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

Molecular Phylogenetics and Evolution 177 (2022) 107609 Available online 11 August 2022 1055-7903/© 2022 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/). Phylogeny and evolution of morphological structures in a highly diverse lineage of fruiting-body-forming amoebae, order Trichiales (Myxomycetes, Amoebozoa) Iv´ an García-Cunchillos a , * , Juan Carlos Zamora b , c , Martin Ryberg d , Carlos Lado a a Real Jardín Bot´ anico, CSIC, Plaza de Murillo 2, 28014 Madrid, Spain b Conservatorie et Jardin Botaniques de la Ville de Gen` eve, Chemin de l’Imp´ eratrice 1, 1292 Chamb´ esy, Switzerland c Museum of Evolution, Uppsala University, Norbyv¨ agen 16, Uppsala 752 36, Sweden d Department of Organismal Biology, Systematic Biology, Uppsala University, Norbyv¨ agen 18D, Uppsala 752 36, Sweden ARTICLE INFO Keywords: Ancestral state reconstructions Evolution Nomenclature Phylogenetics Systematics ABSTRACT 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 withinthan 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). 1. Introduction The supergroup Amoebozoa comprises a highly diverse lineage of ameboid organisms with an exceptional variety of life cycles (Kang et al. 2017). Some amoebozoans can produce fruiting bodies in their life cycles, including the sorocarpic fruiting in dictyostelids (Schaap et al. 2006) and the formation of sporophores in Myxomycetes as well as the protosteloid amoebae (Adl et al. 2019). The latter evolved multiple times in Amoebozoa (Shadwick et al. 2009), and the last common ancestor to the supergroup may also have produced them (Kang et al. 2017). Thus, understanding the evolution of the morphological features in fruiting bodies of these lineages has become one of the main goals in systematics and evolutionary studies in Amoebozoa. Fruiting bodies in Myxomycetes, more commonly referred to as sporophores (Fig. 1), contain the reproductive spores surrounded by an acellular envelope, the peridium, until their release. Sporophores anchor to the substrate by the hypothallus and can be either elevated by a stalk or be sessile. Besides, most species develop a capillitium, a system of sterile filaments intermingled with the spores. While the most common fruiting body morphology is the so-called sporocarp (Fig. 1), there are other three sporophores types: the plasmodiocarps, with elongated morphologies reminiscent of the plasmodium (one of the assimilative stages in Myxomycetes life cycle consisting of a naked, multinucleate mass of protoplasm), and two compound types, the pseudoaethalia and the aethalia (Keller et al. 2022). Despite this simplified definition, Myxomycetes fruiting bodies present highly variable colors, shapes, and sizes (see https://www.myxotropic.org/gallery/). Due to this diversity, Myxomycetes have a long-standing taxonomic tradition (Stephenson et al. 2008), and with 6–9 orders, 13–15 families, 68 genera (Lado and Eliasson 2022; Leontyev et al. 2019), and over 1,000 species (Lado 2005–2022) they are the lineage with more species recognized so far in Amoebozoa (Lara et al. 2020). * Corresponding author. E-mail address: [email protected] (I. García-Cunchillos). Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev https://doi.org/10.1016/j.ympev.2022.107609 Received 29 March 2022; Received in revised form 14 June 2022; Accepted 5 August 2022 Molecular Phylogenetics and Evolution 177 (2022) 107609 2 Within this astonishing diversity, the order Trichiales T. Macbr. stands out as one of the most morphologically diverse orders, with nearly 200 species accepted (Lado 2005–2022). Trichiales are part of the Myxomycetes with bright-colored spores (Fiore-Donno et al. 2005) together with the orders Cribrariales T. Macbr., Reticulariales Leontyev, Schnittler, S.l. Stephenson, Novozhilov & Shchepin, and Liceales E. Jahn (Leontyev et al. 2019). Trichiales species develop unique elastic, threadlike, ornamented capillitium (Fig. 1). Instead, the capillitium is absent in Liceales and Cribrariales, while the species in Reticulariales produce pseudocapillitium, i.e., filiform peridium remnants (Lister 1925). Despite the noticeable diversity, Trichiales systematics has remained stable over the years (e.g., Lister 1925; Martin and Alexopoulos 1969; Nannenga-Bremekamp 1991; Poulain et al. 2011; Rostafi´ nsky 1874, 1875, 1876). These taxonomic treatments assumed some evolutionary hypotheses concerning the capillitium to establish the Trichiales classification. Thus, the distinction between solid threads (family Dianemataceae) and hollow tubules (Arcyriaceae and Trichiaceae) defines the primary division. Differences between the latter two rely on the ornamental elements of the capillitium, consisting of spirals (Fig. 1) in the family Trichiaceae while being cogs, reticula, rings, spines, and verrucae in Arcyriaceae. Generic delimitation depends on other capillitium features, such as the branching pattern, or combinations of multiple macroscopic traits, like stalked vs. sessile sporocarps and evanescent vs. persistent peridium, distinguishing, for example, Arcyria from Arcyodes (Lado and Pando 1997). Some authors recognize a fourth monospecific family, Minakatellaceae, with unique pseudoaethaloid fruiting bodies in Trichiales, questioned by other authors (see Keller et al. 1973). Unexpectedly, the introduction of DNA-based phylogenetic reconstructions in Trichiales (Fiore-Donno et al. 2013) has only confirmed some of the above-mentioned evolutionary hypotheses, while most of them have been refuted. For instance, the dichotomy between solid and hollow capillitium agrees with the phylogeny, although TEM-based studies reported at least four ultrastructural capillitium types of hollow filaments (Ellis et al. 1973; García-Cunchillos et al. 2021a). On the contrary, the spiral capillitium ornamentation seems to have originated independently in multiple clades (Fiore-Donno et al. 2013). Moreover, previous phylogenies recovered most genera in Trichiales as paraphyletic (Fiore-Donno et al. 2013; Leontyev et al. 2019; Ronikier et al. Fig. 1. General structure of a stalked sporocarp in Trichiales. The spores form a mass intermingled with the capillitium, a system of sterile filaments (up to the right, as seen with light microscopy), surrounded by a dehiscent peridium and anchored to the substrate by the hypothallus. This spore mass can be elevated above the substrate by a stalk or be sessile. In Trichiales, stalks can be filled with spore-like bodies (bottom right, as seen with light microscopy) or different refuse materials (not shown). Species: Hemitrichia calyculata. Fig. 2. Spore ornamentation types in Trichiales, as seen with scanning electron microscopy. A Simple reticulate (Hemitrichia calyculata). B Baculate (Prototrichia metallica). C Verrucate (Arcyria denudata). D Baculate (Calonema foliicola). E Baculate (Perichaena quadrata). F Pilate (Metatrichia horrida). G Cristate reticulate (Hemitrichia serpula). H Cristate patched (Oligonema schweinitzii). Scale bar =5 µm. I. García-Cunchillos et al. Molecular Phylogenetics and Evolution 177 (2022) 107609 3 Table 1 Species, specimens, and GenBank accession numbers of the sequences included in Trichiales phylogeny (Fig. 3). Taxonomy followed Lado and Eliasson (2022) and Leontyev et al. (2019). The nomenclatural treatment attended Lado (2005–2022). Species authorships can be consulted in Lado (2005–2022). The species name in bold indicates it as the type species of the genus. Accession numbers starting other than ’ON’ correspond to the sequences retrieved from GenBank. Order Family Species Voucher 18S rRNA EF1A mtSSU Liceales Liceaceae Licea castanea AMFD 102 JX481295.1 JX481329.2 – Licea marginata DWM 7368 JX481296.1 JX481330.1 – Licea parasitica AMFD 341 JX481297.1 JX481331.1 – Reticulariales Reticulariaceae Lycogala epidendrum MA-Fungi 83194 ON713381.1 ON693913.1 ON713288.1 Reticularia jurana AMFD 290 JX481310.1 JX481339.1 – Reticularia jurana MA-Fungi 83010 ON713377.1 – ON713284.1 Reticularia jurana MA-Fungi 83011 ON713378.1 – ON713285.1 Reticularia jurana MA-Fungi 83274 ON713384.1 – ON713291.1 Reticularia lycoperdon AMFD 262 JX481311.1 JX481340.1 – Tubifera ferruginosa AMFD 196 EF513171.1 EF513201.1 – Trichiales Arcyriaceae Arcyodes incarnata Lado 25434 ON713325.1 ON693868.1 ON713237.1 Arcyodes incarnata Lado 25437 ON713326.1 ON693869.1 ON713238.1 Arcyria affinis MA-Fungi 61187 ON713341.1 ON693881.1 ON713250.1 Arcyria affinis MA-Fungi 68912 ON713351.1 ON693886.1 ON713260.1 Arcyria afroalpina MA-Fungi 83613 ON713396.1 ON693927.1 ON713301.1 Arcyria afroalpina MA-Fungi 83614 ON713397.1 ON693928.1 ON713302.1 Arcyria cinerea MA-Fungi 83612 ON713395.1 ON693926.1 ON713300.1 Arcyria cinerea MA-Fungi 87452 ON713403.1 ON693934.1 ON713307.1 Arcyria denudata MA-Fungi 78718 ON713354.1 ON693889.1 – Arcyria denudata MA-Fungi 83327 ON713385.1 ON693916.1 – Arcyria ferruginea MA-Fungi 58962 ON713339.1 ON693879.1 ON713248.1 Arcyria ferruginea MA-Fungi 86476 ON713401.1 ON693932.1 ON713303.1 Arcyria globosa AMFD 252 JX481282.1 JX481318.1 – Arcyria globosa MA-Fungi 52762 ON713335.1 – ON713244.1 Arcyria incarnata MA-Fungi 83426 ON713390.1 ON693921.1 ON713295.1 Arcyria incarnata MA-Fungi 83465 ON713392.1 ON693923.1 ON713297.1 Arcyria insignis MA-Fungi 87847 ON713404.1 ON693935.1 ON713308.1 Arcyria insignis MA-Fungi 87859 ON713405.1 ON693936.1 ON713309.1 Arcyria oerstedii MA-Fungi 58741 ON713337.1 ON693877.1 ON713246.1 Arcyria oerstedii MA-Fungi 61817 ON713342.1 ON693882.1 ON713251.1 Arcyria stipata AMFD 257 EF513170.1 EF513183.1 – Cornuvia serpula MM 29198 JX481285.1 JX481320.1 – Perichaena calongei Lado 25554 ON713327.1 ON693870.1 ON713239.1 Perichaena calongei MA-Fungi 78686 ON713352.1 ON693887.1 ON713261.1 Perichaena calongei MA-Fungi 78692 ON713353.1 ON693888.1 ON713262.1 Perichaena chrysosperma MA-Fungi 63754 ON713345.1 ON693883.1 ON713254.1 Perichaena chrysosperma MA-Fungi 64647 ON713349.1 – ON713258.1 Perichaena corticalis MA-Fungi 68850 ON713350.1 ON693885.1 ON713259.1 Perichaena corticalis MA-Fungi 83138 ON713380.1 ON693912.1 ON713287.1 Perichaena depressa MA-Fungi 83635 ON713398.1 ON693929.1 – Perichaena depressa MA-Fungi 88312 ON713408.1 ON693939.1 – Perichaena dictyonema MA-Fungi 59057 ON713340.1 ON693880.1 ON713249.1 Perichaena liceoides M 0073211 ON713328.1 ON693871.1 – Perichaena liceoides M 0073215 ON713329.1 ON693872.1 – Perichaena megaspora KRAM-M 1765 MT154023.1 MT162162.1 – Perichaena megaspora MA-Fungi 82123 MT154026.2 MT162165.1 ON713281.1 Perichaena nigra MA-Fungi 86774 ON713402.1 ON693933.1 ON713306.1 Perichaena patagonica MA-Fungi 91906 MT154034.2 MT162173.1 ON713317.1 Perichaena patagonica MA-Fungi 91909 MT154035.2 MT162174.1 ON713318.1 Perichaena pedata MA-Fungi 81941 ON713372.1 ON693906.1 ON713280.1 Perichaena quadrata MA-Fungi 88308 ON713406.1 ON693937.1 ON713310.1 Perichaena quadrata MA-Fungi 88310 ON713407.1 ON693938.1 ON713311.1 Perichaena stipitata MA-Fungi 79150 ON713358.1 ON693892.1 ON713265.1 Perichaena stipitata MA-Fungi 79151 ON713359.1 ON693893.1 ON713266.1 Perichaena vermicularis BR 5020025765604 MT154019.1 MT162157.1 – Perichaena vermicularis MA-Fungi 80426 ON713363.1 ON693897.1 ON713270.1 Perichaena vermicularis MA-Fungi 88424 ON713409.1 – ON713313.1 Dianemataceae Calomyxa metallica AMFD 483 JX481284.1 JX481319.1 – Calomyxa metallica IT 560 ON713323.1 ON693865.1 ON713234.1 Calomyxa metallica MA-Fungi 82936 ON713373.1 ON693907.1 – Calomyxa metallica MA-Fungi 82941 ON713375.1 ON693909.1 ON713283.1 Calomyxa metallica MA-Fungi 82942 ON713376.1 ON693910.1 – Dianema corticatum KR-M 0040806 MT154024.1 MT162163.1 – Dianema corticatum KR-M 0040819 MT154025.1 MT162164.1 – Dianema depressum MA-Fungi 80673 ON713367.1 ON693900.1 ON713273.1 Dianema depressum MA-Fungi 82939 ON713374.1 ON693908.1 ON713282.1 Dianema harveyi BR 5020022218059 MT154018.1 MT162156.1 – Dianema harveyi BR 5020210944555V MT154020.1 MT162158.1 – Dianema inconspicuum MM 39161 MT154038.2 MT162177.1 ON713320.1 Dianema mongolicum MM 45002 ON713413.1 ON693944.1 ON713321.1 (continued on next page) I. García-Cunchillos et al. Molecular Phylogenetics and Evolution 177 (2022) 107609 4 2020; Walker et al. 2015). Consequently, it is necessary to investigate other characters previously overlooked to test whether they reflect evolutionary relationships. For example, Fiore-Donno et al. (2013) proposed the spore-like bodies, structures filling the stalks in certain species (Fig. 1), as a diagnostic character of a clade comprising some species of the genera Trichia and Hemitrichia. Other features, traditionally considered variable, such as the spore ornamentation (Fig. 2), could show more straightforward patterns of evolution (García-Cunchillos et al. 2021b). However, there is still an underrepresentation of Trichiales diversity in phylogenetic studies to understand the evolutionary histories of many morphological characters. In this study, we infer a new Trichiales phylogeny with a representative sampling of the morphological diversity encompassed in the order. In this new framework, we trace the evolutionary patterns of the principal morphological features of fruiting bodies, such as the distinct capillitium traits or sporophore types. In particular, we focus on four characters: stalks, spore-like bodies, number of peridium layers, and spore ornamental elements, for which we also conduct, for the first time in Myxomycetes, ancestral state reconstructions to explore and unravel the evolutionary Table 1 (continued) Order Family Species Voucher 18S rRNA EF1A mtSSU Dianema nivale MM 29888 JX481289.1 JX481324.1 – Dianema sp. MA-Fungi 86506 MT154027.2 MT162166.1 ON713304.1 Dianema sp. MA-Fungi 86507 MT154028.2 MT162167.1 ON713305.1 Dianema subretisporum MM 31413 ON713412.1 ON693943.1 ON713319.1 Dianema subretisporum MM 46699 ON713414.1 ON693945.1 ON713322.1 Dianema succulenticola MA-Fungi 80774 – ON693901.1 ON713274.1 Dianema succulenticola MA-Fungi 81387 – ON693903.1 ON713276.1 Dictydiaethalium dictyosporum MA-Fungi 91171 ON713411.1 ON693942.1 ON713314.1 Dictydiaethalium plumbeum MA-Fungi 64421 ON713348.1 ON693884.1 ON713257.1 Licea variabilis MA-Fungi 80591 ON713366.1 ON693899.1 ON713272.1 Licea variabilis MA-Fungi 85637 ON713400.1 ON693931.1 – Prototrichia metallica MA-Fungi 80049 ON713360.1 ON693894.1 ON713267.1 Prototrichia metallica MM 24907 JX481309.1 JX481338.1 – Trichiaceae Calonema foliicola MA-Fungi 50720 ON713333.1 ON693875.1 – Hemitrichia abietina MA-Fungi 58838 ON713338.1 ON693878.1 ON713247.1 Hemitrichia abietina MM 30370 JX481293.1 JX481327.1 – Hemitrichia calyculata MA-Fungi 81807 ON713370.1 – ON713278.1 Hemitrichia calyculata MS 22060 JX481294.1 JX481328.1 – Hemitrichia clavata MA-Fungi 62017 ON713343.1 – ON713252.1 Hemitrichia clavata MA-Fungi 62018 ON713344.1 – ON713253.1 Hemitrichia crassifila MA-Fungi 91880 MT154030.2 MT162169.1 ON713315.1 Hemitrichia crassifila MA-Fungi 91885 MT154031.2 MT162170.1 ON713316.1 Hemitrichia intorta BR 5020003245449 MT154017.1 MT162155.1 – Hemitrichia intorta KR 0022295 MT154021.1 MT162159.1 – Hemitrichia leiocarpa M 0142937 ON713330.1 – ON713240.1 Hemitrichia minor MA-Fungi 80197 ON713361.1 ON693895.1 ON713268.1 Hemitrichia minor U 6369 ON713415.1 ON693946.1 – Hemitrichia pardina MA-Fungi 80413 ON713362.1 ON693896.1 ON713269.1 Hemitrichia serpula MA-Fungi 64060 ON713346.1 – ON713255.1 Hemitrichia serpula MA-Fungi 64068 ON713347.1 – ON713256.1 Metatrichia floriformis MA-Fungi 52989 ON713336.1 – ON713245.1 Metatrichia floriformis MA-Fungi 83204 ON713382.1 ON693914.1 ON713289.1 Metatrichia floripara Lado 25103 ON713324.1 ON693867.1 ON713236.1 Metatrichia horrida MA-Fungi 81778 ON713369.1 ON693904.1 ON713277.1 Metatrichia horrida MA-Fungi 81857 ON713371.1 ON693905.1 ON713279.1 Metatrichia vesparia MA-Fungi 51719 ON713334.1 ON693876.1 ON713243.1 Metatrichia vesparia MA-Fungi 88351 – ON693940.1 ON713312.1 Oligonema schweinitzii MA-Fungi 85559 ON713399.1 ON693930.1 – Oligonema schweinitzii MM 29842 JX481305.1 JX481336.1 – Oligonema sp. MA-Fungi 78856 ON713355.1 ON693890.1 – Oligonema sp. MA-Fungi 78857 ON713356.1 ON693891.1 ON713263.1 Oligonema sp. MA-Fungi 83328 ON713386.1 ON693917.1 ON713292.1 Oligonema sp. MA-Fungi 83357 ON713389.1 ON693920.1 ON713294.1 Trichia affinis Lado 24817 – ON693866.1 ON713235.1 Trichia affinis MA-Fungi 78975 ON713357.1 – ON713264.1 Trichia affinis MA-Fungi 83345 ON713387.1 ON693918.1 – Trichia agaves MA-Fungi 42243 ON713331.1 ON693873.1 ON713241.1 Trichia agaves MA-Fungi 50703 ON713332.1 ON693874.1 ON713242.1 Trichia alpina AMFD 64 JX481312.1 JX481341.1 – Trichia alpina MA-Fungi 80534 ON713364.1 – ON713271.1 Trichia decipiens MA-Fungi 83070 ON713379.1 ON693911.1 ON713286.1 Trichia favoginea MA-Fungi 83229 ON713383.1 ON693915.1 ON713290.1 Trichia lutescens MA-Fungi 83355 ON713388.1 ON693919.1 ON713293.1 Trichia lutescens MA-Fungi 83430 ON713391.1 ON693922.1 ON713296.1 Trichia persimilis – AY643826.1 AY643821.1 – Trichia scabra MA-Fungi 81001 ON713368.1 ON693902.1 ON713275.1 Trichia scabra MA-Fungi 90224 ON713410.1 ON693941.1 – Trichia scabra MS 22055 JX481314.1 JX481343.1 – Trichia sordida AMFD 81 EF513182.1 EF513200.1 – Trichia varia MA-Fungi 80566 ON713365.1 ON693898.1 – Trichia varia MA-Fungi 83469 ON713393.1 ON693924.1 ON713298.1 Trichia verrucosa MA-Fungi 83489 ON713394.1 ON693925.1 ON713299.1 I. García-Cunchillos et al. Molecular Phylogenetics and Evolution 177 (2022) 107609 5 history of these characters. Lastly, we provide a revised systematic classification of the major groups in Trichiales by proposing taxonomic amendments for some taxa to reflect both the phylogenetic affinities and the evolution of the morphological traits. 2. Material and methods 2.1. Samples We obtained new genetic information from three families, 13 genera, and 61 species of Trichiales, plus two genera and two species of Reticulariales as part of the phylogeny outgroup (Supplementary material 1). Whenever possible, we selected two specimens of each species and performed independent DNA extraction shifts to ensure the identity of the new sequences. We further completed the sampling with GenBank public data, with species that available 18S rRNA and EF1A sequences belong to the same voucher (grey-shaded in Table 1), including representatives from the orders Reticulariales and Liceales. The final dataset comprised representatives of almost all families, 73 species, and 132 specimens (Table 1). Besides, we studied 57 samples of 32 species for which DNA obtaining failed; we provide this information since it may be informative for future studies dealing with these species (Supplementary material 2). Unfortunately, we could not obtain specimens of Minakatella longifila, the monospecific genus of the family Minakatellaceae. Different phylogenetic and morphological studies detected striking contrasts when comparing specimens of a presumably single species from the northern and southern hemispheres (Janik et al. 2020, 2021; Ronikier and Lado 2015), particularly between the Neotropics and Europe, the latter often being the origin for the descriptions of multiple species. However, the European specimens we could study were, in most cases, aged, and we could not obtain enough genetic information. To palliate this shortcoming, we have alternatively constructed an 18S rRNA-based phylogeny including all 18S rRNA sequences analyzed in our multilocus phylogeny (Table 1) plus other available sequences in GenBank of the same studied species but from different geographic origins (Supplementary material 3). Our objective with this 18S rRNAbased phylogeny was to confirm that samples of presumably single species but from distant geographical regions were at least nesting in the same principal clades, and then our diagnoses of these groups and nomenclatural changes were not compromised by sampling bias. Taxonomy followed Leontyev et al. (2019) and Lado and Eliasson (2022). The nomenclatural treatment attended Lado (2005–2022). Vouchers of all specimens studied came from the herbaria BR, KR-M, KRAM-M, M, MA-Fungi (https://sweetgum.nybg.org/science/ih/), and private collections of I. Trevi˜ no-Zevallos, C. Lado, M. Meyer, and U. Eliasson. 2.2. DNA Extraction, PCR Amplification, and sequencing Four to eight adjacent sporocarps, or an equivalent portion in plasmodiocarpic and pseudoaethaloid specimens, were selected for total DNA extraction. Each sample was transferred to a safe-lock microcentrifuge tube containing one 3 mm diam. tungsten carbide bead, frozen at −80 ◦C for one hour, and then subjected to mechanical disruption in a TissueLyser II bead mill, according to Fiore-Donno et al. (2012). DNA extraction followed the DNeasy Plant Mini Kit (QIAGEN, Germany) protocol with two minor modifications: i) samples were incubated in the buffer AP1 overnight, and ii) DNA was eluted twice with 80 µL of the buffer AE. To infer the new Trichiales phylogeny, we selected two nuclear (the nuclear small subunit ribosomal RNA or 18S rRNA and the eukaryotic translation elongation factor 1 alpha or EF1A) and one mitochondrial (the mitochondrial small subunit ribosomal RNA or mtSSU) genetic regions. Among the former, the 18S rRNA is the preferred region to study phylogenetic relationships within Myxomycetes (see Schnittler et al. 2017), which, combined with the EF1A, recovered statistically supported clades at various taxonomic ranks (e.g., Fiore-Donno et al. 2005, 2012, 2013, 2018). The mtSSU reported coherent results compared to other genetic regions in the dark-spored Myxomycetes (Lado et al. 2022), but no studies have explored this region in the bright-spored Myxomycetes with phylogenetic purposes. The 18S rRNA sequence length varied 1387–2515 bp due to the presence of introns and the highly variable helices (Fiore-Donno et al. 2012). Consequently, it was amplified and sequenced in three fragments: S1 (509–786 bp), S2 (790–1601 bp), and S3 (707–1279 bp). To apply this so-called primer walking method (Fraser and Fleischmann 1997), the end of each fragment must partially overlap with the beginning of the next one: S1–S2 (187–203 bp overlapping) and S2–S3 (246–249 bp). When S2 or S3 amplifications failed, we completed these positions as Ns (any base) in the alignment, treated as missing data in subsequent analyses. For the amplification of the first fragment (S1), we employed primers SF01, SF02*, and SR01 (Ronikier et al. 2020), in which F and R refer to forward and reverse, respectively, and an asterisk (*) designates its use as inner forward primer in semi-nested PCRs. Following the same nomenclature, we designed new primers (sequences in 5′→ 3′direction) to amplify fragments S2 (SF03: ACGGGTACAGAGGATCAG; SF04*: AGCCTGAGAGATCGCTAC, and SR02: CCTTGTGTGCTCTTCCGT) and S3 (SF05: TAGGGGTGAAATCCGTTGA, SF06*: ACGAAAGTCTGGGGAT, and SR3: TACAAAGAGCAGGGACA). The EF1A was amplified and sequenced as a single amplicon, including the intron present in every Myxomycetes species (Fiore-Donno et al. 2005), and sequences ranged from 876 to 1453 bp. Forward primers for EF1A amplification were EF03 and EF04* (Ronikier et al. 2020), and the reverse primer was KEF_R3 (García-Martín et al. 2018). The mtSSU sequences comprised 334–667 bp, amplified as a single amplicon with the primers pair Kmit_F and Kmit_R (Lado et al. 2022). We did not obtain successful amplification by using inner primers in semi-nested PCRs. Each PCR reaction contained 12.5 µL MyTaq TM DNA Polymerase (BIOLINE, United Kingdom), 1 µL DMSO, 0.5 µL of each primer, forward and reverse (10 mM), 1–3 µL template DNA, and completed with Milli-Q water up to a final volume of 25 µL. Semi-nested PCRs used 1 µL of a dilution 1:10 of the original PCR product. PCR conditions for the amplification of each genetic region included an initial denaturation step (94 ◦C, 1 min), 30 cycles consisting of denaturation (94 ◦C, 1 min), annealing (50–52 ◦C according to each primer, 1 min), and extension (72 ◦C, 3 min), and a final extension step (72 ◦C, 10 min). Successful amplifications were checked through electrophoresis in 1 % agarose gels and 1 ×TAE buffer and purified with the QIAquick Gel Extraction Kit (QIAGEN, Germany). Amplicons were sequenced in both directions, with the same primer pairs, at MACROGEN facilities in Madrid (Spain). 2.3. Alignments The newly obtained sequences, along with those retrieved from GenBank, were aligned for each genetic region with the MAFFT online service (https://mafft.cbrc.jp/alignment/server/) of MAFFT 7 (Katoh et al. 2019), using the L-INS-i strategy (Katoh et al. 2005), and the remaining parameters as default. Every alignment was visually inspected and manually corrected when detecting errors. We discarded introns in the EF1A alignment (positions 226–4708, Supplementary material 4) for further analyses since they were highly divergent to be consistently aligned. Thus, the EF1A dataset consisted of 117 sequences and 1017 bp. Similarly, barely alignable positions in the mtSSU alignment were discarded (289–4004, Supplementary material 5), and the dataset comprised 89 sequences and 375 bp. Highly divergent intron sequences and variable helices of the 18S rRNA resulted in multiple poorly aligned positions. Thus, a mask based on predicted secondary structures of rRNA in the bright-spored Myxomycetes was employed (Fiore-Donno et al. 2013), retaining only those positions under the mask (Supplementary material 6). The new 18S rRNA sequences were added to an existing masked alignment (FioreDonno et al. 2013), including some of the sequences obtained from I. García-Cunchillos et al. Molecular Phylogenetics and Evolution 177 (2022) 107609 6 Fig. 3. Phylogeny of the order Trichiales based on 132 specimens and 73 species (Table 1), including Reticulariales and Liceales as the outgroup. Majority-rule consensus Bayesian tree obtained from the concatenated genetic regions 18S rRNA (1246 bp), EF1A (1017 bp), and mtSSU (375 bp). Phylogenetic supports include Felsenstein’s Bootstrap Proportions and Bayesian Posterior Probabilities, above and below each branch, respectively. Full phylogenetic support, i.e., Felsenstein’s Bootstrap Proportions =100 and Bayesian Posterior Probabilities =1, is represented by a black circle. SH-aLRT and Transfer Bootstrap Expectation are only provided (in grey to the right of the previous ones, above and below, respectively) when the former support values recovered conflictive results. The scale bar indicates the average number of substitutions per site. Double slashes indicate shortened branches. To the right of vouchers, representation of the principal studied morphological features are depicted (see legend): fruiting body types, capillitium architecture, capillitium ornamentation (simplified), spore ornamentation, and peridium number of layers. C Dictydiaethalium plumbeum. D Calomyxa metallica (above), Dianema harveyi (below). E1 Arcyria insignis. E2 Hemitrichia calyculata. F1 Arcyria globosa. F2 Arcyria affinis. G Perichaena calongei. H1 Hemitrichia serpula (above), Trichia affinis (middle), Trichia verrucosa (below). OT Trichia varia. H2 Perichaena patagonica. H3 Metatrichia floriformis. H4 Perichaena stipitata (above), Perichaena depressa (below). I. García-Cunchillos et al. Molecular Phylogenetics and Evolution 177 (2022) 107609 7 Genbank, using the option –add of MAFFT (Katoh and Frith 2012). The final 18S rRNA dataset comprised 128 sequences and 1246 bp. Since we did not detect supported incongruences among the phylogenies inferred from these datasets, a fourth dataset was constructed by concatenating the individual alignments. Missing data in this dataset consisted of Ns. Gaps were treated as missing data in the subsequent analyses. Fig. 3. (continued). I. García-Cunchillos et al. Molecular Phylogenetics and Evolution 177 (2022) 107609 8 Fig. 4. Ancestral state reconstructions for selected characters based on stochastic character mapping simulations. Clade names (to the right of each cladogram) matched those in Fig. 3. A Stalk (red =absent, blue =present). B Spore-like bodies (red =present, blue =absent). Clades C and D were excluded from the analysis (see Material and Methods), and the involved nodes were not inferred (grey circles). C Number of peridium layers (red =single-layered, blue =double-layered). D Spore-ornamental elements (red =verrucae, blue =bacula, green =pila, purple =muri, cristae =yellow), in parentheses, corresponding ornamentation types. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) I. García-Cunchillos et al. Molecular Phylogenetics and Evolution 177 (2022) 107609 9 2.4. Phylogenetic analyses Maximum likelihood trees were inferred with IQ-TREE 2.0 (Nguyen et al. 2015). The best-fit substitution model for each dataset was selected with ModelFinder (Kalyaanamoorthy et al. 2017) under the Bayesian information criterion (BIC). The concatenated dataset consisted of five partitions: 18S rRNA, mtSSU, and three blocks of the EF1A corresponding to the first, second, and third codon positions. For each dataset or partition, model parameters were estimated with edge-linked branch lengths, and the best partition scheme was selected using the greedy search algorithm as implemented in IQ-TREE 2.0 (Chernomor et al. 2016). We conducted ten independent runs for the analyses of each dataset, including the concatenated one, as recommended by Zhou et al. (2018). To further minimize the possibility that the tree search algorithms could get stuck in local optima, we set a smaller value of the parameter perturbation strength for randomized NNI (-pers 0.2) and a higher number of unsuccessful iterations to stop (-nstop 500), as recommended in Nguyen et al. (2015). Phylogenetic trees were also inferred under a Bayesian approach using MRBAYES 3.2.7a (Ronquist et al. 2012) with the parallel Metropolis-coupled Markov chain Monte Carlo ([MC] 3 ) algorithm (Altekar et al. 2004). The same partition blocks as in the maximum likelihood analyses were defined, with unlinked parameters estimation but linked tree topologies. The estimation of DNA models of evolution employed the Reversible Jump Markov Monte Carlo method (Huelsenbeck et al. 2004). Markov chain Monte Carlo (MCMC) simulations consisted of four runs, four chains each, and 20,000,000 generations. Trees were sampled every 1,000 generations, with a 0.25 fraction of the samples discarded as burn-in. Diagnosis of convergence was assessed through the average standard deviation of split frequencies (ASDSF, ideally ≤0.01), the potential scale reduction factors (PSRF ≈1.000), and the inspection of the Effective Sample Sizes (ESS ≥200) of each sampled parameter through TRACER 1.7.1 (Rambaut et al. 2018). Branch phylogenetic supports were assessed with the two most broadly employed methods in phylogenetic studies (Wr´ obel 2008), i.e., the Felsenstein’s bootstrap proportions (FBP) and the Bayesian Posterior Probabilities (BPP). The former was estimated with 1,000 nonparametric replicates in IQ-TREE 2.0 (Chernomor et al. 2016), and the latter in MRBAYES 3.2.7a (Ronquist et al. 2012) after discarding the 0.25 fraction of the sampled trees. While both methods usually agreed in detecting supported (i.e., FBP ≥70 and BPP ≥0.95) and unsupported clades, they recovered conflicting results for some of them. As recommended in Wr´ obel (2008), we estimated a third, approximate likelihood-based measure of branch supports, the SH-aLRT (Anisimova et al. 2011; Guindon et al. 2010), in IQ-TREE 2.0 with 1,000 nonparametric replicates (supported clades when SH-aLRT ≥80). Besides, Lemoine et al. (2018) proposed a modification of the FBP, the transfer bootstrap expectation (TBE), considering the effect of rogue taxa in molecular phylogenies, usually decreasing FBP values. TBE relied on the same 1,000 nonparametric replicates as FBP estimation and were estimated in the online service BOOSTER (https:// booster.pasteur.fr/). Clades were considered supported when TBE ≥0.80. 2.5. Character evolution analyses We reconstructed the character state at ancestral nodes for the following traits: stalk (absent, present), spore-like bodies (present, absent), the number of perdium layers (single-layered, double-layered), and spore ornamental elements (muri, bacula, verrucae, pila, and cristae). Each ornamental element defines a spore ornamentation type (Rammeloo 1974): simple reticulate (muri, strip-like elements with smooth tops, Fig. 2A), baculate (bacula, cylindrical, taller than broad elements with rounded or short-pointed tops, Fig. 2B, D–E), verrucate (verrucae, rounded, broader than tall elements, Fig. 2C), pilate (pila, cylindrical elements supporting spherical head-like structures, Fig. 2F), and cristate (cristae, elements similar to muri but with irregularly shaped tops, Fig. 2 G–H). Cristate ornamentation encompasses two subtypes (see García-Cunchillos et al. 2021b), the cristate reticulate (Fig. 2G) and the cristate patched (Fig. 2H). While, for more detailed information, we refer to both subtypes in the mapping of characters (Fig. 3) and the discussion, we do not consider this distinction in the ancestral state reconstructions (Fig. 4) since the ornamental element is the same, and, in this way, we also minimize the total number of possible states for the ancestral state reconstruction analyses. For the species producing sessile sporophores, the character “spore-like bodies” is unknown, so we established prior probabilities equally divided among all possible states, following Zamora and Ekman (2020). Besides, for this character, we ran the analyses twice: first, considering all the clades, and second, excluding clades C and D, in which states in all taxa are unknown since all species lack stalks (see Results). The reconstructions started from a random sample of 1,000 trees (phylograms) from the total Bayesian posterior tree sampling. Phylograms were pruned to conserve one specimen per species (the one with the shortest branch) and exclude the outgroup. Ancestral state reconstructions relied on stochastic character mappings, performed with SIMMAP (Bollback 2006), using the make.simmap() function implemented in the R package phytools (Revell 2012). Analyses consisted of ten character mapping simulations for each phylogram (Zamora and Ekman 2020). We set an asymmetric model for characters with only two states and a symmetric model for characters with more than two states (see Kistenich et al. 2018). Ancestral state reconstructions based on phylograms (i.e., considering morphological change proportional to genetic change) and chronograms (i.e., morphological change proportional to time) may yield different results (Cusimano and Renner 2014; Litsios and Salamin 2012). Thus, we also conducted the same analyses starting from chronograms. Chronograms were generated from the phylograms with the function chronos() implemented in the R package ape v.5.0 (Paradis and Schliep 2019), which uses penalized likelihoods under a correlated model as described by Paradis (2013). Here, both versions of the analyses (phylograms vs. chronograms) reported slightly different results. However, the differences occurred at nodes for which none of the analyses reported conclusive results. Thus, we only show the results of the reconstructions based on phylograms. 3. Results 3.1. Phylogeny of the order trichiales The ten maximum-likelihood tree searches resulted in highly similar log-likelihood scores, with only marginal differences, for all datasets (results not shown). Nonetheless, we selected the trees with the highest score for further study. The best partition scheme in the EF1A genetic region consisted of three blocks, one for each position in the codontriplets. The best-fitting substitution models for these blocks were TIM3 +I +G, GTR +I +G, and GTR +G, respectively. EF1A was the least informative region and only supported phylogenetic affinities among specimens of the same species or closely related taxa, while the remaining clades received negligible support. The 18S rRNA and the mtSSU regions were comparatively more informative, and both reported highly similar topologies. The best-fitting substitution models for these regions were TIM2e +I +G and GTR +I +G, respectively. While clades in the 18S rRNA tree recovered the higher phylogenetic support, neither resulted in a fully resolved phylogeny. The tree derived from the concatenated dataset, partitioned into five blocks, resulted in mostly well-supported clades, although some relationships remained unsolved. Bayesian results showed a good convergence and thorough sampling (ASDSF <0.005, PSRF =1.000 ± 0.001, ESS >4000 for each parameter). Based on the phylogeny, we recognize eight main clades (A–H) and eight subclades (Fig. 3). The definition of these clades was made to match the current taxonomic treatment and the presence of distinct morphological features as far as possible (see Discussion). Support values in Fig. 3 consist of FBP and BPP. When both estimators reported non-concordant results, i.e., one supported the clade while the other did not, we also provide SH-aLRT I. García-Cunchillos et al. Molecular Phylogenetics and Evolution 177 (2022) 107609 16 4.3.4.4. Subclade H3. ** Metatrichia Ing, Trans. Brit. Mycol. Soc. 47 (1): 51 (1964). Type: Metatrichia horrida Ing, Trans. Brit. Mycol. Soc. 47(1): 51 (1964). Species included here: M. floriformis (Schwein.) Nann.-Bremek., M. floripara (Rammeloo) Rammeloo, M. horrida Ing, M. vesparia (Batsch) Nann.-Bremek. ex G.W. Martin & Alexop. Observations: Subclade H3 encompassed species with pilate spores, except for Trichia varia, with uncertain affinities within clade H. A monophyletic group within H3 comprised all studied species circumscribed in Metatrichia. We consider it premature to propose any taxonomic changes until expanding the sampling of the pilate-spored species in Trichiales phylogeny. 4.3.4.5. Subclade H4. ** Perichaena Fr., in Fries & Lindgren, Symb. gasteromyc., fasc. 2: 11 (1817). Type: Lycoperdon corticale Batsch, Elench. fung. 155 (1783) [≡Perichaena corticalis (Batsch) Rostaf., Sluzowce monogr. 293 (1875)]. =Pyxidium Gray, Nat. arr. Brit. pl. 1: 580 (1821). Type: Pyxidium sessile (Bull.) Gray, Nat. arr. Brit. pl. 1: 580 (1821) [= Perichaena corticalis (Batsch) Rostaf., Sluzowce monogr. 293 (1875)]. =Stegasma Corda, Icon. fung. 5: 20 (1842). Type: Stegasma depressum (Lib.) Corda, Icon. fung. 5: 58 (1842) [≡ Perichaena depressa Lib., Pl. crypt. Arduenna 378 (1837)]. Morphological diagnosis: Sporophores sporocarpic. Peridium dehiscence typically circumscissile. Capillitium tubules of not uniform diameter. Spores ornamented with bacula elements. If dehiscence irregular, then the capillitium poorly developed, consisting of smooth, short tubules, sometimes absent. *** Perichaena agaves (G. Moreno, Liz´ arraga & Illana) GarcíaCunch., J.C. Zamora & Lado, comb. nov. Basionym: Hemitrichia agaves G. Moreno, Liz´ arraga & Illana, in Moreno, Liz´ arraga, Illana, Castillo & Oltra, Rivista. Micol. 43(1): 6 (2000) [≡Trichia agaves (G. Moreno, Liz´ arraga & Illana) Mosquera, Lado, Estrada & Beltr´ an-Tej., in Lado, Cuad. Trab. Fl. Micol. Iber. 16: 82 (2001)]. I. García-Cunchillos et al. Molecular Phylogenetics and Evolution 177 (2022) 107609 17 (MycoBank MB845401). Species included here: P. agaves (G. Moreno, Liz´ arraga & Illana) García-Cunch., J.C. Zamora & Lado, P. corticalis (Batsch) Rostaf., P. depressa Lib., P. dictyonema Rammeloo, P. liceoides Rostaf., P. nigra D. Wrigley, Lado & Estrada, P. quadrata T. Macbr., P. stipitata Lado, Estrada & D. Wrigley. Observations: The genus Perichaena sensu stricto encompasses all the species branching within subclade H4. It includes all traditionally considered Perichaena species with circumscissile dehiscence plus P. liceoides. This genus includes one capillitium lacking species, P. nigra, plus the species P. agaves, previously considered within Trichia. 4.3.4.6. Orphan taxa (OT). ** Cornuvia Rostaf., Vers. Syst. Mycetozoen 15 (1873). Type: Cornuvia serpula (Wigand) Rostaf., in Fuckel, Jahrb. Nassauischen Vereins Naturk. 27–28: 76 (1873). Species included: Cornuvia serpula (Wigand) Rostaf. Observations: Cornuvia is a monospecific genus, and we retain it to highlight its morphological and phylogenetic particularities. ** Trichia Haller, Hist. stirp. Helv. 3: 114 (1768). Type: Trichia ovata Pers., Observ. mycol. 1: 61 (1796) [=Trichia varia (Pers. ex J.F. Gmel.) Pers., Neues Mag. Bot. 1: 90 (1794)]. =? Trichulius Schmidel ex Corda, Icon. fung. 5: 20 (1842). Note: No species names were included in Trichulius by the authors. The identity of the name is based on Schmidel’s (1782) “Trichulius stipitatus globosus”, which illustration and description may apply to several species in Trichiales. Species included (in a strict sense): Trichia varia (Pers. ex J.F. Gmel.) Pers. Observations: Despite presenting pilate spores, the type species of the genus, T. varia, did not branch within subclade H3, although the phylogeny topology at this level is not supported, and we cannot discard alternative groupings. The uncertain phylogenetic position of this species precluded a more precise taxonomic classification of several taxa currently treated under Trichia s.l. Hemitrichia crassifila A. Ronikier & Lado, the third orphan taxon, should be excluded from Hemitrichia s.str.; however, we do not propose any nomenclatural change because it is uncertain to which genus this species belongs. Besides all these taxa, we could not obtain DNA data from the monospecific genus Arcyriatella Hochg. & Gottsb., or study specimens of the also monospecific Minakatella Nann.-Bremek. ex H. Neubert, Nowotny & K. Baumann. Thus, we temporarily retain both genera as possibly distinct, awaiting molecular data that could help to elucidate their phylogenetic relationships. 4.4. Provisional key to the genera of the order Trichiales Our results demonstrate that none of the single genetic regions studied can provide a resolved phylogeny of Trichiales. Even if the combination of multiple regions recovered comparatively more robust I. García-Cunchillos et al. Molecular Phylogenetics and Evolution 177 (2022) 107609 18 results, we should reconsider whether the EF1A is a worthy region to explore phylogenetic relationships in Myxomycetes. Here, we recovered concordant evolutionary histories when considering mitochondrial (mtSSU) and nuclear (18S rRNA, preferred genetic region to infer phylogenies in numerous protist lineages) genetic data. However, phylogenomic studies concerning Myxomycetes (Shchepin et al. 2021), or, at least, including them in a broader context (Kang et al. 2017), are still very incipient. Thus, phylogenomic approaches will become indispensable to determining the still uncertain evolutionary affinities among multiple taxa. Besides, an increasing taxa sampling in our phylogeny has shown evolutionary patterns hitherto unknown for some morphological traits. However, most characters do not define monophyletic groups, and transitions between character states occurred within clades rather than between clades. Ancestral state reconstructions suggested the need to reevaluate some characters and their presumed homology, such as the stalks and their different filling refuse materials in clades G–H or the distinct second peridium layers. Ultrastructural and developmental studies considering Trichiales evolutionary history and morphogenesis of the fruiting bodies will shed light on these emerging questions. The intricate evolutionary scenario hinders the proposal of a complete Trichiales systematics since some relationships remained unsolved, and we still lack phylogenetic information of multiple species. Nevertheless, our phylogeny and revised and updated classification provides a backbone in Trichiales systematics, which serve as a baseline for future studies, and contributes, with a new and broader approach, to the knowledge of the relationships among the species of this lineage in Amoebozoa. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments We thank Emilio Cano for his invaluable help in the tasks of the molecular systematics laboratory and Yolanda Ruiz-Le´ on for her technical support with scanning electron microscopy. We are sincerely grateful to Carlos de Mier for his indispensable assistance with the image processing and Joaquina María García-Martín for her counseling in the methodological aspects of the study. We appreciate the valuable commnets of Enrique Lara, Raquel Pino-Bodas, Pedro Jim´ enez-Mejías, Rub´ en Gonz´ alez-Migu´ ens, and two anonymous reviewers. We also thank the assistance of the staff from the different herbaria: Claes Persson (University of Gothenburg), Dagmar Triebel (Staatliche Naturwissenschaftliche Sammlungen Bayerns), Lisa Castlebury (U.S. National Fungus Collections), and Margarita Due˜ nas (Real Jardín Bot´ anico, CSIC). Besides, we would like to thank Arturo Estrada-Torres, Diana Wrigley de Basanta, Italo F. Trevi˜ no-Zevallos, Marianne Meyer, and Uno Eliasson for their help in obtaining some of the samples. The first author thanks Uppsala University for the support to visit both the Department of Organismal Biology and the Museum of Evolution. Funding This research was supported by the Spanish Government throughout a PhD grant (BES-2015-072763) awarded to the first author, and the Myxotropic Project (grant PGC2018-094660-B-I00, MCIN/AEI/ 10.13039/501100011033/, and “ERDF A way of making Europe”). Appendix A. 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