssal depths. The abyssal sea oor seems to be a mosaic of ronments with a high overlap of the dominant species should semi-isolated habitats, shaped and maintained on larger scales by contribute similarly to ecosystem processes. There is an ongoing diverse environmental gradients. ECOLOGY debate regarding the role of rare species in ecosystem func- Investigating the diversity and distribution of natural microbial ’ tioning, however (e.g., ref. 27). If this rare biosphere turns out to communities in Earth s largest habitat is critical to our under- play an important role, then abundance-based statistics (e.g., standing of global biogeochemical cycles. Unique techniques Bray–Curtis similarity indices) might not adequately describe the (27) and large-scale studies (45), as well as long-term surveys/ underlying communities. Nevertheless, all statistics showed high time series (46), may further elucidate the diverse composition of similarity between the Angola and Guinea Abyssal Plains. Con- deep-sea communities over both space and time (43). sequently, although our sampling was far from exhaustive, and Materials and Methods many more especially rare taxa might be present at the sampling sites, virtually identical communities over large spatial scales, as Sampling Site. Sampling regions, depths, and sample volumes were the western Guinea [Fig. 1 (1); 0° 50’ N5°35’ W; 5,136–5,142 m], eastern Guinea reported here, indicate the prevailing homogeneous environ- [Fig. 1 (2); 0° 0’ S2°25’ W; 5,060–5,066 m), Angola [Fig. 1 (3); 9° 56’ S0°54’ E; fl mental conditions on the abyssal sea oor. Thus, the differences 5,646–5,655 m], and Cape Abyssal Plains (Fig. 1 (4); 28° 7’ S7°21’ E; 5,033– in community structure between the Cape Abyssal Plain and the 5,038 m]. Samples were taken with a multicorer. Each region was sampled by other 2 abyssal plains studied underscore the importance of 3 multicorers; thus, each sampling region consisted of several cores located ecological parameters in shaping community composition. This several kilometers apart. From each multicorer, water from several corers finding is in accordance with, for example, studies of eukaryotic was obtained and directly filtered over polycarbonate filters with a pore size μ picoplankton from marine surface waters of the Southern Ocean of 0.2 m (GTPB; Millipore) and then stored in lysis buffer (47) at -20°C until showing similar community structure over long geographic further treatment. All materials were autoclaved before use. distances and changes between different hydrographic areas fi fi fi DNA Extraction, Puri cation, Cloning and Sequencing of PCR-Ampli ed SSU (40). This might also be supported by the signi cant differences rDNA. Genomic DNA was extracted from the filters using a general phenol/ between several deep-sea clone libraries from different habitats, chloroform/CTAB extraction protocol (47) and further purified by Sepharose as shown in Fig. 4. Keep in mind, however, that we cannot state 4B/PVPP columns (48). Genomic DNA from the same sampling region was with confidence to what extent these differences are the result of pooled together. SSU rDNA was amplified with FastStart Taq DNA poly- PCR biases or represent true differences between the com- merase (Roche Applied Science) under standard conditions as specified by munities (41). the manufacturer. Amplified gene fragments were cloned (StrataClone PCR Studies of metazoans and foraminiferans suggest that 2 of the Cloning Kit; Stratagene) and sequenced (positions 500–1,300) in both di- most important factors controlling deep-sea rections (BigDye Terminator v3.1 Cycle Sequencing Kit; Applied Biosystems) following the manufacturers’ protocols. The primers used are listed in Table are water quality and food supply (42, 43). At the water surface, fl S5 From each sampling region, clone libraries were constructed with general the Angola and Guinea Abyssal Plains lie within the in uence of eukaryotic primers, kinetoplastid-specific primers, cercozoan-specific pri- the South Equatorial Current, whereas the sampling stations at mers, and heterokont-specific primers. the Cape Abyssal Plain are under the influence of the cold, less- productive Benguela Oceanic Current (44). At the sea floor, the Phylogenetic Analysis. The obtained sequences were corrected and assembled Angola and Guinea Abyssal Plains are filled with North Atlantic manually. Chimeric sequences were detected using the Check Chimera (49) Deep Water, modified by injections of Antarctic Bottom Water program and by comparing each clone with its first BLAST hit, both manually through the Mid-Atlantic Ridge System, whereas the Cape and with the Pintail program (50). Multiple alignments for phylogeny were Abyssal Plain is filled with Lower Circumpolar Deep Water (also calculated using SINA Webaligner (22) and phylogenetic analysis with RAxML v7.0.4 (51) under the GTRCAT model with 1,000 bootstrap replicates. referred to as Antarctic Bottom Water), which has lower tem- Phylogenetic trees were drawn using iTOL (52). Representative clones for perature, salinity, and dissolved oxygen (44). All 3 abyssal plains Figs. 2 and 3 were chosen as described in SI Materials and Methods. are furthermore separated by geographic barriers, namely the Guinea Fracture Zone in the North and the Walvis Ridge in the Statistical Analysis. P-distance matrixes were built from pairwise p-distances South (Fig. 1). inferred from pairwise alignments for each possible combination using

Scheckenbach et al. PNAS Early Edition | 5of6 Downloaded by guest on October 4, 2021 ClustalW2 v2.0.10 (53) and distmat (54). Based on these matrixes, pairwise ACKNOWLEDGMENTS. We thank Michael Türkay, Pedro Martínez Arbizu, fi statistics were calculated with Arlequin v3.11 (FST tests) (55), UniFrac (un- Kai George, and the captain, the crew, and the scienti c crew of R / V Meteor weighted UniFrac and P-tests) (56), DOTUR v1.53 (OTU assignment, rar- cruise M63.2 (expedition DIVA2). We also thank Michael Melkonian and Karl-Heinz Linne von Berg for providing laboratory facilities, and Fred Bar- efaction curves,R and richness estimations) (57), SONS v1.0 (OTU-based tlett for helping with the English editing. This work was part of the Census of statistics) (58), -LIBSHUFF v1.22 (59), and PRIMER v6 (PRIMER-E Ltd; OTU- the Diversity of Abyssal Marine Life (CeDAMar) and was supported by the based SIMPROF tests). German Research Foundation (DFG AR 288/10-1,2).

1. Gage JD, Tyler PA (1991) In Deep-Sea Biology: A Natural History of Organisms at the 30. Magurran AE, Henderson PA (2003) Explaining the excess of rare species in natural Deep-Sea Floor, eds Gage JD, Tyler PA (Cambridge Univ Press, Cambridge, UK). species abundance distributions. Nature 422:714–716. 2. Hendrickson JA (1979) In Ecological Diversity in Theory and Practice, eds Grassle JF, 31. Pedrós-Alió C (2006) Marine microbial diversity: Can it be determined? Trends Patil GP, Smith WK, Taille C (International Cooperative Publishing House, Burtonsville, Microbiol 14:257–263. MD), Vol 6, pp 145–158. 32. Moreira D, López-García P (2003) Are hydrothermal vents oases for parasitic protists? 3. Gooday AJ (1999) Biodiversity of foraminifera and other protists in the deep sea: Trends Parasitol 19:556–558. Scales and patterns. Belg J Zool 129:61–80. 33. Lafferty KD, Dobson AP, Kuris AM (2006) Parasites dominate food web links. Proc Natl 4. Arndt H, Hausmann K, Wolf M (2003) Deep-sea heterotrophic nanoflagellates of the Acad Sci USA 103:11211–11216. eastern Mediterranean Sea: Qualitative and quantitative aspects of their pelagic and 34. Marcogliese DJ (2002) Food webs and the transmission of parasites to marine fish. benthic occurrence. Mar Ecol Prog Ser 256:45–56. Parasitology 124 (Suppl):S83–S99. 5. Scheckenbach F, Wylezich C, Mylnikov AP, Weitere M, Arndt H (2006) Molecular 35. Klimpel S, Palm HW, Busch MW, Kellermanns E, Rückert S (2006) Fish parasites in the comparisons of freshwater and marine isolates of the same morphospecies of deep-sea: Poor diversity in pelagic fish species vs. heavy parasite load in a heterotrophic flagellates. Appl Environ Microbiol 72:6638–6643. demersal fish. Deep-Sea Res Pt I 53:1167–1181. 6. DeLong EF, Karl DM (2005) Genomic perspectives in microbial oceanography. Nature 36. Snelgrove PVR, Smith CR (2002) A riot of species in an environmental calm: The 437:336–342. paradox of the species-rich deep sea. In Oceanography and Marine Biology vol. 40, 7. López-García P, Rodríguez-Valera F, Pedrós-Alió C, Moreira D (2001) Unexpected eds Gibson RN, Barnes M, Atkinson RJA (Taylor & Francis, London and New york), pp diversity of small eukaryotes in deep-sea Antarctic . Nature 409:603–607. 311–342. 8. López-García P, Philippe H, Gail F, Moreira D (2003) Autochthonous eukaryotic 37. Danovaro R, et al. (2008) Exponential decline of deep-sea ecosystem functioning diversity in hydrothermal sediment and experimental microcolonizers at the Mid- linked to benthic biodiversity loss. Curr Biol 18:1–8. Atlantic Ridge. Proc Natl Acad Sci USA 100:697–702. 38. Foissner W (2006) Biogeography and dispersal of : A review 9. López-García P, Vereshchaka A, Moreira D (2007) Eukaryotic diversity associated with emphasizing protists. Acta Protozool 45:111–136. carbonates and fluid–seawater interface in Lost City hydrothermal field. Environ 39. Allen JA, Sanders HL (1996) The zoogeography, diversity and origin of the deep-sea Microbiol 9:546–554. protobranch bivalves of the Atlantic: The epilogue. Prog Oceanogr 38:95–153. 10. Edgcomb VP, Kysela DT, Teske A, de Vera Gomez A, Sogin ML (2002) Benthic 40. Díez B, Massana R, Estrada M, Pedrós-Alió C (2004) Distribution of eukaryotic eukaryotic diversity in the Guaymas Basin hydrothermal vent environment. Proc Natl picoplankton assemblages across hydrographic fronts in the Southern Ocean, studied Acad Sci USA 99:7658–7662. by denaturing gradient gel electrophoresis. Limnol Oceanogr 49:1022–1034. 11. Edgcomb V, et al. (2009) Protistan community patterns within the brine and halocline 41. Potvin M, Lovejoy C (2009) PCR-based diversity estimates of artificial and of deep hypersaline anoxic basins in the eastern Mediterranean Sea. Extremophiles environmental 18S rRNA gene libraries. J Eukaryot Microbiol 56:174-181. 13:151–167. 42. Etter RJ, Mullineaux L (2000) Deep-sea communities. In Marine Community Ecology, 12. Stoeck T, Taylor GT, Epstein SS (2003) Novel eukaryotes from the permanently anoxic eds Bertness MD, Gaines S, Hay M (Sinauer, Sunderland, MA), pp 367–393. Cariaco Basin (Caribbean Sea). Appl Environ Microbiol 69:5656–5663. 43. Smith CR, De Leo FC, Bernardino AF, Sweetman AK, Arbizu PM (2008) Abyssal food 13. Countway PD, et al. (2007) Distinct protistan assemblages characterize the euphotic limitation, ecosystem structure and climate change. Trends Ecol Evol 23:518–528. zone and deep sea (2500 m) of the western North Atlantic (Sargasso Sea and Gulf 44. World Atlas. (2005) Available at http://www.nodc.noaa.gov/OC5/WOA05/pr_woa05. Stream). Environ Microbiol 9:1219–1232. html. Accessed 2009. 14. Not F, Gausling R, Azam F, Heidelberg JF, Worden AZ (2007) Vertical distribution of 45. Witman JD, Etter RJ, Smith F (2004) The relationship between regional and local picoeukaryotic diversity in the Sargasso Sea. Environ Microbiol 9:1233–1252. species diversity in marine benthic communities: A global perspective. Proc Natl Acad 15. Takishita K, Yubuki N, Kakizoe N, Inagaki Y, Maruyama T (2007) Diversity of microbial Sci USA 101:15664–15669. eukaryotes in sediment at a deep-sea methane cold seep: Surveys of ribosomal DNA 46. Ruhl HA, Ellena JA, Smith KL, Jr (2008) Connections between climate, food limitation, libraries from raw sediment samples and two enrichment cultures. Extremophiles 11: and carbon cycling in abyssal sediment communities. Proc Natl Acad Sci USA 105: 563–576. 17006–17011. 16. Alexander E, et al. (2009) Microbial eukaryotes in the hypersaline anoxic L’Atalante 47. Clark CG (1992) DNA purification from polysaccharide-rich cells. In Protocols in deep-sea basin. Environ Microbiol 11:360–381. Protozoology 1, eds Lee JJ, Soldo AT (Allen Press, Lawrence, KS), pp D-3.1–D-3.2. 17. Jørgensen BB, Boetius A (2007) Feast and famine: Microbial life in the deep-sea bed. 48. Edel-Hermann V, Dreumont C, Pérez-Piqueres A, Steinberg C (2004) Terminal Nat Rev Microbiol 5:770–781. restriction fragment length polymorphism analysis of ribosomal RNA genes to assess 18. Azam F, Malfatti F (2007) Microbial structuring of marine ecosystems. Nat Rev changes in fungal community structure in soils. FEMS Microbiol Ecol 47:397–404. Microbiol 5:782–791. 49. Maidak BL, et al. (1994) The ribosomal database project. Nucleic Acids Res 22: 19. Pawlowski J, et al. (2007) Bipolar gene flow in deep-sea benthic foraminifera. Mol 3485–3487. Ecol 16:4089–4096. 50. Ashelford KE, Chuzhanova NA, Fry JC, Jones AJ, Weightman AJ (2005) At least 1 in 20 20. Levin LA, et al. (2001) Environmental influences on regional deep-sea species 16S rRNA sequence records currently held in public repositories is estimated to diversity. Annu Rev Ecol Syst 32:51–93. contain substantial anomalies. Appl Environ Microbiol 71:7724–7736. 21. Huber JA, et al. (2007) Microbial population structures in the deep marine biosphere. 51. Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic Science 318:97–100. analyses with thousands of taxa and mixed models. Bioinformatics 22:2688–2690. 22. Pruesse E, et al. (2007) SILVA: A comprehensive online resource for quality-checked 52. Letunic I, Bork P (2007) Interactive Tree Of Life (iTOL): An online tool for phylogenetic and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res 35: tree display and annotation. Bioinformatics 23:127–128. 7188–7196. 53. Larkin MA, et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23: 23. Bass D, Cavalier-Smith T (2004) Phylum-specific environmental DNA analysis reveals 2947–2948. remarkably high global biodiversity of Cercozoa (Protozoa). Int J Syst Evol Microbiol 54. Rice P, Longden I, Bleasby A (2000) EMBOSS: The European Molecular Biology Open 54:2393–2404. Software Suite. Trends Genet 16:276–277. 24. Lara E, Moreira D, Vereshchaka A, López-García P (2009) Pan-oceanic distribution of 55. Excoffier L, Laval G, Schneider S (2005) Arlequin (version 3.0): An integrated software new highly diverse clades of deep-sea diplonemids. Environ Microbiol 11:47–55. package for population genetics data analysis. Evol Bioinform Online 1:47–50. 25. Rex MA, et al. (2005) A source-sink hypothesis for abyssal biodiversity. Am Nat 165: 56. Lozupone C, Hamady M, Knight R (2006) UniFrac: An online tool for comparing 163–178. microbial community diversity in a phylogenetic context. BMC Bioinformatics 7:371. 26. Sogin ML, et al. (2006) Microbial diversity in the deep sea and the underexplored 57. Schloss PD, Handelsman J (2005) Introducing DOTUR, a computer program for “rare biosphere.” Proc Natl Acad Sci USA 103:12115–12120. defining operational taxonomic units and estimating species richness. Appl Environ 27. Caron DA (2009) New accomplishments and approaches for assessing protistan Microbiol 71:1501–1506. diversity and ecology in natural ecosystems. Bioscience 59:287–299. 58. Schloss PD, Handelsman J (2006) Introducing SONS, a tool for operational taxonomic 28. Finlay BJ (2002) Global dispersal of free-living microbial eukaryote species. Science unit-based comparisons of microbial community memberships and structures. Appl 296:1061–1063. Environ Microbiol 72:6773–6779. 29. Grassle JF, Maciolek NJ (1992) Deep-sea species richness: Regional and local diversity 59. Schloss PD, Larget BR, Handelsman J (2004) Integration of microbial ecology and estimates from quantitative bottom samples. Am Nat 139:313–341. statistics: A test to compare gene libraries. Appl Environ Microbiol 70:5485–5492.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.0908816106 Scheckenbach et al. Downloaded by guest on October 4, 2021