Research Article |
Corresponding author: H. Thorsten Lumbsch ( tlumbsch@fieldmuseum.org ) Academic editor: Gerhard Rambold
© 2016 Paul M. Kirika, Pradeep K. Divakar, Ana Crespo, George Mugambi, Elizabeth A. Orock, Steven D. Leavitt, Grace W. Gatheri, H. Thorsten Lumbsch.
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:
Kirika PM, Divakar PK, Crespo A, Mugambi G, Orock EA, Leavitt SD, Gatheri GW, Lumbsch HT (2016) Phylogenetic studies uncover a predominantly African lineage in a widely distributed lichen-forming fungal species. MycoKeys 14: 1-16. https://doi.org/10.3897/mycokeys.14.8971
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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
Parmelinella is a small genus (ca. 10 species) and belongs to the parmelioid clade in the family Parmeliaceae (
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.
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 DNA data matrix was assembled using sequences of nuclear ITS, nuLSU and mitochondrial SSU rDNA of 21 samples, representing 18 specimens of P. wallichiana s. lat. from Africa, Asia and S. America assembled together with DNA sequences of P. aff. wallichiana and P. lindmanii (
Specimens used in this study, with location, reference collection detail and GenBank accession numbers.
Species | Seq/DNA code | Locality | Collector(s) | Voucher specimen | GenBank accession numbers | ||
---|---|---|---|---|---|---|---|
ITS | mtSSU | nuLSU | |||||
Bulbothrix isidiza | 15505 | India: Sikkim | Divakar | MAF | KX341979 | - | KX341998 |
Bulbothrix isidiza | BUIS318it | Congo | Mamush s/n | MAF-Lich 15511 | GQ919262 | GQ919210 | GQ919237 |
Bulbothrix isidiza | BUIS1376I | Madagascar: Col de Tapia N Ambositra | Ertz 12878 (BR) | GQ919263 | GQ919238 | GQ919211 | |
Parmelinella wallichiana | 3122 | Brazil: Curitiba | S. Eliasaro | UPCB | GQ267691 | - | - |
Parmelinella aff. wallichiana | BRYC56001 | Cameroon: Ekona | Orock | BRYC56001 | JQ673451 | - | - |
Parmelinella wallichiana | 7653 | India: Sikkim | Chatterjee & Divakar | MAF-7653 | AY611106 | AY611165 | AY607819 |
Parmelinella wallichiana | 322 | India: Uttaranchal | Divakar | MAF | KX341980 | KX341990 | KX341999 |
Parmelinella wallichiana | 3615 | India: South India | Lumbsch et al. | F | KX341981 | - | - |
Parmelinella wallichiana | 4678 | Kenya: Eastern | Kirika & Lumbsch, 4678 | EA, F, MAF | KX341982 | KX341991 | - |
Parmelinella wallichiana | 4715 | Kenya: Eastern | Kirika & Lumbsch, 4715 | EA, F, MAF | KX341983 | KX341992 | KX342000 |
Parmelinella wallichiana | 9310 | Kenya: Eastern | Kirika, 3432 | EA, F | - | KX341993 | KX342001 |
Parmelinella wallichiana | 9311 | Kenya: Eastern | Kirika, 3487 | EA, F | KX341984 | - | KX342002 |
Parmelinella wallichiana | 9312 | Kenya: Eastern | Kirika, Malombe & Matheka, 3703 | EA, F | KX341985 | - | KX342003 |
Parmelinella wallichiana | 9397 | Kenya: Rift Valley | Kirika, 3334 | EA, F | KX341986 | KX341994 | KX342004 |
Parmelinella wallichiana | 9398 | Kenya: Rift Valley | Kirika, 3145 | EA, F | - | - | KX342005 |
Parmelinella wallichiana | 9552 | Kenya: Rift Valley | Kirika, Mugambi & Lumbsch, 2815 | EA, F | - | KX341995 | KX342006 |
Parmelinella wallichiana | 9592 | Kenya: Rift Valley | Kirika, Mugambi & Lumbsch, 2974 | EA, F | KX341987 | - | - |
Parmelinella aff. wallichiana | 9647 | Kenya: Coast | Kirika & Lumbsch, 4033 | F,EA,MAF | KX341988 | KX341996 | KX342007 |
Parmelinella wallichiana | 9693 | Kenya: Eastern | Kirika, 4280 | F,EA,MAF | KX341989 | KX341997 | KX342008 |
Parmelinella wallichiana | 250204 | China: Yunnan | Crespo, Blanco & Arguello | MAF-L-10411 | DQ279532 | DQ287842 | - |
Parmelinella lindmanii | 3131 | Brazil: Curitiba | S. Eliasaro | UPCB | GQ267190 | - | - |
Total genomic DNA was extracted from small pieces of thallus devoid of any visible damage or contamination using the USB PrepEase Genomic DNA Isolation Kit (USB, Cleveland, OH) in accordance with the manufacturer’s instructions. We generated sequence data from nuclear ribosomal markers, the ITS region and a fragment of the nuLSU, in addition to a fragment of the mtSSU. Polymerase-chain-reaction (PCR) amplifications were performed using Ready-To-Go PCR Beads (GE Healthcare, Pittsburgh, PA, USA) using the dilutions of total DNA. Fungal ITS rDNA was amplified using ITS1F primers (Gardes and Bruns 1993), ITS4 and ITS4A (
New sequences were assembled and edited using GENEIOUS v8.1.7 (
Phylogenetic relationships were inferred using maximum likelihood (ML), and Bayesian inference (BI). Exploratory phylogenetic analyses of individual gene topologies showed no evidence of well-supported (≥ 70% bootstrap values) topological conflict, thus relationships were estimated from a concatenated, three-locus (ITS, nuLSU, mtSSU) data matrix using a total-evidence approach (
Morphological characters, including lobe shape, size and width, cilia and rhizines were studied using a Leica Wild M 8 dissecting microscope. All the specimens of P. wallichiana included in the molecular analysis were evaluated (see Table
Observations and measurements of ascospores were made in water, at 40× (objective) and 10× (eye piece) magnification with a Leica Leitz DM RB microscope. For each species at least 20 spores from different specimens were measured. Mean value (M) and standard deviation (SD) were calculated. In the description of the new species, the results of the measurements are given as (minimum value observed) M ± SD (maximum value observed). M, SD and n (number of spores measured) are expressed within parentheses. Chemical constituents were identified by thin layer chromatography using standard methods (
A total of 28 new DNA sequences of Parmelinella wallichiana were generated for this study (Table
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.
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) (
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 (
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
Morphologically similar to P. wallichiana but differs in having smaller ascospores (5–10 × 5–7.5 µm), being restricted in distribution to Africa and South India, and molecular phylogenetic position (Clade ‘A’; Fig.
The taxon name is in the honor of W.G. Schimper, the first botanist to collect lichens in East Africa.
Thallus foliose, adnate to loosely adnate, 3–7 cm across. Lobes broad, irregularly to subirregularly branched, 3–8 mm wide, rounded crenate, with rotund apices, margins ciliate. Cilia simple, frequent in the lobe axils, 0.1–0.6 mm long. Upper surface grey, grey-green smooth, emaculate, usually pruinose, thallus irregularly cracked towards the centre on older parts, isidiate. Isidia laminal, cylindrical, mostly simple or branched 0.1–0.5 mm high, concolorous with the upper surface. Medulla white. Lower surface black with more than 2 mm broad, brown papillate margins, rhizinate. Rhizines black, evenly distributed, simple, 0.2–1 mm long. Apothecia laminal, adnate to sessile, 1–5 mm in diameter. Disc concave, brown, imperforate. Asci 8-spored. Ascospores ellipsoid to subglobose, 5–10 × 5–7.5 µm (M = 5.5–6.4 × 7.6–8.5 μm, ±SD = 0.7–1.0 × 1.0–2.3 μm, n = 100). Pycnidia absent.
Secondary chemistry – Cortex K+ yellow, UV–; medulla K+ yellow turning red, C–, KC–, P+ orange-red, UV–; upper cortex with secalonic acid A and atranorin, medulla with salazinic acid.
At present the new species is known from Kenya, Cameroon and South India. It occurs in montane regions and in dry woodland areas. It is predominantly corticolous and sometimes saxicolous rarely terricolous, found corticolous on Mangifera indica, Juniperus procera, Podocarpus spp., Lannaea spp. and on Eucalyptus in artificial habitats.
KENYA. Eastern Prov.: Marsabit Co., Marsabit National Park, Lake Paradise, disturbed forest on ridge, 2°16'N 37°56'E, 1434m, on bark, P. Kirika 4678 & H.T. Lumbsch (EA, F, MAF). Eastern Prov.: Marsabit National Park at roadside between Marsabit Lodge and Lake Paradise, forest on slope, 2°18'N 37°57'E, 1513m, on bark, P. Kirika 4715 & H.T. Lumbsch (EA, F, MAF). Eastern Prov.: Tharaka South, Chiakariga, Kijege Hill, Acacia-Commiphora woodland, 00°16'S, 37°50'E, 1160m, on bark, P. Kirika 3432 (EA, F). Eastern Prov.: Mwingi Co., Mumoni Hill, Eucalyptus plantation, 00°31'S, 38°00'E, 1620–1695m, on bark, P. Kirika 3487 & G. Mugambi (EA, F). Eastern Prov.: Tharaka South, Chiakariga, Kijege Hill, Acacia-Commiphora-Encephalartos woodland, 00°16'S, 37°50'E, 1160m, P. Kirika 3436 (EA, F). Rift Valley: Eldama Ravine, Lembus Forest off Eldama Ravine-Eldoret Road, remnant montane forest, 0°13'N, 35°69'E, 2275m, on bark, P. Kirika 2870, G. Mugambi & H.T. Lumbsch (EA, F); 0°16'N, 35°75'E, 2137m, on rock, P. Kirika 2815, G. Mugambi & H.T. Lumbsch (EA, F). Rift Valley: Kericho, James Finlay Tea Estate, Chomogondy, secondary forest, 00°23'S, 35°18'E, 2056m, on bark, P. Kirika 3145 (EA, F). Rift Valley: small disturbed remnant forest in tea plantation, 0°44'S, 35°31'E, 2049m, on bark, P. Kirika 2974 G. Mugambi & H.T. Lumbsch (EA, F). Rift Valley: Bomet , Koiwa, Unilever riparian forest, 00°35S, 35°17'E, 2030m, on bark, P.Kirika 4900 (EA). Rift Valley: Kajiado Co., Ngong Hills, Upland grassland with rocky outcrops, 01°24'S, 36°38'E, 2430m, on soil, P. Kirika 3334 (EA, F). CAMEROON. E of Mount Cameroon, vic. of Ekona, E.A. Orock 56009 (BRY-C). INDIA. S India: Tamil Nadu, Vellore distr., Yellagiri hills, 13°30'N, 79°05'E, 1393m, on Mangifera indica tree trunk, hill side with teak and eucalyptus vegetation, H.T., Lumbsch, P.K. Divakar, D.K. Upreti, J. Tandon 19705a (MAF).
Parmelinella schimperiana is morphologically most similar to P. wallichiana, but differs in having smaller ascospores (5–10 × 5–7.5 µm), whereas the ascospore size in P wallichiana is: 15–20 × 9–14 µm.
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).