How good are global DNA-based environmental surveys for detecting all protist diversity? Arcellinida as an example of biased representation.

Metabarcoding approaches targeting microeukaryotes have deeply changed our vision of protist environmental diversity. The public repository EukBank consists of 18S v4 metabarcodes from 12,672 samples worldwide. To estimate how far this database provides a reasonable overview of all eukaryotic diversity, we used Arcellinida (lobose testate amoebae) as a case study. We hypothesised that (1) this approach would allow the discovery of unexpected diversity, but also that (2) some groups would be underrepresented because of primer/sequencing biases. Most of the Arcellinida sequences appeared in freshwater and soil, but their abundance and diversity appeared underrepresented. Moreover, 84% of ASVs belonged to the suborder Phryganellina, a supposedly species-poor clade, whereas the best-documented suborder (Glutinoconcha, 600 described species) was only marginally represented. We explored some possible causes of these biases. Mismatches in the primer-binding site seem to play a minor role. Excessive length of the target region could explain some of these biases, but not all. There must be some other unknown factors involved. Altogether, while metabarcoding based on ribosomal genes remains a good first approach to document microbial eukaryotic clades, alternative approaches based on other genes or sequencing techniques must be considered for an unbiased picture of the diversity of some groups.

Phylogeny and evolution of morphological structures in a highly diverse lineage of fruiting-body-forming amoebae, order Trichiales (Myxomycetes, Amoebozoa).

Early phylogenetic studies refuted most previous assumptions concerning the evolution of the morphological traits in the fruiting bodies of the order Trichiales and did not detect discernible evolutionary patterns, yet they were based on a limited number of species. We infer a new Trichiales phylogeny based on three independently inherited genetic regions (nuclear and mitochondrial), with a fair taxonomic sampling encompassing its broad diversity. Besides, we study the evolutionary history of some key morphological characters. According to the new phylogeny, most fruiting body traits in Trichiales systematics do not represent exclusive synapomorphies or autapomorphies for most monophyletic groups. Instead, the evolution of the features derived from the peridium, stalk, capillitium, and spores showed intricate patterns, and character state transitions occurred rather within- than between clades. Thus, we should consider other evolutionary scenarios instead of assuming the homology of some characters. According to these results, we propose a new classification of Trichiales, including the creation of a new genus, Gulielmina, the resurrection of the family Dictydiaethaliaceae and the genus Ophiotheca, and the proposal of 13 new combinations for species of the genera Arcyria (1), Hemitrichia (2), Ophiotheca (2), Oligonema (4), Gulielmina (3), and Perichaena (1).

Spore ultrastructural features and significance of their diverse ornamental elements in the evolutionary history of the order Trichiales (Myxomycetes, Amoebozoa).

Spores are the dispersal and reproductive units in Myxomycetes, and their ornamentation, usually at the light microscopy resolution limit, is taxonomically meaningful. Here, we analyze with scanning and transmission electron microscopy the spore ultrastructural features in Trichiales, one of the most morphologically diverse orders. In Trichiales, the spore wall consists of two layers, an outer one, including the ornamentation, and an inner layer subdivided into two sections. The diversity of ornamental elements includes verrucae, bacula, pila, muri, and cristae, the two latter creating reticulate patterns. Each of these elements defines a broadly recognized ornamentation type except cristae, from which we differentiate for the first time the cristate reticulate and patched subtypes. Besides, our results point out a previous incorrect classification of the spores of the species Trichia decipiens and T. scabra. Advances in Trichiales phylogeny showed that the capillitium ornamental elements, such as the spirals, do not reflect phylogenetic relationships among the species. The same seems to apply to some of spore ornamentation types, e.g., baculate. Consequently, we may consider the need to combine multiple characters, like the spore and capillitium ornamentation, so that Trichiales systematics better reflect the still cryptic phylogenetic affinities.

New Approach to the Ultrastructure of the Capillitium in the Order Trichiales (Myxomycetes, Amoebozoa) and its Phylogenetic Implications.

Myxomycetes constitute one of the major lineages within the supergroup Amoebozoa. At the end of their life cycles, most myxomycetes produce spore-bearing fruiting bodies, in which additional structures develop, like the capillitium, a system of sterile filaments intermingled with the spores. The capillitium is a relevant structure in the taxonomy of the order Trichiales, the target group in this study. However, the introduction of molecular phylogenies in Myxomycetes systematics is challenging our comprehension of this structure. We studied the capillitium of 25 species representing nine Trichiales genera, with both scanning and transmission electron microscopy. In this order, the capillitium showed higher diversity than so far recognized. Thus, we distinguished and described five capillitium types and two subtypes based on the presence or absence of a lumen and the wall ultrastructure. These types followed the evolutionary history reported in recent phylogenies, although not all of them defined monophyletic groups. Besides, the spiral ornamentation, which most taxonomists considered to have appeared once, occurred in three different capillitium types. The ultrastructural approaches in Myxomycetes systematics enable the reconsideration of their morphological features in the new phylogenetic scenario.

Nivicolous Trichiales from the austral Andes: unexpected diversity including two new species.

Nivicolous myxomycetes are a group of amoebozoan protists dependent on long-lasting snow cover worldwide. Recent fine-scale analysis of species diversity from the austral Andes revealed high intraspecific variability of most taxa, suggesting independent evolutionary processes and significant differences in species compositions between the Northern (NH) and Southern (SH) Hemispheres. The present study is the second part of this analysis based on representatives of Trichiales. A total of 173 South American collections were studied based on morphological and molecular data, and 15 taxa have been identified. Two of them, Hemitrichia crassifila and Perichaena patagonica, are proposed as new species confirmed by a phylogeny of Trichiales. However, their affinity to the genera in which they are proposed are not confirmed due to polyphyletic character of all genera of Trichiales. Four species, Dianema subretisporum, Trichia contorta var. karstenii, T. nivicola, and T. sordida, are reported for the first time from the Southern Hemisphere. One species, T. alpina, is new for Argentina. Additionally, we provide the first record of Perichaena megaspora from Chile. Specimen frequency and species diversity of Trichiales found at nivicolous localities in the austral Andes are unexpectedly high, exceeding those of Stemonitidales, the most numerous group in the Northern Hemisphere, where Trichiales play a marginal role. By contrast, Trichiales appear the main component of nivicolous assemblages in the Andes. Results of the present work, together with the earlier analysis of Stemonitidales, indicate that the Andes constitute an exceptionally important evolutionary hot spot for nivicolous myxomycetes characterized by an outstanding species diversity.

Didymium azorellae, a new myxomycete from cushion plants of cold arid areas of South America.

A new species of Didymium (Myxomycetes), D. azorellae, isolated from plant debris in a moist chamber, collected during studies of cold arid areas of Argentina and Peru, is described. It can be distinguished by its small size, the tightly packed layer of lime crystals on the peridium, the very scant, or absent, capillitium, and the unique spore ornamentation, especially by scanning electron microscopy. The species developed on dead leaves of cushion plants growing in the extremely harsh environments of the central Andean puna at almost 5000 m elevation and the Andino-Patagonian steppe. Morphology was examined with scanning electron microscopy and light microscopy, and micrographs of relevant details are included here. In order to confirm the identity of the new species described in this paper, a molecular study was conducted based on partial sequences of both the 18S rRNA and the elongation factor 1-alpha gene. Phylogenetic analysis including two specimens from different countries of the newly described species, Didymium azorellae, strongly supports the grouping of these specimens as a separate clade from the rest of the analyzed species.

Population genetics of the westernmost distribution of the glaciations-surviving black truffle Tuber melanosporum.

The black truffle (Tuber melanosporum Vittad.) is an important natural resource due to its relevance as a delicacy in gastronomy. Different aspects of this hypogeous fungus species have been studied, including population genetics of French and Italian distribution ranges. Although those studies include some Spanish populations, this is the first time that the genetic diversity and genetic structure of the wide geographical range of the natural Spanish populations have been analysed. To achieve this goal, 23 natural populations were sampled across the Spanish geographical distribution. ISSR technique demonstrated its reliability and capability to detect high levels of polymorphism in the species. Studied populations showed high levels of genetic diversity (h N = 0.393, h S = 0.678, Hs = 0.418), indicating a non threatened genetic conservation status. These high levels may be a consequence of the wide distribution range of the species, of its spore dispersion by animals, and by its evolutionary history. AMOVA analysis showed a high degree of genetic structure among populations (47.89%) and other partitions as geographical ranges. Bayesian genetic structure analyses differentiated two main Spanish groups separated by the Iberian Mountain System, and showed the genetic uniqueness of some populations. Our results suggest the survival of some of these populations during the last glaciation, the Spanish southern distribution range perhaps surviving as had occurred in France and Italy, but it is also likely that specific northern areas may have acted as a refugia for the later dispersion to other calcareous areas in the Iberian Peninsula and probably France.