A number of lichen-forming fungal species are widely distributed. Here, we investigate biogeographic patterns in a widely distributed isidiate taxon – Parmelinella wallichiana – using molecular sequence data. Our results revealed that Parmelinella wallichina, as currently circumscribed, is not monophyletic but falls into four clades, two of them represented by a sample only. A third clade, occurring in Africa and southern India is described as a new species, Parmelinella schimperiana Kirika & Divakar, sp. nov. Our study adds a further example of previously overlooked, geographically distinct, lineages that were discovered using molecular data.

Africa, genealogical criteria, molecular systematics, new species, Parmeliaceae , Parmelinella , parmelioid lichens, phylogeny, taxonomy

The advent of DNA sequence technologies and advances in molecular phylogenetic methods have revolutionized our understanding on species delimitation and systematics in lichens and fungi in general (reviewed in Crespo and Lumbsch 2010; Lumbsch and Leavitt 2011; Divakar and Crespo 2015; Leavitt et al. 2015a). Molecular sequence data have led to the circumscription of previously overlooked species-level lineages and a number of taxonomic re-evaluations of widely distributed species in lichenized fungi. Diverse examples of widely distributed nominal taxa masking multiple, species-level diversity include: Cladia aggregata (Parnmen et al. 2012), Melanelixia glabra (Divakar et al. 2010a), Melanelixia fuliginosa/M. glabratula (Leavitt et al. 2012), Melanohalea elegantula, M. exasperata (Leavitt et al. 2013a), Montanelia tominii (Leavitt et al. 2015b), Parmelia saxatilis (Molina et al. 2011a), P. sulcata (Divakar et al. 2005, Molina et al. 2011b), Parmelina quercina (Argüello et al. 2007), P. tiliacea (Nuñez-Zapata et al. 2011), Protoparmelia badia (Singh et al. 2015), Physconia distorta (Divakar et al. 2007), Rhizoplaca melanophthalma (Leavitt et al. 2013b), and Sphaerophorus globosus (Högnabba and Wedin 2003). These studies demonstrate that some supposedly cosmopolitan taxa may include previously unrecognized diversity. Thus re-evaluation of widely distributed species applying molecular sequence data is prerequisite to understanding biogeographic patterns in broadly distributed taxa. Furthermore, inaccurate species assessment may have negative consequences for conservation purposes or understanding of diversification patterns.

Parmelinella is a small genus (ca. 10 species) and belongs to the parmelioid clade in the family Parmeliaceae (Divakar et al. 2015). The species included in this genus are characterized by a pored epicortex, isolichenan in the cell walls, subirregular lobes, cylindrical or bifusiform conidia, simple cilia and rhizines, and a yellow-grey upper cortex - containing secalonic acid derivatives and atranorin (Elix 1993; Crespo et al. 2010; Thell et al. 2012). Species in the genus are mainly distributed in subtropical to tropical regions of Africa, Asia, Australasia and South America. Parmelinella chozoubae, P. manipurensis and P. nimandairana are restricted to Asia; P. salacinifera, is reported from Southeast USA, central and south America, and Thailand; P. simplicior occurs in Asia and East Africa; and P. cinerascens, P. lindmanii, P. mutata and P. versiformis are endemic to South America (Elisaro et al. 2010; Benatti 2014). For a long time only four additional Parmelinella species were known from India (Divakar and Upreti 2005), but recent studies added six species to the genus, most of which had previously been known to occur only in South America (Elisaro et al. 2010; Benatti 2014). Of the ten species, only two, P. simplicior and P. wallichiana, have previously been reported from East Africa (Swinscow and Krog 1988; Alstrup et al. 2010).

Parmelinella wallichiana is the only widely distributed species in this genus and is known from Africa, Asia, Australia and South America. While it is widespread in East Africa and Asia, the species is known from a few localities in Australia and South America. Parmelinella wallichiana normally reproduces asexually by isidia and grows in wide range of ecological environments. The species is most frequently epiphytic but also found rarely on rocks. Studies have demonstrated broad, intercontinental distributions of a number of lichen-forming fungi that reproduce via asexual propagules (see e.g. Divakar et al. 2005; Molina et al. 2011a and b; Leavitt et al. 2013a; Roca-Valiente et al. 2013; Divakar et al. 2016).

This study aims to assess biogeographic patterns in the widely distributed, isidiate, lichen-forming fungal species Parmelinella wallichiana. To this end, we generated DNA sequences of nuclear ribosomal internal transcribed spacer region (ITS1, 5.8S and ITS2), large subunit (nuLSU) and mitochondrial small subunit (mtSSU). Phenotypical features were re-evaluated and compared in light of the relationships inferred from the phylogenetic reconstructions.

A total of 28 new DNA sequences of Parmelinella wallichiana were generated for this study (Table 1). These were deposited in GenBank under accession numbers KX341978-KX342008. The dataset included samples from wide geographic regions as Asia, East Africa and South America. The final alignment of the combined data set was 2174 positions in length and was comprised of 458 unambiguously aligned nucleotide position characters in ITS, 844 in the nuLSU, and 872 in the mtSSU. As the topologies of the single locus phylogenies did not show any conflicts they were analyzed in a concatenated data matix (data not shown). The ML and BI analyses were identical in their topology and hence only the ML tree with support values of both analyses is depicted in Figure 1.

Figure 1. 

Phylogenetic relationships among Parmelinella taxa based on a maximum-likelihood (ML) analysis of a concatenated, three locus dataset (ITS, nuLSU & mtSSU rDNA). Since the ML and Bayesian inference topologies were identical, only the ML topology is shown here. Posterior probabilities ≥ 0.95/ ML bootstrap values ≥ 70% are given above the branches.

Specimens representing Parmelinella wallichiana did not form a monophyletic lineage (Fig. 1). This is inconsistent with currently hypothesized species boundaries based on phenotypical features (Divakar and Upreti 2015; Benatti 2014). Species-level polyphylies are commonly found in Parmeliaceae and other groups of lichen-forming fungi (see reviews by Crespo and Lumbsch 2010; Lumbsch and Leavitt 2011).

Specimens representing P. wallichiana s. lat. fell into four distinct well-supported clades. Clade ‘A’ included samples from Kenya, Cameroon, and a single sample from South India. Clade ‘B’ included a single sample from coastal region (Coast Province) of Kenya. Clade ‘C’ included most samples from Asia; and clade ‘D’ was represented by a single sample from South America (Brazil). Specimens in clade ‘A ‘are characterized in having smaller ascospores (5–10 × 5–7.5 µm), whereas they are larger (15–20 × 9–14 µm) in clade ‘C’. Further, the same strongly supported monophyletic clades – ‘A’ and ‘C’ – were recovered in reciprocally monophyletic clades in the independent gene trees (data not shown) (Hudson and Coyne 2002). Presence of the same clades in different single-locus genealogies can be taken as strong evidence that the clades are reproductively and evolutionarily isolated lineages representing distinct species-level lineages (Dettman et al. 2003; Pringle et al. 2005; de Quieroz 2007). The relationships among the clades were well supported (Fig. 1). Clade ‘B’ formed sister-group relationship with clade ‘A’, whereas clade ‘D’ was sister to P. lindmanii, and clade ‘C’ sister to a clade including clade ‘D’ and P. lindmanii. The type material of Parmelinella wallichiana is from Nepal in the Himalayas (Hale 1976a; Divakar and Upreti 2005). Since all samples sequenced by us from the Himalayan regions (China and India) clustered in clade ‘C’, we consider this clade as P. wallichiana s. str.

For clade ‘A’ there are a few potential names available that we studied. For example, Parmelia junodi was described from the Cape Province in South Africa (Steiner 1907) and Parmelia tiliacea var. eximia has been described from Tanzania (Steiner 1888). These taxa have previously been considered synonyms of P. wallichiana (Hale 1976a). However, according to a recent study by Benatti (2014), Parmelia tiliacea var. eximia is a synonym of Parmelinella cinerascens and the type material of Parmelia junodi contained mixture of different species, such as Parmelinopsis minarum or P. horrescens and a fragment to belonged Parmelinella cinerascens. Thus we conclude that those two names are synonyms of Parmelinella cinerascens. The latter is a rare species occurring in South America and until recently was classified in the genus Canoparmelia (Elix et al. 1986). Recently, based on morphological data, Canoparmelia cinerascens was transferred to the genus Parmelinella (see Benatti 2014). Unfortunately, we were unable to sequence this species and hence cannot confirm the phylogenetic position of C. cinerascens. Samples clustered in clade ‘A’ collected from Africa and South India are morphologically similar to Parmelinella wallichiana s. lat. Since there is no name available for this clade, a new species is described below to accommodate samples from Africa and South India (clade ‘A’). Further, the segregation of this new taxon from P. wallichiana s.str. is corroborated by morphological data, discussed below. The new species has a disjunct distribution occurring in Africa and South India. There are abundant examples of this disjunct distribution pattern in flowering plants (see e.g. Mani 1974; Kadereit 2004).

Clades ‘B’ and ‘D’ were each represented by a single specimen from Kenya and Brazil, respectively. The sample from the coastal region of Kenya (clade ‘B’) has a deviating morphology, i.e. very narrow, sublinear and dichotomous lobes, although the specimen from coastal Brazil (clade ‘D’) was more similar to P. wallichiana s. lat. In both cases, study of additional samples will be required before a formal description of these putative species.

Our results add a further example to a growing body of evidence of the existence of distinct lineages hidden under currently circumscribed species (reviewed in Bickford et al. 2007; Crespo and Lumbsch 2010; Lumbsch and Leavitt 2011). Whereas, some studies found no obvious phenotypical differences and interpreted the discovered additional species diversity as cryptic (reviewed in Crespo and Lumbsch 2010; Lumbsch and Leavitt 2011; Hibbett 2016), re-examination of material falling into different clades uncovered previously unrecognized morphological differences. This has been shown in other cases as well (see e.g. Parmelia barrenoae, Divakar et al. (2005); Physconia thorstenii, Divakar et al. (2007); Caloplaca citrina group Vondrák et al. (2009); Melanelixia californica, Divakar et al. (2010); Parmelia mayi, Molina et al. (2011a); Cladia aggregata group, Parnmen et al. (2012); Parmotrema perforatum group, Widhelm et al. in press) and demonstrates the importance of careful re-analysis of morphological and chemical characters in order to phenotypically circumscribe species. Further, the species-level lineages uncovered in this widely distributed isidiate taxon showed biogeographic structure in what was previously believed to be a pantropical species. Although geographical structure of species detection using molecular data has recently been shown to be a common phenomenon in lichenized fungi (Argüello et al. 2007; Divakar et al. 2010a; Otalara et al. 2010; Amo de Paz et al. 2012; Parnmen et al. 2012; Moncada et al. 2014; Leaviit et al. 2015b; Alors et al. 2016); caution must be taken to generalizing for all isidate lichen taxa (Leavitt et al. 2013a; Roca-Valiente et al. 2013; Divakar et al. 2016).

Newly obtained DNA sequences were generated in the Pritzker Laboratory for Molecular Systematics and Evolution at the Field Museum and at the Molecular Laboratory, Department of Biology, Faculty of Pharmacy, Complutense University of Madrid. This study was supported by a grant of the IDP/The Field Museum Africa Training Fund and the Spanish Ministerio de Ciencia e Innovación (CGL2013-42498-P).