endomelanconiopsis, a new anamorph genus in the botryosphaeriaceae

Endomelanconiopsis, a new anamorph genus in the Botryosphaeriaceae

Enith I. RojasEdward Allen Herre

Smithsonian Tropical Research Institute, ApartadoPostal 0843-03092, Balboa, Ancon, Republic ofPanama

Luis C. MejıaDepartment of Plant Biology and Pathology, RutgersUniversity, 59 Dudley Road, Foran Hall, NewBrunswick, New Jersey 08901

A. Elizabeth ArnoldDivision of Plant Pathology and Microbiology,Department of Plant Sciences, University of Arizona,Tucson, Arizona 85721

Priscila Chaverri1

Department of Biology, Howard University, 415 CollegeStreet NW, Washington D.C. 20059

Gary J. Samuels2

United States Department of Agriculture, AgricultureResearch Service, Systematic Mycology & MicrobiologyLaboratory, B-011A, Room 304, 10300 Baltimore Ave.,Beltsville, Maryland 20705

Abstract: A new lineage is discovered within theBotryosphaeriaceae (Ascomycetes, Dothideomycetes,incertae sedis). Consistent with current practice ofproviding generic names for independent lineages,this lineage is described as Endomelanconiopsis gen.nov., with the anamorphic species E. endophytica sp.nov. and E. microspora comb. nov. (5 Endomelanco-nium microsporum). Endomelanconiopsis is character-ized by eustromatic conidiomata and holoblasticallyproduced, brown, nonapiculate, unicellular conidia,each with a longitudinal germ slit. Phylogeneticanalysis of partial sequences of LSU, ITS andtranslation elongation factor 1 alpha (tef1) indicatethat E. endophytica is sister of E. microspora and thatthey are nested within the Botryosphaeriaceae.However because there is no support for the‘‘backbone’’ of the Botryosphaeriacae we are notable to see the interrelationships among the manygenera in the family. Neither species is known to havea teleomorph. Endomelanconiopsis differs from En-domelanconium because conidia of the type species ofEndomelanconium, E. pini, are papillate at the base,

conidiogenous cells proliferate sympodially and thepycnidial wall is thinner; we postulate that theteleomorph of E. pini as yet unknown is aninoperculate discomycete. Endomelanconiopsis endo-phytica was isolated as an endophyte from healthyleaves of Theobroma cacao (cacao, Malvaceae) andHeisteria concinna (Erythroplaceae) in Panama. En-domelanconiopsis microspora was isolated from soil inEurope.

Key words: Austrocenangium, Endomelanconium,endophytic fungi, Heisteria concinna, systematics,Theobroma cacao

INTRODUCTION

Fungal endophytes live inside plant tissues for all orpart of their lives without causing apparent diseasesymptoms (Wilson 1995). Endophytic fungi havebeen found within host tissues at high density anddiversity in all tropical and temperate ecosystemswhere endophytes have been sought (e.g. Arnold andLutzoni 2007, Clay and Holah 1999, Hawksworth2001). Foliar fungal endophytes have been found tobe abundant and diverse in intact tropical forests withhigh plant diversity and low density of individualspecies (Dreyfuss and Petrini 1984, Lodge et al 1996,Rodrigues and Petrini 1997, Arnold et al 2000, vanBael et al 2005), and in mangroves and Theobromacacao agroforestry systems (cacao, Malvaceae, Mal-vales) with low plant diversity and high density ofindividual plant species (Gilbert et al 2002, Suryanar-ayanan et al 1998, Arnold et al 2003).

In most tropical tree species leaves are flushed in alargely endophyte-free (E2) condition (Arnold et al2003, Herre et al 2007). A taxonomically diverseassemblage of airborne spores land on leaf surfaces,some of which initiate endophytic associations. Afterthe wetting of spore-laden leaf surfaces some sporesgerminate and penetrate directly through the cuticleinto the leaf tissue, where hyphae grow between cells(Herre et al 2005, 2007). With time the initiallyendophyte-free leaf tissues become saturated withendophytes, with ca. 100% of sampled leaf fragments(2 3 2 mm) containing cultivable fungi (Arnold et al2003). Data from cohorts of leaves suggest that overtime the species composition of fungal endophyteswithin a leaf changes and that endophyte diversity(number of species per isolate recovered) usuallydeclines as leaves pass from maturity toward senes-cence. Within the leaf the distribution of fungalspecies resembles a quilt-like patchwork: different

1 Current address: AGNR-Plant Science & Landscape Architecture,2112 Plant Sciences Building, University of Maryland, College Park,MD 20742-44522 Corresponding author. E-mail: [email protected]

Mycologia, 100(5), 2008, pp. 760–775. DOI: 10.3852/07-207# 2008 by The Mycological Society of America, Lawrence, KS 66044-8897

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species usually abutting, producing an extremelyheterogeneous mix of fungal species and genotypesat fine scales (Brayford 1990a, b, Lodge et al 1996,Herre et al 2007). Colonization by endophytic fungihas been shown to reduce pathogen susceptibility inthe host plant and affect host photosynthetic andhydraulic physiology in various ways, but general rulesfor tropical plant-endophyte interactions are not yetestablished (Arnold et al 2003, Herre et al 2005, 2007,Arnold and Engelbrecht 2007, Mejıa et al 2003,2008).

Arnold et al (2003) reported 344 endophyticmorphospecies (determined by colony appearancein agar culture) from 126 naturally infected leaves ofT. cacao collected during 2 y from five sites inPanama. Common among these fungal morphospe-cies was one that produced a dark gray or green, tonearly black, colony on potato-dextrose agar. Largeeustromatic pycnidial structures developed in agarcultures; these contained dark brown, holoblasticallyproduced conidia with a longitudinal germ slit.Several representatives of this morphospecies couldbe identified as an Endomelanconium (Corda) Petrakas characterized by Sutton (1980). What appeared tobe the same species was isolated also as an endophytefrom leaves of Heisteria concinna (Erythroplaceae,Santalales), a tree that often grows in association withcacao in lowland moist forests in Panama. Thepublished description of E. microsporum Verkley &van der Aa (Verkley and van der Aa 1997), a speciesisolated from soil in Europe, indicated that it was thesame or very closely related to the endophyte. Thecultures and anamorph morphology of these speciesare strongly suggestive of Botryosphaeria and itsrelatives (Luque et al 2005, Crous et al 2006, Phillips2008). At least some described Endomelanconiumspecies are anamorphs of inoperculate discomycetes,although the teleomorph of the type species, E. pini(Corda) Petrak (Petrak 1940), is not known. Possibleteleomorph relationships and subtle morphologicaldifferences among these various described Endome-lanconium species lead us to suspect that the genus isparaphyletic.

In the present study we examine the phylogeneticposition of the endomelanconium-like endophytes bymeans of molecular sequence data from three loci.We compare them to described species of Endome-lanconium.

MATERIALS AND METHODS

Collection and cultures.—The endophytic cultures reportedhere were obtained from two surveys of endophytic fungi inthe Republic of Panama. The first was carried out in 1999and 2000 in five lowland sites with mixed forest cover across

the Isthmus of Panama, including Barro Colorado Island(BCI, Canal Zone, 09u99350N, 79u509330W) and Nombre deDios (ND, Colon, 09u349N, 79u289W). The mean annualrainfall in ND is 4041 mm, and the average annualtemperature is approximately 26 C (Tosi 1971); at BCIaverage annual temperature is 27 C and rainfall is approx-imately 2600 mm (Leigh et al 1996). In the first surveyleaves of three different ages were sampled from adultTheobroma cacao trees during two seasons (dry and rainy) in1999 and 2000. In the second year of the first survey samplesalso were taken from two co-occurring species, Heisteriaconcinna and Ouratea lucens (Ochnaceae, Malpighiales)(Arnold et al 2003).

The second survey was carried during the rainy season(Jul 2000) at ND and samples were taken only from a singletree of Theobroma cacao. In this survey leaves of five ageclasses (young, 1 wk old leaves; young-mature, 2 wk oldleaves; mature, 4 wk old leaves; mature–old, 8 wk old leaves;and old, . 12 wk old leaves) were sampled to addressquestions regarding colonization patterns. The leaves werecollected at the same time and were processed the day thatthey were collected.

The endomelanconium-like isolates were found duringthe rainy season in asymptomatic foliage of T. cacao and H.concinna at BCI in our first survey; it was the most commonfungus in healthy leaves of cacao for the second survey. Atotal of 15 isolates of endomelanconium-like endophyteswere selected for DNA sequence analyses.

Characterization of colonies and morphology.—After initialisolation on 2% malt extract agar (MEA, Difco, Detroit,Michigan), isolates were cultured on potato-dextrose agar(PDA; BBL, Sparks, Maryland) and simple nutrient agar(SNA, Nirenberg 1976) at 25 C with a 12 h cool whitefluorescent light/12 h dark photoperiod. In addition apiece of sterile filter paper was put on the surface of thesolidified SNA to induce sporulation. Pycnidia typicallyformed within 3 wk on both media.

Growth rates were determined in darkness (with fluores-cent light briefly introduced when the colonies weremeasured) on PDA and SNA (without filter paper) at fivetemperatures (15, 20, 25, 30 and 37 C). Inoculum forgrowth rate studies was taken from young, actively growingcolonies on cornmeal-dextrose agar (Difco cornmeal agar +2% w/v dextrose). A 5 mm diam plug was cut from the edgeof the colony and placed in the middle of the test medium(PDA or SNA). Measurements of colony radius from theedge of the inoculum plug to the edge of the colony weretaken at 24 h intervals. The growth trial was repeated twiceon succeeding weeks and the two measurements wereaveraged.

Morphological descriptions were made from isolatessporulating on PDA or SNA (with filter paper) at 25 C forca. 10 d with photoperiod 12 h darkness/12 h cool whitefluorescent light. Thirty conidia and up to 30 conidiogen-ous cells were measured for each isolate. Sections ofpycnidia for microscopic examination were prepared witha freezing microtome (IEC-CTF Microtome-Cryostat, Inter-national Equipment Co., Needham Heights, Massachu-setts).

ROJAS ET AL: ENDOMELANCONIOPSIS GEN. NOV. 761

Images were captured with a Nikon DXM1200 digitalcamera and Nikon ACT 1 software or with a Nikon Ds-Fi1camera and NIS Elements Basic Research 2.30, SP4(Nikon). Some composite images were made with HeliconFocus version 4.21.5 Pro (Helicon Soft, www.heliconfocus.com). Measurements were made with Scion Image (releaseBeta 4.0.2; Scioncorp, Frederick, Maryland). Data wereanalyzed statistically with Systat 10.0 (SPSS, Chicago,Illinois). Representative cultures of the endophytic endo-melanconium-like species were deposited in the Centraal-bureau voor Schimmelcultures (CBS), Utrecht, the Nether-lands.

DNA extraction, PCR amplification and sequence assembly.—Mycelium for DNA extraction was prepared in potato-dextrose broth (Difco). DNA was extracted following themanufacturer’s protocol for fresh plant tissue with thePureGene genomic DNA isolation kit (Gentra Systems,Minneapolis, Minnesota).

For each isolate 25 mL PCR reactions were employed toamplify sections of the nuclear ribosomal large subunit(LSU), internal transcribed spacers and 5.8 s gene (ITS),and translation elongation factor 1 alpha (tef1). PCRmixtures contained 2.5 mL 103 PCR buffer (New EnglandBiolabs, Ipswich, Massachusetts), 200 mM dNTPs, 25 pmoleeach primer, 1.25 units Taq DNA Polymerase and 10–50 ngtemplate DNA. Primers LROR and LR7 were used toamplify ca. 1100 base pairs of LSU, followed by sequencingof ca. 600 bp with LROR and LR3 (Rehner and Samuels1994). Primers ITS5 and ITS4 (White et al 1990) were usedto amplify the approximately 540 bp ITS, followed bysequencing with the same primers. Primers EF1-728F(Carbone and Kohn 1999) and tef1 rev (Samuels et al2002) were used to amplify and sequence ca. 600 bp of tef1.PCR was carried out on a PT-200 PCR thermal cycler (MJResearch, Waltham, Massachusetts) according to theseconditions: 95 C for 5 min; 35 cycles denaturation at 95 Cfor 30 s, annealing at 51 C for 30 s, extension at 72 C for1 min; and a final extension at 72 C for 10 min. Theconcentration of PCR products in ng/mL was determinedon 1% agarose gel via electrophoresis with Lambda Hind IIIDNA as a marker. PCR products were purified with ExoSAP-ITH reagent following the manufacturer’s instructions (USBCorp., Cleveland, Ohio). Bidirectional sequences wereobtained with BigDye Terminator cycle sequencing kit(Applied Biosystems, Foster City, California). Products wereanalyzed on a 3100 DNA sequencer (Applied Biosystems).Eight representative cultures were sequenced for LSU and15 cultures were sequenced for both ITS and tef1.

Phylogenetic analyses.—Sequences were edited and assem-bled with Sequencher 4.1 (Gene Codes, Wisconsin) andClustal X version 1.8 (Thompson et al 1997) was used toalign sequences, followed by manual adjustment in Mac-Clade 4 (Maddison and Maddison 2000). New DNAsequences were deposited in GenBank (TABLE I).

Two datasets were prepared. One alignment includedLSU sequences from representatives of the major lineageswithin the Botryosphaeriaceae (Crous et al 2006), twospecies representing the endomelanconium-like group, andspecies of Guignardia as outgroup, for a total of 56

terminals. The second dataset included a concatenatedalignment of ITS and tef1 sequences for 15 endomelanco-nium-like isolates from T. cacao and H. concinna,Endomelanconium microsporum (ex type culture, CBS353.97), representative species of the Botryosphaeriaceae,and two species of Guignardia, identified as closely relatedto Botryosphaeria (Schoch et al 2006) as outgroup taxa (totalof 50 terminals).

The LSU dataset was analyzed with PAUP* v. 4.0b10(Swofford 2002) using the neighbor joining (NJ) methodwith Kimura 2-parameter distance calculation and maxi-mum parsimony (MP) using the heuristic search optionwith simple taxa additions and tree bisection and recon-nection (TBR) as the branch-swapping algorithm. Robust-ness of the resulting topology was assessed with 1000bootstrap replicates in both neighbor joining and parsimo-ny analyses.

The ITS and tef1 datasets were analyzed separately and inthe absence of phylogenetic conflict combined for analysiswith NJ and MP as described above and with Bayesianmethods (Rannala and Yang 2005) as implemented with theprogram MrBayes v3.1.2 (Huelsenbeck and Ronquist 2001).MrModeltest 2.2 (Nylander 2004) was used to select the bestmodel, taking into account both the hierarchical likelihoodratio test (LRT) and Akaike information criteria (AIC).Preference was given to the latter (AIC). Bayesian analysiswas performed with models specific to each partition of thedata (GTR + I + G for ITS, and GTR + G for tef). TheBayesian MCMC (Markov chain Monte Carlo) analysis wasrun with three hot chains and one cold chain for 1 500 000generations, sampling every 500 generations. The first 400trees were discarded as burn-in on the basis of likelihoodscores, and PAUP* was used to construct a 50% majorityrule consensus tree from the remaining 5600 trees.

RESULTS

The endomelanconium-like endophyte was foundonly during the rainy season. At Nombre de Dios itwas not found in the first survey, but it was isolatedfrom Theobroma cacao and Heisteria concinna at BCI.It was the most abundant species isolated from thesingle tree (Theobroma cacao) that was the subject ofthe second survey. Of 16 cultures identified as beingthe morphospecies that included the endomelanco-nium-like fungus, nine were confirmed by microscopyto be endomelanconium-like. The remaining sevencultures either had died before they could be fullyidentified or were a different fungus. The endome-lanconium-like fungus was isolated from mature andold leaves; it never was found in leaves younger than1 wk.

Phylogenetic analysis.—BLAST analyses of the NCBIGenBank database with LSU sequences of theendomelanconium-like cultures showed that theclosest matches (maximum identity 97% and se-quence coverage of 100%) were with members of

762 MYCOLOGIA

the Botryosphaeriaceae. We aligned our sequences ofthe endomelanconium-like endophyte with the ex-type culture of E. microsporum CBS 353.97 and withone representative taxon selected from each clade inthe Botryosphaeriaceae tree of Crous et al (2006,TreeBase M2707); we added to this tree Aplosporellaprunicola (Damm et al 2007). Because no variationwas observed among the eight endophytic isolatessequenced for the LSU locus, only two were selectedfor the analysis. The two sequences of the endophytein the tree (FIG. 1) are from cultures that representthe phenotype-groups described below. The LSUdataset had 933 characters, 102 of which wereparsimony informative, 47 were parsimony uninfor-mative and 784 were constant when gaps were treatedas missing characters. A heuristic search found 100equally parsimonious trees (TL 5 339 steps; CI 5

0.522; RI 5 0.787; RC 5 0.411). Neighbor joininganalysis with Kimura 2-parameter distance calculationon the sequence data yielded trees with similartopology (see FIG. 1). The two isolates of theendomelanconium-like endophyte clustered with E.microsporum in a highly supported clade (FIG. 1),indicating that they are congeneric. Although therewas high BS support (FIG. 1) for Botryosphaeria s. lat.,(including Pseudofusicoccum), only internal cladesreceived significant support, and there was nosupport for a backbone. Thus it was impossible todetermine relationships among the clades.

The manually adjusted alignment of 15 isolates ofthe endomelanconium-like species with species of theBotryosphaeriaceae for the concatenated ITS and tef1datasets contained 34 ambiguously aligned charactersin the ITS region (25–42 bp, 54–69 bp insertionfound only in G. mangiferae and G. bidwellii) thatwere excluded, leaving 892 characters. Of these 385were parsimony informative, 46 were parsimonyuninformative and 461 were constant when gaps weretreated as missing characters. A heuristic search foundsix equally parsimonious trees (TL 5 967 steps; CI 5

0.698; RI 5 0.908; RC 5 0.634) and same topology wasfound with Kimura 2-parameters distance calculationsin neighbor joining and Bayesian analyses.

Together these analyses placed the endomelanco-nium-like endophyte isolates and E. microsporum inthe Botryosphaeriaceae (FIG. 2). The 15 endophyticcultures form a highly supported (100% bootstrap,1.0 posterior probability) clade. There was nodistinction between endophytic isolates from T. cacaoand those from H. concinna, indicating that theendomelanconium-like endophytes collected fromboth hosts likely are one species. The endophytesand the ex-type culture of E. microsporum formed astrongly supported clade but E. microsporum differedmarkedly (FIG. 2). The closest morphological com-

parison within the Botryosphaeriaceae is with Sphaer-opsis subglobosa, the anamorph of Neodeightoniasubglobosa Booth in Punithalingham (1969)and which Crous et al (2006) referred to as‘Botryosphaeria’ subglobosa (FIG. 1). It is not closelyrelated to E. microsporum or the endomelanconium-like endophyte (FIG. 1).

Although ITS sequences for all the endophyticcultures were identical, there was slight variation inthe tef1 gene. Two groups were formed with . 0.95Bayesian posterior probability and high NJ and MPbootstrap support, and one isolate (culture 3370 fromT. cacao, BCI, 1999) formed a third group thatdiffered from the other two groups by two bp in thesame locus. These three groups correlated with thesame three groups that were observed based on themorphological characters of their colonies (FIGS. 3–5,see below). There was no obvious relationshipbetween observed groups and their occurrence inparticular sites or host species.

Morphological analyses.—The endomelanonium-likeendophyte was not observed sporulating in nature.Pycnidia (FIGS. 6–9) formed in culture were eustro-matic (Sutton 1980), tending to be cylindrical, largeand the wall was formed of pseudoparenchymatouscells (FIGS. 6–9). The locule was convoluted (FIGS. 8,9) and was lined entirely with conidiogenous cells(FIGS. 8–10). The conidiogenous cells arose directlyfrom the cells of the pycnidial wall (FIG. 11) andpossessed a single, terminal, holoblastic conidiogen-ous locus (FIGS. 11, 12). Macroconidia (FIGS. 13–15)were opaque, brown, unicellular and bilaterallysymmetrical, elliptical in face view and flattened witha narrow edge; a germ slit was positioned on theflattened edge. Often conidia were flattened at thebase at the point of abscission from the conidiogen-ous cell. In addition to these macroconidia, microco-nidia (spermatia?, FIG. 18) formed either in the samepycnidium as the macroconidia or in separatepycnidia (FIG. 16) on SNA and PDA. Microconidiawere variable in shape, ellipsoidal to subcylindrical,hyaline, and arose from phialidic conidiogenous cells(FIG. 17). Two size classes were observed: those withmicroconidia 2.0–4.5 3 1–2 mm (Mean 5 2.9 3

1.3 mm) and those with microconidia 3–10 3 1.0–2.7 mm (Mean 5 6.9 3 1.7 mm).

Two size classes of macroconidia were observed(FIG. 19, TABLE II), designated respectively as Groups1 and 2. Macroconidia of Group 1 were shorter (5.7 6

0.7 3 3.8 6 0.3 mm; 95% confidence interval (CI) 5.6–5.9 3 3.7–3.8 mm), macroconidia of Group 2 werelonger (7.2 6 0.9 3 4.4 6 0.45 mm; 95% CI 7.1–7.3 3

4.3–4.4 mm), the macroconidia of a single culture(3370), referred to below as Group 3 because of its


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764 MYCOLOGIA

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differing growth rate, were not significantly differentin size from others (6.7 6 0.3 3 4.1 6 0.2 mm; 95% CI6.6–6.8 3 4.1–4.2). Macroconidia of E. microsporumwere of intermediate length but wider (FIG. 19; 6.1 6

0.4 3 4.6 6 0.4 mm; 95% CI 6.0–6.3 3 4.4–4.7 mm)than those of either of the endophyte size groups(FIG. 19). No differences were observed in the size ofthe conidiogenous cells of the endophyte groups.

FIG. 1. Parsimony tree based on LSU sequence. The tree was rooted to Guignardia bidwellii, Guignardia sp, and Phyllostictasp. This tree is modified from Crous et al (2006), to which the reader should refer for full discussion of the clades; numbers onthe right side refer to clades in Crous et al (2006). Numbers in the nodes refer to bootstrap support values from 1000 replicatesof NJ and MP respectively.

766 MYCOLOGIA

FIG. 2. Bayesian inference tree (majority rule consensus of 5600 trees) based on the combined ITS and tef1 sequences.Fifteen Endomelanconiopsis isolates from T. cacao and H. concinna were included. Tree was rooted with Guignardia mangiferaeand G. bidwellii. Black lines are $ 0.95 posterior probability coefficients and $ 70% NJ and MP bootstrap support; (*)represents , 0.95 posterior probability coefficients and $ 70% NJ and MP bootstrap support; (**) represents , 0.95 posteriorprobability coefficients and , 70% NJ and MP bootstrap support. Group numbers refer to phenotype-defined groups inEndomelanconiopsis endophytica. Accession numbers in boldface signify ex-type cultures.


Colonies on PDA become dark gray to black andthe quantity of aerial mycelium was variable. Opti-mum temperature for growth on PDA and SNA was30–35 C. Three growth curves were observed after 5 don PDA (FIG. 20). Two of these curves correspondedto the macroconidium-size groups noted above:Group 1 (n 5 5) colony radius at 30 C the same asthe colony radius at 37 C (ca. 50 mm); Group 2 (n 5

9) colony radius at 30 C (ca. 50 mm) that was lessthan the radius at 37 C (55–60 C); Group 3 (n 5 1)colony radius at 30 C (ca. 45 mm) greater than theradius at 37 C (ca. 35 mm). All endophytic isolatesgrew well at 37 C, although isolate 3370 (Group 3)was significantly slower at 30 and 37 C than were theother cultures. Pycnidia usually formed within 1 wk at25 C under alternating light and dark.

In the phylogram (FIG. 2) Group 2 forms a wellsupported clade that is sister of Group1/Group 3.Thus Group 1 isolates had shorter conidia andrelatively slower growth at 37 C while Group 2 isolateshad longer conidia and faster growth at 37 C. Group3, which comprised a single culture, had conidia ofintermediate size and a significantly slower rate ofgrowth than Groups 1 or 2. Despite the phenotypicdifferences, the phylogenetic distance between thesesister groups is small (only five bp difference in tef).We consider the phenotypic differences to representintraspecific variation, but additional collectionsmight support taxonomic subdivision.

Conidia of the endophytic endomelanconium-likespecies are most similar in size to those of E.

microsporum and smaller than the reported measure-ments of all other Endomelanconium species (TA-

BLE II). As noted above conidia of E. microsporumare wider (4.4–4.7 mm) than the endophytes. Afurther difference between the endophytes and E.microsporum is the absence of chlamydospores in theformer (see Verkley and van der Aa 1997). Microco-nidia have not been reported for any other Endome-lanconium species, but the describing authors couldhave overlooked them. We did not notice them in theex-type culture of E. microsporum.

These differences, combined with the respectivedifferences in habitat (leaf endophyte vs. soil) andgeography (Central America vs. southwestern Pacific)and our phylogenetic results, distinguish the endo-phytes from E. microsporum at the species level.

Comparison with Endomelanconium species.—Onlyfive species have been included in Endomelanconium,including E. microsporum discussed above and theunnamed anamorph of Austrocenangium australe(Speg.) Gamundı (TABLE II). The type species ofEndomelanconium, E. pini (Corda) Petrak, has beencollected from dead trunks and branches of Abies fromSwitzerland, Austria, Germany, the Czech Republicand Slovakia but apparently has not been grown inculture. As a result we did not include it in ourphylogenetic analysis. No teleomorph is known for it.

There are small but significant differences betweenE. pini and the two endomelanconium-like speciesnoted above. We studied one specimen of E. pini

FIGS. 3–5. Endomelanconiopsis endophytica, three groups on PDA after10, 25 C, 12 h cool white fluorescent light/12 h dark.3 Group 1 (7394). 4. Group 2 (7463). 5. Group 3 (3370).

R

FIGS. 6–18. Endomelanconiopsis endophytica/Botryosphaeria anamorph. 6–9. Pycnidia. 6. From SNA. 7. From PDA. 8, 9.Pycnidia showing convoluted hymenium in the locule. 10–12. Conidiogenous cells. 10, 11. Conidiogenous cell arising frompycnidial wall cells. 11, 12. Holoblastic conidiogenous locus. 13–15. Macroconidia, opaque brown, unicellular, often flattened

768 MYCOLOGIA

at the base. 14, 15. Germ slit on the flattened edge marked with arrow. 16. Pycnidium producing microconidia on SNA. 17.Phialidic microconidial conidiogenous cells. 18. Microconidia ellipsoidal to subcylindrical, hyaline. FIG. 6 from 9090; 7, 8, 10,14 from 7463; 9 from 9181; 11, 12 from 9188; 13 from 7331; 15 from 3370; 16–18 from 8145. Bars: 6, 7 5 0.5 mm; 8, 9 5

200 mm; 10 5 20 mm; 11–15 5 10 mm; 16 5 0.5 mm; 17, 18 5 10 mm.


(Krieger, Fungi Saxonici 1450; BPI 402518) and madethe following observations. Conidiogenous cells of E.pini tend to proliferate by pushing aside the first-formed conidiogenous cell, which remains as a spur,to form up to two more conidiogenous cells(FIGS. 21–24). Conidiogenesis is holoblastic, but thepoint of attachment of the conidium to the con-idiogenous cell is narrow, which results in the conidiahaving a papillate base when they are discharged(FIGS. 25–28). Papillate conidia also were noted byPetrak (1940) when he proposed Endomelanconium.Conidiogenous cells of E. nanum Gamundı &Arambari (Gamundı and Arambari 1983, Suarez etal 2000) and the unnamed Endomelanconium ana-morph of Austrocenangium australe (Gamundı 1997)are illustrated as being narrowed at the point ofdehiscence of conidia and conidia are papillate.Conidia of E. phoenicicola Yanna et al (Yanna et al1999) have a flat, somewhat protuberant base but theconidiogenous cell is not illustrated as being con-stricted at the point of dehiscence. Pycnidia of E. piniare subcortical and have a convoluted hymenium; thewalls are made of small-celled pseudoparenchyma(Petrak 1940, Sutton 1980) but there is minimalstromatic development (see also Sutton 1980), unliketypical botyosphaeriaceous anamorphs. Even thoughour endomelanconium-like fungi are known onlyfrom cultures their pycnidial walls are formed ofthick-walled pseudoparenchymatous cells (see Verkleyand van der Aa 1997, FIG. 2). Conidia of E. pini areextruded in a slimy, black mass that may extendconsiderably beyond the erumpent pycnidia (alsodescribed by Petrak 1940).

The intimate juxtaposition of E. nanum (Cash)Gamundı and the unnamed Endomelanconium ana-morph with their respective Austrocenangium Ga-mundı teleomorphs (Helotiales) leave no doubt oftheir respective conspecficity (Gamundı et al 1983,

Gamundı 1997). Based on the observations notedabove, the endomelanconium-like fungi that wediscussed are unlikely to be phylogenetically relatedto E. pini, which appears to be more similar to thehelotiaceous anamorphs of Austrocenangium.

The anamorph genus Endomelanconium Petrak isparaphyletic. There are significant differences inpycnidial and conidial morphology and in prolifera-tion of conidiogenous cells among the known‘Endomelanconium’ species (TABLE II). Endomelanco-nium nanum and the unnamed Endomelanconiumanamorph of A. australe (Gamundı and ArambarriGamundı 1997), and most likely E. pini, are helotiac-eous anamorphs. Whether the Austrocenangiumanamorphs are congeneric with E. pini remains tobe proven, and proliferating conidiogenous cells arenot reported for either anamorph. Our endomelan-conium-like endophyte and E. microsporum areobviously Dothideomycetes and not likely to becongeneric with E. pini. However, apart from abroadly circumscribed Endomelanconium, we do notknow of any genus that can accommodate them.Consequently we propose a new genus, Endomelanco-niopsis. The illustrations of E. phoenicicola (Hyde et al1999) suggest that it is also botryosphaeriaceous,possibly a species of Endomelanconiopsis; the describ-ing authors did not report the deposit of cultures ofthis species.

TAXONOMY

Endomelanconiopsis Rojas et Samuels, gen. nov.Genus Endomelanconii Petrak simile sed ad Botryosphaer-

iaceas pertinens et conidia non papillata.Species typica: Endomelanconiopsis endophytica Rojas et

Samuels

Endomelanconiopsis endophytica Rojas et Samuels,sp. nov. FIGS. 3–18Endomelanconio microsporo Verkley & van der Aa simile,

sed conidia (4.7–)5.5–7.5(–10.0) 3 (3.0–)3.5–4.5(–6.2) mmproducens et chlamydosporae absunt; species endophytica.

Holotypus: Ex culto agaro sicco BPI 878370.

Mycobank 11838Optimum temperature at 30–37 C, colony radius

43–55 mm after 5 d (n 5 15) on PDA. Colonies at firstcolorless with hyaline immersed hyphae, after 4 dcolonies olivaceous in center and concentric ringswith irregular shape, after 10 d aerial mycelium densedark olivaceous or gray (Groups 1, 3) or shiny blackwith little aerial mycelium (Group 2). Conidiomataeustromatic, scattered throughout colony, varyingfrom globose to cylindrical, 1–3 cylindrical necks,superficial or immersed in the agar; often cylindricalpapillae protruding from the agar in groups of a few.

FIG. 19. Endomelanconiopsis endophytica. Box plot show-ing conidium length (above, black line) and width (below,gray line) of three phenotype groups. Group 4 isE. microsporum.

770 MYCOLOGIA

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Wall comprising pale brown and black angular cells,becoming hyaline and more hyphal toward theconidiogenous cells. Pycnidial locule convoluted, com-pletely lined with conidiogenous cells. Conidiogenouscells formed from the inner cells all over theconidiomata wall, discrete, determinate, cylindrical,tapered toward the apex, hyaline, holoblastic, rarelywith a single percurrent proliferation, (7.8–23.4 3

0.8–3.5 [apex] 1.3–4.1 [base] mm [Mean 5 14.2 mm 3

1.6 mm apex 3 2.2 mm base]). Conidia ellipsoidal tolimoniform, apex rounded, base flat to rounded,aseptate, hyaline when immature, dark brown with asingle longitudinal slit three-quarters of the length ofthe conidia when mature, (4.7–)5.5–7.5(–10.0) 3

(3.0–)3.5–4.5(–6.2) mm (n 5 399). Microconidiaforming in the same locules as macroconidia fromdensely arranged, enteroblastic, phialidic conidiogen-ous cells, appearing to arise from the inner cells ofthe pycnidial wall. Microconidia ellipsoidal to allan-toid, 2.0–7.0(–10.0) 3 (1.0–2.0(–3.0) mm (n 5 80),formed on PDA and SNA. Chlamydospores notobserved.

Etymology. endophytica, referring to the occurrenceof this fungus as an endophyte.

HOLOTYPE: PANAMA. Nombre de Dios, isolated fromleaves of Theobroma cacao, 2000, E. Rojas, L. Mejıa & Z.Maynard 7463 (BPI 878370, ex-type culture CBS 120379).

Commentary. Additional collections are cited (TA-

BLE I). The description given above is based on severalof these cultures grown on PDA, and it amalgamatesthe diverse measurements of the groups discussed inRESULTS. On SNA there was little mycelial productionand colonies were transparent. Pycnidia formed oftenat the edge of the colony at the interface between theagar and plastic of the Petri plate; they were muchsmaller than on PDA, nearly flask-shaped with one ormore papillae.

Endomelanconiopsis microspora (Verkley & van der Aa)Rojas et Samuels, comb. nov.; Endomelanconium microsporum Verkley & van der Aa,

Mycologia 89:967. 1997.

Mycobank 5111839This species is described and illustrated in Verkley

and Van der Aa (1997).

DISCUSSION

It comes as no surprise that Endomelanconiopsis isderived from within the Botryosphaeriaceae. Rapidlyspreading dark colonies on PDA that characterize E.endophytica and E. microspora (Verkley and van der Aa1997) are typical of Botryosphaeria s.l. (see Phillips2008 and colony descriptions in Punithalingham1978, Samuels and Singh 1986, Alves et al 2004,Luque et al 2005). The eustromatic pycnidia withoften convoluted locules (appearing multiloculate)and holoblastic conidiogenous cells arising directlyfrom cells of the pycnidial wall are further character-istics as are the rather large, unicellular conidia thatare often truncate at the base. Microconidia (sper-matia) usually are not reported for ‘Botryosphaeria’anamorphs (e.g. Pennycook and Samuels 1985,Samuels and Singh 1986, Phillips et al 2005), butthey probably are produced by all species.

Endomelanconiopsis is unusual in having a germ slitin the conidia. In the Botryosphaeriaceae we areaware only of Neodeightonia subglobosa C. Booth(teleomorph Botryosphaeria subglobosa [C. Booth]Arx. & E. Muller) as having similar conidia (seePunithalingham 1969). Crous et al (2006) observedgerm slits in conidia of this species and compared it toLasiodiplodia theobromae, the striations of which werepostulated to be germ slits. Despite the similarity ofconidia of E. endophytica and N. subglobosa, the twospecies are not closely related (FIG. 1).

Botryosphaeria and the Botryosphaeriaceae haveattracted considerable attention in recent years, withthe result that the number of identified species isincreasing rapidly along with the number of associat-ed anamorph generic names (see Crous et al 2006,Damm et al 2007). A process of dismemberingBotryosphaeria based in part on the anamorphphenotype and in part on phylogenetic analysis isunder way (Crous et al 2006, Shenoy et al 2007).There are challenges to defining genera on thesebases. First, as can be seen in (FIG. 1) there is nosupport for the backbone of the Botryosphaeriaceaewhen species of the closely related Guignardia areused as outgroup. The strongly supported lineages(FIG. 1), which is derived from Crous (2006) but witha different outgroup, could be interpreted equally asgenera or as species of Botryosphaeria. That being the

FIG. 20. Endomenanconiopsis endophytica. Growth curvesof three groups on PDA after 72 h in intermittent light.Lines 5 standard error. A total of 5, 9 and 1 isolates weremeasured respectively in Groups 1, 2 and 3.

772 MYCOLOGIA

case the argument for recognizing the lineages mustdepend on phenotype and/or biology. There is auniform biology in the Botryosphaeriaceae (seePhillips 2008); they cause cankers, root diseases andpostharvest fruit rot. Teleomorphs are very similar.Most generic differences are attributed to conidialmorphology, but while there is broad agreement ofanamorph phenotype with clades (see Crous et al2006) this is not consistent. Two primary examplesinclude the distantly related yet apparently morpho-logically indistinguishable Endomelanconiopsis, de-scribed here, and Neodeightonia subglobosa. Similarlythe highly distinctive Lasiodiplodia morphology oc-curs in two or more clades (Crous et al 2006). Thus

whether these lineages represent genera or speciescan be questioned reasonably. We have adopted thenomenclature summarized in Crous et al (2006) andin the Botryosphaeria Website (Phillips 2008) but notwithout misgivings.

Endomelanconiopsis endophytica was the dominantendophytic fungus isolated from mature to old cacaoleaves taken from the one tree that was the subject ofthe second survey at Nombre de Dios in Jul 2000.However it was not encountered in any other tree atNombre de Dios during the first survey, 1999 and2000. Thus it appears to vary greatly in its occurrenceover time, across sites and possibly among hosts. Inthe present work we discuss only endophytic isolates

FIGS. 21–28. Endomelanconium pini. 21. Two immersed pycnidia marked by glistening black masses of extruded conidia. 22–26. Holoblastic conidiogenous cells. 23. A spur-like sympodial proliferation (arrow). 24. A chain of conidiogenous cells. 23, 25,26. Constriction at the point of attachment of the conidium to the conidiogenous cell. 27–28. 27. Conidia, germ slits at arrow. 28.A papillate conidial base at arrow. From Krieger, Fungi Saxonici 1450; BPI 04024518. Bars: 21 5 0.5 mm, 22–28 5 10 mm.


from Theobroma and Heisteria. However isolates withidentical ITS sequences and consistent colony mor-phology have been recovered from additional treesand shrubs representing the Flacourtiaceae, Melia-ceae and Ochnaceae at Barro Colorado Island inPanama (Arnold unpubl). It is likely that E.endophytica has a much wider host and geographicrange than we record here. It also is strongly possiblethat reports of endophytic isolates of species such asDiplodia juglandis, Botryosphaeria sarmentorum, B.viticola or B. iberica, all of which give 97% maximumidentity and 100% sequence coverage (LSU locus),actually could refer to E. endophytica. The wide hostrange is reminiscent of Guignardia mangiferae (anam:Phyllosticta capitalensis), a ubiquitous, cosmopolitanendophyte of many genera of woody plants (Baayen etal 2002), and a species that we also have recovered asan endophyte from leaves of cacao. These fungi areknown only as endophytes in a wide range of hosts.

ACKNOWLEDGMENTS

The authors thank Amy Rossman for comments on thismanuscript; Adnan Ismaiel (BPI) for valuable advice duringthe sequencing phase of this project; Zuleyka Maynard, LuisRamirez and Terri Shirshac for help with the culturecollection; and USDA-ARS-SMML and the SmithsonianTropical Research Institute for research infrastructure andlogistical support. Walter Gams and Orlando Petrinicorrected the Latin diagnoses. Financial support wasprovided by World Cocoa Foundation (WCF), the JohnClapperton Fellowship of Mars Inc., the Andrew W. MellonFoundation and the Smithsonian Tropical Research Insti-tute. ER was the recipient of a Norman E. BorlaugFellowship in support of this work.

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