Mycol. Res. 109 (5): 556–568 (May 2005). f The British Mycological Society 556 doi:10.1017/S0953756205002716 Printed in the United Kingdom.

A re-evaluation of : relationships based on 18S and 28S rDNA1

Jinx CAMPBELL1*, Brigitte VOLKMANN-KOHLMEYER2, Tom GRA¨FENHAN3#, Joseph W. SPATAFORA4 and Jan KOHLMEYER2 1 Department of Coastal Sciences, University of Southern Mississippi, 703 East Beach Drive, Ocean Springs, Mississippi 39564, USA. 2 Institute of Marine Sciences, University of North Carolina at Chapel Hill, Morehead City, North Carolina 28557, USA. 3 Alfred-Wegener-Institute for Polar and Marine Research (AWI), Biological Oceanography, Am Handelshafen 12, 27570 Bremerhaven, Germany. 4 Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331, USA. E-mail : [email protected]

Received 15 November 2004; accepted 19 February 2005.

The Lulworthiales consists of four genera: three that were removed from the Halosphaeriales, namely , , and Kohlmeyeriella; and Spathulospora, reassigned from the Spathulosporales. However, studies have shown that neither Lulworthia nor Lindra are monophyletic genera. This study was therefore undertaken to re-evaluate the genera of the Lulworthiales based on the SSU and LSU rDNA genes. Taxonomic revisions are proposed here for Lulworthia crassa, L. lignoarenaria, L. uniseptata and Lindra marinera: Lulworthia crassa is transferred into the genus Kohlmeyeriella; Lulwoidea gen. nov. is established for L. lignoarenaria; gen. nov. is established for L. uniseptata; and Lindra marinera is reduced to synonymy with L. thalassiae. Taxonomic descriptions are emended for the genus Lulworthia s. str., and for L. grandispora and Lindra thalassiae. A neotype is designated for Lulworthia grandispora.

INTRODUCTION clade, with the two genera Lulworthia and Lindra, was assigned to the new order Lulworthiales and the new The family Halosphaeriaceae was first described by family Lulworthiaceae (Kohlmeyer et al. 2000). Mu¨ ller & von Arx (1962) and initially included six Lulworthia, initially monotypic, became one of the genera. Kohlmeyer (1972) validated and amended the largest genera of marine ascomycetes with 11 accepted family, adding the genera Lulworthia and Lindra, which species and a number of other taxa whose morpho- differed from the other representatives by having fila- logical differences are not sufficiently pronounced to mentous ascospores. Later the order Halosphaeriales allow distinction (Kohlmeyer & Volkmann-Kohlmeyer was described by Kohlmeyer (in Hawksworth & 1991). Until now Lulworthia has been treated as a Eriksson 1986) as the largest of marine ascomycetes monophyletic genus although phylogenetic studies of with over 130 marine (Jones 1995) and six freshwater rDNA have shown that it is paraphyletic (Kohlmeyer species. Molecular studies by Spatafora et al. (1998) et al. 2000, Campbell et al. 2002, Inderbitzin et al. demonstrated, however, that the Halosphaeriales was 2004). Lindra, which consists of six described species, is polyphyletic and comprised two distinct lineages. The the only genus in the Lulworthiales that does not have first clade, Halosphaeriales s. str., closely related to the apical spore chambers filled with mucus. Other genera Microascales, included 11 genera, whereas the second placed in the Lulworthiales based on molecular data include the halosphaerialean genus Kohlmeyeriella (Campbell et al. 2002) and the algicolous genera 1 Dedicated to John Webster on the occasion of his 80th birthday. Spathulospora (Inderbitzin et al. 2004) and Haloguig- * Corresponding author. nardia (Harvey 2004). The placement of Spathulospora # Present address: Federal Biological Research Centre for in the Lulworthiales is particularly important as it has Agriculture and Forestry (BBA), Institute for Plant Virology, Microbiology and Biological Safety, Ko¨ nigin-Luise-Straße 19, long been a phylogenetically enigmatic taxon in the D-14195 Berlin, Germany. systematics of the and was central to J. Campbell and others 557 earlier hypotheses concerning the origin of the Asco- described in Spatafora et al. (1998) or by using the mycota (Kohlmeyer & Kohlmeyer 1979). primers NS0 (5k-TACCTGGTTGATCCTGCC-3k)to With the addition of other genera to the NS8 (White et al. 1990) and NS7a (5k-AAGTTTGA- Lulworthiales, the present phylogenetic study was GGCAATAACAGG-3k) to NL6 (5k-CTATCCTGAG- initiated to re-evaluate the order Lulworthiales by GGAAACTTCG-3k). The PCR products were purified analysing additional isolates from different geo- using either Gene Clean (Bio101) or Prep-A-Gene graphic regions. Specific topics addressed are the matrix (BioRad, Munich), according to the manufac- monophyletic status of the genera Lulworthia and turer’s instructions and sequenced directly using either Lindra, and the position of Kohlmeyeriella, Spathulo- BigDye v2.0 sequencing kits (ABI) or Amersham spora and within the Lulworthiales. Thermo-Sequenase reactions (6 ml scale) and IRD- This investigation is based on phylogenetic analyses labeled primers (generally NS1, NS2, NS3, NS4, NS5, of partial SSU and LSU rDNA sequences for the NS6, NS7, ITS6, NL1, and NL4, occasionally ITS3; largest sampling of isolates that we had available for White et al. 1990, O’Donnell 1992). The sequencing analyses. Morphological characters relevant for the reactions were run on either an ABI 373XL or on a LI- distinction of genera and species are discussed and COR 4000L automated DNA sequencer. Sequences new taxa are proposed as the basis for initiating a were assembled and corrected using Sequencher 4.0.5 phylogenetic classification of the genera of the (Gene Codes, Ann Arbor, MI). Lulworthiales. Phylogenetic analyses MATERIALS AND METHODS Sequences were aligned with published sequence data Isolates and reference material (Table 1) using Clustal X (Thompson et al. 1997), and then refined manually in Se-Al (Rambaut 1996). Isolates were selected to represent as many different Bayesian Metropolis coupled Markov chain Monte species as possible for both Lulworthia and Lindra, Carlo (B-MCMCMC) analyses were performed using including cultures from diverse geographical locations MrBayes 3.0 (Huelsenbeck & Ronquist 2001): searches from around the globe (Table 1). Unfortunately, no were conducted for a total of two million generations voucher specimens have been deposited for a number with phylogenetic trees sampled every 100 generations. of strains by earlier workers, viz. the KMPB and PP Three independent B-MCMCMC analyses were con- isolates. These were included in the study to provide ducted to verify likelihood convergence and burnin a more complete molecular analysis, to link these parameter. Out of 20 000 resulting trees from each cultures with vouchered specimens, and to maximise analysis, the initial 400 trees (40 000 generations) were the amount of data available for future studies. identified as burn in prior to the convergence of like- Material examined: Lindra inflata: UK: Ceredigion: lihoods and were excluded from post-run analyses. In Aberystwyth, on intertidal wood block 28 July 1952, I. M. estimating the likelihood of each tree the general time Wilson 62909, (IMI 62909 – holotype). Lulworthia opaca (as reversible model of substitution (Rodriguez et al. 1990) Halophiobolus opacus): USA: Massachusetts: Provincetown, with invariant sites and gamma distribution (GTR+ on intertidal piling, 24 July 1942, E.S. Barghoorn 14 I+C) was assumed separately for both ribosomal DNA (FH – holotype). Lulworthia cfr opaca: USA: Washington: genes. The majority rule consensus tree from 19 600 San Juan Island, Friday Harbor, on driftwood 18. Feb. 1960, trees was generated along with average branch lengths J. Kohlmeyer [J.K.199] (B). Lulworthia uniseptata: Japan: and posterior probabilities (Fig. 1). Shizuoka Pref., Izu Pen., Shimoda, on submerged wood; 27 Aug. 1982, A. Nakagiri (TKB-F-5050 – holotype). Maximum parsimony analyses of the combined SSU and LSU rDNA sequence data were performed using PAUP 4.0b 10 (Swofford 2002). Gaps were treated as DNA extraction, amplification and sequencing * missing data. Due to the dense taxonomic sampling Cultures were grown in yeast malt broth (O’Donnell, of a number of the terminal clades, i.e., multiple Cigelnik & Nirenberg 1998) using tap water. After representatives of several species, a two-step search filtration small flakes of mycelium were transferred to approach was employed in an attempt to avoid local 1.5 ml tubes and covered with 300 ml CTAB buffer optima (Olmstead et al. 1993). Step-one consisted of (100 mM Tris-HCl pH 8.0 containing: 1.4 M NaCl, 100 heuristic replicates with random starting trees, 25 mM EDTA, 2% (w/v) hexadecyltrimethyl- random stepwise addition and tree-bisection- ammonium bromide, 0.2% (v/v) 2-mercaptoethanol). reconnection branch swapping with MulTrees off; a The mycelium was squeezed several times with a maximum of two trees were saved per replicate. All of micropestle before freezing in liquid nitrogen. During the shortest trees from these initial runs were saved and defrosting the mycelium was crushed with a micro- then used as starting trees for the second step, which pestle. The DNA was then isolated and purified consisted of searches with MulTrees on and the maxi- according to the method described by Turner et al. mum number of trees set to 10 000. Bootstrap values (1997) or Spatafora et al. (1998). The SSU and LSU (Felsenstein 1985) were calculated from 1000 repli- regions of the ribosomal DNA were amplified as cations using a heuristic search on 100 replicates with A re-evaluation of the Lulworthiales 558

Table 1. Fungal species, source code, origin and GenBank accession numbers included in the analyses. Names of taxa sequenced for this study are in bold.

GenBank accession no.

Taxon Sourcea Country of collection SSU LSU

Lulworthiales Anguillospora marina CBS 791.83 USA (California) AY878996 AY878955 Haloguignardia irritans N/A USA (California) AY566252 N/A Kohlmeyeriella tubulata PP 0989 Denmark AY878997 AF491264 K. tubulata PP 1105 Denmark AY878998 AF491265 Lindra crassa ATCC 56663b Belize AY878999 N/A L. marinera J.K. 4743 Puerto Rico AF047580 AF047581 L. marinera J.K. 5091B St. Croix (U.S. Virgin Islands) AY879000 AY878958 L. obtusa IFO 31317b Japan (Hokkaido) AY879002 AY878960 L. obtusa CBS 113030 Germany (Helgoland) AY879001 AY878959 L. thalassiae J.K. 4322A Belize AF195632 AF195633 L. thalassiae J.K. 5090A St.Croix (U.S. Virgin Islands) AF195634 AF195635 Lulworthia crassa IFO 32133b Japan (Niigata) AY879005 AY878963 L. crassa IFO 32134 Japan (Kouchi) AY879006 AY878964 L. fucicola ATCC 64288b Chile (Magallanes) AY879007 AY878965 L. fucicola PP 1235 Denmark N/A AF491270 L. fucicola PP 1249 Kuwait AY879008 AY878966 L. grandispora J.K. 5168Ab Belize AY879012 AY878969 L. grandispora J.K. 4686 Belize AF047582 AF047583 L. grandispora J.K. 5255A Belize AY879013 AY878970 L. grandispora PP 4319 Malaysia N/A AF491271 L. lignoarenaria ATCC 64644 Denmark AY879009 AF491272 L. lignoarenaria IFO 32135 Japan (Hokkaido) AY879010 AY878968 L. medusa J.K. 5581A USA (North Carolina) AF195636 AF195637 L. cfr opaca CBS 218.60 USA (Washington) AY879003 AY878961 L. cfr purpurea CBS 219.60 England (Devon) AY879004 AY878962 L. uniseptata IFO 32137b Japan (Shizuoka) AY879031 AY878988 L. uniseptata PP 4032 England (S. Wales) AY879032 AY878989 L. uniseptata PP 4033 England (S. Wales) AY879033 AY878990 Lulworthia sp. ATCC 64289 Chile (Tarapaca´ ) AY879011 N/A Lulworthia sp. J.K. 4843 Hawaii AF195640 AF195641 Lulworthia sp. J.K. 5332A Australia (Queensland) AY879014 AY878971 Lulworthia sp. J.K. 5393 Moorea (French Polynesia) AF195638 AF195639 Lulworthia sp. J.K. 5401B Moorea (French Polynesia) AY879015 AY878972 Lulworthia sp. KMPB 622 Germany (North Sea) AY879016 AY878973 Lulworthia sp. KMPB 957 Germany (North Sea) AY879017 AY878974 Lulworthia sp. KMPB 958 Germany (North Sea) AY879018 AY878975 Lulworthia sp. KMPB 960 Germany (North Sea) AY879019 AY878976 Lulworthia sp. KMPB 963 Germany (Baltic Sea) AY879020 AY878977 Lulworthia sp. KMPB 965 Norway (Rosfjord) AY879021 AY878978 Lulworthia sp. KMPB 966 Norway (Rosfjord) AY879022 AY878979 Lulworthia sp. KMPB 969 Germany (Helgoland) AY879023 AY878980 Lulworthia sp. KMPB 970 Germany (North Sea) AY879024 AY878981 Lulworthia sp. KMPB 986 Germany (Helgoland) AY879025 AY878982 Lulworthia sp. KMPB 992 Germany (Baltic Sea) AY879026 AY878983 Lulworthia sp. KMPB 994 Germany (North Sea) AY879027 AY878984 Lulworthia sp. KMPB 997 Norway (Rosfjord) AY879028 AY878985 Lulworthia sp. PP 0148 England (Hampshire) AY879029 AY878986 Lulworthia sp. PP 2597 England (Dorset) AY879030 AY878987 Spathulospora adelpha J.K. 5599 Australia (South Australia) AY380314 AY380314 S. antarctica J.K. 3530 Argentina (Santa Cruz) AY380315 AY380315 Zalerion maritimum CBS 167.60 USA (Washington) AY879034 AY878991 Z. maritimum KMPB 82 Germany (Helgoland) AY879037 AY878994 Z. maritimum KMPB 150 Germany (Helgoland) AY879036 AY878993 Z. maritimum KMPB 14648 South Africa (Cape Town) AY879035 AY878992 Z. xylestrix IFO 7836 Canada (Nova Scotia) AY879038 AY878995 Halosphaeriales Aniptodera chesapeakensis ATCC 32818b USA (Maryland) U46870 U46882 Halosphaeria appendiculata CBS 197.60 USA (Washington) U46872 U46885 Hypocreales Bionectria ochroleuca IAM 14569 N/A AB003950 AY283558 CCFC 226708 N/A Hypocrea citrina OSC 100005 USA (Oregon) AY544693 AY544649 J. Campbell and others 559

Table 1. (Cont.)

GenBank accession no.

Taxon Sourcea Country of collection SSU LSU

Microascales Microascus trigonosporous RSA 1942 N/A L36987 U47835 Petriella setifera ATCC 26490 Africa (Kenya) U32421 U48421 Phyllachorales Colletotrichum gloeosporoides N/A N/A M55640 Z18999 Glomerella cingulata FAU 513 N/A U48427 U48428 Pleosporales Leptosphaeria doliolum ATCC 32813 Netherlands U43455 U43475 ATCC 32815 Netherlands Lophiostoma caulium HKU 45291 N/A AF164362 U43469 IMI 143288 England Pleospora herbarum ATCC 11681 USA (Colorado) AF229513 U43476 Setomelanomma holmii PHC 99.4334 France AY161121 AF525678 CBS 110217 USA (Wisconsin) Setosphaeria monoceras CBS 154.26 N/A AY016352 AY016368 Trematosphaeria heterospora CBS 644.86 Switzerland (Zu¨ rich) AY016354 AY016369 Sordariales Cercophora septentrionalis TRTC 42304 N/A U32400 U47823 Chaetomium globosum OSC 38033 N/A U20379 U47825 ATCC 44699 Japan Xylariales Xylaria curta JDR 42375 N/A U32417 U47840 X. hypoxylon ATCC 42768 N/A U20378 U47841

a ATCC, American Type Culture Collection; CBS, Centraalbureau voor Schimmelcultures; CCFC, Canadian Collection of Fungal Cultures; FAU, culture collection of F. A. Uecker; HKU, University of Hong Kong; IAM, Institute of Applied Microbiology, University of Tokyo; IFO, Institute for Fermentation, Osaka, [now NBRC i.e. NITE Biological Resource Center, Chiba]; IMI, CABI Bioscience UK Centre; JDR, culture collection of J. D. Rogers, Washington State University; J. K., herb. J. Kohlmeyer (IMS); KMPB, Kulturensammlung mariner Pilze, Bremerhaven; OSC, Oregon State University Herbarium; PHC, Phabagen Culture Collection, University of Utrecht; PP, University of Portsmouth, Culture Collection of Marine Fungi; RSA, Rancho Santa Ana culture collection; TRTC, Department of Botany, University of Toronto, Canada; N/A, not available. b Ex-type strains. random starting trees, random stepwise addition and even when data is missing (Kluge 1989, Wiens 1998). MulTrees off. Decay indices (Bremer 1988, 1994) were However, under Bayesian analysis the results may be calculated in AutoDecay (Eriksson 1998). Maximum affected by the amount of missing data. To try and parsimony analyses were also performed on constrained alleviate this problem in the Bayesian analyses some topologies. The constraint tree included a clade of all L. regions of the alignment that were particularly rich in fucicola and all L. uniseptata isolates with the rest of the missing data were excluded from the analyses. Align- Lulworthia isolates excluded from Lulworthia s. str. but ments are available from TreeBASE (accession no. not defined into any clade. Heuristic searches were run S1264). with MulTrees off and repeated with MulTrees on, for Of 2391 aligned characters, 190 were excluded due to 100 reps using the same search criteria as for the un- ambiguous alignment; 637 were parsimony informative constrained analyses and keeping trees only compatible (y29%). Maximum parsimony analysis with MulTrees with the constraint tree. A Kishino–Hasegawa (K-H) on generated 10 000 most parsimonious trees; maxi- test (Kishino & Hasegawa 1989) and a Shimodaira- mum parsimony analysis with MulTrees off generated Hasegawa (S-H) test (Shimodaira & Hasegawa 1999) 354 trees. All trees were of length 2240, with a consist- was performed as implemented in PAUP to determine ency index (CI) of 0.52, retention index (RI) of 0.78 and if the trees were significantly worse than the trees from rescaled consistency index (RC) of 0.40. The trees the unconstrained analyses. differed in the relative position of the taxa within the terminal clades; the grouping of taxa in these clades was constant among all the trees. The best tree (as determined by a K-H test) of the 354 trees generated RESULTS with MulTrees off is presented in Fig. 2. Support The current data set is a blend of several studies. A measured by bootstrapping (>50%) and decay indices consequence of this is the amount of missing data, (d) is given at the corresponding branches. which results in a lack of overlap among taxonomic Although there are differences in overall tree top- partitions. Studies have shown that under maximum ology between the Bayesian and parsimony analyses, parsimony analysis it is more beneficial and a more the topologies among and within the well-supported robust tree is produced when all characters are included, terminal clades remain constant. The genus Lulworthia A re-evaluation of the Lulworthiales 560

0.98 Lindra marinera JK4743 0.99 Lindra thalassiae JK4322A 1.00 Lindra marinera JK5091A K Lindra thalassiae JK5090A 1.00 Lindra crassa ATCC 56663 Lulworthia sp. JK5332A 1.00 Lulworthia sp. JK5393 H Lulworthia sp. JK4843 1.00 Spathulospora adelpha Spathulospora antarctica 1.00 Kohlmeyeriella tubulata PP0989 0.99 Kohlmeyeriella tubulata PP1105 1.00 1.00 Lulworthia crassa IFO32133 G Kohlmeyeriella Lulworthia crassa IFO32134 Lulworthia grandispora JK4686 Lulworthia grandispora JK5255A 1.00 Lulworthia grandispora JK5168A F Lulworthia grandispora PP4319 Lulworthia sp. JK5401A 1.00 Lulworthia lignoarenaria ATCC 64644 Lulworthia lignoarenaria IFO 32135 I Lulwoidea 0.92 Lulworthia fucicola ATCC 64288 Lulworthia fucicola PP1235 Lulworthia fucicola PP1249 Lulworthia sp. KMPB957 0.95 Lulworthia sp. KMPB958 A Lulworthia sensu stricto Lulworthia sp. KMPB960 Lulworthia sp. KMPB963 Lulworthia sp. KMPB992 1.00 Lulworthia sp. KMPB994 Lulworthia sp. KMPB622 Lulworthia sp. KMPB965 1.00 Lulworthia sp. KMPB966 Lulworthia sp. KMPB997 D 1.00 0.99 Lulworthia cfr opaca CBS 218 60 1.00 Lulworthia sp. ATCC 64289 Lulworthia sp. PP0148 Lulworthia sp. KMPB969 E 1.00 Lulworthia sp. KMPB986 Lulworthia sp. KMPB970 Zalerion maritimum CBS167 60 Zalerion maritimum KMPB14648 Zalerion maritimum KMPB150 1.00 Zalerion maritimum KMPB82 1.00 Zalerion xylestrix IFO7836 B Lulwoana Lulworthia uniseptata IFO32137 Lulworthia uniseptata PP4032 1.00 Lulworthia uniseptata PP4033 1.00 1.00 Lulworthia cfr purpurea CBS 219 60 Lulworthia sp. PP2597 C Lulworthia medusa JK5581A Haloguignardia irritans 1.00 Anguillospora marina CBS 791 83 Lindra obtusa CBS 113030 J Lindra obtusa IFO 31317 1.00 Aniptodera chesapeakensis 1.00 Halosphaeria appendiculata 1.00 0.99 0.97 Microascus trignosporus 1.00 Petriella setifera 1.00 Colletotrichum gloeosporoides Glomerella cingulata 1.00 Bionectria ochroleuca Hypocrea citrina 1.00 Cercophora septentrionalis Chaetomium globosum 1.00 Xylaria curta Xylaria hypoxylon 1.00 Pleospora herbarum Setosphaeria monoceras Leptosphaeria doliolum 1.00 Setomelanomma holmii Lophiostoma caulium 1.00 Trematosphaeria heterospora 10 changes Fig. 1. B-MCMCMC consensus tree from 19 600 trees inferred from Bayesian analyses of the combined 18S and 28S rDNA data. Posterior probability values are given at the corresponding nodes. is inferred as polyphyletic, a finding consistent with and L. medusa; Zalerion maritimum is placed in a clade previous phylogenetic studies (Kohlmeyer et al. 2000, with its teleomorph, L. uniseptata. Sequence data are Campbell et al. 2002). Lulworthia s. str. contains L. consistent with Z. maritimum being conspecific with Z. fucicola and some isolates named as Lulworthia sp. The xylestrix, which is in agreement with Anastasiou (1963). remaining Lulworthia species are dispersed in several Maximum parsimony analysis on the constrained clades: L. crassa is in a clade with Kohlmeyeriella topology, which forced the monophyly of Lulworthia tubulata; L. lignoarenaria is in a separate clade distinct uniseptata with Lulworthia s. str., generated 12 trees of from the other species of Lulworthia; Haloguignardia is length 2268 with MulTrees off and 1365 trees of the a sister taxon to the clade containing L. cfr purpurea same length with MulTrees on. A K-H and S-H test J. Campbell and others 561

71/d1 Lulworthia grandispora JK4686 96/d17 89/d10 Lulworthia grandispora JK5255A Lulworthia grandispora JK5168A F 99/d1 Lulworthia sp.JK5401A d3 Lulworthia grandispora PP4319 100/d100 Spathulospora adelpha d3 100/ Spathulospora antarctica 67/ d28 Kohlmeyeriella tubulata PP0989 d5 Kohlmeyeriella tubulata PP1105 100/d63 Lulworthia crassa IFO32133 G Kohlmeyeriella 82/d9 Lulworthia crassa IFO32134 76 /d2 Lindra marinera JK4743 51 /d1 92/d3 Lindra thalassiae JK4322A 100/d14 Lindra marinera JK5091A K 86/ Lindra thalassiae JK5090A d14 Lindra crassa ATCC 56663 97 /d5 Lulworthia sp. JK5332A 100/d30 Lulworthia sp. JK5393 H 100/ Lulworthia sp. JK4843 50/d1 d35 Lulworthia lignoarenaria ATCC 64644 Lulworthia lignoarenaria IFO 32135 I Lulwoidea 85/ Lulworthia sp. KMPB965 d17 Lulworthia sp. KMPB966 Lulworthia sp. KMPB997 53 100/ D d24 Lulworthia sp. KMPB622 65 / Lulworthia cfr opaca CBS 218 60 88/d2 d2 Lulworthia sp. ATCC 64289 68/d2 Lulworthia sp. PP0148 94/d6 Lulworthia sp. KMPB986 E d15 Lulworthia sp. KMPB969 Lulworthia sp. KMPB970 Lulworthia fucicola ATCC 64288 64/d1 Lulworthia fucicola PP1235 98/d8 Lulworthia fucicola PP1249 Lulworthia sp. KMPB957 Lulworthia sp. KMPB992 A Lulworthia sensu stricto Lulworthia sp. KMPB958 Lulworthia sp. KMPB963 Lulworthia sp. KMPB994 Lulworthia sp. KMPB960 Zalerion maritimum KMPB82 Zalerion xylestrix IFO7836 75/d2 Zalerion maritimum KMPB150 Zalerion maritimum KMPB14648 89/d6 Zalerion maritimum CBS167 60 B Lulwoana Lulworthia uniseptata IFO32137 100 68/d1 98 / Lulworthia uniseptata PP4032 94/d4 d4 Lulworthia uniseptata PP4033 54 Lulworthia cfr purpurea CBS 219 60 Lulworthia sp. PP2597 C 97/d10 Lulworthia medusa JK5581A Haloguignardia irritans Anguillospora marina CBS 791 83 100/d25 Lindra obtusa CBS 113030 J 100/ Lindra obtusa IFO 31317 d21 Xylaria curta Xylaria hypoxylon 98/d13 Aniptodera chesapeakensis 98/d9 72 /d4 Halosphaeria appendiculata 78/d3 Microascus trignosporus 98/d9 Petriella setifera 89/d6 Colletotrichum gloeosporoides Glomerella cingulata 55/ Bionectria ochroleuca d3 Hypocrea citrina 100/d28 Cercophora septentrionalis Chaetomium globosum 93/d32 Leptosphaeria doliolum 92/d32 Setomelanomma holmii 100 Pleospora herbarum 99/ 98/d10 /d21 Setosphaeria monoceras d32 Lophiostoma caulium Trematosphaeria heterospora 10 changes

Fig. 2. Phylogram of the best tree (length 2240, CI=0.52, RI=0.78, RC=0.40 and HI=0.48) inferred from the maximum parsimony analyses of the combined 18S and 28S rDNA data with MulTrees off. Bootstrap values greater than 50% and decay indices (d) are given at the corresponding nodes. comparing the 12 trees from the MulTrees off analysis marinera and Li. thalassiae. The data are consistent and the MP tree from the unconstrained analysis in- with Li. marinera and Li. thalassiae being treated as dicated that the constrained trees were significantly conspecific. The second clade contains Lindra obtusa worse than the tree generated in the unconstrained with its anamorph, Anguillospora marina. The first analysis (P=0.000 [K-H], P=0.0001 [S-H]). The genus clade is more closely related to Lulworthia grandispora, Lindra is also polyphyletic, dispersed in two widely se- Lul. crassa, K. tubulata and Spathulospora, than it is to parated clades. One clade contains Lindra crassa, Li. Lindra obtusa. A re-evaluation of the Lulworthiales 562

DISCUSSION 1956, Kohlmeyer 1958, Lloyd & Wilson 1962, Kohlmeyer 1963). Nakagiri (1984) established through culturing that Zalerion maritimum is the anamorph of Clade A: Lulworthia sensu stricto L. uniseptata, and this is confirmed by the sequence data (Figs 1–2). We also verified Anastasiou’s (1963) Efforts to locate type material of Lulworthia fucicola, finding that Z. maritimum and Z. xylestrix are con- type species of the genus Lulworthia described by specific (Figs 1–2). Sutherland (1916) have been unsuccessful (Kohlmeyer Based on morphology and the molecular results a 1968a). The L. fucicola impasse has been discussed in new genus, Lulwoana, is established for L. uniseptata. detail elsewhere (Kohlmeyer et al. 2000) and a neotype was subsequently established by Campbell (2005). Lulwoana Kohlm., Volkm.-Kohlm., J. Campb., Spata- Eleven species of Lulworthia are currently accepted, but fora & Gra¨ f., gen. nov. many Lulworthia collections could not be identified because characteristic morphological features were Etym.: From Lulworthia, and the Latin suffix-ana,to lacking. For practical purposes, unidentifiable species indicate the close connection of the new genus to Lulworthia. with ascospore lengths between 150–500 mm had Ascomata superficialia vel partim immersa in ligno, been lumped together in the past as Lulworthia sp. globosa ad subglobosa, ostiolata, coriacea, atro-brunnea vel (Kohlmeyer & Volkmann-Kohlmeyer 1991). nigra. Papilla vel rostrum cylindrica, recta vel irregularis. The phylogenetic analyses indicate that Lul- Peridium bistratosum; stratum externum e cellulis parvis worthiales s. str. consists of only one named species, multangulis vel irregularis, atris; stratum internum e cellulis L. fucicola, represented in this study by the ex-neotype planis vel rhomboideis, hyalinis constans. Hamathecium culture (ATCC 64288). It is characterised by ascospore absens; centra ascomatum immaturorum e cellulis pseudo- dimensions of (66–) 77–110 (–121)r4–6 mm and dark parenchymaticis, multangularibus, leptodermis, hyalinis brown, globose ascomata with long necks. In addition compositum. Asci octospori, fusiformes, curvati, leptodermi, unitunicati, mox deliquescentes. Ascosporae filiformes, hya- to the neotype, the L. fucicola clade includes two linae, uniseptatae, utrinque cameram conicam continentes, identified strains from Denmark (PP1235) and Kuwait mucum exsudantem. Status anamorphosus: Zalerion mar- (PP1249) and six isolates from the North Sea (KMPB itimum. 957, 958, 960, 963, 992, 994) listed so far as Lulworthia Type species: Lulwoana uniseptata (Nakagiri) Kohlm., sp. (Figs 1–2). Except for the Kuwait strain (PP1249), Volkm.-Kohlm., J. Campb., Spatafora & Gra¨ f., comb. nov. whose origin remains questionable, all have in common [Basionym: Lulworthia uniseptata Nakagiri, Trans. mycol. that they occur in temperate areas. The six KMPB Soc. Japan 25: 382 (1984).] strains are not documented by vouchers to permit Ascomata superficial or partly immersed in wood, morphological comparison, but based on sequence globose to subglobose, ostiolate, coriaceous, dark L. fucicola data could be considered conspecific with . brown or black. Papilla or neck cylindrical, straight or The remaining Lulworthia are distributed over several irregular. Peridium two layered; outer layer composed clades throughout the tree and their morphological of small, polygonal or irregular dark cells; inner differences or lack thereof will be discussed here. layer composed of flat or rhomboid hyaline cells. Hamathecium absent; immature ascomata filled with a pseudoparenchyma of polygonal, thin-walled and Clade B: Lulworthia uniseptata hyaline cells. Asci eight-spored, fusiform, curved, thin- This clade contains the well-defined species Lulworthia walled, unitunicate, deliquescing early. Ascospores uniseptata and its anamorph Zalerion maritimum (Figs filiform, hyaline, one-septate, with a conical chamber at 1–2). Molecular analyses indicate that L. uniseptata each end, releasing mucus from the tip. Anamorph: does not belong in Lulworthia s. str. (Figs 1–2, Clades A Zalerion maritimum. and B). Furthermore, K-H and S-H tests on the The anamorph is discussed further by Bills et al. constrained topology forcing the monophyly of L. (1999). uniseptata with Lulworthia s. str. indicate that the con- strained analysis is a significantly worse phylogenetic Clade C: Lulworthia medusa and L. cfr purpurea hypothesis for the data as compared to the trees inferred in the unconstrained maximum parsimony analyses. Ascomata of Lulworthia medusa are found in salt Lulworthia uniseptata differs from all other species marshes of the USA on decomposing Spartina spp. of Lulworthia by its one-septate ascospores. It is (Kohlmeyer et al. 2000). This species has no specific characterised by a single septum approximately in the morphological features to distinguish it from the middle of the ascospore and an ascospore length of Lulworthia s. str. clade (Clade A) except for its asco- 94–148 mm. Ascospore septation occurs in three species spore length (350–526 mm). However, this ascospore of Lulworthia, viz. L. uniseptata, L. lindroidea (9–12 length overlaps completely with L. grandispora (Clade septa) and L. lignoarenaria (31–38 septa). However, the F); the only difference is the substrate, since L. medusa tendency to form septa is also found in normally non- is restricted to Spartina, while L. fucicola and L. septate Lulworthia species during germination (Wilson grandispora occur on wood. Two other isolates from J. Campbell and others 563 wood collected in the UK and identified as L. purpurea Meyers (1957) gives the ascospore length as (CBS 219.60) and Lulworthia sp. (PP2597), group with 565–756 mm based on one collection from Cedar Key, L. medusa (Figs 1–2, Clade C), but no reference Florida. From the molecular results it becomes appar- material was available for comparison. For the same ent that a broader range in ascospore length must be reason we prefer to call the L. purpurea strain L. cfr acknowledged. Three of the collections (J.K. 4686, purpurea. The placement of the L. purpurea clade 5168, 5255) identified as L. grandispora (Figs 1–2) are (Clade C) as a sister clade to the L. uniseptata clade from different locations in Belize with carbonaceous (Clade B) was consistent in both the parsimony and ascomata, and ascospore lengths of up to 645 mm. Bayesian analyses, however, the placement of the sole Another collection (J.K. 5401A) is from French isolate of Haloguignardia was unstable in these analyses Polynesia (Moorea) and has only 345–395 mm long (Figs 1–2). For this reason, we do not currently include ascospores. It had been identified as Lulworthia sp., but L. purpurea in the new genus Lulwoana, although molecular analysis shows that it can clearly be con- future studies involving additional sampling of sidered as belonging to L. grandispora. For the fifth Lulworthialean taxa may support this classification. L. grandispora (PP 4319) from Malaysia no ascospore measurements are available. Apparently, L. grand- ispora is widely distributed in tropical waters around Clade D: Lulworthia cfr opaca and four isolates of the globe and further studies on material from outside Lulworthia sp. the Caribbean are warranted. The isolate Lulworthia cfr opaca was collected in As no type material is available, and in accordance Washington State (USA) and tentatively identified as with the Code, we herewith establish a neotype for L. opaca (J.K.199, Kohlmeyer 1960). Our recent L. grandispora with a specimen on dead Rhizophora thorough comparison of this voucher material with the mangle proproot collected in Belize in 1988 by Jan type specimen of L. opaca (as Halophiobolus opacus in Kohlmeyer and B. Volkmann-Kohlmeyer. This speci- FH) revealed that they agreed morphologically; the men was chosen as the neotype as there are numerous ascospore measurements being 155–190r3.5–4 mmin ascomata in good condition and this collection is the the type versus 131–180r5 mm. However, we found an best preserved of all other specimens of L. grandispora important difference, namely the presence of chla- examined (i.e. J.K. 4686, J.K. 5255, J.K. 5401). mydospores in the type, but they were absent in the Cultures and voucher specimens are deposited at collection from Washington and therefore we chose to IMS; another culture is being deposited at CBS. call this isolate L. cfr opaca. For the other isolates Furthermore, we herewith amend the original descrip- (KMPB 622, 965, 966, 997), no reference herbarium tion for L. grandispora as having ascospore lengths material was available. A feature common to all from 345–756 mm. members of the clade is their occurrence in temperate habitats. Bayesian analyses suggest that this clade (D) Lulworthia grandispora S. P. Meyers, Mycologia 49: may be best accommodated in Lulworthia s.str. based 513 (1957). on its relationship with L. fucicola, although the monophyly of this grouping was not supported in Type: Belize: Man-of-War Cay, 16x 53k 00a N, maximum parsimony analyses. 88x 06k 15a W, on dead Rhizophora mangle proproot, 19 Oct. 1988, J. Kohlmeyer & B. Volkmann-Kohlmeyer J.K. 5168 (IMS – neotypus hic designatus). Clade E: Five isolates of Lulworthia sp. Like clade D, clade E (Figs 1–2) is another group of five Clade G: Kohlmeyeriella tubulata and Lulworthia isolates (ATCC 64289, KMPB 969, 970, 986, PP0148) crassa that appears to be closely related to Lulworthia s. str (Clade A). The only isolate out of this clade with Corollospora tubulata was transferred by Jones, deposited herbarium material is ATCC 64289 (C32-1) Johnson & Moss (1983) to a new genus, Kohlmeyeriella, from Chile. It has all the morphological characteristics Halosphaeriales. On the basis of phylogenetic analyses of Lulworthia s. str., but ascospores are 110–236 mm of the 28S rDNA, Campbell et al. (2002) removed long and 4 mm wide. Like L. fucicola these isolates all Kohlmeyeriella from the Halosphaeriales and placed come from temperate waters. it in the Lulworthiaceae, Lulworthiales. Also on mor- phological grounds K. tubulata fits well within the Lulworthiales, mainly because of its well-developed Clade F: Lulworthia grandispora ascospore end chambers, filled with mucus (Campbell Lulworthia grandispora has been distinguished from et al. 2002). L. fucicola mainly on the basis of its extremely long Lulworthia crassa is phylogenetically closely related ascospores. Unfortunately, the type material of to K. tubulata: in the Bayesian analyses (Fig. 1, Clade L. grandispora could not be located in the Farlow G) L. crassa is in a clade with K. tubulata with a pos- Herbarium (FH) nor in the New York Botanical terior probability of 0.99; in the parsimony analyses the Garden (NY) where it was supposed to be deposited. ‘best’ maximum parsimony tree groups L. crassa and A re-evaluation of the Lulworthiales 564

K. tubulata in a clade with 67% bootstrap support. Papilla conica, prope subiculum basale. Peridium bistratosum; Also morphologically, L. crassa closely resembles K. stratum externum e cellulis magnis luminibus grandibus, tubulata. Both species are arenicolous, with carbon- stratum internum e cellulis planis luminibus angustis constans. aceous ascomata developing on sand grains. Short Hamathecium absens; centra ascomatum immaturorum papillae are close to the basal subiculum, and the e cellulis pseudoparenchymaticis, multangularibus, lepto- dermis, hyalinis compositum. Asci octospori, clavati, ascomatal venter is initially filled with a thin-walled pedunculati, leptodermi, unitunicati, mox deliquescentes. pseudoparenchyma. Most striking is the similarity of Ascosporae filiformes, hyalinae, multiseptatae, utrinque the ascospores, which are one-celled and fusiform, with cameram conicam continentes, mucum exsudantem. a mucilage-filled, tapering tube at each end (Kohlmeyer Type: Lulwoidea lignoarenaria (Jørg. Koch & E. B. G. 1968b, Nakagiri 1984). This apical tube is homologous Jones) Kohlm., Volkm.-Kohlm., J. Campb., Spatafora & with the end chambers of Lulworthia. Therefore, on the Gra¨ f., comb. nov. [Basionym: Lulworthia lignoarenaria Jørg. basis of morphology and confirmed with the molecular Koch & E. B. G. Jones, Mycotaxon 20: 389 (1984).] results we hereby transfer Lulworthia crassa to Ascomata superficial, mostly on grains of sand, sub- Kohlmeyeriella. globose to ellipsoidal, subiculate, carbonaceous, black. Papilla conical, near the basal subiculum. Peridium Kohlmeyeriella crassa (Nakagiri) Kohlm., Volkm.- Kohlm., J. Campb., Spatafora & Gra¨ f., comb. nov. two-layered; outer layer composed of large cells with large lumina; inner layer composed of flat cells with Basionym: Lulworthia crassa Nakagiri, Trans. mycol. narrow lumina. Hamathecium absent; immature asco- Soc. Japan 25: 378 (1984). mata filled with a pseudoparenchyma of polygonal, thin-walled and hyaline cells. Asci eight-spored, Clade H: Three isolates of Lulworthia sp. from the tropics clavate, pedunculate, thin-walled, unitunicate, early All three isolates in Clade H are from tropical deliquescing. Ascospores filiform, hyaline, multiseptate, environments, i.e. Hawaii (J.K. 4843), Moorea (J.K. with a conical chamber at each end, releasing mucus 5393) and Queensland, Australia (J.K. 5332A). from the tip. However, despite their position on the tree these isolates are morphologically too different to warrant The genus Lindra description as a single species: two are from wood (J.K. 4843, 5332A) and have ascospore lengths of about Six species of Lindra have currently been described: 250–340 mm, while isolate J.K. 5393 has 420–530 mm Lindra crassa, L. hawaiiensis, L. inflata, L. marinera, long ascospores and very unusual, curved ascomata L. obtusa and L. thalassiae (Kohlmeyer & Volkmann- that develop on bark. Kohlmeyer 1991). The genus is based on the type spe- cies, L. inflata, and differs from members of Lulworthia Clade I: Lulworthia lignoarenaria mainly by the absence of apical mucus-filled chambers of the ascospores; all Lindra species have septate The arenicolous species Lulworthia lignoarenaria, first ascospores. Although Wilson (1956) described the described from Denmark (Koch & Jones 1984) has also ascospores of L. inflata as appendaged, examination of been found frequently in Japan (Nakagiri 1989). The the type material (IMI 62909) revealed that the tips are molecular data indicate that L. lignoarenaria is in a inflated but cannot be considered ‘appendaged’. They separate clade clearly apart from the other Lulworthia do not contain or release mucus and can be considered species (Figs 1–2, Clade I). The filamentous ascospores homologous with the apical chambers of Lulworthia have the characteristic apical, mucus-filled chambers, but with a loss of function (Campbell et al. 2002), but are distinguished from Lulworthia s. str.by which is also supported by the molecular data as Lindra numerous septa (31–38). Morphologically, L. lignoar- is nested terminally within Lulworthia (Figs 1–2). enaria shows adaptations to its arenicolous mode of Drawings of ascospore tips by Wilson (1956: figs life, namely carbonaceous ascomata attached with 29–30) and a photograph in Hyde, Jones & Moss (1986, subicula to grains of sand and necks located near the fig. 1) show a gelatinous film that attaches the asco- base, characters that are also found in Lindra obtusa, spore to the substrate. No other Lindra species has Kohlmeyeriella tubulata and Corollospora species. ascospore tips like L. inflata. Unfortunately, no isolates Based on morphological features and molecular data a or fresh material of the rarely collected L. inflata have new genus, Lulwoidea, is established for Lulworthia become available to us; therefore, no revision at the lignoarenaria. genus level can be proposed at this time, especially since the phylogenetic analyses revealed the genus Lulwoidea Kohlm., Volkm.-Kohlm., J. Campb., Lindra to be polyphyletic. Spatafora & Gra¨ f., gen. nov. Etym.: From Lulworthia and -oideus indicating re- semblance. Clade J: Lindra obtusa Ascomata superficialia, plerumque in granis arenae, sub- Lindra obtusa, collected only from temperate environ- globosa ad ellipsoidea, subiculata, carbonacea, nigra. ments, and its anamorph, Anguillospora marina, are not J. Campbell and others 565 monophyletic with the L. thalassiae clade (Figs 1–2). Lindra crassa, with an ascospore length of Molecular analyses under both Bayesian (posterior 320–520 mm and 15–23 septa, is closely related to probability of 1.00) and maximum parsimony (boot- L. thalassiae. It had originally been described as strap of 100%) algorithms indicate that L. obtusa is L. thalassiae var. crassa, but was raised to species level the most basal lineage of the Lulworthiales, which is by Kohlmeyer & Volkmann-Kohlmeyer (1991). It can interesting in the context of its unique ascospore be distinguished by its wider ascospores (8–10 mm vs morphology. Ascomata of L. obtusa are remarkably 3–7 mminLindra thalassiae) and considerably longer similar to those of Corollospora, a genus of the ascospores, although there is some overlap in ascospore Halosphaeriales. Adaptation to the habitat, viz. sandy length. All isolates of L. thalassiae/L. crassa collected beaches, has caused in both genera, as well as in over the years have come from tropical areas and ap- Kohlmeyeriella tubulata, a parallel evolution of pear closely related to the three isolates of Lulworthia carbonaceous ascomata with ostioles near the basal sp. (J.K. 4843, 5332, 5393) from the tropics. subiculum, attachment to sand grains, and a pseudo- parenchyma with pit-connections initially filling the Teleomorph–anamorph connections in the Lulworthiales fruiting bodies (Kohlmeyer 1968b, Nakagiri & Tubaki Teleomorph–anamorph connections have been rec- 1983, Kohlmeyer & Volkmann-Kohlmeyer 1987a). ognized in two species of the Lulworthiales, viz. Lindra obtusa and Lulwoana uniseptata (Nakagiri 1984, as Lulworthia uniseptata). Anguillospora marina, the ana- Clade K: Lindra thalassiae, Lindra marinera and morph of Lindra obtusa, has filiform, 9–13(–19) septate Lindra crassa conidia that resemble the ascospores of its teleomorph So far, Lindra thalassiae and L. marinera had been (Nakagiri & Tubaki 1983). Zalerion maritimum, the distinguished solely on the basis of ascospore length anamorph of Lulwoana uniseptata, is characterized by and septation (Orpurt et al. 1964, Meyers 1969). Lindra coiled, fuscous or almost black conidia (Nakagiri thalassiae ascospores are (220–) 230–390 mm long and 1984). The teleomorph has been rarely reported, whereas 14–26 septate, L. marinera ascospores are 152–230 mm Z. maritimum is frequently collected in temperate long and 6–17 septate (Kohlmeyer & Volkmann- waters. In addition to the two named anamorphs there Kohlmeyer 1991). The data overlap, however, and are reports of chlamydospores associated with two variability of these features made a distinction difficult additional Lulworthiales. Linder (in Barghoorn & and unreliable. Sequence data of the two species are Linder 1944) describes ‘chains of spores’ occurring in consistent with treating the two taxa as conspecific cultures of Lulworthia opaca (as Halophiobolus opacus), (Figs 1–2); therefore L. marinera is reduced to syn- and we confirmed that such chlamydospores, viz. onymy. We amend the original description for chains of ellipsoidal, light brown cells, are contained in L. thalassiae as having ascospores that are 152–390 mm the type material (slide Barghoorn, No. 14 at FH). long, with 6–24 septa. Similar chains, often associated with ascomata, have

Key to the members of the Lulworthiales 1 Ascospores with mucilage-containing, cell-like processes at each end ...... 2 Ascospores without such apical chambers or appendages ...... 20 2(1) Saprobes, in wood, seagrasses or marsh plants; ascomata of some species on sand grains or in/on coralline algae ...... 3 Parasites of uncalcified Rhodophyta or Phaeophyta; not in or on coralline algae . . . . . 15 3(2) Ascomata on hard substrates (sand grains, corals, coralline algae) ...... 4 Ascomata in/on other substrates ...... 6 4(3) Ascomata superficial on corals ...... Lulworthia calcicola Ascomata immersed in coralline algae ...... 5 5(4) Ascospores 120–180r4–6 mm...... Lulworthia curalii Ascospores 200–270r2.5–6 mm...... Lulworthia kniepii 6(3) Ascomata on sand grains (arenicolous species) ...... 7 Ascomata mostly in/on wood, also in seagrasses, saltmarsh plants or decomposing algae . . . 10 7(6) Ascospores without septa ...... 8 Ascospores septate ...... 9 8(7) Ascospore diameter less than 10 mm...... Kohlmeyeriella crassa Ascospore diameter more than 10 mm ...... Kohlmeyeriella tubulata 9(7) Ascospores 1 septate, shorter than 150 mm; anamorph Zalerion maritimum ...Lulwoana uniseptata Ascospores 31–38 septate, longer than 400 mm...... Lulwoidea lignoarenaria 10(6) Ascospores 9–12 septate; ascospores 170–240 mm long ...... Lulworthia lindroidea Ascospores without septa ...... 11 A re-evaluation of the Lulworthiales 566

11(10) Ascomata in Spartina culms; ascospores 350–525 mm long ...... Lulworthia medusa Ascomata in other hosts or substrates ...... 12 12(11) Ascomata in Zostera rhizomes and leaves; ascospores 260–310 mm long . . . Lulworthia halima Ascomata in wood or decaying algae ...... 13 13(12) Ascospores 65–120 mm long ...... Lulworthia fucicola Ascospores longer ...... 14 14(13) Inhabiting tropical waters; ascospores 345–755 mm long . ....Lulworthia grandispora Inhabiting temperate waters; without morphological or ecological characters; ascospores less than 345 mm long ...... Lulworthia sp. 15(2) Producing galls in Cystoseira and Halidrys spp. (Phaeophyta), on the west coast of North America ...... Haloguignardia irritans In Ballia spp. (Rhodophyta), in the Southern Hemisphere; not producing galls . . . . . 16 16(15) From Antarctic and subantarctic habitats; ascomata without hairs . . . Spathulospora antarctica From Australia or New Zealand; ascomata with hairs ...... 17 17(16) Ascospores longer than 65 mm...... 18 Ascospores usually shorter than 65 mm ...... 19 18(17) Ascospore diameter less than 14 mm, spathulate to spoon-shaped at apices; few antheridia on long hairs ...... Spathulospora phycophila Ascospore diameter more than 14 mm; each end with a conical mucus-filled chamber; many antherida on short stalks ...... Spathulospora adelpha 19(17) Ascomata enclosed by long, curved hairs; ascospore tips spathulate; appendages subterminal ...... Spathulospora lanata Ascomata bare, except for a few short hairs around the ostiole; ascospores with subglobose, basally flat, terminal appendages ...... Spathulospora calva 20(1) Ascospore diameter 8–10 mm, length 320–520 mm ...... Lindra crassa Ascospore diameter less than 8 mm...... 21 21(20) Arenicolous species; ascomata with subicula; anamorph Anguillospora marina ...Lindra obtusa Not arenicolous, without subiculum, usually immersed in the substrate ...... 22 22(21) Temperate species in wood; ascospores with 30 or more septa ...... Lindra inflata Tropical species in wood, sea grasses or algae; ascospores with less than 30 septa . . . . . 23 23(22) In wood; ascospore length 100–190 mm ...... Lindra hawaiiensis In decomposing algae or sea grasses; ascospore length 150–390 mm .... Lindra thalassiae been reported for Lindra hawaiiensis (Kohlmeyer & ascospore septation, as seen in Lulwoidea lignoarenaria, Volkmann-Kohlmeyer 1987b). Lulwoana uniseptata and Lulworthia lindroidea. Therefore, although we have a number of molecularly well-defined clades, it is not possible to actually trans- late these results into new genera (with the above CONCLUSION exceptions) due to a lack of consistent morphological These molecular analyses have shed some light on the differences. However, it cannot be excluded that a phylogeny of the Lulworthiales. They have enabled thorough morphological comparison of large amounts us to: (1) move Lulworthia crassa into Kohlmeyeriella; of freshly collected material (including ascospore (2) establish two new genera, Lulwoidea for L. lignoar- length/width, end chambers, peridium structure and enaria, and Lulwoana for L. uniseptata; (3) discover ascoma color) may yield the necessary information for the identity of some of unidentified Lulworthia strains; a revision of these Lulworthia sp. (4) reduce Lindra marinera to synonymy with L. tha- Recent molecular studies led to the surprising dis- lassiae; (5) confirm Zalerion xylestrix is conspecific covery that the two algicolous genera Spathulospora with Zalerion maritimum; and (6) confirm teleomorph- and Haloguignardia are members of the Lulworthiales anamorph relationships for Lulworthia uniseptata (Harvey 2004, Inderbitzin et al. 2004). These genera and Zalerion maritimum, and Lindra obtusa and were therefore included in the present analyses Anguillospora marina. But the dilemma of morphologi- (Figs 1–2). This placement of the genera Spathulospora cally indistinguishable taxa for the genus Lulworthia, and Haloguignardia, parasites of Rhodophyta and represented in this study by clades D, E, and H, re- Phaeophyta, respectively, emphasizes the special mains unresolved. Ascospore length and width cannot importance of the mucus-filled apical structures of the be used at the generic level and only rarely at the species ascospores as a shared, derived character within the level. Ascoma features including neck length appear to Lulworthiales. Furthermore, these findings empha- be equally unreliable in most cases (for example Clade size the need to include additional algicolous taxa in H, Lulworthia sp. J.K. 5332A), since their dimensions future molecular systematic studies of the Lulworthiales. are variable and often dependent on the substrate. The Unfortunately, the algae inhabiting species of Lul- only character that offers some degree of reliability is worthia, L. curalii and L. kniepii, both from coralline J. Campbell and others 567 algae and the original L. fucicola from Fucus were not Houghton, G. C. Llewelyn & C. E. O’Rea, eds): 584–589. CAB available for this study. As discussed earlier, L. fucico- International, Slough. la, described by Sutherland (1916), has only been found Inderbitzin, P., Lim, S. R., Volkmann-Kohlmeyer, B. & Kohlmeyer, J. (2004) The phylogenetic position of Spathulospora based on once on Fucus, and the other two species cannot be DNA sequences from dried herbarium material. Mycological cultivated. Direct sequencing of the ascomata may be a Research 108: 737–748. possibility in the future. A discussion of the close re- Jones, E. B. G. (1995) Ultrastructure and taxonomy of the aquatic lationship of Haloguignardia and Spathulospora with ascomycetous order Halosphaeriales. Canadian Journal of Botany other members of the Lulworthiales would therefore at 73 (Suppl. 1): S790–S801. Jones, E. B. G., Johnson, R. G. & Moss, S. T. (1983) Taxonomic present be premature. studies of the Halosphaeriaceae: Corollospora Werdermann. The polyphyly of the genus Lindra is equally re- Botanical Journal of the Linnean Society 87: 193–212. markable, since there are no morphological differences Kishino, H. & Hasegawa, M. (1989) Evaluation of the maximum that would suitably distinguish the L. obtusa and likelihood estimate of the evolutionary tree topologies from DNA L. thalassiae clades at the generic level. Moreover, the sequence data, and the branching order of Hominoides. Journal of Molecular Evolution 29: 170–179. genus Lindra lacks the important character of a mucus- Kluge, A. (1989) A concern for evidence and a phylogenetic hypoth- filled apical chambers in spite of its close relationship to esis among Epicrates (Booidae, Serpentes). Systematic Zoology 38: Lulworthia. It must therefore follow that the loss of the 7–25. apical chambers has not only occurred once but twice Koch, J. & Jones, E. B. G. (1984) Lulworthia lignoarenaria, a new in the Lulworthiales. marine pyrenomycete from coastal sands. Mycotaxon 20: 389–395. Kohlmeyer, J. (1958) Beobachtungen u¨ ber mediterrane Meerespilze sowie das Vorkommen von marinen Moderfa¨ ule–Erregern in ACKNOWLEDGEMENTS Aquariumszuchten holzzersto¨ render Meerestiere. Berichte der Deutschen Botanischen Gesellschaft 71: 98–116. We thank Karsten Schaumann at AWI for making all the KMPB Kohlmeyer, J. (1960) Wood-inhabiting marine fungi from the Pacific isolates available; the curators of herbaria B, FH, IMI, and A. Northwest and California. Nova Hedwigia 2: 293–343. Nakagiri for the loan of material; and the American Type Culture Kohlmeyer, J. (1963) Parasitische und epiphytische Pilze auf Collection, Centraalbureau voor Schimmelcultures, and the Institute Meeresalgen. Nova Hedwigia 6: 127–146. for Fermentation for provision of cultures. Detailed information Kohlmeyer, J. (1968a) Revisions and descriptions of algicolous by C. A. Shearer on her collections from Chile is gratefully ac- marine fungi. Phytopathologische Zeitschrift 63: 341–363. knowledged. Appreciation is expressed to Alga Zuccaro for providing Kohlmeyer, J. (1968b) Da¨ nische Meerespilze. Berichte der Deutschen additional information on her environmental sampling from Fucus. Botanischen Gesellschaft 81: 53–61. Kohlmeyer, J. (1972) A revision of Halosphaeriaceae. Canadian Journal of Botany 50: 1951–1963. REFERENCES Kohlmeyer, J. & Kohlmeyer, E. (1979) Marine Mycology: the higher fungi. Academic Press, New York. Anastasiou, C. J. (1963) The genus Zalerion Moore et Meyers. Kohlmeyer, J., Spatafora, J. W. & Volkmann-Kohlmeyer, B. (2000) Canadian Journal of Botany 41: 1135–1139. Lulworthiales, a new order of marine Ascomycota. Mycologia 92: Barghoorn, E. S. & Linder, D. H. (1944) Marine fungi: their 453–458. taxonomy and biology. Farlowia 1: 395–467. Kohlmeyer, J. & Volkmann-Kohlmeyer, B. (1987a) Reflections on Bills, G. F., Platas, G., Pala´ ez, F. & Masurekar, P. (1999) the genus Corollospora (Ascomycetes). Transactions of the British Reclassification of a pneumocandin – producing anamorph, Glarea Mycological Society 88: 181–188. lozoyensis, previously identified as Zalerion arboricola. Mycological Kohlmeyer, J. & Volkmann-Kohlmeyer, B. (1987b) Marine fungi Research 103: 179–192. from Aldabra, the Galapagos, and other tropical islands. Canadian Bremer, K. (1988) The limits of amino acid sequence data in Journal of Botany 65: 571–582. angiosperm phylogenetic reconstruction. Evolution 42: 795–803. Kohlmeyer, J. & Volkmann-Kohlmeyer, B. (1991) Illustrated key to Bremer, K. (1994) Branch support and tree stability. Cladistics 10: the filamentous higher marine fungi. Botanica Marina 34: 1–61. 295–304. Lloyd, L. S. & Wilson, I. M. (1962) Development of the perithecium Campbell, J. (2005) Neotypification of Lulworthia fucicola. in Lulworthia medusa (Ell. & Ev.) Cribb & Cribb, a saprophyte on Mycologia: in press. Spartina townsendii. Transactions of the British Mycological Society Campbell, J., Shearer, C. A., Mitchell, J. I. & Eaton, R. A. (2002) 45: 359–372. Corollospora revisited: a molecular approach. In Fungi in Marine Meyers, S. P. (1957) Taxonomy of marine pyrenomycetes. Mycologia Environments (K. D. Hyde, ed.): 15–33. Fungal Diversity Press, 49: 475–528. Hong Kong. Meyers, S. P. (1969) Thalassiomycetes XI. Further studies of the Eriksson, T. (1998) AutoDecay. Vers. 4.0. Department of Botany, genus Lindra with a description of L. marinera, a new species. Stockholm University, Stockholm. Mycologia 61:486–495. Felsenstein, J. (1985) Confidence limits on phylogenies: an approach Mu¨ ller, E. & Arx, J. A. von (1962) Die Gattungen der didymosporen using the bootstrap. Evolution 39: 783–791. Pyrenomyceten. Beitra¨ge zur Kryptogamenflora der Schweiz 11 (2): Harvey, J. B. J. (2004) Phylogenetic studies of the marine brown algae 1–922. Cystoseira and Halidrys including co-evolution with the associated Nakagiri, A. (1984) Two new species of Lulworthia and evaluation of fungal endophyte Haloguignardia irritans. [Dissertation Abstracts genera-delimiting characters between Lulworthia and Lindra International 65 (5).] Proquest Information and Learning, Ann (Halosphaeriaceae). Transactions of the Mycological Society of Arbor, MI. Japan 25: 377–388. Hawksworth, D. L. & Eriksson, O. (1986) The names of accepted Nakagiri, A. (1989) Marine fungi in sea foam from Japanese orders of ascomycetes. Systema Ascomycetum 5: 175–184. coast. Institute for Fermentation Research Communications 14: Huelsenbeck, J. P. & Ronquist, F. (2001) MRBAYES: Bayesian 52–79. inference of phylogenetic trees. Bioinformatics 17:754–755. Nakagiri, A. & Tubaki, K. (1983) Lindra obtusa, a new marine Hyde, K. D., Jones, E. B. G. & Moss, S. T. (1986) How do fungal ascomycete and its Anguillospora anamorph. Mycologia 75: spores attach to surfaces? In Biodeterioration 6 (S. Barry, D. R. 487–497. A re-evaluation of the Lulworthiales 568

O’Donnell, K. (1992) Ribosomal DNA internal transcribed spacers Swofford, D. L. (2002) PAUP*: phylogenetic analysis using parsi- are highly divergent in the phytopathogenic ascomycete Fusarium mony (*and other methods). Version 4. Sinauer Associates, sambucinum (Gibberella pulicaris). Current Genetics 22: 213–220. Sunderland, MA. O’Donnell, K., Cigelnik, E. & Nirenberg, H. I. (1998) Molecular Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & systematics and phytogeography of the Gibberella fujikuroi species Higgins, D. G. (1997) The Clustal-X windows interface: flexible complex. Mycologia 90: 465–493. strategies for multiple sequence alignment aided by quality analysis Olmstead, R. G., Bremer, B., Scott, K. M. & Palmer, J. D. (1993) tools. Nucleic Acids Research 25: 4876–4882. A molecular systematic analysis of the Asteridae sensu lato based Turner, D., Kovacs, W., Kuhls, K., Lieckfeldt, E., Peter, B., Arisan- on rbcL sequences. Annals of the Missouri Botanical Garden 80: Atac, I., Strauss, J., Samuels, G. J., Bo¨ rner, T. & Kubicek, C. P. 700–722. (1997) Biogeography and phenotypic variation in Trichoderma Orpurt, P. A., Meyers, S. P., Boral, L. L. & Simms, J. (1964) sect. Longibrachiatum and associated Hypocrea species. Thalassiomycetes V. A new species of Lindra from turtle grass, Mycological Research 101: 449–459. Thalassia testudinum Ko¨ nig. Bulletin of Marine Science of the Gulf White, T. J., Bruns, T., Lee, S. & Taylor, J. W. (1990) Amplification and Caribbean 14: 405–417. and direct sequencing of fungal ribosomal RNA genes for phylo- Rambaut, A. (1996) Se-Al: Sequence Alignment Editor. vers. 1.0 alpha genetics. In PCR Protocols: guide to methods and amplifications 1.0. University of Oxford, UK. (M. A. Innis, D. H. Gellfand, J. J. Sninsky & T. J. White, eds): Rodriguez, F., Oliver, J., Marin, A. & Medina, J. (1990) The general 315–322. Academic Press, New York. stochastic model of nucleotide substitution. Journal of Theoretical Wiens, J. (1998) Does adding characters with missing data increase or Biology 142: 485–502. decrease phylogenetic accuracy? Systematic Biology 47: 625–640. Shimodaira, H. & Hasegawa, M. (1999) Multiple comparisons of Wilson, I. M. (1956) Some new marine Pyrenomycetes on wood or log-likelihoods with applications to phylogenetic inference. rope: Halophiobolus and Lindra. Transactions of the British Molecular Biology and Evolution 16: 1114–1116. Mycological Society 39: 401–415. Spatafora, J. W., Volkmann-Kohlmeyer, B. & Kohlmeyer, J. (1998) Independent terrestrial origins of the Halosphaeriales (marine Ascomycota). American Journal of Botany 85: 1569–1580. Sutherland, G. K. (1916) Additional notes on marine Pyrenomycetes. Transactions of the British Mycological Society 5: 257–263. Corresponding Editor: H. T. Lumbsch