﻿Three new species of Trimmatothelopsis (Acarosporales, Acarosporaceae) from southwestern North America

﻿Abstract The discovery and study of three new species of Trimmatothelopsis from Southwestern North America, T.californica, T.mexicana, and T.novomexicana, adds not only to the diversity of the genus and family but generated new insights into the occurrence of two ascus types in the genus and the variety of conidiogenous cells and conidia. Trimmatothelopsis now includes 15 species with a mainly Holarctic distribution (Asia, Europe, North America) and one species in Australia. A key is supplied to the genus. An overview of the genus Trimmatothelopsis is given, including differentiation from other genera of Acarosporaceae. The monotypic genus Thelocarpella is considered to be a synonym of Trimmatothelopsis. The new combination Trimmatothelopsiswirthii is proposed. The ascus type is shown to be variable in the genus with species with two types being intermixed with each other in our phylogeny.


Introduction
There are estimated to be approximately 416 described species of Acarosporaceae worldwide (Knudsen et al. 2023).Before these new discoveries in this paper, the genus Trimmatothelopsis included 11 crustose lichens, which occur on calcareous and non-calcareous rocks or in soil crusts in Asia, Australia, Europe, and North America (Knudsen and Lendemer 2016;Knudsen et al. 2021a).In southwestern North America the genus was only known from California and Nevada.For a history of the genus see Knudsen et al. 2021a.
The main form of the thallus in Trimmatothelopsis is areoles or squamules dispersed and/or with congregations of thalline units but not forming an areolate crust.Trimmatothelopsis americana, for instance differs in having carbonized lecideoid apothecia with its algal layer occurring in a biofilm at its base.The terricolous species, T. benedarensis, T. rhizobola, and T. terricola have a hypothallus of well-developed rhizohyphae with the two latter species having rhizohyphae in root-like bundles.In the genus, the areoles or squamules are either pale with a reddish brown circle around the apothecia (some specimens of T. dispersa and T. schorica), or light or dark brown.The apothecia of Trimmatothelopsis have a disc usually 0.5 mm or less in width (Knudsen and Lendemer 2016).In all species the hymenium is 150-350 µm high, globose, and widest at the equator.The paraphyses are thin, 1-2 µm wide.The asci contain 200-300 ascospores or more.The ascospores are usually ellipsoid and not longer than 5 µm, except in T. schorica in which they are spherical, 7-10(-12) µm, or broadly ellipsoid 7-9 × 5-7 µm.
The asci in the genus are functionally unitunicate but some variation is found among the species.While most of the species of the genus have an IKI-Acarospora-type ascus stain (Hafellner 1993), T. americana and T. gordensis have an ascus with IKI+ light blue tholus and space between the inner and outer wall of the ascus with a darker blue area in the upper layers of the tholus.Trimmatothelopsis montana has a blue ascus stain but in the type specimen no darker layer was observed in the upper layers of the tholus.
The subhymenium is either IKI+ blue and euamyloid or hemiamyloid, blue turning red.The hypothecium is usually narrow 10-40 μm continuous with a narrow parathecium of usually the same width that merges into the cortex.The algal layer is usually continuous and not interrupted, extending down the sides of the apothecia.The medulla is usually ca.200 μm thick.No secondary metabolites have been detected with thin-layer chromatography.
The IKI+ blue ascus stain has some similarity to the ascus stains in the genera Timdalia and Pleopsidium but neither have a darker blue area in the upper layers of the tholus (Hafellner 1993;Hafellner and Türk 2001).Both genera are monophyletic with Timdalia also differing in producing psoromic acid and Pleopsidium in having yellow thalli (Westberg et al. 2015).Because of the high hymenium the species of Trimmatothelopsis are easily confused with the monophyletic genus Myriospora which has an Acarospora-type ascus stain (Westberg et al. 2011).In determining specimens, one must first determine the appropriate genus.The high hymenium and globose apothecia easily separate Trimmatothelopsis species from most of the species of the non-monophyletic Sarcogyne and Acarospora groups, both which have Acarospora-type ascus stains.
Our objective is the taxonomic and phylogenetic study of the rich diversity of Acarosporaceae in southwestern North America where occur 93 described species and where new taxa are still being discovered (Knudsen et al. 2021b;Knudsen et al. 2023).This study of diversity is laying the foundation for phylogenomic work which we have begun with our current study of the Acarospora strigata group and possible hybridization and introgression in the evolution of the family.

Herbarium study
We studied our recent collections and specimens in SBBG (UCR lichen herbarium transferred to SBBG in 2022 and 2023), at OBI, and in the private herbarium of Jana Kocourková and Kerry Knudsen (hb. K&K).This continues our study of the species included in this genus since 2011 (Knudsen et al. 2011;Knudsen andLendemer 2016. Knudsen et al. 2021a).The morphology of specimens was examined with dissecting microscopes.At 1000× with compound microscopes the anatomy of hand sections was examined and measured in water.Ascospore and conidia measurements of species are indicated as (min-)(x ¯ − SD)-x ¯-(x ¯ + SD)(-max), where 'min' and 'max' are the extreme values observed, x ¯ the arithmetic mean and SD the corresponding standard deviation.They are followed by the number of measurements (n); the length/breadth ratio of ascospores is indicated as l/b and given as x ¯ the arithmetic mean value.The amyloid reaction of the hymenial gel and subhymenium was tested with fresh undiluted IKI (Merck's Lugol for the gram staining method, Sigma-Aldrich 1.09261) (see protocol in Knudsen and Kocourková 2018).The ascus stain was studied in IKI (Hafellner 1993).Thin-layer chromatography (TLC) in solvents A, B', C was performed to identify secondary metabolites (Orange et al. 2001).On completion of the study holotype, isotype and paratype material was placed in BYU-C, SBBG and PRM.

Imaging
Macrophotographs were taken with the digital camera Olympus DP74 mounted on Olympus SZX 16 stereomicroscope using PROMICRA QuickPHOTO CAMERA 3.3 software and stacked using Olympus DeepFocus 3.5 module for increasing the depth of field.Microphotographs were taken with a digital camera Olympus DP74 mounted on an Olympus BX51 light microscope fitted with Nomarski interference contrast and using PROMICRA QuickPHOTO CAMERA 3.

DNA extraction, PCR amplification and sequencing
DNA was extracted from 12 dried herbarium specimens via (Suppl.material 1) the Invisorb Spin Plant Mini Kit, according to the manufacturer's protocol with slight modifications (i.e.eluted in 50 μL of DNA, instead of 100 μL, and incubated in buffer for 15 minutes before final centrifuging).Total extracted DNA was stored at -20 °C.The quality and yield of DNA isolated was checked on a 1% agarose gel and DNA concentration and purity were then measured precisely using a UVS-99 spectrophotometer (ACTGene).The selected markers for this study were the internal transcribed spacer (ITS; White et al. 1990), the large subunit of the nuclear ribosomal DNA (nrLSU; Vilgalys and Hester 1990), and the small subunit of the mitochondrial ribosomal DNA (mtSSU; Zoller et al. 1999).The ITS, nrLSU, and mtSSU regions were amplified via polymerase chain reaction (PCR).
Each reaction contained 1 μL (20-25 ng) of extracted genomic DNA, 10 μL of 2x MyTaq Red DNA Polymerase (Bioline), 8.2 μL of water, 0.4 μM of forward/reverse primer (10 μM) for a total reaction volume of 20 μl.Conditions for nrITS, mtSSU nrDNA: initial denaturation 95 °C for 5 min, followed by five cycles (95 °C for 33 s, 56 °C for 30 s, and 72 °C for 30 s), then ten cycles (95 °C for 30 s, 54 °C for 30 s, and 72 °C for 30 s), and twenty cycles (95 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s) with a final extension 72 °C for 15 min.Conditions for the nLSU: initial denaturation 95 °C for 1 min, followed by five cycles (95 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s) and finally 30 cycles (95 °C for 30 s, 52 °C for 30 s, and 72 °C for 60 s), with a final extension 72 °C for 10 min.Before sequencing, the PCR products were purified using the enzymatic method ExoSap-ITTM Express Reagent provided by Thermo Fisher (Scientific, Inc.) according to the manufacturer's protocol.PCR products were run on a 1.0% agarose gel via electrophoresis and stained with ethidium bromide for 20 min.Purified PCR products, water, and forward primer (8 μL in total volume) were sequenced by BIOCEV, Vestec, Czech Republic.

Sequence alignment and phylogenetic analysis
Sequences were checked against the UNITE and NCBI databases for contamination.All newly generated sequences were deposited in GenBank (Suppl.material 1).The sequences were proofread and concatenated manually into a single data set using SEQUENCHER version 5.4.6 (GeneCodes).Sequences were aligned using the multiple sequence alignment online service MAFFT version 7 with 'G-INS-1' strategy (Katoh and Toh 2008).Indels longer than 1 bp were coded by the simple gap coding method (Simmons and Ochoterena 2000) as implemented in SEQSTATE 1.4.1 (Müller 2005).A partition homogeneity test (ILD) with heuristic search was performed under one thousand replicates between the ITS, nLSU, and mtSSU sequences by PAUP* version 4.0a169 (Swofford 2002) to determine whether the partitions were homogeneous for test of congruence.The final alignments are accessible at TreeBASE database (https:// treebase.org/)under submission ID 29625.For phylogenetic analyses two trees were generated (i.e.ITS + mtSSU + nLSU and only mtSSU data sets), the GTR+I model was selected as the best-fitting model of nucleotide substitution based on the Akaike Information Criterion using JMODELTEST 2.1.10for each gene (Darriba et al. 2012).Phylogenetic trees were constructed using MRBAYES 3.2.2(Ronquist and Huelsenbeck 2003).Input data was formatted for MRBAYES via the FABOX (Villesen 2007) with slightly modification (i.e.analyses were executed under the GTR+GAMMA nucleotide substitution model).Three replicate analyses with four chains each were computed 30,000,000 generations, sampling every 1000 th generation.After this number of runs, the average standard deviation of split frequencies reached a value lower than 0.01, indicating that convergence was reached.The data were additionally analyzed using maximum likelihood (ML) method.Tree searches for ML analyses were executed under the GTR+GAMMA nucleotide substitution model (general time reversible substitution model with a gamma model of rate heterogeneity) in RAxML v.8.2.10 (Stamatakis 2014).The Bayesian inference tree with posterior probabilities and ML phylogenetic tree with 1000 replicates were visualized using Fig-TREE v1.4.4 (Rambaut 2012).Sequences of Pycnora sorophora was the outgroup and five of the six recognized genera of the Acarosporaceae [three monophyletic genera (Myriospora, Pleopsidium, Timdalia) and selected specimens representing the non-monophyletic Acarospora and Sarcogyne groups] were used to recover the monophyletic Trimmatothelopsis clade.The Trimmatothelopsis clade contained all available sequences of the species in the genus.The results recover the same results as a large family tree in Knudsen et al. (2023).
The final alignment contained 1834 concatenated characters, consisting of 1-436 (ITS), 437-1084 (mtSSU), 1068-1834 (nLSU) nucleotide positions.Of these characters, 1338 were constant, 179 were variable and parsimony-uninformative and 317 were parsimony-informative.The topology of the ML tree confirmed the tree topology obtained from the Bayesian Inference and, therefore, only the Bayes tree is presented (Fig. 1).The MCMC analysis of the three concatenated genes was run for 30,000,000 generations, resulting in trees.The alignment contained a total of 538 unique site patterns.The analyses identified a well-supported Trimmatothelopsis clade with 100 percent bootstrap support (BPP) in the combined data set (Fig. 1).The new species were nested within the Trimmatothelopsis clade.Species relationships within the clades are resolved, and relationships amongst all clades were resolved with strong confidence.
In the phylogeny (Fig. 1), the species with an IKI+ ascus stain are recovered intermixed with species with an Acarospora-type ascus, suggesting that the variation in the amyloidity of the ascus is of limited phylogenetic importance.
The three new species of Trimmatothelopsis described here and the placement of Trimmatothelopsis wirthii (C.Roux) K. Knudsen & Kocourk. in the genus, brings the total number of species to fifteen.Diagnosis.Similar to Trimmatothelopsis oreophila but differing in having regularly elevated apothecia mostly 0.5 mm wide in a dark brown crown, in having areoles with an elevated mycelial base instead of being squamulose with a stipe, and in having short conidia (1.8-)2.0-2.26-2.5(-2.8)× (0.8-)0.82-0.98-1.1(-1.3)µm, l/b 2.3.
Habitat and distribution.Notes.Trimmatothelopsis californica differs from other species in the genus in having short conidia.It can easily be confused with Acarospora elevata H. Magn., a species often on granite at high elevations from California to the Rockies (Knudsen 2007).Acarospora elevata has an elevated parathecial crown but has usually a lower hymenium than T. californica (ca 60-120 µm) as well as dark blue euamyloid hymenial gel vs. IKI+ hemiamyloid hymenial gel.
Specimens of Trimmatothelopsis can be misidentified as Myriospora.Myriospora differs in having shorter conidia usually less 3 µm long.Both are well-supported as separate genera (Fig. 1; Westberg et al. 2011;Knudsen et al. 2021c).Because of the height of hymenium and a poor understanding of Myriospora taxonomy at that time in North American lichenology, T. californica was identified as Acarospora scabrida Hedl.ex H. Magn.(Knudsen 2005(Knudsen , 2007)).The circumscription of A. scabrida is heterogenous in Knudsen (2007).We do not recognize Myriospora scabrida (Hedl.ex H. Magn.)K. Knudsen & L. Arcadia as occurring in California.
Etymology.This is the first species of the genus discovered in Mexico and is named in honor of the work of all the Mexican lichenologists in North America.
Ecology and distribution.On siliceous red sandstone, known only from the type locality at Nuevo León, Sabinas Hidalgo, Presa Sombretillo, at an altitude of 385 m.
Notes.Trimmatothelopsis mexicana has the same IKI ascus stain with a light blue tholus and blue space between inner and outer layer of the ascus as in five other species of Trimmatothelopsis.The blue stain in upper tholus was observed once.It differs from all species in genus in having the longest conidia and the second longest conidiogenous cells.Trimmatothelopsis rhizobola has the longest conidiogenous cells (Knudsen and Lendemer 2016).They are filiform, 15-20(-40) × 1 μm.
We were expecting Trimmatothelopsis mexicana in the Chihuahuan Desert in New Mexico, but did not find it.Instead we discovered another new species, T. novomexicana.Trimmatothelopsis mexicana is currently known only from the type locality.We are sure someone will collect this distinctive species in Mexico in the future, and it may occur at least in New Mexico or Texas in the United States.
Etymology.This species is named after the state of New Mexico where it was discovered.
Ecology and distribution.The holotype was collected on scattered limestone rocks in full sun on a low crest in the Chihuahuan Desert in New Mexico at an elevation of 1850 m.It was growing in pinyon-juniper woodland on uplifted and eroded Permian reefs.It was associated with Acarospora peltastica and Circinaria contorta.We expect it to be locally frequent and to occur in the adjacent Guadalupe National Park in pinyon-juniper areas.It was definitely rare about 100 miles from the type locality at the Carrizozo Malpais lava beds, on calcareous reconsolidated sandstone, growing with Peltula obscurans var.deserticola, at the base of a north-facing sandstone slope with junipers at elevation of 1950 m.We studied this area extensively and only collected it once.Trimmatothelopsis novomexicana often occurred in small patches with a few apothecia among other lichens on rough limestone.
Notes.Four other species of Trimmatothelopsis have similar ascus stains with a darker blue stain in upper layers of a pale blue tholus: T. americana, T. gordensis, T. mexicana and T. wirthii.Trimmatothelopsis novomexicana differs from T. americana especially in having apothecia lacking a carbonized outer surface.Trimmatothelopsis novomexicana differs from T. gordensis especially in having areolate thallus vs. a thallus of discreet areoles.Trimmatothelopsis novomexicana differs from T. mexicana in having longer conidia and in lacking a stipe.Trimmatothelopsis novomexicana differs from T. wirthii especially in having areoles vs. large squamules up to 7 mm wide.
The narrow morphological and genetic differences between these similar species are probably based on a long geographical isolation from each other.One mystery of the protologue of Thelocarpella gordensis was a description of periphyses in the ostiole of the ascomata (see the drawing in Navarro-Rosinés et al. 1999).Our colleague Martin Westberg borrowed the holotype from Claude Roux.He was told he could photograph it (a photograph we used in this study) but could not dissect any of the areoles (Westberg, pers. comm.)Later, based on its recovery in the Acarosporaceae, the description of periphyses in gordensis was treated as a misinterpretation of the incurving melanized hyphae of the parathecium merging with the cortex around the punctiform discs (Knudsen and Lendemer 2016).In studying Trimmatothelopsis novomexicana we discovered the source of the misinterpretation of T. gordensis as a perithecioid lichen.The authors had mistaken the elongated black tips of the terminal cells of ostiole hyphae of the pycnidia for being hyphae of a perithecia (see Fig. 4J).In the specimens of T. novomexicana there is also no evidence apothecia grow out of stromata that contained pycnidia as in Sarcogyne similis H. Magn.(see pictures of the synonym Sarcogyne reebiae K. Knudsen in Knudsen et al. 2011).In the recent description of Trimmatothelopsis wirthii (see below) it is stated there are no periphyses in the "ostiole".Roux also describes pycnidia with dark ostiole hyphae.Roux described melanized horizontal hyphae incurving from the parathecium and merged with the cortex around the punctiform apothecia as pseudopapillae (Roux 2021).d'andésite, 27. Aug. 2020, V. Wirth, herb. Cl. Roux (holotype, n.v., isotype, SNMS, n.v.), syn.nov.
Description.See Roux (2021).Roux treats Trimmatothelopsis wirthii as having a perithecioid ascomata based upon a morphological genus concept we have already rejected (Knudsen and Lendemer 2016).Also see notes below and for Trimmatothelopsis novomexicana.
Ecology and distribution.Known only from the type collection from France massif des Vosges, département du Haut-Rhinn, collected on calcareous andesite on a vertical rock face at 1060 m.The size, shape and pale color is similar to some montane specimens of T. oreophila which differs in having an Acarospora-type ascus stain.
Notes.Thelocarpella is a morphological genus concept which treats two species of Acarosporaceae as perithecioid lichens with periphyses or pseudoparaphyses (Navarro-Rosinés et al. 1999;Roux 2021).We treat these two species as Acarosporaceae with paraphyses.Roux (2021) has accused us of promoting a phylogenetic concept of Trimmatothelopsis.Our concept of the genus Trimmatothelopsis is integrative, based on the congruence between phylogenetic data and classic taxonomic analysis (Leavitt et al. 2018;Lücking et al. 2021).In the results section we discuss the distribution IKI+ blue ascus in Trimmatothelopsis which does not support the idea that the stain distinguishes Thelocarpella from all other species in Trimmatothelopsis.For further discussion of T. wirthii see discussion of T. novomexicana above.
Unfortunately, the type of Trimmatothelopsis wirthii was not sequenced but it would be in the Trimmatothelopsis clade based on its anatomy and morphology.Its large squamules up to 7 mm in width distinguish Trimmatothelopsis wirthii from all other species in genus.Though disagreeing with Roux about the genus Thelocarpella we praise him for his excellent description of A. wirthii despite treating it as a perithecioid lichen.

World Key of Trimmatothelopsis with taxonomic citations
3 software.The figure plates were processed with the module Figure Maker fitted to the same software.

Figure 1 .
Figure1.Bayesian inference tree obtained by phylogenetic analysis using a combined data set of ITS, mtSSU and nLSU sequences of 27 members of Acarosporaceae.Bayesian posterior probability (BPP ≥ 0.95) and maximum likelihood bootstrap values (ML ≥ 70%) are indicated above branches (BPP/ML).Pycnora sorophora was used as outgroup.In bold are species with IKI+ blue asci.The remaining species of Trimmathelopsis not in bold have IKI-asci.