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Species diversity in Penicillium and Talaromyces from herbivore dung, and the proposal of two new genera of penicillium-like fungi in Aspergillaceae

Coprophilous fungi are saprotrophic organisms that show great diversity, mainly on herbivore dung. The physico-chemical characteristics of this peculiar substrate combined with the high level of fungal adaptation to different environmental conditions offer the perfect setting for discovering new taxa. This study focused on the species diversity of penicillium-like fungi isolated mainly from herbivore dung collected at different Spanish locations. From 130 samples, a total of 104 isolates were obtained, and 48 species were identified. Preliminary identifications were based on morphology and partial β-tubulin (tub2) gene sequences. Putative new taxa were characterized by a multi-gene sequencing analysis testing the tub2, the internal transcribed spacer rDNA (ITS), calmodulin (cmdA), and RNA polymerase II second largest subunit (rpb2) genes, and a detailed phenotypic study. Using this polyphasic approach and following the genealogical concordance phylogenetic species recognition (GCPSR) method, we propose the new genera Penicillago (for Penicillium nodositatum) and Pseudopenicillium (for Penicillium megasporum and P. giganteum) in the family Aspergillaceae, and 11 new species, including seven Penicillium, three Talaromyces and one Pseudopenicillium. A lectotype and epitype are designed for Penicillium nodositatum. Our results show that the species diversity of penicillium-like fungi on herbivore dung has not been widely studied and that this substrate seems to be a good reservoir of interesting Eurotialean fungi.


INTRODUCTION
Herbivore dung is a complex substrate that has a high amount of readily available carbohydrates with high nitrogen content, water-soluble vitamins, growth factors and mineral ions (Bell 1983). Due to the nutritional properties, together with its physical structure, pH, and the varying moisture content, it is an excellent substrate for a great diversity of fungi to grow (Bell 1983, Richardson 2001, Herrera et al. 2011. Ascomycetes accounts for the largest number of species found on dung from herbivorous animals; genera such as Ascobolus, Lasiobolus, Podospora or Thelebolus practically only occur on this substrate (Webster 1970, Bell 1983, Richardson 2001. On the contrary, other ascomycetes like Eurotialean penicillium-like fungi are not considered predominantly coprophilous (Krug et al. 2004), but they can be commonly found on this substrate when its soluble materials are exhausted, and its relative humidity has reduced to below 85 % (Kuthubutheen & Webster 1986). However, species diversity of this latter group has been poorly studied on herbivore dung. Although Seifert et al. (1983) provided a list of near 20 Penicillium species isolated from animal faeces, their identification was based exclusively on phenotypic features.
Traditionally, based on similarities in ecology, morphology and extrolite production, most of the Penicillium species considered coprophilous (i.e. P. brevistipitatum, P. clavigerum, P. concentricum, P. coprobium, P. coprophilum, P. formosanum, P. glandicola and P. vulpinum) were classified in series Claviformia (Seifert & Samson 1985, Frisvad & Samson 2004, Wang & Zhuang 2005. Currently, the new concept of the genus, which is based on molecular taxonomy, coprophilous penicillia belongs in section Robsamsonia , which also includes other species previously assigned to series Urticicolae (Frisvad & Samson 2004). Furthermore, other species of Penicillium previously found on dung currently belong to the genus Talaromyces . Coprophilous species reported in this latter genus are T. atroroseus, T. helicus, T. flavus, T. muroii and T. trachyspermus, and two others T. dupontii and T. emersonii, which have been transferred to respectively, Thermomyces and Rasamsonia (Masunga et al. 2006, Frisvad et al. 2013, Yilmaz et al. 2014. The taxonomy of penicillium-like fungi has changed dramatically due to phylogenetic data.  demonstrated that Penicillium is phylogenetically moreclosely related to Aspergillus (family Aspergillaceae) than to Talaromyces (family Trichochomaceae). In the last decade, the species of these genera have been delineated on the basis of a polyphasic approach that has mainly included the evaluation of morphological and multi-locus sequence analyses testing with the internal transcribed spacer region rDNA (ITS), and the β-tubulin (tub2), calmodulin (cmdA), and/or the DNAdependent RNA polymerase II largest subunit (rpb2) genes (Peterson 2008, Houbraken et al. 2014a, Samson et al. 2014, Yilmaz et al. 2014). In addition, tub2 was recommended as an identification marker in Penicillium  and Talaromyces (Yilmaz et al. 2014).

Molecular identification and phylogenetic analysis
Isolates were cultured on PDA or malt extract agar (MEA; Difco, Detroit, USA) for 7─14 d at 25 °C. DNA was extracted using the FastDNA® kit protocol (MP Biomedicals, Solon, OH) and for the lysis step done with a FastPrep® FP120 cell disrupter (Thermo Savant, Holbrook, NY).
Preliminary species identifications were carried out BLASTing tub2 DNA sequences using GenBank. In the case of putative new species, the ITS region, including the 5.8S rRNA gene, and fragments of cmdA and/or rpb2 genes were also amplified and sequenced. The primer pairs used were: ITS5/ITS4 for ITS (White et al. 1990), Bt2a/Bt2b for tub2 (Glass & Donaldson 1995), CMD5/CMD6 for cmdA (Hong et al. 2006), and RPB2-5F/ RPB2-7Cr for rpb2 (Liu et al. 1999). The amplification protocol and PCR conditions were performed using methods and primers previously described (Peterson 2008, Samson et al. 2014, Yilmaz et al. 2014. The amplified products were purified and sequenced at Macrogen Corp. Europe (Amsterdam, the Netherlands) with a 3730xl DNA analyzer (Applied Biosystems, Foster City, CA). Consensus sequences were obtained using SeqMan v. 7.0.0 (DNASTAR, Madison, WI). Newly generated sequences and their GenBank/EMBL accession numbers are summarized in Table 1.
Sequences were retrieved from GenBank taking into account the last update of the database of the International Commission of Penicillium and Aspergillus (http://www. aspergilluspenicillium.org), which includes all the accepted species for Penicillium and Talaromyces (Samson et al. 2014, Yilmaz et al. 2014. Single and concatenated phylogenetic analyses were performed to delineate putative new species and the phylogenies corresponding to each section of those genera were properly reconstructed.
Data sets for each locus were aligned individually using ClustalW (Thompson et al. 1994), in MEGA v. 6.0 software (Tamura et al. 2013), refined with MUSCLE (Edgar 2004) under the same platform, and manually adjusted when needed. Larger alignments including different sections of each genus was performed using the MAFFT tool in the EMBL-EBI Web Services portal (https://www.ebi.ac.uk/Tools/msa/mafft/) and manually adjusted in MEGA v. 6.0. Phylogenetic reconstructions by maximum likelihood (ML) and Bayesian inference (BI) were carried out using MEGA v. 6.0 and MrBayes v. 3.1.2 (Huelsenbeck & Ronquist 2001, Ronquist & Huelsenbeck 2003, Tamura et al. 2013, respectively. For ML analyses, the trees were inferred using Nearest-Neighbour-Interchange as a heuristic method and gaps were treated as partial deletion with a 95 % site coverage cut-off. Phylogenetic support for internal branches was assessed by 1 000 ML bootstrapped pseudoreplicates and bootstrap support (bs) ≥ 70 % was considered significant. The BI analyses were performed using five million Markov chain Monte Carlo (MCMC) generations, with two runs (one cold and three heated chains) and samples were stored every 1 000 generations. The 50 % majority-rule consensus tree and posterior probability values (pp) were calculated after discarding the first 25 % of the samples. A pp value ≥ 0.95 was considered significant. The best substitution models for each data partition were estimated using jModelTest v. 2.1.3 according to the Akaike criterion (Darriba et al. 2012, Guindon & Gascuel 2003. The length, number of variable and phylogenetic informative sites, and substitution models for each data partition are summarized in Table 2. The resulting trees were plotted using FigTree v. 1.3.1 (http://tree.

Phenotypic characterisation
Phenotypic characterization was carried out using standard growth conditions suggested by Visagie et al. (2014b) and Yilmaz et al. (2014). Briefly, isolates were cultured onto MEA 2 %

RESULTS
During the study, 130 dung samples were processed and 104 isolates of penicillium-like fungi were recovered, including three from soil (Table 1). Preliminary identification based on morphology and tub2 sequences showed that most of the isolates belonged to Penicillium (n = 91; 87.5 %) and a few to Talaromyces (n = 9; 8.65 %), while four of them (3.85 %) could not be assigned to any genus despite exhibiting a penicillium-like morphology.
To maximize the quality of the analyses, three separate tub2 alignments were done for the different genera studied. We also carried out additional analyses of single-gene ITS, cmdA and rpb2 and combined datasets from the different genes, corresponding to the genera or sections where putative new species were included ( Table 2). The topology of the trees was similar for both methods, with ML trees used to represent the results. Bootstrap and BI posterior probability values are indicated on relevant branches. Final identification of the isolates, resulting from the phylogenetic and phenotypic characterization, is shown in Table 1.
In section Roquefortorum (92 % bs/1 pp) three isolates (FMR 15031, FMR 15032, FMR 15188) were clustered in the same terminal clade. This is a small section of closely related species (i.e. P. carneum, P. paneum, P. psychrosexualis and P. roqueforti) commonly associated with the cheese industry . Our isolates were related to P. roqueforti, but formed an independent and distant branch that could represent an undescribed species. In order to evaluate possible intra-and inter-specific variability within the species and confirm the phylogenetic position of our putative new species, we performed an additional analysis with tub2 gene (see Fig. S1 in supplemental material) with more sequences of the species available in GenBank. This demonstrated that P. roqueforti is divided in two clades with an intra-specific variability of around 0.08 %, with our isolates forming a separate branch from the P. roqueforti complex. The concatenated analysis using ITS, tub2, cmdA and rpb2 (Fig. 2) supported the novelty of these three isolates, being therefore proposed as the new species P. mediterraneum. Although, P. mediterraneum and P. roqueforti have Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Inst E-mail: p.crous@westerdijkinstitute.nl identical ITS barcodes, both species have unique tub2, cmdA and rpb2 sequences.
In section Chrysogena (74 % bs/--pp), which includes wellknown species usually isolated from soil and also from indoor environments , two dung isolates were identified as P. chrysogenum and P. flavigenum, respectively. Whereas the two isolates of the section Penicillium (--% bs/1 pp), a group that currently mainly includes plant pathogenic penicillia , were both identified as P. expansum.
Isolates of section Robsamsonia (88 % bs/0.97 pp) were identified as P. brevistipitatum (n = 1), P. concentricum (n = 2), P. coprobium (n = 2), P. coprophilum (n = 1), and P. griseofulvum (n = 7); however, five isolates (FMR 15192, FMR 15210, FMR 15211, FMR 16481 and FMR 16491) could not be assigned to any known species. The preliminary tub2 analysis, but also the concatenate phylogeny ( Fig. 3) including the currently accepted species in the section, showed that the five isolates grouped together in a supported and undescribed lineage closely related to the ex-type strain of P. glandicola. An additional tub2 analysis (see Fig. S2 in supplemental material) with more sequences of P. glandicola showed that our isolates and P. glandicola were 97.4 % similar. The genetic differences and morphological peculiarities observed in this group of dung isolates justify the description of a new species, namely P. synnematicola.
The only isolate (FMR 15041) of section Turbata (95 % bs/1 pp) did not fit with any species of the section, although it was closely related to P. bovifimosum. In Paradoxa (94 % bs/--pp) 10 isolates were included; four were identified as P. atramentosum, one as P. magnielliptisporum, and the rest (FMR 15040, FMR  Fig. 3. ML tree of Penicillium section Robsamsonia inferred from the combined ITS, tub2, cmdA, and rpb2 loci. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above 70 %/0.95 are indicated on the nodes. The tree is rooted to P. brevicompactum CBS 257.29. The name in red is the new species described in this study. T = ex-type strain.

Editor-in-Chief
Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: p.crous@westerdijkinstitute.nl 15104, FMR 15107, FMR 15191 and FMR 15196) were allocated in three single branches which represent three putative new species for the genus. The concatenated phylogeny of sections Paradoxa and Turbata with the six unidentified isolates is shown in Fig. 4. In the former section, the isolates FMR 15107 and FMR 15040 formed a terminal clade separated from other one formed by FMR 15191 and 15196. The lineages of these two clades were in turn distant from the two terminal branches where FMR 15104 and the ex-type strain of P. atramentosum were located, respectively. Genetic differences of the three undescribed lineages with respect to P. atramentosum (97.07 % and 98.23 % similar with tub2 and concatenate dataset for FMR 15107 and FMR 15040, respectively; 96.2 % and 96.2 % with tub2 and concatenate dataset for FMR 15191 and FMR 15196; 94.68 % and 95 % with tub2 and concatenate dataset for FMR 15104) justifies them as distinct taxa. The three species are proposed below as P. ibericum, P. balearicum and P. fimosum. An additional phylogenetic analysis with tub2 including more sequences of the most closely related species confirms our proposal (see Fig.  S3 in supplemental material). In section Turbata, P. bovifimosum and FMR 15041 were located in the same clade, but the latter formed a long terminal branch that proved to be a distinct species, described below as P. caprifimosum.
Section Ramosa (97 % bs/0.98 pp) only included the unidentified isolate FMR 15038. Currently, the section comprises 13 species (Visagie et al. 2014a, Rong et al. 2016, although two of them, P. lanosum and P. kojigenum, were considered conspecific by . The concatenate analysis performed with ITS, tub2, and cmdA ( Fig.  5), rpb2 was not included due to the lack of sequences for all species in the section, showed that FMR 15038 was placed in an independent branch between two clades, one with P. kojigenum and P. lanosum, and the other with P. jamesonlandense and P.
swiecickii. The similarity among our isolate and its phylogenetic sisters was 98.3 %, proving that our isolate is an undescribed species, which is proposed below as P. beceitense.
The only isolate of section Brevicompacta (99 % bs/1 pp) was identified as P. brevicompactum, a species commonly inhabiting in soil and decaying vegetation, but also described from food, cereals, textiles, clinical specimens and faeces of snake (Pitt 1979, Guevara-Suarez et al. 2016. The seven isolates classified in section Canescentia (98 % bs/1 pp) were identified as P. canescens (n = 1), P. murcianum (n = 3) and P. radiatolobatum (n = 3). Despite tub2 being unable to discriminate between P. radiatolobatum and P. murcianum, these two species can be recognized with the cmdA and rpb2 barcodes.
Section Exilicaulis (99 % bs/1 pp) included eight of our isolates. It is noteworthy that the ML general tree based only on tub2 presented some doubtful results in the species identification of some isolates, especially those belonging to the P. restrictum-clade. However, when we carried out a restricted alignment with only members of the section and reconstructing the ML tree, most of them were satisfactory identified (see Fig.  S4 in supplemental material). These were P. arabicum (n = 2), P. burgense (n = 1), P. cinereoatrum (n = 1), P. momoii (n = 1), and P. rubefaciens (n = 2). The only isolate that could not be identified was FMR 15841, precisely located in the P. restrictumclade. The most recent review of the section by Visagie et al. (2016b) indicates that such a clade needs further revision due to the high variability between strains currently belonging to the clade. Thus, the identification of FMR 15841 remains uncertain until its taxonomy is resolved.
Section Lanata-Divaricata (92 % bs/1 pp) included three dung isolates. One of them was identified as P. brasilianum, a widespread species commonly found in soil (Pitt 1979) and recently also reported from human clinical specimens (Guevara-  . The other two isolates were identified as P. cremeogriseum, a species previously found in forest soil in Ukraine , Visagie et al. 2016a). To our knowledge, this is the first report of P. brasilianum and P. cremeogriseum associated with herbivore dung.
The only species identified in our study belonging to section Stolkia (99 % bs/1 pp) was P. canariense. This species was described from soil in Canary Islands (Peterson & Sigler 2002), the same geographical origin as our isolate. This is the second isolate of the species found so far.
Isolates of section Citrina (98 % bs/1 pp) were identified as P. citrinum (n = 5) and P. sizovae (n = 2). Although we have not found previous records of these species from dung, they have a worldwide distribution, being isolated from soil, foodstuff, and many other types of substrates . This section comprises nearly 40 species, but only those identified here were included in the analysis to simplify the tub2 phylogenetic tree (Fig. 1).
In sections Sclerotiora (77 % bs/--pp) and Cinnamopurpurea (96 % bs/--pp) P. lilacinoechinulatum (n = 1) and P. cvjetkovicii (n = 1) were respectively identified. Although the former species was previously considered synonym of P. bilaiae, they are currently recognised as distinct penicillia (Visagie et al. 2013). P. lilacinoechinulatum is an uncommon species which type was collected from Japanese soil (Abe 1956). Penicillium cvjetkovicii is a recently described from indoor air samples in the USA (Peterson et al. 2015).
Aspergilloides (99 % bs/1 pp) was the last section in Penicillium that included dung isolates. The species identified were P. glabrum (n = 3), P. frequentans (n = 2), P. roseoviride (n = 1), and P. rudallense (n = 1). Although the former two are currently accepted as distinct species, they were considered synonyms for a long time . Penicillium frequentans together with P. spinulosum and P. glabrum are generally the most common species in the section, being isolated from a wide range of substrates, including soil, food, bark, and indoor environments ).

Talaromyces phylogeny
The phylogenetic tree based on the tub2 locus (Fig. 6) shows the relationships of the nine Talaromyces isolates included in the study. The aligned dataset was 388 bp long, from which 217 were variable sites and 170 phylogenetic informative. The ML substitution model was K2+G+I, while for BI it was HKY+G+I. Two main clades were formed, representing sections Talaromyces (98 % bs/1 pp) and Trachyspermi (90 % bs/1 pp). Only three sect. Talaromyces species were identified, namely T. muroii (FMR 15496), T. ruber (FMR 15839) and T. sayulitensis (FMR 15842); the rest (FMR 15489, FMR15490, FMR 15199, FMR 15035 and FMR 15307) could not be identified with this single marker, just like the only isolate (FMR 16441) included in section Trachyspermi.
To resolve the taxonomy of these unidentified isolates, we calculated a phylogeny for each section using sequences of the ITS, tub2 and cmdA genes from the accepted species in both sections, except for T. amyrossmaniae in section Trachyspermi. This is a recently described species that morphologically differs from our isolates and species of Trachyspermi by producing short determinate synnemata on both the natural substrate and in vitro (Rajeshkumar et al. 2019). Although rpb2 was sequenced for all our isolates (Table 1), this marker was not incorporated in the concatenated analysis due to the lack of sequences for all ex-type strains. The combined phylogeny of the members of section Talaromyces is shown in Fig. 7. This analysis showed that the unidentified isolates FMR 15489 and FMR 15490 exhibited both a similarity of 99.42 % with respect to the ex-type strain of T. angelicus, a monotypic species described by Sang et al.    2013). Although most of their morphological features matched with those of the protologue of T. angelicus, we observed some phenotypic variation with respect to the description of the species by Yilmaz et al. (2014); i.e, production of diffusible red pigment in one of our isolates (FMR 15490), the colonies of our isolates were deep turquoise and pastel red rather than yellow to white on MEA, they grew faster at 37 °C (on CYA 34-37 mm diam in 7 d vs 25-27 mm in Yilmaz et al. 2014), and exhibited stipes up to 100 μm long (up to 120 μm in Yilmaz et al. 2014). On the other hand, while FMR 15199 was located in an independent branch clearly separated from the other species of the section; FMR 15035 and FMR 15307 clustered together and were closely related to T. curcubitiradicus and T. funiculosus, but with enough genetic difference to be considered a distinct species (see also Fig. S5 in supplemental material). These genetic differences and the particular phenotypic features observed in the three FMR isolates allow us to propose the following new species, T. coprophilus (FMR 15199) and T. pseudofuniculosus (FMR 15035 and FMR 15307). Both can be easily identified with the tub2 marker.

Sect. Talaromyces
The combined phylogenetic analysis of the section Trachyspermi (Fig. 8) showed that the isolate FMR 16441 was located in a single branch within the same clade as T. albobiverticillius, T. erythromellis, T. heiheensis, and T. solicola (88 % bs/0.99 pp). Considering the phylogenetic position of our isolate and the morphological differences observed, the new species T. catalonicus is proposed.

Phylogeny of miscellaneous Aspergillaceae
According to the tub2 phylogeny, isolates FMR 15296, FMR 16442, FMR 14718 and FMR 15299 were related to some penicillia currently excluded from the genus Penicillium (i.e. P. giganteum, P. megasporum and P. nodositatum), but belonging to the family Aspergillaceae (Fig. 9). Previous molecular data on these fungi suggested that they could represent distinct genera in the family (Peterson et al. 2010, Visagie et al. 2013. The aligned dataset of our analysis was 393 bp long, from which 242 were variable sites and 216 phylogenetic informative. The best substitution model for ML was K2 +G+I and for BI it was GTR +G+I. While FMR 15296 and FMR 16442 were located in a wellsupported clade along with the ex-type strain of P. nodositatum (CBS 333.90), the other two (FMR 14718 and FMR 15299) were in a fully supported distant clade together with the ex-type strains of P. giganteum (NRRL 3553) and P. megasporum (NRRL 2232). The phylogeny inferred with sequences of the ITS, tub2 and rpb2 genes (Fig. 10), including the isolates under study and members of different genera of Aspergillaceae, confirms the proposal of two novel genera, which are described below as Penicillago (Pgo.), typified by Penicillium nodositatum, and Pseudopenicillium (Pse.) with Penicillium giganteum and Penicillium megasporum, this latter species here selected as the type. The former genus is phylogenetically more related to Penicillium, whereas the latter to the genus Hamigera.
The analysis of ITS and tub2 showed that the ex-type strain of P. nodositatum (CBS 333.90) and the isolates FMR 15296 and FMR 16442 have practically identical sequences. However, CBS 333.90 was not included in the concatenated phylogeny with the three markers (Fig. 10) since its rpb2 sequence was not available for comparison. Despite this, we observed some cultural difference between our isolates respect to the protologue of P. nodositatum (Valla et al. 1989 and 34-36 mm diam in 7 d, respectively, vs 13-19 mm and 16-30 mm in CBS 333.90) and showed the ability to grow at 37 °C. Considering the phylogenetic position of our isolates and their morphological similarity with P. nodositatum, they were the base to propose the new combination Pgo. nodositata. Other fungus excluded from Penicillium s. str. and closely related to Pgo. nodositata was P. kabunicum (Visagie et al. 2013). Since only the ITS barcode was available for the ex-type strain of the latter species (CBS 575.90) in the GenBank database (MH862240), we constructed an ITS phylogenetic tree with different genera in Aspergillaceae showing that P. kabunicum clustered with Pgo. nodositata (see Fig. S6 in supplemental material). The isolate FMR 14718 matched morphologically and molecularly with P. giganteum, whereas FMR 15299 was placed in a new lineage into the Pseudopenicillium clade (Figs 9, 10). The morphological differences observed, such as having smaller conidia (up to 6 μm) than those of the other two species in the genus (conidia up to 12 μm in Pse. giganteum, and up to 10 μm in Pse. megasporum), and the genetic differences obtained (98 % similar to Pse. giganteum, and 98.16 % similar to Pse. megasporum) allow us to propose the new species Pse. cervifimosum.
Etymology: Name refers to the geographical area (the Balearic Islands) where the species was found.
Distinguishing characters: Penicillium balearicum is resolved in the P. atramentosum-clade containing six species, P. atramentosum, P. balearicum, P. fimosum, P. ibericum (the latter three described here), P. magnielliptisporum and P. mexicanum (Visagie et al. 2014a). Penicillium atramentosum, P. balearicum, P. magnielliptisporum and P. mexicanum have smooth-walled stipes and conidia, and the conidia of the latter two are larger (5 × 4 μm in P. magnielliptisporum, 4 × 3.5 μm in P. mexicanum); while P. atramentosum can be differentiated by its production of red soluble pigment on CYA and by the lack of growth at 30 °C (Pitt 1979, Frisvad & Samson 2004. Penicillium balearicum differs from P. fimosum and P. ibericum mainly by having longer stipes (up to 500 μm long), by the production of abundant brown exudate droplets on YES, and by the moderately strong acid production on CREA.
Etymology: Name refers to Beceite, the vilage where the fungus was found.
Distinguishing characters: Penicillium beceitense is closely related to P. lanosum and P. kojigenum, two species that are indistinguishable according to our phylogeny of the section Ramosa (Fig. 5). To date, there is no updated description for either P. lanosum or P. kojigenum. The protologue of P. kojigenum described conidia that are rough-walled, measuring 2.2-2.6 μm (Smith 1961). The absence of growth at 30 °C is the relevant feature to distinguish P. beceitense from other species of the section, such as P. chroogomphum, P. jamesonlandense, P. lanosum, P. ribium, and P. soppii (Rong et al. 2016).
Etymology: From the Latin capra = "goat", and fimosum = "dungdwelling", describing the substrate from where the species was isolated.
Distinguishing characters: All the species in section Roquefortorum show fast growth on CYA and MEA and produce rough-walled conidiophores. Penicillium roqueforti is the closestrelated species to P. mediterraneum, and it can be distinguished by a more restricted growth on CYA at 30 °C (5-15 mm diam) and by its blackish green colony reverse on YES (Frisvad & Samson 2004.
Etymology: Name refers to the formation of synnemata.
Distinguishing characters: Section Robsamsonia includes some species that produce synnemata, e.g. P. coprophilum, P. glandicola and P. vulpinum. Penicillium coprophilum and P. vulpinum can be differentiated from our new species by their smooth-walled stipes . Penicillium synnematicola is closely related to P. glandicola, a species with world-wide distribution isolated from oak trees and forest soil, including dungy soil and mouse dung , and both produce coarsely roughened synnematous Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: p.crous@westerdijkinstitute.nl conidiophores. However, P. glandicola can be distinguished by having shorter phialides (7.5-10.5 µm) and ellipsoidal and yellow-green conidia. In addition, the colonies of P. glandicola show orange-brown to brown and bright orange-red reverse on CYA and YES, respectively, and tend to have a more restricted growth on both media (17-30 mm on CYA; 19-36 mm on YES) .
Etymology: Name refers to the phylogenetic closeness to Penicillium (Fig. 10).
Diagnosis: Morphologically differs from Penicillium by its symmetrically biverticillate conidiophores, ampulliform to acerose phialides with a fine long neck, and conidia with conspicuous disjunctors. Conidiophore structure resembles Talaromyces, but phylogenetically distant.
Mycelium superficial and immersed, composed of septate, branched, canary yellow to chrome yellow hyphae. Conidiophores symmetrically branched, penicillium-like, composed of long, septate, hyaline stipes, often terminating in a small vesicle giving rise to a verticil of metulae, occasionally irregularly branched; metulae appressed to divergent, cylindrical to somewhat obpyriform, bearing a compact verticil of conidiogenous cells. Conidiogenous cells phialidic, ampulliform to acerose, with a fine and long neck. Conidia dry, catenate, with conspicuous disjunctors, subglobose to ellipsoid, coarsely echinulate, subhyaline to brown; conidial chains short to moderately long. Sexual morph unknown.

Notes:
The genus Penicillago is introduced to accommodate Penicillium nodositatum. It is noteworthy that this species has had a confusing taxonomic history. Initially, Valla et al. (1989) classified P. nodositatum in the subgenus Biverticillium from its morphological affinity with members of the series Islandica (Pitt 1979). Later, based on molecular data, it was considered a member of the Penicillium section Sclerotiora, and tentatively placed in synonymy with P. bilaiae by , despite morphological differences in the conidiophore structure (biverticillate in P. nodositatum vs monoverticillate in P. bilaiae). However, Visagie et al. (2013) noticed that in the previous study that synonymy was based on a culture of P. bilaiae (CBS 330.90) mislabeled as P. nodositatum. The origin of the problem was probably introduced by  since they erroneously designated on pag. 66 "No. 330.90 (CBS)" as neotype of P. nodositatum. In fact, the International Commission of Penicillium and Aspergillus (http://www. aspergilluspenicillium.org/) included the living ex-type strain of the herbarium specimen of P. nodositatum as CBS 333.90, this therefore being the correct ex-type strain for the species. Newly sequence data of that strain showed that P. nodositaum was closely related to P. kabunicum, and neither of them belonged in Penicillium s. str. , Visagie et al. 2013. We observed that the only sequence available for P. kabunicum (CBS 574.90, ITS sequence GenBank MH862240) showed a 99.64 % of similarity with that of P. nodositatum (CBS 333.90, ITS sequence GenBank NR_103703) (Fig. S6 in supplemental material), suggesting they could be conspecific. However, the protologue of P. kabunicum (Baghdadi 1968) disagree with that of P. nodosotatum (Valla et al. 1989) in features such as the conidial morphology (globose, smooth-walled and hyaline in P. kabunicum vs ellipsoid to subglobose, echinulate and brown in P. nodosotatum). Therefore, further comparative studies will be needed to determine whether both species should be treated as synonym.
The possibility of examining two Spanish isolates that fit morphologically with P. nodositatum and their DNA sequences coincide with those of the ex-type strain of this species, has allowed us to delineate a new genus of penicillium-like fungi in the family Aspergillaceae (Figs 9, 10). Because herbarium type material for P. nodositatum is lost according to Visagie et al. (2013), the illustrations included in the protologue of the species (Valla et al. 1989) are selected here as lectotype, and the metabolically inactive culture CBS 333.90 as epitype deposited in the Westerdijk Fungal Biodiversity Institute.
Etymology: Pseudomeaning "false"-, name refers to the morphological similarity but not belonging to Penicillium.
Diagnosis: Phylogenetic closeness to Hamigera, but differs in the lack of sexual morph and by its brown globose conidia with conspicuous disjunctors. Morphologically differs from Penicillium and Penicillago by its short and often irregularly branched conidiophores producing large conidia in short chains.
Mycelium superficial and immersed, composed of septate, branched, hyaline to subhyaline hyphae. Conidiophores undifferentiated, reduced to conidiogenous cells arising directly from the main hyphae, or differentiated composed of short to moderately long, aseptate or few septate, hyaline to brown stipes, terminating in an irregular penicilli; penicilli varying from monoverticillate to partly biverticillate, with few metulae bearing each a verticil of conidiogenous cells; metulae terminal or subterminal, generally divergent, cylindrical or apically inflated. Conidiogenous cells phialidic, ampulliform, with swollen base and tapering abruptly to a slender neck of variable length. Conidia dry, catenate, with conspicuous disjunctors, globose to subglobose, with spinulose walls, brown, dark in masses; conidial chains short. Sexual morph unknown. Notes: The genus Pseudopenicillium is proposed to accommodate a new species and two species previously classified in the genus Penicillium, P. giganteum and P. megasporum, the latter being selected as the type since it was the first described. Penicillium giganteum and P. megasporum were recovered from soil samples in India and the UK, respectively. Despite their different geographical origin, both were considered conspecific and classified in series Megasporus (Pitt 1979). Penicillium asperosporum, also included in this series, was later shown to be a synonym of P. montanense in section Aspergilloides . Multi-gene phylogenies (Peterson et al. 2010 demonstrated that P. megasporum and P. giganteum were distinct species, closely related to the genus Hamigera. Our phylogeny confirms this relationship and shows that Hamigera and the afore-mentioned Penicillium species comprise two sister clades with enough genetic differences to be considered distinct genera (Figs 9, 10).
Distinguishing characters: Talaromyces coprophilus is characterised by the production of orange-red ascomata with spiny ellipsoidal ascospores, the lack of growth on DG18, and a good growth on other culture media tested. Phylogenetically, it forms an independent and distant branch in an unsupported clade where the ex-type strains of T. cnidii, T. flavovirens, T. siamensis, and T. xishaensis are found (Fig. 7). Morphologically, T. coprophilus resembles T. flavovirens in the production of a sexual morph; however, the latter differs in slower growth on CYA at 25 °C (19-20 mm after 7d), and larger ascospores (4-7 × 3-4 μm) (Yilmaz et al. 2014).
Distinguishing characters: Talaromyces cucurbitiradicus, recently described by Su & Niu (2018), and T. funiculosus are the closest related species to T. pseudofuniculosus. The former can be distinguished from the other two by the production of chlamydospores and absence of growth at 37 °C. Talaromyces funiculosus differs from the new species by its divergent metulae (appressed in T. pseodofuniculosus) and by the pigmentation of the conidiophores, being olivaceous in T. funiculosus and brownish yellow in T. pseudofuniculosus. Moreover, T. pseudofuniculosus has a restricted growth on CREA (2-4 mm diam; 20-30 mm diam in T. funiculosus) and does not produce funiculose colonies, a typical feature of T. funiculosus mainly on MEA and OA (Yilmaz et al. 2014).

DISCUSSION
In recent years, the phylogeny of the genera Penicillium, Talaromyces and other members of family Trichocomaceae sensu lato was studied in order to clarify their taxonomic relationships. Subsequently, many species were re-classified and new taxonomic approaches proposed for species delineation and identification. Considering that several of these fungi have been discovered from specific and poorly studied substrates using modern taxonomic techniques , Barbosa et al. 2018), we explored the species diversity of penicillium-like fungi from herbivore dung collected in different Spanish regions (Andalusia, Balearic and Canary Islands, Castile and Leon, Catalonia and Galicia) using the gene markers recommended for their study , Yilmaz et al. 2014.
Our results agree with other studies in which a large set of isolates of penicillium-like fungi could be identified with the tub2 sequence analysis (Guevara-Suarez et al. 2016, Visagie et al. 2014a, Chen et al. 2016, Barbosa et al. 2018. It is a good marker, easy to amplify and sequence, and useful for the classification at the sectional and species level, and even for detection of putative undescribed species. However, in addition to the ITS and tub2 barcodes, the characterization of penicillia requires multi-gene analyses along with other markers such as fragments of cmdA and/ or rpb2 loci. Based on this molecular approach, we were able to find a remarkable diversity of penicillium-like fungi that have not been described on herbivore dung so far. Among the 104 isolates recovered, we identified a total of 48 species, 38 Penicillium including seven new taxa (i.e. P. balearicum, P. beceitense, P. caprifimosum, P. fimosum, P. ibericum, P. mediterraneum, and P. synnematicola), seven Talaromyces including three new taxa (T. catalonicus, T. coprophilus, and T. pseudofuniculosus), and three species in the newly prosposed genera Penicilago (Pgo. nodositata) and Pseudopenicillium (Pse. giganteum, and the new species Pse. cervifimosum). In addition, of note is that the highest number of species identified (34 out of 48) and novel taxa described (9 out of 11) were recovered from samples collected in the Mediterranean area, namely Catalonia and the Balearic island of Mallorca (Table 1). This is not surprising if we consider that this area is recognized as one of the most species rich regions in Southern Europe (Cuttellot et al. 2008, Rundel et al. 2018).
Among Penicillium isolates, those of the sections Fasciculata and Robsamsonia were the most frequently isolated, accounting nearly 20 % of the isolates in each section. It is of note, however, that in Fasciculata most isolates identified were of P. crustosum, while in Robsamsonia, as expected, we found the most species diversity among the sections included here. This latter section currently comprises 11 species ) and we identified five of these taxa, in addition to the new P. synnematicola. This species would seem to be a common coprophilous fungus in the Mediterranean area, as its isolates were recovered exclusively from samples collected in different locations from Mallorca and Catalonia. As mentioned before, Houbraken et al. (2016) restricted the majority of coprophilous Penicillium species in section Robsamsonia. However, our results clearly show that the coprophilous penicillia are distributed across different and phylogenetically distant sections of the genus (Fig. 1). In sections such as Aspergilloides, Exilicatus and Paradoxa, we even found a similar diversity to the section Robsamsonia. Of particular note is Paradoxa with the highest number of new penicillia proposed in this study (P. ibericum, P. fimosum and P. balearicum). However, we also identified P. atramentosum and P. magnielliptisporum as first records on dung. The former species is commonly associated to cheese and P. magnielliptisporum was described from house dust (Visagie et sal. 2014a). The only species in the section reported previously from animal faeces was P. paradoxum, an aspergilluslike Penicillium collected in different countries such as Denmark, India, New Zealand, the Netherlands and UK , Rajeshkumar et al. 2016. Paradoxa is a very small section (nine species, including our new proposal) in comparison to sections Aspergilloides and Exillicaulis, which have 50 species each, but without any species reported from dung (Houbraken et al. 2014d, Visagie et al. 2016. The most Talaromyces isolates found in our survey were from section Talaromyces. This is the largest section in the genus, with nearly 60 species, 22 of them described in the last four years from different substrates, including human clinical specimens (Visagie et al. 2015, Crous et al. 2016, Cheng et al. 2016, Wang et al. 2016a, b, 2017, Yilmaz et al. 2016, Guevara-Suarez et al. 2017, Barbosa et al. 2018, Jiang et al. 2018. We added two more new taxa in the section (T. coprophilus and T. pseudofuniculosus), but also identified several interesting species firstly reported from dung, such as T. angelicus, T. muroii, T. ruber, and T. sayulitensis. Unlike T. ruber, which is a worldwide species isolated from soil, air and fuel (Yilmaz et al. 2012), the other three are considered uncommon fungi previously found in the roots of Angelica gigas in Korea (Sang et al. 2013), in soil in Taiwan (Chen et al. 2016), and in house dust in Mexico (Visagie et al. 2014a), respectively. The other new Talaromyces species described is T. catalonicus in section Trachyspermi. This is a much smaller section than Talaromyces, with 19 species including our new one (Yaguchi et al. 1996, Yilmaz et al. 2014, Chen et al. 2016, Luo et al. 2016, Wang et al. 2016b, Guevara-Suarez et al. 2017, Barbosa et al. 2018, Rajeshkumar et al. 2019). The most recently described are T. aerius from indoor air in China (Chen et al. 2016), T. amyrossmaniae from decaying fruits in India (Rajeshkumar et al. 2019), T. brasiliensis from honey and nest of bee in Brazil (Barbosa et al. 2018), T. heiheensi from rotten wood in China (Wang et al. 2017), T. minnesotensis from human specimens in the USA (Guevara-Suarez et al. 2017), and T. rubrifaciens from air outlets of public buildings also in China (Luo et al. 2016), although this latter was considered a synonym of T. albobiverticillius by Chen et al. (2016). To date, however, there are no reports of species in either of these two sections isolated from dung samples.
Penicilago nodositata seems to be a common fungus usually associated with the roots of Alnus in Europe (Valla 1989, Sequerra et al. 1997; it is not surprising therefore to recover this fungus from animal dung, in our case from a wild pig, which feeds on a wide variety of plant material including roots, shoots and fruit. Conversely, Pse. giganteum is a rare species only known from its type collection, a garden soil sample from India (Roy & Singh 1968). In addition to the latter species, we also recovered from herbivore dung other uncommon species of Penicillium and Talaromyces, which are monotypic or have very few isolates available for investigation. These are P. canariense (section Stolkia), P. cremeogriseum (section Lanata-Divaricata), P. momoii (section Exilicaulis), and P. roseoviride (section Aspergilloides), including the three Talaromyces species mentioned before from section Talaromyces (i.e. T. angelicus, T. muroii and T. sayulitensis). Therefore, our study highlights that herbivore dung is a good substrate for recovering interesting species of penicillium-like fungi, although their ecological role on that substrate remains unclear and would merit further investigation. Fig. S1. ML tree of selected Penicillium section Roquefortorum species, inferred from tub2. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above 70 %/0.95 are indicated on the nodes. The tree is rooted to P. sansonianum AS 3.5403 and P. osmophilum CBS 462.72. The name in red is the new species described in this study. T = ex-type strain. Bootstrap support values/Bayesian posterior probability scores above 70 %/0.95 are indicated on the nodes. The tree is rooted to P. brevicompactum CBS 257.29. The name in red is the new species described in this study. T = ex-type strain. Fig. S3. ML tree of selected Penicillium section Paradoxa species, inferred from tub2. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above 70 %/0.95 are indicated on the nodes. The tree is rooted to species of section Tubata. The names in red are the new species described in this study. T = ex-type strain. Fig. S4. ML tree of selected Penicillium section Exilicaulis species, inferred from tub2, including the isolates belonging to this section recovered in this work. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above 70 %/0.95 are indicated on the nodes. The tree is rooted to P. trzebinskii CBS 382.48. T = ex-type strain.