Crustose Calicioid Lichens and Fungi in Mountain Cloud Forests of Tanzania

A total of 26 crustose calicioid lichens and fungi were found in Tanzania. Most of them belong to a group of species with wide distributions in cool areas of both hemispheres and occasional occurrence in high mountains at low latitudes. In Tanzania calicioids mainly occur in the middle and upper forest zones and their niches are found on the bark of old trees and on lignum, most of them restricted to mountain cloud forests. Calicioids are rare and often red-listed, and are also bioindicators of long forest continuity. Consequently, they form an important biota in mountain cloud forests and deserve attention in the context of preserving biodiversity and developing conservation policies. One new species, Chaenothecopsis kilimanjaroensis, is described. Chaenotheca hispidula and Pyrgillus cambodiensis are reported as new to Africa and Calicium lenticulare and Chaenothecopsis debilis are reported as new to Tanzania.


Introduction
Calicioid lichens and fungi represent an artificial, antiquated concept and not a natural grouping of species. Very early on the "Calicioidea" [1][2][3] were recognized as a group characterized by having a mazaedium (mature spores being collected on the surface of the fruit body) and typically they were crustose lichens with stalked fruit bodies. With this circumscription, they were also later to be considered a prime example of a natural group, often ranked as an order, Caliciales [4]. This approach was later codified, e.g., by Keissler [5] as "Coniocarpinae". The emphasis on the cardinal character of a mazaedium also led to the inclusion of fruticose lichens (e.g., Sphaerophorus Pers.). In addition, minute fungi with apothecia superficially similar to those originally included in the "Calicioidea" came to be added, for example, genera like Chaenothecopsis Vain. and Stenocybe Nyl. ex Körb., both of which later were transferred to Mycocaliciales [6]. That the calicioids are not monophyletic was first suggested by [7] based on morphological and secondary chemistry data, a conclusion later supported by molecular data in later studies [8,9]. The calicioids are thus derived from various ascomycete clades, such as Arthoniales, Lecanorales, and Pyrenulales, and primarily characterized by the parallel development of passive spore dispersal and/or a superficial [7,9] morphological similarity with archetypic "Calicioidea" in which the ascomata are differentiated into a stalk and distinct capitulum [8]. The ascomata accumulate mature spores on the surface of the fruit bodies as a thick, often black or brown layer, the mazaedium, and unlike most ascomycetes the spore dispersal is passive [7,8]. Calicioids, mazedium-forming and non-mazaediate alike, are often found together in similar habitats, mostly on bark and wood in undisturbed primary forests [10,11]. Calicioids are frequently used in nature conservation as bioindicators of species-rich areas with long forest continuity [11,12]. They are mainly rare and amplifications of the Internal transcribed spacer (nuITS) region. Primers ITS1f [42] and ITS4 [43] were used. The AccuPower PCR PreMix (Bioneer, Daejeon, Korea) was used, adding 3 µL diluted DNA, 1.5 µL of each primer (10 mM), and water to a total volume of 20 µL. The PCR protocol was: initial denaturation for 4 min at 95 • C, followed by 35 cycles of 1 min at 94 • C, 1 min at 54 • C, 45 s at 72 • C, and final elongation for 5 min at 72 • C. The PCR products were visualized by electrophoresis on 1.5% agarose gels. Products were purified using Illustra™ ExoStar buffer diluted 10×, following the manufacturer's protocol. Sequencing was conducted by Macrogen (Available online: www.macrogen.com).

Alignments and Phylogenetic Reconstructions
Five newly generated sequences were aligned along with 37 sequences downloaded from GenBank (Table 1) by using MAFFT v.7 (Available online: https//mafft.cbrc.jp/alignment/server/), after assessment of their quality. The selected DNA sequences downloaded from GenBank, along with the newly produced sequences (Table 1) were assembled and edited using AliView (Available online: https://ormbunkar.se/aliview/) [44]. Phylogenetic relationships and their posterior probabilities (PP) were inferred using a Bayesian approach, and additional support values were estimated using maximum likelihood bootstrap support (MLbs). For the Bayesian analyses, the most likely models of evolution were estimated using the Akaike Information Criterion (AIC) as implemented in Modeltest 3.7 [45]. The GTR + I + G model of evolution was employed.
The Bayesian analysis was executed using MrBayes 3.2.6 [46], where two analyses of two parallel runs were carried out for 10 M generations. Each run included four chains, and trees were sampled every 1000 generations and 25% were discarded as burn in. All runs converged on the same average likelihood score and topology. Maximum likelihood estimates were carried out by RAxML version 8.2.10 using the GTR + G + I model of site substitution [47]. The branch support was acquired by maximum likelihood bootstrapping (MLbs) of 1000 replicates [48], and MLbs ≥ 70% were considered to be significant.

Morphological and Chemical Studies
The morphological study of the thallus and the fruit body was performed on freezing microtome sections 10 to 15 µm thick and on squash preparations under the microscope. The sections were mounted in cotton blue and glycerin.
The color reactions of the thallus and apothecia was tested using a 10% aqueous potassium hydroxide solution (K).

Calicium chlorosporum F. Wilson
First reported from Tanzania by [21] and later also by [41], but so far only known from high altitudes in the Arusha region. Habitat: At the base of old Podocarpus in montane cloud forest. Distribution: A subtropical species described from Australia but widely distributed also in mountainous areas of Africa with occurrences in Ethiopia, Kenya, Madagascar, the Mascarene Islands, South Africa, and Uganda. Additionally reported from Asia (India and Nepal), Australasia [25], and North America.

Calicium diploellum Nyl.
Reported from Tanzania, Morogoro Region, (Uluguru Mts, Luhangalo) by [40]. Habitat: On bark in montane cloud forests. Distribution: Earlier reported from Kenya by [21]. Additionally known from Europe.  [39] as occurring in the Nguru Mts. in the Morogoro region. Subsequently [21] also reported from the Uluguru and Usambara Mts. Further records from Tanzania: Iringa Region (Idete, Udzungwa Mts, Massisiwe); Morogoro Region (Uluguru Mts, Luhangalo) were given by [40] and in addition from the Iringa region, Udzungwa Mts., and Kilimanjaro region (Mt. Kilimanjaro) by [41]. The species is widely distributed in tropical to warm temperate areas in Europe, Asia, the Americas and Australia and/or Asia. Habitat: On wood and bark in montane cloud forests. Distribution: Widely distributed in temperate to warm temperate areas. Known from the Americas, Asia, Australasia and Europe. Previously reported from Burundi [39] as C. subquercinum Asah. and from Kenya [50]. New to Tanzania.

Calicium salicinum Pers.
Habitat: On wood and bark in montane cloud forests. Distribution: First recorded from Kilosa and Morogoro Districts (Morogoro Region) of Tanzania [39], and subsequently from Arusha [21] and Massisiwe (Morogoro Region) by [40].  [39]. Widely distributed and also known from Europe, Asia and South America. Note: Chaenotheca deludens belongs to group of species closely related to C. stemonea. Originally described from New Zealand, Chaenotheca deludens is characterized by having very long-stalked apothecia with flexuous stalks with a slight reddish brown pruina that dissolves in K, rather than forming violet crystals as in C. gracillima (Vain.) Tibell and differing from the latter also in lacking an excipulum. The material from Tanzania SGT 432, however, is morphologically quite different. The Tanzanian material has rather short-stalked non-pruinose apothecia with straight stalks and no K+ reaction was observed. Like C. deludens the material does have a finely granular thallus and catenulate asci. The Tanzanian material is too scanty for an extensive assessment of its morphology. Although the nuITS of the Tanzanian material in a network analyses of the nuITS is close to C. deludens (e.g., to GenBank no. AF408678 from New Zealand, not shown) there are numerous SNPs between them. Material currently referred to C. deludens probably form a species complex of morphologically similar species and this should be further studied.  [21]. Widely distributed in the Northern and Southern Hemisphere. Note: Chaenotheca furfuracea displays intraspecific genetic variation in nuITS sequences. It is characterized by the minutely verrucose spore surface and in this respect Tanzanian material agrees well with material from Europe. (Figure 1). Chaenotheca confusa (described from South America) is similar, but has spores with an ornamentation of minute areolae. Considerable variation is, however, found in the nuITS sequences of C. furfuracea from various areas (not shown here) and this name may hide several cryptic species.
GenBank no. AF408678 from New Zealand, not shown) there are numerous SNPs between them. Material currently referred to C. deludens probably form a species complex of morphologically similar species and this should be further studied.  [21]. Widely distributed in the Northern and Southern Hemisphere. Note: Chaenotheca furfuracea displays intraspecific genetic variation in nuITS sequences. It is characterized by the minutely verrucose spore surface and in this respect Tanzanian material agrees well with material from Europe. (Figure 1). Chaenotheca confusa (described from South America) is similar, but has spores with an ornamentation of minute areolae. Considerable variation is, however, found in the nuITS sequences of C. furfuracea from various areas (not shown here) and this name may hide several cryptic species.
Chaenotheca olivaceorufa (Vain.) Zahlbr. Habitat: Occurring on trunks of podocarps in the upper cloud forest region. Distribution: First reported from Africa (Tanzania, Kilimanjaro Region, Kilimanjaro) by [40]. Widely distributed in Central and South America and Australasia. Note: This species is closely related to C. hispidula and forms part of the critical complex mentioned above under the latter species, and the naming here is provisional. One collection (SGT 425) comes close to material from India and China that may be identified as C. nepalensis A. Schmidt, which is similar to C. olivaceorufa (described from Brazil) [32] This, however, cannot so far be supported by molecular data since no relevant sequences of South American material is available.

Chaenotheca sphaerocephala Nádv.
Habitat: On trunk of old podocarp in rain-shaded situations in mountain cloud forest at 2500-2800 m altitude. Distribution: This species was reported from Mt. Kilimanjaro by [41]. Habitat: On trunk in rain-shaded situations of mountain cloud forest at 2300-2800 m altitude, in Rwanda on Hagenia abyssinica. Distribution: From Africa this species was first reported from Rwanda [52], and subsequently also from Tanzania [41]. Note: This species is part of the C. stemonea complex, and the naming here is provisional since C. stemonea in this wide sense may harbor several cryptic species.  [41] but prior to that reported from Africa also from the DR of Congo, Kenya, and Rwanda [21]. Widely distributed in temperate areas of both the Northern and Southern Hemisphere (Eurasia, Americas, and Australasia). In areas of low latitudes only on high mountains.  [21]. New to Tanzania.
Chaenothecopsis kilimanjaroensis Temu and Tibell sp. nov. (Figure 2) Chaenothecopsis with a very short stalk which are K-, with single or aggregated capitula formed on the same thallus. The stalk is medium brown towards the base and translucent in water. Spores are uniseptate and uniseriately arranged in asci. Spore ornamentation consisting of elongated blister-like verrucae. Commensal/parasite on sterile lichen crusts and/or the thallus of Chaenotheca chloroxantha on bark in montane cloud forests. The attacked host thallus loses pigmentation and turns slightly mauve grey (Figure 2A,B). Apothecia 0.21-0.27 mm high, very short-stalked or with medium-long, olivaceous brown stalks ( Figure 2C). Capitulum single (Figure 2A) and lenticular to broadly obconical, 0.021-0.027 mm diam. (Figure 2A), or 2-5 aggregated capitula ( Figure 2B) are formed on the same and then usually short stalk ( Figure 2B). Excipulum dark brown ( Figure 2D), 6-10 µm thick, consisting of 2-3 layers of periclinally orientated intertwined hyphae measuring 2-3 µm diam. Hypothecium poorly developed, convex, pale with brown/greenish brown hyphae invading from the base. Stalk medium brown, particularly towards the base pale and translucent in water, 0.01 mm diam., consisting of intertwined, periclinal hyphae, K-, Asci cylindrical with uniseriately arranged spores ( Figure 2E) and a thickened apex penetrated by a fine canal, 37.4-44.3 µm long and 2.1-2.8 µm wide. Spores ( Figure 2F) ellipsoidal to narrowly ellipsoidal, pale brown, 1-septate, 6-6.9 µm long and 2.1-2.6 µm wide, with a rather poorly pigmented septum and a minutely verrucose ornamentation ( Figure 2H) barely visible under the light microscope, but under SEM seen to be formed from minute, often slightly elongated blister-like verrucae ( Figure 2H).  Figure 2F) ellipsoidal to narrowly ellipsoidal, pale brown, 1-septate, 6-6.9 µm long and 2.1-2.6 µm wide, with a rather poorly pigmented septum and a minutely verrucose ornamentation ( Figure 2H) barely visible under the light microscope, but under SEM seen to be formed from minute, often slightly elongated blisterlike verrucae ( Figure 2H).  (Table 1), is presented in Figure 3. The outgroup was chosen with reference to the phylogeny [9]. Five sequences of Chaenothecopsis kilimanjaroensis have as a clade strong support in both analyses (PP = 1, MLbs = 100) and the most closely related species is Chaenothecopsis debilis.  (Table 1), is presented in Figure 3.
The outgroup was chosen with reference to the phylogeny [9]. Five sequences of Chaenothecopsis kilimanjaroensis have as a clade strong support in both analyses (PP = 1, MLbs = 100) and the most closely related species is Chaenothecopsis debilis.  Note. Like some other species in Chaenothecopsis, e.g., C. consociata (Nádv.) A.F.W. Schmidt, C. epithallina Tibell and C. formosa Titov, this species is a parasite/commensal on lichen thalli, which eventually seems to kill the host thallus. It is unusually variable in its ascoma morphology and the apothecia seem to either develop to being relatively long-stalked and carrying one capitulum only, or, alternatively, several more or less aggregated capitula are formed on a short stalk. When welldeveloped the stalk is distinctly pale olivaceous brown and more or less translucent in water-mount, a feature very unusual in the genus. Chaenothecopsis pusilla (Ach.) A.F.W. Schmidt has occasionally been observed to have a stalk that is rather pale towards the base, but then usually with a greenish or greenish blue tinge [22]. In having irregularly aggregated capitula it also recalls C. amurense Titov [10] but differs i.e., in host (Trentepohlia for the latter) and in having 1-septate, rather than non-septate, spores. It might be noted that C. pusilla is not monophyletic in the analysis. This species as currently construed probably harbors a complex of morphologically similar but genetically different species that have not yet been studied in detail.  [41], but from Africa previously known from the Ivory Coast [21]. Additionally occurring in Australia and South America. Note: The unusual features and problematic placement of Heterocyphelium among the calicioids have earlier been pointed out [7] and recently it has, based on sequence data, been shown to belong in Arthoniales [54].

Mycocalicium victoriae (F. Wilson) Tibell
Reported from Tanzania and Madagascar [21]. Note. Like some other species in Chaenothecopsis, e.g., C. consociata (Nádv.) A.F.W. Schmidt, C. epithallina Tibell and C. formosa Titov, this species is a parasite/commensal on lichen thalli, which eventually seems to kill the host thallus. It is unusually variable in its ascoma morphology and the apothecia seem to either develop to being relatively long-stalked and carrying one capitulum only, or, alternatively, several more or less aggregated capitula are formed on a short stalk. When well-developed the stalk is distinctly pale olivaceous brown and more or less translucent in water-mount, a feature very unusual in the genus. Chaenothecopsis pusilla (Ach.) A.F.W. Schmidt has occasionally been observed to have a stalk that is rather pale towards the base, but then usually with a greenish or greenish blue tinge [22]. In having irregularly aggregated capitula it also recalls C. amurense Titov [10] but differs i.e., in host (Trentepohlia for the latter) and in having 1-septate, rather than non-septate, spores. It might be noted that C. pusilla is not monophyletic in the analysis. This species as currently construed probably harbors a complex of morphologically similar but genetically different species that have not yet been studied in detail.

Discussion
Twenty-six (26) crustose calicioids reported here occur in Tanzania, most of them in mountain cloud forests, but some lowland species have also been included to make the list of crustose calicioids of Tanzania complete. Most species belong to Calicium, Chaenotheca, and Chaenothecopsis.

Habitats of Cloud Forest Crustose Calicioids
Calicioids have been shown to occur mainly in undisturbed forests with a long forest continuity [11,57], and they have frequently been used as bioindicators of habitats with high species diversity and as indicators of "key habitats [12]. In addition, many are included in Red lists of several countries [58,59]. In Tanzania this pattern is also clearly discernable and calicioids mainly occur in mountain cloud forests (middle and upper forest zones) and their niches are found on the bark of old trees and on lignum.

Distribution of Tanzanian Cloud Forest Crustose Calicioids
Most of the Tanzanian crustose calicioids belong to a group of species with wide distributions in cool areas of both hemispheres and occasional occurrence in high mountains of low latitudes. Consequently, they form an important biota in mountain cloud forests and deserve attention in the context of preserving biodiversity and developing conservation policies. The wider distribution of Tanzanian cloud forest calicioids may be detailed on several levels. On the most general level two patterns may be discerned: 4.2.1. Species with a Main Distribution in Cool Temperate Areas-"Temperate Species" Many of the species are widely distributed, with high frequencies in temperate to cool temperature areas of both hemispheres, as well as at high altitudes in mountains at lower latitudes. Thus several are, apart from on the high mountains of Africa, also found along the Andes, but at low latitudes only in its high mountains, e.g., in Equador and Venezuela [27]. Similarly, some of these species occur in high altitudes along the Himalaya and its continuation to the Tasman Belt, e.g., in Papua-New Guinea [31]. Calicium species, such as C. indicum Tibell, C. laevigatum Tibell, C. nobile Tibell, C. pyriforme Tibell, C. tenuisporum Tibell and C. verrucosum Tibell, are representatives of this group.
In Africa occurrences of this "cool temperate element" are found from the Atlas, at high altitudes of the African volcanoes and the East Arch Mountains down to lower altitudes in Southern Africa [21]. Some of these distributions nowadays appear very disjunct, but dispersal might have happened during Ice Age temperature depressions and associated widening of areas with temperate forests over large parts of Africa in connection with the quaternary glaciations.