Taxon-rich transcriptomics supports higher-level phylogeny and major evolutionary trends in Foraminifera.

Foraminifera, classified in the supergroup Rhizaria, are a common and highly diverse group of mainly marine protists. Despite their evolutionary and ecological importance, only limited genomic data (one partial genome and nine transcriptomic datasets) have been published for this group. Foraminiferal molecular phylogeny is largely based on 18S rRNA gene sequence analysis. However, due to highly variable evolutionary rates of substitution in ribosomal genes plus the existence of intragenomic variation at this locus, the relationships between and within foraminiferal classes remain uncertain. We analyze transcriptomic data from 28 species, adding 19 new species to the previously published dataset, including members of the strongly under-represented class Monothalamea. A phylogenomic reconstruction of Rhizaria, rooted with alveolates and stramenopiles, based on 199 genes and 68 species supports the monophyly of Foraminifera and their sister relationship to Polycystinea. The phylogenomic tree of Foraminifera is very similar to the 18S rRNA tree, with the paraphyletic single-chambered monothalamids giving rise to the multi-chambered Tubothalamea and Globothalamea. Within the Monothalamea, our analyses confirm the monophyly of the giant, deep-sea xenophyophores that branch within clade C and indicate the basal position of monothalamous clades D and E. The multi-chambered Globothalamea are monophyletic and comprise the paraphyletic Textulariida and monophyletic Rotaliida. Our phylogenomic analyses support major evolutionary trends of Foraminifera revealed by ribosomal phylogenies and reinforce their current higher-level classification.

Assessing SSU rDNA Barcodes in Foraminifera: A Case Study using Bolivina quadrata.

The Small Subunit Ribosomal RNA gene (SSU rDNA) is a widely used tool to reconstruct phylogenetic relationships among foraminiferal species. Recently, the highly variable regions of this gene have been proposed as DNA barcodes to identify foraminiferal species. However, the resolution of these barcodes has not been well established, yet. In this study, we evaluate four SSU rDNA hypervariable regions (37/f, 41/f, 43/e, and 45/e) as DNA barcodes to distinguish among species of the genus Bolivina, with particular emphasis on Bolivina quadrata for which ten new sequences (KY468817-KY468826) were obtained during this study. Our analyses show that a single SSU rDNA hypervariable sequence is insufficient to resolve all Bolivina species and that some regions (37/f and 41/f) are more useful than others (43/e and 45/e) to distinguish among closely related species. In addition, polymorphism analyses reveal a high degree of variability. In the context of barcoding studies, these results emphasize the need to assess the range of intraspecific variability of DNA barcodes prior to their application to identify foraminiferal species in environmental samples; our results also highlight the possibility that a longer SSU rDNA region might be required to distinguish among species belonging to the same taxonomic group (i.e. genus).

The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote small sub-unit rRNA sequences with curated taxonomy.

The interrogation of genetic markers in environmental meta-barcoding studies is currently seriously hindered by the lack of taxonomically curated reference data sets for the targeted genes. The Protist Ribosomal Reference database (PR(2), http://ssu-rrna.org/) provides a unique access to eukaryotic small sub-unit (SSU) ribosomal RNA and DNA sequences, with curated taxonomy. The database mainly consists of nuclear-encoded protistan sequences. However, metazoans, land plants, macrosporic fungi and eukaryotic organelles (mitochondrion, plastid and others) are also included because they are useful for the analysis of high-troughput sequencing data sets. Introns and putative chimeric sequences have been also carefully checked. Taxonomic assignation of sequences consists of eight unique taxonomic fields. In total, 136 866 sequences are nuclear encoded, 45 708 (36 501 mitochondrial and 9657 chloroplastic) are from organelles, the remaining being putative chimeric sequences. The website allows the users to download sequences from the entire and partial databases (including representative sequences after clustering at a given level of similarity). Different web tools also allow searches by sequence similarity. The presence of both rRNA and rDNA sequences, taking into account introns (crucial for eukaryotic sequences), a normalized eight terms ranked-taxonomy and updates of new GenBank releases were made possible by a long-term collaboration between experts in taxonomy and computer scientists.

New xenophyophores (Foraminifera, Monothalamea) from the eastern Clarion-Clipperton Zone (equatorial Pacific).

Xenophyophores are large, agglutinated foraminifera that dominate the benthic megafauna in some parts of the deep sea. Here, we describe an assemblage of largely fragmentary specimens from the Clarion-Clipperton Zone (CCZ), an area of the eastern abyssal Pacific hosting large, commercially significant deposits of polymetallic nodules. We recognised 18 morphospecies of which eight yielded DNA sequences. These include two new genera and three new species, Claraclippia seminuda gen. & sp. nov., Stereodiktyoma mollis gen. & sp. nov., and Aschemonella tani sp. nov., three that are assigned to known species, Abyssalia foliformis, Aschemonella monilis and Shinkaiya contorta, and two assigned to open nomenclature forms Abyssalia aff. foliformis and Stannophyllum aff. granularium. An additional ten forms are represented only by morphology. The following seven are placed in known genera, species and open-nomenclature forms: Aschemonella? sp., Homogammina sp., Psammina multiloculata, P. aff. multiloculata, P. aff. limbata form 1 sensu Gooday et al., 2018, P. aff. limbata form 2 sensu Gooday et al., 2018, and Stannophyllum spp. The other three could not be identified to genus level. This new collection brings the total of described and undescribed species and morphotypes from the CCZ to 27 and 70, respectively, reinforcing the already high diversity of xenophyophores known from this part of the Pacific.

Novel contributions to the molecular and morphological diversity of freshwater monothalamid foraminifera: Description of six new species.

Non-marine monothalamous foraminifera are common in freshwater and soil habitats. They comprise a poorly-known group lacking sufficient information about diversity, morphology, reproduction, distribution and ecology. Based on an integrative morphological and molecular approach we describe a new family, a new genus and six new species of freshwater monothalamids from different localities in the Netherlands and France. We establish Astroperula as a new genus of organic walled freshwater foraminifera that contains two species, Astroperula dumacki and Astroperula parvipila. Furthermore, two new agglutinated freshwater monothalamids are described and illustrated, Limnogromia leanneae and Lacogromia pawlowskii, one new organic walled, Velamentofex dujardini, and a new naked monothalamid, Haplomyxa retiforma. Additional information is provided about a special form of cell division in Claparedellus lachmannii and L. leanneae, and feeding behavior in cultures of V. dujardini. Morphological observations are added for an unidentified Limnogromia sampled from the Netherlands and two types of Lacogromia sampled from an alpine region in the French Pyrenees and a karst sinkhole in Bosnia and Herzegovina. The present study provides additional insight into the ecology, diversity and occurrence of freshwater monothalamids and emphasizes the fact that a combination of morphological and molecular methods is necessary to clearly distinguish species in this group.

Three new species of Gromia (Protista, Rhizaria) identified from the Romanian Black Sea shelf.

The protist genus Gromia was first described in 1835 by Dujardin and while gromiids are prominent in the marine environment, Gromia oviformis was, for a long time, the only valid species regularly recorded. To date, 16 species that are morphologically and/or genetically distinct have been described. While recent studies are documenting their diversity and their ecological importance, G. oviformis has been the sole gromiid species identified in the Black Sea, although unnamed Gromia species have also been recorded. We collected sediment samples from the Romanian continental shelf at varying depths (48 - 58 m) to study the morphological and genetic diversity of gromiids in this part of the Black Sea. Three new species, Gromia bugnae sp. nov., Gromia dianae sp. nov. and Gromia fabi sp. nov., were identified based on an integrative taxonomic approach, thus bringing the total described gromiid species to 19. Analysis of partial SSU rRNA gene sequences confirms that these are distinct species. Additionally, an undescribed species is represented by a sequence from the northern part of the Black Sea (Sevastopol, Kazachya Bay). The study provides further evidence of the diversity of gromiids in the Black Sea and underlines the importance of this little-known group in marginal seas.

Shared ancestry of algal symbiosis and chloroplast sequestration in foraminifera.

Foraminifera are unicellular organisms that established the most diverse algal symbioses in the marine realm. Endosymbiosis repeatedly evolved in several lineages, while some engaged in the sequestration of chloroplasts, known as kleptoplasty. So far, kleptoplasty has been documented exclusively in the rotaliid clade. Here, we report the discovery of kleptoplasty in the species Hauerina diversa that belongs to the miliolid clade. The existence of kleptoplasty in the two main clades suggests that it is more widespread than previously documented. We observed chloroplasts in clustered structures within the foraminiferal cytoplasm and confirmed their functionality. Phylogenetic analysis of 18S ribosomal RNA gene sequences showed that H. diversa branches next to symbiont-bearing Alveolinidae. This finding represents evidence of of a relationship between kleptoplastic and symbiotic foraminifera.. Analysis of ribosomal genes and metagenomics revealed that alveolinid symbionts and kleptoplasts belong to the same clade, which suggests a common ancestry.

Carterina labinea sp. nov. - A new alien foraminifer from the Southeastern Mediterranean shelf.

The foraminiferal order Carterinida is characterized by agglutinated tests consisting of calcareous spicules. Four species have so far been described from the tropical Pacific and the Caribbean Sea. We report here the first occurrence of Carterina from the Southeastern Levantine Shelf of the Mediterranean Sea. Based on molecular and morphological results, we describe Carterina labinea sp. nov., which is characterized by a trochospiral test with a conical, tapered spiral side and a concave umbilical side. The test is composed of elongate fusiform calcareous spicules. Tests have a mean diameter of 720 µm, which is twice the vertical height, and spicules vary in length and width from 112/15 µm to 73/14 µm. Our results show that the genus Carterina extends its distribution beyond tropical Seas and might be more widely distributed than previously thought. Moreover, the appearance of the new species in the study area suggests it is a new colonizer as continuous biomonitoring studies in the area show that it is absent in sediment samples taken before 2020. Our results highlight the importance of ongoing monitoring programs in high-risk basins such as the Levantine to gain more information about the biodiversity changes and improve environmental conservation of Eastern Mediterranean littoral areas progressively colonized by tropical species.

Molecular and morphological diversity of monothalamous foraminifera from South Georgia and the Falkland Islands: Description of four new species.

Based on molecular and morphological data, we describe three new genera and four new species of monothalamids from the sublittoral zone (21-250 m) in South Georgia fjords that belong to different monothalamid clades. Limaxia alba gen. nov. sp. nov. (Clade A) has an elongate, subcylindrical test, 359-688 µm long, with some detritus attached to the organic wall. Hilla argentea gen. nov. sp. nov. (Clade Y) has a cylindrical, finely agglutinated test, 535-755 µm long. Pseudoconqueria lenticularis gen. nov. sp. nov. branches separately. It has a spindle-shaped, finely agglutinated test, 280-574 µm long. Bathyallogromia olivacea sp. nov. (Clade C) has an ovate organic-walled test, 369-433 µm long. We present the first genetic data on two monothalamid species originally described from South Georgia, Hippocrepinella alba (Clade C) and Hippocrepinella hirudinea (Clade D), as well as a single sequence for C. delacai (Clade J) originally described from McMurdo Sound, Antarctica. In addition, we report nine undescribed species branching in six different monothalamid clades (A, B, BM, C, J, Y), eight of them sampled around South Georgia and one collected from the Falkland Islands near Stanley.

Morphological and molecular diversity of monothalamids (Rhizaria, Foraminifera), including two new species and a new genus, from SW Greenland.

Single-chambered (monothalamous) foraminifera are poorly known compared to their multichambered relatives. In this first study of monothalamids from Greenland, we describe one new genus and two new species belonging to different clades from the Nuuk fjord system. Nujappikia idaliae Gooday & Holzmann gen. nov. sp. nov. (Clade Y) has a bottle-shaped test terminating in a single aperture located on a short neck. The flexible wall is basically organic but with a very fine agglutinated veneer. Bathyallogromia kalaallita Gooday & Holzmann sp. nov. (Clade C) has a broadly ovate test with an organic wall and a mound-like apertural structure. It is larger and genetically distinct from the two other Bathyallogromia species, both from the Southern Ocean. A survey of the morphological diversity of monothalamids in our samples revealed 49 morphospecies, of which 19, including the two new species, yielded DNA sequences. Five were assigned to the genera Bathysiphon, (Clade BM), Micrometula. (Clade BM), Psammophaga. (Clade E), Hippocrepinella (Clade D) and Crithionina (Clade J). The remaining twelve represented unknown taxa branching in clades A, C, F, and Y and one new clade. Our results add to growing evidence that monothalamids are common and diverse in fjords and other high-latitude settings.

Mitochondrial cytochrome c oxidase subunit I (COI) metabarcoding of Foraminifera communities using taxon-specific primers.

Foraminifera are a species-rich phylum of rhizarian protists that are highly abundant in most marine environments. Molecular methods such as metabarcoding have revealed a high, yet undescribed diversity of Foraminifera. However, so far only one molecular marker, the 18S ribosomal RNA, was available for metabarcoding studies on Foraminifera. Primers that allow amplification of foraminiferal mitochondrial cytochrome oxidase I (COI) and identification of Foraminifera species were recently published. Here we test the performance of these primers for the amplification of whole foraminiferal communities, and compare their performance to that of the highly degenerate LerayXT primers, which amplify the same COI region in a wide range of eukaryotes. We applied metabarcoding to 48 samples taken along three transects spanning a North Sea beach in the Netherlands from dunes to the low tide level, and analysed both sediment samples and meiofauna samples, which contained taxa between 42 µm and 1 mm in body size obtained by decantation from sand samples. We used single-cell metabarcoding (Girard et al., 2022) to generate a COI reference library containing 32 species of Foraminifera, and used this to taxonomically annotate our community metabarcoding data. Our analyses show that the highly degenerate LerayXT primers do not amplify Foraminifera, while the Foraminifera primers are highly Foraminifera- specific, with about 90% of reads assigned to Foraminifera and amplifying taxa from all major groups, i.e., monothalamids, Globothalamea, and Tubothalamea. We identified 176 Foraminifera ASVs and found a change in Foraminifera community composition along the beach transects from high tide to low tide level, and a dominance of single-chambered monothalamid Foraminifera. Our results highlight that COI metabarcoding can be a powerful tool for assessing Foraminiferal communities.

Cenozoic climatic changes drive evolution and dispersal of coastal benthic foraminifera in the Southern Ocean.

The Antarctic coastal fauna is characterized by high endemism related to the progressive cooling of Antarctic waters and their isolation by the Antarctic Circumpolar Current. The origin of the Antarctic coastal fauna could involve either colonization from adjoining deep-sea areas or migration through the Drake Passage from sub-Antarctic areas. Here, we tested these hypotheses by comparing the morphology and genetics of benthic foraminifera collected from Antarctica, sub-Antarctic coastal settings in South Georgia, the Falkland Islands and Patagonian fjords. We analyzed four genera (Cassidulina, Globocassidulina, Cassidulinoides, Ehrenbergina) of the family Cassidulinidae that are represented by at least nine species in our samples. Focusing on the genera Globocassidulina and Cassidulinoides, our results showed that the first split between sub-Antarctic and Antarctic lineages took place during the mid-Miocene climate reorganization, probably about 20 to 17 million years ago (Ma). It was followed by a divergence between Antarctic species ~ 10 Ma, probably related to the cooling of deep water and vertical structuring of the water-column, as well as broadening and deepening of the continental shelf. The gene flow across the Drake Passage, as well as between South America and South Georgia, seems to have occurred from the Late Miocene to the Early Pliocene. It appears that climate warming during 7-5 Ma and the migration of the Polar Front breached biogeographic barriers and facilitated inter-species hybridization. The latest radiation coincided with glacial intensification (~ 2 Ma), which accelerated geographic fragmentation of populations, demographic changes, and genetic diversification in Antarctic species. Our results show that the evolution of Antarctic and sub-Antarctic coastal benthic foraminifera was linked to the tectonic and climatic history of the area, but their evolutionary response was not uniform and reflected species-specific ecological adaptations that influenced the dispersal patterns and biogeography of each species in different ways.

Broad sampling of monothalamids (Rhizaria, Foraminifera) gives further insight into diversity of non-marine Foraminifera.

Non-marine foraminifera are among the least known groups of protists and only a handful of species have been described since the 19th century. We collected one naked and five morphologically almost identical organic-walled monothalamid species from freshwater and terrestrial environments from Germany and Austria. One of the species was identified as Lieberkuehnia wageneriClaparède and Lachmann, 1859. As its original description is ambiguous and its type specimen has been lost, a neotype is proposed. We describe four new organic-walled monothalamous foraminifera and a novel Reticulomyxa species both morphologically and genetically. Analyses of molecular data of the different isolates revealed that they are distributed across six different clades. Two new genera, Claparedellus gen. nov. and Velamentofex gen. nov., and five new monothalamous families, Lacogromiidae fam. nov., Limnogromiidae fam. nov., Lieberkuehniidae fam. nov., Edaphoallogromiidae fam. nov. and Velamentofexidae fam. nov., are established.

Trichinella infection in wild animals from endemic regions of Argentina.

Natural infection with Trichinella has been described in more than 150 mammalian species. However, few reports of Trichinella infection in wild animals have come from Argentina. In this study, muscle tissue was obtained from wild animals in Argentina with the aim of evaluating the presence of Trichinella. A total of 169 muscle samples were collected to determine the presence of Trichinella larvae by artificial digestion. The 169 muscle samples originated from 12 species including 36 opossums (Didelphis albiventris), 19 armadillos (Chaetophractus villosus), 9 capybaras (Hydrocaeris hydrocaeris), 1 puma (Puma concolor), 3 grey fox (Lycalopex gymnocercus), 6 coypus (Myocastor coypus), 6 skunks (Conepatus chinga), 2 ferrets (Galictis cuja), 66 rats (Rattus norvegicus), 6 mice (Mus musculus), 12 wild boars (Sus scrofa), and 3 wild cats (Felis geoffroyi). Trichinella infection was detected in 1 puma [2 larvae per gram (LPG)], 3 wild boars (8-420 LPG), 3 armadillos (0.04-0.08 LPG), and 9 rats (0.1 to 150 LPG). Only 3 Trichinella isolates, of 1 rat and 2 wild boars from Neuquén, were identified as Trichinella spiralis by nested PCR. The presence of Trichinella infection among wild animal populations suggests a sylvatic cycle of transmission in Argentina, which can serve as a reservoir for humans and domestic animals. Further, evidence of high prevalence in rats emphasizes the need to improve pig management, mainly in small individual farms without adequate technology, to enhance the quality of feeds, and to improve veterinary services to avoid exposure of pigs to Trichinella.

Xenophyophores (Rhizaria, Foraminifera), including four new species and two new genera, from the western Clarion-Clipperton Zone (abyssal equatorial Pacific).

The Clarion-Clipperton Zone (CCZ) occupies a vast swathe of the Pacific with extensive polymetallic nodule deposits. Eastern and central parts host diverse assemblages of xenophyophores (megafaunal agglutinated foraminifera). Here we describe xenophyophores obtained using a Remotely Operated Vehicle from the western CCZ. Eleven distinct forms include two known species, Stannophyllum zonarium Haeckel, 1888 and Aschemonella monile Gooday and Holzmann in Gooday et al., 2017b. Another four are described as new species based on morphological and genetic data. In Abyssalia foliformis gen. nov., sp. nov. and Abyssalia sphaerica sp. nov. the flattened or spherical test comprises a homogeneous framework of sponge spicules. Psammina tenuis sp. nov. has a delicate, thin, plate-like test. Moanammina semicircularis gen. nov., sp. nov. has a stalked, fan-shaped test and is genetically identical to 'Galatheammina sp. 6' of Gooday and co-workers from the eastern CCZ. Sequence data revealed a spherical 'mudball', which disintegrated and cannot be formally described, to be a novel xenophyophore. Finally, four morphospecies are represented by dead tests: Psammina spp., Reticulammina sp., and an unknown genus with a unique test structure. This collection enhances our knowledge of Pacific xenophyophore diversity and provides the first genetic confirmation of wide geographic ranges for abyssal species.

The Foreign Oligochaete Species Quistadrilus multisetosus (Smith, 1900) in Lake Geneva: Morphological and Molecular Characterization and Environmental Influences on Its Distribution.

The presence of the oligochaete species Quistadrilus multisetosus (Smith, 1900) originating from North America has been mentioned for several decades in Europe, the Middle East and Russia. Its distribution and abundance in Europe is still unknown but it can be considered as potentially invasive. This species was recently discovered in Lake Geneva (Switzerland/France) and three other Swiss lakes. The aims of the present work are to report its repartition and abundance in Lake Geneva, to study its ecology and to determine its invasive potential in this lake. We also provide an identification key for correctly differentiating Q. multisetosus from the closely related species Spirosperma ferox Eisen, 1879 and Embolocephalus velutinus (Grube, 1879), and study the phylogenetic position of Q. multisetosus within several Tubificinae lineages based on the cytochrome c oxidase (COI) marker. Twenty-eight sites have been monitored since 2009 in Lake Geneva. In several sites, the COI sequence corresponding to this species was also searched for in sediment samples using high-throughput sequencing. In addition, we examined specimens collected in this lake before 2009 likely to belong to Q. multisetosus and to have been misidentified. We found that Q. multisetosus was only present in the lake downstream of a wastewater treatment plant and a combined sewer overflow in the Vidy Bay (near Lausanne) and at a site located nearby. These results confirmed the high tolerance of this species to organic matter pollution. Q. multisetosus was already present in this location in 1974 (misidentified as Spirosperma ferox), which suggests that Q. multisetosus has a limited capacity to disseminate in this lake. However, we recommend continuing monitoring its presence in Lake Geneva in the future, especially in the context of warming of waters that could contribute to the expansion of this species.

Xenophyophores (Rhizaria, Foraminifera) from the Eastern Clarion-Clipperton Zone (equatorial Pacific): the Genus Psammina.

Xenophyophores are important megafaunal organisms in the abyssal Clarion-Clipperton Zone (CCZ; equatorial Pacific), a region hosting commercially significant deposits of polymetallic nodules. Previous studies assigned those with attached, fan-like tests to Psammina limbata, a species described from the central CCZ based on morphology. Here, we redescribe the holotype of P. limbata and then show that limbata-like morphotypes collected in the eastern CCZ include three genetically distinct species. Psammina aff. limbata is closest morphologically to P. limbata. The others are described as P. microgranulata sp. nov. and P. rotunda sp. nov. These fan-shaped species form a well-supported clade with P. tortilis sp. nov., a morphologically variable species exhibiting features typical of both Psammina and Semipsammina. A second clade containing Psammina sp. 3, and two species questionably assigned to Galatheammina branches at the base of this group. The genus Psammina includes another 9 described species for which there are no genetic data, leaving open the question of whether Psammina as a whole is monophyletic. Our study increases the number of xenophyophore species described from the eastern CCZ from 8 to 11, with a further 25 morphotypes currently undescribed. Many additional species of these giant foraminifera undoubtedly await discovery in abyssal settings.

Cytochrome c oxidase barcodes for aquatic oligochaete identification: development of a Swiss reference database.

INTRODUCTION: Aquatic oligochaetes represent valuable indicators of the quality of sediments of watercourses and lakes, but their difficult identification based on morphological criteria compromises their more common use for eco-diagnostic analyses. This issue could be overcome by using DNA barcodes for species identification. A 10% threshold of cytochrome c oxidase (COI) divergence was proposed for differentiating between oligochaete species based on molecular and morphological data. A Swiss database of COI sequences of aquatic oligochaetes was initiated in 2012. The aim of this study is to complement the Swiss oligochaete database of COI sequences and to confirm the relevance of this threshold for species delimitation. METHODS: We sequenced the COI sequence of 216 specimens collected in different regions of Switzerland and ITS2 region of some lineages whose delimitation with COI data was doubtful. RESULTS: We distinguished 53 lineages, among which 34 were new for Switzerland and 17 sequenced for the first time. All the lineages were separated by more than 10% of COI variation, with the exception of some species within Nais and Uncinais. In these two genera, the threshold was lowered to 8% to be congruent with the morphological analysis. The total number of lineages reported so far for Switzerland is 75, including 59 morphospecies or unidentified species and 16 cryptic species. DISCUSSION: Our study shows that the threshold of 10% of COI divergence is generally appropriate to distinguish aquatic oligochaete lineages, but that it must be adjusted for some species. The database reported here will be complemented in the future in parallel to the development of genetic oligochaete indices.

Taxonomic revision of freshwater foraminifera with the description of two new agglutinated species and genera.

Most foraminifera inhabit marine habitats, but some species of monothalamids have been described from freshwater environments, mainly from Swiss water bodies over 100 years ago. Recent environmental DNA surveys revealed the presence of four major phylogenetic clades of freshwater foraminifera. However, until now only one of them (clade 2) has been associated to a morphologically described taxon-the family Reticulomyxidae. Here, we present morphological and molecular data for the genera representing the three remaining clades. We describe two new agglutinated freshwater genera from China and the Netherlands, Lacogromia and Limnogromia, which represent clades 3 and 4, respectively. We also report the first ribosomal DNA sequences of the genus Lieberkuehnia, which place this genus within clade 1. Our study provides the first morphotaxonomic documentation of molecular clades of freshwater foraminifera, showing that the environmental DNA sequences correspond to the agglutinated monothalamous species, morphologically similar to those described 100 years ago.