Research Article |
Corresponding author: Josepa Gené ( josepa.gene@urv.cat ) Academic editor: Thorsten Lumbsch
© 2021 Isabel Iturrieta-González, Dania García, Josepa Gené.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Iturrieta-González I, García D, Gené J (2021) Novel species of Cladosporium from environmental sources in Spain. MycoKeys 77: 1-25. https://doi.org/10.3897/mycokeys.77.60862
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Cladosporium is a monophyletic genus in Cladosporiaceae (Cladosporiales, Dothideomycetes) whose species are mainly found as saprobes and endophytes, but it also includes fungi pathogenic for plants, animals and human. Species identification is currently based on three genetic markers, viz., the internal transcribed spacer regions (ITS) of the rDNA, and partial fragments of actin (act) and the translation elongation factor 1-α (tef1) genes. Using this phylogenetic approach and from morphological differences, we have recognized six new species originating from soil, herbivore dung and plant material collected at different Spanish locations. They are proposed as Cladosporium caprifimosum, C. coprophilum, C. fuscoviride and C. lentulum belonging in the C. cladosporioides species complex, and C. pseudotenellum and C. submersum belonging in the C. herbarum species complex. This study revealed that herbivore dung represented a reservoir of novel lineages in the genus Cladosporium.
Cladosporiales, Cladosporiaceae, hyphomycetes, phylogeny, Spain, taxonomy
Cladosporium is a ubiquitous genus in the family Cladosporiaceae of the recently proposed order Cladosporiales in the Dothideomycetes (
Cladosporium is morphologically characterized mainly by its asexual morph, which shows differentiated conidiophores producing acropetal chains of conidia from mono- or polyblastic conidiogenous cells. Both conidiogenous cells and conidia exhibit conidiogenous loci (scars) with a unique coronate structure, which is composed of a central convex dome surrounded by a raised periclinal rim, usually thickened, refractive and darkly pigmented (
While aiming to explore the diversity of microfungi from Spain, several interesting Cladosporium isolates have been recovered from different environmental samples. Using the above mentioned polyphasic approach and following the Genealogical Phylogenetic Species Recognition (GCPSR) criterion (
Samples of soil, plant debris and herbivore dung were collected between 2016 and 2018 at various Spanish locations. Dilution plating methods were used for isolating fungi from soil and dung samples following the procedure described by
Among the cladosporium-like fungi found, we recovered eight isolates in pure culture which did not match any of the currently accepted species within the genus Cladosporium (Table
Cladosporium species, strain information and GenBank accession numbers for sequences obtained in this study.
Species | Strain number1 | Substrate | GenBank nucleotide accession no. for2: | ||
ITS | act | tef1 | |||
C. caprifimosum | FMR 16532T | Goat dung | LR813198 | LR813205 | LR813210 |
C. coprophilum | FMR 16101 | Unidentified herbivore dung | LR813199 | LR813204 | LR813211 |
FMR 16164T | Unidentified herbivore dung | LR813201 | LR813207 | LR813213 | |
C. fuscoviride | FMR 16385T | Garden soil | LR813200 | LR813206 | LR813212 |
C. lentulum | FMR 16288T | Unidentified leaf litter | LR813203 | LR813209 | LR813215 |
FMR 16389 | Unidentified herbivore dung | LR813202 | LR813208 | LR813214 | |
C. pseudotenellum | FMR 16231T | Garden soil | LR813145 | LR813146 | LR813196 |
C. submersum | FMR 17264T | Submerged plant material | LR813144 | LR813195 | LR813197 |
Genomic DNA was extracted from cultures growing on potato dextrose agar (PDA; Pronadisa, Spain) after 7 days of incubation at 25 °C, following the modified protocol of
The sequences obtained were compared with other fungal sequences deposited in the NCBI database through the BLASTn tool. Alignment of those sequences and the phylogenetic analysis for each locus were performed with the MEGA (Molecular Evolutionary Genetics Analysis) program v. 6.0. (
Phylogenetic reconstructions were made with the three phylogenetic markers (ITS, act and tef1) recommended for an accurate identification at the species level (
Determined by Mega software v. 6.0., the best nucleotide substitution model for ML analysis of the C. cladosporioides complex was General Time Reversible with Gamma distribution and invariant sites (GTR+G+I), and for the C. herbarum complex the best was the Kimura 2-parameter with Gamma distribution and invariant sites (K2+G+I). Bootstrap support value (MLBS) ≥ 70% was considered significant (
The MP analysis was performed using the heuristic search option with TBR (tree bisection and reconnection) branch swapping and 1,000 random sequence additions. Tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RCI) were calculated. Bootstrap analysis was based on 1,000 replications. Maximum parsimony bootstrap support value (PBS) ≥ 70% was considered significant (
Determined by jModelTest (
Final sequence alignments and trees generated in this study were registered in TreeBASE under the submission number S27350 (http://treebase.org).
Microscopic features of the Cladosporium isolates were obtained from cultures growing on synthetic nutrient-poor agar (SNA; 1 g of KH2PO4, 1 g of KNO3, 0.5 g of MgSO4 × 7H2O, 0.5 g of KCl, 0.2 g of glucose, 0.2 g of sucrose, 14 g of bacteriological agar, 1 L of distilled water) after 7 to 14 days at 25 °C in the dark, mounted onto semi-permanent slides with Shear's solution (
Three individual phylogenies (ITS, tef1 and act), carried out for the C. cladosporioides and C. herbarum species complexes, were visually very similar and the ILD test showed that the three loci datasets were congruent in both complexes (P = 0.16) and could be combined. Phylogenies obtained by ML, MP and BI also showed a visual topological congruence and were similar to that obtained by other authors (
Maximum likelihood (ML) tree obtained from the combined analysis of ITS, tef1 and act sequences of 101 strains from the C. cladosporioides complex. The tree is rooted with C. sphaerospermum
For the C. herbarum species complex, the alignment comprised 1,057 bp (ITS 503 bp, tef1 309 bp and act 245 bp) with 407 bp variable sites (ITS 101 bp, tef1 186 bp and act 120 bp) and 240 bp phylogenetically informative sites (ITS 27 bp, tef1 123 bp and act 90 bp), using Cercospora beticola (
Maximum likelihood (ML) tree obtained from the combined analysis of ITS, tef1 and act sequences of 58 strains from C. herbarum complex. The tree is rooted with Cercospora beticola
The eight unidentified isolates did not match any known lineage of Cladosporium species, six were related to the C. cladosporioides species complex and two to the C. herbarum complex, and together they represented six new phylogenetic species in the genus.
In the combined phylogeny of the C. cladosporioides complex, 71 species were delineated (Fig.
In the C. herbarum complex, 40 species were phylogenetically well-delimited, including two novel lineages each represented by FMR 16231 and FMR 17264 (Fig.
The percentages of identity between the six putative new Cladosporium species and their relatives are summarized in Table
Percentage of identity between the novel Cladosporium and their closest species.
Species | Closest taxa | Loci | ||
---|---|---|---|---|
ITS | act | tef1 | ||
C. caprifimosum (FMR 16532) | C. asperulatum 1 | 100 | 99.12 | 89.02 |
C. coprophilum (FMR 16101 and 16164) | C. chasmanthicola | 100 | 97.22 | 96.79 |
C. sinuatum 1 | 100 | 97.65 | 97.50 | |
C. fuscoviride (FMR 16385) | C. alboflavescens | 100 | 97.79 | 96.75 |
C. lentulum (FMR 16288 and 16389) | C. exasperatum | 100 | 92.2 | 81.4–82.7 |
C. longicatenatum | 100 | 95.75 | 90.87 | |
C. parapenidielloides | 100 | 95.28 | 90.48 | |
C. pseudotenellum (FMR 16231) | C. tenellum 1 | 100 | 97.5–100 | 83.2–84.1 |
C. submersum (FMR 17264) | C. subcinereum | 100 | 98.57 | 95.98 |
The name refers to goat dung, the substrate where the species was isolated (capra = goat and fimus = dung, with the adjectival Latin suffix -osus, indicating abundance or full or marked development).
Spain, Catalonia, Tarragona province, La Fatarella, from goat dung, Mar. 2017, I. Iturrieta-González, M. Guevara-Suarez & J. Guarro (holotype
Mycelium in vitro superficial and immersed, composed of septate, branched, subhyaline, smooth to verruculose hyphae, 1–2 μm wide. Conidiophores dimorphic, micronematous or macronematous, arising from lateral or terminal hyphae, erect to slightly flexuous, non-nodulose, septate, branched or unbranched, 8–137 μm long, 2–4 μm wide, pale brown, slightly verrucose. Conidiogenous cells integrated, terminal, cylindrical, sometimes geniculate at the apex, 22–44 × 3–4 μm, bearing up to four conidiogenous loci, darkened and refractive. Ramoconidia aseptate, almost cylindrical, 10–24 × 2–4 μm [av. (± SD) 15.8 (± 3.4) × 3.1 (± 0.45)], olive to pale brown, smooth. Conidia forming branched chains, with up to five conidia in the terminal unbranched part, aseptate, olive to pale brown, smooth; small terminal conidia ellipsoidal to obovoid, 3–7 × 2–3.5 μm [av. (± SD) 5.7 (± 0.83) × 2.4 (± 0.43)]; intercalary conidia ellipsoidal to somewhat fusiform, 6–11.5 × 2–3 μm [av. (± SD) 7.8 (± 1.06) × 2.6 (± 0.39)]; secondary ramoconidia ellipsoidal to almost cylindrical, 9–14 × 2.5–3.5 μm [av. (± SD) 11.3 (± 1.6) × 2.9 (± 0.26)].
(14 d at 25 °C). Colonies on OA reaching 24–25 mm diam., dark green (30F8), flat, slightly dusty, aerial mycelium scarce, margin regular; reverse dark green (30F8) to black. On PDA attaining 34–35 mm diam., olive (3E6/3F4), slightly umbonate, radially folded, velvety, aerial mycelium scarce, margin slightly lobate; reverse dark green (30F8) to olive (3E4). On SNA reaching 25–26 mm diam., olive (3E8), flat, dusty, aerial mycelium scarce, margin regular; reverse dark green (30F8) to black.
Optimum 20 °C, maximum 30 °C, minimum 5 °C.
Spain.
Although C. caprifimosum clearly belongs to the C. cladosporioides species complex, our multi-locus analysis did not reveal any phylogenetic relationship with other species in the complex. It is represented by a single branch placed distance from other Cladosporium species (Fig.
Name refers to the substrate where the species was isolated, unidentified herbivore dung (ancient Greek, kópros = dung + phílos = loving).
Spain, Extremadura, Badajoz province, Granja de Torrehermosa, unidentified herbivore dung, Jan. 2017, J. Cano (holotype
Mycelium in vitro superficial and immersed, composed of septate, branched, pale brown, smooth hyphae, 3–5 μm wide. Conidiophores macronematous, arising laterally or terminally from hyphae, erect to slightly flexuous, non-nodulose, septate, unbranched, up to 124 μm long, 3–4 μm wide, pale brown, smooth. Conidiogenous cells integrated, terminal, rarely intercalary, cylindrical, (7–)14–33 × (2–)3–4 μm, bearing up to 3 conidiogenous loci, slightly darkened and refractive. Ramoconidia 0(–1)-septate, subcylindrical to cylindrical, 9–19 × 3–5 μm [av. (± SD) 12.3 (± 2.8) × 3.9 (± 0.54)], pale brown, smooth. Conidia forming branched chains, with up to five conidia in the terminal unbranched part, aseptate, pale brown, smooth to verruculose; small terminal conidia ellipsoidal to slightly obovoid, 4.5–7 × 2.5–4 μm [av. (± SD) 6 (± 0.64) × 3.1 (± 0.31)]; intercalary conidia ellipsoidal, 6–10.5 × 2.5–4 μm [av. (± SD) 7.7 (± 1.32) × 3.3 (± 0.37)]; secondary ramoconidia subcylindrical to cylindrical, 7–12.5 μm long × 3–5 μm [av. (± SD) 9.6 (± 1.7) × 4.2 (± 0.51)].
(14 d at 25 °C). Colonies on OA reaching 21–22 mm diam., olive (2F6) to black, dark green margin (30F4), flat, slightly dusty at the center, aerial mycelium scarce, margin regular; reverse dark green (30F8) to black. On PDA attaining 36–37 mm diam., olive (2F6/2E3), greenish gray margin, slightly depressed and irregularly folded at the center, velvety, aerial mycelium scarce, margin regular; reverse dark green (30F8/27F3). On SNA reaching 27–28 mm diam., olive (3F6/2F8), flat, slightly dusty, aerial mycelium scarce, margin regular; reverse dark green (30F8) to black.
Optimum 20 °C, maximum 25 °C, minimum 5 °C.
Spain.
Spain, Extremadura, Badajoz province, Granja de Torrehermosa, unidentified herbivore dung, Mar. 2017, J. Cano (FMR 16101).
Based on the multi-locus analysis (Fig.
Name refers to the dark green reverse of the colonies of the species growing in all agar media tested (fuscus = dark brown, blackish or figuratively dull and viridis = green).
Spain, Catalonia, Tarragona province, Cambrils, Samà Park, garden soil, Feb. 2017, I. Iturrieta-González & J. Gené (holotype
Mycelium in vitro superficial and immersed, composed of septate, branched, subhyaline to pale brown, smooth to verruculose hyphae, 1–3 μm wide. Conidiophores semi-macronematous to macronematous, arising laterally and terminally from hyphae, sometimes reduced to conidiogenous cells, septate, erect to slightly flexuous, branched or unbranched, sometimes geniculate at the apex, up to 56 μm long, 3–4 μm wide, pale brown, smooth to verruculose. Conidiogenous cells terminal and subterminal, cylindrical to slightly clavate, 8–27 × 3–4 μm, bearing up to 4 conidiogenous loci, darkened and refractive. Ramoconidia 0–1(–3)-septate, subcylindrical to ellipsoidal, 7.5–22 × 2.5–4 μm [av. (± SD) 12.8 (± 3.9) × 3 (± 0.43)], pale brown, smooth to verruculose. Conidia in branched chains with up to 4 conidia in the terminal unbranched part, pale brown, smooth to verruculose, with protuberant, slightly darkened and refractive hila; small terminal conidia aseptate, globose, subglobose to obovoid, 3–6 × 2–3.5 μm [av. (± SD) 4.5 (± 0.66) × 3 (± 0.39)]; intercalary conidia aseptate, ellipsoidal to somewhat limoniform, 4.5–7 × 2.5–4 μm [av. (± SD) 5.7 (± 0.70) × 3.2 (± 0.36)]; secondary ramoconidia 0(–1)-septate, subcylindrical to ellipsoidal 6–11.5 × 2.5–4 μm [av. (± SD) 8.8 (± 1.64) × 3.1 (± 0.40)].
(14 d at 25 °C). Colonies on OA reaching 31–32 mm diam., olive (3F8) to dark green (30F5), olive final edge (2F8), flat, velvety, aerial mycelium scarce, margin regular; reverse dark green (30F5) to black. On PDA attaining 44–46 mm diam., gray to olive to olive yellow (3D1/2E5/2C6), white at the final edge, flat, velvety, aerial mycelium scarce, margin regular; reverse dark green (30F8) to black, with a whitish final edge. On SNA reaching 34–35 mm diam., olive (3F8), flat, velvety, aerial mycelium scarce, margin regular; reverse dark green (30F8), olive final edge (3F3).
Optimum 25 °C, maximum 30 °C, minimum 5 °C.
Spain.
Cladosporium fuscoviride is closely related to C. alboflavescens (Fig.
Name refers to its slower growth with respect to the phylogenetically related species (lentus = figuratively slow, with Latin adjectival suffix -ulus = diminutive).
Spain, Catalonia, Tarragona province, Tarragona, unidentified leaf litter, Feb. 2017, I. Iturrieta-González (holotype
Mycelium in vitro superficial and immersed, composed of septate, branched, subhyaline to yellowish brown, smooth to verruculose hyphae, 1–4 μm wide. Conidiophores macronematous, arising laterally and terminally from hyphae, septate, erect to slightly flexuous, unbranched, sometimes geniculate at the apex, occasionally branched, up to 406 μm long, 3–4 μm wide, pale brown to brown, smooth to verrucose. Conidiogenous cells integrated, terminal and subterminal, cylindrical to subcylindrical, 11–27 × 2–4(–5) μm, bearing up to 5 conidiogenous loci, darkened and refractive. Ramoconidia 0(–2)-septate, subcylindrical to cylindrical, 10.5–23 × 2.5–4.5 μm [av. (± SD) 14.2 (± 2.61) × 3.2 (± 0.52)]; pale brown, smooth to verruculose. Conidia forming branched chains with up to 5 conidia in the unbranched part of the chain, pale brown, smooth to slightly verruculose, with protuberant, slightly darkened and refractive hila; small terminal conidia aseptate obovoidal to ellipsoidal, 4.5–7.5 × 1.5–2.5 μm [av. (± SD) 5.8 (± 0.81)) × 2.7 (± 0.29)]; intercalary conidia 0(–1)-septate, ellipsoidal to subcylindrical, 6–10.5 × 2–3 μm [av. (± SD) 8.4 (± 1.31) × 2.3 (± 0.34)]; secondary ramoconidia 0(–1)-septate, ellipsoidal to subcylindrical, slightly constricted at septum when present, 7.5–14.5 × 2–3 μm [av. (± SD) 10.5 (± 2.05) × 2.5 (± 0.30)].
(14 d at 25 °C). Colonies on OA reaching 19–20 mm diam., olive (3F8), flat, velvety, aerial mycelium scarce, margin regular; reverse dark green (30F8) to black. On PDA attaining 28–36 mm diam., dark green (27F8), with a whitish final edge, slightly umbonate, radially folded, velvety, aerial mycelium scarce, margin slightly lobulate; reverse olive brown (4E4), whitish at the edge. On SNA reaching 22–23 mm diam., olive (3F5), flat, slightly dusty, aerial mycelium scarce, margin fimbriate; reverse dark green (30F8) to black.
optimum 20 °C, maximum 30 °C, minimum 5 °C.
Spain.
Spain, Catalonia, Tarragona province, Poblet, unidentified herbivore dung, Mar. 2017, I. Iturrieta-González, M. Guevara-Suarez & J. Guarro (FMR 16389).
Our phylogeny shows C. lentulum included in a well-supported terminal clade together with the ex-type strains of C. exasperatum, C. parapenidielloides and C. longicatenatum, three species all described from plant material collected in Australia (
The name refers to “C. tenellum”, the closest phylogenetic species.
Spain, Catalonia, Tarragona province, Reus, garden soil, Feb. 2017, I. Iturrieta-González (holotype
Mycelium in vitro superficial and immersed, composed of septate, branched, subhyaline to pale brown, smooth-walled, occasionally tuberculate and with abundant swellings, hyphae, 2–3(–4.5) μm wide. Conidiophores macronematous, arising laterally or terminally from hyphae, erect to slightly flexuous, non-nodulose, occasionally geniculate at the apex, septate, unbranched, occasionally branched, up to 146 μm long, 2.5–3 μm wide, pale brown, smooth to slightly verruculose. Conidiogenous cells integrated, terminal or intercalary, cylindrical, sometimes geniculate, 15–32 × 2.5–3 μm, with up to five conidiogenous loci, thickened, darkened and refractive, often crowded at the apex. Ramoconidia rarely formed, 0(–1)-septate, ellipsoidal to subcylindrical, 9–14.5 × 4–5.5 μm [av. (± SD) 11.6 (± 1.60) × 4.6 (± 0.44)], pale brown, verruculose. Conidia forming branched chains, with up to four conidia in the terminal unbranched part, aseptate, pale brown, verruculose to verrucose; small terminal conidia subglobose to obovoid, 4–7 × 3–5 μm [av. (± SD) 5.8 (± 0.77) × 3.9 (± 0.60)]; intercalary conidia ellipsoidal to limoniform, 6–8.5 × 3–5 μm [av. (± SD) 7.4 (± 0.73) × 3.8 (± 0.50)]; secondary ramoconidia 0(–2)-septate, ellipsoidal to subcylindrical, 7–12.5 × 4–5 μm [av. (± SD) 9.6 (± 1.76) × 4.4 (± 0.33)] with 1–3 distal hila.
(14 d at 25 °C). Colonies on OA reaching 21–22 mm diam., olive (2F8/2F4), flat, velvety, aerial mycelium scarce, margin fimbriate; reverse dark green (30F8) to black. On PDA attaining 29–30 mm diam., olive gray (3E2/3F2), paler at the periphery, radially folded, velvety, aerial mycelium scarce, margin slightly lobate; reverse dark green (30F8) to black. On SNA reaching 21–22 mm diam., olive (2F8), flat, slightly powdery, aerial mycelium scarce, margin fimbriate; reverse dark green (30F8) to black.
Optimum 20 °C, maximum 30 °C, minimum 5 °C.
Spain.
Based on the phylogeny of the C. herbarum complex (Fig.
Name refers to the aquatic habitat where the substrate (submerged plant material) of the fungus was collected (submersus = submerged, verb in participle, from submergere).
Spain, Catalonia, Tarragona province, Cornudella del Montsant, Siurana’s swamp, submerged plant material, Feb. 2018, I. Iturrieta-González, E. Carvalho & J. Gené (holotype
Mycelium in vitro superficial and immersed, composed of septate, branched, subhyaline to pale brown, smooth-walled to verruculose hyphae, 1–3 μm wide. Conidiophores dimorphic, micronematous or macronematous, arising laterally and terminally from hyphae, erect to slightly flexuous, nodulose, geniculate at the apex, septate, unbranched, occasionally branched with small prolongations just below the septum, up to 77 μm long, 3–5 μm wide, pale brown to brown, smooth to verruculose. Conidiogenous cells integrated, terminal and intercalary, geniculate, nodulose, 11–28 × 3–6 μm, bearing up to five conidiogenous loci, darkened and refractive. Ramoconidia rarely formed, 0(–1)-septate, sometimes constricted at the septum when present, cylindrical to subcylindrical, 10.5–24 × 4.5–7 μm [av. (± SD) 16 (± 3.6) × 6.1 (± 1.03)], pale brown, verruculose to verrucose. Conidia forming short branched chains, pale brown, verrucose, occasionally verruculose, with protuberant and slightly darkened hila; small terminal conidia aseptate, ovoid to ellipsoidal, 6–12.5 × 3.5–7 μm [av. (± SD) 7.8 (± 1.63) × 4.8 (± 0.79)]; intercalary conidia and secondary ramoconidia 0–1-septate, ellipsoidal or subcylindrical, 7.5–16 × 4.5–8 μm [av. (± SD) 11 (± 2.18) × 5.7 (± 0.99)].
(14 d at 25 °C). Colonies on OA reaching 22–23 mm diam., brownish gray to olive brown (4E2/4E4), umbonate, velvety, aerial mycelium scarce, margin slightly irregular and fimbriate; reverse dark green to olive brown (6F8/4E3). On PDA attaining 26–28 mm diam., olive (3F3/1F5), slightly umbonate, radially folded, velvety, aerial mycelium scarce, margin irregularly undulate; reverse dark green (30F9) to black with brownish red (9C6) areas observed between 15 and 20 °C and a white edge. On SNA reaching 21–22 mm diam., olive (3E3), slightly umbonate, loosely cottony, margin fimbriate; reverse dark olive brown to golden gray (3E3/4C2).
Optimum 20 °C, maximum 35 °C, minimum 5 °C.
Spain.
Cladosporium submersum is related to C. subcinereum, and morphologically differentiated by having shorter conidiophores (up to 77 μm vs up to 140 μm), shorter conidiogenous cells (11–28 vs 16–38 μm), shorter ramoconidia (10.5–24 vs 19–59 μm), and longer terminal conidia (6–12.5 vs 5–7 μm), which are ovoid to ellipsoidal in our species and globose to subglobose in C. subcinereum (
Cladosporium is a well-delineated genus, the taxonomic structure and phylogenetic relationships of its species have been investigated in several studies over the last decade, so far giving rise to a genus of more than two hundred well-established species (
To our knowledge, Cladosporium species as dung inhabiting fungi have been reported in a very few studies, C. cladosporioides and C. herbarum being the most reported species (
Although no temperature studies have been systematically applied to characterize most Cladosporium species (
The authors are grateful to Lei Cai of Institute of Microbiology of the Chinese Academy of Sciences (Beijing, China) for sending the sequences of C. tianshanense included in this study. This study was supported in part by the Spanish Ministerio de Economía y Competitividad, grant CGL2017-88094-P.
Table S1
Data type: Species name and strains data
Explanation note: Species, strain information and GenBank accession numbers of the sequences included in the phylogenetic analyses.