Biodiversity and biogeography of Radiolaria in the world oceans through metabarcoding 

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Comparative molecular phylogeny and morphological taxonomy

Final molecular phylogeny is composed of 90 distinct nassellarian specimens generated by 61 sequences of the 18S rDNA gene and of 57 sequences of the partial 28S (D1 & D2 regions) rDNA gene (Supplementary Material Table S2). From the 61 final sequences of the 18S rDNA gene, 38 were obtained in this study and 23 were previously available, of which 16 have been morphologically identified and 7 are environmental. Regarding the partial 28S rDNA gene, 55 new sequences were obtained in this study and 2 were previously available and morphologically described. The final alignment matrix has 32.4% of invariant sites, and in total includes 67 new sequences. All of these specimens cover 7 superfamilies (Acanthodesmoidea, Acropyramioidea, Artostrobioidea, Cannobotryoidea, Eucyrtidioidea, Plagiacanthoidea, Pterocorythoidea, and three undefined families), based on morphological observations performed with light microscopy (LM; Supplementary Material Fig. S2), scanning electron microscopy (SEM) and/or confocal microscopy (CM) on the exact same specimens for which we obtain a sequence. Overall, molecular phylogeny is consistent with morphological classification at the superfamily level, although there are some specific discrepancies. The phylogenetic analysis shows 11 clades (Fig. 1) clearly differentiated with high values of ML bootstrap (BS > 99) and posterior probabilities (PP > 0.86).
Clade A holds the most basal position with 16 sequences of which 10 are novel sequences, 5 were previously available and one is environmental. All specimens clustering within this clade (Fig. 2.A, Fig. 3.A) have a simple and round cephalis, an apical horn, a small ventral rod and multisegmented (cephalis, thorax and several abdomen) skeleton with distinctive inner rings, that correspond to the Superfamily Eucyrtidioidea. All multisegmented nassellarians with spherical cephalis encountered in our study belong to this superfamily, both morphologically and phylogenetically. The rest of the clades group together with a high BS (100) and PP (1) values. Thereafter clades B, C, D and E cluster together in lineage II. Clades X, F and G constitute the lineage III, highly supported (100 BS and 1 PP) as sister group of the lineage IV. This last lineage it is composed by the clades H, as the basal group and clades I and J highly related phylogenetically (100 BS and 1 PP).
Within lineage II, clade B is represented by only one sequence from this study (Osh128), and its morphology matches with the superfamily Acropyramioidea (Fig. 2.B, Fig. 3.B) exhibiting a pyramidal skeleton constituted of a reduced cephalis and thorax. Clade C is composed by two novel sequences (Fig. 2.C, Fig. 3.C). Their overall round morphology and a characteristic small and flat cephalis not well distinguished from the thorax agrees with the undetermined family Carpocaniidae (Petrushevskaya, 1971a; De Wever et al., 2001). Its sister clade, the Clade D is constituted by three new (Ses58, Mge17-70 and Mge17-9) and one environmental sequences, and the morphology of these specimens (Fig. 2.D, Fig. 3.D) agrees with the definition of the Superfamily Artostrobioidea. They share a multisegmented skeleton without significant inner rings, a hemispherical cephalis and an important ventral rod. The last clade of the lineage II, clade E, gathers four new sequences, one and two environmental sequences. This clade includes all the monocyrtid (cephalis) nassellarians where the spines A and V are merged forming the so-called D-ring (Acanthodesmoidea). There are representatives for two out of three families (Stephaniidae is the missing family), yet no phylogenetic differences were found for the included families, Acanthodesmiidae (Fig. 2.E1, Fig. 3.E1) and Triospyrididae (Fig. 2.E2-E4, Fig. 3.E2).
In lineage III, clades X and F are highly supported as sister clades (100 BS and 1 PP). Clade X is established by two novel sequences morphologically identified as Archipilium johannismonicae (Fig. 2.X1, Fig. 3.X1) and Enneaphormis enneastrum (Fig. 2.X2, Fig. 3.X2). These two specimens have a very short or missing median bar (MB) allowing the development of three large feet with a three-pointed star (dorsal and lateral, left and right, rays) shape forming a significant circular frame where they build the thorax when present. In Clade F there are two novel sequences (Ses59 and Mge17-94) and a previously available sequence. These specimens are morphologically identified in the genus Eucecryphalus (Fig. 2.F, Fig. 3.F) and a third specimen as Tricolocamptra (AB246685). The former genus belongs to the undetermined family Theopiliidae (De Wever et al., 2001; Matsuzaki et al., 2015) with a hat shaped morphology and pronounced segmentation. The genus Eucecryphalus, the senior synonym of Theopilium, is the type genus of the family Theopiliidae (Matsuzaki et al., 2015), and thus Clade F is automatically specified as the “true” Theopiliidae. The taxonomic position of the second genus, Tricolocamptra, varies through references and the available image for the specimen lacks taxonomical resolution, therefore it is not possible to establish the link with any described morphological group.

Molecular dating

The molecular clock dated the diversification between Spumellaria and Nassellaria (the root of the tree) with a median value of 512 Ma (95% Highest Posterior Density -HPD-: between 600 and 426 Ma) (Fig. 4). From now on, all dates are expressed as median values followed by the 95% HPD interval. Despite the large uniform distribution given to the ingroup (U [180, 500]), the first diversification of Nassellaria was settled at 423 (500-342) Ma. Clade A had its first radiation dated in 245 (264-225) Ma. The common ancestor to all the other clades diversified at around 340 (419-271) Ma into two main groups, the so-called lineage II and the lineages III-IV splitting into two other lineages soon afterwards. Within lineage II, the first diversification occurs at 276 (354-209) Ma, and clades C and D diversified 197 (250-160) Ma. Thereafter in this lineage the phylogenetic relationships are dubious, however clade D diversified from any other clade 248 (315-191) Ma. Diversification within these clades was 28 (49-4) Ma for clade C, 175 (194-155) Ma for clade D and 77 (95-60) Ma for clade E. The lineages III and IV diversified from each other 274 (344-215) Ma. Lineage III rapidly diversified 243 (304-197) Ma when clades F and X split from clade G, followed by the fast diversification of clade G 196 (241-170) Ma. It was 168 (243-76) Ma when clade F separate of clade X, and 86 (157-32) Ma and 40 (106- 4) Ma when they respectively diversified. The last lineage (IV) diversified between clades H, I and J 206 (287-129) Ma, and clades I and J 138 (206-88) Ma. Despite this early divergence between clades, radiation within clades was more recent, being 87 (171-26) Ma, 70 (87-53) Ma and 73 (90-58) Ma for clades H, I and J, respectively.

Post-hoc analyses

The lineages through time analysis (Fig. 5) shows a classic exponential diversification slope, with a 0.0109 rate of speciation (Ln(lineages)·million years-1). The first diversification of extant Nassellaria (~423 Ma) corresponds to the divergence between clade A and the rest of the clades. However, the first increase in the slope (up to: 0.017, t-test: p<0.001) occurs at ~275 Ma, when the main lineages start expanding; lineage II splits between clade B and clades C, D and E, clade A diversifies and the evolutionary lineages III and IV diverged from each other followed by the rapid diversification of lineage III. After this sudden increase of the main lineages, a second diversification event happened (ca. 198 Ma) where both the evolutionary lineage IV and the clade G diversify and clade C splits from clade D. Thereafter, the diversification seems to be stepped and separate in different periods of time, were only lineage III keep diversifying. The last and relatively uninterrupted diversification occurred ~82 Ma corresponding to the speciation within the already present clades and the first diversification of the clades H, F, E, J, I, X and C.

Environmental genetic diversity of Nassellaria

A total of 229 18S rDNA and two 28S rDNA environmental sequences affiliated to Nassellaria were retrieved from public database and placed in our reference phylogenetic tree (Fig. 7; Supplementary Material Table S3). Most of the environmental sequences (151) belonged to clade G, while 41 other sequences were scattered between clades A, D, E, F, I and J (2, 6, 26, 1, 2 and 4 sequences respectively). The rest of the sequences could not be placed within any existing clade. From those, 28 were closely related to clade E, and the others at basal nodes mainly over lineage II. These 37 sequences were then included in a phylogenetic tree (with a GTR+G+I model, 4 invariant sites and 1000 bootstraps) and 3 new and highly supported clades appeared (Supplementary Material Fig. S3). The first clade, constituted by 31 sequences, was assigned to Collodaria in Biard et al. (2015). A second clade formed by 2 sequences (BS: 94) was highly related to clades B and C (BS: 96). And the last clade, constituted as well of 2 sequences (BS: 100) was basal to the node formed by clades C, D and the previous mentioned environmental clade (BS: 58). The 2 remaining sequences were highly related to clades I (BS: 84) and to the node comprising clades I and J (BS: 100). Finally, 2 sequences were aligned within clade D and the last sequence was closely related to clade B, C and D. Regarding the sequences blasted for the partial 28S matrix, 2 sequences were extracted and mapped within clades E and G respectively.


Morpho-molecular classification of living Nassellaria

In our phylogenetic classification the overall morphology of Nassellaria, rather than the initial spicular system, is the most accurate feature to differentiate clades at the Superfamily level (Supplementary Material Fig. S1). We could not find any pattern in the initial spicular system that enables to separate clades, suggesting that the complexity of the initial spicular system is not related to Nassellaria phylogeny (e.g., Acanthodesmoidea, Plagiacanthoidea). Yet, this morphological character has been used to discriminate families and higher taxonomical levels (Petrushevskaya, 1971b). As well, the arches connecting these spicules were used in previous classifications and, in some cases, to discern at genus, and partly family levels (Petrushevskaya, 1971b; De Wever et al., 2001). In addition to our results, it has never been hypothesized the evolution of the initial spicular system complexity through the radiolarian fossil record. The use of the overall morphology in Nassellaria classification makes recognition of live cells easier under a light microscope facilitating morphology based ecological studies.
The extent Nassellaria included in our study can be divided in 11 morpho-molecular clades, grouped in four main evolutionary lineages based on phylogenetic clustering support, common morphological features and molecular dating: Eucyrtidioidea in lineage I; Acropyramioidea, Carpocaniidae, Artostrobioidea and Acanthodesmoidea in lineage II; Archipilioidea, Theopiliidae and Plagiacanthoidea in lineage III, and Cycladophora, Lychnocanoidea and Pterocorythoidea in lineage IV. The unified morpho-molecular framework here proposed reveals a partial agreement between the traditional taxonomy and the molecular classification. Such discrepancies have already been reported not only in other Radiolaria groups (Decelle et al., 2012b; Biard et al., 2015) but as well in other SAR taxa such as Foraminifera (Pawlowski and Holzmann, 2002), Phaeodaria (Nakamura et al., 2015) or Tintinnida (Bachy et al., 2012), being a common issue in protists classification (Schlegel and Meisterfeld, 2003; Caron, 2013).
Our revised morpho-molecular classification confirms the monophyly of the ancient Superfamilies Eucyrtidioidea and Acropyramioidea, represented by the clades A and B respectively, as well as the undetermined Family Carpocaniidae, the Superfamilies Artostrobioidea and Acanthodesmoidea, (clades C, D and E, respectively). The Superfamily Plagiacanthoidea is however paraphyletic, appearing in two different clades (X and G). Similarly, previously proposed families are scattered within clade G or even including the Superfamily Cannobotryoidea. Clade F and H both display an identical architecture of the initial spicular system, yet they are clustered based in the overall morphology in two different clades, the family here proposed as Theopiliidae (clade F) and Cycladophora-like specimens (clade H). As the genus Eucecryphalus (clade F) is the genus of the family Theopiliidae (see Matsuzaki et al., 2015), clade F holds the name Theopiliidae. Regarding clade H, a new family Cycladophoridae is established herein as defined in the taxonomic note. Regarding clades I and J, Matsuzaki et al. (2015) include them within the same Superfamily (Pterocorythoidea) due to the strong phylogenetic relationship reported by Krabberød et al. (2011). Yet due to the evolutionary patterns and the phylogenetic distance, these two clades should be considered as two different superfamilies, Pterocorythoidea and Lychnocanoidea (Lychnocanoidea Haeckel, 1882, sensu Kozur and Mostler, 1984). In our study the undetermined family Bekomidae was scattered within clade I, showing no phylogenetic differences. Re-examination of the cephalic structure in Lamprotripus concludes the assignation of the genus to the Superfamily Lychnocanoidea, yet intergeneric morphological differences remain still elusive. Further molecular analyses must reveal these discrepancies between morphological and molecular classification.

Evolutionary history of Nassellaria

The morphological evolution of Nassellaria is marked by their dubious appearance in the fossil record, whether it happened with the primitive nassellarian forms (in the Upper Devonian) or with the first multi-segmented nassellarians (in the Early Triassic) (De Wever et al., 2001; Suzuki and Oba, 2015). Our results showed that the first diversification of Nassellaria agrees in both time (~423 Ma; 95% HPD: 500-342 Ma; Figs. 4, 5) and reconstructed morphology (two- or multi-segmented last common ancestor) with the primitive nassellarians. Thus, the most likely scenario, proposed by Petrushevskaya (1971a) and continued by Cheng (1986), is where Nassellaria originated during the Devonian from primitive radiolarian forms and not during the Triassic. This hypothesis can explain the sudden appearance of forms in the Middle Triassic already pointed out by De Wever et al. (2003) and confirmed by our results.
The oldest known nassellarian like fossils are Popofskyellidae and Archocyrtiidae from the Devonian (Cheng, 1986). The former family shares with lineage I a multisegmented nature of the skeleton whereas the Archocyrtiidae has a unique and large cephalic segment and three long feet, derived characteristics that can be found over lineages II, III and IV. Such similarities in the morphology and the accordance of the molecular clock in the branching times with the fossil record allow us to hypothesis these two families as possible ancestors of living Nassellarians; as already suggested by Cheng (1986). Thereafter, the evolutionary connection of Popofskyellidae and lineage I is debatable, due to a fragmented fossil record along the Permian (Isakova and Nazarov, 1986; De Wever et al., 2003). Regarding lineage II, it is likely that diverged from Archocyrtiidae by reduction of the feet, though phylogenetic relationships within this lineage remain still unclear. A second morphological modification might have happened in the separation of the cephalis and thorax and the appearance of lineage IV (probable candidate is the Ultranaporidae). Therefore, Archocyrtiidae could be a direct ancestor of lineage III due the strong similarities within the members of this lineage and the Palaeozoic Archocyrtiidae.

Table of contents :

1. Unicellular plankton diversity in the marine environment 
2. Nassellaria and Spumellaria in the radiolarian context 
2.1. The fossil record
2.2. The advent of molecular phylogenetic analysis
2.3. Nassellaria, Spumellaria and the molecular clock
2.4. Distribution and biogeography
3. Challenging the species concept: morphology, DNA and the strength of teamwork 
Chapter 1 – Molecular diversity of Nassellaria and Spumellaria 
1.1 – Time calibrated morpho-molecular classification of Nassellaria (Radiolaria)
1.2 – A morpho-molecular classification of Spumellaria dated with the fossil record
Chapter 2 – Radiolaria classification and evolution, an integrative approach 
Chapter 3 – From intracellular variability to community ecology 
3.1 – Intracellular gene variability in Nassellaria and Spumellaria
3.2 – Biodiversity and biogeography of Radiolaria in the world oceans through metabarcoding
Discussion and perspectives
1. Towards an integrative classification: limitations and advantages
2. A new framework for the evolution and classification of Radiolaria
3. Approaching a better understanding of the molecular environmental diversity


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