Phylogenetic patterns of morphological and chemical ... - INBio

Systematics and Biodiversity 6 (1): 31–41 doi:10.1017/S1477200007002629 Printed in the United Kingdom

Issued 29 February 2008 C The Natural History Museum

Phylogenetic patterns of morphological and chemical characters and reproductive mode in the Heterodermia obscurata group in Costa Rica (Ascomycota, Physciaceae) 1

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Robert L¨ ucking , Ruth del Prado , H. Thorsten Lumbsch , Susan Will-Wolf , André Aptroot , Harrie J. M. Sipman , 5 5 Loengrin Umaña & José Luis Chaves 1 Department of Botany, The Field Museum, 1400 South Lake Shore Drive, Chicago, Illinois, 60605-2496, USA. Email: rlucking@fieldmuseum.org 2 Department of Botany, University of Wisconsin-Madison, Madison, Wisconsin 53706-1381 USA. Email: [email protected] 3 ABL Herbarium, G.v.d. Veenstraat 107, NL-3762 XK Soest, The Netherlands. Email: [email protected] 4 Botanisches Museum Berlin Dahlem, K¨ onigin-Luise-Strasse 6-8, D-14191 Berlin, Germany. Email: [email protected] 5 Laboratorio de Hongos, Instituto Nacional de Biodiversidad (INBio), Apdo. 22-3100, Santo Domingo de Heredia, Costa Rica. Email: [email protected]; [email protected]

submitted January 2006 accepted March 2006

Contents Abstract

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Introduction 32 Materials and methods 34 Chemical analysis 34 Molecular analysis 34 Results

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Discussion and conclusions Acknowledgements References

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Abstract Species delimitation in lichens of the Heterodermia obscurata group (sect. Polyblastidium) is based on reproductive mode, lobe underside pigmentation, and medullary chemistry as main character complexes. However, these characters are inconsistently used by different workers, and phenotypes with different medullary chemistry or underside pigmentation are often lumped into single species. In addition, molecular studies in other genera of Physciaceae have shown that phenotypes reproducing either sexually or asexually do not necessarily form monophyletic clades, which does not support the species pair concept postulating that vegetatively reproducing species form monophyletic sister clades to sexually reproducing species. Obviously, these deviating concepts greatly influence the number of species recognised in biotic inventories, as is the case in the Costa Rican Lichen Biodiversity Inventory (Ticolichen). Here, depending on which species concepts are used, the number of taxa recognised in the H. obscurata group varies between 10 and 25. We therefore tested the phylogenetic patterns of distribution of morphological and chemical characters and reproductive mode in apotheciate versus sorediate taxa of the H. obscurata group in Costa Rica by means of molecular phylogenetic analysis of ITS sequences. Based on our results, the use of both medullary chemistry and underside pigmentation for the distinction of species is supported, which is in contrast to the concepts used in recent treatments of the genus. The data are ambiguous, however, with regard to the species pair concept: in the case of H. flabellata vs. H. obscurata, the species pair concept does not seem to hold, while in two other supposed species pairs (Heterodermia ‘rottbollii’ vs. H. ‘pseudobscurata’ and H. ‘violacea’ vs. H. ‘reagens’), the sorediate forms appear not to be sister taxa of the apotheciate forms. Key Words lichens, species concept, species pairs, secondary chemistry, Ticolichen 31

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Robert L¨ ucking et al.

Introduction The lichen genus Heterodermia (Physciaceae) is among the most common in tropical regions and particularly abundant in perhumid climates at mid-elevations (Kurokawa, 1962, 1973; Swinscow & Krog, 1976; Krog, 2000; Büdel et al., 2000; Moberg, 2004). The genus is distinguished from other foliose genera in the Physciaceae chiefly by its prosoplectenchymatous upper cortex in combination with atranorin as a cortical substance. Most species are also characterised by lacking a lower cortex and producing abundant marginal cilia often resembling rhizines, and norstictic and salazinic acids are common medullary substances (Poelt, 1965; Hafellner et al., 1979). Apart from its abundance in wet tropical and subtropical ecosystems with oceanic character, species of Heterodermia do have ethno-lichenological importance and have been screened for antimicrobial and antiviral activity (Saklani & Uptreti, 1992; Negi & Kareem, 1996; Upreti, 1996; Cohen et al., 1996; Jäger et al., 1997; Shahi et al., 2001; Falcao et al., 2002). Species of Heterodermia can be assigned to four major groups or sections (Kurokawa, 1962, 1973; Schumm, 2000, 2001; Will-Wolf et al., in prep.), based on the type of lobe branching, cilia morphology, presence of a lower cortex, and presence of accessory lumina (sporoblastidia) in the ascospores. The most abundant and speciose group is the H. obscurata group (sect. Polyblastidium of Kurokawa, 1973), whose species have more or less loosely appressed lobes with anisotomous branching and usually black, squarrose branched cilia, lack a lower cortex, and have ascospores with sporoblastidia. The main characters employed in species circumscription within the Heterodermia obscurata group are: (1) mode of reproduction (apothecia vs. soralia or isidia); (2) pigmentation of the (ecorticate) lobe underside; and (3) medullar chemistry. The different modes of sexual versus vegetative reproduction are often adscribed to the species pair concept (Poelt, 1970, 1972), which assumes that otherwise identical lichens that differ in their reproductive mode evolved from a common ancestor and are to be regarded as specifically distinct (e.g. H. flabellata with apothecia, H. obscurata with soralia, H. crocea with isidia). An alternative hypothesis regards such forms as representing different stages of a complex life cycle, with multiple, independent evolution of asexual strains (Tehler, 1982). Recent molecular studies suggest that even more complex evolutionary patterns exist: while in some cases, such as in other genera of Physciaceae, the taxonomic importance of sexual versus vegetative reproductive modes have been overestimated (Lohtander et al., 1998; Myllys et al., 1999, 2001), other studies showed different reproductive groups as distinct, either confirming the species concept or even indicating that sexual and vegetative forms represent more than one species each (Hoffmann & DePriest, 2000; Kroken & Taylor, 2001, Printzen et al., 2003). In many species of Heterodermia that lack a lower cortex, yellow to red pigments or pigmented layers of loose, arachnoid tissue are produced on the underside of the lobe. The pigments involved were shown to be anthraquinones in H. obscurata and H. flabellata (Cohen & Towers, 1995; Din et al., 2002), but in other species they are currently not identified, although the

K− reaction in some of them suggest that pulvinic acid derivates may also be involved (Kurokawa 1962). Species of the H. obscurata group have lobes with their underside either pure white (sometimes dark violet grey towards the centre), sparsely spotted with yellow (K+ purple or K−; e.g. in H. casarettiana, H. corallophora, H. corcovadensis, H. lamelligera, H. ‘rottbollii’), or completely covered with an arachnoid, ochraceous red, K+ purple tissue (e.g. in H. flabellata, H. obscurata and H. crocea). The colour, pattern and K+ reaction of the underside pigments has been considered as specific by Kurokawa (1962, 1998) and some other authors (Trass, 1992), but more recent treatments merge, for example, H. propagulifera with yellow spots with H. japonica completely white (Swinscow & Krog, 1976; Moberg & Purvis, 1997; Moberg & Nash, 2002). All species of Heterodermia contain zeorin and other terpenoids as medullary compounds. Many taxa also produce norstictic and/or salazinic acid. Some authors recognise such chemically deviating phenotypes as different species (Kurokawa, 1962; Schumm, 2000, 2001), while others regard them as chemotypes without taxonomic value (Swinscow & Krog, 1976; Moberg & Purvis, 1997; Brodo et al., 2001; Moberg & Nash, 2002). Species of the Heterodermia obscurata group often produce substances of the norstictic acid chemosyndrome. Most of these chemotypes are morphologically indistinguishable, and thus are often merged with those lacking the acid (Moberg & Purvis, 1997; Trass, 2000; Moberg & Nash, 2002; Moberg, 2004) or treated at the infraspecific level (e.g. as varieties; Kurokawa, 1962; Table 1). We found also that, in all specimens of the H. obscurata group studied by us that contained norstictic acid (as well as in those of the H. podocarpa group), the acid was produced only at the lobe tips and not in the entire medulla. This phenomenon is easily observed in inadequately dried specimens wherein the decomposing acid causes the lobe tips and apothecial margins to become ferrugineous red. Possible implications of the restricted distribution of the medullary compounds within the lichen thallus have apparently never been reported in the literature, but might account for misidentifications if spot tests are performed on the medulla of the inner lobes. In traditional Heterodermia taxonomy (here considering the apotheciate and sorediate taxa only), the three main character sets ‘reproductive mode’ (apothecia versus soralia), ‘medullary chemistry’ (norstictic acid present or absent), and ‘underside pigmentation’ have been used in combination to circumscribe species (Table 2). Consequently, evaluation of the phylogenetic pattern of these characters is essential for an understanding of species delimitation in this group and has important consequences for assessing the diversity of these tropical lichens. The parallel use of different, conflicting species concepts, either emphasising or downweighting chemical or reproductive characters, is certainly not a desirable solution (Hawksworth, 1976; Lumbsch, 1998). One way of assessing the validity of chemical characters for taxonomic purposes is the use of taxa that are well characterised by a set of morphological and anatomical features. Such taxa include H. crocea, H. flabellata, H. lutescens, H. obscurata, H. squamulosa, H. verruculifera, and H. vulgaris (Kurokawa, 1962, 1998; Esslinger & Bratt, 1998). The fact that these species exhibit a

Phylogenetic patterns of morphological and chemical characters

Medullary chemistry

Underside pigmentation

Apothecia

Soralia

zeorin only

arachnoid ochraceous red, K+ purple spotted yellow, K+ purple spotted yellow, K− white (to violet grey) arachnoid ochraceous red, K+ purple spotted yellow, K+ purple spotted yellow, K− white (to violet grey)

flabellata ‘rottbollii’ lamelligera appendiculata corcovadoensis dentritica (unnamed) ‘violacea’

obscurata ‘pseudobscurata’ (unnamed) japonica (unnamed) propagulifera casarettiana ‘reagens’

norstictic acid plus zeorin

Table 1

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Taxa in the Heterodermia obscurata group producing either apothecia or soralia and otherwise distinguished by their underside pigmentation and medullar chemistry (after Kurokawa, 1962, 1998; Trass, 1992). Three character combinations have not yet been found and are hence unnamed; two other new taxa were discovered in the Costa Rican material studied here and will be formally described in a separate paper, together with the new combinations of the epithets ‘reagens’ and ‘rottbollii’ (Will-Wolf et al., in prep., indicated as quotation marks).

Species name

Coll. No.

Location

GenBank acc. no.

Heterodermia appendiculata (Kurok.) Swinscow & Krog Heterodermia appendiculata (Kurok.) Swinscow & Krog Heterodermia appendiculata (Kurok.) Swinscow & Krog Heterodermia boryi (Fée) Hale Heterodermia casarettiana (A. Massal.) Trevis. Heterodermia casarettiana (A. Massal.) Trevis. Heterodermia casarettiana (A. Massal.) Trevis. Heterodermia flabellata (Fée) D.D. Awasthi Heterodermia flabellata (Fée) D.D. Awasthi Heterodermia flabellata (Fée) D.D. Awasthi Heterodermia flabellata (Fée) D.D. Awasthi Heterodermia japonica (M. Satô) Swinscow & Krog Heterodermia japonica (M. Satô) Swinscow & Krog Heterodermia japonica (M. Satô) Swinscow & Krog Heterodermia japonica (M. Satô) Swinscow & Krog Heterodermia japonica (M. Satô) Swinscow & Krog Heterodermia lamelligera (Taylor) Trass Heterodermia leucomela (Fée) Swinscow & Krog Heterodermia leucomela (Fée) Swinscow & Krog Heterodermia obscurata (Nyl.) Trevis Heterodermia obscurata (Nyl.) Trevis Heterodermia obscurata (Nyl.) Trevis Heterodermia ‘pseudobscurata’ Heterodermia ‘reagens’ Heterodermia ‘reagens’ Heterodermia ‘reagens’ Heterodermia ‘rottbollii’ Heterodermia ‘violacea’

10252c

Costa Rica: Carrizal Village

DQ337311

15525b

Costa Rica: Carrizal Village

DQ337312

2270A

Costa Rica: Sarchi Village

DQ337320

unknown 10006f 10017c 15441a unknown 15094 15146 17578 15096a 15132c 15206 15265 16597a 2046G unknown 10020e unknown 15184d 485a 15517 15148b 15171i 595E 15455 16606a

unknown Costa Rica: Las Tablas Protection Zone Costa Rica: Las Tablas Protection Zone Costa Rica: Paraiso near Cartago unknown Costa Rica: Las Tablas Protection Zone Costa Rica: Las Tablas Protection Zone Costa Rica: Las Tablas Protection Zone Costa Rica: Las Tablas Protection Zone Costa Rica: Las Tablas Protection Zone Costa Rica: La Amistad International Park Costa Rica: La Amistad International Park Costa Rica: Tapanti National Park Costa Rica: Las Tablas Protection Zone unknown Costa Rica: Las Tablas Protection Zone unknown Costa Rica: La Amistad International Park Costa Rica: Las Tablas Protection Zone Costa Rica: Irazu Volcano National Park Costa Rica: Las Tablas Protection Zone Costa Rica: Las Tablas Protection Zone Costa Rica: Las Tablas Protection Zone Costa Rica: Cerro de la Muerte Costa Rica: Tapanti National Park

AJ421419 DQ337304 DQ337318 DQ337305 AF540519 DQ337307 DQ337308 DQ337309 DQ337313 DQ337322 DQ337314 DQ337315 DQ337316 DQ337319 AF540520 DQ337321 AY449726 DQ337323 DQ337310 DQ337324 DQ337325 DQ337306 DQ337317 DQ337326 DQ337327

Table 2

Taxa and specimens used in the cladistic analyses. New sequences are indicated by numbers in bold with the prefix DQ and series 337304-337327.

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uniform medullary chemistry makes it questionable whether species including both forms with and without norstictic acid should be assumed in all other instances. Here, we address the issue of character evaluation using ITS sequence data to infer a phylogeny of species in the Heterodermia obscurata group in Costa Rica. This group is highly diverse and includes forms producing apothecia or soralia, with and without norstictic acid, and with different types of underside pigmentation. Although this study encompasses a geographically restricted sample, it allows for comparison of chemically different forms collected at the same localities. Of the 13 forms currently distinguished (Table 1), 12 were found in Costa Rica (except H. dentritica), and ten are included in this analysis.

Materials and methods Chemical analysis Specimens were chemically analysed using standardised thin layer chromatography (TLC) (Culberson & Kristinsson, 1970, Lumbsch, 2002) using solvent system C. All specimens were also subjected to spot tests with potassium hydroxide (K) of the medulla and lobe underside to assess the distribution of medullar substances within the lichen thallus. The colour and distribution of lobe underside pigments was studied under a stereomicroscope (Leica MS5), and spot tests (K) were applied to check for the presence or absence of a K+ purple reaction.

Molecular analysis New nuITS rDNA sequences were obtained from 23 samples collected in Costa Rica, representing ten putative species of the Heterodermia obscurata group delimitated by their reproductive mode, underside pigmentation, and medullary chemistry. A data matrix was assembled using these 23 sequences and two additional sequences of H. flabellata and H. obscurata downloaded from GenBank. An additional new sequence of H. leucomela and one downloaded from GenBank were included as outgroup together with one of H. boryi (Table 2). Total DNA was extracted from freshly collected material and herbarium specimens, using the DNeasy Plant Mini Kit (Qiagen). Dilutions (10−1 up to 10−3 ) or undiluted DNA was used for PCR amplifications of the gene coding for the nuclear ITS rDNA. Primers for amplification were ITS1F (Gardes & Bruns, 1993) and ITS4 (White et al., 1990). The 25 µL PCR reactions contained 2.5 µL buffer, 2.5 µL dNTP mix, 2 µL of each primer (20 µM), 5 µL BSA, 2 µL Taq, 2.5 µL genomic DNA extract and 6.5 µL distilled water. Thermal cycling parameters were: initial denaturation at 94 ◦ C for 3 minutes, followed by 34 cycles of: 94 ◦ C for 45 seconds, 52 ◦ C for 1 minute, 72 ◦ C for 1.5 min, and a final elongation at 72 ◦ C for 10 min. Amplification products were observed on 1% agarose gels stained with ethidium bromide and subsequently purified following the gelease cleanup protocol. Fragments were sequenced using the Big Dye Terminator reaction kit (ABI PRISM, Applied Biosystems). Sequencing

and PCR amplifications were performed using the same sets of primers. Cycle sequencing was executed with the following program: initial denaturation for 1 min at 96 ◦ C followed by 31 cycles of: 96 ◦ C for 15 s, 50 ◦ C for 10 s, 60 ◦ C for 4 min. Sequenced products were precipitated with 10 µL of sterile dH2 O, 2 µL of 3 M NaOAc, and 50 µL of 95% EtOH, before they were loaded on an ABI 3100 (Applied Biosystems) automatic sequencer. Sequence fragments obtained were assembled with SeqMan 4.03 (DNASTAR) and manually adjusted. The nuITS data set contains sequence portions that are highly variable. Standard multiple alignment programs, such as Clustal (Thompson et al., 1994) become less reliable when sequences show a high degree of divergence. Therefore we employed an alignment procedure that uses a linear Hidden Markov Model (HMM) as implemented in the software SAM (Sequence Alignment and Modelling system; Karplus et al., 1998) for the alignment. Regions that were not aligned with statistical confidence were excluded from the cladistic analysis. The alignment was analysed by maximum parsimony (MP) and by a Bayesian approach (B/MCMC) (Larget & Simon, 1999; Huelsenbeck et al., 2001). MP analysis was undertaken with PAUP∗ (Swofford, 2003). A heuristic search of 1000 random taxon addition replicate searches was conducted with TBR branch-swapping and MulTrees option in effect, equally weighted characters, and gaps treated as missing data. A strict consensus tree was computed from 15 equally parsimonious trees obtained in the heuristic search. Bootstrap (Felsenstein, 1985) was performed to estimate the robustness of the clades, based on 1000 replicates with the same settings as in the heuristic search. Only clades that received bootstrap support equal or above 75% were considered as strongly supported. B/MCMC analyses were conducted using MrBayes 3.0 (Huelsenbeck & Ronquist, 2001). Posterior probabilities were approximated by sampling trees using a Markov chain Monte Carlo (MCMC) method. The posterior probabilities of each branch were calculated by counting its occurrence in trees that were visited during the course of the MCMC analysis. For all data sets the general time-reversible model of nucleotide substitution was used (Rodriguez et al., 1990), including estimation of invariant sites and assuming a discrete gamma distribution with six rate categories (GTR+I+G), and parameters were calculated for each partition separately as proposed by Nylander et al. (2004). MrBAYES was run on the data set producing 2 000 000 generations. Twelve chains were run simultaneously. Trees were sampled every 100 generations for a total of 20 000 trees. The first 200 000 generations (i.e. the first 2000 trees) were deleted as the ‘burn in’ of the chain. We plotted the log-likelihood scores of sample points against generation time using TRACER 1.0 (http://evolve.zoo.ox.ac.uk/software.html?id=tracer) to ensure that stability was achieved after the first 200 000 generations by checking whether the log-likelihood values of the sample points reached a stable equilibrium value (Huelsenbeck & Ronquist, 2001). Of the remaining 18 000 trees, a majority rule consensus tree with average branch lengths was calculated using the sumt option of MrBayes. Posterior probabilities were


Figure 1

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Strict consensus of 15 equally most parsimonious trees as result of the MP analysis (outgroup of 1 taxon). Supported branches are indicated by thick lines, with Jackknife/Bootstrap support values given. Numbers above branched indicate number of character state changes.

obtained for each clade. Clades that received posterior probabilities of 0.95 and above were considered as strongly supported. All trees were visualised using the program Treeview (Page, 1996).

Results We generated 24 new nuITS rDNA sequences for this study, including one of Heterodermia leucomela as part of the outgroup. Four further sequences were downloaded from GenBank, one each of H. flabellata, H. obscurata, H. leucomela, and H. boryi (as part of the outgroup). Several other Heterodermia sequences downloaded from GenBank were eventually omitted from the analysis due to obvious problems with their taxonomic identification. The sequences were aligned to produce a matrix of 485 unambiguously aligned nucleotide positions of which 138 were variable and 103 parsimony-informative. The alignment is available in TreeBase (http://www.treebase.org/ treebase/). The likelihood parameters in the sample had the following mean values (±variance): LnL = −2123.087

(±0.224), base frequencies π(A) = 0.266 (±0.0003), π(C) = 0.246 (±0.0002), π(G) = 0.212 (±0.0002), π(T) = 0.277 (±0.0002), rate matrix r(AC) = 0.12 (±0.0004), r(AG) = 0.173 (±0.0007), r(AT) = 0.101 (±0.0004), r(CG) = 0.03595 (±0.0003), r(CT) = 0.551 (±0.001), r(GT) = 0.01866 (±0.0002), the gamma shape parameter alpha = 0.831 (±0.01) and the proportion of invariable site p(invar) = 0.431 (±0.003). Strong support under MP and Bayesian criteria were defined as equal or more than 75% MP bootstrap and equal or more than 0.95 posterior probability, respectively. Maximum parsimony analysis retained 15 equally parsimonious trees of 274 steps in length (CI = 0.67, RI = 0.86). The strict consensus (Fig. 1) shows strong support for the two main branches, one including H. appendiculata, H. flabellata and H. obscurata, and the other including H. japonica and its close allies. The Bayesian analysis (Fig. 2) shows a rather similar topology for most parts of the tree, except for the position of H. ‘pseudobscurata’ and H. ‘rottbollii’. In both analyses, the specimens identified as H. appendiculata cluster on a strongly supported clade sister to another

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Figure 2

Majority rule consensus of Bayesian analysis. Supported branches are indicated by thick lines.

strongly supported clade including H. flabellata and H. obscurata. The latter two share an arachnoid, orchaceous lobe underside and are considered a species pair, producing either apothecia (H. flabellata) or soralia (H. obscurata). They also differ slightly in lobe morphology, with H. flabellata having closely appressed, dichotomously branched lobes, while H. obscurata features loosely appressed, anisotomously branched lobes similar to those of H. japonica (Fig. 3). H. flabellata and H. obscurata are nested within each other and do not form separate, monophyletic clades. The second main branch includes all taxa similar to Heterodermia japonica, with more compact, white lobe underside often becoming violet-grey towards the centre and sometimes producing spots of yellow, K− or K+ purplish pigment at the lobe tips (Fig. 4). Heterodermia japonica and H. ‘reagens’ share the same lobe morphology and underside pigmentation and differ only in the absence vs. presence of norstictic acid in the medulla of the lobe tips. Both taxa form separ-

ate, monophyletic clades, although H. japonica is not supported in either analysis. Heterodermia casarettiana is similar to H. ‘reagens’ except that the lobe underside features yellow pigment spots (K−; probably pulvinic acid derivates); this taxon forms a paraphyletic and non-supported grade together with the single sample of H. ‘violacea’ in the parsimony analysis and also including H. ‘pseudobscurata’ and H. ‘rottbollii’ in the Bayesian analysis. Heterodermia lamelligera takes a basal position in both analyses. The latter three lack norstictic acid (as in H. japonica) but produce yellow pigment spots (as in H. casarettiana); they differ in that the yellow pigments are K+ purple (H. ‘pseudobscurata’ and H. ‘rottbollii’) and/or in the production of apothecia instead of soralia (H. lamelligera and H. ‘rottbollii’). Heterodermia ‘violacea’ agrees with H. casarettiana in producing norstictic acid, but lacks yellow pigment spots and produces apothecia instead of soralia; it would thus rather fit the concept of the primary species of H. ‘reagens’. Removing H. ‘violacea’ from the


Figure 3

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Habit of Heterodermia species. A–D. H. flabellata, thallus lobes with apothecia and arachnoid underside (in B). E–F. H. obscurata, thallus lobes with soralia and arachnoid underside (in E).

parsimony analysis increases the support for the H. casarettiana clade. Although most of the analysed samples originate from Costa Rica, some remarkable geographic patterns can be seen (Fig. 5), in particular the almost identical ITS sequences of one sample of H. flabellata from northwestern Costa Rica and one sample of H. obscurata from the southeastern part of the country. Also, the two sequences downloaded from GenBank of H. flabellata and H. obscurata merge with the sequences generated from the Costa Rican specimens. Specimens of H. japonica, H. ‘reagens’, and H. casarettiana were partly collec-

ted at the same localities, which did not affect their clustering according to morphological and chemical features in the analysis.

Discussion and conclusions Our analysis suggests that lobe morphology, underside pigmentation, and medullar chemistry are important for recognising monophyletic groups in the Heterodermia obscurata group and thus support a narrow species concept as partly

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Figure 4

Habit of Heterodermia species. A–B. H. japonica, thallus lobes with soralia and violet underside (in B). C–D. H. casarettiana, thallus lobes with soralia and yellow pigment spots on lobe undersides (in D). E–F. H. ‘violacea’, thallus lobes with apothecia and violet underside (in F).

advocated by Kurokawa (1962, 1998) and Trass (2000). For example, H. japonica and H. ‘reagens’ were considered conspecific by recent workers (Swinscow & Krog, 1976; Moberg & Purvis, 1997; Moberg & Nash, 2002), but our samples of these species form separate monophyletic clades, suggesting that the presence of norstictic acid allows to discriminate otherwise morphologically similar and closely related taxa. The same applies to yellow pigment spots, which in our analysis appear to be taxon specific but in recent treatments of Het-

erodermia have not been accepted as specific character, with the inclusion of H. propagulifera (very similar to H. casarettiana but producing anthraquinones instead of pulvinic acid derivates) in H. japonica s.lat. (Swinscow & Krog, 1976; Moberg & Purvis, 1997; Moberg & Nash, 2002). An alternative interpretation of the observed phylogenetic patterns would be that intraspecific populations are highly structured due to past bottleneck situations and with restricted gene flow due to isolation mechanisms. However, such an


Figure 5

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Geographical distribution in Costa Rica of samples of species of the Heterodermia obscurata group used in the present analysis. Thin lines connect samples with very similar or identical sequences receiving strong support.

interpretation seems highly unlikely given the origin of the examined samples, with several specimens of H. japonica, H. ‘reagens’, and H. casarettiana originating from the same localities and even from mixed collections. The two other taxa containing norstictic acid besides H. ‘reagens’, viz. H. casarettiana and H. ‘violacea’, form a monophyletic group, again supporting the taxonomic importance of medullary chemistry in this group of species. The position of H. ‘violacea’ with respect to H. casarettiana is unresolved, although morphologically it differs by the absence of yellow pigment spots and the formation of apothecia instead of soralia. Regarding the presence of yellow pigment spots on the lobe underside, the two analyses provide ambiguous results. In the Bayesian analysis, three of the four taxa featuring yellow spots (H. casarettiana, H. ‘pseudobscurata’, H. ‘rottbollii’) are found on a clade together with H. ‘violacea’ (yellow spots absent), while in the maximum parsimony analysis, the positions of H. ‘pseudobscurata’ and H. ‘rottbollii’ are unresolved. There is no clear separation of taxa with anthraquinones (H. ‘pseudobscurata’, H. ‘rottbollii’) versus pulvinic acid derivates (H. casarettiana, H. lamelligera). Yet, the fact that the single samples of H. lamelligera, H. ‘pseudobscurata’ and H. ‘rottbollii’ are not nested within other taxa in the cladogram suggests that they represent independent species. The use of contrasting reproductive modes to distinguish species is ambiguously resolved

in our analysis. In two cases, viz. Heterodermia ‘rottbollii’ (apothecia) vs. H. ‘pseudobscurata’ (soralia) and H. ‘violacea’ (apothecia) vs. H. ‘reagens’ (soralia), the fertile vs. sorediate counterparts do not merge (except for the unresolved position of H. ‘pseudobscurata’ and H. ‘rottbollii’ in the Bayesian analysis). However, H. flabellata (with apothecia) and H. obscurata (with soralia) clearly merge. Since we have only employed a single data set, we cannot distinguish whether the cause of this lack of separation is that only one species is involved, or whether processes such as incomplete lineage sorting are responsible. Additional studies are necessary to resolve the taxonomic status of the two reproductively deviating forms. It is remarkable that in this case the two reproductive forms also exhibit morphological differences. H. flabellata has closely appressed, almost dichotomously branched lobes, while H. obscurata features loosely appressed, anisotomously branched lobes. Because of its limited geographic range and sampling size, this study cannot provide a thorough solution to the application of characters to circumscribe species in Heterodermia. In some widespread and common species (e.g. H. flabellata vs. H. obscurata) the situation might be more complex, with strains of cryptic species not recognised by morphological and chemical features alone, such as in the genus Letharia (Kroken & Taylor, 2001). However, our study shows

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that forms distinguished by medullary chemistry or lobe underside pigmentation alone cannot be simply lumped into single species but should, at least for the time being, be recognised as independent. This requires great care in the identification of specimens, since the medullary depsidone norstictic acid is often found in the lobe tips only and the yellow spots on the lobe underside and their K-reaction are not easily observed. This implicates, for example, that H. ‘rottbollii’, which was considered a variety of H. flabellata by Kurokawa (1962) and merged with that species by later authors, should be recognised at the species level. The taxon features scattered yellow spots instead of an ochraceous red arachnoid tissue and is clearly not closely related to H. flabellata, as obvious from this analysis. A similar pattern is observed in H. ‘pseudobscurata’, which differs in the same way from H. obscurata and is more closely related to H. japonica and its allies.

Acknowledgements The stay of the second author at The Field Museum was financed by a fellowship of the Fulbright Postdoc-Program of the Spanish Ministery of Science and Education. The Ticolichen biodiversity inventory in Costa Rica is supported by a grant from the National Science Foundation (DEB 0206125 to The Field Museum; PI Robert Lücking) and by funds from the World Bank and the Dutch Government to the Instituto Nacional de Biodiversidad (INBio). We appreciate the support ´ of the Sistema Nacional de Areas de Conservación (SINAC) and the Ministerio de Ambiente y Energ´ıa (MINAE) in receiving collection permits and those necessary for taxonomic DNA analysis.

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